o0

USP 43 THE UNITED STATES PHARMACOPEIA

NF 38 THE NATIONAL FORMULARY

Volume 1 Byauthority of the United States Pharmacopeial Convention.

Prepared by the CounCil of Experts and its Expert Committees

Offkm/rromMay 1, 2020

The designation on the cover of this publication, "USP NF 2020," is for ease of
identification only. The publication contains two separate compendia: The United
States Pharmacopeia, Forty-Third Revision, and The National Formulary,
Thirty-Eighth Edition.

THE UNITED STATES PHARMACOPEIAL CONVENTION

12601 Twinbrook Parkway, Rockville, MD 20852

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 SIX-MONTH IMPLEMENTATION GUIDELINE
The UnitedStates Pharmacopeia-National Formulary and itssupplements become official six months after being releasedto the
public. The USP-NF, which is released on November 1 of each year, becomes official on May 1 of the following year. This
six-month implementation timing gives users more time to bring their methods and procedures into compliance with new
and revised USP-NF requirements.
The table below describesthe official dates of the USP-NF and itssupplements. The 2019 USP 42-NF 37, and its supplements,
Interim Revision Announcements (lRAs) and Revision Bulletins to that edition, will be official until May 1, 2020, at which time the
USP 43-NF 38 becomes official.

Publication Release Date Official Date Official Until

USP43-NF38 November 1, 2019 May 1,2020 May 1, 2021 (except as superseded by supplements, IRAs, and
Revision Bulletins)
First Supplement to the February 1, 2020 August 1, 2020 May 1,2021 (except as superseded by Second Supplement, IRAs,
USP43-NF 38 and Revision Bulletins)
Second Supplement to the june 1, 2020 December 1, 2020 May 1, 2021 (except as superseded by IRAs and Revision Bulletins)
USP 43-NF 38
USP 2021 Issue 1 November 1, 2020 May 1,2021 May 1, 2022 (except as superseded by supplements, IRAs, and
Revision Bulletins)

The table below gives the details of the IRAs that will apply to USP 43-NF 38.

IRA PF Posting Date Comment Due Date IRAPosting Date IRA Official Date
46(1) january 2, 2020 March 31, 2020 May 29,2020 july 1,2020
46(2) March 2, 2020 May 31, 2020 july 31, 2020 September 1, 2020
46(3) May 1, 2020 july 31, 2020 September 25,2020 November 1, 2020
46(4) july 1, 2020 September 30, 2020 November 20, 2020 january 1, 2021
46(5) September 1, 2020 November 30, 2020 january 31, 2021 March 1, 2021
46(6) November 2, 2020 january 31, 2020 March 27, 2021 May 1, 2021

Revision Bulletins are published on the USP website and the USP-NF Online product and become official on the date specified
in the Revision Bulletin.

NOTICE AND WARNING

Concerning U.S. Patent or Trademark Rights-Substances, products, or equipment referenced or described in the USP-NF may
be associated with intellectual propertyrights, including but not limited to, registered trademarks,patents, and/or trade secrets.
Nothing inthe USP-NFshould be construedas a representation asto such intellectual property rights. Furthermore, the inclusion
in the USP-NF of a monograph, general chapter, or other reference addressing any substance, product, or equipment with
respect to which intellectual property rights may exist shall not be deemed, and is not intended as, a grant of, or authority to
exercise, any right or privilege protected by such patent, trademark,and/or trade secret.All such rightsand privileges are vested
intheir respective owners,and no other person mayexercise the same without expresspermission, authority, or license secured
from such owner.
Concerning Use of USP-NF Text-Attention iscalledto the fact that USP-NFtext isfully copyrighted. Authors and others wishing
to use portions of the text should request permission to do so from the Legal Department of the USPC, [email protected].
Copyright © 2019 The United States Pharmacopeial Convention 12601 Twinbrook Parkway, Rockville, MD 20852
All rights reserved.
ISSN 0195-7996 (print)
ISSN 1930-2932 (online)
ISBN 978-1-936424-95-5
Printed in the United States by United Book Press, Inc., Baltimore, MD

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 USP 43 iii

nte
VOLUME 1 NF 38
Admissions................................................................................. 5581
Mission Statement and Preface......................................................... v Articles Admitted to NF38 by Supplement.................................. 5581
People 2015-2020 Revision Cycle...................................................... ix New Articles Appearing in NF38 That Were Not Included in NF
Officers................................................................................................ ix 37 Including Supplements....................................................... 5581

Board of Trustees................................................................................. ix New Articles Appearing in NF38................................................. 5581

Council of Experts....................... ix Annotated List................................................................... 5582

Expert Committees................................. x USPand NFExcipients........................... 5584

In Memoriam....................................................................................... xvii NF38 Monographs.................................................................... 5597

Members and Delegates of the United States Pharmacopeial Con- Index.......................................................................................... 1-1
vention, as of May 31, 2019............................................................ xviii
2018 Recognition of Monograph and Reference Material VOLUME 4
Donors............................................................................................. xxv
Articles of Incorporation.................................................................... xxvii General Notices and Requirements ".......... 6111
USPGovernance........................ xxviii Guide to General Chapters....................................................... 6121
Bylaws................... xxviii Reagents, Indicators, and Solutions......................................... 6127
Rules and Procedures......... xxviii Reagent Specifications................................................................. 6131
USP Policies.......................................................................................... xxviii Indicators and Indicator Test Papers 6219
Admissions........... xxxi Buffer Solutions...... 6223
Articles Admitted to USP43 by Supplement..................................... xxxi ColorimetricSolutions......................... 6224
New Articles Appearing in USP 43 That Were Not Included in 'USP 42 Test Solutions.............................................................................. 6225
Including Supplements...................................................................... xxxi
VolumetricSolutions................................................................... 6237
Articles Included in USP 42 But Not Included in USP 43....................... xxxl
Chromatographic Columns......................................................... 6252
Annotated List............ xxxiii
Reference Tables....................................................................... 6259
General Notices and Requirements................................................... 3
Containers for Dispensing Capsules and Tablets.......................... 6259
Guide to General Chapters ·.... 13
Description and Relative Solubility of USP and NFArticles........... 6273
USP43 Approximate Solubilities of USP and NFArticles.......................... 6334
USP 43, A-I Monographs................................................................... 19 Atomic Weights.................................. 6344
Index 1-1 Half-Lives of Selected Radionuclides............................................ 6345
AlcoholometricTable.................................................................. 6346
VOLUME 2 IntrinsicViscosity Table............................................................... 6349
General Chapters
General Notices and Requirements. 2485
See page 6121 for detailedcontents
Guide to General Chapters ........................... 2495
i
General Tests and Assays............................................................. 6405
USP 43, J-Z Monographs................................................................... 2501
General Requirements for Tests and Assays.................................. 6405
Index................................................................................................. 1-1
Apparatus for Tests and Assays.................................................... 6445

VOLUME 3 Microbiological Tests.................................................................. 6449

Biological Tests and Assays.......................................................... 6488
General Notices and Requirements.................................................. 4725 Chemical Tests and Assays.......................................................... 6587
Guide to General Chapters............................................................... 4735 Physical Tests and Determinations............................................... 6819
Global Health Monographs.............................................................. 4741 Index.......................................................................................... 1-1
Dietary Supplements Monographs 4745

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iv USP 43

VOLUME 5

General Notices and Requirements.......................................... 7211

Guide to General Chapters....................................................... 7221
General Information Chapters..................................................... 7227
DietarySupplement Chapters..................................................... 8687
Index '-1

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 USP 43 Mission and Preface v

•IS •I nand r f e
F3 and Suppl m nt
Thissection provides background information on the New and revised monographs and general chapters and
United States Pharmacopeial Convention (USP), as well as omitted monographs from this edition are indicated in the
general information about the 43rd revision of the United Admissions section of the print publication and in the USP
States Pharmacopeia (USP 43) and the 38th edition of the Admissions List section of the USP-NF Online.
National Formulary (NF-38) Online (USP-NF Online or USP- USP-NF Organization-The USP-NF is published online
NF). Whether a document isofficial or not official will no longer and printed as a five-volume set. To facilitate convenient use
be linked to when a specific publication such as the main and reference, all five volumesincludethe combined index, as
USP-NF edition or one of the Supplements becomes official, well as the USP General Notices and the Guide to General
but instead will be based on the individual document. USP will Chapters. Volume 1 includes front matter (Mission and Preface,
continue to publishthe USP-NF standards on a set schedule, People, governance pages and websites, and Admissions/
and each individual document will be associatedwith the Annotations) and USP monographs A-I. Volume 2 includes USP
indicated official date. Learn more about USP's transition to monographs J-Z. Volume 3 includes Global Health
document-centric official dates on our website (https:// monographs, DietarySupplements monographs, NF
www.uspnf.com/new-uspnf-online-faqs/end-user#irg 1). Admissions/Annotations, Excipients, and NF monographs.
Volume 4 includes general chapters numbered below 1000
MISSION STATEMENT (GeneralTests and Assays-including chapter charts),
Reagents, and Reference Tables. Volume 5 includes general
USP-NF is published in continuing pursuit of the mission of chapters numbered above 1000 (General Information) and
USP: To improve global health through public standards and Dietary Supplements general chapters. General chapters
relatedprograms that helpensure the quality, safety, and benefit specific to dietarysupplementsare includedin numerical order
of medicines and foods. with the rest of the general chapters in USP. Excipient
monographs are usually presented in NF but also may appear
HISTORY in USP with suitablecross-referencing when they are also drug
USP has a rich history, dating back to 1820, when 11 substances. The Excipients section (Volume 3) presents a
physicians met in the Senate Chamber of the U.S. Capitol tabulation of excipients byfunctional category.
building to establish a pharmacopeia for the United States. [NOTE-USP43-NF38 is the last publication that will be
Learn more about USP's historyand major milestones on the available in print as a five-volume set. Future publications will
USP website (https://www.usp.org/about/usp-timeline). not be printed; only the online format will contain all current
USP-NF content.]
CONTENT OF USP-NF Revisions to USP-NF-USP-NFis continuously revised by
an exceptional process of public involvement and substantial
USP-NF contains official substance (ingredient) and interaction between USP and its stakeholders, both
product monographs for official articles recognized in USP-NF domestically and internationally. Revisions are presented in
(see General Notices 2.20 OfficialArticles). USP-NFalso includes the USP-NFthree times per year in November, February, and
monographs for compounded preparations. With few June. Accelerated Revisions [Errata, Interim Revision
exceptions, such as articles covered by Global Health Announcements (IRAs), and Revision Bulletins] are published
monographs, the intent isthat all articles for which periodically and with greater frequency.
monographs are provided in USP-NF are legally marketed in Standard Revisions--USP's standard revision process calls
the UnitedStatesor are contained in legally marketed articles. for publication of a proposed revision in the Pharmacopeial
Global Health monographs are providedfor articles that are Forum (PF) for a 90-day notice and comment period and,
not approved or legally marketed inthe United States, but that after the revision is approved by the relevant USP Expert
have been approved by a stringent regulatory authority [as Committee, publication in the USP-NF.
defined by the World Health Organization (WHO)] and are AcceleratedRevisions--The Accelerated Revision process is
used for essential purposes in other parts of the world. used to make revisions to USP-NF official more quickly than
A USP-NF monograph for an official substance, product, or through USP's StandardRevision process. Learn more about
preparation may consistof various components, including the Revision Bulletins, Interim Revision Announcements (IRA),
article's name; definition; packaging, storage, and other Errata, and the criteria for and implementation of each on
requirements; and a specification. General chapters provide the USP website (http://www.uspnf.com/official-text).
frequently cited procedures, sometimes with acceptance Modification of Compendial References-USP and its
criteria, in order to compile into one location repetitive Expert Committees periodically deem it necessary to modify
information that is applicable to many monographs. See general chapter titlesor similar text that may be referenced in
General Notices 3.10 Applicability of Standards for more other standards.throughout the USP-NF. When this occurs,
information about standards contained in USP-NF USP staff undertake a rigorous processfor identifying and
monographs and general chapters. updating such references. These updates may occur through
a routine revision, or, in cases in which an update appears to

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vi Mission and Preface USP 43

present no significant change in the affected standard, comment. If comments are received, they are considered and
through a direct update of the reference in that standard incorporated as appropriate by the Expert Committee(s). In
without providingan opportunity for notice and comment. In cases where proposals advance to official status without
allcases, USP will publishon itswebsitea notice indicating the republication in PF, a summaryof comments received and the
source change, any resulting references, and whether those appropriate Expert Committee's responses are published in
references will be updated through a routine revision or a the Commentarysection of the USP website at the time the
direct update. revision is published.
Updating Chemical Information-Updates to the . The Commentary is not part of the official text and is not
Chemical Information section at the beginning of monographs intended to be enforceable by regulatory authorities. Rather,
occur on an ongoing basisand are not identified with revision it explains the basis of the Expert Committee's response to
symbols. Chemical names and molecular weights are updated publiccomments. If there isa difference between the contents
when a monograph undergoes revision to match the official of the Commentary and the official text, the official text
source, United States AdoptedNames (USAN). Chemical prevails. In case of a dispute or question of interpretation, the
structures are updated on a continuous basis. language of the official text, alone and independent of the
Chemical names typically reflectthe naming conventionsat Commentary, shall prevail.
the time of the monograph development or revision. If the Print and Electronic Presentations-See General Notices
nomenclature rulesof CAS or IUPAC are significantly changed, 2.70 Official Textfor more information about USP-NF product
the chemical names can be revised or added to implement formats. USP43-NF38 is the last publication that will be
those rules. Molecular weights are derivedfrom the chemical available in print or on a flash drive. Future publications will
formula and are based on the table of atomic weights.Atomic not be printed or on flash drives; onlythe online format will
weights are recommended by the IUPAC and reflect the contain all current USP-NF content.
isotopic composition of normal terrestrial material. When the USP-NF Translations-Translations of the USP-NF are
IUPAC recommended valuesare changed, it isunderstood that available in Spanish, Russian, and Chinese. The Spanish
the changes in molecularweights will be made in due course. translation iscurrent; other translations are based on previous
Graphical representation of the chemical compound revisions of the USP-NF.
structures isintended as a visual aid to help establish chemical USP Reference Standards-The use of USP Reference
identity and is understood to represent one of many possible .Standards promotes uniform qualityof drugs and supports
ways to depict the molecule. Addition of a graphical reliability and consistency by those performing compliance
representation or changes in such representation, that result testing and other users of USP-NF, including manufacturers,
in the same chemical information, e.g., a flipped chiral buyers, and regulatory authorities. USP Reference Standards
molecule or adding a moleculestructure, may be introduced are referencedin specific proceduresin both monographs and
outside of the revision process. It isalsounderstood that in the general chapters. USP advances this materialvia careful
case of tautomerism, the moleculedepicted may be one of the characterization studiesand collaborative testing, followed by
tautomers, but it is intended to represent all isomers in review and approval of the compendial use of the reference
equilibrium. Stereogenic centers depicted with plain bonds material by Expert Committees of the Council of Experts. This
imply mixtures of pertinent stereomers-enantiomer, program benefitsfrom the Widespread voluntarycontribution
diastereomers, epimers (anomers), etc. . of suitable materials and test data from pharmaceutical
Depending on the timing of these updates, users may see a manufacturers. The USP Catalog, which lists the collection of
differencein a chemicalstructure between the publications in USP Reference Standards, and more information about use
PFand USP-NF, and between the USP-NF and the USP-NF and storage, can be accessed on USP's website (https://
Online. www.usp.org/reference-standards/
Shading and Symbols-Shading is used to identify text reference-standards-cataloq).
that has been modified, added, or deleted since it was last
published. Symbols identify the beginning and end of each USP GOVERNING, STANDARDS-SEn-ING, AND
revision or nonharmonized text. The following table ADVISORY BODIES
summarizesthe types ofsymbolsand the associatedsubscripts USP's governing, standards-setting, and advisory bodies
used in USP publications: include the USP Convention, the Board of Trustees, the
Council of Experts and its Expert Committees, Expert Panels,
Revision Type Revision Symbol and Text Subcommittees, Joint Standard-Setting Subcommittees, and
4
4 (USPl-May-2020)
staff. Additional volunteer bodies include StakeholderForums
In Process Revision (Book or
and ProjectTeams, which offer stakeholdersthe opportunity
Supplement)
4
4 (NF l-May-2020) to contribute, through advice and recommendations, to the
4
4 (GH l-May-2020)
advancement of USP's standards and processes. Learn about
the composition and work of the USP Convention, Board of
Interim Revision Announcement 4
1-)u~2020)
4(IRA Trustees, Expert Committees, and Expert Panels on the USP
website (https://www.usp.org/about/
..
4
Revision Bulletin 4(RB 1-1ul-2020)
convention-membership). Learn more about Stakeholder
Harmonization Forums and Project Teamson the USP website (https://
4
www.usp.org/get-involved/provide-input/
Errata HU~2020)
4(ERR
stakeholder-forums). Alisting of all current Voting Delegates
4
4(CN 1-)ul-2Q20) of the USP Convention and members and Government
Chapter References
4
Liaisons to the Council of Experts and its Expert Committees
4(Offidall-JuI-2020)
and Expert Panels is included in the People section.
Pending 4
4 (TBD)
Working with the Food and Drug Administration-USP
works with the Secretary of the Department of Health and
Human Services, and the principal agency in the Department
Commentary-For revisions that are publishedfor public for this work is the Food and Drug Administration (FDA). USP
reviewand comment in PF, the proposal may advance to works in many wayswith the agency, but the primary
official status or be republished in PFfor further notice and interaction is through the Government Liaison Program. The

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 USP 43 Mission and Preface vii

Government Liaison Program allows representatives of FDA that such requirements apply to a particular biologic product.
and other government agencies to participate in Expert This is equally so for biologics approved under the
Committee and Expert Panel meetings, enabling interactions long-standing PHS Act "351(a)" pathway, as well as the new
between government staffand Expert Committees.Staffinthe "351 (k)" pathway for biosimilars added by the 2010
FDA Centers who are responsible for review of compendial healthcare reform legislation (Biologics Price Competitionand
activities providespecific links and opportunities for exchange Innovation Act,Title VII, Subtitle Aof the Patient Protection
of comments. The Office of Policy for Pharmaceutical Quality and Affordable Care Act, Public Law 111-148).
in the Center for Drug Evaluation and Research provides a Dietary Supplements-The Dietary Supplement Health
primarycompendial point of contact between FDA and USP. and Education Actof 1994 amendments to the FD&C Act
provide that a dietary supplement may be deemed a
RULES AND PROCEDURES misbranded food if it is covered by the specifications of an
official compendium (e.g., USP-NF), is represented as
Governing Documents-USP-NF standards are conforming to the specifications of an official compendium,
recognized widelybecause they are authoritative and and fails to so conform. This contrasts with pharmaceutical
science-based and are established by a transparent and products,wherein conformance to applicablecompendial
credible process.Seethe Articles of Incorporation section inthis standards is mandatory, whether or not the product claims to
book; the Bylaws (https://www.usp.org/about/ conform.
convention-membership/bylaws), and the Rules and Compounding-USP providesgeneral chapters and
Procedures of the Council of Experts (https://www.usp.org/ monographs for compounded preparations, as well as
about/leadership/policies-rules). Collectively, these monographs for bulksubstances used in compounding. USP
documents serve USP volunteers and staffas the governing standardsare recognizedinvariousprovisions ofthe FD&C Act
principles for USP's standards-setting activities. and such provisions do not differentiate between
manufactured and compounded medicines. Moreover, the
LEGAL RECOGNITION FD&C Act and the 2013 Drug Quality and Security Act
Recognition of USP-NF-USP-NFis recognized by lawand specifically reference USP standards for compounding. Learn
custom in many countriesthroughout the world. In the United more about compounding on the USP website (https://
States, the Federal Food, Drug, and CosmeticAct(FD&C Act) www.usp.org/compounding). '
definesthe term "official compendium" asthe official USP, the Medical Devices-Section 201(h) of the FD&C Actdefines
official NF, the official Homeopathic Pharmacopeia of the United a deviceas an instrument, apparatus, similar article, or
States, or any supplement to them. USP-NF standards playa component thereof recognized in USP-NF. Section 502(e) of
role in the adulteration and misbranding provisions of the the FD&C Act definesthe established name of a device in the
FD&C Act, which apply as wellto biologics, a subset of drugs, absence of an FDA designation of the official name as the
under the Public Health Service Act (see General Notices 2.30 official title in an official compendium. Despite these statutory
Legal Recognition). provisions, there isno comparable recognitionof USP's rolein
FDA requiresthat namesfor articles that are not official must establishing compendial standards for medical devices as
be clearly distinguished and differentiated from any name exists for drugs and biologics. Under authority granted by the
recognized in an official compendium. Drugs with a name Foodand DrugAdministration Modernization Actof 1997, the
recognized in USP-NF alsowill be considered misbranded Centerfor Devices and Radiological Health recognizes national
unless they meet compendial standards for packaging and and international standards, includingsome USP tests and
labeling (see General Notices 3.10.10 Applicability of Standards assays, for medical devices.
to Drug Products, Drug Substances, and Excipients for more Nomenclature-For information on the nomenclature
information). . development process, the Nomenclature and Labeling. Expert
Drugs-USP's goal isto have substance and drug product Committee, and USP's work with United States Adopted
monographs in USP-NFfor FDA-approved drugs inthe United Names(USAN), see the USP website (https://www.usp.org/
States, including chemicaland biologic medicines, and their expert-committees/
ingredients. USP also provides monographs in USP-NFfor nomenclature-and-Iabeling-expert-committee-work-plan).
legally marketed therapeutic products not approved by FDA, Chemical Names and CAS Registry Numbers-Chemical
e.g., pre-1938 drugs, over-the-counter(OTC) drugs marketed subtitles given in the monographs are index names used by
under FDA's OTC Monograph system, dietary supplements, the Chemical Abstracts Service (CAS) of the American
and compounded preparations. The GlobalHealth section of Chemical Society. They are provided only in monographs in
USP-NF contains monographs for articles that are not which the titles specify substances that are definable chemical
approved or legally marketed in the United States, but that entities. Thefirstsubtitle isthe invertedform of the systematic
have been approved by a stringent regulatory authority (as chemical name developed by CAS for the purpose of the
defined by WHO) and are used for essential purposes in other Collective Index (CI). The second subtitle, given in uninverted
parts of the world. Conformance with a USP-NF monograph, form, isa preferred IUPAC name (PIN) sanctioned and used by
ifapplicable, is requiredat alltimes in the life of an articlefrom the International Union of Pure and Applied Chemistry
production to expiration. (IUPAC). Preferred IUPAC names also are used by the WHO.
Biologics-In the United States,biologics are consideredto Occasionally a third subtitle is supplied for historical reasons
be a subset of drugs, whether they were approved by FDA or when the synonym uses an alternative, but equivalent,
under the FD&C Act [and received a new drug application naming convention. Monographs with chemical subtitles also
(NDA)] or under the Public Health Service Act[PHS Act, where generally carry CAS registry numbers. These bracketed
they receive a biologics license application (BLA)]. As of March numbersfunction independently of nomenclatureas invariant
23,2020, applicationsfor biological products approved under numerical designators of unique, unambiguous chemical
the FD&C Act will be deemed to De licenses for the biological substances in the CAS registry, and thus are convenient and
products under the PHS Act. All PHS Act biologics are subject widely used.
to the applicable drug regulatoryrequirements of the FD&C
Act, which means they are required to comply with the HARMONIZATION ACTIVITIES
adulteration and misbranding provisions of the FD&C Act, USP participates in several collaborative activities with
including USP-NF compendial requirements, to the extent global pharmacopeias in both bilateral and multilateral

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 viii Mission and Preface USP 43

settings. Examples of USP's current activities includethe formats. Proposed revisions to FCC are available for public
following. viewing and comment through the FCC Forum; The FCC Forum
Pharmacopeial Discussion Group-USP harmonizes can be accessed free of charge at http://
pharmacopeiaI excipient monographs and general chapters www.foodchemicalscodex.org.
~hrough the Pharma.copeial Discussion Group (PDG), which
Inc!udes representatives from the European, Japanese, and OTHER USP RESOURCES
United .States pharmacop~i~s~ and WHO (as an observer).
According to the PDG definition, "a pharmacopeial general Chromatographic Columns-This comprehensive
chapter or other pharmacopeial document is harmonized reference, previously titled Chromatographic Reagents
when a pharmaceutical substance or product tested by the provides detailed information needed to conduct '
document's harmonized procedure yields the same results chromatographic procedures found in USP-NF.
and the same accept/reject decision is reached." Informati~n Chromatographic Columns lists the brand names of the column
regarding PDG, including history, the PDG working reagents cited in every proposal for new or revised gas- or
procedure, a glossary, and lists of monographs and general liquid-chromatographic analytical procedures that have been
chapters that have completed stages 1-4 of the published in PF since 1980. Chromatographic Columns also
pharmacopeiaI harmonization process, resulting in an helpsto track which column reagents were used to validate
approved USP Stage 4 Harmonization text is available on analytical procedures that have become official. The branded
USP's webs~te (https:/ /~ww.usp.org/harm~nized-standards). column reagents listis updated bimonthlyand maintained on
Internatl~nal Meetmg of the World Pharmacopeias-
USP's website.
USP workswith WHO and global pharmacopeial partners on USP Dictionary-The USP Dictionaryof USAN and
the s~rategy and establishment of Good Pharmacopoeial International Drug Names is an online resource containing the
m~s~ up-to-date United States Adopted Namesof drugs;
)ractlce~ (GPhP) a~ a set of guiding principles for the
appropriate establishment of pharmacopeial standards. offlclal USP-NF names; nonproprietary, brand, and chemical
Adopt!Adapt Agreements-USP grants the rightsto copy names; graphic formulas; molecularformulas and weights;
and/or adapt USP standards for use in other pharmacopeias CAS registry numbers and code designations; drug
through this formal mechanism. manufacturers; and pharmacologic and therapeutic '
Bilat~~al Agreements-USP partners with pharmacopeias
categories. The Dictionary helps to ensure the accuracyof the
for the JOint development of pharmacopeial standards uslnq following: product labeling; reports, articles, and
this informal process. correspondence; FDA regulatory filings; and pharmaceutical
USP Exchan.ge ~~ograms-USP regularly welcomes the package inserts. It is publishedannually. Formore information
exchange of scientific personnel through this program with about the Dictionary see the USP website (https://
the g~al ~f sh~ring scie!1tific knowledge among global' www.usp.org/products/usp-dictionary).
organizations Involved In standards setting and the effective
use of standards.

OTHER USP COMPENDIA

USP Co.mp?unding .Compendium:- The USP Compounding
Compendium IS an online compendium that includes all
compounding-related general chapters from the USP-NF as
well as the supporting general'chapters that are referenced in
the compounding general chapters and in GENERAL NOTICES
AND REQI!IRE~ENTS. ~he purpose of ~he USP Compounding
Compendium IS to provide compoundinq practitioners with
convenient access to associated general chapters. .
USP Herbal MedicinesCompendium-The USP Herbal
Medicines Compendium (HMC) is an online compendium that
helps ensure the quality of the herbal ingredients used in
herb~! m~dicines. HMC monographs provide quality
s~eclflC~tlons-tests, procedures, and acceptance criteria-
with validated analytical procedures and allied reference
materials that aid in conformity assessment. HMC can help
ingredient manufacturers, herbal product manufacturers
regulatory agencies, and other stakeholders to assess '
conformance of herbal medicinal ingredientswith
independent public standards and to control the quality of
articles moving in international commerce. The HMC is
available at https://hmc.usp.org.
USP Dietary Supplements Compendium-The Dietary
Supplements Compendium isan online compendium that
combines USP-;NF stan~ards for dietary supplements,
standards and inforrnation from the Food Chemicals Codex
regulatory and industry documents, and other tools and '
resources. I

Food Chemicals Codex-The Food Chemicals Codex (FCC) is

a compendium of internationally recognized monograph
standards and tests for the purity and qualityof food
ingr~dients, e.9., pre~ervatives, flavorings, colorings, and
nutrren.ts. FCC IS published everytwo years, with supplements
everySIX months, and is available in print and electronic

www.ilovepharma.com
USP 43 People / Committees ix

2015-2020 Revision Cycle

Officers of the USP Convention, Board of Trustees,
and the Council of Experts, Expert Committees,
Expert Panels, and Advisory Groups

Bethesda, MD
Officers (2015-2020) Susan C. Winckler, R.Ph., J.D.
Trustee At-Large
Jesse L. Goodman, M.D., M.P.H. Alexandria, VA
President Ron T. Piervincenzi, Ph.D.
Washington, DC ChiefExecutive Officer, February 2014-present
Timothy R. Franson, B.S. Pharm., M.D. (ex-officio)
Past President Rockville, MD
Indianapolis, IN
John E. Courtney, Ph.D.
Treasurer Council of Experts (2015-2020)
Bethesda, MD
Susan S. de Mars, J.D. Jaap Venema, Ph.D.
Secretary Chair, Council of Experts
Rockville, MD Rockville, MD
Pascal Anger, Ph.D.
Chair, Biologics Monographs 4-Antibiotics
Board of Trustees (2015-2020) Verrleres-le-Bulsson, France
Richard A. Blessing, M.S.
Thomas R. Temple, B.S. Pharm., M.S., F.A. Ph.A. Chair, Chemical Medicines Monographs 1
Chair North Chicago, IL .
Trustee At-Large Edward K. Chess, Ph.D.
Des Moines, IA Chair, Biologics Monographs 3-Complex Biologics
Gregory E. Amidon, Ph.D. McHenry,lL
Trustee Representing the Pharmaceutical Sciences Stephanie Y. Crawford, Ph.D., M.P.H.
Ann Arbor, MI Chair, Nomenclature and Labeling
Laura Herman, M.B.A., M.A. Chicago,lL
Trustee Representing the Public Gigi S. Davidson, B.S. Pharm., DICVP
New Canaan, CT Chair, Compounding
Robert J. Meyer, M.D. Plttsboro.. NC
Trustee Representing the Medical Sciences Michael R. De Felippis, Ph.D.
Charlottesville, VA . Chair, Biologics Monographs I-Peptides and Insulins
Marilyn K. Speedie, Ph.D. Indianapolis, IN
Trustee Representing the Pharmaceutical Sciences James E. De Muth, Ph.D., R.Ph.
Minneapolis, MN Chair, General Chapters-Dosage Forms
Stephen P. Spielberg, M.D., Ph.D. Madison, WI
Trustee Representing the MedicalSciences Jonathan W. DeVries, Ph.D.
Upeer Gwynedd, PA Chair, Food Ingredients
Gad R. Wilensky, Ph.D. . Coon Rapids, MN
Trustee At-Large Dennis E. Doherty, M.D., FCCP
Chair, Healthcare quality and Safety

www.ilovepharma.com
x Committees / People USP 43

Lexington, KY Pharm.D.; Caroline R. Weinstein-Oppenheimer,

Mary G. foster, Pharm.D., BfA Pharm.D.
Chair, General Chapters-Packaging and Distribution
Fort Collins, CO
Dennis K.J. Gorecki, B.S.P., Ph.D. Expert Committees for the United
Chair, Non-Botanical DietarySupplements
Saskatoon, Saskatchewan, Canada States Pharmacopeia
Xlaorong He, Ph.D.
Chair, General Chapters-Physical Analysis Nomenclature and Labeling
Ridgefield, CT STEPHANIE Y. CRAWFORD, PH.D., M.P.H., Chair
Mary C. Houck, Ph.D. Mary B. Baker, Pharm.D.; Dawn M. Boothe, D.V.M., Ph.D.;
Chair, Excipient Monographs 2 Mike Cohen, M.S.; Elizabeth Igne Ferreira, Ph.D.; Karen
FortCollins, CO Hauda,J.D., M.S.; KentJohnson, M.Pharm.; Armen
David Hussong, Ph.D. Melikian, Ph.D.; Ginette A. Pepper, R.N., Ph.D., FAAN;
Chair, General Chapters-Microbiology Thomas P. Reinders, Pharm.D.; Joanne G. Schwartzberg,
Kensington, MD M.D.; Maged Sharaf, Ph.D.; Eric Sheinin, Ph.D.; Thomas
Kim C. Huynh-Ba, M.S. R. Tice, Ph.D.; Claudia Vincenzi, Ph.D., Pharm.D.; Gillian
Chair, Chemical Medicines Monographs 4 Woollett, Ph.D.
Newark, DE
Amy J. Karren, B.S. Pronunciation Expert Panel
Chair, Chemical Medicines Monographs 5 WIlliAM M. HELLER, PH.D., Chair
Flagstaff, AZ Mary B. Baker, Pharm.D.; Stephanie Y. Crawford, Ph.D.,
Nancy Lewen, B.S. M.P.H.; Doreen J. Elston, Pharm.D.; Kent Johnson,
Chair, General Chapters-ChemicalAnalysis M.Pharm.; Joan c. May, Ph.D.; Anthony Palmieri,
Owasso, OK Ph.D.; Ginette A. Pepper, R.N., Ph.D., FAAN; Thomas
Robin J. Maries, B.S., M.S., Ph.D. P. Reinders, Pharm.D. .
Chair, Botanical DietarySupplements and Herbal Medicines
Ottawa, Ontario, Canada Healthcare Quality and Safety
Michael G. Mulkerrln, Ph.D. DENNIS DOHERTY, M.D., FCCP, Chair
Chair, Biologics Monographs 2-Proteins TimothyAlbertson, Ph.D.; Phil Ayers, Pharm.D.; Danial E.
Hillsborough, CA Baker, Pharm.D.; Mark Decerbo, Pharm.D.; Peter
Eric J. Munson, Ph.D. Glassman, M.B.B.S.; Roy Guharoy, Pharm.D., M.B.A.;
Chair, Excipient Monographs 1 Raymond Hohl, M.D.; Duane Kirking, Ph.D.; Raymond
West Lafayette, IN Love, Pharm.D.; Melody Ryan, Pharm.D.; Joanne
Bernard A. Olsen, Ph.D. Schwartzberg, M.D.; Patricia Sokol, J.D.; J. Russell
Chair, Chemical Medicines Monographs 3 Teagarden, D.M.H.; .JeanneTuttle,.B.S. Pharm.; Terri
Wake Forest, NC Warholak, Ph.D.; HSiang Shonna Yin, M.S.
Ernest Parente, Ph.D. Drug Allergy and Intolerance Classification Expert
Chair, Chemical Medicines Monographs 2 Panel
Overland Park, KS RAYMOND LOVE, PHARM.D., Chair
Robert R. Singer, M.S. Dennis Doherty, M.D., FCCP; Gay Dolin, M.S.N.; Roy
Chair, General Chapters-Statistics Guharoy, Pharrn.D; M.B.A.; Robert Hausam, M.D.;
Union City, CA Russell leftwich, M.D.; Robert McClure, M.D.; Gerald
Reinhard Walter, Ph.D. McEvoy, Pharm.D.; Tejal Patel, Pharm.D.; Seth Powsner,
Chair, Chemical Medicines Monographs 6 Ph.D.; GeorgeAllen Robinson, B.S. Pharm.; Shelly Spiro,
Whippany, NJ B.S. Pharm.
Wesley E. Workman, Ph.D.
Chair, General Chapters-BiologicalAnalysis Exchange of Compounded Drug Preparation
Saint Charles, MO Information in Health IT System Expert Panel
ROY GUHAROY, PHARM.D., M.B.A., ANDRICHARD PARRISH, PH.D.,
Co-Chairs
Expert Committees (2015-2020) David Aguero, Pharm.D.; Sandra Benavides, Pharm.D.;
Scott Louis Ciarkowski, Pharm.D.; Catherine Graeff,
[NOTE-The following listing of Expert Committees M.S.; John T. Kilbourne, M.D.; Leanne Murray,
includes the Expert Panels that serve in an advisory Pharm.D.; Tammy.P. Powell, M.S.; Seth Strawbridge,
capacityto the specific Expert Committee. The listing Pharm.D.; Kathy Vleson, Pharm.D.
of Expert Panels and their membership represents Health Literacy Expert Panel
those that have been fully formed and approved as of JOANNE G. SCHWARTZBERG, M.D., Chair
July 2015. Expert Panels are continuouslyformed and CindyBrach, MPP; Joseph Chebli, Pharm.D.; Terry Davis,
concluded throughout the USP revision cycle, and PIi.D.; Radhika Devraj, Ph.D.; Dennis Doherty, M.D.,
other membership listings will appear in the future.] FCCP; Gerald McEvoy, Pharm.D.; Juliet Nguyen,
Pharm.D.; Ruth Parker, M.D.; Seth Powsner, Ph.D.; N.
Lee Rucker, M.S.P.H.; Patricia Sokol, J.D.; J. Russell
Expert Panels for the Council of Teagarden, D.M.H.; Keith Trettin, B.S. Pharm.; Hsiang
Shonna Yin, M.S.
Experts Executive Committee
Drug Classification Expert Panel
Spanish Translation Expert Panel DUANE M. KIRKING, PH.D., Chair
OSCAR QUATIROCCHI, M.S., Chair _.: Danial E. Baker, Pharm.D.; Dennis Doherty, M.D., FCCP;
Ofelia Espejo Gonzalez, Ph.D.; Monica I. Hirschhorn, M.S.; Lauren Hoffman, Pharrn.Dc Gerald McEvoy, Pharm.D.;
Francisco Kuribrena, Ph.D.; Pilar Pais, Ph.D.; Jose Marfa Seth Powsner, Ph.D.; N. Lee Rucker, M.S.p.H.; Brian
Parisi, M.S.; Luisa Fernanda Ponce De Leon, Pharm.D.; Solow, M.D.; J.Russell Teagarden, D.M.H.; JeanneTuttle,
Mauricio A. Seigelchifer, Ph.D.; Ofelia Villalva-Rojas, B.S. Pharm.; Dennis West, Ph.D.

www.ilovepharma.com
USP 43 People / Committees xi

Parenteral Nutrition Safety Expert Panel Aurelie Mieze-Richard, Pharm.D.; Patrick Noland, M.S.;
PHIL AYERS, PHARM.D., Chair Scott Roberts
Mary B. Baker, Pharm.D.; Elizabeth Bobo, M.S.; Denise
Bohrer, Ph.D.;Joseph Boullata, Pharm.D.; MarkDecerbo, Radioactive Drugs Expert Panel
Pharm.D.; Dennis Doherty, M.D., FCCP; Kathleen M. JAMES A. PONTO, M.S., Chair
Gura, Pharm.D.; Deborah Houston, Pharm.D.; Matthew Corinne Bensimon, Ph.D.; jonathan M. Fitzsimmons, Ph.D.;
T. jenkins, M.S.; Gordon S. Sacks, Pharm.D.; Maureen , Un:!esh Gangadharmath, P~.D.; Kim C. Huynh-Ba, M.S.;
Schanck, Pharm.D.; David S. Seres, M.D.; Connie Rae Thijs Kroon, Pharm.D.; Adrian Nunn, Ph.D.; David Pipes,
Sullivan, B.S. Pharm.; Patricia Worthington, M.S.N. Ph.D.; Kara Weatherman, Pharm.D.; Martin Williamson
Ph.D.; Steve Zigler, Ph.D. '
Chemical ,Medicines Monographs 1
RICHARD A. BLESSING, M.S., Chair Chemical Medicines Monographs 5
Gennady A!1anchenko, Ph.D.; Elizabeth (Betsy) Cariello, AMY j. KARREN, B.S., Chair
B.A.; Alain Duguet, Ph.D.; Rupa Iyer, M.S.; Monika Jain D.J. poan, M.S; S.ushil Gangwal, Ph.D.; Min Li, Ph.D.; Judy
Ph.D.; Greg Kaster, M.S.; Jasmina Novakovic, Ph.D.; jeff lin, M.S.; Pau!lne McGregor, Ph.D.; Marian Meyer,
Rohrer, Ph.D.; David Schuck, Ph.D.; jan Srbek, Ph.D.; Ph.D., t\1:B.A., Jonathan Parks, B.S.; Justin Pennington,
Giordano Trazzi, Ph.D. Ph.D.; Vljaya Ramesh, B.Pharm.; Gurvinder Singh Rekhi,
Ph.D.; Iffaaz Salahudeen, Ph.D.; MaryW.Seibel B.S.'
Chemical Medicines Monographs 2 Hameraj Singh, Ph.D.; Michael Skibic, M.S. ' ,
ERNEST PARENTE, PH.D., Chair
Mahmoud AlOmari, Ph.D.; Allan Bokser, Ph.D.; Matthew Chemical Medicines Monographs 6
Borer, Ph.D.; jama Elmi, Ph.D.; Michael Koberda, Ph.D.; REINHARD WALTER, PH.D., Chair
joan C. May, Ph.D.; Beth L. Minter, B.S.; Maria Ines Seamus Boland, B.S.; RobertGraham Buice, Ph.D., M.B.A.;
Santoro, Ph.D.; Jeff Schwartzenhauer, M.S.; Dennis Timothy Gilmer, Ph.D.; Carmen Gonzalez, Ph.D.; John
Stephens, Ph.D.; Luciano Virgili, Ph.D.; Zhenyu Wang joseph Hemes, Ph.D.; Todd Lewis, M.S.; William Long
Ph.D.; joseph Yakupkovic, Ph.D.; Patrick Vat, Ph.D. ' Ph.D.; Phil Nethercote, Ph.D.; Raphael Ornaf, Ph.D.; ,
Davi~ H. Rogers, Ph.!:?; .Thomas Rosanske, ~h.D.; Donna
Chemical Medicines Monographs 3 S. Seibert, Ph.D.; Christina Szabo, Ph.D.; Xiaopinq
BERNARD A. OLSEN, PH.D., Chair Wang, Ph.D.; Kylen Whitaker, Ph.D.; Zeena Williams,
Samuel Akapo, Ph.D.; Bianca Avramovitch, Ph.D.; Amy Ph.D.
Barker, Ph.D.; Lynn Blessing, M.S.; Thomas Broadbent
Ph.p.; Ian Chung, P~.D.; Debashis Das, Ph.D.; jeffrey' Acetaminophen OTC Expert Panel-CONCLUDED
Heitman, Ph.D.; Yurl Goldberg, Ph.D.; Eric Kesslen, KYLEN WHITAKER, PH.D., Chair
Ph.D.; Pauline Lacroix, M.S.; Donald Parsons, Ph.D.; Tina M. Engel, Ph.D.; David A. Fay, Ph.D.; Saulius A. Gylys'
David Reed, M.B.A.; Murugan Saravanan, M.S.; joseph Michael T. Rankin, M.S.; David H. Rogers, Ph.D.; GregorY
Stowell, Ph.D. K. Webster, Ph.D.; jonathan Zeszotarski, Ph.D.
Chemical Medicines Monographs 4 Biologics Monographs 1-Peptides and Insulins
KIM C. HUYNH-BA, M.S., Chair MIKE DE FELIPPIS, PH.D., Chair
josep M~ria d~ Ciurana Gay" M.S.; Simona Dragan, Ph.D.; Wilfried Arz, Ph.D.; Chaim Eidelman, Ph.D.; GyongyiGratzl,
Natalia Borisovna Epshteln, Pharm.D., M.S.N.; Quanyin Ph.D.; Gerhard Haas, Ph.D.; Morten Hach, M.S.; Marion
Gao, Ph.D.; jerome M. Lewis, Ph.D.; Oscar Liu, Ph.D.; King, Ph.D.; Peter Larsson; Jean-Marc Poudrel, Ph.D.;
Annarapu Malleswarareddy, Ph.D.; Mariann Harold Rode, Ph.D.; Raimon Rubires, Ph.D.;Ved
Neverovitch, M.S.; Patrick Noland, M.S.; james A. Ponto, Srivastava, Ph.D.; Michael Verlander, Ph.D.
M.S.; Hemant KumarSharma, Ph.D.; William Clatiramer Expert Panel
Taraszewski, Ph.D.; Martin Williamson, Ph.D.; Min Xia GYONGYI GRATZL, PH.D., Chair
Ph.D.; Steve S. Zigler, Ph.D. ' Joseph Louis Glajch, Ph.D.; Satyanarayana Kota, Ph.D.;
(821) Identification and Assay of Radionuclides and Barbara Mulloy, Ph.D.; Todd A. Osiek, Ph.D.; Marcia
(1821) Radioactivity Theory and Practice and (1823) Cecilia Rusjan, Ph.D.; Rakesh Singh Shekhawat, Ph.D.;
Drugs for Positron Emission Tomography Expert ReneThuermer, Ph.D.; Patrick Vallano, Ph.D.; Michael
Panel-CONCLUDED Verlander, Ph.D; Vera Weinstein, Ph.D.
SALLY W. SCHWARZ, M.S., Chair Clucagon Expert Panel.....CONCLUDED
Cathy Sue Cutler, Ph.D.;jonathan M. Fitzsimmons, Ph.D.' HAROLD RODE, PH.D., Chair
Paula M. Jacobs, Ph.D.; Thijs Kroon, Pharm.D.; Jerome' jan Amstrup, Ph.D.; Matthew W. Borer, Ph.D.; Gerhard
M. Lewis, Ph.D.; Roger Moroney, M.S.; james A. Ponto Manfred Haas, Ph.D.; Anne Munkjespersen, Ph.D.;
M.S.; Duann V. Thistlethwaite, B.S.; Steven S. Zigler, ' Elizabeth Kramer, Ph.D.
Ph.D.
Insulin Expert Panel
(825) Radiopharmaceuticals Compounding Chapter HEATHER BOUX, PH.D., Chair
JAMES PONTO, M.S., Chair JanAmstrup, Ph.D.; Wilfried Arz, Ph.D.; Chris Burns, Ph.D.'
David Barnes, B.S. Pharm.; Allegra DePietro, M.S.; Wendy jill Crouse-Zeineddini, Ph.D.; Morten Hach, M.S.; ,
Galbraith, Pharm.D.; Fred Patrick Gattas, Pharm.D.; Elizabeth Kramer, Ph.D.; Ned Mozier, Ph.D.; Karthik
Richard Lewis Green, B.S. Pharm.; Kim C. Huynh-Ba, Ramani, Ph.D.; Sarah Richer; Harold Rode, Ph.D.
M.S.;Brenda Suejensen, M.A.; Patricia C. Kienle, M.P.A.;
Vivian Sue Loveless, Pharm.D.; Paul Barry Mahan, B.S. Biologics Monographs 2-Proteins
Pharm.; Rezaul H. Mannan, Ph.D.; Steve Zigler, Ph.D. MICHAEL MULKERRIN, PH.D., Chair
Gregory Beck, Ph.D.; Heather Boux, Ph.D.; Chris Burns
Non-Radioactive Imaging Agents Expert Panel- Ph.D.; Frederic Carriere, Ph.D.; Jill Crouse-Zeineddin'j
CONCLUDED Ph.D.; Michel Girard, Ph.D.; Anne Munk jespersen, B:S.;
JEROME M. LEWIS, PH.D., Chair Sridevi K~ambhampaty, Php.; Robert Mayer, Ph.D.;
Francisco Aguilar-Parrilla, Ph.D.; james Walter Brodack, Ned MOZier, Ph.D.; DhananlayPatankar, Ph.D.;Mauricio
Ph.D.; Dilip R. Ch?udh~ry, Ph.D.; Francette Delaloge, Seigelchifer, Ph.D.
Ph.D.; Joseph LoUIS Glalch, Ph.D.; ErnestVictorGroman,
Ph.D.; Gordon Craig Hill, Ph.D.; Kim C. Huynh-Ba, M.S.;

www.ilovepharma.com
xii Committees / People USP 43

Enzyme Expert Panel Cell Banking Expert Panel

FREDERIC CARRIERE, PH.D., Chair ROBERT BELL, PH.D., Chair
Gregory Be~k,. Ph.D.;.Fran~is Dwulet, Ph.D.; Olaf Friedrich, Jeri Ann Boose, Ph.D.; Sunil Gairola, Ph.D.; Luhong He,
Ph.D.; LUIgi G. Ghidorsi: Christopher Hosty, M.S.; Ph.D.; Ruud Hulspas, Ph.D.; Jette Dina Kreiberg, Ph.D.;
Andreas Koerner, Ph.D.; Thomas K. Langdon, B.S.; Michael Laird, Ph.D.; Archie Lovatt, Ph.D.; Sam
jeffrey Lessman Yaghmour, M.S.; Earl Zablackis, Ph.D.
Fe Function Assays Expert Panel Residual DNA in Biotechnology-Derived Products
JILL CROUSE-ZEINEDDINI, PH.D., Chair Expert Panel
Shan Chung, Ph.D.; Marina Feschenko, Ph.D.; Scott Kuhns, WESLEY E. WORKMAN, PH.D., Chair
Ph.D.; LeeAnn Machiesky, M.S.; Bhavin Parekh, Ph.D.; Pascal Anger, Ph.D.;Jon R. Borman; Scott Kuhns, Ph.D.;
Teresa Surowy, Ph.D.; MaxTejada, Ph.D. judith Shimoni, Ph.D.;Weihong Wang,·Ph.D.
Biologics Monographs 3-Complex Biologics Vaccine Pol}'saccharide NMR Identity Testing
EDWARD K. CHESS, PH.D., Chair Expert Panel
Mehrshid Alai, Ph.D.; Pascal Anger, Ph.D.; Parastoo Azadi, CHRISTOPHER JONES, PH.D., Chair
Ph.D.; Svetlana Bergelson, Ph.D.; Barbara Blum, Ph.D., Yves Aubin, Ph.D.; Francesco Berti, Ph.D.;CristianaCampa,
M.P.H.; Mirella Ezban, Ph.D.; Elaine Gray, Ph.D.; Donald Ph.D.;Thomas P.jacques, Ph.D.;Michael T.Jones, Ph.D.;
MacLean, Ph.D.; Nicole Provost, Ph.D.;Elizabeth I. Read, jeremy P. Kunkel, Ph.D.;Neil Ravenscroft, Ph.D.;Philippe
M.D.; Peter Vandeberg, Ph.D.; Christian Viskov, Ph.D.; Talaga, Ph.D.; Earl Zablackis, Ph.D.
Darin Weber, Ph.D.
Validation of Commercial Test Kits Expert Panel-
Bovine Unfractionated Heparin Expert Panel- CONCLUDED
CONCLUDED KENNETH MILLER, PH.D., Chair
WESLEY E. WORKMAN, PH.D., Chair Heather Boux, Ph.D.; David Good, M.S.; Wendy
TaniaAndrade, B.S. Pharm.; Irene Bartoli, B.S.; MariaLeticia Saffell-Clemmer, M.S.
Bertot, Pharm.D.; Elaine Gray, Ph.D.; Marco Guerrini,
Pharm.D.; Kristian johansen, Ph.D.; Robert Linhardt, Viral Vaccines Expert Panel
Ph.D.; Barbara Mulloy, Ph.D.; Marilene Nuss Rangel; EARL ZABLACKIS, PH.D., Chair
Zachary Shriver, Ph.D.; PearleTorralba, Ph.D.; Christian Luca Benetti, Ph.D.; Sunil Gairola, Ph.D.; Lucy Gisonni-Lex,
Viskov, Ph.D. M.S.! Keith Howard, Ph:,o.; Christophe~ Jones, Ph.D.;
Archie Lovatt, Ph.D.; BrI) Patel, Ph.D.; Sllke
CD-34 Positive Cells Expert Panel-CONCLUDED Schepelmann, Ph.D.; Vaneet Sharma, Ph.D.; Mark Van
NICOLE M. PROVOST, PH.D., Chair Ooij, Ph.D.
Ruud Hulspas, Ph.D.; Elizabeth I. Read, M.D.; Michael D.
Rosu-Myles, Ph.D.; Luisa Saraiva, Ph.D.; Richard j. General Chapters-Chemical Analysis
Stebbings, Ph.D.; D. Robert Sutherland, M.S.; Lili Wang, NANCY LEWEN, B.S., Chair
Ph.D.;Albertus W. Wognum, Ph.D. Anthony Bevilacqua, Ph.D.; Christopher Burgess, Ph.D.;
Robert T. Cambron, Ph.D.; Thomas DiFeo, Ph.D.; john
Low Molecular Weight Heparins Expert Panel Dolan, Ph.D.; joseph Louis Glajch, Ph.D.; John P.
ELAINE GRAY, PH.D., AND EDWARD K. CHESS, PH.D., Co-Chairs Hammond, FRSC; John Hinshaw, Ph.D.; Brent Kleintop,
Christopher P. Bryant, Ph.D.; Ishan Capila, Ph.D.; Gyongyi Ph.D.; Steven Leinbach, M.S.; Gregory Martin, M.S.;
Gratzl, Ph.D.; Kristian Johansen, Ph.D.;Annarapu Nuno Matos, B.S.; Oscar Quattrocchi, M.S.; Helmut
Malleswa~areddy, Ph.D.; Barbara Mu.lloy, Ph:D.; Anna Rockstroh, Ph.D.; MarkG. Schweitzer, Ph.D.; Timothy
K.Y. Nordin; Bruna Parma, M.S.; Christian Viskov, Ph.D. Shelbourn, M.S.; Rostyslaw O. Siabicky, B.S.; Teri Soli,
Ph.D.; Kevin A Swiss, Ph.D., Timothy j. Wozniak, Ph.D.
Biologics Monographs 4-Antibiotics
PASCAL ANGER, PH.D., Chair . Analytical Methodologies Based on the Light
Elizabeth (Betsy) Bruce Cariello, B.A; Sheila Deneau, B.S.; Scattering Phenomena Expert Panel
Colleen Guthrie, B.S.; Robert Klasson, M.S.; Mark G. KEVIN SWISS, PH.D., Chair
Papich, M.S.; Cindy Reid, B.S.; Jennifer [aye Schimmel, Emilia Byrne, M.S.; Nila Das, Ph.D.;Joseph Louis Glajch,
M.D.; Jonathan A Stewart, B.S.; Yaozuo Yuan, Ph.D. Ph.D.; Thomas Gonyon, B.S.; John P. Hammond, FRSC;
Stephen.Hussey; Chris jones, Ph.D.;jonathan Kingsbury,
General Chapters-Biological Analysis Ph.D.; Richard Meury, B.S.; Helmut Rockstroh, pn.D.;
WESLEY E. WORKMAN, PH.D., Chair Jack Saad, B.S.; Zhigang Sun, Ph.D.; William F. Weiss,
Robert Bell, Ph.D.; Mahesh Bhalgat, Ph.D.; Christopher Ph.D.; Eloise Welfare, Ph.D.; Renliang Xu; Earl Zablackis,
Jones, Ph.D.; Jeremy Kunkel, Ph.D.; Huijuan Li, Ph.D.; Ph.D.
Kenneth Miller, Ph.D.;Anthony Mire-Sluis, Ph.D.;
Anthony AG. Ridgway, Ph.D.; Wendy Saffell-Clemmer, Chemometrics Expert Panel-CONCLUDED
M.S.; Teruhide Yamaguchi, Ph.D.; Earl Zablackis, Ph.D. PEl CHEN, PH.D., AND NUNO MATOS, Co-Chairs
Chunsheng Cai, Ph.D.; RobertT. Cambron, Ph.D.; Peter de
Bioassay General Chapter B. Harrington, Ph.D.; Mark l- Henson, Ph.D.; Yang
DAVID LANSKY, PH.D., Chair (Angela) Liu, Ph.D.; Zhenqi(Pete) Shi, Ph.D.; Yvan C.D.
Jan Amstrup, Ph.D.; Walter Hauck, Ph.D.; Bhavin Parekh, Vander Heyden, Ph.D.; Stanislav O. Zakharkin, Ph.D.; Lin
Ph.D.;Andrew Rugaiganisa, M.S.; Perceval Sondag, Zhang, Ph.D.
M.S.; Ralf Stegmann, Ph.D.; Ryan Yamagata, M.S.;
Lingmin Zeng, Ph.D. Elemental Impurities Expert Panel
NANCY LEWEN, B.S., Chair
Biologics Stability Expert Panei Charles Barton, Ph.D., DABT; Courtney M. Callis, M.P.H.,
ANTHONY MIRE-SLUIS, PH.D., Chair DABT; Steven j. Dentali, Ph.D.;Anna M. Fan, Ph.D.,
Kimberly Cheung, M.S.; Nila Das, Ph.D.; Stephanie Ferrari, DABT; Edward j. Fletcher, B.S.; Bruce A Fowler, Ph.D.,
M.S.; [ens Krogh Rasmussen, M.S.; joseph Kutza, Ph.D.; AT.S.; Roland Froetschl, Ph.D.; Richard Ko, Pharm.D.,
Lori A McCaig, Ph.D.; Nausheen Rahman, Ph.D.; Ph.D.; Timothy L. Shelbourn, M.B.A, M.S.
Camilla Santos, Ph.D.;Michael Walsh, M.S.; Allison Wolf,
M.S.

www.ilovepharma.com
USP 43 People / Committees xiii

Good Documentation Practices Expert Panel- General Chapters-Dosa!Jje Forms

CONCLUDED . JAMES E. DE MUTH, PH.D., Chair
KIM C. HUYNH-BA, M.S., Chair Emmanuel Akala, Ph.D.;lIgaz Akseli, Ph.D.; Scott Aldrich,
Kathleen V. Brady, B.S.; Frank J. Diana, Ph.D.; Lisa Ann Fink, B.S.; Susan Cady, M.S.; Paul Curry, Ph.D.; Mario
M.B.A.; Craig Hamilton, Ph.D.; judy Lin, M.S.; Anjan K. Gonzalez, Ph.D.; Vivian A. Gray, B.S.; Ralph Heasley,
Mittal, M.Pharm.; Kevin A. Swiss, Ph.D. Ph.D.; Anthony Hickey, Ph.D.; MunirA. Hussain, Ph.D.;
Johannes Kraemer, Ph.D.; Stefan Leiner, Ph.D.; John
Modernization of Identification Tests Expert Panel Mauger, Ph.D.; Colin Minchom, Ph.D.; Jolyon Mitchell,
NANCY LEWEN, B.S., Chair Ph.D.; Pierre-Alain Muller, Ph.D.; Guirag Poochikian,
Anthony C. Bevilacqua, Ph.D.; Geoffrey P. R. Carr, Ph.D.; Ph.D.; Chetan Pujara, Ph.D.; Shobhan Sabnis, Ph.D.;
Pei Chen, Ph.D.; jonathan W. DeVries, Ph.D.; Maryna John Shabushnig, Ph.D.; Raymond D. Skwierczynski,
Dmitriieva, Ph.D.; Michael Hornig, Ph.D.; BernardA. Ph.D.; Jason Suggett, Ph.D., M.B.A.; Monica Tejwani,
Olsen, Ph.D.; Jeffrey S. Rohrer, Ph.D. Ph.D.; Thomas R. Tice, Ph.D.; Kevin Warner, Ph.D.;
(461) Residual Solvents Expert Panel Mehran Yazdanian, Ph.D.
OSCAR A. QUATIROCCHI, M.S., Chair (771) Ophthalmic Preparation Expert Panel
Coleman C. Chasteen, M.S.; john Connelly, Ph.D.; jeffrey ASHIM K. MITRA, PH.D., Chair
Fleitman, Ph.D.; john V. Hinshaw, Ph.D.; Bruce P. Dale S. Aldrich, Ph.D.~ Martin Coffey, Ph.D.; Paul Curry,
johnson, Ph.D.; Eric C. Kesslen, Ph.D.; Brent Kleintop, Ph.D.; Jeffrey S. Heitman, Ph.D.; John Mauger, Ph.D.;
Ph.D.; Elizabeth Kovacs, B.S.; PaulW. Lockwood, M.S.; Seshadri Neervannan, Ph.D.; Stacey M. Platzer; Chetan
Gregory P. Martin, M.S.; Kevin A. Swiss, Ph.D.; Yuwen Pujara, Ph.D.; Satish K. Singh, Ph.D.; Monica Tejwani,
Wang, Ph.D. Ph.D.; Thomas R. Tice, Ph.D.
Quality Standards for Pharmaceutical Continuous (788) Particulate Matter in Injections Expert Panel
Manufacturing Expert Panel DALE S. ALDRICH, PH.D., Chair
NUNO MATOS, B.S., Chair Dan Berdovich; Mary Lee Ciolkowski, Ph.D.; Kevin Dahl,
Shaukat AIi,Ph.D.; Ahmad Almaya; Brian Carlin, Ph.D.; Ph.D.; linda Narhi, Ph.D.; KentA. Peterson;Dean Ripple,
Thomas P. Garcia, Ph.D.; Douglas Hausner, Ph.D.; Eric Ph.D.
jayjock, Ph.D.; Keith Jensen, PIl.D.; johannes Khinast;
Pramod Kotwal, Ph.D.; Marcus U. Krumme, Ph.D.; Kim Uquid-Filled Capsules Expert Panel
Lamey, Ph.D.; Fernando J. Muzzio, Ph.D.; William G. VIVIAN A. GRAY, B.S., Chair
Randolph, Ph.D.; MarkG. Schweitzer, Ph.D.; Raymond joe Fotso, Ph.D.; Munir A. Hussain, Ph.D.; Stephen C.
Skwierczynski, Ph.D.; Kelly Swinney, Ph.D.; Bernhardt Tindal; Madhusudan Vudathala, M.Pharm., M.B.A.
Tout, Ph.D.; AmyWalia, M.A.
Performance Test for Semisolid Dosage Forms
Validation and Verification Expert Panel- Expert Panel
CONCLUDED KAlLAS THAKKER, PH.D., Chair
GREGORY P. MARTIN, M.S., Chair Bryan Crist; james E. De Muth, Ph.D.; Geoffrey N. Grove,
Kimber l. Barnett, Ph.D.; Christopher Burgess, Ph.D.; Paul Ph.D.; l. Thomas Hall, Ph.D.; John S. Heaney; Patricia l.
D. Curry, Ph.D.; Gyongyi Gratzl, Ph.D.; john P. Lee, M.S.; Patrick C. Mahn; William M. Rosenthal; Steve
Hammond, FRSC; Elizabeth Kovacs; David J: LeBlond, W. Shaw
Ph.D.; Rosario LoBrutto, Ph.D.; Pauline l. McGregor,
Ph.!?; Phil Nethercote, Ph.D.; Allen C. ~empleton, Ph.D.; Solubility Criteria for Veterinary Drugs Expert
David P. Thomas, Ph.D.; M.l. jane Weitzel Panel
SUSAN CADY, M.S., Chair
Water for Analytical and Pharmaceutital Purposes Bryan Crist; MarioA. Gonzalez, Ph.D.; MarkG. Papich,
Expert Panel M.S., D.V.M.; Alan F. Parr, Pharm.D., Ph.D.; Monica
ROSTYSLAW O. SLABICKY, B.S., Chair . Tejwani, Ph.D.
Anthony C. Bevilacqua, Ph.D.; Lucia Clontz, D.H.S., M.S.;
Max S. Lazar, B.A.; Nancy Lewen, B.S.; Bruno Rossi, Sutures Expert Panel
M.D.; Teri C. Soli, Ph.D. JAMES E. DE MUTH, PH.D., Chair
Edwin Anderson, M.S.; John C.
Chen; Frank Corniello;
General Chapters-Physical Analysis Nomi Steen
XIAORONG HE, PH.D., Chair
Shaukat Ali, Ph.D.; Lawrence H. Block, Ph.D.; Geoff P. R. Use of Enzymes in the Dissolution Testing of Gelatin
Carr, Ph.D.; Martin J. Coffey, Ph.D.;Tim Freeman, B.A.; Capsules Expert Panel
David J. Goldfarb, Ph.D.; Bruno Hancock, Ph.D.; Stephen VIVIAN A. GRAY, B.S., Chair
Hoag, Ph.D.; Mario Hubert, Ph.D.; Richard Meury, B.S.; EwartCole, Ph.D.; Luigi Ghidorsi; Ilan-Hwa Guo, Ph.D.;
Matthew Mullarney, M.S.; Prabu Nambiar, Ph.D.; Myke Feixue Han, Ph.D.; Jian-Hwa Han, Ph.D.; Christopher T.
Scoggins, Ph.D.; Changquan Sun, Ph.D.; Zhigang Sun, Hosty; johannes Kraemer, Ph.D.; Thomas Langdon;
Ph.D.; Allen Templeton, Ph.D.; Eloise Welfare, Ph.D.; Steven R. Leinbach; Stefan Leiner, Ph.D.; Gregory P.
Dale Wurster, Ph.D.; Bing-Shiou Yang, Ph.D.; Geoff Martin, M.S.; Steven M. Meyerhoffer, Ph.D.; Richard C.
Zhang, Ph.D. Moreton, Ph.D.; Edward Shneyvas, Ph.D.; jason A.
Suggett, Ph.D.; Stephen C. Tindal; Madhusudan
Impurities in Drug Substance and Drug Products Vudathala, M.Pharm., M.B.A.; Hu Wang, M.S.
Expert Panel
PRABU NAMBIAR, PH.D., M.B.A., Chair Visual Inspection of Parenterals Expert Panel
Shaukat Ali, Ph.D.; Steven W. Baertschi, Ph.D.; Judy P. RUSSELL E. MADSEN, M.S., Chair
Boehlert, Ph.D.; Robert G. Buice, Ph.D.; Xiaorong He, Dale S. Aldrich, Ph.D.; John D. Ayres, M.D., J.D.; Roy
Ph.D., M.B.A.; Kim C. Huynh-Ba, M.S.; Michael Koberda, Cherris; John G. Shabushnig, Ph.D.; Deborah Shnek,
Ph.D.; Robert E. Osterberg, R.Ph., Ph.D., Fellow-ATS; Ph.D.
ErnestParente, Ph.D.;Oscar Quattrocchi, M.S.; David H~ General Chapters-Microbiology
Rogers, Ph.D.; Mark G. Schweitzer, Ph.D.; MaryW. DAVID HUSSONG, PH.D., Chair
Seibel; Rostyslaw O. Siabicky, B.S.; Teri C. Soli, Ph.D.; james Agalloco, M.S.; James Akers, Ph.D.; Dilip Ashtekar,
Kevin A. Swiss, Ph.D. Ph.D.; Anthony M. Cundell, Ph.D.; Dennis Guilfoyle,

www.ilovepharma.com
xiv Committees / People USP 43

Ph.D.; Rajesh Gupta, Ph.D.; Russell Madsen, M.S.; Karen (661.3) Plastic Systems Used for Manufacturing
McCullough, M.S.; Robert Mello, Ph.D.; David Roesti, Pharmaceutical Products Expert Panel
Ph.D.; Donald Singer, M.S.; Paul Stinavage, Ph.D.; DENNIS R. JENKE, PH.D., Chair
EdwardTidswell, Ph.D. Weibing Ding, Ph.D.; Michael N. Eakins, Ph.D.; MaryG.
Modern Microbiological Methods Expert Panel Foster, Pharm.D., BFA; james Hathcock, Ph.D.; jerold
ANTHONY M. CUNDEll, PH.D., AND EDWARD C. TIDSWEll, PH.D.,
(Jerry) M. Martin, M.S.; Diane M. Paskiet, M.S.; Robert
Co-Chairs Steininger; Cheryl L. M. Stults, Ph.D.; Ken M. Wong,
ThierryBonnevay, Ph.D.; Ralph Breton, B.S. Pharm.; M.S.
Claudio Denoya, Ph.D.; Garydu Moulin, Ph.D.; john (662) Metal Packaging Components and Systems-
Duguid, B.S.; Rajesh K. Gupta, Ph.D.; David Hussong, CONCLUDED
Ph.D.; Matthew jenkins, M.S.; Amy Lynn McDaniel, CHERYll. M. STULTS, PH.D., Chair
Ph.D.; Michael Miller, Ph.D.; Felix Montero Julian, Ph.D.; Peter Claessens; Benjamin jeyaretnam, Ph.D.; Ralph Lessor,
David Newton, Ph.D.; Kuldip Patel; Steven Richter, Ph.D.; Sara Miller, M.S.; Gaby Reckzuegel; john
Ph.D.; David Roesti, Ph.D.; Yongqiang Zhang, Ph.D.; Willenbrock, B.S.
Steve S. Zigler, Ph.D.
General Chapters-~tatistics
General Chapters-Packaging and Distribution ROBERT R. SINGER, M.S., Chair \
MARY G. FOSTER, PHARM.D., BFA, Chair Bruno Boulanger, Ph.D.; Richard Burdick, Ph.D.; David
ChrisAnderson, M.A.; Bettine Boltres, Ph.D.; Glaucia K. Christopher, M.S.; David Lansky, Ph.D.; Dave LeBlond,
Braga, Ph.D.; jeffrey Carrico, Pharm.D.; Chris Chandler, Ph.D.; Juris Meija, Ph.D.; Anthony Okinczyc, M.P.H.;
Pharm.D.; Michael N. Eakins, Ph.D.; Dana Guazzo, Peter Rigsby, M.S.; Dennis Sandell, Ph.D.; Timothy
Ph.D.; Renaud Janssen, Ph.D.; Dennis jenke, Ph.D.; Schofield, M.A.; CharlesTan, Ph.D.; Edwin van den
Wendy Mach, B.S.; Dan Malinowski; Daniel L. Norwood, Heuvel, Ph.D.; jane Weitzel; Harry Yang, Ph.D.
Ph.D.; Diane Paskiet,.M:S.; RobertSeevers, Ph.D.;Cheryl
L. M. Stults, Ph.D.; LI Xiong, Ph.D.; Gao Yonghua, B.S. Content Uniformity with Large Sample Sizes
Pharm. Expert Panel
DENNIS SANDEll, PH.D., Chair
(381) Elastomeric Closure for Injections Expert IIgaz Akseli, Ph.D.; james S. Bergum, Ph.D.; Paul Curry,
Panel Ph.D.; Walter Hauck, Ph.D.; jeffrey Hofer, M.S.; Gregory
DIANE M. PASKIET, M.S., ANDRENAUD JANSSEN, PH.D., Co-Chairs L. Lamer, M.S.; Raymond Skwierczynski, Ph.D.
Douglasj. Ball, M.S., DABT; Michael N. Eakins, Ph.D.; Dana
Guazzo, Ph.D.; Dennis R. jenke, Ph.D.; Douglas Kiehl,
M.S., B.S.;. Heinz Kirchmeyer, Ph.D'i Philippe LeGall, Expert Committees for the National
M.S.; Daniel L. Norwood, Ph.D.; MIChael A. Ruberto,
Ph.D.; Lisa M. Yoest, M.S. Formulary
(659) Packaging and Storage Requirements Expert
Panel-CONCLUDED Excipients Monographs 1
CHRIS CHANDLER, PHARM.D., Chair ERIC MUNSON, PH.D., Chair
Glaucia K. Braga, B.S.; jeffrey Carrico, Pharrn.Dc MaryG. Thiago Carvalho, Ph.D.; Brian Carlin, Ph.D.; Richard
Foster, Pharm.D., BFA; Eleanor Freeman, B.S.; Wendy Cawthorne, Ph.D.; Richard Creekmore, Ph.D.; Vivek
Mach, B.S.; Devinder Pal, M.Pharm.; Robert H. Seevers, Dave, Ph.D.; Felicitas Guth; Otilia Koo, Ph.D.; Phil
Ph.D.; Gao Yonghua, B.S. Pharm.; Li Xiong, Ph.D. Merrell, Ph.D.; Dominic Moore; Chris Moreton, Ph.D.;
jasmine Musakhanian, M.S.; Charles Vesey, M.S.;
(660) Containers-Glass Expert Panel Richard Wendt, Ph.D.; jin Zhao, Ph.D.
BETIINE BOlTRES, PH.D., ANDMICHAEL EAKINS, PH.D., Co-Chairs
Michael E. Akers, B.A.; Alberto Biavati, Ph.D.; juan (1059) Chapter Excipient Performance Expert
Cerdan-Diaz; Carol Rea Flynn, M.S.; Emanuel Panel
Guadagnino, M.D.; Daniel Edward Haines, Ph.D.; Kevin GREGORY AMIDON, PH.D., AND RICHARD MORETON, PH.D.,
McLean, B.S.; Kelly Murphy, Ph.D.; Volker Rekowski; Co-Chairs
jennifer Martell Roark, B.S.; Holger Roehl, Ph.D.; Gao IIgaz Akseli; Shaukat Ali, Ph.D.; Lawrence Block, Ph.D.;
Yonghua, B.S. Pharm. Thiago Cardoso Carvalho, Ph.D.; Brian Carlin, Ph.D.;
Richard Creekmore, Ph.D.; Stephen Hoag,Ph.D.;
Biocompatibility of Materials Used in Packaging johannes Khinast; jasmine Musakhanian, M.S.;
Systems, Medical Devices, and Implants Expert Natarajan Rajagopalan, Ph.D.; Hiroko Shibata, Ph.D.;
Panel jiasheng Tu, pn.D.; Katherine Ulman, B.S.; Maureen
DANIELl. NORWOOD, PH.D., Chair TaylorVander Fliet, M.S.
Douglas j. Ball, M.S.; Stephen A. Barat, Ph.D.;William P.
Beierschmitt, Ph.D.; Denise Bohrer, Ph.D.; Michael N. (1059) Excipient Performance Expert Panel-
Eakins, Ph.D.; Jill A. Glosson, B.A.; john Iannone, B.S.; CONCLUDED
Renaud Janssen, Ph.D.; Douglas ~. Kiehl, M.S.; Wendy ERIC A. SCHMITI, PH.D., Chair
Mach, B,S.; Robert Przygoda; AnitaY. Sawyer, M.S.; Abdullah M. AI-Mohizea, Ph.D.; Shaukat Ali, Ph.D.;
Cheryl L. M. Stults, Ph.D. Lawrence H. Block, Ph.D.; Patrick Deluca, Ph.D.; Carl
Frey, M.S.; Xiaorong He, Ph.D., M.B.A.; .Stephen-W.
CUnical Trial Materials (GDP) Expert Panel- Hoag, Ph.D.; Michelle A. LonSJ' Ph.D.; Richard C.
CONCLUDED Moreton, Ph.D.; PrabuNarnbiar, Ph.D., M.B.A.; james
MARY G. FOSTER, PHARM.D., BFA, Chair A. Ponto, M.S.; KentStemitzke, Ph.D.; Kevin A. Swiss,
Christopher Anderson, M.A.; Rafik H. Bishara, Ph.D.; _ Ph.D.; Sean V. Taylor, Ph.D.
Glaucia K. Braga, Ph.D.; jeffrey Carrico,Pharm.D.;Steven
A. jacobs, M.B.A.; Martin jeiven, M.S.; Claude jolicoeur, (1197) Good Distribution Practices for 'Bulk
B.S.; Gao Yonghua, B.S. Pharm. Pharmaceutical Excipients Expert Panel-
CONCLUDED
RICHARD c. MORETON, PH.D., Chair
Lawrence H. Block, Ph.D.; William DaleCarter, M.S.; Zak
T. Chowhan, Ph.D.; Marc Fages; Elizabeth

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USP 43 People / Committees xv

Ferguson-Brown; Mary G. Foster, Pharm.D., BFA; Tu, Ph.D.; WanyingWu, Ph.D.; Zhongzhen Zhao,
Linda A. Herzog, M.B.A;Ashok V. Katdare, Ph.D.; Ph.D.
Zakiya Kurdi, Ph.De Edward G. Malawer, Ph.D.,CQA;
FrankMilek, Ph.D.; Becc~ Mitchell; Dwight Mutchler; Herbal Medicines Compendium (HMC) South
Garnet E. Peck, Ph.D.; Mike Schultz, R.Ph.; Alexa Asia Expert Panel
Smith, M.S.; Glenn Sokoloski; Kelly Taylor; jiasheng SUKHDEV S. HANDA, PH.D., FNAIM, FNA.SC, Chair
Tu, Ph.D. AmitAgarwal, Ph.D.; Rasadah Mat Ali, Ph.D.; Mohamed
Zahir Mohammed Farhad; C.K. Katiyar, Ph.D.;
Excipients Monographs 2 Bhawanishankar Madhira, Ph.D.; Ami Fazlin Syed
MARY C. HOUCK, PH.D., Chair Mohamed; Ph.D.; A Nagarajan, Ph.D.; D.G. Naik,
lawrence H. Block, Ph.D.; Andrew Bluj; Tim Cabelka, Ph.D.; Ph.D.; Sankaran Natarajan, Ph.D.; M.K. Raina, Ph.D.;
Arya jayatilaka, Ph.D.; Russell Maus, Ph.D.; Robert E. J.L.N. Sastry, Ph.D.; Rajeev Kr. Sharma, Ph.D.; R.
Osterberg, R.Ph., Ph.D., Fellow-ATS; julie Warner Pier, Sundaram, Ph.D.; NeerajTandon, Ph.D.; Surapote
M.S.; Anisul Quadir, Ph.D.; Gwen Rucker; Barbara Serr, Wongyai, Ph.D.
Ph.D.; Huimin Sun, Ph.D.;jiasheng Tu, Ph.D.; jacqueline
Tordik; FanWu, Ph.D.; Timothy Yasika Dietary Supplements Safety Modeling-
CONCLUDED
Glycerin Expert Panel MARY L. HARDY, M.D., Chair
TIM B. CABELKA, PH.D., Chair V.A Shiva Ayyadurai, Ph.D.; MaryA Fox, Ph.D., M.P.H.;
Frances K. Byrne, M.S.; Ian A Duncan, Ph.D.; Tanja Scott A. Jordan, Ph.D.; Mkaya Mwamburi, M.D., Ph.D.,
Natterer; Marian J. Rinken, Ph.D.; Gwen E. Rucker, M.A (Econ); DianeR. Mould, Ph.D.; RobertE. Osterberg,
B.S.; David A. Sharknas, B.S.; Hong Zhou, Ph.D. R.Ph., Ph.D., Fellow-ATS; CharlieYoe, Ph.D.
Povidones Expert Panel-CONCLUDED Cannabis Expert Panel
BERNHARD D. FUSSNEGGER, PH.D., AND CARL PERINI, M.S., IKHLAS A. KHAN, PH.D., Chair
Co-Chairs Paula N. Brown, Ph.D.; Lawrence Deyton, M.D.; Mahmoud
Feng Chen, Ph.D.; DavidJ. Fillar, M.B.A.; Edward G. A. Elsohly, Ph.D.; Sytze Elzinga, M.S.; Christopher
Malawer,Ph.D.;SyedAA Rizvi, Ph.D.; JohnW. Spink, Hudalla, Ph.D.; Holly Johnson, Ph.D.; Robin Maries,
Ph.D.; Fan Wu, Ph.D. Ph.D.; jeremy Melanson, Ph.D.; Ethan Russo, M.D.;
. Gordon Vrdoljak, Ph.D.; AndrewWaye, Ph.D.; joshua
Talc Methods Expert Panel Wurzer, B.S.
JULIE WARNER PIER, M.S., AND MARTIN RUTSTEIN, PH.D.,
Co-Chairs Non-Botanical Dietary Supplements
Daniel Crane; Sean M. Fitzgerald, B.S.; Mickey E. Gunter, DENNIS K.J. GORECKI, B.S.P., PH.D., Chair
Ph.D.; Don Halterman, M.S.; Mary C. Houck, Ph.D.; joseph M. Betz, Ph.D.; Michael S. Bradley, M.S.; james
lee Poye, B.S.; Matthew S. Sanchez, Ph.D.; Alan M. Brooks, Ph.D.; Bill Gurley, Ph.D.; Chung Hyun, M.S.; joy
Segrave, B.S.; Gary P. Tomanino, B.S.; Drew R.Van A joseph, M.S.; Raimar Lobenberq, Ph.D.; Richard
Ordern, M.A.; james Webber, Ph.D. Myers, Ph.D.; james Neal-Kababick, B.S.; Peter Rice,
Pliarm.D., Ph.D.;Amy Roe, Ph.D.; Aniko Solyom, Ph.D.;
Karunakar Sukuru, Ph.D.; Darryl Sullivan, Ph.D.
Expert Committees for the USP and (2251) Adulteration of Dietary Supplements with
the Dietary Supplements Compendium Drugs and Drug Analogs Expert Panel-
CONCLUDED
DENNIS K.J. GORECKI, B.S.P., PH.D., Chair
Botanical Dietary Supplements and Herbal Joseph ~. Betz, Ph.D.; Pei Che~, Ph.D.; Hwee-Lin9, Koh,
Medicines Ph.D., Ikhlas A Khan, Ph.D., james Neal-Kababkk,
ROBIN J. MARLES, PH.D., Chair . B.S.; John Spink, Ph.D.; Darryl Sullivan, Ph.D.
Thomas Brendler, B.A; josef A Brinckmann; Paula Naomi
Brown, Ph.D.; Angela Calderon, Ph.D.; Steven Dentali, Extended-Release Dietary Supplements Expert
Ph.D.; Edward Fletcher, B.A;StefanGafner, Ph.D~; joerg Panel-CONCLUDED
Gruenwald, Ph.D.; De-an Guo, Ph.D.; Sukhdev Swami JOY A. JOSEPH, M.S., Chair
Handa, M.Pharm., Ph.D.; James Harnly, Ph.D.; Craig Charles Barton, Ph.D., DABT; joseph F.Borzelleca, Ph.D.;
H.opp, Ph.D.; Scott A jordan, P.h.D.; Ikhlas Khan, PliD.; Michael S. Bradley, M.S.; james R. Brooks, Ph.D.;
Richard Ko, Pharm.D., Ph.D.; Tleraona low Dog, M.D.; Marion Ehrich, Ph.D.; Vivian A Gray, B.S.; Carol
Mirtha Navarro, Ph.D.; Pilar Pais, Ph.D.; Guido Pauli, Johnston, Ph.D., R.D.; Raimar tobenberq, Ph.D.;
Pharm.D., Ph.D.; Eike Reich, Ph.D.; Paul Schiff, Ph.D.; Alexander G. Schauss, Ph.D., FACN; Elizabeth A
Shangmei Shi, B.S. Yetley, Ph.D.
Creen Tea Extract Hepatotoxicity Expert Panel Probiotics Expert Panel
RICHARD KO, PHARM.D., AND AMY LYNNE ROE, PH.D., MARY ELLEN SANDERS, Chair
Co-Chairs Mike Bradley, M.S.; Marie-Eve Boyte; james Brooks,
joseph M. Betz, Ph.D.; Bill Gurley,Ph.D.; Kan He, Ph.D.; Ph.D.; Pierre Burguiere, Ph.D.; Scott jackson, Ph.D.;
Scott Jordan, Ph.D.; Mahendra P. Kapoor, Ph.D.; David Keller; Marco Pane, M.S.; Amy Roe, Ph.D.;Jean
Tieraona Low Dog, M.D.; Robin James Maries, Ph.D.; Schoeni, Ph.D.; Butry Stahl, B.S.; Cliristina Skovgaard
Victor Navarro, M.D.; Mary Paine, Ph.D.; Pradyumna Vegge, Ph.D.
Theertham Rao, Ph.D.; Cynthia Rider, Ph.D.
Herbal Medicines Compendium (HMC) East Asia
Expert Panel
DE-AN GUO, PH.D., Chair
Yuan-shiun Chang, Ph.D.; ShiLin Chen, Ph.D.; Takashi
Hakamatsuka, Ph.D.; Shen ji, Ph.D.; Veong Shik Kim,
Ph.D.; Hwee-Ling Koh, Ph.D.; Clara Bik San Lau, Ph.D;
Ping Li, Ph.D.; Qing Li, Ph.D.; Shuangcheng Ma,
Ph.D.; Shangmei Shi; Viet Hung Tran, Ph.D.; Pengfei

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xvi Committees / People USP 43

Deborah Houston, Pharm.D.; BrendaJensen, M.A.;

Expert Committees for the Food Patricia C. Kienle, M.P.A.; William A. Mixon, M.S.; John
Chemicals Codex Musil, Pharm.D.; David Newton, Ph.D.; Alan Parr,
Pharm.D., Ph.D.; Abby Roth; Robert Shrewsbury, Ph.D.;
Connie Rae Sullivan, B.S. Pharm.; JamesT. Wagner;
Food Ingredients BrendaYuzdepski, B.S. Pharm.
JONATHAN W. DEVRIES, PH.D., Chair
Richard C. Cantrill, Ph.D.;[unshl Chen, M.D.; Hwei-Fang Compounding with Hazardous Drugs Expert
Cheng, Ph.D.; HenryChin, Ph.D.; Grady Chism, Ph.D.; Panel-CONCLUDED
Robin Churchill, Ph.D.; RogerClemens, DrPH; John Clos, PATRICIA C. KIENLE, M.P.A., Chair
Ph.D.; Helen Darling, Ph.D.; Andrew Ebert, Ph.D.; [aap Thomas H. Connor, Ph.D.; Eric Kastango, M.B.A., B.S.
Evers, Ph.D.; Carl Frey, M.S.; Einat Haleva, Ph.D.; Lori Pharm.; Melissa A. McDiarmid, M.D., M.P.H.;
Klopf, Ph.D.; Hemant G. Koshia, Ph.D.; Dana Krueger, Kenneth R. Mead, Ph.D.; Martha Polovich, Ph.D.;
B.S.; Diane McColl, J.D.; Bert Popping, Ph.D.; Yoko Lucille A. Power; James T. Wagner
Uematsu, Ph.D.; YongningWu, Ph.D.; Liangli Yu, Ph.D.
Dietary Proteins Expert Panel Government Liaisons to Expert
SNEH D. BHANDARI, PH.D., Chair
Spencer Carter, Ph.D.;Jonathan W. DeVries, Ph.D.; Committees and Expert Panels
Melanie Downs, Ph.D.; [aap Evers, Ph.D.; Christophe
Fuerer, Ph.D.; Boyan Gao, Ph.D.; Philip Haselberger, Jibril Abdus-Sarnad, Ph.D.; Rajiv Agarwal, Ph.D.;
B.A.; XiaoPingHuang, Ph.D.; Philip E. Johnson, Ph.D.; Mohammed Ahmed, M.S.; Om Anand, Ph.D.; Shalini
Steve Holroyd, Ph.D.; AndrewT. Mackey, Ph.D.; Anand, Ph.D.; Kristen Anderson, Ph.D.; Matthew Barlow,
James Neal-Kababick, B.S.; Reto Portmann, Ph.D.; RN, BSN; Julie N. Barrows, Ph.D.; JacintaBatson, M.B.A.;
Haowei Song, Ph.D.;Chao Wu, Ph.D.; JinchuanYang, Eden Bermingham, DVM, M.S., DACVCP; Ashwinkumar
Ph.D.; We Zhu, Ph.D. Bhirde, Ph.D.; Jonathan Bray, B.S.; Michael Brent;
Michael Brewer, Ph.D.; Daniel Brown; Janice Brown,
Food Adulteration Expert Panel M.S.; Lana Bruney, Ph.D.; Teresa Cain, Ph.D.; Steven
HENRY CHIN, PH.D., Chair Casper, Ph.D.; Wiley Chambers, M.D.; Jane Chang,
Grant Abernethy, Ph.D.; David Bolliet; Richard C. Ph.D.; Richard Chang, Ph.D.; Anissa Cheung, Ph.D.;
Cantrill, Ph.D.; Christophe Cavin, Ph.D.; Hwei-Fang Donna Christner, Ph.D.; John Cipollo, Ph.D.; David
Cheng, Ph.D.; Robin Churchill, Ph.D.; Helen Darling, Claffey, Ph.D.; Maegen Colehour, M.S.; Celia Noemi
Ph.D.; Jonathan W. DeVries, Ph.D.; Andrew Ebert, Cruz, Ph.D.; Mike Darj, Ph.D.; Swapan De, Ph.D.; Ian
Ph.D.; Kim Huynh-Ba, M.S.; Shaun Kennedy; Dana DeVeau, Ph.D.;Julie Dohm, Ph.D.; Zedong Dong, Ph.D.;
Krueger, B.S.; MicheleLees, Ph.D.; Fernando Antunes Jason Dreabit, M.A.; Sheila Dreher-lesnick, Ph.D.;
Lopes; Bert Popping, Ph.D.; Lars Reimann, M.S.; Stephanie Emory, Ph.D.; Okponanabofa Eradiri, Ph.D.;
Roman Romero, Ph.D.;. John Spink, Ph.D.; Thomas Erika E. Englund,Ph.D.;Cory Evans, Ph.D.; Raafat Fahmy,
Tarantelli, B.S.; Yoko Uematsu,Ph.D.;Saskia van Ruth, Ph.D.; Karen Farizo, Ph.D.; Adam Fisher, Ph.D.; Daniel
Ph.D.; Carl Winter, Ph.D.; Yongning Wu, Ph.D. Folmer, Ph.D.; Rick Friedman, M.S.; Michael Furness,
Food Adulterants Hazard Identification Expert M.S.; Christopher Galliford, Ph.D.; Zongming Gao,
Panel-CONCLUDED Ph.D.; Mary Papa Ghods, R.Ph.; Mohamed Ghorab,
HENRY CHIN, PH.D., Chair Ph.D.; Tapash Ghosh, Ph.D.; DevinderGill, Ph.D.;
AndrewEbert, Ph.D.; Richard Lane, Ph.D.; Diane McColl, Gurpreet Gill-Sangha, Ph.D.; Lillie Golson, Pharm.D.;
J.D.; Bert Popping, Ph.D.; Joseph Scimeca; Carl Jennifer Goode, Ph.D.; Edisa Gozun, Pharm.D.; Yin GUo,
Winter, Ph.D. Ph.D.; William Hallett, Ph.D.; Blake Hamann, Ph.D.;
Bruce Harris, Ph.D.; Danielle Marie Harris, Pharm.D.;Joel
Non-Targeted Methods for Milk Ingredients Hathaway, Ph.D.; lijuan He, Ph.D.; Mohammad
Expert Panel-CONCLUDED Heidaran, Ph.D.;William Hess, B.S. Pharm.; Yong Hu,
ROBERT MAGALETIA, PH.D., Chair Ph.D.; Gloria Huang, Ph.D.; Laura Huffman, M.S.;
Sned D. Bhandari, Ph.D.; Jonathan W. DeVries, Ph.D.; GregoryHunter, Ph.D.; Latiff Hussain, Ph.D.; MaiHuynh,
Gerard Downey, Ph.D.; Stephen Ellison, Ph.D.; James Ph.D.; Karthik Iyer, Ph.D.; Edwin [ao, Ph.D.; YoungJhon;
M. Harnly, Ph.D.; Elizabeth Hobbs; Steven Holroyd, Jacquin L. Jones, MS, BSN, RN; Ravindra Kasliwal, Ph.D.;
Ph.D.; Gregory A. Israelson, B.S.; Joseph David Keire, Ph.D.; Michael Kennedy, Ph.D.; James
Katzenmeyer, Ph.D.;AndrewT. Mackey, Ph.D.; Kenney, Ph.D.; Saeed Khan, Ph.D.; Erin Kim, Ph.D.;
Benjamin B. Perston, Ph.D.; Jianwei Qin, Ph.D.; Kathryn E. King, Ph.D.; Anna Kooser, Ph.D.; Bogdan
Roman Romero, Ph.D.; PaulWehling; Thomas Wheat, Kurtyka, Ph.D.; David Lau, M.A.; Hyoung S. Lee, Ph.D.;
Ph.D.; Steven Zbylut, Ph.D. Sau lee, Ph.D.; David Lewis, Ph.D.; Xihao Li; jing Li,
Ph.D.; Jennifer Liang, Ph.D.; June Liang, Ph.D.;Tsai-Lien
Olive on Authenticity and Quality Expert Panel Lin, Ph.D.; Heather Lombardi, Ph.D.; Ewa Marszal, Ph.D.;
RICHARD C. CANTRILL, PH.D., Chair Marilyn Martinez, Ph.D.; Timothy McGovern, Ph.D.;
Diego Luis Garcia-Gonzalez, Ph.D.; Claudia Guillaume, Jeffrey Medwid, Ph.D.; Randa Melhem, Ph.D.; John
M.S.; Zohar Kerem, Ph.D.; Paul H. Miller, B.A.; Agusti Metcalfe, Ph,D.; Yana Mille, B.S. Pharm.; Adil
[ordl Romero, Ph.D.; Selina Wang, Ph.D. Mohammad, Ph.D.; Magdi Mossoba, Ph.D.; Laura
Moussa, Ph.D.; Karunakar Neelam, Ph.D.; Nina Ni,
Ph.D.; Pallavi Nithyanandan, Ph.D.; Scott EdwardNorris,
Expert Committees for the USP and Ph.D.; Sarai Obando, Ph.D.; Thomas O'Connor, Ph.D.;
Steven Oh, Ph.D.; Andrea Ottesen, Ph.D:; Frank Perrella,
USP on Compounding Ph.D.; Erika Pfeiler, Ph.D.; Zhihao Peter Qiu, Ph.D~;
Radhika Rajagopalan, Ph.D.; MuthukumarRarnaswarny,
Compounding Ph.D.; Sam G. Raney, Ph.D.; Ashutosh Rao,Ph.D.;
GIGI S. DAVIDSON, B.S. PHARM., DICVP, Chair Shahnaz Read, Ph.D.; Bhagwant Rege, Ph.D.; JamesRice,
Lisa Ashworth, B.S. Pharm., R.Ph.; Gus Bassani, Pharm.D.; Ph.D.; Jason Rodriguez, Ph.D.;Sara Rothman; Allen
Edmund J. Elder, Jr., Ph.D.; Ryan Forrey, Pharm.D., M.S.; Rudman, Ph.D.; Tracy Rupp,: Ph.D.; R.D. Satzger, Ph.D.;

www.ilovepharma.com
USP 43 People / Committees xvii

Zuben Erach Sauna, Ph',O.; Deborah Schmiel, Ph.D.;

Suzanne Sechen, Ph.D.; Hamid Shafiei, Ph.D.; Rakhi
Shah, Ph.D.; Millie Shah, Ph.D.; Balajee Shanmugam,
Ph.D.; Meiyu Shen, Ph.D.; Akhtar Siddiqui, Ph.D.; Mark
Skasko, Ph.D.; Fenhong Song, Ph.D.; Charudharshini
Srinivasan, Ph.D.; [annavi Srinivasan, Ph.D.; Marla
Stevens-Ril~y, Ph.£?;Ann Stohlman, V.M:D.; Yichun Sun,
Ph.D.; Jennifer SWisher, Ph.D.; Frank SWitzer, Ph.D.;
Neeru Takiar, M.S.; Carmen Tartera, Ph.D.; Jennifer
Thomas, Ph.D.; Colleen Thomas, Ph.D.; Yiying Tsai,
Pharm.D.; SalehTurujman, Ph.D.; Katherine Tyner,
Ph.D.; Ubrani Venkataram, Ph.D.; Steve Wolfgang,
Ph.D.; Jeffrey Woodruff, Ph.D.; Bingyuan Wu, Ph.D.;
Geoffrey Wu, Ph.D.; Larisa Wu, Ph.D.; [o Wyeth,
Pharm.D.; jingyue Yang, Ph.D.; Yuda long, Ph.D.
Other Government Liaisons
Agency for Healthcare Research and Quality
Diane D. Cousins,R.Ph.; Deborah G. Perfetto, Pharm.D.,
M.S.
U.S. Centers for Disease Control and Prevention
Christopher A. Elkins, Ph.D.; Nadine Shehab, Pharm.D.,
M.P.H.; Melissa Schaefer, M.D.
Health Canada
Matthew Decan, Ph.D.; Jessica Priem, M.S.; Nana
Bafi-Yeboa, Ph.D.
National Administration of Medicines, Foods,
and Medical Technology (ANMAT)
CarolinaAbba, Pharm.D.
National Institutes of Health
Rao Rapaka, Ph.D.; Cynthia Dyann Davis, Ph.D.
National Institute of Food and Drug Safety
Evaluation
Kwangmoon Lee, Ph.D.
Saudi Food and Drug Authority
Ali Mohammed Alhomaidan, Ph.D.;Ali M.Alsamil, Ph.D.

In Memoriam
USP would like to acknowledge the following Expert .
Volunteersand Government Liaisons who have passed
away during the 2015-2020 Cycle:
Stefan Christians, Ph.D. (Biologics Monographs 2-Proteins)
Scott V. W. Sutton, Ph.D. (General Chapters-Microbiology
ExpertCommittee)

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xviii Convention Members / People USP 43

an I a so
the Unit tes
ma opeial Con ntion
as of ay 31, 2019
Academic Institutions and Associations Thereof- Lama LindaUniversity School of Medicine, john N. Buchholz, Ph.D.
Academic Associations
Louisiana StateUniversity School of Medicine, Kurt l- Varner, Ph.D.

American Association of Colleges of Nursing, Ginette A. Pepper, Ph.D. Loyola University Chicago StritchSchool of Medicine, Jawed Fareed, Ph.D.

American Association of Colleges of Osteopathic Medicine, Anthony[, Silvagni, Mayo MedicalSchool, PeterJ. Post, Pharm.D., R.Ph.
D.O., Pharm.D., M.Sc., FACOFP, FAFPE MedicalUniversity of South Carolina College of Medicine, KennethD.Tew,Ph.D.
A~~g~an Association of Colleges of Pharmacy, Lynette R. Bradley-Baker, R.Ph., MeharryMedicalCollege School of Medicine, Clivel G. Charlton, Ph.D.

Association of American MedicalColleges, David W. Nierenberg, M.D. Mercer University School of Medicine, WayneGlasgow, Ph.D.
Morehouse School of Medicine, Ward Kirlin, Ph.D.
Association of American Veterinary MedicalColleges, Dawn M. Boothe, D.V.M.,
Ph.D., M.S. Mount Sinai School of Medicine, Joel S. Mindel, M.D.,Ph.D.
Association of Faculties of Pharmacy of Canada, Raimar l.oebenberq, Ph.D. New York MedicalCollege, MarioA. Inchiosa, Ph.D.
New York University School of Medicine, Lewis S. Nelson,M.D.
Academic Institutions and Associations Thereof-
NortheastOhioMedicalUniversity College of Medicine, Moses O. Oyewumi,
Colleges and Schools of Medicine B.Pharm., Ph.D.
Northwestern University Feinberg School of Medicine, Steven M. Belknap, M.D.
Baylor College of Medicine, Lynn C. Yeoman, Ph.D.
OhioState University College of Medicine, Robert j. Weber, Pharm.D., M.S.
Boston University School of Medicine, CarolT. Walsh, Ph.D.
Pennsylvania StateUniversity College of Medicine, Kelly D. Karpa, Ph.D., R.Ph.
Brody Schoolof Medicineat EastCarolina University, Abdel A.Abdel-Rahman,
Ph.D., FAHA Rutgers~ the StateUniversity of Newjersey, NewjerseyMedicalSchool, Deborah
Lazzanno, Ph.D.
Case Western Reserve University School of Medicine, WalterB. Geho, M.D., Ph.D.
Rutgers, the StateUniversity of Newjersey, Robert WoodjohnsonMedicalSchool
Chicago MedicalSchoolat Rosalind Franklin University of Medicine and Science, Susan Goodin, Pharm.D., FCCP, BCOP ,
Ann K. Snyder,Ph.D.
SaintLouis University School of Medicine, HeatherMacarthur, Ph.D., B.Sc.
Columbia University College of Physicians andSurgeons, Rudina Odeh-Ramadan,
Pharm.D. San juan Bautista School of Medicine, Marielis E. Rivera Ruiz, Ph.D.
Creighton University School of Medicine, PeterW.Abel, Ph.D. Sidney Kimmel MedicalCollege at Thomas jefferson University, WalterKraft,
M.D., M.S., FACP
Duke University School of Medicine, Sharon L. Ellison, Pharm.D.
Southern IllinoisUniversity School of Medicine, Carl Faingold, Ph.D.
East Tennessee State University james H. Quillen College of Medicine, Kenneth E.
Ferslew, Ph.D. SUNY at Buffalo School of Medicine and Biomedical Sciences, PaulJ. Kostyniak,
Ph.D., DABT
Eastern Virginia MedicalSchool, Arun MehnaatamaiMohanram, M.D.
SUNY Downstate MedicalCenter College of Medicine, JacobV.Aranda, M.D.,
Florida State University College of Medicine, Graham A. Patrick, Ph.D. Ph.D., FRepc .
Geisel School of Medicine at Dartmouth, Lionel D. Lewis, M.D. SUNY Upstate MedicalUniversity, David F. Lehmann, M.D., Pharm.D.
Georgetown University School of Medicine, ThomasG. Sherman, Ph.D. Temple University School of Medicine, AlanCowan, Ph.D.
HowardUniversity College of Medicine, Robert E. Taylor, M.D., Ph.D., FACP Texas A&M Health Science CenterCollege of Medicine, D. Samba Reddy, Ph.D.,
joan C. Edwards Schoolof Medicine MarshallUniversity, GaryO. Rankin, Ph.D. R.Ph.

johns Hopkins University School of Medicine, Daniel M. Ashby, M.S., FASHP Texas Tech University Health Sciences CenterSchool of Medicine, Michael P.
Blanton, Ph.D.
Keck Schoolof Medicine of University of Southern California Paul D. Holtom
M.D. " The Warren AlpertMedicalSchool of BrownUniversity, Wayne D. Bowen, Ph.D.

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USP 43 People / Convention Members xix

Tufts University School of Medicine, Ross W.Thompson, M.S., R.Ph. Washington University School of Medicine in St. Louis, Evan D. Kharasch, M.D.,
Ph.D.
Tulane University School of Medicine, David W. Busija, Ph.D.,M.D. (Hon.)
Wayne State University School of Medicine, Stephen A.lerner, M.D.
Universidad Central delCaribe School of Medicine, Harry Mercado, M.D.
WrightState University Boonshoft School of Medicine, Khalid M. Elased, Ph.D.,
University ofAlabama at Birmingham School of Medicine, Richard j. Whitley, R.Ph.
M.D.
Yale University School of Medicine, Seth Powsner, M.D.
University of Arizona College of Medicine, Hillary A. Franke, M.D., M.S.
University of Arkansas for Medical Sciences College of Medicine, Paul L. Prather,
Ph.D., B.S. Academic Institutions and Associations Thereof-
Colleges and Schools of Pharmacy
University of California Davis School of Medicine, TimothyE. Albertson, M.D.,
M.P.H., Ph.D.
AlbanyCollege of Pharmacy andHealth Sciences, Robert A. Hamilton, Pharm.D.,
University of California San Francisco School of Medicine, Linda L Liu, M.D. M.P.H.
University of Chicago Pritzker School of Medicine, Michael L. Maitland, M.D., AuburnUniversity Harrison School of Pharmacy, jayachandraRamapuram, Ph.D.
Ph.D.
Butler University College of Pharmacy and Health Sciences, SudipK. Das, Ph.D.
University ofCincinnati College ofMedicine, MarianneF.Ivey, Pharm.D., M.P.H.,
FASHP Campbell University School of Pharmacy, jinsong Hao, Ph.D.
University of Connecticut HealthCenter School of Medicine, Kimberly l- Metcalf, Chicago State University College of Pharmacy, Due P. Do, Ph.D.
Pharm.D.
Creighton University School of Pharmacy and Health Professions, [, Christopher
University of HawaiiJohn A. Burns School of Medicine, Robert A. Nichols, Ph.D. Bradberry, Pharm.D.
University of Iowa Carver College of Medicine, Donald E. letendre, Pharm.D. Drake University College of Pharmacy and Health Sciences, AbebeE. Mengesha,
Ph.D.
University of Kansas School of Medicine, Sam l- Enna,Ph.D.
Duquesne University School of Pharmacy, IraS. Buckner, Ph.D.
University of Kentucky College of Medicine, Lisa A. Cassis, Ph.D.
Ernest MarioSchool of Pharmacy at Rutgers University, longqin Hu, Ph.D.
University of Louisville School of Medicine, DemetraAntimisiaris, Pharm.D., CGP,
FASCP Ferris State University College of Pharmacy, Kim E. Hancock, Ph.D.

University of MarylandSchool of Medicine, Margaret M. McCarthy, Ph.D. Florida A & M University College of Pharmacy and Pharmaceutical Sciences, Man-
dip Sachdeva,Ph.D.
University of Massachusetts Medical School, Roy Guharoy, Pharm.D., M.B.A.,
FCP, FCC, FASHP Hampton University School of Pharmacy, Chengan Du, Ph.D., M.P.H.

University of Michigan Medical School, PaulF. Hollenberg, Ph.D. HowardUniversity College of Pharmacy, Nursing andAlliedHealth Sciences, Em-
manuel O. Akala, Ph.D.
University of Mississippi School of Medicine, Deborah S. Minor, Pharm.D., FAHA
IdahoState University College of Pharmacy, Kevin W. Cleveland, Pharm.D.
University of Missouri-Kansas CitySchool of Medicine, james M. Wooten,
Pharm.D. Lake Erie College of Osteopathic Medicine School of Pharmacy, SachinS. Devi,
B.Pharm., Ph.D.
University of Nevada School of Medicine, lain L. Buxton, Pharm.D.
Loma Linda University School of Pharmacy, Victoria Maskiewicz, Ph.D.
University of New Mexico Health Sciences Center School of Medicine, Arti Prasad,
M.D. Long IslandUniversity Arnoldand MarieSchwartz College of Pharmacy and
Health Sciences, DavidR.Taft, B.S.Pharm., Ph.D.
University of North Dakota School of Medicine and Health Sciences, JamesE. Por-
ter, Ph.D. Massachusetts College of Pharmacy and Health Sciences-Boston, David A. Wil-
liams, Ph.D.
University of Oklahoma College of Medicine, Pramod K. Chetty, M.D.
Massachusetts College of Pharmacy and Health Sciences School of Pharmacy-
University of Pennsylvania School of Medicine, Patrick l. Brennan, M.D. Worcester, RobertCampbell,Ph.D.,M.S.
University of Pittsburgh School of Medicine, DennisP. Swanson,M.S. Mercer University College of Pharmacy and Health Sciences, Ayman Akil, Ph.D.
University of Puerto Rico School of Medicine, MiguelA. Marrero, M.D. Midwestern University Chicago College of Pharmacy, Anna Kabakov, Pharm.D.
University of South Alabama College of Medicine, jack A. DiPalma, B.S., M.D. Midwestern University College of Pharmacy-Glendale, Bill J. Bowman, Ph.D.,
R.Ph.
University of South Carolina School of Medicine, KennethB.Walsh, Ph.D.
North Dakota State University College of Pharmacy, Nursing andAlliedSciences,
University of South Florida Morsani College of Medicine, Shufeng Zhou, M.D., jagdish Singh, Ph.D.
Ph.D.
Northeastern University Bouve College of Health Sciences School of Pharmacy,
University of Tennessee, Memphis College of Medicine, TrevorW. Sweatman, MansoorM. Amiji, Ph.D.
Ph.D.
NOVA Southeastern University College of Pharmacy, HamidH.Omidian, Ph.D.,
University of Texas Medical School at Houston, GaryC. Rosenfeld, Ph.D. M.Sc., B.Sc.
University of Utah School of Medicine, Richard [, Sperry, M.D.,Ph.D. OhioNorthern University Raabe College of Pharmacy, Yousif B. Rojeab, B.Sc.,
Ph.D.
University 9f Virginia School of Medicine, Matthew T. jenkins, M.S., Pharm.D.
University of Washington School of Medicine, S~zanne M. Allen, M.D.,M.P.H.
Oregon State University College of Pharmacy, l- Mark Christensen, Ph.D.
Pacific University College of Health Professions School of Pharmacy, Reza Karimi,
University of Wisconsin School of Medicine and Public Health, F. Michael Hoff- Ph.D., R.Ph.
mann, Ph.D.
Palm Beach AtlanticUniversity Gregory School of Pharmacy, AdwoaO. Nornoo,
Vanderbilt University School of Medicine, C. Michael Stein, M.D. B.Pharm., M.S., Ph.D.
Virginia Commonwealth University School of Medicine, DominicA. Sica, M.D. Purdue University School of Pharmacy and Pharmaceutical Sciences, Stephen R.
Wake Forest University School of Medicine, Mary L. Bennett, B.S., Pharm.D. Bym, Ph.D.

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xx Convention Members / People USP 43

Roseman University of Health Sciences College of Pharmacy, Mark C. Decerbo, University of Oklahoma College of Pharmacy, VibhuduttaAwasthi, Ph.D.,
Pharm.D., BCPS, BCNSP M.Pharm.
SamfordUniversity McWhorterSchool of Pharmacy, John J.Arnold, Ph.D., B.S. University of Pittsburgh School of Pharmacy, Michael A. Zemaitis, Ph.D.
Shenandoah University Bernard j. DunnSchool of Pharmacy, Nina Hengen, M.D., University of Rhode IslandCollege of Pharmacy, David R. Worthen, Ph.D.,J.D.
Ph.D.
University of Sao Paulo College of Pharmacy, Prof. Terezinhade JesusAndreoli
South Carolina College of Pharmacy, JamesJ. Sterrett, Pharm.D., BCPS Pinto
South DakotaState University College of Pharmacy, David L. Helgeland, University of Southern California School of Pharmacy, Stan G. louie, Pharm.D.
B.S.Pharm., M.B.A., Ed.D.
University of Tennessee HealthScience Center College of Pharmacy, HassanAI-
South University School of Pharmacy, S. Craig Dyar, Ph.D. moazen, Ph.D.
Southern IllinoisUniversity Edwardsville School of Pharmacy, William M. Kolling, University of the Pacific Thomas j. Long School of Pharmacy and HealthSciences,
Ph.D. . Xiaoling Li, Ph.D.
Southwestern OklahomaState University School of Pharmacy, Shelly Stockton, University of the Sciences in Philadelphia, Philadelphia College of Pharmacy, Ed-
Ph.D. ward F. Foote, Pharm.D., BCPS, FCCP
St. john's University College of Pharmacy and AlliedHealthProfessions, Abu Ser- University of Utah College of Pharmacy, John W. Mauger, Ph.D.
ajuddin, Ph.D. .
University of Washington School of Pharmacy, Thomas K. Hazlet,Pharm.D.,
St. Louis College of Pharmacy, John Pieper, Pharm.D. Dr.P.H.
SUNY at Buffalo School of Pharmacy and Pharmaceutical Sciences, Alfred T. Re- University of Wisconsin-Madison School of Pharmacy, James E. DeMuth, Ph.D.,
iman, R.Ph. R.Ph.
Temple University School of Pharmacy, Michael Borenstein, Ph.D. University of Wyoming School of Pharmacy, Kurt Dolence, Ph.D.
Texas Southern University College of Pharmacy and HealthSciences, Dong liang, Virginia Commonwealth University/Medical College of Virginia School of Pharma-
Ph.D. cy, BarbaraJ. Exum, Pharm.D.
Texas Tech University HealthSciences CenterSchool of Pharmacy, ArthurA. Nel- Washington StateUniversity College of Pharmacy, Danial E. Baker, Pharm.D.,
son, Ph.D., R.Ph. FASHP, FASCP
TheOhio StateUniversity College of Pharmacy, RobertW. Brueggemeier, Ph.D. Wayne StateUniversity Eugene Applebaum College of Pharmacy and HealthSci-
ences, SheilaM. Wilhelm, Pharm.D.
TheUniversity of ArizonaCollege of Pharmacy, TerriWarholak, Ph.D.,R.Ph.
West VirginiaUniversity School of Pharmacy, Arthur I. [acknowltz, Pharm.D.
The University of Iowa College of Pharmacy, Mickey L. Wells, Ph.D.
Western University of HealthSciences College of Pharmacy, SunilPrabhu,
TheUniversity of Louisiana at MonroeCollege of Pharmacy, Sarnl M. Nazzal, Pharm.D., R.Ph.
Ph.D.
Wilkes University Nesbitt School of Pharmacy, Harvey A. Jacobs, Ph.D.
The University of North Carolinaat Chapel Hill Eshelman School of Pharmacy,
Dennis M. Williams, Pharm.D. XavierUniversity of Louisiana College of Pharmacy, Tarun K. Mandai, Ph.D.
TheUniversity of Texas at Austin College of Pharmacy, Janet C. Walkow, Ph.D.
The University of Toledo College of Pharmacy, KennethS.Alexander, R.Ph., Ph.D. Academic Institutions and Associations Thereof-
Colleges and Schools of Osteopathic Medicine .
Touro University California College of Pharmacy, Alison McCormick, Ph.D.
University of Arkansas for MedicalSciences College of Pharmacy, Melanie Rein- NOVA Southeastern University Dr. Kiran C. PatelCollege of Osteopathic Medi-
hardt, Pharm.D. . cine, lynne lafferty, Pharm.D., N.D., M.B.A.
University of California SanDiegoSkaggs School of Pharmacy and Pharmaceuti-
cal Sciences, Grace M. Kuo, Pharm.D., Ph.D., M.P.H. . Consumer and Other Organizations Representing the
University of California San Francisco School of Pharmacy, leslie Z. Benet, Ph.D. Public Interest
University of Cincinnatijames L. Winkle College of Pharmacy, Pankaj B.Desai,
Ph.D. AARP, leigh Purvis
University of Colorado Skaggs School of Pharmacy, PeterJ.Rice, Pharm.D., Ph.D. Alzheimer's Association, William Thies, Ph.D.
University of Florida College of Pbormac», RhondaCooper-DeHoff, Pharm.D., American Autoimmune Related Diseases Association, Virginia T. ladd, R.T.
M.S., FAHA
American Cancer Society, Inc., leonard Lichtenfeld, M.D., MACP
University of Georgia College of Pharmacy, GurvinderSingh Rekhi, Ph.D., M.S.
American HeartAssociation, Robertlee Page II, Pharm.D.,M.S.P.H., FCCP, FA-
University of Houston College of Pharmacy, F. LamarPritchard, Ph.D. HA, FASHP, FASCP, BCPS, CGP
University of Illinoisat Chicago College of Pharmacy, Stephanie Y. Crawford, Arthritis Foundation, John H. Klippel, M.D.
Ph.D., M.P.H. '
Center for Science in the Public Interest, David G. Schardt, M.S.
University of Kansas School of Pharmacy, Christian Schoneich, Ph.D.
Consumers Union, Lisa L. Gill, B.A.
University of Maryland School of Pharmacy, NatalieD. Eddington, Ph.D.
ECRllnstitute, MarcusSchabacker, M.D., Ph.D.
University of Michigan College of Pharmacy, Gregory E. Amidon, B.S., Ph.D.
Institute for Safe Medication Practices, Michael R. Cohen, R.Ph., M.S., 5c.D.,
University of MinnesotaCollege of Pharmacy, Lynda Welage, Pharm.D.,FCCP FASHP
University of Mississippi School of Pharmacy, Michael A. Repka, D.D.S., Ph.D. National Organizationfor Rare Disorders, Ronald J. DeBellis, B.S., Pharm.D.

University of Missouri-Kansas City School of Pharmacy, Cydney E. McQueen,

Pharm.D. Governmental Bodies or Divisions or Associations
University of NebraskaMedicalCenter College of Pharmacy, MichaelF. Powell, Thereof
B.S. Pharm., M.S.
Agency for Healthcare Research and Quality, Scott R. Smith, Ph.D.

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USP 43 People / Convention Members xxi

Argentine Pharmacopoeia, Hector A. Giuliani, Pharm.D. Permanent Commission of the Pharmacopoeia of the UnitedMexican States, Ma-
ria del Carmen Becerril-Martinez
Association of Food and Drug Officials, Cynthia T. Culmo
Pharmacopoeia of Chinese Taipei, law-lou Kang, Ph.D.
Brazilian Pharmacopoeia Commission, Monica da Luz CarvalhoSoares
Pharmacy Council of India, Bhojraj Suresh, M.Pharm., Ph.D.,D.Sc.
Brazil's NationalInstituteof QualityControlin Health, Filipe SoaresQuirinoSilva,
Ph.D. Philippine Pharmacopeia, Maria lourdes e. Santiago, B.S.Pharm., M.S.Pharm.
British Pharmacopoeia Commission, James Pound Saudi Food and DrugAuthority, Abdullah S.Alhomoud, M.S.
Centers for Disease Control and Prevention, Julian Jolly, Pharm.D., R.Ph. StateAdministration of Ukraine on Medicinal Products, Andrii V. Shovkovyi
Centers for Medicare & Medicaid Services, Jeffrey A. Kelman, M.D. Tanzania Food and Drugs Authority, Yonah Hebron Mwalwisi, M.Sc., B.Pharm.
ChinaNational Centerfor Food Safety Risk Assessment, Junshi Chen, M.D. ThaiPharmacopoeia Committee, Kornvika Charupant, Ph.D.
Chinese Pharmacopoeia Commission, Zhang Wei The Pharmacy Board of Sierra Leone, Wiltshire CN. Johnson,B.Pharm. (Hons.),
M.Sc, MPSSL, FPCPharm
European Directorate for the Qualityof Medicines and HealthCare, SusanneKeitel
Therapeutic Goods Administration of Australia, Vivienne Christ, B.App.Sc.,
FDA Centerfor Biologics Evaluation and Research, Ewa Marszal, Ph.D., M.S. MASM
FDA Centerfor Devices and Radiological Health, Jose A. Centeno, Ph.D., FRSC Ukrainian Scientific Pharmacopoeial Center for Quality'ofMedicines, OleksandrI.
Gryzodub, Ph.D.
FDA Centerfor Drug Evaluation and Research, Pallavi Nithyanandan, Ph.D.
UnitedStates Agency for InternationalDevelopment, AnthonyF.Boni
FDA Centerfor Food Safety and Applied Nutrition, Daniel E. Folmer, Ph.D.
UnitedStates Air Force-Office of the Surgeon General, Deborah Myers, R.Ph.,
FDA Centerfor Veterinary Medicine, Hilary Hoffman, M.S., Ph.D. M.B.A.
Federal Commission for Protection AgainstSanitary Risks, Rocio del CarmenAla- UnitedStates Army-Office of theSurgeon General, John Spain, Pharm.D., BCPS
torre Eden-Wynter, M.e.
UnitedStates Department of Health and Human Services, DonaldWright,M.D.,
Federal Service on Surveillance in Healthcare of Russian Federation, Valentina F. M.P.H.
Kosenko, Ph.D., Pharm.
UnitedStates Food and DrugAdministration Office of theCommissioner, Hany W.
Food and Drugs Authority, Ghana, Eric Karikari-Boateng, M.S. Demian,M.S.
Food, Medicine and Health Care Administration and ControlAuthorityof Ethiopia, UnitedStates Navy Bureau of Medicine and Surgery-Office of theSurgeon Gen-
Yehulu DenekewAlamneh, B.Sc., M.A. eral, ThinhV. Ha, Pharm.D., M.B.A., M.S.
General Directorate of Medicines, Supplies and Drugs, Laura Ceron, Pharm.D. UnitedStates Public Health Service-Office of the Surgeon General, David J.Skan-
Health Canada, Karen Reynolds chy, Ph.D., B.S. .

HealthCanada, Food Directorate, Barbara Lee Vietnamese Pharmacopoeia Commission, Luc ThiThu Hang, Ph.D.

Indian Pharmacopoeia Commission, G.N. Singh,Ph.D., M.Pharm.

Other Health Practitioner, Professional and
Indonesian Pharmacopoeia Commission, Augustine Zaini, M.S.' Scientific Associations
InternationalTrade Administration-U.S. Department of Commerce, James D.
Rice, B.A., M.A. MCC International, Amy Hope
Japan's Ministryof Health Labourand Welfare, Pharmaceuticals and MedicalDe- Academy of Managed Care Pharmacy, Afton Wagner, Pharm.D.
vices Agency, Nobuo Uemura .
Academy of Nutrition and Dietetics, Ann Erickson, M.A., R.D.
Jordan Food and DrugAdministration, Dr. Lubna R. Qusous
American Academy of Neurology, JackJ. Chen, Pharm.D., FASCP, FCCP
Ministry of Food and DrugSafety, Dr. Jin-Ho Wang
American Academy of Nurse Practitioners, JanTowers, Ph.D.,NP-C
Ministry of Healthof the Republic of Belarus, liudmila Reutskaya, Pharm.D.
American Academy of Ophthalmology, Suber S. Huang,M.D.,M.B.A.
Ministry of Healthof the Republic of Uzbekistan, Mirzohidjon Kodirov, Ph.D.
American Academy of Physician Assistants, Marie-Michele Leger, M.P.H., PA-C
Ministry of Healthof Turkey, Filiz BGtev Koc, Ph.D.
American Academy of Veterinary Pharmacology and Therapeutics, Carol A. Davis,
Morocco's Directorate of Medicines and Pharmacy, Laila Hakkou Ph.D.
National Agency for Food and DrugAdministration and Control, Dr. MonicaH. American Association of Pharmaceutical Scientists, ChristopherMcCurdy, Ph.D.,
Eimunjeze R.Ph.
National Association of Boards of Pharmacy, Elizabeth Scott Russell, B.S.Pharm. American Botanical Council, Mark Blumenthal
National Center for Quality Control, Ofeliadel Rosario Villalva Rojas, Q.F. American Chemical Society, MaryM. Kirchhoff, Ph.D.
National Centre for Expertise of Drugs, Medical Products and MedicalEquipment, American College of Clinical Pharmacology, PeterWiernik, M.D.
Ardak U.Tulegenova, Ph.D. .
American College of Clinical Pharmacy, Michael S. Maddux, Pharm.D., FCCP
National DrugAuthorityof Uganda, Gordon KatendeSematiko
American College of Physicians, DouglasS. Paauw, M.D.
National Health Surveillance Agency, lais Santana Dantas, B.S.
American DentalAssociation, Kathleen Ziegler, Pharm.D.
National Institutefor Biological Standards and Contro~ Adrian F. Bristow, Ph.D.
American DentalEducation Association, Jeffrey Stewart,D.D.S., M.S.
National Instituteof Drugand Food Surveil/ance, EduardoVergel Bayona
American Geriatrics Society, Todd P. Semla, M.S., Pharm.D., BCPS, FCCP, AGSF
National Instituteof Metrology, Qualityand Technology, Valnei smarcero da
Cunha, Ph.D. American Medical Association, Amy B. Cadwallader, Ph.D.
National Instituteof Standards and Technology, Michael J. Tarlov, Ph.D. American Nurses Association, Rita MunleyCallaqher, Ph.D., R.N.
National Institutes for Food and Drug Control, Bo li, Ph.D. American Optometric Association, Jimmy D. Bartlett, O.D.
National Institutes of Health, RobertDeChristoforo, R.Ph., M.S., FASHP

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xxii Convention Members / People USP 43

American Pharmacists Association, ThomasE.Menighan,R.Ph., M.B.A., FAPhA Kansas MedicalSociety, Tracie Collins, M.D.,M.P.H.
American Public HealthAssociation, DeborahK. Walker, Ed.D. MaineMedicalAssociation, StevanGressitt, M.D.
American Society for Clinical Pharmacology and Therapeutics, Patricia W.Slat- Massachusetts MedicalSociety, BruceG. Karlin, M.D.
tum, Pharm.D., Ph.D.
MedChi-Maryland StateMedical Society, Peter H. Rheinstein, M.D.,J.D., M.S.
American Society for Microbiology, AmyL. leber, Ph.D.
M~dical Association of the State of Alabama, Allan R.Goldstein, M.D.
American Society for Nutrition, Robert M. Russell, M.D.
Medical Society of NewJersey, joseph N. Micale, M.D.
American Society for Pharmacology and Experimental Therapeutics, MaryPaine,
Ph.D. Medical Society of the Districtof Columbia, Kim A.Bullock, M.D.

American Society for Quality, Donald C. Singer,M.S. Medical Society of the Stateof New York, Richard S. Blum, M.D.

American Society of Anesthesiologists, Elizabeth Rebello, M.D. Missouri StateMedicalAssociation, Patrick J. Mills

American Society of Consultant Pharmacists, Arnold E. Clayman, Ph.D., FASHP Montana MedicalAssociation, Jean Branscum

American Society of Health-System Pharmacists, Paul W.Abramowitz, Pharm.D., New Mexico Medical Society, Jerry D. Mclaughlin, M.D.
FASHP
North DakotaMedicalAssociation, Robert (Rob)W. Beattie,M.D.
American Veterinary MedicalAssociation, Michael A.Strobel, D.V.M., M.S.
Pennsylvania Medical Society, RalphSchmeltz, M.D., FACP, FACE
Argentine Association of IndustrialPharmacy and Biochemistry, FedericoMontes
de Oca, M.B.A. Puerto Rico MedicalAssociation, Rolance G. Chavier-Roper, M.D.

Brazilian National Association of Compounding Pharmacists, Mariado Carmo Rhode IslandMedical Society, Peter A. Hollmann, M.D.
Garcez SouthDakotaStateMedicalAssociation, E. PaulAmundson, M.D.
Calibration and Validation Group, Herman lam, Ph.D. Tennessee MedicalAssociation, John J. Ingram III, M.D.
Canadian Society for Pharmaceutical Sciences, Neal M. Davies, B.Sc. Pharm., Texas MedicalAssociation, Kevin H. McKinney, M.D., FACE, FACP
Ph.D., R.Ph.
Utah MedicalAssociation, Michelle S. McOmber,B.S., M.B.A.
Drug InformationAssociation, Katie N. Truong, B.A.
West Virginia StateMedicalAssociation, Kevin W.Yingling, M.D., R.Ph., FACP
European Federation for Pharmaceutical Sciences, Hendrik J. de Jong, Ph.D.
Wisconsin MedicalSociety, Thomas M. Derrig, M.D.
Infusion Nurses Society, Mary C. Alexander, C.R.N.I., M.A., R.N., CAE, FAAN
Instituteof Food Technologists, William Fisher, M.S.
Health Practitioner, Professional and Scientific
InternationalAcademy of Compounding Pharmacists, Gary E. McCrory, Associations-U.S. State Pharmacy Associations
B.S.Pharm.
InternationalCouncil of Nurses, DavidC.Benton,RGN, RMN, BSC, MPhil, FFNF, Alabama Pharmacy Association, Valerie A.Oakley, Pharm.D.
FRCN
Alaska Pharmacists Association, Sara Doran-Atchison, Pharm.Di, M.P.H., NCPS
InternationalSociety for Cellular Therapy, Joseph c. laning, Ph.D.
ArizonaPharmacy Association, Kelly Fine, R.Ph.
InternationalSociety for Pharmaceutical Engineering, Stephen M. Tyler, M.S.
Arkansas Pharmacists Association, Kristen G. Riddle, Pharm.D.
Korea Biomedicine IndustryAssociation, JungTae Park
California Pharmacists Association, Jon R.Roth,CAE
National Alliance of StatePharmacy Associations, RebeccaP, Snead, R.Ph.,
B.S.Pharm. Colegio de Farmaceuticos de Puerto Rico, Milagros Morales, R.Ph.
National CommunityPharmacists Association, Ronna B.Hauser, Pharm.D. Colorado Pharmacists Society, JenniferDavis, Pharm.D., M.B.A.
National Pharmaceutical Association, Barry A. Bleidt, Ph.D., Pharm.D. Connecticut Pharmacists Association, PeterJ. Sposato, R.Ph., B.C.N.P.
National Pharmacy Technician Association, Michael J. Johnston, CPhT Delaware Pharmacists Society, Kenneth(Kevin) Musto, R.Ph.
NewJersey Pharmaceutical QualityControl Association, BarbaraJ. Ferguson, B.A. Florida Pharmacy Association, Michael A.Mone, J.D., R.Ph., FAPhA
PanAmerican Pharmaceutical Federation, Maria Elena Girard-Cuesy Georgia Pharmacy Association, larry l. Braden, B.S., R.Ph., D.Sc.
Parenteral Drug Association, Inc., Tina S. Morris, Ph.D. HawaiiPharmacists Association, Ronald T. Taniguchi, Pharm.D.
Regulatory AffairsProfessional Society, Paul Brooks Idaho State Pharmacy Association, Pam Eaton
Society of CriticalCare Medicine, TimothyS. Yeh, M.D., FCCM IllinoisPharmacists Association, Norman A. Hoback, B.S.Pharm.
Society of NuclearMedicine and Molecular Imaging, Jeffrey P. Norenberg, IndianaPharmacists Alliance, Daniel D. Degnan III, Pharm.D., M.S.
Pharm.D.
Iowa Pharmacy Association, Thomas R. Temple, R.Ph., M.S.
Western Compendial Discussion Group, Assad J. Kazeminy, Ph.D.
Kansas Pharmacy Association, Alison Smith, Pharm.D.
Kentucky Pharmacists Association, Don B. Kupper, R.Ph., M.B.A.
Health Practitioner, Professional and Scientific
Associations-U.S. State Medical Societies Louisiana Pharmacists Association, Randall Brooks, B.S.
Maine Pharmacy Association, laurier A.lamie, R.Ph.
Connecticut StateMedicalSociety, HenryJacobs, M.D., J.D.
MarylandPharmacists Association, Matthew G. Shimoda, Pharm.D.
HawaiiMedicalAssociation, [one Geimer-Flanders, D.O.
Massachusetts Pharmacists Association, Steven D.Geoffroy, R.Ph.
lIfinois StateMedicalSociety, Maitreyi Janarthanan, M.D.
Michigan Pharmacists Association, Eric D. Roath,Pharm.D.
IndianaStateMedicalAssociation, Daria Schooler, M.D., R.Ph.
Minnesota Pharmacists Association, lowell J. Anderson, D.Sc., FAPhA, FFIP
Iowa MedicalSociety, Harold W. Miller, M.D.
Mississippi Pharmacists Association, Kimsey O'Neal, Pharm.D.

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USP 43 People / Convention Members xxiii

MissouriPharmacy Association, Ron L Fitzwater, CAE, M.B.A. Korea Pharmaceutical and Bio·Pharma Manufacturers Association, Hee-Mok
Won, Pharm.D., M.S.
Montana Pharmacy Association, Lori Morin, M.B.A., Pharm.D.
Mexico's National Chamberof the Pharmaceutical Industry, [. Rivelino Flores,
Nebraska Pharmacists Association, Samuel C. Augustine, Pharm.D., FAPhA M.Sc.
New Hampshire Pharmacists Association, Elizabeth A. Robertson, R.Ph. National Association of Chain DrugStores, Alex [, Adams, Pharm.D.
NewJersey Pharmacists Association, Steve H. Zlotnick, Pharm.D. Personal Care Products Council, BethAnne Lange,Ph.D., B.S.
New Mexico Pharmacists Association, William G. Troutman, Pharm.D. SaoPauloStatePharmaceutical Manufacturers Association, Nelsondos Santos,
jr.
North Carolina Association of Pharmacists, Stephen F.Eckel, Pharm.D., M.H.A.
TheJoint Commission, jeannell M. Mansur, B.S. Pharm., Pharm.D.
North DakotaPharmacists Association, Michael D. Schwab
TheJordanian Association of Manufacturers of Pharmaceuticals & MedicalAppli-
Ohio Pharmacists Association, Amelia S. Bennett, R.Ph. ances, Hanan J. Sboul, M.B.A., CAE
Oklahoma Pharmacists Association, Wiley L. Williams, j.D.
Oregon State Pharmacy Association, Tiffany Pye, Pharm.D., BCPS Nongovernmental Standards-Setting and Conformity
Pennsylvania Pharmacists Association, George R.Haynes, Pharm.D., Ph.D.
Assessment Bodies
Pharmacy Society of Wisconsin, Susan M. Kleppin, R.Ph., FASHP Accreditation Council for Pharmacy Education, Dimitra V. Travlos, Pharm.D.,
Rhode IslandPharmacists Association, John Grossomanides, Pharm.D. BCPS

SouthCarolina Pharmacy Association, CraigBurridge, M.S., CAE American Type Culture Collection, Elizabeth j. Kerrigan, B.A.

SouthDakotaPharmacists Association, Eric Grocott, Pharm.D., B.S. Association for the Advancement of MedicalInstrumentation, joseph CarlLew-
elling, B.A., M.A.
Tennessee Pharmacists Association, MicahCost, Pharm.D., M.S.
ChileanPharmacopeia Foundation, Caroline R. Weinstein-Oppenheimer, Ph.D.
Texas Pharmacy Association, DebbieGarza,R.Ph.
Clinicaland Laboratory Standards Institute, Glen Fine, M.S., M.B.A., CAE
Utah Pharmacists Association, Adam W. jones
VirginiaPharmacists Association, William T. Lee, D.Ph, M.P.A, FASCP
Washington D.C. Pharmacy Association, Michael j. Kim, Pharm.D.
Washington State Pharmacy Association, jeffRochon, Pharm.D.
West Virginia Pharmacists Association, Pattyjohnston, B.S.
Wyoming Pharmacy Association, William H. Rathburn, R.Ph.

Manufacturer, Trade and Affiliated Associations

American Chemistry Council, Michael P. Walls, Esq.

American HerbalProducts Association, Holly E. johnson, Ph.D.
American HospitalAssociation, john R.Combes, M.D.
Animal HealthInstitute, Richard A.Carnevale, V.M.D.
Association for Accessible Medicines, David R. Gaugh, R.Ph.
Association of the European Self-Medication Industry, Christelle Anquez-Traxler
Biotechnology InnovationOrganization, Earl S. Dye,Ph.D.
BulkDrug Manufacturers Association, Airra Krishna Reddy, B.Sc.
Compressed GasAssociation, Michael B. Tiller, B.S., B.A.
Consumer Health Products Canada, Adam C. Kingsley, B.Sc., NP,CAE
Consumer Healthcare Products Association, john Punzi, Ph.D.
Councilfor Responsible Nutrition, james C. Griffiths, Ph.D.
Egyptian Chamber of Pharmaceutical Industry, Abdulla Molokhia, Ph.D.
European Generic Medicines Association, julieMarechal-jamal, M.S.
Flavor and ExtractManufacturers Association, john H.Cox, j.D.
Generic Animal Drug Alliance, Stephanie Batliner
Indian Drug Manufacturers'Association, Daara B. Patel, B.Com., D.J.M.M.
International Dairy Foods Association, john T. Allan III, B.S., M.S.
InternationalFederation of Pharmaceutical Manufacturers and Associations, Car-
oline Mendy
InternationalFoodAdditives Council, RobertRankin
InternationalGeneric and Biosimilar Medicines, Nicholas Cappuccino,Ph.D.
InternationalPharmaceutical Excipients Council of theAmericas, Priscilla Zawislak

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www.ilovepharma.com
USP 43 People / Donors xxv

USP acknowledges and appreciates the support of the st.John Fisher College - School of Pharmacy
following companies that participated in the USP Sun Vat-Sen University
standards-setting process by providing information for the
Teva
development of new monographs or by donating reference ·1

standard candidate materials. The companies listed are UCB Pharma

those that either provided monographs that were published West-Ward
as proposals in 2018 or donatecf reference material Zydus Cadila
candidates that were released as USP Reference Standards
in 2018. Gold Level Donors
Ajinomoto
Platinum Level Donors
Albemarle
AbbVle
Bachem
Ajanta
Biogen
Amneal
Chiesi
Apotex
Cipla
AstraZeneca
Dow Chemical
Aurobindo
Dr. Reddy's
Bayer
Eastman
Boehringer Ingelheim
Fresenius Kabi
Cambrex
Gland Pharma
China Pharmaceutical University
HRA Pharma
Croda
Indena
DSM
Jubilant
DuPont
Koster Keunen
Evonik
Lundbeck
FMC Corporation
Mallinckrodt
Glaxosmith Kline
Merz
Glenmark
PCCA
Himalaya
Pfizer
ICL Performance
Pharmavite
Impax
Scinopharm
Janssen
Sicor
Lupin
SMS Pharmaceuticals
Medichem
Sun Pharma
Medisca
Welch, Holme &: Clark
Merck
Xavier University of Louisiana
Moehs Iberica
Mylan Silver Level Donors
Natural Remedies Aarti Industries
Novartis ACS Dofbar
Par Pharma Aizant
Roche Alembic
Sanofi-Aventis Almirall
Shanghai Institute of Materia. Medica Alzo
Shire Amano Enzyme

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xxvi Donors / People USP43

AMRI Kashiv Pharma

AMSA Katra Phyto Chem
Apicore Katwijk Chernie
Arbor Kongo Chemical
Arevipharma Kyowa Hakko
Aspen lilly
BASF l'Oreal
Baxter lusochimica
Beijing Gingko Mangalam
Beneo Marksans
Bergstrom Nutrition Menadiona
Bimeda Micro Advanced Research Centre
BioPQQ Monteloeder
Biospectra Mutchler
Blue California New Francisco (YunFu City) Biotechnology
Bristol-Myers Squibb Noramco
Budenheim Iberica Novitium
Cargill Novo Nordisk
CBC Olon
Cellmark Orchid
Changzhou Pharmaceutical Factory Otsuka
Chemische Fabrik Pfanstiehl
CKD Bio Piramal
Clariant Proto Chemicals
Corden Purecircle
Daiichi Sankyo Quimica Sintetica
Dipharma RPG life Sciences
Dishman Sabinsa
DMV-Fonterra Septodont
Dottikon Sichuan long March
Eisai Sichuan Xieli
Emcure Siegfried
Enzyme Development Sintetica-Bioren
Eon labs SPI Polyols
Ethypharm SST Corporation
Excella T&R Chemical
Excelvite Taiyo
Fabbrica Italiana Sintetici Taro
Ferrer Tennants
FineTech Thermo Fisher
Flamma Tianjin Tianyao
Flavor and Extract Manufacturers Association Trifarma
Formosa Unichem
Fujian Fukang Unique Pharmaceutical labs
Gattefosse Upsher-Smith
GE Healthcare Vanderbilt
Helsinn Vertellus
Herbalife Wilmar
Hovione Wockhardt
Hunan Dongting Yamasa
Icrorn Zhejiang Apeloa jianyuan
Impossible Foods Zhejiang Hisoar
Indivior Zhejiang MedicineCo
Indoco Zhejiang University
Interquim Zoetis
jB Chemicals
jiaherb
K.A. Malle
K.D. Pharma
Kaneka

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 USP 43 Articles of Incorporation / Donors xxvii

I• I of Inc p r •
I
In May of 1900, the USP Board of Trustees was directed by
the Conventionto incorporatethe USP organization under the
laws of the District of Columbia. Because the District of
Columbia required that the majority of officers subscribingto
the Certificate of Incorporation be residents, the filing was
slightly delayed in order to appoint appropriate
representatives. Nevertheless, the articles of incorporation
were prepared, appropriatelysigned, and finally filed on July
11 of that year. The original certificate reads as follows:

Certification of Incorporation
Thisisto certify that we, whose names are hereunto
subscribed, citizens of the United States, of full age, and a
majoritycitizens of the District of Columbia, do associate
ourselves together pursuant to the provisions of sections 545-
552 inclusive of the Revised Statutes of the United States
relating to the District of Columbia and of the Actof Congress
to amend the same, approved the twenty-third day of April
1884, under the corporate name of The United States
Pharmacopoeial Convention.
ThisAssociation is organizedfor a period of nine hundred
and ninety-nineyears. The particularobjects and business of
this Association are the encouragement and promotion of the
science and art of medicine and pharmacy by selecting by
research and experiment and other proper methods and by
naming such materials as may be properly used as medicines
and drugs with formulas for their preparation; by establishing
one uniform standard and guide for the use of those engaged
in the practice of medicineand pharmacy in the UnitedStates
whereby the identity, strength, and purityof allsuch
medicines and drugs may be accurately determined, and for
other like and similar purposes; and by printing and
distributing at suitable intervals such formulas and the results
of such and similar selections, names and determinations
among the members of this Association, pharmacists, and
physicians generally in the United Statesand others interested
in pharmacy and medicine.
The management and control of the affairs, funds, and
property of this Association for the firstyear of its existence
shall be vested in a Board of Trustees consisting of the seven
following persons:

ALBERT E. EBERT GEORGE W. SLOAN

SAMUELA.D. SHEPPARD HORATIOC. WOOD

WILLIAM S. THOMPSON CHARLES RICE

CHARLES E. DOHME

In testimony whereof we have hereunto set our hands and affixed our sealsthis seventh day of July, 1900.
WILLIAM S. THOMPSON [SEAL] WILLIAM M. MEW [SEAL]

G. LLOYD MAGRUDER [SEAL] FRANKM. CRISWELL . [SEAL]

JOHN T. WINTER [SEAL] MURRAYG. MOTIER [SEAL]

THOMAS C. SMITH [SEAL]

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xxviii Donors / USP Governance USP 43

USP Governan
Bylaws
The USP 2015-2020 Bylaws are availableat http://
www.usp.org/about-usp/leadership/bylaws.

Rules and Procedures

The Rules and Procedures of the 2015-2020 Council of
Experts are available at http://www.usp.org/about/leadership/
policies-rules/council-experts.

USP Policies
The USP policiesare available at http://www.usp.org/
about-usp/leadership/policies-rules.

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 THE UNITED STATES
SP4 PHARMACOPEIA
Official from May 1, 2020

FORTY·THIRD REVISION

www.ilovepharma.com
Copyright ©2019 The United States PharmacopeiaJ Convention
12601 Twinbrook Parkway, Rockville, MD 20852
All rights reserved.

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 USP 43 USP Admissions xxxi

u •
I I•

Articles Admitted to USP 43

First Supplement (August 1, 2019) USP MONOGRAPHS
Alosetron Hydrochloride
GENERAL CHAPTERS Alosetron Tablets
(64) Probiotic Tests Amiodarone Hydrochloride Tablets
(644) Conductivity of Solutions Amlodipine and Olmesartan Medoxomil Tablets
USP MONOGRAPHS Rotigotine
Azelastine Hydrochloride Ophthalmic Solution DIETARY SUPPLEMENTS
Benzphetamine Hydrochloride Chinese Skullcap Root
Benzphetamine Hydrochloride Tablets Chinese Skullcap Root Dry Extract
Clindamycin Hydrochloride Compounded Oral Solution Chinese Skullcap Root Powder
Desvenlafaxine Cobamamide
Desvenlafaxine Fumarate l-alpha-Glycerylphosphorylcholine
Desvenlafaxine Succinate Omega-3 Free Fatty Acids
Diclofenac Sodium Topical Solution
Estriol Compounded Vaginal Cream New Articles Appearing in USP 43 That
Famciclovir Tablets
Itraconazole Capsules Were Not Included in USP 42 Including
Leucovorin Calcium for Injection Suppl~ments
Naproxen Compounded Oral Suspension
Phytonadione Compounded Oral Suspension [NoTE-The articles included in this list are noted in the
Tranexamic Acid Tablets book with the following symbols 6. 4USP 1.May.2020' This applies
to new articles aswell as sections of existing items that have
DIETARY SUPPLEMENTS been revised.]
Cyanocobalamin Chewable Gels
GENERAL CHAPTERS
Second Supplement (Dec~mber 1, (856) Near-Infrared Spectroscopy
(1850) Evaluation of Screening Technologies for Assessing
2019) Medicine Quality
GENERAL CHAPTERS USP 43
(60) Microbiological Examination of Nonsterile Products- Arginine Hydrochloride Compounded Oral Solution
Tests for Burkholderia Cepacia Complex Carboxymethylcellulose Sodium Compounded
(509) Residual DNA Testing , Intraperitoneal Solution, Veterinary
(825)· Radiopharmaceuticals-Preparation, Compounding, Chlorambucil Compounded Oral Suspension
Dispensing, and Repackaging Chloramphenicol Compounded Oral Suspension, Veterinary
(1071) Rapid Microbial Testsfor Release of Sterile Short-Life Clonidine Hydrochloride Compounded Oral Suspension
Products: A Risk-Based Approach Diclofenac Potassium for Oral Solution
(1085) Guidelines on the Endotoxin Tests Hydrochlorothiazide Compounded Oral Suspension
(1229.16) Prion Inactivation Phenytoin Compounded Topical Gel
(1236) Solubility Measurements Rotigotine Transdermal System
(1430) Analytical Methodologies Based on Scattering Sodium Benzoate Compounded Oral Solution
Phenomena-General Torsemide Compounded Oral Suspension
(1430.1) Analytical Methodologies Based on Scattering Vancomycin Hydrochloride Compounded Oral Solution
Phenomena-Static Light Scattering DIETARY SUPPLEMENTS
(1430.2) Analytical Methodologies Based on Scattering
Phenomena-Light Diffraction Measurements of Creatine
Particle Size
(1430.4) Analytical Methodologies Based on Scattering Articles Included in USP 42 But Not
Phenomena-Electrophoretic Light Scattering Included in USP 43
(Determination of Zeta Potential)
(1430.5) Analytical Methodologies Based on Scattering USP MONOGRAPHS
Phenomena-Small Angle X-Ray Scattering and Small Aminobenzoate Potassium for Oral Solution
Angle Neutron Scattering Aminobenzoate Potassium Tablets
Aminoglutethimide
Aminoglutethimide Tablets

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 xxxii USP Admissions USP 43

Aminosalicylate Sodium Methylprednisolone Acetate Cream

Aminosalicylate Sodium Tablets Methysergide Maleate Tablets
Aminosalicylic Acid Tablets Metyrapone Tablets
Astemizole Mezlocillin Sodium
Astemizole Tablets Mezlocillin for Injection
Atropine Sulfate Tablets Miconazole Injection
Azelastine Hydrochloride Ophthalmic Solution Monobenzone Cream
Cefazolin Ophthalmic Solution Moricizine Hydrochloride Tablets
Chromium Cr 51 Edetate Injection Morrhuate Sodium Injection
Cocaine and Tetracaine Hydrochlorides and Epinephrine Nafcillin Sodium Capsules
Topical Solution Nafcillin Sodium for Oral Solution
Dehydrocholic Acid Tablets Nafcillin Sodium Tablets
Demeclocycline Oral Suspension Nalidixic Acid
Demeclocycline Hydrochloride Capsules Nalidixic Acid Oral Suspension
Dexamethasone Sodium Phosphate Cream Nalidixic Acid Tablets
Dexamethasone Sodium Phosphate Ophthalmic Ointment Netilmicin Sulfate
Dextroamphetamine Sulfate Capsules Netilmicin Sulfate Injection
Diazepam Capsules Norfloxacin
Diazepam Extended-Release Capsules Norfloxacin Ophthalmic Solution
Dicloxacillin Sodium for Oral Suspension Norfloxacin Tablets
Diethylstilbestrol Injection Omeprazole Oral Suspension
Diethylstilbestrol Tablets Oxacillin Sodium Capsules
Emetine Hydrochloride Oxacillin Sodium for Oral Solution
Emetine Hydrochloride Injection Oxazepam Tablets
Ergoloid Mesylates Sublingual Tablets Oxprenolol Hydrochloride Tablets
Indium In 111 Ibritumomab Tiuxetan Injection Oxtriphylline Oral Solution
Indium In 111 Satumomab Pendetide Injection .Oxtriphylline Tablets
lobenguane I 131 Injection Oxytetracycline and Nystatin Capsules
Iodinated I 131 Albumin Aggregated Injection Oxytetracycline and Nystatin for Oral Suspension
lodohippurate Sodium I 123 Injection Oxytetracycline Calcium
lodohippurate Sodium I 131 Injection Oxytetracycline Calcium Oral Suspension
Rose Bengal Sodium I 131 Injection Pantoprazole Oral Suspension
Krypton Kr 81m Phenmetrazine Hydrochloride
Levodopa Capsules Phenmetrazine Hydrochloride Tablets
Levodopa Tablets Phensuximide
Loracarbef Phensuximide Capsules
Loracarbef Capsules Chromic Phosphate P 32 Suspension
Loracarbef for Oral Suspension Sodium Phosphate P 32 Solution
Magaldrate Oral Suspension Potassium Perchlorate Capsules
Magaldrate Tablets Probucol
Mazindol Tablets Probucol Tablets
Menadiol Sodium Diphosphate Injection Propyliodone Injectabfe Oil Suspension
Menadiol Sodium Diphosphate Tablets Sisomicin Sulfate
Menadione Injection Sisomicin Sulfate Injection
Manganese Sulfate Injection Suprofen
Meclocycline Sulfosalicylate Suprofen Ophthalmic Solution
Meclocycline Sulfosalicylate Cream Technetium Tc 99m Albumin Injection
Mephenytoin Technetium Tc 99m Albumin Colloid Injection
Mephenytoin Tablets Technetium Tc 99m Apcitide Injection
Meprednisone Technetium Tc 99m Arcitumomab Injection
Meprobamate Oral Suspension Technetium Tc 99m Depreotide Injection
Mesoridazine Besylate Technetium Tc 99m Etidronate Injection
Mesoridazine Besylate Injection Technetium 99mTc Fanolesomab Injection
Mesoridazine Besylate Oral Solution Technetium Tc 99m Gluceptate Injection
Mesoridazine Besylate Tablets Technetium Tc 99m Lidofenin Injection'
Metaproterenol Sulfate Inhalation Solution Technetium Tc 99m Nofetumomab Merpentan Injection
Metaraminol Bitartrate Thioridazine Oral Suspension
Methacycline Hydrochloride Capsules Xenon Xe 127
Methdilazine Hydrochloride Xenon Xe 133 Injection
Methdilazine Hydrochloride Oral Solution
Methdilazine Hydrochloride Tablets
Methenamine Oral Solution
Methenamine Mandelate for Oral Solution
Methenamine Mandelate Oral Suspension
Methenamine Mandelate Delayed-Release Tablets
Methylbenzethonium Chloride Ointment
Methylbenzethonium Chloride Topical Powder
Methyldopa Oral Suspension .
Methyldopa and Chlorothiazide Tablets

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USP 43 USP Annotated List xxxiii

p n te I•

General Notices, Monographs, General

Chapters, Reagents, and Tables Affected by
Changes Appearing in USP 43
Page citations refer to thepages of USP 43. Note-In thelists below, if a section is neworifa subsection isadded to ordeleted
from anexisting section itislabeled assuch inparentheses after thesection orsubsection name. Items onthis list thatappear without
the designation "new", "added", or"deleted" are items in which changes havebeen madeto existing official text.

General Chapters General Information

(1010) Analytical Data-Interpretation and Treatment,
General Tests and Assays 7233
General Requirements for Tests and Assays (1043) Ancillary Materials for Cell, Gene, and
(7) Labeling (Revision Bulletin official May 1, 2019), Tissue-Engineered Products, 7381
6435 (1046) Cell-Based Advanced Therapies and Tissue-Based
LABELS AND LABELING FOR DRUG PRODUCTS Products, 7400
AND COMPOUNDED PREPARATIONS (1047) Gene Therapy Products, 7426
EXPRESSED AS ACTIVE MOIETY IN NAME AND
INTRODUCTION
STRENGTH
MANUFACTURING OVERVIEW
LABELS AND LABELING FOR INJECTABLE
PRODUCTS . MANUFACTURING OF GENETHERAPY PRODUCTS
LABELS AND LABELING FOR PRODUCTS IN ON-SITE PREPARATION AND ADMINISTRATION
OTHER CATEGORIES ANALYTICAL METHODS
LABELS AND LABELING FORANIMAL DRUG GLOSSARY
PRODUCTS APPENDIX
Biological Tests and Assays (1099) Limit on Number of Large Deviations When
(121) Insulin Assays (Revision Bulletin official May 1, Assessing Content Uniformity in Large Samples
2019), 6547 (Revision Bulletin official March 1, 2019), 7744
ASSAY (11 36) Packaging and Repackaging-Single-Unit
Containers (deleted), 7919
Rabbit Blood Sugar Method-Quantitative
(1177) Good Packaging Practices (deleted), 8003
ADDITIONAL REQUIREMENTS
(1850) Evaluation of Screening Technologies for
USP Reference Standards Assessing Medicine Quality (new), 8626
Chemical Tests and Assays
(1856) Near-Infrared Spectroscopy-Theory and
(203) High-Performance Thin-Layer Chromatography Practice, 8658
Procedure for Identification of Articles of Botanical
Origin, 6602 Reagents, Indicators, and Solutions
INTRODUCTION
EQUIPMENT REAGENT SPECIFICATIONS
PROCEDURE Glyceryl Monostearate (new), 6168
(591) Zinc Determination, 6817 Lecithin (new), 6175
INTRODUCTION Lysolecithin (new), 6176
PROCEDURE Manganese Chloride Tetrahydrate (new), 6177
Physical Tests and Determinations Manganese Sulfate (new), 6176
(701) Disintegration (Stage 4 Harmonization official May Methyl Red (deleted), 6179
1, 2020), 6940 Polypeptone (new), 6192
(800) Hazardous Drugs-Handling in Healthcare Polysorbate 20 (new), 6192
Settings (Revision Bulletin official May 1, 2019), 7071 Sodium Butyrate (new), 6202
(856) Near-Infrared Spectroscopy (new), 7161 Sodium Oleate (new), 6204
Sodium Propionate (new), 6205

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xxxiv USP Annotated List USP 43

Sodium Taurocholate (new), 6206 ADDITIONAL REQUIREMENTS

Sodium Taurodeoxycholate (new), 6206 Bendamustine Hydrochloride (Revision Bulletin official
INDICATORS March 1, 2019), 493
, IMPURITIES
Methyl Red, 6221
OrganicImpurities
Xylenol Orange, 6221
Betaxolol Hydrochloride, 557
TEST SOLUTIONS IDENTIFICATION
18 N Phosphoric Acid TS (new), 6233 ASSAY
50% Sodium Hydroxide TS (new), 6235 IMPURITIES
Xylenol Orange TS, 6237 OrganicImpurities
ADDITIONAL REQUIREMENTS
Reference Tables USP Reference Standards
CONTAINER SPECIFICATIONS FOR CAPSULES AND Betaxolol Tablets, 559
TABLETS IDENTIFICATION
Diazoxide Capsules (deleted), 6262
Test 8 (added)
ASSAY
Oxprenolol Hydrochloride Tablets, Extended-Release
(deleted),6268 PERFORMANCE TESTS
Oxycodone and Acetaminophen Capsules (deleted), Dissolution
6268 IMPURITIES (added)
Piperazine Citrate Tablets (deleted), 6268 ADDITIONAL REQUIREMENTS
Pyrvinium Pamoate Tablets (deleted), 6269 USP Reference Standards
Ritodrine Hydrochloride Tablets (deleted), 6269 Calcium Acetate Capsules (Revision Bulletin official May
Scopolamine Hydrobromide Tablets (deleted), 6270 1,2019),680 .
PERFORMANCE TESTS
DESCRIPTION AND RELATIVE SOLUBILITY OF USP AND NF . Dissolution
ARTICLES ADDITIONAL REQUIREMENTS
Anise Oil (new), 6276 Labeling (added)
Glyceryl Mono and Dicaprylate (new), 6297 Carboxymethylcellulose Sodium Compounded
Glyceryl Mono and Dicaprylocaprate (new), 6297 Intraperitoneal Solution; Veterinary.(new), 775
Medium-Chain Triglycerides, 6331 Chlorambucil Compounded Oral Suspension (new), 925
Neohesperidin Dihydrochalcone (new), 6309 Chloramphenicol Compounded Oral Suspension,
Polypropylene Glycol 11 Stearyl Ether (new), 6317 Veterinary (new), 930
Cholecalciferol, 985
Monographs (USP 43) IDENTIFICATION
Allopurinol Compounded Oral Suspension, 133 ASSAY
DEFINITION Cidofovir (Revision Bulletin official June 1, 2019), 999
ASSAY (added)
SPECIFIC TESTS IMPURITIES
ADDITIONAL REQUIREMENTS Residue on Ignition (deleted)
Clonidine Hydrochloride, 1101
Packaging and Storage, Beyond-Use Date, and
USP Reference Standards (added) DEFINITION
Alprazolam Tablets (Revision Bulletin official March 1, IDENTIFICATION
2019), 146 Test A and Test B
IMPURITIES (added) ASSAY
Amitriptyline Hydrochloride, 261 IMPURITIES
IDENTIFICATION OrganicImpurities
Test A Clonidine Hydrochloride Compounded Oral Suspension
IMPURITIES (new), 1103 .
OrganicImpurities Clonidine Transdermal System (Revision Bulletin official
June 1, 2019), 110 5 ·
Apomorphine Hydrochloride Tablets (deleted), 358
PERFORMANCE TESTS
Arginine Hydrochloride Compounded Oral Solution
(new),372 Drug Release
Aurothioglucose Injectable Suspension (deleted), 434 Demeclocycline Hydrochloride, 1248
CHEMICAL INFORMATION
Azatadine Maleate (deleted), 437
IDENTIFICATION
Azatadine Maleate Tablets (deleted), 438
Azathioprine Tablets, 441 ASSAY
Demeclocycline Hydrochloride Tablets, 1249
IDENTIFICATION
IDENTIFICATION
ASSAY
Test 8 (added)"
PERFORMANCE TESTS
ASSAY
Dissolution
ADDITIONAL REQUIREMENTS
IMPURITIES (added)
Packaging and Storage

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USP 43 USP Annotated List xxxv

Deslanoside (deleted), 1258 ADDITIONAL REQUIREMENTS

Deslanoside Injection (deleted), 1259 USP Reference Standards
Diazoxide Capsules (deleted), 1333 Fluticasone Propionate and Salmeterol Inhalation
Diazoxide Injection (deleted), 1334 Powder (Revision Bulletin official February 8, 2019), 2010
Diclofenac Potassium for Oral Solution (new), 1344 ASSAY
Diflunisal Tablets, 1376 PERFORMANCE TESTS
IDENTIFICATION IMPURITIES
Test B SPECIFIC TESTS
ASSAY Foreign Particulate Matter
PERFORMANCE TESTS ADDITIONALREQUIREMENTS
IMPURITIES (added) Labeling (added) and USP Reference Standards
ADDITIONAL REQUIREMENTS Hydrochlorothiazide Compounded Oral Suspension
Packaging and Storage (new),2221
Diltiazem Hydrochloride Extended-Release Capsules Hydrocortisone Gel, 2230
(Revision Bulletin official May 1, 2019), 1399 IDENTIFICATION
PERFORMANCE TESTS ASSAY
Dissolution IMPURITIES (added)
IMPURITIES SPECIFIC TESTS (added)
Diphenhydramine Hydrochloride Oral Powder (new), 1428 ADDITIONAL REQUIREMENTS
Dobutamine Hydrochloride, 1466 USP Reference Standards
IDENTIFICATION Indomethacin Extended-Release Capsules, 2325
ASSAY IDENTIFICATION
IMPURITIES ASSAY
Organic Impurities PERFORMANCE TESTS
SPECIFIC TESTS IMPURITIES
Colorof Solution (deleted) ADDITIONALREQUIREMENTS
ADDITIONAL REQUIREMENTS Insulin (Revision Bulletin official May 1, 2019), 2335
USP Reference Standards CHEMICAL INFORMATION
Donepezil Hydrochloride Tablets(Revision Bulletin DEFINITION
official March 1, 2019), 1488 IDENTIFICATION
IDENTIFICATION Test A
UltravioletAbsorption, Test A ASSAY
ASSAY PRODUCT-RELATED SUBSTANCES AND
PERFORMANCE TESTS IMPURITIES
Dissolution Product-Related Substances
IMPURITIES OTHER COMPONENTS
Doxycycline Compounded Oral Suspension, Veterinary, 1530 ADDITIONAL REQUIREMENTS
DEFINITION Labeling and USP Reference Standards
ASSAY Insulin Injection (Revision Bulletin official May 1, 2019), 2338
SPECIFIC TESTS IDENTIFICATION
ADDITIONAL REQUIREMENTS ASSAY
Packaging and Storage and Beyond-Use Date OTHER COMPONENTS
Drospirenone and Ethinyl Estradiol Tablets (Revision ADDITIONAL REQUIREMENTS
Bulletin official April 1, 2019), 1556 Labeling and USP Reference Standards
IMPURITIES IsophaneInsulin Suspension (Revision Bulletin official
Ergocalciferol, 1665 May 1, 2019), 2351
IDENTIFICATON IDENTIFICATION
ASSAY ASSAY
ADDITIONAL REQUIREMENTS ADDITIONAL REQUIREMENTS
USP Reference Standards Labeling and USP Reference Standards
Fenofibrate Capsules (Revision Bulletin official May 1, Insulin Zinc Suspension (Revision Bulletin official May
2019), 1834 ' 1, 2019), 2356
PERFORMANCE TESTS IDENTIFICATION
Fentanyl,1846 ASSAY
DEFINITION ADDITIONAL REQUIREMENTS
IDENTIFICATION Labeling and USP Reference Standards
Test A Extended Insulin Zinc Suspension (Revision Bulletin
ASSAY official May 1, 2019), 2357
IMPURITIES IDENTIFICATION
Organic Impurities ASSAY

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xxxvi USP AnnotatedList USP 43

ADDITIONAL REQUIREMENTS IMPURITIES

Labeling and USP Reference Standards OrganicImpurities
Prompt InsulinZinc Suspension (Revision Bulletin official Naproxen Sodium and Pseudoephedrine Hydrochloride
May 1, 2019), 2359 . Extended-Release Tablets (Revision Bulletin official
IDENTIFICATION March 1, 2019), 3084
ASSAY IMPURITIES
ADDITIONAL REQUIREMENTS Nicardipine Hydrochloride Injection, 3149
Labeling and USP Reference Standards DEFINITION
Itraconazole Capsules (Revision Bulletin official August 1, IDENTIFICATION
2019),2468 Test B (added)
PERFORMANCE TESTS ASSAY
Dissolution IMPURITIES
IMPURITIES OTHER COMPONENTS
ADDITIONAL REQUIREMENTS SPECIFIC TESTS
Labeling (added) Bacterial Endotoxins Test
Loperamide Hydrochloride, 2658 ADDITIONAL REQUIREMENTS
IDENTIFICATION Packaging and Storage and USP Reference
Test A Standards
ASSAY Olmesartan Medoxomil Tablets (Revision Bulletin official
IMPURITIES March 19, 2019), 3244 -
Organic Impurities PERFORMANCE TESTS
ADDITIONAL REQUIREMENTS Dissolution
USP Reference Standards Oxprenolol Hydrochloride Extended-Release Tablets
(deleted), 3324
Manganese Chloride for Oral Solution (deleted), 2732
Oxycodone and Acetaminophen Capsules (deleted),
Mazindol (deleted), 2742
3341
Metformin Hydrochloride, 2821
Pantoprazole Sodium Delayed-Release Tablets (Revision
DEFINITION Bulletin official May 1, 2019), 3391
IDENTIFICATION PERFORMANCE TESTS
Test C (added) Dissolution
ASSAY Parachlorophenol (deleted), 3399
IMPURITIES Phenytoin Compounded Topical Gel (new), 3525
Organic Impurities Physostigmine Salicylate Ophthalmic Solution (deleted),
ADDITIONAL REQUIREMENTS 3536 .
USP Reference Standards . Piperazine Citrate Tablets(deleted), 3575
Metformin Hydrochloride Extended-Release Tablets Prednisolone Cream (deleted), 3660
(Revision Bulletin official March 1, 2019), 2824 Primidone Oral Suspension (deleted), 3684
PERFORMANCE TESTS Procaine and Tetracaine Hydrochloridesand
Dissolution Levonordefrin Injection (deleted), 3695
Methacholine Chloride, 2833 Promazine Hydrochloride Syrup (deleted), 3713
DEFINITION Propafenone Hydrochloride Extended-Release Capsules
IDENTIFICATION (Revision Bulletin official March 8, 2019), 3729
Test A PERFORMANCE TESTS
ASSAY Dissolution
IMPURITIES IMPURITIES
Organic Impurities Propoxycaine Hydrochloride (deleted), 3743
SPECIFIC TESTS Propoxycaine and Procaine Hydrochlorides and
ADDITIONAL REQUIREMENTS Levonordefrin Injection (deleted), 3744
USP Reference Standards Propoxycaine and Procaine Hydrochlorides and
Methotrexate Injection (Revision Bulletin official May 1, Norepinephrine Bitartrate Injection (deleted), 3745
2019),2857 Propranolol Hydrochloride, 3746
IMPURITIES DEFINITION
Miconazole Nitrate (Revision Bulletin official March 1, IDENTIFICATION
2019),2947 Tet A
SPECIFIC TESTS ASSAY
OpticalRotation IMPURITIES
Minocycline Hydrochloride Oral Suspension (deleted), OrganicImpurities (added)
2966 SPECIFIC TESTS
Minoxidil, 2972 Melting Range or Temperature (deleted) and
IDENTIFICATION OpticalRotation (deleted)
ASSAY ADDITIONAL REQUIREMENTS
USP Reference Standards

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USP 43 USP Annotated List xxxvii

Propyliodone (deleted), 3754 Taurine (Revision Bulletin official March 1, 2019), 4223
Pseudoephedrine Hydrochloride Extended-Release ASSAY
Tablets (Revision Bulletin official March 1, 2019), Tetracycline Hydrochloride, 4303
3764 IDENTIFICATION
PERFORMANCE TESTS Test A and Test C
Dissolution IMPURITIES
Pyrvinium Pamoate (deleted), 3789 SPECIFIC TESTS
Pyrvinium Pamoate Oral Suspension (deleted), 3789 Loss on Drying
Pyrvinium Pamoate Tablets(deleted), 3790 Thioridazine Hydrochloride Oral Solution (deleted),
Quetiapine Extended-Release Tablets (Revision Bulletin 4354
official April 1, 2019), 3795 Torsemide Compounded Oral Suspension (new), 4438
ASSAY Vancomycin Hydrochloride Compounded Oral Solution
PERFORMANCE TESTS (new),4589
Dissolution Vancomycin Injection (Revision Bulletin official March 1,
IMPURITIES 2019),4585
Risedronate Sodium, 3900 DEFINITION
IDENTIFICATION ASSAY
Test C (added) SPECIFIC TESTS
ASSAY Composition of Vancomycin, pH, and Particulate
IMPURITIES Matter in Injections
SPECIFIC TESTS ADDITIONAL REQUIREMENTS
ADDITIONAL REQUIREMENTS Packaging and Storage and Labeling
USP Reference Standards Vitamin A Tablets, 4635
Ritodrine Hydrochloride (deleted), 3910 ASSAY
Ritodrine Hydrochloride Injection (deleted), 3911 PERFORMANCE TESTS
Ritodrine Hydrochloride Tablets (deleted), 3911 Dissolution
Rosuvastatin Tablets (Revision Bulletin official April 1, ADDITIONAL REQUIREMENTS
2019), 3950 Labeling
PERFORMANCE TESTS
Dissolution Monographs (Dietary Supplements)
Rotigotine Transdermal System (new), 3955 Calcium with Vitamin D Tablets, 4843
Saccharin Calcium IDENTIFICATION (added)
DEFINITION STRENGTH
IDENTIFICATION Vitamin D (Cholecalciferol or Ergocalciferol),
Test A Method 7 and Vitamin D (Cholecalciferol or
ASSAY Ergocalcifero!), Method 2 (added)
SPECIFIC TESTS Calcium and Vitamin D with MineralsTablets, 4845
Readily Carbonizable Substances Test and Color IDENTIFICATION (added)
. of Solution . STRENGTH
ADDITIONAL REQUIREMENTS Vitamin D (Cholecalciferol or Ergocalcifero!),
USP Reference Standards Method 7 and Vitamin D (Cholecalciferol or
Ergocalcifero!), Method 2 (added)
Scopolamine Hydrobromide Injection (deleted), 4012
ADDITIONAL REQUIREMENTS
Scopolamine Hydrobromide Ophthalmic Solution
(deleted), 4013 Labeling
Scopolamine Hydrobromide Tablets(deleted), 4013 Creatine (new), 4920
Secobarbital (deleted), 4013 Oil-Soluble Vitamins Tablets, 5356
Sodium Benzoate Compounded Oral Solution (new), DEFINITION
4056 IDENTIFICATION (added)
Sodium Fluoride Gel, 4071 STRENGTH
IDENTIFICATION Vitamin A, Method 7; Vitamin A, Method 2;
ASSAY VitaminA, Method 3; Vitamin D
(Cholecalciferol or Ergocalcifero!), Method 7;
ADDITIONAL REQUIREMENTS Vitamin D (Cholecalciferol or Ergocalcifero!),
Packaging and Storage and USP Reference Method 2; Vitamin D (Cholecalciferol or
Standards Ergocalcifero!), Method 3; Vitamin E, Method
Sodium Fluoride Tablets, 4073 7; Vitamin E, Method 2; Vitamin E, Method 3
IDENTIFICATION and Vitamin A, Vitamin D, and Vitamin E,
Test B Method 4; Phytonadione, Method 3 (added)
ASSAY ADDITIONAL REQUIREMENTS
ADDITIONAL REQUIREMENTS Labeling and USP Reference Standards
Packaging and Storage and USP Reference Oil-Soluble Vitamins with MineralsTablets, 5378
Standards DEFINITION

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xxxviii USP Annotated List USP43

IDENTIFICATION (added) Acid, Niacin or Niacinamide, Pyridoxine

STRENGTH Hydrochforide, Calcium Pantothenate,
Vitamin A, Method 1; Vitamin 0 (Cholecalciferol Riboflavin, and Thiamine, Method 4 (added)
or Ergocalciferol), Method 1; Vitamin E, ADDITIONAL REQUIREMENTS
Method 1; Phytonadione, Method 1; Vitamin Labeling and USP Reference Standards
A, Vitamin 0, Vitamin E, and Phytonadione, Water-Soluble Vitamins Tablets, 5512
Method 2 (added) and Beta Carotene DEFINITION
ADDITIONAL REQUIREMENTS IDENTIFICATION (added)
Labeling and USP Reference Standards STRENGTH
Oil- and Water-Soluble VitaminsTablets, 5419 Ascorbic Acid, Calcium Ascorbate, and Sodium
DEFINITION Ascorbate; Cyanocobalamin, Method 2; Folic
IDENTIFICATION (added) Acid, Method 1; Folic Acid, Method 2; Calcium
STRENGTH Pantothenate, Method 2; Niacin or
Vitamin A, Method 1; Vitamin A, Method 2' Niacinamide, Pyridoxine Hydrochloride,
Vitamin A, Method 3; Vitamin 0 ' Riboflavin, and Thiamine, Method 1; Niacin,
(Cholecalciferol or Ergocalciferol), Method 1; Method 2; Niacinamide, Method2;
Vitamin 0 (Cholecalciferol or Ergocalciferol), Riboflavin, Method 2; Thiamine, Method 2'
Method 2; Vitamin 0 (Cholecalciferol or Niacin or Niacinamide, Pyridoxine '
Ergocalciferol), Method 3; Vitamin E, Method Hydrochloride, Riboflavin, and Thiamine,
1; Vitamin E, Method 2; Vitamin E, Method 3; Method 3 and Folic Acid, Method 3; Ascorbic
Vitamin A, Vitamin 0 (Cholecalciferol or Acid, Niacin or Niacinamide, Pyridoxine
Ergocalciferol), Vitamin E, Method 4; Hydrochloride, Calcium Pantothenate,
Phytonadione, Method 3 (added); Ascorbic Riboflavin, and Thiamine, Method 4 (added)
Acid, Calcium Ascorbate, and Sodium Water-Soluble Vitamins with MineralsTablets, 5552
Ascorbate; Biotin, Method2; DEFINITION
Cyanocobalamin, Method 2; Folic Acid, IDENTIFICATION (added)
Method 1; Folic Acid, Method 2; Caldum STRENGTH
Pantothenate, Method 2; Niacin or Ascorbic Acid, Calcium Ascorbate, and Sodium
Niacinamide, Pyridoxine Hydrochloride, Ascorbate; Folic Acid, Method 1; Folic Acid,
Riboflavin, and Thiamine, Method 1; Niacin, Method 2; Calcium Pantothenate, Method2;
Method 2; Niacinamide, Method 2; Niacin or Niacinamide, Pyridoxine
Riboflavin, Method 2; Thiamine, Method 2; Hydrochloride, Riboflavin, and Thiamine,
Niacin or Niacinamide, Pyridoxine Method 1; Niacin, Method 2; Niacinamide,
Hydrochloride, Riboflavin, and Thiamine, Method 2; Riboflavin, Method 2; Thiamine,
Method 3; and Folic Acid, Method 3; Ascorbic Method 2; Niacin or Niacinamide, Pyridoxine
Acid, Niacin or Niacinamide,' Pyridoxine Hydrochloride, Riboflavin, and Thiamine,
Hydrochloride, Calcium Pantothenate, Method 3 and Folic Acid, Method 3; Ascorbic
Riboflavin, and Thiamine, Method 4 (added) Acid, Niacin or Niacinamide, Pyridoxine
ADDITIONAL REQUIREMENTS Hydrochloride, Calcium Pantothenate,
Labeling and USP Reference Standards Riboflavin, and Thiamine, Method 4 (added)
Oil- and Water-Soluble Vitamins with Minerals Tablets ADDITIONAL REQUIREMENTS
5476 . ' USP Reference Standards
DEFINITION
IDENTIFICATION (added)
STRENGTH
Vitamin A, Method 1; Vitamin A, Method 2;
Vitamin A, Method 3; Vitamin 0
(Cholecalciferol or Ergocalciferol), Method 1;
Vitamin 0 (Cholecalciferol or Ergocalciferol),
Method 2; Vitamin 0 (Cholecalciferol or
Ergocalciferol), Method 3; Vitamin E, Method
1; Vitamin E, Method 2; Vitamin E, Method 3;
Vitamin A, Vitamin 0 (Cholecalciferol or
Ergocalciferol), and Vitamin E, Method 4;
Phytonadione, Method 3 (added); Ascorbic
Acid, Calcium Ascorbate, and Sodium
Ascorbate; Biotin, Method2;
Cyanocobalamin, Method 2; Folic Acid,
Method 1; Folic Acid, Method 2; Calcium
Pantothenate, Method 2; Niacin or
Niacinamide, Pyridoxine Hydrochloride,
Riboflavin, and Thiamine, Method 1; Niacin,
Method 2; Niacinamide, Method2;
Riboflavin, Method 2; Thiamine, Method 2;
Niacin or Niacinamide, Pyridoxine
Hydrochloride, Riboflavin, and Thiamine,
Method 3; and Folic Acid, Method 3; Ascorbic

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 USP 43 General Notices 1

L
RE NT
Applying to Standards, Tests, Assays, and Other
Specifications of the United States Pharmacopeia

1. TITLE AND REViSiON....... 3 8. TERMS AND DEFINITIONS......................................... 9

8.10. Abbreviations.......................... 9
2. OFFICIAL STATUS AND LEGAL RECOGNITION........ 3 8.20. About.................................................................. 9
2.10. Official Text......................................................... 3 8.30. Alcohol Content................................................ 10
2.20. Official Articles..................................................... 3 8.40. Atomic Weights................................. 10
2.30. Legal Recognition................................................ 3 8.50. Blank Determinations........................................ 10
8.60. Concomitantly................................. 10
3. CONFORMANCE TO STANDARDS..................... ........ 4 8.70. Desiccator......................................................... 10
3.10. Applicability of Standards.................................... 4 8.80. Logarithms........................................................ 10
3.20. Indicating Conformance...................................... 5 8.90. Microbial Strain................................................. 10
8.100. Negligible..................................... 10
4. MONOGRAPHS AND GENERAL CHAPTERS.............. 5 8.110. NLT/NMT........................................................ 10
4.1 O. Monographs ~........... 5 8.120. Odor............................................................... 10
4.20. General Chapters................................................. 5 8.130. Percent............................................................ 10
8.140. Percentage Concentrations.............................. 10
5. MONOGRAPH COMPONENTS................................... 6 8.150. Pressure........................................................... 10
5.10. Molecular Formula ,.............. 6 8.160. Reaction Time.................... 10
5.20. Added Substances............................................... 6 8.1 70. SpecificGravity............................... 10
5.30. Description and Solubility ~.... 6 8.180. Temperatures.................................................. 10
5.40. Identification....................................................... 6 8.190. Time................................................................ 10
5.50. Assay................................................................... 6 8.200. Transfer........................................................... 11
5.60. Impurities and Foreign Substances....................... 7 8.210. Vacuum........................................................... 11
5.70. Performance Tests............................................... ,7 8.220. Vacuum Desiccator.......................................... 11
5.80. USP Reference Standards..................................... 7 8.230. Water........................................... 11
8.240. Weights and Measures..................................... 11
6. TESTING PRACTICES AND PROCEDURES................. 7
6.10. Safe Laboratory Practices..................................... 7 9. PRESCRIBING AND DiSPENSiNG............................. 12
6.20. Automated Procedures........................................ 7 9.10. Use of Metric Units............................................ 12
6.30. Alternative and Harmonized Methods and
Procedures............... 7 10. PRESERVATION, PACKAGING, STORAGE, AND
6.40. Dried, Anhydrous, Ignited, or Solvent-Free Basis.. 8 LABELING................................................ 12
6.50. Preparation of Solutions....................................... 8 10.10. Packaging and Storage.................................... 12
6.60. Units Necessary to Complete a Test..................... 8 10.20. Labeling.......................................................... 12
6.70. Reagents............................................................. 8
6.80. Equipment...... 9

7. TEST RESULTS :................................................. 9

7.10. Interpretation of Requirements............................ 9
7.20. Rounding Rules................................................... 9

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 USP 43 General Notices 3

L NOTI
REQUIREMENT
Routine revisions are published in the USP-NF Online and
The General Notices and Requirements section (the General become official on the date indicated, usually six months after
Notices) presents the basic assumptions, definitions, and publication. Accelerated Revisions supersede the U5P-NF
default conditionsfor the interpretation and applicationofthe Online and become official on the date indicated. links to
United 5tates Pharmacopeia (U5P) and the National Formulary Accelerated Revisions on the USP website can be found in any
(NF). . superseded monograph or general chapter in the USP-NF
Requirements stated in these General Notices applyto all Online.
articles recognized in the U5P and NF (the "compendia") and Printand USB flash driveversions of the U5P and NFalso are
to all general chapters unlessspecifically stated otherwise. available. Routine revisions are providedwith the same timing
as the U5P-NF Online. Official text published in 5upplements
1. TITLE AND REVISION supersedes that in the previously pu~lished print or USB flash
driveversions of U5P-NF. These versions also are superseded
The full title of this publication (consisting of five volumes by- Accelerated Revisions as described above.
and including its 5upplements), is The Pharmacopeia of the In the event of any disparity between the print or USB flash
United5tatesof America, Forty-Third Revision and the National drive versions and the U5P-NF Online, the U5P-NF Online will
Formulary, Thirty-Eighth Edition. These titles may be be deemed to apply.
abbreviated to U5P 43, to NF 38, and to U5P 43-NF 38. The 2.20. OfficialArticles
United 5tates Pharmacopeia, Forty-Third Revision, and the An officialarticle isan article that isrecognizedin U5P or NF.
National Formulary, Thirty-Eighth Edition, supersede all earlier An article is deemed to be recognized and included in a
revisions. Wherethe terms"U5P," "NF," or "U5P-NP' are used compendium when a monograph for the article is published
without further qualification during the period in which these in the compendium and an official date is generally or
compendia are official, they refer only to U5P 43, NF 38, and specifically assigned to the monograph.
any Supplement(s) thereto. The same titles, with no furt~er The title specified in a monograph isthe official title for such
distinction, apply equallyto print or electronic. presentation of article. Other names considered to be synonyms of the official
these contents. Although U5P and NF are publishedunder one titles may not be used as substitutes for official titles. Fordrug
cover and share these General Notices, they are separate products that incorporate a sensor to detect that the product
compendia. has been administered, the official title shall be the title
This revision is official beginning May1, 2020 unless specified in the relevant drug product monograph plus the
otherwise indicated in specific text. words "with sensor".
5upplements to USP and NF are published periodically. Official articles include both official substances and official
Accelerated Revisions, published periodically on the Official products. An official substance is a drug substance, excipient,
Text section of USP's website (http://www.usp.org/usp-nf/ dietary ingredient, other ingredient, or component of a
official-text), are designed to make revisions offici~1 r:nore finished device for which the monograph title includes no
quickly than through the routine processfor publishing indication of the nature of the finished form.
standards in the USP-NF. Interim Revision Announcements are An official product is a drug product, dietary supplement,
Accelerated Revisions to USP and NFthat contain official compounded preparation, or finished device for which a
revisions and their effective dates. monograph is provided.
Revision Bulletins are Accelerated Revisions to official text or 2.30. Legal Recognition
postponements that requir~ expedited publicat!on. Th~~ The U5P and NF are recognized in the laws and regulations
generallyare official immediately unlessotherwise specified of many countries throughout the world. Regula~ory
in the Revision Bulletin. authorities may enforce the standards presented In the U5P
Errata are Accelerated Revisions representing corrections to and NF, but because recognition of the USP and NF may vary
items erroneously published. Announcements of the by country, users should understand applicable laws and
availability of new USP Reference Standards and regulations. Inthe UnitedStatesunder the Federal Food, Drug,
announcements of tests or procedures that are held in and CosmeticAct(FDCA), both U5P and NF are recognizedas
abeyance pending availability of required USP Reference official compendia. Adrug with a name recognized in U5P-NF
Standards are also available on the "Official Text" tab of USP's must comply with compendial identitystandards or be
website. deemed adulterated, misbranded, or both. See, e.g., FDCA §
501(b) and 502(e)(3)(b); also FDA regulations, 21 CFR §
2. OFFICIAL STATUS AND LEGAL RECOGNITION 299.5(a&:b). To avoid being deemed adulterated, such drugs
2.10. Official Text must also comply with compendial standards for stre.ngth,.
Official text of the U5P and NF is published in the U5P-NF quality, and purity,unlesslabeledto show allrespects In which
Online (www.uspnt.corn) in the edition identified as the drug differs. See, e.g., FDCA § 501(b) and 21 CFR §
"CURRENTLY OFFICIAL" and in Accelerated Revisions that 299.5(c). In addition, to avoid being deemed misbranded,
supersede the U5P-NF Online as described below. drugs recognized in U5P-NFmust also be packaged and

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4 General Notices USP 43

labeled in compliancewith compendial standards. See FDCA similarity to statistical procedures may seem to sugg.est an .
§ 502(g). . intent to make inference to some largergroup of units, but In
A dietary supplement represented as conforming to .. all cases statements about whether the compendial standard
specifications in USP will be deemed a misbranded food If It is met apply only to the u.nits tested. Repea.ts, replicates,
fails to so conform. See FDCA § 403(s)(2)(D). . statistical rejection of outliers, or extrap~latlons of resul~s to
Enforcement of USP standards is the responsibility of FDA . larger populations, as well as the necessity and appropriate
and other government authoritiesin the U.S. and elsewhere. frequency of batch testing, are neither specified nor
USP has no role in enforcement. proscribedby the compendia;such decisio~s are based o~ the
objectives of the testing. Frequency of testing and sampling
3. CONfORMANCE TO STANDARDS are leftto the preferences or direction of those ~erfor~ing
compliance testing, and other usersof USP-NF, Including
3.10. Applicabilityof Standards manufacturers, buyers, or regulatory authorities.
Standards for an article recognized inthe compendia (USP- Official products are prepar~d accor~ing to recognized
NF) are expressed in the article's monograph, applicable principles of good manufactunng practice and from
general chapters, and General Notices. The identity, stren~~h, ingredients that meet USP or NF standards, where standar~s
quality and purityof an article are determined by the official for such ingredientsexist(fordietarysupplements, see section
tests, procedures, and acceptance criteria, and ?ther . 3.70.20. Applicability of Standards to MedicalDevices, Dietary
requirements incorporated In the monograph, Inapplicable Supplements, and Their Components and Infjredients). .
general chapters, or in the General Notices. "Applicable Official substances are prepared according to recognized
general chapters" means general chapters numbered below principles of good manufacturing practice and from
1000 or above 2000 that are made applicable to an article ingredients complying with specifications designed to ensure
through reference in General Notices, a monograph, or that the resultant substances meet the requirements of the
another applicablegeneral chapter numbered below 1000. compendial monographs.
Where the requirements· of a monograph differfrom the 3.70.70. Applicability of Standardsto Drug Products, Drug
requirements specified in these General Notices o~ an Substances, and Excipients
applicablegeneral chapter, the monograph requirements The applicable USP or NF standard applies to any article
apply and supersede the requirements of the General Notices marketed in the United States that (1) is recognized in the
or applicablegeneralchapters, whether or not the monograph' compendium and (2) isintended or labeledfor use as a drug
explicitly states the difference. or as an ingredient in a drug. Such articles (drug products,
General chapters numbered 1000 to 1999 are for drug substances, and excipients) includeboth human drugs
informational purposes only. They cont~in no mandator:y . (whether dispensed by prescription, "over the counter," or
tests, assays, or other requirementsapplicable to any official otherwise),as well as animaldrugs. The applicablestandard
article regardless of citation in a general chapter numbered applies to such articles whether or not the added
below'1000 a monograph, or these General Notices. General designation "USP" or "NF" is used. The standards apply
chapters nu~bered above 2000 apply only to articles that are equallyto articles bearing ~h~ ?fficial titlesor ~~me~ derived
intended for use as dietary ingredients and dietary by transpositionof the definitive words of official titles or
supplements. General chapter citations in NF monographs transpositionin the order of the names of two or more drug
referto USP general chapters. . substances inofficial titles, or where there isuse of synonyms
Early adoption of revised standards in advance ofthe official with the intent or effectof suggesting a significant degree
date is allowed by USP u~less specified otherwise.a~ the tiJ!le of identity with the official title or name.
of publication. Where revised standards for an existinq article 3.70.20. Applicability of Standards to Medical Devices,
have been published as final approved "offlclal text" (as Dietary Supplements, and TheirComponents and Ingredients
approved in section 2.70. Official Text) but have ~ot yet An article recognized in USP or NFshall comply with the
reached the official date (6 months after publication, unless compendial standards ifthe arti~le is a ~edi~al device,
otherwisespecified; see "official date", section 2.20. Official component intende~ for a ~edlcal devlc~, dlet~ry .
Articles), compliancewith the revised standard shall not supplement, dietary Ingredient, or other Ingredient that IS
preclude a finding or indication of conf?rmance wi.th intended for incorporation into a dietary supplement, and
compendial standards, unless USP specifies otherwise by is labeled as conforming to the USP or NF.
prohibiting early adoption in a particularstandard. Generally, dietary supplements are prepared from
The standards in the relevant monograph, general ingredients that meet USP, NF, or Food Chemicals Codex
chapter(s), and General Notices apply at all times in the life of standards. Where such standards do not exist, substances
the articlefrom production to expiration. It is also noted that may be used indietarysupplements ifthey have been shown
the manufacturer's specifications, and manufacturing to be of acceptable food grade quality using other suitable
practices(e.g., Quality by Design, Process Analytical procedures.
Technology and Real Time Release Testing initiatives), 3.70.30. Applicability of Standardsto the Practice of
generally ar~ followed to ensure that the articlewill comply Compounding. .
with compendial standards until its expiration date, when USP compounding practice standards, Pharmaceutical
stored as directed. Every compendialarticle in commerce shall Compounding-Nonsterile Preparations (795) and
be so constituted that when examined in accordance with Pharmaceutical Compounding-SterilePreparations (797), as
these assays and test procedures, it meets all applicable appropriate, apply to compounding practice or activity
pharmacopeial requirements (General Notices, monographs, regardless of whether a monograph existsfor the
and general chapters). Thus, an~ official article is exp~~ted to compounded preparation or these chapters are referenced
meet the compendial standards If tested, and any offklal in such a monograph. In the United States, (795~ ~nd (797)
articleactuallytested as directed in the relevant monograph are not applicableto drugs compounded by entities
must meet such standards to demonstrate compliance. registered with FDA as outsourcing facilities as defined by
Some tests, such as those for Dissolution and Uniformityof FDCA § 503B becausesuch facilities are required to comply
Dosage Units, require multiple dosage un!ts i~ conjunction with FDA's c~rrent good manufacturi~g pr~ctice .
with a decision scheme. These tests, albeit usmq a number of requirements. Compounded preparations, Including drug
dosage units, are infact one determination. These procedures products compounded byoutsourcingfacilities, mayalsobe
should not be confused with statistical sampling plans.The

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 USP 43 General Notices 5

subject to applicable monographs; see section 2.20. Official instances, users may wishto ascertain functional equivalence
Articles and section 4.70. Monographs. or determine such characteristics before use.
3.20. Indicating Conformance 4. 10.10. Applicability of Test Procedures
Adrug product, drug substance, or excipient may use the A single monograph may include more than one test,
designation "USP" or "NF" in conjunctionwith itsofficial title procedure, and/or acceptance criterion for the same
or elsewhere on the labelonly when (1) a monograph is attribute. Unless otherwisespecified in the monograph, all
provided in the specified compendium and (2) the article tests are requirements. In some cases, monograph
complies with the identity prescribed in the specified instructions allow the selection of tests that reflect attributes
compendium. of differentmanufacturers' articles, such as different
When a drug product, drug substance, compounded polymorphic forms, impurities, hydrates, and dissolution.
preparation, or excipientdiffers from the relevant USP or NF Monograph instructions indicatethe tests, procedures, and/
standard of strength, quality, or purity, as determined by the or acceptance criteria to be used and the required labeling.
application of the tests, procedures, and acceptance criteria The order in which the tests are listed in the monograph
set forth in the relevantcompendium, its difference shall be is based on the order in which they are approved by the
plainly stated on its label. relevant Expert Committee for inclusion in the monograph.
When a drug product, drug substance, compounded Test 1 is not necessarily the test for the innovatoror for the
preparation, or excipientfails to comply with the identity reference product. Depending on monograph instructions,
prescribed in USP or NF or contains an added substance that a labeling statement isnot typically requiredifTest1 isused.
interferes with the prescribed tests and procedures, the article 4.10.20. Acceptance Criteria
shall be designated by a name that isclearly distinguishing and The acceptance criteria allow for analytical error,for
differentiating from any name recognized in USP or NF. unavoidable variations in manufacturing and
A medical device, dietarysupplement, or ingredient or compounding, and for deterioration to an extent
component of a medical deviceor dietarysupplement mayuse considered acceptable under practical conditions. The
the designation "USP" or "NF" in conjunction with its official existenceof compendial acceptance criteria does not
title or elsewhere on the labelonlywhen (1) a monograph is constitute a basis for a claim that an official substance that
provided in the specified compendium and (2) the article more nearly approaches 100% purity "exceeds"
complies with the monograph standards and other applicable compendial quality. Similarly, the fact that an article has
standards in that compendium. been prepared to tighter criteria than those specified in the
The designation "USP" or "NF" on the label may not and monograph does not constitute a basis for a claim that the
does not constitute an endorsement by USP and does not article "exceeds" the compendial requirements.
representassuranceby USP that the articleisknown to comply Anofficial product shall be formulated with the intent to
with the relevantstandards. USP may seek legal redress ifan provide 100% of the quantity of each ingredientdeclared
article purports to be or is represented as an official article in on the label. Where the minimum amount of a substance
one of USP's compendia and such claim isdetermined by USP present in a dietarysupplement is required by lawto be
not to be made in good faith. higher than the loweracceptance criterion allowed for inthe
The designation "USP-NF" may be usedon the label of an monograph, the upper acceptance criterion contained in
article provided that the label also bears a statement such as the monograph may be increased by a corresponding
"Meets NFstandards as published by USP," indicating the amount.
particular compendium to which the article purports to apply. The acceptance criteria specified in individual
Whenthe letters"USP," "NF,"or "USP-NF" are used on the monographs and in the general chapters for compounded
label of an article to indicate compliancewith compendial preparationsare based on such attributesof quality as might
standards, the letters shall appear in conjunction with the be expected to characterize an article compounded from
official title of the article. The letters are not to be enclosed in suitable bulkdrug substances and ingredients, using the
any symbol such as a circle, square, etc., and shall appear in procedures providedor recognized principles of good
capital letters. compounding practice, as described in these compendia.
If a dietarysupplement does not complywith all applicable 4.20. General Chapters
compendial requirementsbut contains one or more dietary Each general chapter isassigned a number that appears in
ingredientsor other ingredientsthat are recognized in USP or angle brackets adjacent to the chapter name (e.g.,
NF, the individual ingredient(s) may be designated as Chromatography (621 ». General chapters may contain the
complying with USP or NFstandards or being of USP or NF following:
quality providedthat the designation is limited to the • Descriptions of tests and proceduresfor application
individual ingredient(s)and does not suggest that the dietary through individual monographs,
supplement complies with USP standards. • Descriptions and specifications of conditions and
practices for pharmaceutical compounding,
4. MONOGRAPHS AND GENERAL CHAPTERS • General information for the interpretation of the
4.10. Monographs compendial requirements,
Monographs set forth the article's name, definition, • Descriptions of general pharmaceutical storage,
specification, and other requirements related to packaging, dispensing, and packaging practices, or
storage, and labeling. The specification consists of tests, • General quidance to manufacturers of official substances
procedures, and acceptance criteria that help ensure the or official products.
identity,strength, quality, and purityof the article. Forgeneral When a general chapter is referenced in a monograph,
requirements relating to specific monograph sections, see acceptance criteria may be presented after a colon.
section 5. MONOGRAPH COMPONENTS. Somechaptersmay serveas introductoryoverviews ofa test
Because monographs may not provide standards for all or of analytical techniques. Theymay reference other general
relevantcharacteristics, some official substances may conform chapters that contain techniques, detailsof the procedures,
to the USP or NFstandard but differ with regard to and, at times, acceptance criteria.
nonstandardized propertiesthat are relevantto their use in
specific preparations. To assuresubstitutability in such

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6 GeneralNotices USP43

5. MONOGRAPH COMPONENTS manufacture of official preparations intended for internal or

5.10. Molecular Formula topical use, providedthat the denaturant isvolatile and does
The use of the molecularformula for the official not remain in the finished product. A preparation that is
substance(s) named in defining the required strength of a intended for topicalapplication to the skin may contain
compendialarticleisintended to designatethe chemical entity specially denatured alcohol, providedthat the denaturant is
or entities, as given in the complete chemical name of the either a usual ingredient in the preparation or a permissible
article, having absolute (100%) purity. added substance; in either case the denaturant shall be
5.20. Added Substances identified on the labelof the topical preparation. Where a
Added substances are presumed to be unsuitable for process is given in the individual monograph, any
inclusion in an official articleand therefore prohibited, if their preparation compounded using denatured alcohol shall be
presence impairs the bioavailability, therapeutic efficacy, or identical to that prepared by the monograph process.
safetyof the official article; or they interfere with the assays 5.20.20.2. In Dietary Supplements
and tests prescribed for determining compliancewith the Additional ingredients may be added to dietary
compendial standards (see section 3.20. Indicating supplement products provided that the additional
Confurmanc~. . ingredients (1) complywith applicable regulatory
The a.ir in a container of an official article may, where requirements, and (2) do not interfere with the assays and
appropriate, be evacuated or be replaced by carbon dioxide, tests prescribedfor determining compliancewith
helium, argon, or nitrogen, or by a mixtureofthese gases.The compendial standards.
use of such gas need not be declared in the labeling. 5.30. Description and Solubility
5.20.10. Added Substances in Official Substances Only where a quantitativesolubility test is given in a
Official substances may contain only the specific added monograph and is designated as such is it a test for purity.
substances that are permitted by the individual monograph. A monograph may include information regarding the
Such added substances shall not exceed the quantity article'sdescription. Information about an article's
required for providingtheir intended effect. Wheresuch "descriptionand solubility" also is provided in the reference
addition is permitted, the labelshall indicate the name(s) table Description and Relative Solubility of USP and NFArticles.
and amount(s) of any added substance(s). The referencetable merelydenotes the properties of articles
5.20.20. Added Substances (Excipients and Ingredients) in , that comply with monograph standards. The reference table
Official Products isintended primarily for those who use, prepare, and dispense
Suitablesubstances and excipients such as antimicrobial drugs and/or related articles. Although the information
agents, pharmaceutical bases, carriers, coatings,flavors, provided in monographs and the information in the reference
preservatives, stabilizers, and vehicles may be added to an table may indirectly assistin the preliminary evaluation of an
official product to enhance its stability, usefulness, or article, it is not intended to serve as a standard or test for
elegance, or to facilitate its preparation, unless otherwise purity.
specified in the individual monograph. The approximate solubility of a compendial substance is
Added substances and excipients employed solely to indicated by one of the following descriptive terms:
impart color may be incorporatedjnto official products
other than those intended for parenteralor ophthalmic use, Parts of Solvent Required
in accordance with the regulations pertaining to the use of Descriptive Term for 1 Part of Solute
colors issued by the FDA, providedsuch added substances Very soluble less than 1
or excipients are otherwise appropriate in all respects. (See
also Injections and Implanted Drugs Products (1), Product Freely soluble From 1 to 10
Quality Tests Common to Parenteral Dosage Forms, Specific Soluble From10 to 30
Tests, Vehicles and addedsubstances, Addedsubstances.)
The proportions of the substancesconstituting the base Sparingly soluble From 30 to 10O
in ointment and suppositoryproducts and preparations may Slightly soluble From 100 to 1,000
be varied to maintain a suitable consistency underdifferent
climatic conditions, provided that the concentrations of Very slightly soluble From 1,000 to 10,000
drug substances are not varied and providedthat the Greaterthan or equal to
bioavailability, therapeutic efficacy, and safetyof the Practically insoluble, or Insoluble 10,000 " ,
preparation are not impaired.
5.20.20.1. In Compounded Preparations ' 5.40. Identification
Compounded preparationsfor which a complete Acompendial test titled Identification is provided as an aid
composition is given shallcontain only the ingredients in verifying the identityof articles as they are purported to be,
named in the formulas unless specifically exempted herein e.g., those taken from labeled containers, and to establish
or in the individual monograph. Deviation from the whether it is the article named in USP-NF. The Identification
specified processesor methods of compounding, although test for a particular article may consistof one or more
not from the ingredients or proportions thereof" may occur procedures. When a compendialldentification test is
provided that the finished preparation conformsto the undertaken, all requirements of allspecified procedures in the
relevant standards and to preparations produced by test must be met to satisfy the requirementsof the test. Failure
following the specified process. of an article to meet all the requirementsof aprescribed
Where a monograph for a compounded preparation calls Identification test (l.e., failure to meet the requirements of all
for an ingredient in an amount expressedon the dried basis, of the specified procedures that are components of that test)
the ingredient need not be dried beforeuse ifdue allowance indicatesthat the article is mislabeled and/or adulterated.
is made for the water or other volatile substances present in 5.50. Assay
the quantity taken. Assay tests for compounded preparationsare not intended
Specially denatured alcohol formulas are available for use for evaluating a compounded preparation before dispensing,
in accordance with federal statutes and regulations of the but instead are intended to serveasthe official test inthe event
Internal Revenue Service. Asuitableformula of specially of a question or dispute regarding the preparation's
denatured alcohol may be substituted for Alcohol in the conformance to official standards.

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 USP 43 GeneralNotices 7

5.50.10. Units of Potency (Biological) 5.60.20. Residual Solvents in USP and NFArticles
Forsubstances that cannot be completelycharacterized All USP and NF articles are subject to relevant control of
by chemical or physical means or that need confirmation of residual solvents, even when no test is specified in the
functionality or tertiarystructure, it may be necessary to individual monograph. If solvents are used during
express quantities of biological activity in units of biological production,they must be of suitablequality. Inaddition, the
potency, each defined by an authoritative, designated toxicity and residual level of each solventshall be taken into
reference standard. In caseswhere international reference consideration, and the solventslimited according to the
materials have been discontinued, international units of principles defined and the requirementsspecified in Residual
potency may be defined interms of molecular mass, such as Solvents (467), usingthe general methods presented therein
in the cases of vitamins A, D, and E. or other suitable methods.
Where available, World Health Organization (WHO) 5.60.30. Elemental Impurities in USP Drug Products and
international biological standards define the International Dietary Supplements
Units (IU). USP monographs referto the units assigned by Elemental impurities in official drug products are
USP Reference Standards either directly as International controlled according to the principles defined and
Units (IU) or as "USP Units." Forsome biological products, requirementsspecified in Elemental Impurities-Limits (232).
units of potency are valueassigned against a corresponding Elemental contaminants in official dietary supplements are
U.S. Standard established by FDA, whether or not controlled according to the principles defined and
International Units or USP Units have been defined (see requirements specified in Elemental Contaminants in Dietary
Biologics (1041». Note that product-related labeling, e.g., Supplements (2232).
on containers, need not use the full phrase "USP [product 5.70. Performance Tests
name] Units" that appears in many USP monograph labeling Where content uniformity determinations have been made
sections. The term "USP Units" can be used on product using the same analytical methodology specified in the Assay,
labeling consistent with USP compendial requirements, with appropriate allowances made for differences in sample
provided it is clearfrom the context that the potency is preparation, the average of all of the individual content
stated in terms of USP [product name] Units. In such uniformity determinations may be used as the Assay value.
circumstances it should be clear that "USP Units" and "USP 5.80. USP Reference Standards
[product name] Units" share the same meaning. USP Reference Standardsare authentic specimensthat have
5.60. Impurities and Foreign Substances been approved as suitablefor use as comparison standards in
Testsfor the presence of impurities and foreign substances USP or NFtests and assays. (See USP Reference Standards (11 ).)
are provided to limitsuch substances to amounts that are Where USP or NF tests or assays call for the use of a USP
unobjectionable under conditions in which the article is Reference Standard, only those results obtained using the
customarily employed (see also Impurities in Drug Substances specified USP Reference Standard are conclusive. Where a
and Drug Products (1086». procedure calls for the use of a compendial article rather than
Nonmonograph tests and acceptance criteria suitablefor for a USP Reference Standard as a material standard of
detecting and controlling impurities that may resultfrom a reference, a substance meeting all of thecompendial
change in the processing methods or that may be introduced monograph requirementsfor that articleshall be used. Ifany
from external sources should be employed in addition to the new USP or NF standard requiresthe use of a new USP
tests provided in the individual monograph, where the Reference Standardthat isnot yet available, that portion of the
presence of the impurityis inconsistentwith applicablegood standard containing the requirement shall not be official until
manufacturing practices or good pharmaceutical practices. the specified USP reference material is available.
5.60.10. Other Impurities in USP and NFArticles Unless a Reference Standard label bears a specific potency
If a USP or NF monograph includes an assayor organic or content, assume the Reference Standard is 100.0% pure in
impuritytest based on chromatography, other than a test the official application. Unless otherwise directed in the
for residual solvents, and that monograph procedure does procedure in the individual monograph or in a general
not detect an impurity present inthe substance, the amount chapter, USP Reference Standards are to be used in
and identity of the impurity, where both are known,shall be accordance with the instructionson the labelof the Reference
stated in the labeling (certificate of analysis) of the official Standard.
substance, under the heading Other Impurity(ies).
The presence of any unlabeledother impurity inan official 6. TESTING PRACTICES AND PROCEDURES
substance is a variancefrom the standard ifthe content is
0.1% or greater. The sum of all Other Impurities combined 6.10. Safe laboratory Practices
with the monograph-detected impurities may not exceed In performing compendial procedures, safe laboratory
2.0% (see OrdinaryImpurities (466», unless otherwisestated practicesshall be followed, includingprecautionary measures,
in the monograph. protective equipment, and work practicesconsistentwith the
The following categories of drug substances are chemicals and procedures used. Before undertaking any
excluded from Other Impurities requirements: procedure described in the compendia, the analystshould be
aware of the hazards associated with the chemicals and the
• Fermentation products and semi-synthetics derived . techniques and means of protecting against them. These
therefrom, compendia are not designed to describe such hazardsor
• Radiopharmaceuticals, protective measures.
• Biologics, 6.20. Automated Procedures
• Biotechnology-derived products, Automated and manual procedures employing the same
• Peptides, basic chemistryare considered equivalent provided the
• Herbals, and automated system is properly qualified as being suitableto
execute the compendial manual method and the analytical
• Crude products of animal or plant origin. procedure isverified under the new equipment conditions.
Any substance knownto be toxicshall not be listed under 6.30. Alternative and Harmonized Methods and
Other Impurities. Procedures
An alternative method or procedure is definedas any
method or procedure other than the compendial method or

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8 General Notices USP 43

procedure for the article in question. The alternative method 6.50.20. Solutions
or procedure must be fully validated (see Validation of Unless otherwise specified, all solutions shall be prepared
Compendial Procedures (1225» and must produce comparable with Purified Water. Solutions forquantitative measures shall
results to the compendial method or procedure within be prepared using accurately weighed or accurately
allowable limits established on a case-by-case basis. Alternative measured analytes (see section 8.20. About).
methods or procedures can be developedfor anyone of a An expression such as "(1 in 10)" means that 1 part by
number of reasons not limited to simplification of sample volume of a liquid shall be diluted with, or 1 part by weight
preparation, enhanced precision and accuracy, improved of a solidshall be dissolved in, a sufficient quantity of the
(shortened) run time, or being better suited to automation diluent or solventto make the volumeof the finished
than the compendial method or procedure.Onlythose results solution 10 parts by volume. For example,a 1 in 10 solution
obtained by the methods and proceduresgiven in the is prepared by diluting 1 mL of a liquid or dissolving 1 g of
compendia are conclusive. a solid in sufficient solventto make 10 mL of the solution.
Forevaluation as a potentialreplacementor addition to the An expression such as "(20:5:2)" means that the respective
standard, alternative methods and procedures should be numbers of parts, byvolume, of the designated liquids shall
submitted to USP (see section 4.10. Monographs). be mixed, unless otherwise indicated.
Certain general chapters contain a statement that the text 6.50.20.1. Adjustments to Solutions
in question is harmonizedwith the corresponding text of the When a specified concentration iscalledfor in a
European Pharmacopoeia and/or the Japanese Pharmacopoeia procedure, a solution of other normality or molarity may be
and that these texts are interchangeable. Therefore, ifa used, provided that allowance is made for the difference in
substance or preparation isfound to complywith a concentration and that the change does not increase the
requirement using an interchangeable method or procedure error of measurement.
from one of these pharmacopeias, it should complywith the Proportionately largeror smaller quantities than the
requirementsof the USP-NF. Whena difference appears, or in specified weights and volumes of assay or test substances
the event of dispute, only the resultobtained by the method and Reference Standards may be taken, providedthe
and/or procedure given in the USP-NF is conclusive. measurement is made with at leastequivalentaccuracy.
6.40. Dried, Anhydrous, Ignited, or Solvent-Free Basis Unless otherwise indicated, analyteconcentrationsshall
All calculations in the compendia assumean "as-is" basis be prepared to within ten percent (10%) of the indicated
unless otherwise specified. value. In the case in which a procedure is adapted to the
Test procedures may be performed on the undried or working range of an instrument, solution concentrations
unignited substance and the results calculated on the dried, may differ from the indicated value by more than ten
anhydrous, or ignited basis, provided a test for Loss on percent (10%), with appropriate changes in associated
Drying, or WaterDetermination, or Loss on Ignition, calculations. Any changes shall fall within the validated
respectively, is given in the monograph. Where the presence range of the instrument.
of moistureor other volatile material may interfere with the When adjustment of pH is indicated with either an acid
procedure, previous dryingof the substance isspecified in the or base and the concentration is not indicated, appropriate
individual monograph and isobligatory. concentrationsof that acid or base may be used.
The term "solvent-free" signifies that the calculation shall 6.50.20.2. Test Solutions
be correctedforthe presenceof known solvents as determined Information on TestSolutions (TS) is provided in the Test
using the methods described in (467) unless a test for limitof Solutions portion of the Reagents, Indicators, and Solutions
organic solvents is provided in the monograph. sectionof the USP-NF. Use of an alternative TestSolution or
The term "previously dried" without qualification signifies a change in the Test Solution used may requirevalidation.
that the substance shall be dried as directed under Loss on 6.50.20.3. Indicator Solutions
Drying (731) or WaterDetermination (921) (gravimetric Wherea procedure specifies the use of an indicator TS,
determination). approximately 0.2 mL, or 3 drops, of the solutionshall be
Where drying in vacuum over a desiccantisdirected, a added unless otherwise directed.
vacuum desiccator, a vacuum drying pistol, or other suitable 6.60. Units Necessary to Complete a Test
vacuum drying apparatus shall be used. Unless otherwisespecified, a sufficient number of units to
6.40.10. Ignite to Constant Weight ensure a suitableanalytical resultshall be taken.
"Ignite to constant weight" means that ignition shall be 6.60.10. Tablets
continued at 800 ± 25°, unless otherwise indicated, until Where the procedure of a Tablet monograph directsto
two consecutiveweighings, the second of which is taken weigh and finely powder not fewerthan a given number of
after an additional period appropriate to the nature and Tablets, a counted number of Tablets shall be weighed and
quantity of the residue, do not differ by more than 0.50 mg reduced to a powder. The portion of the powdered Tablets
per g of substance taken. taken shall be representative of the wholeTablets and shall,
6.40.20. Dried to Constant Weight in turn, be weighed accurately.
"Dried to constant weight" means that drying shall be 6.60.20. Capsules
continued until two consecutive weighings, the second of Wherethe procedure of a Capsule monograph gives
which is taken after an additional drying period appropriate direction to remove, as completely as possible, the contents
to the nature and quantity of the residue, do not differ by of not fewerthan a given number ofthe Capsules, a counted
more than 0.50 mg per g of substance taken. number of Capsules shall be carefully opened and the
6.50. Preparation of Solutions contents quantitatively removed, combined, mixed, and
6.50.10. Filtration weighedaccurately. Theportionof mixedCapsules contents
Where a procedure gives direction to "filter" without taken shall be representative of the contents of the Capsules
further qualification, the liquid shallbe passed through and shall, in turn, be weighed accurately.
suitablefilter paper or equivalentdevice until the filtrate is 6.70. Reagents
clear. Due to the possibility of filter effects, the initial The proper conduct of the compendial proceduresand the
volumes of a filtrate may be discarded. reliability ofthe results depend, in part, upon the qualityof the
reagents used in the performance of the procedures. Unless
otherwisespecified, reagentsconforming to the specifications

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USP 43 General Notices 9

set forth in the current edition of Reagent Chemicals published of Testing and Materials (ASTM) standards E1 for
by the American Chemical Society (ACS) shall be used. Where Iiquid-in-glass thermometers.
such ACS reagent specifications are not available or where the
required puritydiffers, compendial specifications for reagents 1. TEST RESULTS
of ~cceptable quality are provided (see the Reagents
Indicators, and Solutions section of the USP-NF). Reagents not 7.10. Interpretation of Requirements
coyered by any of these specifications should be of a grade Analytical resultsobserved in the laboratory(or calculated
suitable to the proper performance of the method of assayor from·experimental measurements) are compared with stated
test involved. acceptance criteria to determine whether the article conforms
Li~ting of these reagents, including the indicators and to compendial requirements.
solutions employed as reagents, in no way implies that they The reportable value, which often is a summaryvalue for
have therapeutic utility; furthermore, any reference to USP or several individual determinations, iscompared with the
NF in their labeling shall include also the term "reagent" or acceptance criteria. The reportablevalue isthe end result of a
"reagent grade." USP may supply reagents ifthey otherwise completed measurement procedure, as documented.
may not be generally commercially available. Whereacceptance criteria are expressednumerically herein
6.80. Equipment through specification of an upper and/or lower limit,
Unless otherwisespecified, a specification for a definitesize permitted values includethe specified values themselves, but
or type of container or apparatus in a procedure isgivensolely no valuesoutside the Iimit(s). Acceptance criteria are
~s a recomm~ndation. Other dimensionsor types maybe used considered significant to the last digit shown.
If they are suitable for the intended use. 7.10.5. Nominal Concentrations in Equations
6.80.10. Apparatus for Measurement Where a "nominal concentration" is specified, calculate
YVh~re volume~ric flasks or other exact measuring, the concentration based on the labelclaim. In assay
wel~hlng, or sorting devices are specified, this or other procedures, water correction is typically stated in the
equipment of at least equivalent accuracyshall be Definition and on the label of the USP Reference Standard.
employed. . Forother procedures, correction for assayed content,
6.80.10.1. Pipet/Pipette potency, or both is made prior to using the concentration
Wher~ a pipet/pipette isspecified, a suitable buret may in the equation provided in the monograph.
be s~l;>stltuted: Where a "to contain" pipet/pipette is 7.10.10. Equivalence Statements in Titrimetric Procedures
specified, a suitable volumetric flask may be substituted. The directionsfor titrimetric procedures conclude with a
6.80.10.2. light Protection statement of the weight of the analyte that is equivalentto
Where low-actinic or light-resistant containers are each ml of the standardized titrant. In such an equivalence
specified, either containers specially treated to protect statement, the number of significant figures in the
contents from light or clear containers that have been concentration of the titrant should be understood to
render~d opaque by application of a suitable coating or correspond to the number of significant figures in the
wrappmg may be used. weight of the analyte. Corrections to calculations based on
6.80.20. Instrumental Apparatus the blank determination are to be made for all titrimetric
. An instru.ment may be substituted for the specified assays where appropriate (see Titrimetry (541 »).
Instrument If the substitute uses the same fundamental 7.20. Rounding Rules
prin~ip~es of operation and is of equivalent or greater
The observed or calculated values shall be rounded offto
sensitivity and accuracy. These characteristics shall be the number of decimal places that is in agreement with the
qualified as appropriate. Where a particularbrand or source limit expression. Numbersshould not be rounded until the
of a material, instrument, or piece of equipment, or the final calculations for the reportable value have been
name and address of a manufacturer or distributor is completed. Intermediate calculations (e.g., slope for linearity)
me~tioned (ordinarily in a footnote), this identifica'tion is . may be rounded for reporting purposes, but the original (not
furnished solely for informational purposes as a matter of rounded) value should be used for any additional required
convenience, without implication of approval, calculations. Acceptancecriteria arefixednumbers and are not
endorsement, or certification. rounded.
6.80.20.1. Chromatographic Tubes and Columns When rounding is required, consider only one digit in the
The term "diameter" refers to internal diameter (10). decimal place to the right of the last place in the limit
6.80.20.2. Tubing . expression. Ifthis digit is smaller than 5, it is eliminated and
The term "diameter" refers to outside diameter (00). the preceding digit is unchanged. Ifthis digit is equal to or
6.80.20.3. Steam Bath weater than 5, it is eliminated and the preceding digit is
Where use of a steam bath isdirected, useactively flowing Increased by 1.
steam or another regulated heat source controlled at an 8. TERMS AND DEFINITIONS
equivalent temperature. 8.10. Abbreviations
6.80.20.4. Water Bath • RS refers to a USP Reference Standard.
Awater bath requires vigorously boiling water unless • CS refers to a Colorimetric Solution.
otherwise specified. • TS refers to a Test Solution.
6.80.30. Temperature Reading Devices
Temperature reading devices suitable for pharmacopeial • VS refers to a Volumetric Solution that is standardized in
tests conform to specifications that are traceable to a accordance with directionsgiven in the individual
National Instituteof Standards and Technology (NIST) monograph or in the Reagents, Indicators, and Solutions
standard ~r e~u!valent. Temperature reading devices may section of USP-NF.
be of the hq~ld-!n-glass type or an analog or digital 8.20. About
temperature I~dlcator type, such as a resistance temperature "About" indicates a quantity within 10%.
device, thermistor, or thermocouple. Standardization of If the measurement is stated to be "accuratelymeasured"
thermometers isperformed on an established testing or "accuratelyweighed," follow the statements in Volumetric
frequency with a temperature standard traceable to NIST. Apparatus (31) and Balances (41), respectively.
For example, referto the current issueof American Society

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 ~ .•

10 General Notices USP 43

Illustration of Rounding Numerical Values

for Comparison with Requirements
Compendial Requirement Unrounded Value Rounded Result Conforms
Assaylimit 2:98.0% 97.96% 98.0% Yes
97.92% 97.9% No
97.95% 98.0% Yes
. Assaylimit ~1 01.5% 101.55% 101.6% No
101.46% 101.5% Yes
101.45% 101.5% Yes
Limittest ~0.02% 0.025% 0.03% No
0.015% 0.02% Yes
0.027% 0.03% No
Limittest ~3 ppm 3.5 ppm 4 ppm No
3.4 ppm 3 ppm Yes
2.5 ppm 3 ppm Yes

8.30. Alcohol Content quantity of freshly opened material after exposure to the air
Percentages of alcohol, such as those under the heading for 15 minutes. An odor designation is descriptive only and
Alcohol Content, refer to percentage by volume of C2H sOH at should not be regarded as a standard of purity for a particular
15.56°. Where a formula, test, or assay calls for alcohol, ethyl lot of an article.
alcohol, or ethanol, the USP monograph article Alcohol shall 8.130. Percent
be used. Where reference is made to "C2H sOH," absolute "Percent" used without qualification means:
(100%) ethanol is intended. Where a procedure calls for • For mixtures of solids and semisolids, percent weight in
dehydrated alcohol, alcohol absolute, or anhydrous alcohol, weight;
the USP monograph article Dehydrated Alcohol shall be used. • For solutions or suspensions of solids in liquids, percent
8.40. Atomic Weights weight in volume;
Atomic weights used in computing molecular weights and • For solutions of liquids in liquids, percent volume in
the factors in the assays and elsewhere are those established volume;
by the IUPAC Commission on Isotopic Abundances and • For solutions of gases in liquids, percent weight in
Atomic Weights. . volume.
8.50. Blank Determinations
Where it is directed that "any necessary correction" be For example.a 1 percent solution is prepared by dissolving
made by a blank determination, the determination shall be 1 g of a solid or semisolid, or 1 mL of a liquid, in sufficient
conducted using the same quantities of the same reagents solvent to make 100 mL of the solution.
treated in the same manner as the solution or mixture 8.140. Percentage Concentrations
containing the portion of the substance under assay ortest, Percentage concentrations are expressed as follows:
but with the substance itself omitted. • Percent Weight in Weight (w/w) is defined as the number
8.60. Concomitantly of g of a solute in 100 g of solution.
"Concomitantly" denotes that the determinations or • Percent Weight in Volume (w/v) is defined as the number
measurements are to be performed in immediate succession. of g of a solute in 100 mL of solution.
8.70. Desiccator • Percent Volume in Volume (v/v) is defined as the number
The instruction "in a desiccator" indicates use of a tightly of mL of asolute in 100 mL of solution.
closed container of suitable size and design that maintains an 8.1 SO. Pressure
atmosphere of low moisture content by means of a suitable Pressure is determined by use of a suitable manometer or
desiccant such as anhydrous calcium chloride, magnesium barometer calibrated in terms of the pressure exerted by a
perchlorate, phosphorus pentoxide, or silica gel. See also column of mercury of the stated height.
section 8.220. Vacuum Desiccator. 8.160. Reaction Time
8.80. Logarithms Reaction time is 5 minutes unless otherwise specified.
Logarithms are to the basel O. 8.170. Specific Gravity
8.90. MlcroblalStraln Specific gravity is the weight of a substance in air at 25°
A microbial strain cited and identified by its American Type divided by the weight of an equal volume of water at the same
Culture Collection (ATCC) catalog number shall be used temperature. .
directly or, if subcultured, shall be used not more than five 8.180. Temperatures .
passages removed from the original strain. Temperatures are expressed in centigrade (Celsius)
8.100. Negligible degrees, and all measurements are made at 25° unless
"Negligible" indicates a quantity not exceeding 0.50 mg. otherwise indicated. Where moderate heat is specified, any
8.110. NLT/NMT temperature not higher than 45° (113° F) is indicated.
"NLT" means "not less than." "NMT" means "not more 8.19.0. Time . .
than." Unless otherwise specified, rounding rules, as described in
8.120. Odor section 7.20. Rounding Rules, apply to any time specified.
"Odorless," "practically odorless," "a faint characteristic
odor," and variations thereof indicate evaluation of a suitable

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USP 43 General Notices 11

8.200. Transfer Units Symbol Notes

"Transfer" indicates a quantitative manipulation.
8.210. Vacuum gram g
"Vacuum" denotes exposure to a pressureof less than 20 milligram mg
mm of mercury (2.67 kPas), unless otherwise indicated.
8.220. Vacuum Desiccator The syrnbol pq is used in the
USP and NFto represent rnl-
"Vacuum desiccator" indicates a desiccator that maintains crograms, but micrograms
a low-moisture atmosphere at a reduced pressureof not more may be represented as
than 20 mm of mercury(2.67 kPas) or at the pressure "mcg" for labelingand pre-
designated in the individual monograph. scribingpurposes.The term
"gamma," symbolized by y,
8.230. Water frequently is used to repre-
8.230.10. Water as an Ingredient in an Official Product sent microgramsin blo-
As an ingredient in an official product, water meets the microgram IJg chemicalliterature.
requirements of the appropriate water monograph in USP nanogram ng
or NF.
8.230.20. Water in the Manufacture of Official Substances picogram pg
When used in the manufacture of official substances, Also referred to as the unified
water shall meet the requirementsfor drinking water as set atomic mass unit and is
forth in the U.S. Environmental Protection Agency National equal to 1/12 times the mass
Primary Drinking Water Regulations or in the drinking water dalton Da of the free carbon 12 atom.
regulations of the European Union or of Japan, or in the kilodalton kDa
World Health Organization'sGuidelines for Drinking Water
Time
Quality. Additional specifications may be required in
monographs. second s
8.230.30. Water in a Compendial Procedure
minute min
When water is calledfor in a compendial procedure, the
USP monograph article Purified Water shall be used unless hour h
otherwise specified. Definitions for other types of water are Volume
provided in Reagents, Indicators, and Solutions and in Water
for Pharmaceutical Purposes (1231). 1 Lis equal to 1000 cm3 (cu-
8.240. Weights and Measures liter L biccentimeters)
In general, weights and measures are expressed in the deciliter dL
International 'System of Units (SI) as established and revised
by the Conference qenerate des poids et mesures. For 1 mLisequal to 1 ern', some-
milliliter mL times referredto as cc
compendial purposes, the term "weight" is considered to be
synonymous with "mass." microiiter IJL
Molality is designated by the symbol m preceded by a Tempera-
number that represents the number of moles of the ture
designated solute contained in 1 kilogram of the designated
solvent. Celsius °C
Molarity is designated by the symbol M preceded by a Amount of
number that represents the number of moles of the Substance
designated solute contained in an amount of the designated Historically referredto as
solvent that is sufficient to prepare 1 literof solution. , gram-molecular weight or
Normality is designated by the symbol N preceded by a mole mol gram-atomicweight
number that represents the number of equivalents of the millimole mmol
designated solute contained in an amount of the designated
solvent that is sufficient to prepare 1 literof solution. micromole urnol
The symbol for degrees e) without a qualifying unit of femtomole fmol
measure represents degrees Celsius.
Chart of Symbols and Prefixes commonly employed for SI Also referredto as
metric units and other units: gram-,equivalent weight. It is
used in the calculation of
substance concentration in
Units Symbol Notes units of normality. This unit
isno longer preferredfor use
length in analytical chemistryor
equivalent Eq metrology.
meter m
milli equiva-
centimeter cm lent mEq
millimeter mm Osmoticpressure of a solu-
tion, related to substance
Previously referred to as a mi- osmole Osmol concentration.
micrometer IJm cron
milliosmole mOsmol
Previously the symbolrnu (for
nanometer nm millimicron) was used Pressure
Angstrom A Equal to 0.1 nm pascal Pa
Mass kilopascal kPa
kilogram kg

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12 General Notices USP 43

Units Symbol Notes 9. PRESCRIBING AND DISPENSING

pounds per
9.10. Use of Metric Units
square inch psi Prescriptions for compendial articles shall be written to state
the quantity and/or strength desired in metric units unless
millimeter of otherwise indicated in the individual monograph [see also
mercury mmHg Equal to 133.322 Pa
section 5.50.10. Units of Potency (Biological) above]. If an
Electrical amount is prescribed by any other system of measurement,
units only an amount that is the metric equivalent of the prescribed
ampere A amount shall be dispensed. Abbreviations forthe terms
"Units" or "International Units" shall not be used for labeling
volt V or prescribing purposes. Apothecary unit designations on
millivolt mV labels and labeling shall not be used.
9.20. Changes in Volume
hertz Hz Unit of frequency In the dispensing of prescription medications, slight
kilohertz kHz changes in volume owing to variations in room
temperatures may be disregarded.
megahertz MHz

electron volt eV 10. PRESERVATION, PACKAGING, STORAGE, AND-

kilo-electron LABELING
volt keV 10.10. Packaging and Storage
mega-elec- All articles in USP or NF are subject to the packaging and
tron volt MeV storage requirements specified in Packaging and Storage
Requirements (659), unless different requirements are provided
Radiation
in an individual monograph.
SI unit of activity for radionu- 10.20. Labeling
becquerel Bq elides All articles in USP or NF are subject to the labeling
kilobecquerel kBq requirements specified in Labeling (7), unless different
requirements are provided in an individual monograph.
megabec-
querel MBq

gigabecquer-
el GBq

Non-SI unit of activity for ra-

curie Ci dionuclides

millicurie mCi

microcurie IJCi
nanocurie nCi

Other

acceleration
due to grav- Used to express rate of centri-
ity 9 fugation

revolutions Used to express rate of centri-

per minute rpm fugation

Selected 51 Prefixes
Name Symbol Factor

giga G 109
mega M 106
kilo k 103
deci d 10-1

centi c 10-2

milli m 10-3
I

micro IJ 10-6
nano n 10-9

pico P 10-12
femt6 f 10-15

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 USP 43 Guide to General Chapters 13

I• e r
(Forcomplete alphabeticallistof all general chapters in this Biological Tests and Assays
Pharmacopeia, see under "General chapters" in the index.)
(92) Growth Factors and Cytokines Used in CellTherapy
Manufacturing......................................................................... 6539
GENERAL TESTS AND ASSAYS
General Requirements for Tests and Assays (111) Design and Analysis of Biological Assays............................. 6543

(1) Injections and Implanted Drug Products (Parenterals)- (115) Dexpanthenol Assay.......................................................... 6547
Product Quality Tests............................................................... 6405 (121) InsulinAssays..................................................................... 6547
(2) Oral Drug Products-Product Quality Tests............................ 6410 (121.1) PhysicochemicalAnalytical Procedures for Insulins.......... 6550
(3) Topical and Transdermal Drug Products-Product Quality (123) Glucagon BioidentityTests................................................. 6552
Tests........................................................................................ 6415
(124) Erythropoietin Bioassays..................................................... 6557
(4) Mucosal Drug Products-Product QualityTests..................... 6423
(126) Somatropin BioidentityTests.............................................. 6558
(5) Inhalation and Nasal Drug Products-General Informationand
Product Quality Tests............................................................... 6427 (127) FlowCytometric Enumeration of Cd34+ Cells.................... 6560
(7) labeling................................................................................. 6435 (129) Analytical Procedures for Recombinant Therapeutic
Monoclonal Antibodies............. 6565
(11) USP Reference Standards 6442
(130) Protein A QualityAttributes................................................ 6570

Apparatus for Tests and Assays (151) Pyrogen Test. 6577

(17) Prescription Container labeling........................................... 6445 (161) Medical Devices-Bacterial Endotoxin and Pyrogen Tests.. 6579

(31) Volumetric Apparatus.......................................................... 6447 (162) Diphtheria Antitoxin Potency Testing for Human Immune
Globulins.................................................................................. 6582
(41) Balances.............................................................................. 6448
(165) Prekallikrein Activator......................................................... 6583
(171) Vitamin B12Activity Assay................................................. 6584
Microbiological Tests
(51) Antimicrobial Effectiveness Testing....................................... 6449
Chemical Tests and Assays
(55) Biological Indicators-Resistance Performance Tests............. 6452
IDENTIFICATION TESTS
(60) Microbiological Examination of Nonsterile Products-Tests 6454
For BurkholderiaCepacia Complex , .. (181) Identification-Organic Nitrogenous Bases........................ 6587

(61) Microbiological Examination of Nonsterile Products: (191) Identification Tests-GeneraL............................................ 6587

Microbial Enumeration Tests 6457
(193) Identification-Tetracyclines.............................................. 6592
(62) MicrobiologicalExamination of Nonsterile Products:Testsfor
Specified Microorganisms 6463 (197) Spectroscopic Identification Tests 6593

(63) Mycoplasma Tests 6471 (198) Nuclear Magnetic Resonance Spectroscopy Identity
Testing of Bacterial Polysaccharides Used in Vaccine
(64) Probiotic Tests..................................................................... 6477 Manufacture............................................................................ 6596

(71) SterilityTests........................................................................ 6480 (201) Thin-layer Chromatographic Identification Test................. 6600

(202) Identification of Fixed Oils by Thin-Layer
Chromatography...................................................................... 6601
Biological Tests and Assays
(203) High-PerformanceThin-layer Chromatography Procedure
(81) Antibiotics-Microbial Assays............................................... 6488 for Identification of Articlesof BotanicalOrigin......................... 6602
(85) Bacterial Endotoxins Test..................................................... 6508 LIMIT TESTS
(87) Biological Reactivity Tests, in Vitro....................................... 6514 (206) Aluminum........................ 6605
(88) Biological ReactivityTests, in Vivo........................................ 6516 (207) Test for l,6·Anhydro Derivativefor Enoxaparin Sodium..... 6605
(89) Enzymes Used as Ancillary Materials in Pharmaceutical Man- (208) Antl-FactorXa and Anti-Factor lIa Assays for
ufacturing................................................................................ 6521 Unfractionated and low Molecular Weight Heparins................ 6611
(89.1) Collagenase 1.................................................................... 6524 (209) low MolecularWeight Heparin Molecular Weight
Determinations........................................................................ 6614
(89.2) Collagenase II................................................................... 6528
(210) Monosaccharide Analysis 6616
(90) Fetal BovineSerum-Quality Attributes and Functionality
Tests 6533 (211) Arsenic............................................................................... 6621
(91) Calcium Pantothenate Assay................................................ 6536 (212) Oligosaccharide Analysis.................................................... 6623
(221) Chloride and Sulfate........................................................... 6636

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 14 Guide to General Chapters USP 43

Chemical Tests and Assays Chemical Tests and Assays

(223) Dimethylaniline................................................................. 6636 (551) VitaminEAssay.................................................................. 6767
(226) 4.Epianhydrotetracycline................................................... 6636 (561) Articles of BotanicalOrigin................................................. 6774
(227) 4-Aminophenol in Acetaminophen-Containing (563) Identificationof Articles of BotanicalOrigin........................ 6788
Drug Products.......................................................................... 6637
(565) BotanicalExtracts............................................................... 6798
(228) Ethylene Oxide and Dioxane.............................................. 6639
(571) Vitamin A Assay................................................................. 6801
(232) Elemental Impurities-limits.............................................. 6641
(580) VitaminC Assay................................................................. 6805
(233) Elemental Impurities-Procedures...................................... 6645
(581) Vitamin D Assay................................................................. 6808
(241) Iron................................................................................... 6648
(591) Zinc Determination............................................................ 6817
(251) Lead.................................................................................. 6649
(261) Mercury ~ ;............. 6650 Physical Tests and Determinations
(267) Porosimetry by Mercury Intrusion...................................... 6653 (601) Inhalation and Nasal Drug Products: Aerosols, Sprays, and
(268) Porosity by Nitrogen Adsorption-Desorption................... 6655 Powders-Performance Quality Tests 6819

(271) Readily Carbonizable Substances Test................................ 6660 (602) Propellants 6844

(281) Residue on Ignition............................................................ 6660 (603) TopicalAerosols................................................................. 6845

(291) Selenium........................................................................... 6661 (604) LeakRate........................................................................... 6846

OTHER TESTS ANDASSAYS (610) Alternative Microbiological Sampling Methods for

Nonsterile Inhaled and Nasal Products..................................... 6846
(301) Acid-NeutralizingCapacity................................................. 6661
(611) Alcohol Determination....................................................... 6848
(311) Alginates Assay.................................................................. 6662
(616) BulkDensity and Tapped Density of Powders..................... 6850
(341) Antimicrobial Agents - Content........................... 6664
(621) Chromatography.............................................................. 6853
(345) Assayfor Citric Acid/Citrate and Phosphate............ 6668
(630) Visual Comparison............................................................. 6865
(351) Assayfor Steroids............................................................... 6669
(631) Color and Achromicity....................................................... 6865
(381) Elastomeric Closuresfor Injections..................................... 6669
(641) Completeness of Solution.................................................. 6866
(391) Epinephrine Assay.............................................................. 6676
(643) Total Organic Carbon........................................................ 6867
(401) Fats and Fixed Oils 6676
(644) Conductivity of Solutions................................................... 6868
(411) FolicAcid Assay 6689
(645) Water Conductivity............................................................ 6871
(413) Impurities Testing in Medical Gases 6692
(651) Congealing Temperature................................................... 6874
(415) Medical Gases Assay......................................................... 6693
(659) Packaging and Storage Requirements................................ 6876
(425) lodometric Assay- Antibiotics............................................ 6696
(660) Containers-Glass.............................................................. 6881
(429) Light Diffraction Measurement of ParticleSize.................... 6697
(661) Plastic Packaging Systems and Their Materialsof
(431) Methoxy Determination 6702 Construction............................................................................ 6887
(441) Niacin or Niacinamide Assay ; 6704 (661.1) PlasticMaterialsof Construction..................................... 6893
(451) Nitrite Titration.................................................................. 6709 (661.2) Plastic Packaging Systems for Pharmaceutical Use........... 6912
(461) Nitrogen Determination.................................................... 6709 (670) Auxiliary Packaging Components....................................... 6916
(466) Ordinary Impurities............................................................ 6710 (671) Containers-Performance Testing...................................... 6922
(467) Residual Solvents............................................................... 6712 (691) Cotton............................................................................... 6929
(469) Ethylene Glycol, Diethylene Glycol, and Triethylene Glycol (695) Crystallinity........................................................................ 6931
in Ethoxylated Substances........................................................ 6733
(696) Characterization of CrystallineSolids ByMicrocalorimetry
(471) Oxygen FlaskCombustion................................................. 6735 and Solution Calorimetry.......................................................... 6931
(481) Riboflavin Assay................................................................. 6735 (697) Container Content for Injections........................................ 6934
(501) Salts of Organic Nitrogenous Bases.................................... 6741 (698) DeliverableVolume............................................................ 6935
(503) Acetic Acid in Peptides....................................................... 6741 (699) Density of Solids................................................................ 6938
(503.1) TrifluoroaceticAcid (TFA) in Peptides.............................. 6743 (701) Disintegration.................................................................... 6940
(507) Protein Determination Procedures..................................... 6744 (705) Quality Attributes of Tablets Labeled as Having a
Functional Score...... 6944
(509) Residual DNATesting........................................................ 6748
(711) Dissolution......................................................................... 6945
(511) Single-Steroid Assay........................................................... 6750
(721) Distilling Range.................................................................. 6955
(525) Sulfur Dioxide....;............................................................... 6751
(724) Drug Release...................................................................... 6957
(531) Thiamine Assay.................................................................. 6756
(729) Globule Size Distribution in Lipid Injectable Emulsions ...... 6963
(541) Titrimetry............................................................................ 6764
(730) Plasma Spectrochemistry................................................... 6967

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 USP 43 Guide to General Chapters 15

Physical Tests and Determinations Physical Tests and Determinations

(731) Losson Drying................................................................... 6970 (941) Characterization of Crystallineand PartiallyCrystalline
Solids by X-Ray· Powder Diffraction(XRPD)............................... 7201
(733) Losson Ignition................................................................. 6970
(735) X-Ray Fluorescence Spectrometry...................................... 6971 GENERAL INFORMATION
(736) Mass Spectrometry............................................................ 6975 (1004) Mucosal Drug Products-Performance Tests.................... 7227
(741) Melting Range or Temperature.......................................... 6980 (1005) Acoustic Emission............................................................. 7230
(755) Minimum Fill..................................................................... 6982 (101 0) Analytical Data-Interpretation and Treatment................ 7233
(761) Nuclear Magnetic Resonance Spectroscopy....................... 6984 (1024) BovineSerum.................................................................. 7260
(771) Ophthalmic Products-Quality Tests.................................... 6993 (1025) Pancreatin........................................................................ 7272
(776) Optical Microscopy............................................................ 6998 (1027) FlowCytometry 1............................................ 7281
(781) Optical Rotation................................................................. 7001 (1029) Good Documentation Guidelines............ 7296
(782) Vibrational Circular Dichroism Spectroscopy...................... 7002 (1030) BiologicalAssayChapterS-Overview and Glossary.......... 7300
(785) Osmolality and Osmolarity 7008 (1031) The Biocompatibility of Materials Used in Drug
Containers, Medical Devices, and implants..... 7313
(786) Particle Size Distribution Estimation by Analytical Sieving... 7010
(1032) Design and Development of Biological Assays.................. 7320
(787) Subvisible Particulate Matter in Therapeutic Protein
Injections................................................................................. 7014 (1033) Biological AssayValidation ,... 7337
(788) Particulate Matter in Injections........................................... 7017 (1034) Analysis of Biological Assays............................................. 7351
(789) Particulate Matter in Ophthalmic Solutions........................ 7020 (1039) Chemometrics................................................................. 7363
(790) Visible Particulates in Injections.......................................... 7021 (1041) Biologics.......................................................................... 7379
(791) pH..................................................................................... 7022 (1043) Ancillary Materials for Cell, Gene, and Tissue-Engineered
Products.......................................................................... 7381
(795) Pharmaceutical Compounding-Nonsterile Preparations... 7025
(1044) Cryopreservation of Cells................................................. 7389
(797) Pharmaceutical Compounding-Sterile Preparations 7037
(1046) Cell-based Advanced Therapies and Tissue-based Products 7400
(800) Hazardous Drugs-Handling in Healthcare Settings........... 7071
(1047) Gene Therapy Products.................................................... 7426
(801) Polarography..................................................................... 7089
(1048) Quality of Biotechnological Products: Analysis of The
(811) Powder Fineness................................................................ 7093 Expression Construct in Cells Used for Production of R-DNA
Derived Protein Products.......................................................... 7452
(821) Radioactivity...................................................................... 7094
(1049) Quality of Biotechnological Products: StabilityTesting of
(823) Positron EmissionTomography Drugs for Compounding, . Biotechnological/Biological Products........................................ 7454
Investigational, and Research Uses............ 7100
(1050) Viral Safety Evaluationof Biotechnology Products
(825) Radiopharmaceuticals-Preparation, Compounding, Dis- Derived from Cell Linesof Human or Animal Origin................. 7459
pensing, and Repackaging....................................................... 7109
(1050.1) Design, Evaluation, and Characterization Of Viral Clear-
(831) Refractive Index................................................................. 7137 ance Procedures....................................................................... 7472
(841) Specific Gravity.................................................................. 7137 (1051) Cleaning Glass Apparatus................................................. 7481
(846) SpecificSurface Area.......................................................... 713'9 (1052) Biotechnology-Derived Articles-Amino AcidAnalysis...... 7481
(852) Atomic Absorption Spectroscopy....................................... 7142 (1053) Capillary Electrophoresis 7492
(853) Fluorescence Spectroscopy................................................ 7146 (1054) Biotechnology-Derived Articles-Isoelectric Focusing....... 7499
(854) Mid-Infrared Spectroscopy................................................. 7151 (1055) Biotechnology-Derived Articles-Peptide Mapping.......... 7501
(855) Nephelometry and Turbidimetry........................................ 7155 (1056) Biotechnology-Derived Articles-Polyacrylamide Gel
(856) Near-Infrared Spectroscopy............................................... 7161 Electrophoresis......................................................................... 7508

(857) Ultraviolet-Visible Spectroscopy......................................... 7166 (1057) Biotechnology-Derived Articles-Total Protein Assay........ 7515

(861) Sutures-Diameter............................................................. 7175 (1058) Analytical Instrument Qualification................................... 7522

(871) Sutures-Needle Attachment............................................. 71 78 (1059) Excipient Performance..................................................... 7528

(881) Tensile Strength................................................................. 7179 (1061) Color-Instrumental Measurement.................................. 7553

(891) Thermal Analysis................................................................ 7180 (1062) Tablet Compression Characterization............................... 7556

(90S) Uniformity of Dosage Units................................................ 7183 (1063) Shear Cell Methodology for Powder FlowTesting............ 7566

(911) Viscosity-Capillary Methods............................................. 7187 (1064) Identification of Articles of BotanicalOrigin by High-

Performance Thin-LayerChromatography Procedure............... 7578
(912) Viscosity-Rotational Methods.................................... ....... 7189
(1065) Ion Chromatography....................................................... 7588
(913) Viscosity-Rolling BallMethod........................................... 7193
(1066) Physical Environments That Promote Safe Medication Use 7590
(914) Viscosity-Pressure Driven Methods................................... 7195
(1071) Rapid MicrobialTests for Releaseof Sterile Short-LifeProd-
(921) Water Determination......................................................... 7196 ucts: A Risk-Based Approach...................................................... 7602
(1072) Disinfectants and Antiseptics............................................ 7608

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 16 Guide to General Chapters USP 43

GENERAL INFORMATION (continued) GENERAL INFORMATION (continued)

(1074) Excipient Biological SafetyEvaluation Guidelines.............. 7613 (1"30) Nucleic Acid-Based Techniques-Approaches for
DetectingTrace Nucleic Acids (Residual DNA Testing).............. 7897
(1~7~) Good Manufacturing Practices for Bulk Pharmaceutical Ex-
clplents.................................................................................... 7617 (1132) Residual HostCell Protein Measurementin
Biopharmaceuticals..................................... 7900
(1079) Good Storage and Distribution Practices for Drug
Products................................................................................... 7635 (1136) Packaging and Repackaging-Single-Unit Containers...... 7919
(1079.1) Storage and Transportation of Investigational Drug (1151) Pharmaceutical DosageForms......................................... 7929
Products................................................................................... 7644
(1152) Animal Drugsfor Usein Animal Feeds.............................. 7952
(1080) Bulk Pharmaceutical Excipients-Certificate of Analysis.... 7647
(1160) Pharmaceutical Calculations in Pharmacy Practice............ 7953
(1084) Glycoprotein and Glycan Analysis-General
Considerations 7655 (1163) Quality Assurance in Pharmaceutical Compounding ....... 7979

(1085) Guidelines on the Endotoxins Test................................... 7665 (1168) Compoundingfor PhaseI Investigational Studies............. 7985

(1086) Impurities in Drug Substances and Drug Products............ 7678 (1174) PowderFlow.................................................................... 7993

(1087) Apparent Intrinsic Dissolution-Dissolution Testing (1176) Prescription Balances and Volumetric Apparatus Usedin 7997
Proceduresfor Rotating Disk and Stationary Disk...................... 7681 Compounding .
(1088) In Vitro and in Vivo Evaluation of Dosage Forms.............. 7684 (1177) Good Packaging Practices................................................ 8003

(1090) Assessment of Solid Oral Drug Product Performance (1178) Good Repackaging Practices................. 8006
and Interchangeability, Bioavailability, Bioequivalence, and Dis-
solution.................................................................................... 7695 (1180) Human Plasma 8008

(1091) Labeling of Inactive Ingredients....................................... 7706 (1181) Scanning Electron Microscopy......................................... 8025

(1092) The Dissolution Procedure: Developmentand Validation. 7707 (1184) Sensitization Testing l.......... 8035

(1094) Capsules-Dissolution TestingAnd Related Quality (1191) Stability Considerations in Dispensing Practice................. 8044
Attributes................. 7725 (119~) .Significant Change Guidefor Bulk Pharmaceutical
(1097) Bulk PowderSampling Procedures................................... 7732 Exciplents ,.,................. 8049

(1099) Limit on Number of LargeDeviations When Assessing (119~) .Good Distribution Practices for Bulk Pharmaceutical
Content Uniformity in Large Samples....................................... 7744 Exciplents.i.c. 8060

(1102) Immunological Test Methods-General Considerations... 7746 (1207) Package IntegrityEvaluation-SterileProducts 8079

(1103) Immunological Test Methods-Enzyme-Linked (1207.1) PackageIntegrityTesting in the Product Life Cycle-Test 8086
Immunosorbent Assay (Elisa) ~.................... 7753 Method Selection and Validation .

(1104) Immunological Test Methods-Immunoblot Analysis~...... 7763 (1207.2) PackageIntegrityLeak TestTechnologies...................... 8097

(1105) Immunological Test Methods-Surface Plasmon (1207.3) Package SealQuality TestTechnologies........................ 8111
Resonance................................................................................. 7773 (1208) Sterility Testing-Validation of Isolator Systems................ 8113
(1106) Immunogenicity Assays-Design And Validation of (1210) Statistical Tools for Procedure Validation.......................... 8117
Immunoassays to Detect Anti-Drug Antibodies......................... 7788
(1211) Sterility Assurance............................................................ 8129
(1106.1) Immunogenicity Assays-Design AndValidation of
Assays to Detect Anti-Drug Neutralizing Antibody.......;............ 7802 (1216) Tablet Friability................................................................ 8137
(1111) Microbiological Examination of Nonsterile Products: (1217) Tablet Breaking Force...................................................... 8138
AcceptanceCriteria for Pharmaceutical Preparations and .
Substancesfor Pharmaceutical Use........................................... 7819 (1222) Terminally Sterilized Pharmaceutical Products-c-Pararnet- 8142
ric Release .
(1112) Application of WaterActivity Determination to
Nonsterile Pharmaceutical Products....... 7820 (1223) Validation of Alternative Microbiological Methods........... 8144
(1.113) Microbial Characterization, Identification, and StrainTyp- (1223.1) Validation of Alternative Methods to Antibiotic
Ing........................................................................................... 7823 Microbial Assays....................................................................... 8157
(1115) Bioburden Control of Nonsterile Drug Substances and (1224) Transfer of Analytical Procedures...................................... 8164
Products................................................................................... 7827
(1225) Validation of CompendiaI Procedures.............................. 8166
(1116) Microbiological Control and Monitoring of Aseptic
Processing Environments.......................................................... 7833 (1226) Verification of CompendialProcedures............................. 8171

(111 7) Microbiological BestLaboratory Practices.................. ....... 7845 (1227) Validation of Microbial Recovery FromPharmacopeial
Articles..................................................................................... 8172
(1118) Monitoring Devices-Time, Temperature, and Humidity.. 7850
(1228) Depyrogenation............................................................... 8176
(1120) RamanSpectroscopy....................................................... 7856
(1228.1) DryHeat Depyrogenation............................................. 8181
(1121) Nomenclature.................................................................. 7862
(1228.3) Depyrogenation by Filtration........................................ 8184
(1125) NucleicAcid-Based Techniques-General........................ 7865
(1228.4) Depyrogenation by Rinsing........................................... 8187
(1126) Nucleic Acid-Based Techniques-Extraction, Detection
and Sequencing....................................................................... 7870 (1228.5) Endotoxin Indicators for Depyrogenation...................... 8190

(1127) Nucleic Acid-Based Techniques-Amplification................ 7879 (1229) Sterilization of CompendialArticles.................................. 8194

(1128) NucleicAcid-Based Techniques-Microarray.................... 7887 (1229.1) Steam Sterilization by DirectContact............................ 8199

(1129) NucleicAcid-Based Techniques-Genotyping.................. 7893 (1229.2) MoistHeat Sterilization of AqueousLiquids........... 8202

(1229.3) Monitoring of Bioburden.............................................. 8206

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 USP 43 Guide to General Chapters 17

GENERAllNFOIRMATION (continued) GIENIERAllNFOIRMATION (continued)

(1229.4) Sterilizing Filtration of Liquids....................................... 8209 (1664) Assessment of DrugProductLeachables Associated With
Pharmaceutical Packaging/Delivery Systems..... 8455
(1229.5) Biological Indicators for Sterilization.............................. 8216
(1664.1) Orally Inhaled and Nasal Drug Products........................ 8467
(1229.6) Liquid-Phase Sterilization.............................................. 8219
(1724) Semi-Solid Drug Products-PerformanceTests................. 8473
(1229.7) GaseousSterilization..................................................... 8222
(1730) Plasma Spectrochemistry-Theoryand Practice............... 8484
(1229.8) DryHeat Sterilization.................................................... 8225
(1735) X-Ray Fluorescence Spectrometry-Theory and Practice.. . 8490
(1229.9) Physicochemical Integrators and Indicators for
Sterilization '.......... 8227 (1736) Applications of Mass Spectrometry.................................. 8508
(1229.10) Radiation Sterilization................................................. 8228 (1761) Applications of Nuclear Magnetic Resonance
Spectroscopy............................................................................ 8529
(1229.11) VaporPhaseSterilization............................................. 8231
(1771) Ophthalmic Products-Performance Tests....................... 8549
(1229.12) New Sterilization Methods.......................................... 8233
(1782) Vibrational Circular Dichroism Spectroscopy-Theory and
(1229.13) Sterilization-In-Place.................................................... 8233 Practice............................................................ 8549
(1229.14) Sterilization Cycle Development.................................. 8235 (1787) Measurementof Subvisible Particulate Matter In
TherapeuticProtein Injections.................................................. 8562
(1229.15) Sterilizing Filtration of Gases....................................... 8238
(1788) Methodsfor the Determination of Particulate Matterin In-
(1229.16) Prion Inactivation '......................... 8239 jectionsand OphthalmicSolutions........................................... 8575
(1230) Waterfor Hemodialysis Applications 8240 (1790) Visual Inspection of Injections 8587
(1231) Waterfor Pharmaceutical Purposes........... &242 (1821) Radioactivity-Theory and Practice............................ 8604
(1234) Vaccines for HumanUse-Polysaccharide and (1823) Positron Emission TomographyDrugs·lnformation.......... 8617
Glycoconjugate Vaccines.......................................................... 8276
(1850) Evaluation of Screening Technologies for Assessing Medi-
(1235) Vaccines for Human Use-General Considerations........... 8291 cine Quality.............................................................................. 8626
(1236) Solubility Measurements.................................................. 8306 (1852) Atomic Absorption Spectroscopy-Theory and Practice... 8632
(1237) Virology Test Methods..................................................... 8319 (1853) Fluorescence Spectroscopy-Theory and Practice............ 8641
(1238) Vaccines for HumanUse-Bacterial VaccineS................... 8338 (1854) Mid-Infrared Spectroscopy-Theory and Practice............. 8650
(1240) Virus Testing of Human Plasma for FurtherManufacture.. 8350 (1856) Near-Infrared Spectroscopy-Theory and Practice........... 8658
(1241) Water·Solid Interactions in Pharmaceutical Systems......... 8359 (1857) Ultraviolet-Visible Spectroscopy-Theory and Practice..... 8670
(1251) Weighing on an Analytical Balance.................................. 8363 (1911) Rheometry....................................................................... 8679
(1265) Written Prescription Drug Information-Guidelines.......... 8368
(1285) Preparation of Biological Specimens for Histologic and lin- DIETARY SUPPLEMENTS
munohistochemical Analysis..................................................... 8370 (2021) Microbial Enumeration Tests-Nutritional and Dietary
Supplements 8687
(12~5.1) Her:natoxyl!n a~d Eosin Staining Of SectionedTIssue for
Microscopic Examination......................................................... 8374 (2022) Microbiological Procedures for Absence of Specified
MicroorganismS-Nutritional and Dietary Supplements........... 8692
(1430) Analytical Methodologies Based on ScatteringPhenomena
-General................................................................................. 8376 (2023) Microbiological Attributes of Nonsterile Nutritional and
Dietary Supplements 8698
(1430.1) Analytical Methodologies Based on Scattering Phenom-
ena-Static Light Scattering..................................................... 8379 (203.0~Supplemental Information for Articles of Botanical
Origin 8702
(1430.2) Analytical Methodologies Based on ScatteringPhenom-
ena-Light Diffraction Measurements of Particle Size............... 8386 (2040) Disintegration and Dissolution of Dietary Supplements... 8711
(1403.4) Analytical Methodologies Based on Scattering Phenom- (2091) WeightVariation of Dietary Supplements......................... 8718
ena-Electrophoretic Light Scattering (Determination of Zeta
Potential) :................................ 8389 (2232) Elemental Contaminantsin Dietary Supplements............. 8719
(1430.5) Analytical Methodologies Based on ScatteringPhenom- (2250) Detectionof Irradiated Dietary Supplements.................... 8722
ena-Smail Angle X-Ray Scattering and Small Angle Neutron
Scattering................................................................................. 8392 (2251) Screening for Undeclared Drugs and DrugAnalogues ..... 8725
(1467) Residual Solvents-Verification of Compendial Procedures (2750) Manufacturing Practices for Dietary Supplements............ 8742
and Validation of Alternative Procedures................................... 8404
(1601) Productsfor Nebulization-Characterization Tests 8407
(1602) Spacersand Valved Holding ChambersUsedWith
Inhalation Aerosols-Characterization Tests.............................. 8411
(1644) Theoryand Practice of Electrical Conductivity
Measurements of Solutions................................................ ....... 8423
(1660) Evaluation of The InnerSurface Durability Of Glass
Containers.................. 8429
(1661) Evaluation of Plastic Packaging Systems and Their
Materials of Construction WithRespect to TheirUser Safety lm-
pact......................................................................................... 8434
(1663) Assessment of Extractables Associated With
Pharmaceutical Packaging/Delivery Systerns.i., 8442

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www.ilovepharma.com
USP 43 Official Monographs / Abacavir 19

• •
I I I

Analysis
Abacavir Oral Solution Samples: Standard solution and Sample solution
DEFINITION Calculate the percentage of C14H1SN60 in the portion of
Abacavir Oral Solution contains NLT90.0% and NMT 110.0% Oral Solution taken:
of the labeled amount of abacavir (C14H1SN60).
Result = (ru/rs) x (Cs/Cu) x (Mr,/Mrz) x 100
IDENTIFICATION
• The retention time of the major peak of the Sample solution = peak area of abacavir from the Sample solution
corresponds to that of the Standard solution, as obtained in = peak area of abacavir from the Standard solution
the Assay. = concentration of USP Abacavir Sulfate RS in the
Standardsolution(mg/mL)
ASSAY = nominal concentration of abacavir in the Sample
• PROCEDURE solution (mg/mL)
Solution A: Trifluoroacetic acid and water (0.05: 99.95) = molecular weight of abacavir mutiplied by 2,
Solution B: Methanol and water (17:3) 572.66
Diluent: 1 ml of phosphoric acid diluted with water to 1000 = molecular weight of abacavir sulfate, 670.74
mL
Mobile phase: See the gradient table below. Acceptance criteria: 90.0%-110.0%
Time SolutionA Solution B PERFORMANCE TESTS
(min) (%) (0/0) • DELIVERABLE VOLUME (698): Meets the requirements
0 95 5 IMPURITIES
ORGANIC IMPURITIES
20 70 30
• Procedure
35 10 90 Solution A, Solution B, Diluent, Mobile phase, System
40 10 90 suitability solution, Standard solution, Sample solution,
Chromatographic system, and System suitability:
41 0 100 Proceed as directed in the Assay.
50 0 100 Sensitivity solution: 0.2 IJg/mL of USP Abacavir Sulfate RS in
Diluent, from the Standard solution. [NOTE-The
51 95 5 concentration of this solution is 0.05% of the nominal
55 95 5 concentration of the Sample solution.]
Analysis
Samples: Diluent, Standard solution, Sample solution, and
System suitability solution: 0.2 mg/mL of USP Abacavir Sensitivity solution. [NoTE-In the Sample solution disregard
System Suitability Mixture RS in Diluent- any peaks corresponding to peaks identified in the Diluent
Standard solution: 0.46 mg/mL of USP Abacavir Sulfate RS and any peak with a peak area less than the abacavir peak
in Diluent area in the Sensitivity solution.]
Sample solution: Equivalent to 0.4 mg/mL of abacavir in Calculate the percentage of each impurity in the portion of
Diluent, from Oral Solution. [NoTE-Sonicate, if necessary.] Oral Solution taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = (ru/rs) x (Cs/Cu) x (1 IF) x (Mr1/ Mrz) x 100
Mode: LC
Detector: UV254 nm = peak area of abacavir from the Sample solution
Column: 3.9-mm x 15-cm; 5-lJm packing L1
Column temperature: 30°
=peak area of abacavir from the Standard solution
= concentration of USP Abacavir Sulfate RS in the
Flow rate: 0.8 mL/min Standard solution(mg/mL)
Injection size: 10 f.JL = nominal concentration of abacavir in the Sample
System suitability . solution (mg/mL)
Samples: System suitability solution and Standard solution F = relative response factor for each impurity from
Suitability requirements Impurity Table 7
Resolution: NLT1.5 between abacavir and = molecular weight of abacavir mutiplied by 2,
trans-abacavir, System suitability solution 572.66
Relative standard deviation: NMT 2.0%, Standard = molecular weight of abacavir sulfate, 670.74
solution

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20 Abacavir / OfficialMonographs USP 43

Acceptance criteria Table 1 (continued)

Individual impurities: See Impurity Table 7. Time Solution A Solution B
Total impurities: NMT 2.0% (min) (%) (0/0)

41 95 5
Impurity Table 1
Relative Relative Acceptance 50 95 5
Retention Response Criteria,
Name Time Factor NMT(%)
System suitability solution: 0.2 mg/mL of USP Abacavir
Cyclopropyldiaminopur- System Suitability Mixture RS in Diluent
ine abacavtr- 0.57 1.4 0.3 Standard solution: 0.21 mg/mL of abacavir sulfate in
Descyclopropyl abacavtr" 0.68 1.0 0.8 Diluent (equivalent to 0.18 mg/mL of abacavir), from USP
Abacavir Sulfate RS
Abacavir 1.00 - - Sample stock solution: Transfer the equivalent to 1500 mg
trans-Abacavir" 1.04 1.0 - of abacavir, from a portion of Tablets, into a 250-mL
volumetric flask. Add 150 mL of Diluent. Shake
Any individual unspeci- mechanically for 45 min. Dilute with Diluent to volume. Pass
fied impurity . - 1.0 0.2
a portion through a suitable filter of 0.45-lJm or finer pore
a N6-Cyclopropyl-9H-purine-2,6-diamine. size. Discard the first 3 mL of the filtrate.
b [(1 S,4R)-4-(2,6-Diamino-9H-purin-9-yl)cyclopent-2-enyl]methanoI. Sample solution: 0.18 mg/mL of abacavir in Diluent using
c {(1R,4R)-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-cyc1opent-2-enyl) the filtrate obtained in the Sample stock solution
methanol. It is a process impurity and monitored in the drug substance. Chromatographic system
(See Chromatography (621), System Suitability.)
SPECIFIC TESTS Mode: LC
o MICROBIAL ENUMERATION TESTS (61) and TESTS FOR Detector: UV 254 nm
SPECIFIED MICROORGANISMS (62): The total aerobic Column: 3.9-mm x 15-cm; packing L1
microbial count does not exceed 100 cfu/mL, and the total Flow rate: 0.8 mL/min
combined molds and yeast count does not exceed 10 cfu/ Injection volume: 10 IJL
mL. It also meets the requirement for absenceof Escherichia System suitability
coli. Samples: System suitability solution and Standardsolution
o pH (791): 3.8-4.5 Suitability requirements
Resolution: NLT 1.5 between abacavir and
ADDITIONAL REQUIREMENTS . trans-abacavir, System SUitability solution
o PACKAGING AND STORAGE: Preserve in well-closed Relative standard deviation: NMT 2.0%, Standard
containers. Store at controlled room temperature. solution
o USP REFERENCE STANDARDS (11) Analysis
USP Abacavir Sulfate RS Samples: Standardsolution and Sample solution
USP Abacavir System Suitability Mixture RS Calculate the percentage of the labeled amount of
A mixture containing abacavlr sulfate and 1rans-abacavir abacavir (C14H1SN60) in the portion of Tablets taken:

Result = (ru/rs) x (Cs/Cu) x (M,rlM'2) x 100

Abacavlr Tablets ru = peak response of abacavir from the Sample
solution
DEFINITION rs = peak response of abacavir from the Standard
Abacavlr Tablets contain Abacavir Sulfate equivalent to NLT solution
90.0% and NMT 110.0% of the labeled amount of abacavir C, =concentration of abacavlr sulfate in the Standard
(C14H1SN60).
solution (mg/mL)
Cu = nominal concentration of abacavir in the Sample
IDENTIFICATION solution (mg/mL)
o A. The retention time of the major peak of the Sample M'I = molecular weight of abacavir multiplied by 2,
solution corresponds to that of the Standardsolution, as 572.66
obtained in the Assay. M'2 = molecular weight of abacavir sulfate, 670.74
ASSAY Acceptance criteria: 90.0%-110.0%
o PROCEDURE
Diluent: 1.0 mL of phosphoric acid in 1 L of water PERFORMANCE TESTS
Solution A: Trifluoroacetic acid and water (0.05: 99.95) o DISSOLUTION (711)
Solution B: Methanol and water (85:15) Medium: 0.1 N hydrochloric acid; 900 mL
Mobile phase: See Table 7. Apparatus 2: 75 rpm
Time: 15 min
Table 1 Standard solution: 0.39 mg/mL of USP Abacavir Sulfate RS
Time Solution A Solution B in Medium
(min) (%) (0/0) Sample solution: Pass a portion of the solution under test
through a suitable filter of 0.45-lJm pore size.
0 95 5
Instrumental conditions
20 70 30 Mode: UV
Analytical wavelength: 254 nm
35 10 90
Blank: Medium
40 10 90

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USP 43 OfficialMonographs / Abacavir 21

Calculate the percentage of the labeled amount of Table 2 (continued)

abacavir (C14H1SN60) dissolved: Relative Relative Acceptance
Retention Response Criteria,
Result =(AulAs) x (Csf L) x (M,d M,2) x V x 100 Name Time Factor NMT (%)
Total impurities - - 1.0
Au =absorbance of the Sample solution
As =absorbance of the Standard solution a N6-Cydopropyl-9H-purine-2,6-diamine.
c, =concentration of the Standard solution(mg/mL) b [(1S,4R)-4-(2,6-Diamino-9H-purin-9-yl)-cyclopent-2-enyl]methano!.
L = label claim (mg/Tablet) c {(1 R,4R)-4-[2-Amino-6-(cyciopropylamino)"9H-purin-9-yl]-cyciopent-2-enyl)
methanol.
M" = molecular weight of abacavir multiplied by 2, d Processimpuritymonitored in the drug substance and not included in the total
572.66 impurities.
= molecular weight of abacavir sulfate, 670.74 e N6-Cyclopropyl-9-{(1 R,4S)-4-[(2,5-diamino-6-chloropyrimidin-4-yloxy)
=volume of Medium, 900 mL methyl]cyclopent-2-enyl}-9H-purine-2,6-diamine.

Tolerances: NLT 80% (Q) of the labeled amount of abacavir ADDITIONAL REQUIREMENTS
(C14H1SN60) is dissolved. • PACKAGING AND STORAGE: Preserve in well-closed
• UNIFORMITY OF DOSAGE UNITS (90S): Meet the containers. Store at room temperature.
requirements • USP REFERENCE STANDARDS (11)
USP Abacavir Sulfate RS
IMPURITIES USP Abacavir System Suitability Mixture RS
• ORGANIC IMPURITIES -A mixture of abacavir sulfate and trans-abacavir.
Diluent, Solution A, Solution B, Mobile phase, System
suitability solution, Standard solution, Sample solution,
and Chromatographic system: Proceed as directed in the
Assay.
Analysis Abacavir and Lamivudine Tablets
[Nora-Record the chromatograms for 2.5 times the
retention time of abacavir.] DEFINITION
Samples: Standard solution and Sample solution Abacavirand Lamivudine Tablets contain an amount of
Calculatethe percentage of each impurityin the portion abacavir sulfate and lamivudine equivalent to NLT 90.0%
of Tablets taken: and NMT 110.0% of the labeled amount of abacavir
(C14H1SN60) and NLT 90.0% and NMT 110.0% of the
Result = (rulr s) x (CslCu) x (1 IF) x (M,dM'2) x 100 labeled amount of lamivudine (CSH llN 303S), respectively.
ru = peak response of each impurityfrom the Sample IDENTIFICATION
solution • A. The retention times of the major peaks of the Sample
rs = peak response of abacavir from the Standard solution correspond to those of the Standard solution, as
solution obtained in the Assay.
Cs = concentration of USP Abacavir Sulfate RS in the
Standardsolution (mg/mL) ASSAY
Cu = nominal concentration of abacavir in the Sample • PROCEDURE
solution (mg/mL)
Diluent: 0.1 N hydrochloric acid
F = relative response factor for each impurity(see Solution A: Water and trifluoroacetic acid (2000:1)
Table 2) Solution B: Acetonitrile, methanol, and trifluoroacetic acid
M'I = molecular weight of abacavir multiplied by 2, (1000: 1000: 1)
572.66 Mobile phase: See Table 1. [NoTE-Return to original
M'2 = molecular weight of abacavir sulfate, 670.74 conditions and re-equilibrate the system for about 7 min.]
Table 1
Acceptance criteria: See Table 2.
Time Solution A Solution B
(min) (%) (%)
Table 2
Relative Relative Acceptance 0 100 0
Retention Response Criteria,
Name Time Factor NMT (%) 4 100 0

Cyclopropyldiaminopurine 12 70 30
abacavir" 0.57 1.4 0.2 12.1 40 60
Descyclopropyl abacavlr" 0.68 1.0 0.2 13.1 40 60
Abacavir 1.0 - - 13.2 100 0
trans-Abacavi,.e. d 1.04 - -
o-~rimidine derivative abaca- System suitability solution: Dissolve the contents of one
vi ,e 1.24 - - vial of USP Lamivudine Resolution Mixture C RS in 2.5 mL
of Diluent. [NoTE-One vial of USP Lamivudine Resolution
Anyother individual impurity - 1.0 0.2 Mixture C RS contains 0.8 mg of USP Lamivudine
Resolution Mixture C RS.]
Standard solution: 0.35 mg/mL of USP Abacavir Sulfate RS
and 0.15 mg/mL of USP Lamivudine RS in Diluent. Sonicate
to dissolve prior to final dilution.

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22 Abacavir / OfficialMonographs USP 43

Sample stock solution: Nominally 3 mg/mL of abacavirand PERFORMANCE TESTS

1.5 mg/mL of lamivudine in Diluent prepared as follows. • DISSOLUTION (711)
Transfer NLT 5 Tablets to a suitable volumetricflask. Add Medium: 0.1 N hydrochloric acid; 900 mL
Diluent to about 50% ofthe final volumeand shakefor NMT Apparatus 2: 75 rpm
30 min to disperse the Tablets. Dilute with Diluent to Time: 30 min
volume. Passthrough a suitablefilter. Standard solution 1: 0.79 mg/mL of USP Abacavir Sulfate
Sample solution: Nominally 0.3 mg/mL of abacavlr and RS in Medium. Sonicate to dissolve prior to final dilution.
0.15 mg/mL of lamivudine in Diluent from Sample stock Standard solution 2: 0.33 mg/mL of USP Lamivudine RS in
solution Medium. Sonicate to dissolve prior to final dilution.
Chromatographic system Sample solution: Pass a portion of the solution under test
(See Chromatography (621), System SUitability.) through a suitable filter.
Mode: LC Instrumental conditions
Detector: UV 270 nm Mode: UV
Column: 4.6-mm x 15-cm; 3.5-J.Jm packing L1 Wavelength: 240-320 nm
Column temperature: 40° Cel/length: 0.2 mm
Flow rate: 1.5 mL/min Blank: Medium
Injection volume: 10 J.JL Analysis: The calculations of the percentages dissolved are
System suitability performed using multi-component analysis software.
Samples: System suitability solution and Standardsolution Tolerances: NLT 80% (Q) of the labeled amount of abacavir
[NOTE-The relative retention times for and lamivudine is dissolved.
lamivudine-S-oxide and lamivudine-R-oxide, in • UNIFORMITY OF DOSAGE UNITS (905): Meet the
relation to the lamivudine peak, are 0.31 and 0.36, requirements
respectively; the relative retention times for
lamivudine diastereomer and lamivudine are 0.88 IMPURITIES
and 1.0, respectively; System suitabilitysolution.] • ORGANIC IMPURITIES
Suitability requirements Diluent, Solution A, Solution B, Mobile phase, System
Resolution: NLT 1.0 between lamivudine-S-oxide and suitability solution, Standard solution, Sample stock
lamivudine-R-oxide; NLT 1.0 between lamivudine solution, Sample solution, Chromatographic system,
diastereomer and lamivudine, System suitability solution and System suitability: Proceed as directed in the Assay.
Relative standard deviation: NMT 1.5% each for Analysis
abacavir and lamivudine, Standardsolution Sample: Sample solution
Analysis Calculatethe percentage of each individual abacavirrelated
Samples: Standard solution and Sample solution impurity and each unspecified impurity in the portion of
Calculatethe percentage of the labeled amount of abacavir Tablets taken:
(C14H1SN60) in the portion of Tablets taken: Result = (rulrr) x 100
Result = (rulrs) x (CsICu) x (MrdMr:z.) x 100 ru = peak response of each abacavir related impurity
tu = peak response of abacavirfrom the Sample
or unspecified impurity· .
solution rr = sum of the peak responses of abacavir, allabacavir
ts = peak response of abacavirfrom the Standard
related impurities, and all unspecified impurities
solution Calculatethe percentage of each lamivudine related
Cs = concentration of USP Abacavir Sulfate RS in the
impurity in the portion of Tablets taken:
Standardsolution (mg/mL)
Cu = nominal concentration of abacavir in the Sample Result = (rulrr) x 100
solution (mg/mL)
Mr7 = molecular weight of abacavir multiplied by 2, t» = peak response of each lamivudine related
572.66 impurity
Mr2 = molecular weight of abacavirsulfate, 670.74 rr = sum of the peak responses of lamivudine and all
lamivudine related impurities
Acceptance criteria: 90.0%-110.0% of the labeled amount
of abacavir Acceptance criteria: See Table 2. Disregard any peak less
Calculate the percentage of the labeled amount of than 0.05%.
lamivudine (CSH llN 303S) in the portion of Tablets taken:
Table 2
Result = (rulrs) x (CslCu) x 100 Relative Acceptance
Retention Criteria,
tu = peak response of lamivudine from the Sample Name Time NMT(%)
solution
Cytosine" 0.12 0.2
rs = peak response of lamivudine from the Standard
solution l.amlvudlne-Seultoxlde'' 0.19 0.2
Cs = concentration of USP Lamivudine RS in the
Lamlvudlne-z-sultoxlde- 0.21 0.2
Standardsolution (mg/mL)
Cu = nominal concentration of lamivudine in the Lamivudine-carboxylic acld" 0.49 _e
Sample solution (mg/mL)
Lamivudine diastereomer(Larnivu- _e
dlne-rrons)' 0.52
Acceptance criteria: 90.00/0-110.0% of the labeled amount
of lamivudine Lamivudine 0.60 -
Lamivudine-uracil derlvatives 0.78 0.2

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USP 43 OfficialMonographs / Abacavir 23

Table 2 (continued)
Relative Acceptance
Abacavir Sulfate
Retention Criteria,
Name Time NMT(%)
cyclopro~yldiaminopurine
abacavi 0.80 0.2
Descyclopropyl abacavlr' 0.85 0.2
3-Hydroxyabacavi~ 0.89 _e (C14H1SN60)2' H2S04 670.74
_e
2-Cyclopentene-1-methanol, 4-[2-amino-6-(cyclopropyl
Abacavir 1.0 amino)-9H-purin-9-yl]-, (1 S-ds)-, sulfate (salt) (2:1);
Anyindividual unspecified (1S,4R)-4-[2-Amino-6-(cyclopropylamino)-9 H-purin-9-yl]-
impurity - 0.2 2-cyclopentene-1-methanol sulfate (salt) (2:1) [188062-
Totallamivudinerelated 50-2].
Irnpurltles- - 0.6
DEFINITION
Total abacavirrelated Abacavir Sulfate contains NLT 97.0% and NMT 102.0% of
lrnpurltles' - 1.0 (C14H1SN60h· H2S04, calculated on the anhydrous and
solvent-free basis.
a4-Aminopyrimidin-2(1 H)-one(Iamivudine related impurity).
b 1-[(2R,3S,SS)-2-(Hydroxymethyl)-1, 3-oxathiolan-S-yl]cytosine S-oxide. IDENTIFICATION
c 1-[(2R,3R,SS)-2-(Hydroxymethyl)-1 ,3-oxathiolan-S-yl]cytosine S-oxide.
d (2RS,SSR)-S-(Cytosine-l-yl)-1 ,3-oxathiolane-2-carboxylic acid.
e Process impuritymonitored in the drug substance.
f 1-[(2S,SS)-2-(Hydroxymethyl)-1 ,3-oxathiolan-S-yl]cytosine.
9 1-[(2RS,SSR)-2-(Hydroxymethyl)-1 ,3-oxathiolan-S-yl]uracil. , ..)
h N6-Cyclopropyl-9H.purine-2,6-diamine. • B. The retention time of the major peak of the Sample
i [(lS,4R)-4-(2,6-Diamino-9H-purin-9-yl)cyclopent-2-enyl]methanol.
solution corresponds to that of the System sUitability
j (1R,2R,4 S)-2-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-4-
(hydroxymethyl)cyclopentan-1-ol. solution, obtained as directed in the test for Organic
kIncludes alliamivudinerelated impurities. Impurities, Procedure 2.
'Includes allabacavirrelatedand all individual unspecified impurities. • C. IDENTIFICATION TESTS-GENERAL, Sulfate (191)
Sample solution: 5 mg/mL
ADDITIONAL REQUIREMENTS ASSAY
• PACKAGING AND STORAGE: Preserve in tight, light-resistant • PROCEDURE
containers. Store at controlled room temperature. Mobile phase: Acetonitrile, phosphoric acid, and water
• USP REFERENCE STANDARDS (11) (20:1 :180)
USP Abacavir Sulfate RS Standard solution: 0.04 mg/mL of USP Abacavir Sulfate RS
USP Lamivudine RS in water
USP Lamivudine Resolution Mixture C RS Sample solution: 0.04 mg/mL of Abacavir Sulfate in water
This is a mixture of lamivudine and the following Chromatographic system
impurities (other impurities may also be present). (See Chromatography (621), System Suitability.)
Uracil: Pyrimidine-2,4(1 H,3H)-dione. . Mode: LC
C4H4N202 112.09 Detector: UV 254 nm
Lamivudine-uracil derivative: 1-[(2RS,5SR)-2- Column: 4.6-mm x 5-cm; 5-~m packing L1
(Hydroxymethyl)-1,3-oxathiolan-5-yl]uracil. Column temperature: 30°
CSH lON 204S 230.24 Flow rate: 1 mL/min
Cytosine: 4-Aminopyrimidin-2(1 H)-one. Injection size: 20 ~L
C4HsN30 111.10 System suitability
Lamivudine-S-sulfoxide: 1-[(2R,3S,5S)-2- Sample: Standard solution
(Hydroxymethyl)-1 ,3-oxathiolan-5-yl]cytosine S-oxide. Suitability requirements
CSH11N 304S 245.26 Relative standard deviation: NMT 1.5%
Lamivudine-R-sulfoxide: 1-[(2R,3R,5S)-2- Analysis
(Hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine S-oxide. Samples: Standardsolution and Sample solution
CSH llN 304S 245.26 Calculate the percentage of (C14H1SN60)2 . H2S04 in the
Lamivudine carboxylic acid: (2RS,5SR)-5-(Cytosine-1-yl)- portion of Abacavir Sulfate taken:
1,3-oxathiolane-2-carboxylic acid.
CSH 9N 304S 243.24 Result = (ru/rs) x (Cs/Cu) x 100
Lamivudine diastereomer: 1-[(2S,5S)-2-(Hydroxymethyl)-
1,3-oxathiolan-5-yl]cytosine. = peak area of abacavir from the Sample solution
CSHllN303S 229.26 = peak area of abacavir from the Standard solution
Salicylic acid: 2-Hydroxybenzoic acid. =concentration of USP Abacavir Sulfate RS in the
C7H 603 138.12 Standard solution (mg/mL)
Cu =concentration of Abacavir Sulfate in the Sample
solution (mg/mL)

Acceptance criteria: 97.0%-102.0% on the anhydrous and

solvent-free basis

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24 Abacavir / Official Monographs USP 43

IMPURITIES Impurity Table 1 (continued)

INORGANIC IMPURITIES Relative Acceptance
• Residue on Ignition (281): NMT 0.2% Retention Criteria,
ORGANIC IMPURITIES Name Time NMT(%)
• Procedure 1: Related Compounds Anyunspecified impurity - 0.1
Solution A: Trifluoroacetic acid and water (0.05: 99.95)
Solution B: Methanol and water (17:3) a [(1S,4R)-4-(2,6-Diamino-9H-purin-9-yl)cyclopent-2-enyl]methanoI.
Mobile phase: See the gradient table below. b {(1 R,4R)-4-[2-Amino-6-(cyclopropylamino)-9H-purin-9-yl]-cyclopent-2-enyl}
methanol.
c N6-Cyclopropyl-9-{(1 R,4S)-4-[(2,5-diamino-6-chloropyrimidin-4-yloxy)
Time Solution A Solution 8 methyl]cyclopent-2-enyl}-9H-purine-2,6-diamine.
(min) (%) (%) d 9-[(1 R,4S)-4-( tert-Butoxymethyl)cyclopent-2-enyl]-N6-cyclopropyl-9 H-
0 95 5 purine-2,6-diamine.

20 70 30 • Procedure 2: Enantiomeric Purity

35 10 90 Solution A: Heptane, 2-propanol, and diethylamine
(850:150:1 ).
35.1 95 5 Solution B: Heptane and 2-propanol (1:1)
50 95 5 Mobile phase: See the gradient table below.

System suitability solution: 0.25 mg/ml of USP Abacavir Time Solution A Solution 8 Flow Rate
(min) (%) (%) (ml/min)
Related Compounds Mixture RS in water
Sample solution: 0.25 mg/ml of Abacavir Sulfate in water 0 100 0 1.0
Chromatographic system
25 100 0 1.0
(See Chromatography (621), System Suitability.)
Mode: lC 27 0 100 0.8
Detector: UV 254 nm 37 0 100 0.8
Column: 3.9-mm x 15-cm; 5-J..Im packing II
Column temperature: 30° 39 100 0 1.0
Flow rate: 1 ml/min 55 100 0 1.0
Injection size: 20 J..Il
System suitability
Sample: System suitability solution Diluent: Methanol and trifluoroacetic acid (200: 1)
Suitability requirements System suitability solution: Transfer a quantity of USP
Resolution: NlT 1.5 between abacavir and trans-abacavir Abacavir Stereoisomers Mixture RS to a suitable volumetric
Analysis flask, add a volume of Diluent equivalent to 30% of the final
Sample: Sample solution volume, and sonicate until the solid is fully dissolved. Add a
Calculate the percentage of each impurity in the portion of volume of 2-propanol equivalent to about 30% of the final
Abacavir Sulfate taken: volume, mix, and dilute with heptane to volume to obtain
0.4 mg/ml of USP Abacavir Stereoisomers Mixture RS.
Result = (ru/rr) x 100 Sample solution: Transfer 4 mg of Abacavir Sulfate to a
1O-ml volumetric flask. Add 3 ml of Diluent, and sonicate
ru =peak area of each impurity from the Sample until the solid isfullydissolved. Add 3 ml of 2-propanol, mix,
solution and dilute with heptane to volume.
rr = sum of the areas of all the peaks from the Sample Chromatographic.system
solution (See Chromatography (621), System Suitability.)
Mode: lC
Acceptance criteria Detector: UV 286 nm
Individual impurities: See Impurity Table 1. Column: 4.6-mm x 25-cm; 10-J..Im packing l51
Total impurities: NMT 0.8% Column temperature: 30°
Injection size: 20 J..Il
Impurity Table 1 System suitability
Relative Acceptance [NOTE-The relative retention times for trans-abacavir,
Retention Criteria, abacavir enantiomer, and abacavir are 0.8, 0.9, and 1.0,
Name Time NMT(%) respectively.]
Descyclopropyl abacavir' 0.65 0.2 Sample: System suitabilitysolution
Suitability requirements
Abacavir 1.00 - Resolution: NlT 1.0 between trans-abacavir and abacavir
trans-Abacavirb 1.04 0.2 enantiomer; NlT 1.5 between abacavir enantiomer and
abacavir
O-Pyrimidine derivativeabacavlr" 1.33 0.2 Analysis
t-Butyl derivativeabacavird 1.67 0.2 Sample: Sample solution
Calculate the percentage of abacavir enantiomer in the
portion of Abacavir Sulfate taken:
Result = (ru/rr) x 100
ru =peak area of abacavir enantiomer from the Sample
solution

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 USP 43 Official Monographs / Abiraterone 25

= total peak areas of abacavir and abacavir Table 1 (continued)

enantiomer from the Sample solution Time Solution A Acetonitrile Ethanol
(min) (%) (%) (%)
Acceptance criteria
70 50 20 30
Individual impurities: NMT 0.3% of abacavir enantiomer
SPECIFIC TESTS [NOTE-Protect solutions from light.]
• WATER DETERMINATION, Method Ie (921): NMT 0.5% System suitability solution: 0.625 mg/mL of USP
ADDITIONAL REQUIREMENTS Abiraterone System SUitability Mixture RS in acetonitrile.
• PACKAGING AND STORAGE: Preserve in well-closed [NOTE-See Table 2 for relative retention times of the main
containers. Store at room temperature. components of the mixture.]
• USP REFERENCE STANDARDS (11)
USPAbacavir Sulfate RS Table 2
USP Abacavir Stereoisomers Mixture RS Relative
A mixture of abacavir sulfate, abacavir enantiomer, and Retention
Name Time
trans-abacavir.
USPAbacavir Related Compounds Mixture RS 7-Ketoabiraterone acetate 0.42
A mixture of abacavir glutarate, O-pyrimidine derivative u-Epoxyabiraterone acetate 0.62
abacavir, descyclopropyl abacavir, trans-abacavir, and
t-butyl derivative abacavir. ~-Epoxyabiraterone acetate 0.66
Ablraterone 0.69
3-Deoxy-3-acetyl abiraterone-3-ene 0.85
Abiraterone acetate 1.0
Ablraterone Acetate
Abiraterone ethyl ether 1.18
Abiraterone isopropyl ether 1.26
Anhydro abiraterone 1.29
3-Deoxy 3-chloroabiraterone 1.31
O.Chlorobutylabiraterone 1.33

C26H33N02 391 .55 Standard solution: 0.625 mg/mL of USP Abiraterone

Androsta-5, 16-dien-3-01, 17-(3-pyridinyl)-, acetate (ester), Acetate RS in acetonitrile
(3~); Sample solution: 0.625 mg/mL of Abiraterone Acetate in
17-(Pyridin-3-yl)androsta-5, 16-dien-3~-yl acetate [154229- acetonitrile
18-2]. Chromatographic system
(See Chromatography (621), System Suitability.)
DEFINITION Mode: LC
Abiraterone Acetate contains NLT 98.0% and NMT 102.0% of Detector: UV 254 nm
abiraterone acetate (C26H nN02), calculated on the as-is Column: 3-mm x 15-cm; 3-l.Im packing L1
basis. Column temperature: 15°
IDENTIFICATION Flow rate: 0.45 mL/min
Injection volume: 10 I.IL
System suitability
Samples: System suitabilitysolution and Standard solution
• A.~~~~~Sl'~~~~(J~i Suitability requirements
SPlFctrosc()PY:.l?7~J..(cN,.•May''':.!U;/:U) Resolution: NLT 1.0 between anhydro abiraterone and
• B. The retention time of peak of the Sample 3-deoxy 3-chloroabiraterone peaks, System suitability
solution corresponds to that of Standard solution, as solution
obtained in the Assay. Relative standard deviation: NMT 0.73%, Standard
solution
ASSAY Analysis
• PROCEDURE Samples: Standardsolution and Sample solution
Solution A: 10 mM of ammonium acetate in water Calculate the percentage of abiraterone acetate
Mobile phase: See Table 1. (Cz6H33NOz) in the portion of Abiraterone Acetate taken:
Table 1 Result = (rulrs) x (CsICu) x 100
Time Solution A Acetonitrile Ethanol
(min) (%) (%) (%) to = peak response from the Sample solution
0 50 20 30 rs = peak response from the Standard solution
Cs = concentration of USP Abiraterone Acetate RS in
40 15 55 30
the Standard solution(mg/mL)
47 0 20 80 Cu = concentration of Abiraterone Acetate in the
Sample solution (mg/mL)
58 0 20 80
60 50 20 30 Acceptance criteria: 98.0%-102.0% on the as-is basis

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26 Abiraterone / Official Monographs USP 43

IMPURITIES ADDITIONAL REQUIREMENTS

• RESIDUE ON IGNITION (281): NMT 0.1 % • PACKAGING AND STORAGE: Preserve in well-closed
• ORGANIC IMPURITIES containers. Store at controlled room temperature and
[NOTE-Protect solutions from light.] protect from light.
Solution A, Mobile phase, System suitability solution, • USP REFERENCE STANDARDS (11)
Standard solution, Sample solution, and USP Abiraterone Acetate RS
Chromatographic system: Proceed as directed in the USPAbiraterone System Suitability Mixture RS
Assay. It contains Abiraterone Acetate and small amounts of the
Sensitivity solution: 0.3 J.Jg/mL of USP Abiraterone Acetate following:
RS in acetonitrile, from Standard solution Abiraterone
System suitability 17-(pyridin-3-yl)androsta-5,16-dien-3p-ol.
Samples: System suitability solution, Standard solution, and C24H31NO 349.52
Sensitivity solution Abiraterone ethyl ether
Suitability requirements 3p-Ethoxy-17-(pyridin-3-yl)androsta-5,16-diene.
Resolution: NLT 1.0 between anhydro abiraterone and C26H3SNO 377.57
3-deoxy 3-chloroabiraterone peaks, System suitability Abiraterone isopropyl ether
solution 3p-lsopropoxy-17-(pyridin-3-yl)androsta-5,16-diene.
Signal-to-noise ratio: NLT 10, Sensitivity solution C27H37NO 391.60
Relative standard deviation: NMT 0.73%, Standard Anhydro abiraterone
solution 17-(Pyridin-3-yl)androsta-3,5,16-triene.
Analysis C24H29N 331.50
Samples: Standard solution and Sample solution O-Chlorobutylabiraterone
Calculate the percentage of each impurity in the portion of 3P-(4-Chlorobutoxy)-17-(pyridin-3-yl)androsta-5,16-
Abiraterone Acetate taken: diene.
C2sH3sCINO 440.07
Result =(rulrs) x (CslCu) x (1IF) x 100
3-Deoxy-3-acetyl abiraterone-3-ene
to = peak area of each impurity from the Sample 1-[17-(Pyridin-3-yl)androsta-3,5, 16-trien-3-yl]ethanone.
solution C26H31NO 373.53
rs = peak area of abiraterone acetate from the 3-Deoxy 3-chloroabiraterone
3p-Chloro-17-(pyridin-3-yl)androsta-5,16-diene.
Standard solution
Cs = concentration of USP Abiraterone Acetate RS in C24H30ClN 367.96
the Standard solution (mg/mL) a-Epoxyabiraterone acetate
Cu = concentration of Abiraterone Acetate in the 17-(Pyridin-3-yl)-16a,17a-epoxyandrost-5-en-3p-yl
Sample solution (mg/mL) acetate.
F = relative response factor for each individual C26H33N03 407.55
impurity (see Table 3) P-Epoxyabiraterone acetate
1 7-(Pyridin-3-yl)-16P,17p-epoxyandrost-5-en-3p-yl
Acceptance criteria: See Table 3. Disregard any peak less acetate.
than 0.05%. C26H33N03 407.55
7-Ketoabiraterone acetate
Table 3 7-0xo-17-(pyridin-3-yl)androsta-5, 16-dien-3p-yl acetate.
I Relative Relative Acceptance C26H31N03 405.54
Retention Response ,Criteria,
Name Time Factor NMT (%)
a-Epoxyabiraterone
acetate 0.62 0.26 0.25
~-Epoxyabiraterone
Abiraterone Acetate Tablets
acetate 0.66 0.26 0.25
DEFINITION
Abiraterone 0.69 1.0 0.20 Abiraterone Acetate Tablets contain NLT90.0% and NMT
Abiraterone
110.0% of the labeled amount of abiraterone acetate
acetate 1.0 - - (C26H33N02)'
Abirateroneethyl IDENTIFICATION
ether 1.18 1.0 0.20 • A. The retention time of the major peak of the Sample
Abiraterone solution corresponds to that of the Standard solution, as
isopropyl obtained in the Assay.
ether 1.26 1.0 0.20 • B. The UV spectrum of the major peak of the Sample
Unspecified solution corresponds to that of the Standard solution, as
impurity - 1.0 0.10 obtained in the Assay.
Total impurities - - 0.80 ASSAY
• PROCEDURE
SPECIFIC TESTS Solution A: 10 mM of ammonium acetate in water
• WATER DETERMINATION, Method Ic (921): NMT 0.25% Mobile phase: See Table 1.

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USP 43 Official Monographs / Abiraterone 27

Table 1 Calculate the percentage of the labeled amount of

Time Solution A Acetonitrile Ethanol abiraterone acetate (Cz6H nNO z) in the portion of Tablets
(min) (%) (%) (%) taken:
0 50 20 30
Result = (rulr s) x (CsICu) x 100
40 15 55 30
47 0 20 80 t» = peak response from the Sample solution
ts = peak response from the Standardsolution
58 0 20 80 Cs = concentration of USP Abiraterone Acetate RS in
60 50 20 30 the Standardsolution (mg/mL)
Cu = nominal concentration of abiraterone acetate in
70 50 20 30 the Sample solution (mg/mL)

[NOTE-Protect solutions from light.] Acceptance criteria: 90.0%-110.0%

System suitability solution: 0.625 mg/mL of USP
Abiraterone System Suitability Mixture RS in acetonitrile. PERFORMANCE TESTS
• DISSOLUTION (711 >
[NOTE-See Table 2 for relative retention times of the
[NOTE-Protect solutions from light.]
main components of the mixture.]
Buffer: 56.5 mM of monobasic sodium phosphate in water.
Table 2 Adjust with 5 N sodium hydroxide or phosphoric acid to a
pH of 4.5.
Relative Medium: 0.25% of sodium lauryl sulfate in Buffer; 900 mL
Retention
Name Time Apparatus 2: 50 rpm
Time: 45 min
7-Ketoabiraterone acetate 0.42 Standard solution: 0.3 mg/mL of USP Abiraterone Acetate
a-Epoxyabiraterone acetate 0.62 RS in Medium prepared as follows. Transfer USP Abiraterone
Acetate RS into a suitable volumetric flask. Add 4% of the
P-Epoxyabiraterone acetate 0.66 flask volume of acetonitrile to dissolve, and dilute with
"- Medium to volume.
Abiraterone 0.69
Sample solution: Pass a portion of the solution under test
3-Deoxy-3-acetyl abiraterone-3-ene 0.85 through a suitable filter of 10-lJm pore size. Use the filtrate.
Abiraterone acetate 1.0 Mobile phase: Acetonitrile, formic acid, and water (55:
0.05: 45)
Abiraterone ethyl ether 1.18 Chromatographic system
Abiraterone isopropyl ether 1.26 (See Chromatography (621 >, System Suitability.)
Mode: LC
Anhydro abiraterone 1.29
Detector: UV 252 nm
3-Deoxy 3-chloroabiraterone 1'.31 Column: 4.6-mm x 3-cm; 5-lJm packing L1
Flow rate: 1 mL/min
O-Chlorobutylabiraterone 1.33
Injection volume: 10 IJL
System suitability
Standard solution: 0.625 mg/mL of USP Abiraterone Sample: Standardsolution
Acetate RS in acetonitrile Suitability requirements
Sample solution: Nominally equivalent to 0.625 mg/mL of Tailing factor: NMT 2.0
abiraterone acetate in acetonitrile, prepared from NLT20 Relative standard deviation: NMT 2.0%
powdered Tablets as follows. Transfer the powder to a Analysis
suitable volumetric flask. Add 50% of the flask volume of Samples: Standardsolution and Sample solution
acetonitrile, shake by mechanical means for 30 min, and Calculate the percentage of the labeled amount of
dilute with acetonitrile to volume. Pass a portion of the abiraterone acetate (Cz6H33NOz) dissolved:
solution through a suitable filter of 0.45-lJm pore size, and
use the clear solution for analysis. (rufr s) x (CsIL) x Vx 100
Chromatographic system
(See Chromatography (621 >, System Suitability.) t» = peak response from the Sample solution
Mode: LC rs = peak response from the Standardsolution
Detector: UV 254 nm or diode array. [NOTE-Usediode Cs = concentration of the Standard solution (mg/mL)
array detector to perform Identification test B.] L = label claim (mg/Tablet)
Column: 3-mm x 15-cm; 3-lJm packing L1 V = volume of Medium, 900 mL
Column temperature: 150
Flow rate: 0.45 mL/min Tolerances: NLT 85% (Q) of the labeled amount of
Injection volume: 10 IJL abiraterone acetate (Cz6H33NOz) is dissolved.
System suitability . • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Samples: System suitability solution and Standardsolution requirements
Suitability requirements
Resolution: NLT 1.0 between anhydro abiraterone and IMPURITIES
3-deoxy 3-chloroabiraterone peaks, System suitability • ORGANIC IMPURITIES
solution [NOTE-Protect solutions from light.]
Relative standard deviation: NMT 2.0%, Standard Solution A, Mobile phase, System suitability solution,
solution Standard solution, Sample solution, and
Analysis Chromatographic system: Proceed as directed in the
Samples: Standardsolution and Sample solution Assay.

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 28 Abiraterone / Official Monographs USP43

Sensitivity solution: 0.3 J,Jg/ml of USP AbirateroneAcetate 17-(Pyridin-3-yl)androsta-5,16-dien-3p-ol.

RS in acetonitrile from Standard solution Cz4H31NO 349.52
System suitability Abiraterone ethyl ether
Samples: System suitability solution, Standard solution, and 3p-Ethoxy-17-(pyridin-3-yl)androsta-5,16-diene.
Sensitivity solution Cz6H3SNO 377.57
Suitability requirements Abiraterone isopropyl ether
Resolution: NlT 1.0 between anhydro abiraterone and 3p-lsopropoxy-l7-(pyridin-3-yl)androsta-5,16-diene.
3-deoxy 3-chloroabiraterone peaks, System suitability Cz7H37NO 391.60
solution Anhydro abiraterone
Signal-to-noise ratio: NlT 10, Sensitivity solution 17-(Pyridin-3-yl)androsta-3,5,16-triene.
Relative standard deviation: NMT 2.0%, Standard Cz4Hz9N 331.50
solution O-Chlorobutylabiraterone
Analysis 3P-(4-Chlorobutoxy)-17-(pyridin-3-yl)androsta-5,16-
Samples: Standard solution and Sample solution diene.
Calculate the percentage of each impurityin the portion of CZSH3SCINO 440.07
Tablets taken: 3-Deoxy-3-acetyl abiraterone-3-ene
Result = (rulrs) x (CslCu) x (1 IF) x 100 1-[17-(Pyridin-3-yl)androsta-3,5,16-trien-3-yl]ethanone.
Cz6H31NO 373.53
ru = peak area of each impurity from the Sample 3-Deoxy 3-chloroabiraterone
solution 3p-Chloro-17-(pyridin-3-yl)androsta-5,16-diene.
rs = peak area of abiraterone acetate from the Cz4H30CIN 367.96
Standard solution a-Epoxyabiraterone acetate
Cs =concentration of USP AbirateroneAcetate RS in 17-(Pyridin-3-yl)-16a,17a-epoxyandrost-5-en-3p-yl
the Standard solution (mg/ml) acetate.
Cu = nominal concentration of abiraterone acetate in CZ6H33N03 407.55
the Sample solution (mg/ml) P-Epoxyabiraterone acetate
F = relative response factor for each individual 17-(Pyridin-3-yl)-16p,17p-epoxyandrost-5-en-3p-yl
impurity (see Table 3) acetate.
CZ6H33N03 407.55
Acceptance criteria: See Table 3. Disregard any peak less 7-Ketoabiraterone acetate
than 0.05%. 7-0xo-17-(pyridin-3-yl)androsta-5,16-dien-3p-yl acetate.
C26H31N03
Table 3
Relative Relative Acceptance
Retention Response Criteria,
Name Time Factor NMT(%)
7-Ketoabiraterone Acamprosate Calcium
acetate 0.42 1.4 0.50
a-Epoxyabiraterone
acetate 0.62 0.26 0.80
~-Epoxyabiraterone
acetate 0.66 0.26 0.80 CloHzoCaNzOsSz 400.48
Abiraterone 0.69 1.0 0.40 1-Propanesulfonic acid, 3-(acetylamino)-, calcium salt (2:1);
Calcium 3-(acetylamino)propane-l-sulfonate [77337-73-6].
Abiraterone
acetate 1.0 - - DEFINITION
Abirateroneethyl Acamprosate Calcium contains NlT 98.0% and NMT 102.0%
ether' 1.18 - - of acarnprosate calcium (CloHzoCaNzOsSz), calculated on
the dried basis.
Abiraterone
isopropyl - - IDENTIFICATION
ether- 1.26
Unspecified
impurity - 1.0 0.20
Total impurities - - 2.0 • A·!~~~E.~T~~~~
§p~c:tr()sc:opY:;l9.. ... . .
aThisisa process impurityand is controlledin the drug substance monograph. • B. The retention time of the major peak of the Sample
It is included in the table for identification only,and it is not to be reported in solution corresponds to that of the Standard solution, as
the total impurities. obtained in the Assay.
• C. IDENTIFICATION TESTS-GENERAL (191), Chemical
ADDITIONAL REQUIREMENTS Identification Tests, Calcium: Meets the requirements
• PACKAGING AND STORAGE: Preserve in tight containers, and
store at controlled room temperature. ASSAY
• USP REFERENCE STANDARDS (11) • PROCEDURE
USP Abiraterone Acetate RS Mobile phase: Add 5.0 ml of triethylamineper 1 l of water
USP Abiraterone System Suitability Mixture RS and adjust with phosphoric acid to a pH of 4.0.
It contains Abiraterone Acetate and small amounts of the System suitability solution: 10 mg/ml of USP
following: Acamprosate Calcium RS and 0.005 mg/ml each of USP
Ablraterone

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 USP 43 OfficialMonographs / Acamprosate 29

Acamprosate Related Compound BRS and glacial acetic Standard solution: Transfer 3.0 mL of Standard stock
acid in water. Sonication may be used to aid in dissolution. solution B to an appropriate container. Add 0.15 mL of
Standard solution: 0.3 mg/mL of USP Acamprosate Solution A and shake vigorously for 30 s. Heat in a water
Calcium RS in water. Sonication may be used to aid in bath at 50 for 30 min. Cool under a stream of cold water,
0

dissolution. centrifuge, and pass the supernatant through a suitable

Sample solution: 0.3 mg/mL of Acamprosate Calcium in membrane filter.
water. Sonication may be used to aid in dissolution. Sample stock solution A: 20 mg/mL of Acamprosate
Chromatographic system Calcium in water
(See Chromatography (621), System Suitability.) Sample stock solution B: 2000 IJg/mL of Acamprosate
Mode: LC Calcium from Sample stock solutionA in Diluent
Detector: UV 210 nm Sample solution: Transfer 3.0 mL of Sample stock solution B
Column: 4.6-mm x 25-cm; 5-lJm packing L1 to an appropriate container. Add 0.15 mL of Solution A and
Flow rate: 0.7 mL/min shakefor 30 s. Heat in a water bath at 50 for 30 min. Cool
0

Injection volume: 20 IJL under a stream of cold water, centrifuge, and pass the
Run time: NLT 2 times the retention time of the supernatant through a suitable membrane filter.
acamprosate peak Chromatographic system
System suitability (See Chromatography (621), System SUitability.)
Samples: System sultabllitv solution and Standard solution Mode: LC
[NoTE-See Table 1 for the relative retention times.] Detector: UV 261 nm
Suitability requirements Column: 4.6-mm x 15-cm; 3- or 5-J.lm packing L1
Resolution: NLT 1.5 between acetic acid and Flow rate: 1 mL/min
acamprosate related compound B; NLT 1.3 between Injection volume: 20 IJL .
acamprosate related compound Band acamprosate, Run time: NLT 2 times the retention time of acamprosate
System suitability solution related compound A
Tailing factor: NMT 2.0, Standard solution System suitability
Relative standard deviation: NMT 0.73%, Standard Sample: Standard solution
solution [NOTE-The relative retention times for fluorescamine
Analysis and acamprosate related compound Aare about 0.5
Samples: Standardsolution and Sample solution and 1.0, respectively. Acamprosatecalcium is not
Calculatethe percentage of acamprosate calcium . detected by this chromatographic system.]
(ClOHzoCaNzOsSz) in the portion of Acamprosate Calcium Suitability requirements
taken: Resolution: NLT 2.0 between fluorescamine and
acamprosate related compound A
Result = (rulrs) x (CsiCu) x 100 Relative standard deviation: NMT 5.0% for
acamprosate related compound A
t» = peak response from the Sample solution Analysis
rs = peak response from the Standard soiution Samples: Standard solution and Sample solution
Cs = concentration of USP Acamprosate Calcium RS in Calculate the percentage of acamprosate related
the Standardsolution(mg/mL) compound A in the portion of Acamprosate Calcium
Cu = concentration of Acamprosate Calcium in the taken:
Sample solution (mg/mL)
Result = (rulr s) x (CsIC u) x 100
Acceptance criteria: 98.0%-102.0% on the dried basis
tu = peak responsefrom the Sample solution
IMPURITIES rs = peak responsefrom the Standardsolution
c, = c~Q~.~.Qt~~~.i~r10f!~~i~r~~~I~rq~~t~R~I~t~g
~9~P9I..1ngt\iB~!i!(~~Rq.@e¢".?()l!J) in the Standard
• LIMIT OF ACAMPROSATE RELATED COMPOUND A solution(J.lg/mL)
Solution A: 5 gIL offluorescamine in acetonitrile. Use within Cu = concentration of Acamprosate Calcium in the
24 h of preparation. Sample solution (lJg/mL)
Buffer: 13.8 gIL of monobasic sodium phosphate prepared
asfollows. Transfer a suitableamount of monobasicsodium Acceptance criteria: NMT 0.05%
phosphate to a volumetric flask. Dissolve in 90% ofthe final • ORGANIC IMPURITIES
flask volume of water. Adjust with 10 N sodium hydroxide Mobile phase: Add 5.0 mL of triethylamine per 1 Lof water
TS or phosphoric acid to a pH of 6.5. Dilute with water to and adjust with phosphoric acid to a pH of 4.0.
volume. System suitability solution: 10 mg/mL of USP
Mobile phase: Acetonitrile, methanol, and Buffer Acamprosate Calcium RS and 0.005 mg/mL each of USP
(10:10:80) Acamprosate Related Compound B RS and glacial acetic
Diluent: 24.6 gIL of boric acid prepared asfollows. Transfer acid in water. Sonication may be used to aid in dissolution.
a suitable amount of boric acid to an appropriate Standard solution: 0.Oq5 mg/mL of USP Acamprosate
volumetric flask. Dissolve in 90% of the final flask volume Calcium RS in water. Sonication may be used to aid in
of water. Adjustwith 10 N sodium hydroxide TS to a pH of dissolution.
10.4. Dilute with water to volume. Sample solution: 10 mg/mL of Acamprosate Calcium in
Standard stock solution A: 250 IJg/mL of USP Acamprosate water. Sonication may be used to aid in dissolution.
Related Compound A RS in water Chromatographic system
Standard stock solution B: 1 IJg/mL of USP Acamprosate (See Chromatography (621), System Suitability.)
Related Compound A RS from Standard stock solution A in Mode: LC
Diluent Detector: UV 210 nm
Column: 4.6-mm x 25-cm; 5-lJm packing L1

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 30 Acamprosate / OfficialMonographs USP 43

Flow rate: 0.7 mL/min • USP REfERENCE STANDARDS (11)

Injection volume: 20 IJL USP Acamprosate Calcium RS
Run time: NLT 6 times the retention time of the USP Acamprosate Related Compound A RS
acamprosate peak 3-Aminopropane-l-sulfonic acid.
System suitability C3H gN0 3S 139.17
Samples: System suitability solution and Standard solution USP Acamprosate Related Compound B RS
[NOTE-Therelative retention time for acetic acid is O.7i Calcium 3-formamidopropane-1-sulfonate.
see Table 7 for the other relative retention times.] CSH16CaN20SS2 372.42
Suitability requirements
Resolution: NLT 1.5 between acetic acid and
acamprosate related compound Bi NLT 1.3 between
acamprosate related compound Band acamprosate,
System suitability solution Acarbose
Tailing factor: NMT 1.5 for acamprosate, Standard
solution
Relative standard deviation: NMT 15.0% for acetic
acid, System SUitability solution; NMT 5% for
acamprosate, Standard solution
Analysis
Samples: Standard solution and Sample solution C2sH43N01S 645.60
Calculate the percentage of each impurity in the portion of o-Glucose, 0-4,6-dideoxy-4-[[[15-(1 a,4a,5~,6a)]-4,5,6­
Acamprosate Calcium taken: trihydroxy-3-(hydroxymethyl)-2-cycJohexen-l-yl]amino ]-a-
Result =(ru/rs) x (Cs/Cu) x 100 o-glucopyranosyl-(1 ~4)-O-a-o-glucopyranosyl-(1 ~4)-i
0-4,6-Dideoxy-4-{[(15,4R,55,65)-4,5,6-trihydroxy-3-
tu = peak response of each impurity from the Sample (hydroxymethyl)-2-cycJohexen-1-yl]amino}-a-o-
solution glucopyranosyl-(1 ~4)-O-a-o-glucopyranosyl-(1 ~4)-o­
ts = peak response of acamprosate from the Standard glucose [56180-94-0].
solution DEFINITION
Cs = concentration of USP Acamprosate Calcium RS in Acarbose is produced by certain strains of Actinoplanes
the Standard solution (mg/mL) utahensis. It contains NLT 95.0% and NMT 102.0% of
Cu =concentration of Acamprosate Calcium in the acarbose (C2sH43N01S), calculated on the anhydrous basis.
Sample solution (mg/mL)
IDENTIFICATION
Acceptance criteria: See Table 7.
Table 1
Relative Acceptance • A.
Retention Criteria, ~»" '""" ,i '
Name Time NMT(%) • B. The retention time of the major peak of the Sample
Calcium" 0.4 - solution corresponds to that of the Standard solution, as
obtained in the Assay.
Acamprosate related
compound B 0.8 0.05 ASSAY
• PROCEDURE
Acamprosate 1.0 - Solution A: 0.6 mg/mL of monobasic potassium phosphate
N-Methyl and 0.35 mg/mL of dibasic sodium phosphate in water
acarnprosate" 1.9 0.05 Mobile phase: Acetonitrile and Solution A (3:1)
Anyindividual System suitability solution: 20 mg/mL of USPAcarbose
unspecified - System SUitability Mixture RS in water
impurity 0.05 Standard solution: 20 mg/mL of USP Acarbose RS in water
Totalimpurities' - 0.5 Sample solution: 20 mg/mL of Acarbose in water
Chromatographic system
a Includedfor identification only.Thispeak isdue to the calcium counterionand (See ~hromatography (621), System Suitability.)
hence is not an impurity. Mode: LC
b 3-(N-Methylacetamido)propane-l-sulfonate. Detector: UV 210 nm
C The sum of acamprosate related compound Afrom the Limit of Acamprosate Column: 4-mm x 25-cmi packing L8
Related Compound A test and all impurities from the test for OrganicImpurities. Column temperature: 35°
Flow rate: 2 mL/min
SPECIFIC TESTS Injection volume: 10 IJL
• pH (791) System suitability
Sample solution: 0.05 g/mL of Acamprosate Calcium in Sample: System suitability solution
carbon dioxide-free water Identify the acarbose peak and the peaks due to the
Acceptance criteria: 5.5-7.0 impurities listed in Table 7.
• Loss ON DRYING (731 ) Suitability requirements
Analysis: Dry at 105° for 3 h. Peak-to-valley ratio: The ratio of the height of the
Acceptance criteria: NMT 0.4% impurity A peak to the height of the valley between the
ADDITIONAL REQUIREMENTS impurity A peak and the acarbose peak is NLT 1.2.
• PACKAGING AND STORAGE: Store in tight containers. Chromatogram comparability: The chromatogram
obtained is similar to the chromatogram provided with

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USP 43 Official Monographs / Acarbose 31

USP Acarbose System Suitability Mixture RS for the Table 1 (continued)

known impurities found. Relative Relative Acceptance
Analysis Retention Response Criteria,
Samples: Standard solution and Sample solution Name Time Factor NMT (%)
Calculate the percentage of acarbose (C2sH43N018) in the Total impurities - - 3.0
portion of Acarbose taken:
a 0-4,6-Dideoxy-4-([(1 S,4R,5S, 65)-4,S, 6-trihydroxy-3-(hydroxymethyl)
Result =(r vIr s) x (C sICv) x 100 cyclohex-2-enyl]amino}-a-0-glucopyranosyl-(1-4)-0-a-0-glucopyranosyl-
(1-4)~0-arabino-hex-2-ulopyranose.

ru = peak response from the Sample solution b (1R,4R,5 5,6 R)-4, S,6-Trihydroxy-2-(hydroxymethyl)cyclohex-2-enyl4-0-[4,6-
dideoxy-4-([(1 S,4R,5S,65)-4,S,6-trihydroxy-3-(hydroxymethyl)cyclohex-2-
rs = peak response from the Standard solution enyl]amino}-a-o-glucopyranosyl]-a-o-glucopyranoside.
Cs = concentration of USP Acarbose RS in the Standard c a-o-Glucopyranosyl 4-0-[4,6-dideoxy-4-([(1 5,4 R,S 5, 65)-4,S,6-trihydroxy-3-
solution (mg/mL) (hydroxymethyl)cyclohex-2-enyl]amino}-a-o-glucopyranosyI]-a-o-
Cu = concentration of the Sample solution (mg/mL) glucopyranoside.
d 4-0-[4,6-Dideoxy-4-{[(1 S,4R,SS, 65)-4,S, 6-trihydroxy-3-(hydroxymethyl)
cyclohex-2-enyl]amino}-a-o-glucopyranosyl]-0-glucopyranose.
Acceptance criteria: 95.0%-102.0% on the anhydrous e 0-4, 6-Dideoxy-4-{[(15,4 R,S 5, 65)-4,S,6-trihydroxy-3-(hydroxymethyl)
basis cyclohex-2-enyl]amino}-a-o-glucopyranosyl-(1-4)-O-a-o-glucopyranosyl-
(1-4)-0-a-o-glucopyranosyl.(1-4)-0~arabino-hex-2-ulopyranose (4-0-a-
IMPURITIES acarbosyl-o-fructopyranose).
• RESIDUE ON IGNITION (281) f 0-4,6-Dideoxy-4-{[(1 S,4R,SS,65)-4,S,6-trihydroxy-3-(hydroxymethyl)
Sample: 1.0 g cyclohex-2-enyl]amino}-a-o-glucopyranosyl-(1_4).0-a-o-glucopyranosyl-
(1-+4)-0-a-o-glucopyranosyl-(1-+4)-0-glucopyranose (4-0-a-acarbosyl-o-
Acceptance criteria: NMT 0.2% glucopyranose).
• CHROMATOGRAPHIC PURITY 9 a-o-Glucopyranosyl 0-4,6-dideoxy-4-{[(lS,4R,SS,65)-4,S,6-trihydroxy-3-
Mobile phase, System suitability solution, Sample (hydroxymethyl)cyclohex-2-enyl]amino}-a-o-glucopyranosy1-(1-+4)-0-a-O-
solution, and Chromatographic system: Proceed as glucopyranosyl-(1->4)-0-a-o-glucopyranoside (a-o-glucopyranosyl a-
directed in the Assay. acarboside).
h 0-4,6-Dideoxy-4-{[(1 S,4R,SS,65)-4,S,6-trihydroxy-3-(hydroxymethyl)
Diluted sample solution: Dilute 1.0 mL of the Sample cyclohex-2-enyl]amino}-a-o-glucopyranosyl-(1-4)-0-6-deoxy-a-o-
solution with water to 100.0 mL. glucopyranosyl-(1-+4)-0-glucopyranose.
Analysis
Samples: Sample solution and Dilutedsample solution SPECIFIC TESTS
Calculate the percentage of each impurity in the portion of • OPTICAL ROTATION, Specific Rotation (781 S)
Acarbose taken: Sample solution: 10 mg/mL in water
Acceptance criteria: +168 0 to +183 0
Result =(r vir A) x (1 IF) x 100 • pH (791)
Sample solution: 50 mg/mL
ru = peak response of each impurity from the Sample Acceptance criteria: 5.5-7.5
solution • WATER DETERMINATION, Method Ic (921): NMT 4.0%
rA = peak response of the main acarbose peak from
the Diluted sample solution ADDITIONAL REQUIREMENTS
F = relative response factor for each impurity (see • PACKAG.ING AND STORAGE: Preserve in tight containers.
Table 7) • USP REFERENCE STANDARDS (11)
USP Acarbose RS
Acceptance criteria: See Table 7. USP Acarbose System SUitability Mixture RS

Table 1
Relative Relative Acceptance
Retention Response Criteria,
Name Time Factor NMT (%)
ImpurityA" 0.9 1 0.6
A AcarboseTablets
Impurity Bb 0.8 1.6 0.5
DEI=INITION
ImpurityCC 1.2 1 1.5 Acarbose Tablets contain NtT 90:0% and' NMT 1'10.0% of the
ImpurityDd 0.5 1.33 1.0 labeled amount of acarbose (C2sH43N018)'
Impurity Ee 1.7 0.8 0.2
Impurity Ff 1.9 0.8 0.3
ImpurityG9 2.2 0.8 0.3
Impurity Hh 0.6 1 0.2
Anyindividual
unknownlrnpur- - -
ity 0.2

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32 Acarbose / OfficialMonographs USP 43

ASSAY
.. PROCEDURE
B ," . ,'6mg/mLo '. sphate and.
g/lTI Lof c)ter. Filter
s1

5,.~m pacKing 1..8

I.;
.5 times tfw retention time of acarbose

+
ountoracaroose
~ • "- -- - '- , ' - - '" '.

Result = (r ulrs) x Csx(l / L) X. Vx·' (j()

,u := peal< response from,the.$ample~6Iution
's tandara
so
=co
. solution , a ...
solution C (mg/mL)
L = labelclaim. (m let)
V = volumeof M~dlu .' 900 mL
= peak response of aca'rb6se fromthe Sample
solution Tolerances: NLT
= peakresponse of acarbose fromfheStandard acar e (Ci s H4
solution • UNIFORMITY OF : Meefthe

Cu
=conce
soluti
= nomin
)
SPAcarDos~RS'in fheStandariJ
. ..
..' ..
' ..... .
,

entration ofacarbose.iri the Sample

... requirements

so/uti /Il1L)
Acceptance criteria: 90.0o/~"1 0.0%
PE TS
-DI S

S P
Acarb
soluti
to vo
Syste bilit}t. ,. ,.. '. ,. .
M . (5:9'5) Sa . ystem suitability s07utionJtanda,d~sol1Jti9r(1~
Standard stock solution:. Imt ()f USP Acar~ose RSin and Sensitivity solution
Medium" . . . Suitability requirE!menfs .. . _'.'
Peak~to,.valley ratio:'The ratio.of the he
impurity A peak to the height of the va, l i t t l e

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USP 43 Official Monographs / Acebutolol 33

Acebutolol Hydrochloride

- o V;~~yCH'
I '-': . HCI
~ ~ CH, CH,
H,C ~
o
ti1rfdaia s'olLitionl ClsH2sN204 . HCI 372.89
l,rrlPur'ity In the portion of Butanamide, N-[3.;acetyl-4-[2-hydroxy-3-[(l-methylethyl)
amino]propoxy]phenyl]-, monohydrochloride, (±)-;
(±)-3'-Acetyl-4'-[2-hydroxy-3-(isopropylamino)propoxy]-
R~~uJ,t~ (Tulfs}k(CS/C;u)Y<OIf) x1g<> butyranilide monohydrochloride [34381-68-5].
fu =peak(esponse of:any individuaJirriplIrity from'the DEFINITION
Sample solution 'C"'c' , " " " ' . ".' , '
Acebutolol Hydrochloride contains NLT 98.0% and NMT
= peakJespons~ of acaJPose, frQrpS,ta,nda!cf solutio" 102.0% of acebutolol hydrochloride (ClsH2sN204 . HCI),
1 calculated on the dried basis.
~'C(lOcentra1:i6nofUSP'Acarbose RS, in' Stanqdid
IDENTIFICATION
solution 7 (mg/mL) ',,' '" ' , _'," --c

=no, . ' 'onofacarbose in the'Sample

solu f
= relq1Neres seeTetbJe}): OSCOPIC IDEi\4TIFICATI()N TESTS (197),lnfrared
Accept~nc~ criteri,a:,See Table}. is " " , 0 y:,197K", (CN1-May-2020)
• B. The retention time of the major peak of the Sample
Table 1
solution corresponds to that of the Standard solution, as
obtained in the Assay.
~e'athie Relative Acceptance .. C. IDENTIFICATION TESTS-GENERAL (191), Chloride: Meets
Retention Re$p'~~se Crit rll, the requirements of tests A and B
Name "Time ,Factor N %}
Impul'hyDa 0.5 l.a ~1:2
ASSAY
• PROCEDURE
Impul'itY<Bb 0.8 1'.6 0;5' Mobile phase: Methanol, glacial acetic acid, and 0.3%
aqueous solution of sodium dodecyl sulfate (675:20:325).
Imp"llQj;}iAC ().9 1;0 t'.~
Make adjustments if necessary to achieve a retention time
Impl,ll"tt;}Jc<! 1.2 1.0 1.0 for acebutolol of between 4 and 7 min.
Standard solution: 0.14 mg/mL of USP Acebutolol
Anyunspecified
Impurity
~

Lo O.~
Hydrochloride RS in water
Sample solution: 0.14 mg/mL of Acebutolol Hydrochloride
T()tql ,impurities ~
- 3.0 in water
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 2 mL/min
Injection volume: 10!JL
System suitability
Sample: Standard solution
Suitability requirements
Column efficiency: NLT 1500 theoretical plates
Tailing factor: NMT 2.5
Relative standard deviation: 0.73%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of acebutolol hydrochloride
(ClsH2sN204' HCI) in the portion of Acebutolol
Hydrochloride taken:

Result = (r vir s) x (C siC v) x 100

ru = peak response of acebutolol from the Sample

. solution
r5 = peak response of acebutolol from the Standard
solution
C5 = concentration of USP Acebutolol Hydrochloride
RS jn the Standard solution (mg/mL)
Cu = concentration of Acebutolol Hydrochloride in the
Sample solution (mg/mL)

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34 Acebutolol / Official Monographs USP 43

Acceptance criteria: 98.00/0-102.0% on the dried basis ru = peak response of acebutolol related compound A
or acebutolol related compound B or acebutolol
IMPURITIES related compound I from the Sample solution
• RESIDUE ON IGNITION (281): NMT 0.1% rs = peak response of acebutolol related compound A
• ORGANIC IMPURITIES or acebutolol related compound B or acebutolol
Solution A: Mix 2.0 mL of phosphoric acid and 3.0 mL of related compound I from Standard solution B,
triethylamine, and dilute with water to 1 L. Standard solution C, or Standard solution 0
Solution B: Acetonitrile and Solution A (1 :1) Cs = concentration of USP Acebutolol Related
Standard stock solution 1: 0.2 mg/mL of USP Acebutolol Compound A RS or USP Acebutolol Related
Related Compound A RS prepared as follows. Dissolve a Compound B RS or USP Acebutolol.Related
suitable amount of USP Acebutolol Related Compound A Compound I RS in Standard solution B, Standard
RS in a suitable volumetric flask,in 50% of the total volume solution C, or Standard solution D (mg/mL)
of acetonitrile, and dilute with Solution A to volume. Cu = concentration of Acebutolol Hydrochloride in the
Standard stock solution 2: 0.2 mg/mL of USP Acebutolol Sample solution (mg/mL)
Related Compound B RS prepared as follows. Dissolve a
suitable amount of USP Acebutolol Related Compound B Calculate the percentage of any unspecified impurity in the
RS in a suitable volumetric flask, in 50% of the total volume portion of the sample taken:
of acetonitrile, and dilute with Solution A to volume.
Standard solution A: 0.002 mg/mL of USP Acebutolol Result = (r vir s) x (C siCv) x 100
Hydrochloride RS in Solution A
Standard solution B: 0.004 mg/mL of USP Acebutolol = peak response of any unspecified impurity from
Related Compound I RS in Solution A the Sample solution
Standard solution C: 0.002 mg/mL of USPAcebutolol =peak response of acebutolol from Standard
Related Compound A RS in Solution A from Standard stock solution A
solution 7 =concentration of USP Acebutolol Hydrochloride
Standard solution D: 0.004 mg/mL of USP Acebutolol RS in Standard solution A (mg/mL)
Related Compound B RS in Solution A from Standard stock =concentration of Acebutolol Hydrochloride in the
solution 2 Sample solution (mg/mL)
System suitability solution: 0.4 fJg/mL each of USP
Acebutolol Hydrochloride RS and USP Acebutolol Related Acceptance criteria: See Table 2. Disregard peaks below
Compound I RS in Solution A from Standard solution A and 0.05%.
Standard solution B
Sample solution: 2 mg/mL of Acebutolol Hydrochloride in Table 2
Solution A Relative Acceptance
Mobile phase: See Table 7. Retention Criteria,
Name Time NMT(%)
Table 1 Acebutolol related
Time Solution A Solution B compound B' 0.72 0.2
(min) (0/0) (0/0)
Acebutolol related
0 98 2 compound Ib 0.91 0.2

2 98 2 Acebutolol 1.00 -
30.5 10 90 Acebutolol related
compound AC 1.48 0.1
41 10 90
Any unspecified impurity - 0.10

Chromatographic system Total Impurities? - 0.5

(See Chromatography (621), System Suitability.)
Mode: LC a N-{3-Acety/-4-[2-hydroxy- 3-(isopropylamino)propoxy]phenyl}acetamide.
Detector: UV 240 nm b N-{3-Acetyl-4-[3-(ethylamino)-2-hydroxypropoxy]phenyl}butyramide.
Column: 4.0-mm x 12.5-cm; 5-fJm packing L1 c N-(3-Acetyl-4-hydroxyphenyl)butyramide.
dTotal impurities include specified and unspecified impurities.
Column temperature: 40°
Flow rate: 1.2 mL/min
Injection volume: 25 fJL SPECIFIC TESTS
System suitability • pH (791)
Samples: Standard solution A and System suitability solution Sample: 10 mg/mL of Acebutolol Hydrochloride in water
Suitability requirements Acceptance criteria: 4.5-7.0
Resolution: NLT 7.0 between acebutolol and acebutolol • Loss ON DRYING (731)
related compound I, System suitability solution Analysis: Dry at 105° for 3 h.
Relative standard deviation: NMT 2.0%, Standard Acceptance criteria: NMT 1.0%
solution A ADDITIONAL REQUIREMENTS
Analysis • PACKAGING AND STORAGE: Preserve in tight containers, and
Samples: Standard solution A,Standard solution B, Standard store at controlled room temperature.
solution C, Standard solution D, and Sample solution • USP REFERENCE STANDARDS (11)
Calculate the percentage of acebutolol related compound USP Acebutolol Hydrochloride RS
A, acebutolol related compound 8, and acebutolol related USP Acebutolol Related Compound A RS
compound I in the portion of the sample taken: N-(3-Acetyl-4-hydroxyphenyl)butyramide.
C12H1SN03 221.25
Result = (r vir s)x (C siCv) x 100

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USP 43 OfficialMonographs / Acebutolol 35

USP Acebutolol Related Compound B RS Cu =nominal concentration of acebutolol from the

N-{3-Acetyl-4-[2-hydroxy-3-(isopropylamino)propoxy] Sample solution (mg/mL)
phenyl}acetamide. MrI =molecular weight of acebutolol, 336.43
C16H24N204 308.37 Mr2 =molecular weight of acebutolol hydrochloride,
USP Acebutolol Related Compound I RS 372.89
N-{3-Acetyl-4-[3-(ethylamino)-2-hydroxypropoxy]
phenyl}butyramide. Acceptance criteria: 90.0%-110.0%
C17H26N204 322.40
PERFORMANCE TESTS
• DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 2: 50 rpm
Acebutolol Hydrochloride Capsules Time: 30 min
Standard solution: A known concentration of USP
DEFINITION Acebutolol Hydrochloride RS in Medium
Acebutolol Hydrochloride Capsules contain the equivalent of Sample solution: Pass a portion of the solution under test
NLT 90.0% and NMT 110.0% of the labeled amount of through a suitable filter.
acebutolol (ClsH2sN204)' Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
IDENTIFICATION Mode: UV
• A. The UV absorption spectra of the major peak of the Analytical wavelength: 232 nm
Sample solution exhibit maxima and minima at the same Analysis
wavelengths as those of the corresponding peak of the Samples: Standardsolution and Sample solution
Standardsolution, as obtained in the Assay. Calculate the percentage of the labeled amount of
• B. The retention time of the major peak of the Sample acebutolol (ClsH2SNz04) dissolved:
solution corresponds to that of the Standardsolution, as
obtained in the Assay. (Au/As) x c, x V x (1 /L) x (M,dM'2) x 100
ASSAY Au =absorbance of the Sample solution
• PROCEDURE As =absorbance of the Standardsolution
Buffer: Dissolve 2.4 9 of sodium I-decanesultonate in 1000
mL of water. Adjust with glacial acetic acid to a pH of 3.5. Cs =concentration of acebutolol in the Standard
Mobile phase: Methanol and Buffer (60:40) solution (mg/mL)
Standard solution: 0.22 mg/mL of USP Acebutolol V =volume of Medium, 900 mL
Hydrochloride RS in methanol. [NOTE-This is equivalent to L =label claim (mg/Tablet)
0.2 mg/mL of acebutolol.] M'I =molecular weight of acebutolol, 336.43
Sample stock solution: Nominally 1 mg/mL of acebutolol Mr2 = molecular weight of acebutolol hydrochloride,
prepared asfollows. Transfer an equivalent to 200 mg of 372.89
acebutolol, from the contents of NLT 20 Capsules, to a
200-mL volumetric flask. Add 180 mL of methanol, and stir Tolerances: NLT 80% (Q) of the labeled amou nt of
by mechanical means for 30 min. Dilute with methanol to acebutolol (ClsH2sNz04) is dissolved.
volume. • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Sample solution: Nominally 0.2 mg/mL of acebutolol in requirements
methanol from Sample stock solution IMPURITIES
Chromatographic system • ORGANIC IMPURITIES
(See Chromatography (621), System Suitability.) Test 1
Mode: LC Buffer: Prepare as directed in the Assay.
Detector: UV 254 nm. For IdentificationA, use a Mobile phase: Methanol and Buffer (44:56)
diode-array detector in the range of 200-400 nm. Diluent: Methanol and Buffer (50:50)
Column: 3.9-mm x 15-cm; 4-J.lm packing L1 Standard stock solution: 0.6 mg/mL of USP Acebutolol
Flow rate: 1 mL/min Hydrochloride RS prepared as follows. To a suitable
Injection volume: 20 J.lL amount of USP Acebutolol Hydrochloride RS in a suitable
System suitability volumetric flask, add methanol, about 24% of the flask
Sample: Standardsolution volume, swirl to dissolve, and dilute with Diluent to
Suitability requirements volume.
Tailing factor: NMT 1.5 Standard solution: 1.4 J.lg/mL of USP Acebutolol
Relative standard deviation: NMT 2.0 Hydrochloride RS in Diluent from Standardstocksolution
Analysis Sample stock solution: Nominally 2.5 mg/mL of
Samples: Standardsolution and Sample solution acebutolol prepared asfollows. Transfer a portion of the
Calculate the percentage of the labeled amount of contents of 20 opened Capsules, equivalent to 250 mg of
acebutolol (ClsH2SN204) in the portion of Capsules taken: acebutolol, to a 1OO-mL volumetric flask. Add 25 mL of
methanol and shake by mechanical means for 15 min.
Result =(ru/rs) x (CslCu) x (M,dMr2) x 100 Dilute with Diluent to volume. Centrifuge a portion of this
solution and use the supernatant.
=peak response of acebutolol from the Sample Sample solution: Nominally 250 J.lg/mLof acebutolol in
solution Diluent from Sample stock solution
= peak response of acebutolol fromthe Standard Chromatographic system
solution (See Chromatography (621), System Suitability.)
=concentration of USP. Acebutolol Hydrochloride Mode: LC
RS in the Standardsolution (mg/mL) Detector: UV 240 nm

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36 Acebutolol / OfficialMonographs USP 43

Column: 3.9-mm x 15-cm; 4-lJm packing L1 = peak response of acebutolol from the Standard
Flow rate: 1 mL/min solution
Injection volume: 35 IJL = concentration of USP Acebutolol Hydrochloride
Run time: NLT 2 times the retention time of acebutolol RS in the Standard solution (lJg/ m L)
System suitability = nominal concentration of acebutolol from the
Sample: Standardsolution Sample solution (lJg/mL)
Suitability requirements = molecular weight of acebutolol, 336.43
Relative standard deviation: NMT 6.0% = molecular weight of acebutolol hydrochloride,
Analysis 372.89
Samples: Diluent, Standardsolution, and Sample solution
Calculate the percentage of each impurity eluting before Acceptance criteria
the acebutolol peak in the portion of Capsules taken: Test 2: NMT 0.5% of any individual impurity. Disregard
any peaksfrom the Mobilephase.
Result = (ru/rs) x (CslCu) x (Mr 1/Mr2 ) x 100 Sum of impurities from Test 1 and Test 2: NMT 1.0%
to = peak response of each individual impurity from ADDITIONAL REQUIREMENTS
the Sample solution • PACKAGING AND STORAGE: Preserve in tight containers, and
rs = peak response of acebutolol from the Standard store at controlled room temperature.
solution • USP REFERENCE STANDARDS (11)
Cs = concentration of USP Acebutolol Hydrochloride USP Acebutolol Hydrochloride RS
RS in the Standardsolution (uq/rnl.)
Cu = nominal concentration of acebutolol in the
Sample solution (uq/rnt)
Mr1 =molecular weight of acebutolol, 336.43 Acepromazine Maleate
Mr2 = molecular weight of acebutolol hydrochloride,
372.89

Acceptance criteria: NMT 0.5% of any individual

impurity. Disregard any peaksfrom the Diluent.
Test 2
Buffer and System suitability: Proceed as directed in Test
¢:o
7.
Mobile phase: Methanol and Buffer (50:50) C19H22N20S . C4H 404 442.53
Standard stock solution: 0.6 mg/mL of USP Acebutolol Ethanone, 1-[1 0-[3-(dimethylamino)propyl]-
Hydrochloride RS prepared as follows. To a suitable 10H-phenothiazin-2-yl]-, (Z)-2-butenedioate (1:1);
amount of USP Acebutolol Hydrochloride RS in a suitable 10-[3-(Dimethylamino)propyl]phenothiazin-2-yl methyl
volumetric flask, add methanol, about 24% of the flask ketone maleate (1:1) [3598-37-6].
volume, swirl to dissolve, and dilute with Mobilephase to
volume. DEFINITION
Standard solution: 1.4 IJg/mL of USP Acebutolol Acepromazine Maleate contains NLT 98.0% and NMT 101.0%
Hydrochloride RS in Mobile phase from Standardstock of acepromazine maleate (C19H22N20S . C4H404) , calculated
solution on the anhydrous basis.
Sample stock solution: Nominally 2.5 mg/mL of Throughout the following procedures, protect samples, the
acebutolol prepared as follows. Transfer a porti.on of the USPReference Standard, and solutions containing them, by
contents of 20 opened Capsules, equivalent to 250 mg of conducting the procedures without delay, under subdued
acebutolol, to a 1OO-mL volumetric flask. Add 25 mL of light, or using low-actinic glassware.
methanol and shake by mechanical means for 15 min.
Dilute with Mobile phase to volume. IDENTIFICATION
Sample solution: Nominally 250 IJg/mL of acebutolol in
Diluent from Sample stocksolution prepared asfollows.
Centrifuge a portion of Sample stock solution, and transfer
10.0 mL of the clear supernatant to a 1OO-mL volumetric
flask. Dilute with Mobile phase to volume.
Chromatographic system peak for acepromazine
(See Chromatography (621), System Suitability.) of the Sample solution corresponds to that of the Standard
Proceed as directed in Test 7 except for the Injection solution, as obtained in the Assay.
volume and Run time. ASSAY
Injection volume: 70 IJL • PROCEDURE
Run time: NLT 3 times the retention time of acebutolol Buffer: Add 6 mL of triethylamine to 700 mL of water, and
Analysis adjust with phosphoric acid to a pH of2.5.
Samples: Mobile phase, Standardsolution, and Sample Mobile phase: Acetonitrile and Buffer(300:700)
solution Standard stock solution: 1 mg/mL of USP Acepromazine
Calculate the percentage of each impurity eluting after Maleate RS in 0.05 N hydrochloric acid
the acebutolol peak.in the portion of Capsules taken: Standard solution: 0.1 mg/mL of USP Acepromazine
Maleate RS in water from. Standardstock solution
Result = (rufrs) x (Cs/Cu) x (MrdMr) x 100 Sample stock solution: 1 mg/mL of Acepromazine Maleate
in 0.05 N hydrochloric acid
t» = peak response of each individual impurity from Sample solution: 0.1 mg/mL of Acepromazine Maleate in
the Sample solution water from Sample stock solution

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 USP 43 OfficialMonographs / Acepromazine 37

Chromatographic system ADDITIONAL REQUIREMENTS

(See Chromatography (621), System Suitability.) • PACKAGING AND STORAGE: Preserve in well-closed
Mode: LC containers, protected from light. Store at room
Detector: UV 280 nm temperature.
Column: 4-mm x 15-cm; 5-~m packing L7 • LABELING: Label it to indicate that it isfor veterinary use
Flow rate: 1 mL/min only.
Injection volume: 10 ~L • USP REFERENCE STANDARDS (11)
System suitability USP Acepromazine Maleate RS
Sample: Standardsolution
Suitability requirements
Column efficiency: NLT 1500 theoretical plates
Tailing factor: NMT 2.5
Relativestandard deviation: NMT 2.0% Acepromazine Maleate Injection
Analysis
Samples: Standardsolution and Sample solution DEFINITION
Calculate the percentage of acepromazine maleate Acepromazine Maleate Injection isa sterile solution of
(C19HzzNzOS . C4H 4 0 4) in the portion of Acepromazine Acepromazine MaleateinWaterfor Injection. Itcontains NLT
Maleatetaken: 90.0% and NMT 110.0% of the labeled amount of
acepromazine maleate (C19HzzNzOS . C4H 4 0 4) .
Result =(r v/r s) x (C siC v) x 100 Throughout the following procedures, protect samples, the
USP Reference Standard, and solutions containing them, by
rv = peak area responsefrom the Sample solution conducting the procedures without delay, under subdued
rs = peak area responsefrom the Standardsolution light, or using low-actinic glassware.
Cs = concentration of USP Acepromazine Maleate RS IDENTIFICATION
in the Standardsolution (mg/mL)
Cu = concentration of the Sample solution (mg/mL)

Acceptance criteria: 98.0%-101.0% on the anhydrous 19.~I~~...~.··.(J • 9}Z)~;hlf'lTrd..,.ecJ

basis 20)

IMPURITIES a of Injection, equivalentto 20 mg of

• RESIDUE ON IGNITION (281): NMT 0.2% acepromazine maleate, add 2 mL of water and 3 mLof 2 N
• ORGANIC IMPURITIES sodium hydroxide, and extract with two 5-mLportions of
Conduct this test without exposure to daylight, and with the cyclohexane. Combine the cyclohexane extracts, and
minimum necessaryexposure to artificial light. evaporate to dryness under vacuum, using gentle heat if
Diluent: Methanol and diethylamine (19:1) necessary.
Sample solution: 20.0 mg/mL of Acepromazine Maleatein Acceptance criteria: Meetsthe requirements
Dnuent . • B. The retention time of the major peak of the Sample
Standard solution: 0.1 mg/mLof Acepromazine Maleatein solution corresponds to that of the Standardsolution, as
Diluent from the Sample solution obtained in the Assay.
Chromatographic system ASSAY
(See Chromatography (621), Thin-Layer Chromatography.) • PROCEDURE
Mode: TLC Buffer: Add 6 mL of triethylamine to 700 mL of water, and
Adsorbent: 0.25-mm layerof chromatographic silica gel adjust with phosphoric acid to a pH of 2.5.
mixture Mobile phase: Acetonitrile and Buffer (300:700)
Application volume: 10 ~L Standard stock solution: 1 mg/mLof USP Acepromazine
Developing solvent system: n-Heptane, isobutyl alcohol, Maleate RS in 0.05 N hydrochloric acid
and diethylamine (75:17:8) Standard solution: 0.1 mg/mLof USP Acepromazine
Analysis Maleate RS in water from Standard stock solution
Samples: Standardsolution and Sample solution Sample stock solution: 1 mg/mLof Acepromazine Maleate
Develop the chromatogram in the Developing solvent in 0.05 N hydrochloric acid from an appropriately diluted
system until the solventfront has moved three-fourths volume of Injection
the length of the plate. Remove the plate from the Sample solution: Nominally 0.1 mg/mLof Acepromazine
chamber and allowto air dry. Examine the plate under Maleate in water from Sample stock solution
short-wavelength UV light. Chromatographic system
Acceptance criteria: 0.5%; no spot, other than the principal (See Chromatography (621), System Suitability.)
acepromazine spot and any at the origin, observed in the Mode: LC
chromatogram of the Sample solution is more intense than Detector: UV 280 nm
the principal spot observed in the chromatogram of the Column: 4-mm x 15-cm; 5-~m packing L7
Standardsolution. Flow rate: 1 mL/min
SPECIFIC TESTS Injection volume: 10 ~L
• MELTING RANGE OR TEMPERATURE (741): 136°-139° System suitability
• pH (791) Sample: Standardsolution
Sample solution: 10 mg/mL of Acepromazine Maleate in Suitability requirements
water . Column efficiency: NLT 1500 theoretical plates
Acceptance criteria: 4.0-5.5 Tailing factor: NMT 2.5
• WATER DETERMINATION, Method I (921): NMT 1.0% Relative standard deviation: NMT 2.0%
Analysis
Samples: Standardsolution and Sample solution

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38 Acepromazine / OfficialMonographs USP 43

Calculate the percentage of acepromazine maleate Standard stock solution: 1 mg/mL of USP Acepromazine
(C'9HzzNzOS . C4H4 0 4) in the portion of Injection taken: Maleate RS in 0.05 N hydrochloric acid
Standard solution: 0.1 mg/mL of USP Acepromazine
Result = (r vir s) x (C siC v) x 100 Maleate RS in water from Standardstocksolution
Sample stock solution: TransferNLT 10 Tablets to a 200-mL
= peak area from the Sample solution volumetric flask, add 100 mL of 0.05 N hydrochloric acid,
=peak area from the Standardsolution and sonicate for 10 min. Shakeby mechanical means for 30
=concentration of USP Acepromazine Maleate RS min, and dilute with 0.05 N hydrochloric acid to volume.
in the Standardsolution (mg/mL) Sample solution: Nominally 0.1 mg/mL of Acepromazine
= nominal concentration of the Sample solution Maleate in water from Sample stocksolution. Pass a portion
(mg/mL) of this solution through a filter of 0.5-l..Im or finer pore size.
Chromatographic system
Acceptance criteria: 90.0%-110.0% (See Chromatography (621), System Suitability.)
Mode: LC
SPECIFIC TESTS Detector: UV 280 nm
• pH (791): 4.5-5.8 Column: 4-mm x 15-cm; 5-l..Im packing L7
• BACTERIAL ENDOTOXINS TEST (85): NMT 4.5 USP Endotoxin Flow rate: 1 mL/min
Units/mg of acepromazine maleate Injection volume: 10 I..IL
• STERILITY TESTS (71): It meets the requirements when System suitability
tested as directed for Test for Sterilityof the Product to Be Sample: Standardsolution
Examined, Membrane Filtration. Suitability requirements
• OTHER REQUIREMENTS: It meets the requirements in Column efficiency: NLT 150Q theoretical plates
Injections and Implanted Drug Products (1). Tailing factor: NMT 2.5
ADDITIONAL REQUIREMENTS Relative standard deviation: NMT 2.0%
• PACKAGING AND STORAGE: Preserve in tight, light-resistant, Analysis
single-dose or multiple-dose containers as described in Samples: Standardsolution and Sample solution
Packaging and Storage Requirements (659), Injection Calculate the percentage of acepromazine maleate
Packaging. Store at controlled room temperature. (C19HzzNzOS . C4H4 0 4) in the portion of Tablets taken:
• LABELING: Label it to indicate that it is for veterinary use
only. Result =(rvlrs) x (CslCv) x 100
• USP REFERENCE STANDARDS (11)
USP Acepromazine Maleate RS ru = peak area from the Sample solution
ts = peak area from the Standardsolution
Cs = concentration of USP Acepromazine Maleate RS
in the Standard solution (mg/mL)
Cv = nominal concentration of the Sample solution
Acepromazine Maleate Tablets (mg/mL)
DEFINITION Acceptance criteria: 90.0%-110.0%
Acepromazine Maleate Tablets contain NLT 90.0% and NMT
110.0% of the labeled amount of acepromazine maleate ADDITIONAL REQUIREMENTS
(C'9Hzz NzOS . C4H404 ) . .
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
Throughout the following procedures, protect samples, the containers, and store at controlled room temperature.
USP Reference Standard, and solutions containing them, by • LABELING: Label the Tablets to indicate that they are for
conducting the procedures without delay, under subdued veterinary use only.
light, or using low-actinic glassware. • USP REFERENCE STANDARDS (11)
USP Acepromazine Maleate RS
IDENTIFICATION

• A •.. ~!~~~9'~~~~~~i~<I~~N~I~~~~~~ON?"'~~J:ii(jl~6)~i,rijff;gl~(J Acetaminophen

Spectro.scOpy:;l SJ,!.K. a (CN.l'-MiW·2020)
Sample: To a quantity of powdered Tablets, equivalent to
20 mg of acepromazine maleate, add 2 mL of water and 3
mL of 2 N sodium hydroxide, and extract with two 5-mL
portions of cyclohexane. Combine the cyclohexane
extracts, and evaporate to dryness under vacuum, using CSH9NO z 151.16
gentle heat if necessary. Acetamide, N-( 4-hydroxyphenyl)-;
Acceptance criteria: Meet the requirements 4'-Hydroxyacetanilide [103-'90-2].
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as DEFINITION
obtained in the Assay. Acetaminophen contains NLT 98.0% and NMT 102.0% of
acetaminophen (CSH9NO z), calculated on the dried basis.
ASSAY
• PROCEDURE
Buffer: Add 6 mL of triethylamine to 700 mL of water, and
adjust with phosphoric acid to a pH of 2.5.
Mobile phase: Acetonitrile and Buffer (300:700)

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 USP 43 Official Monographs / Acetaminophen 39

IDENTIFICATION Standard solution: 1.25 IJg/mL of USP 4-Aminophenol RS

in methanol
Sample solution: 25 mg/mL of Acetaminophen in
methanol
~~j9~~§~I~.;;I~~~\·. System suitability
Spec k)SCOPY;~91'I<A.(CN1.. ) Sample: Standard solution
• B. The retention time of the major peak of the Sample [NOTE-Therelative retention times for 4-aminophenol
solution corresponds to that of the Standard solution, as and acetaminophen are 0.6 and 1.0, respectively.]
obtained in the Assay. Suitability requirements
Relative standard deviation: NMT 5.0%
ASSAY
Analysis
• PROCEDURE
Samples: Standard solution and Sample solution
Use low-actinic glassware for preparation of the Sample Calculate the percentage of 4-aminophenol in the portion
solution.
of Acetaminophen taken:
Solution A: 1.7 giL of monobasic potassium phosphate and
1.8 giL of dibasic sodium phosphate, anhydrous Result =(r vir s) x (C siCv) x 100
Solution B: Methanol
Mobile phase: See Table 7. ru = peak response of 4-aminophenol from the
Sample solution
Table 1 rs = peak response from the Standard solution
Time
(min)
Solution A
(%)
Solution B
(%)
Cs =concentration of USP 4-Aminophenol RS in the
Standard solution (lJg/mL)
0.0 99 1 Cu = concentration of Acetaminophen in the Sample
solution (lJg/mL)
3.0 99 1
7.0 19 81 Acceptance criteria: NMT 0.005%
• ORGANIC IMPURITIES
7.1 99 1 Solution A: Methanol, water, glacial acetic acid (50:950:1)
10.0 99 1 Solution B: Methanol, water, glacial acetic acid
(500:500:1)
Standard solution: 0.1 mg/mL of USP Acetaminophen RS Mobile phase: See Table 2.
in methanol
Table 2
Sample solution: 0.1 mg/mL of Acetaminophen in
methanol Time Solution A Solution B
(min) (%) (%)
Chromatographic system
(See Chromatography (621), System Suitability.) 0 82 18
Mode: LC .
8 82 18
Detector: UV 230 nm
Column: 4.6-mm x 10-cm; 3.5-lJm packing L7 53 0 100
Column temperature: 35°
58 0 100
Flow rate: 1.0 mL/min
Injection volume: 5 IJL 59 82 18
System suitability 73 82 18
Sample: Standardsolution
Suitability requirements
Tailing factor: NMT 2.0 Diluent: Methanol
Relative standard deviation: NMT 1.0% System suitability solution: 20 IJg/mL of USP
Analysis Acetaminophen RS and 80 IJg/mL each of USP
Samples: Standardsolution and Sample solution Acetaminophen Related Compound B RS and USP
Calculate the percentage of acetaminophen (CSH9NO z) in Acetaminophen Related Compound C RS in Diluent
the portion of Acetaminophen taken: Standard solution: .1.25 IJg/mL of USP Acetaminophen
Related Compound D RS and 0.25 IJg/mL of USP
Result = (r vir s) x (C siC v) x 100 Acetaminophen Related Compound J RS in Diluent
Sample solution: 25 mg/mL of Acetaminophen in Diluent
ru = peak response from the Sample solution Chromatographic system
rs = peak response from the Standardsolution (See Chromatography (621), System Suitability.)
Cs =concentration of USP Acetaminophen RS in the Mode: LC
Standardsolution (mg/mL) Detector: UV 254 nm
Cu =concentration of Acetaminophen in the Sample Column: 4.6-mm x 25-cm; 5-lJm packing L7
solution (mg/mL) Flow rate: 0.9 mL/min
Column temperature: 40°
Acceptance criteria: 98.0%-102.0% on the dried basis Injection volume: 5 IJL
System suitability
IMPURITIES Samples: System suitability solution and Standard solution
• RESIDUE ON IGNITION (281): NMT 0.1 % [NOTE-See Table 3 for relative retention time values.]
• LIMIT OF FREE 4-AMINOPHENOL Suitability requirements
Solution A, Solution B, Mobile phase, and Tailing factor: NMT 2.0 for acetaminophen related
Chromatographic system: Proceed as directed in the compound D, Standardsolution
Assay.

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 40 Acetaminophen / Official Monographs USP 43

Resolution: NLT2.0 between acetaminophen and SPECIFIC TESTS

acetaminophen related compound B; NLT 1.5 between • Loss ON DRYING (731)
acetaminophen related compound Band Analysis: Dry at 105° to constant weight.
acetaminophen related compound C, System suitability Acceptance criteria: NMT 0.5%
solution
Relative standard deviation: NMT 5.0% for ADDITIONAL REQUIREMENTS
acetaminophen related compound D, Standard solution • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Analysis ' containers, and store at room temperature. Protect from
Samples: Standard solution and Sample solution moisture and heat.
Calculate the percentage of acetaminophen related • USP REFERENCE STANDARDS (11)
compound J in the portion of Acetaminophen taken: USPAcetaminophen RS
U5P Acetaminophen Related Compound B RS
Result = (r vIr s) x (C sIC v) x 100 N-(4-Hydroxypheny/)propanamide.
C9H"N02 165.19
ru = peak response of acetaminophen related USPAcetaminophen Related Compound C RS
compound J from the Sample solution N-(2-Hydroxyphenyl)acetamide.
r5 = peak response of acetaminophen related CgH 9N02 151.16
compound J from the Standard solution USPAcetaminophen Related Compound D RS
C5 = concentration of USP Acetaminophen Related N-Phenylacetamide.
Compound J RS in the Standard solution CgH 9NO 135.17
(uq/rnl.) USPAcetaminophen Related Compound J RS
Cu = concentration of Acetaminophen in the Sample N-(4-Chlorophenyl)acetamide (p-chloroacetanilide).
solution (lJg/mL) CgHgCINO 169.61
USP4-Aminophenol RS
Calculate the percentage of acetaminophen related C6H 7NO 109.13
compounds B, C, and D and any unspecified impurity in
the portion of Acetaminophen taken:
Result = (r vIr s) x (C sIC v) x (1 IF) x 100
Acetaminophen Capsules
ru = peak response of each specified or unspecified
impurity from the Sample solution DEFINITION
r5 = peak response of acetaminophen related Acetaminophen Capsules contain NLT90.0% and NMT
compound D from the Standard solution 110.0% of the labeled amount of acetaminophen
C5 = concentration of USP Acetaminophen Related (CgH 9 N0 2 ) ·
Compound D RS in the Standard solution
(uq/rnl) IDENTIFICATION
Cu =concentration of Acetaminophen in the Sample • A. The retention time of the major peak of the Sample
solution (lJg/mL) solution corresponds to that of the Standard solution, as
F = relative response factor for each impurity shown obtained in the Assay.
in Table 3 • B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201)
Acceptance criteria: See Table 3. [NoTE-The relative Sample solution: 1 mg/mL of acetaminophen prepared as
retention times and relative response factors in Table 3 follows. Triturate from contents of the Capsules in
(where applicable) are calculated relative to those of methanol. Filter, and use the clear filtrate.
acetaminophen related compound D.] Chromatographic system
Developing solvent system: Methylene chloride and
Table 3 methanol (4:1)
Relative Relative Acceptance Acceptance criteria: Meet the requirements
Retention Response Criteria,
Name Time Factor NMT (%) ASSAY
• PROCEDURE
Acetaminophen 0.43 - - Mobile phase: Methanol and water (1:3)
Acetaminophen Standard solution: 0.01 mg/mL of USP Acetaminophen RS
related compound Ba 0.67 1.2 0.05 in Mobile phase
Sample stock solution: Weigh the contents of NLT 20
Acetaminophen
related compound Cb 0.71 0.38 0.05
Capsules, and calculate the average weight of the contents
of each Capsule. Mix the combined contents of the
Acetaminophen Capsules, and transfer a portion, equivalent to 100 mg of
related compound DC 1.0 1.0 0.05 acetaminophen, to a 200-mL volumetric flask. Add 100
Acetaminophen mL of Mobile phase, shake by mechanical means for 10 min,
related compound Jd 1.73 - 0.001 and dilute with Mobile phase to volume. Transfer 5.0 mL of
Individual unspecified im-
this solution to a 250-mL volumetric flask, and dilute with
purity - 1.0 0.05 Mobile phase to volume. Pass a portion of this solution
through a filter of 0.5-lJm or finer pore size, discarding the
Total impurities - - 0.1 first 10 mL of the filtrate.
Sample solution: Nominally 0.01 mg/mL of
a N-(4-Hydroxyphenyl)propanamide.
b N-(2-Hydroxyphenyl)acetamide.
acetaminophen from the Sample stock solution in Mobile
C N-Phenylacetamide. .
phase. Pass a portion of this solution through a filter of
d N-( 4-Chlorophenyl)acetamide (p-chloroacetanilide).

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 USP 43 Official Monographs / Acetaminophen 41

0.5-J.lm or finer pore size, discarding the first 10 mL of the

filtrate. Acetaminophen Oral Solution
Chromatographic system DEfiNITION
(See Chromatography (621), System Suitability.) Acetaminophen Oral Solution contains NLT 90.0% and NMT
Mode: LC
110.0% of the labeled amount of acetaminophen
Detector: UV 243 nm
(CS H9N02 ) ·
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 1.5 mL/min IDENTIFICATION
Injection volume: 10 IJL • A. The retention time of the major peak of the Sample
System suitability solution corresponds to that of the Standard solution, as
Sample: Standard solution obtained in the Assay.
Suitability requirements • B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Column efficiency: NLT 1000 theoretical plates (201)
Tailing factor: NMT 2 Sample solution: Nominally 1 mg/mL of acetaminophen in
Relative standard deviation: NMT 2.0% methanol from the Oral Solution
Analysis Chromatographic system
Samples: Standard solution and Sample solution Developing solvent system: Methylene chloride and
Calculate the percentage of the labeled amount of methanol (4:1)
acetaminophen (CSH9N02) in the portion of Capsules Acceptance criteria: Meets the requirements
taken:
ASSAY
Result = (rulrs) x (CslCu) x 100 • PROCEDURE
Mobile phase: Methanol and water (1:3)
ru = peak response from the Sample solution Standard solution: 0.01 mg/mL of USP Acetaminophen RS
rs = peak response from the Standardsolution in Mobilephase
Cs =concentration of USP Acetaminophen RS in the Sample stock solution: Nominally 2 mg/mL in Mobile
Standard solution (mg/mL) phase, prepared as follows. Transfer 500 mg of
Cu = nominal concentration of acetaminophen in the acetaminophen from a measured volume of Oral Solution
Sample solution (mg/mL) to a 250-mL volumetric flask, and dilute with Mobilephase
to volume.
Acceptance criteria: 90.00/0-110.0% Sample solution: Nominally 0.01 mg/mL of
acetaminophen in Mobilephasefrom the Sample stock
PERFORMANCE TESTS solution. Pass a portion of this solution through a filter of
• DISSOLUTION (711) 0.5-J.lm or finer pore size, discarding the first 10 mL of the
Medium: Water; 900 mL filtrate. Use the clear filtrate.
Apparatus 2: 50 rpm Chromatographic system
Time: 45 min (See Chromatography (621), System Suitability.)
Standard solution: A known concentration of USP Mode: LC
Acetaminophen RS in Medium Detector: UV 243 nm
Sample solution: A filtered portion of the solution under Column: 3.9-mm x 30-cm; packing L1
test, suitably diluted with Medium to obtain a concentration Flow rate: 1.5 mL/min
similar to that of the Standard solution Injection volume: 10 J.lL
Instrumental conditions System suitability
Mode: UV Sample: Standard solution
Analytical wavelength: 249 nm Suitability requirements
Analysis Column efficiency: NLT 1000 theoretical plates
Samples: Standard solution and Sample solution Tailing factor: NMT 2
Calculate the percentage of the labeled amount of Relative standard deviation: NMT 2.0%
acetaminophen (CSH9N02) dissolved. Analysis
Tolerances: NLT 75% (Q) of the labeled amount of Samples: Standard solution and Sample solution
acetaminophen (CSH9N02 ) is dissolved. Calculate the percentage of the labeled amount of
• UNIFORMITY OF DOSAGE UNITS (905): Meet the acetaminophen (CSH9N02) in the portion of Oral Solution
requirements taken:
IMPURITIES Result =(ru/rs) x (CslCu) x 100
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
PRODUCTS (227): Meet the requirements = peak responsefrom the Sample solution
ADDITIONAL REQUIREMENTS = peak responsefrom the Standard solution
• PACKAGING AND STORAGE: Preserve in tight containers, and =concentration of USP Acetaminophen RS in the
store at controlled room temperature. Standard solution (mg/mL)
• USP REFERENCE STANDARDS (11) =nominal concentration of acetaminophen in the
USP Acetaminophen RS Sample solution (mg/mL)

Acceptance criteria: 90.0%-110.0%

PERFORMANCE TESTS
• DELIVERABLE VOLUME (698): Meets the requirements for
oral solutions packaged in multiple-unit containers

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42 Acetaminophen / OfficialMonographs USP43

• UNIFORMITY OIF DOSAGE UNITS (90S): Meets the System suitability

requirements for oral solutions packaged in single-unit Sample: Standard solution
containers Suitability requirements
Column efficiency: NLT 1000 theoretical plates
IMPURITIES
Tailing factor: NMT 2.0
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG Relative standard deviation: NMT 2.0%
PRODUCTS (227): Meets the requirements
Analysis
SPECIFIC TESTS Samples: Standard solution and Sample solution
• pH (791): 3.8-6.1 Calculate the quantity, in g, of acetaminophen (CSH9N02)
• ALCOHOL DETERMINATION, Method /I (611) (if present) in 100 g of Acetaminophen for Effervescent Oral Solution
Analysis: Determine by the gas-liquid chromatographic taken:
procedure, using acetone as the internal standard.
Acceptance criteria: 90.0%-115.0% of the labeled amount Result =(rufrs) x (CslC u) x (1 IF,) x Lx F2
of alcohol (C2H sOH)
ru = peak response from the Sample solution
ADDITIONAL REQUIREMENTS ts = peak response from the Standard solution
• PACKAGING ANDSTORAGE: Preserve in tight containers, and Cs = concentration of USP Acetaminophen RS in the
store at controlled room temperature. Standard solution (mg/mL)
• USP REFERENCE STANDARDS (11) Cu = nominal concentration of acetaminophen in the
USP Acetaminophen RS Sample solution (mg/mL)
F, = conversion factor, 1000 mg/g
L = label claim (mg/unit)
F2 = conversion factor, 100, based on the label claim
of g of acetaminophen per 100 g of sample
Acetaminophen for Effervescent Oral
Solution Acceptance criteria: 5.63-6.88 g
DEFINITION , PERFORMANCE TESTS
Acetaminophen for Effervescent Oral Solution contains, in • MINIMUM FILL (755): Meets the requirements for solids
each 100 g, NLT 5.63 g and NMT 6.88 g of acetaminophen packaged in multiple-unit containers
(CSH9 N0 2 ) · • UNIFORMITY OF DOSAGE UNITS (90S): Meets the
requirements for solids packaged in single-unit containers
IDENTIFICATION
IMPURITIES
• A. A 1O-g portion dissolves, with effervescence, in water
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
when dissolved as directed for the Sample solution in the
PRODUCTS (227): Meets the requirements
Assay. ' .
• B. The retention time of the major peak of the Sample ADDITIONAL REQUIREMENTS
solution corresponds to that of the Standard solution, as •. PACKAGING AND STORAGE: Preserve in air-tight containers,
obtained in the Assay. and store at controlled room temperature.
• C. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST • USP REFERENCE STANDARDS (11)
(201) USPAcetaminophen RS
Sample solution: Triturate 0.4 g of the powder with 25 mL
of methanol, and filter.
Chromatographic system
Developing solvent system: Methylene chloride and
methanol (4:1) Acetaminophen Oral Suspension
Acceptance criteria: Meets the requirements
DEFINITION
ASSAY Acetaminophen Oral Suspension is a suspension of
• PROCEDURE Acetaminophen in a suitable aqueous vehicle. It contains
Mobile phase: Methanol and water (1:3) NLT90.0% and NMT 110.0% of the labeled amount of
Standard solution: 0.01 mg/mL of USP Acetaminophen RS acetaminophen (CSH 9N02) .
in Mobilephase
Sample stock solution: Dissolve 109 of Acetaminophen for IDENTIFICATION
Effervescent Oral Solution in 200 mL of water in a 1000-mL
volumetric flask, using gentle heat if necessary, until
effervescence subsides, and then dilute with water to
volume.
Sample solution: Nominally 0.16 mg/mL of )
acetaminophen in Mobilephasefrom the Sample stock Sample: Transfer a volume of Oral Suspension, equivalent
solution. Pass a portion of this solution through a filter of to 240 mg of acetaminophen, to a separator. Add 50 mLof
0.5-l.Im or finer pore size, discarding the first 10 mL of the ethyl acetate, and shake. Filter the ethyl acetate extract
filtrate. through a funnel containing glass wool and 10 g of
Chromatographic system anhydrous sodium sulfate. Collect the filtrate in a beaker,
(See Chromatography (621), System SUitability.) and evaporate on a steam bath to dryness. Dry the residue
Mode: LC under vacuum over silica gel.
Detector: UV 243 nm Acceptance criteria: The crystals so obtained meet the
Column: 3.9-mm x 30-cm; packing L1 requirements.
Flow rate: 1.5 mL/min
Injection volume: 10 I.IL

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 USP 43 Official Monographs / Acetaminophen 43

It B. The retention time of the acetamihophen peak of the Table 1 (continued)

Sample solution correspondsto that of the Standard Time SofutionA $olu'tioili:l
solution, as obtained in the Assay. (l11hi) (%) . '(%)

ASSAY fl;~ roo 0

$;0 0 100
Addfhe following:
6.0 100 0
A.PROCEDURE
10.0 1.00 0
Mob'
S
Diluent:'Methanol,:phospnoric add; arid ,Water (50: O. f:
50)
St

s6lutlorriiiM6b71e
hrough a filter' of ..
he first 10 mlofthe

, system SuitaQi/ity.) S
aceta '. '
Ch~o .• ic system
.(See Chromatography (621),'System SUitability.)
Mode::L~

.,ooothe<iretkiU' plates

'. d Sample solution

e percentage labeledamnt ()f
inophen (CSH9 NOi) in the portion ral
sion taken: . .
Result = (rulrs) x (CslCu) x 10Q
=.peakresponse from the SampJesoliJti9n Result = (rulrs) x (esICu) x 100
~. peak'response from fhe'StdndiJrdsoJutron
~ co tamin()Rh~n,RSin the acetami~ophen from the
St
ev == n etamir'l0phen'inthe :>f, aicet:atninoph~mfrQrTI' the
Sample so/uti
Acceptance criteria: .90
110.0%A.A. (RB J-Aug-2018) -Jul-2019)

Change'f,
Acceptance criteria: 90.0%-110.0%
• PROCEDURE PERFORMANCE TESTS
A.Solution A:' Acetonitrile,.trifluoroacetic acid, and water • UNIFORMITY OF DOSAGE UNITS (905)
(14:0.1 : 86). .' . '. . . . .. For single-unit containers: Meetsthe requirements
,Solution B: Acetonitrile, ififluoroacetl<add, and'Water~(90: • DELIVERABLE VOLUME (698)
0.1: .10) For multiple-unit containers: Meets the requirements
Mobile phase: See TClble't ~
Table 1
Time sciMiollJ\ S()h,ltio,rrl:\
(Win) (%) (%)
0.0 100 ()

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44 Acetaminophen / Official Monographs USP43

IMPURITIES Analysis, '.'." _ _ , ~ _ ",.. . ' _

SarTIple~~- $tcmdardsolution ~nd S.aniple'~dlutio,., , , ,,,_. __ ,_
Delete the following: Calculate the percentage of4-:-citniriophenol in the portion
of Oral Suspension taken:
J..o 4-AMINOPHENOL-U~ ACET~\I\iiINOPHEN:tONTAININCfDiiiG
PRODUCTS (227): Mee,ts th~ _• _ - - - Re~ul{ =,(rulrs) x(C;rCu)x10~
requirements.... (Postponed ~n 1.Au972(118)

Add the following: -

Ai ORGANlC IMPURl"rlES
Solution A:. 0.20/0trlfluoroatetic acid in-w~ter
Solution B:0.20/0trifluoroacetic acid in acetonitrile
Mobile phase: See Table 2. - , ~ ,

Table 2
Time SolutiClnA Solution 0 Calculate the percentage,of acetaminophendimeroriny
(min) (%) (0/0) unspecified impurity in the portion of Oral Suspension
0.0 98 :2 taken:
1.0 98 :2 Result == (rulrs) x (CsICurx 100
8.0 80 20
rlJ = p-eakresponse ..
9.0 5 ~.5 unspecified i
10.0 5 95 ts = peak response of ace
,S.tandard solution
10.5 98 :2 Cs = concen P Acetaminopher'-RS iotfle
13.0 98 :2 Standa u mL)
.tu = nominal c'oncentratl acetaminophenirfthe
Sample solution (o1g
Buffer: 10 mM sodium citr dih-
prepared byadding 1.1 9 sodi Acceptance criteria: See Table 3. The reporting threshol~ is
1.3g of citric acid monohydrate o.OS% for any impurities.
dissolving, and diluting with W.
sodium citrate dihydrate to in Table 3
acid monohydrate to decrease
A<CJl!~
Relative
achieve a pH of 4.0. Retention Cri
Diluent: Acetonitrile and Buffe Name Time NM )
Sensitivity solution: 0.16
4~Aminophenol 0;28 O~l5'
and 0.08 JJg/rhL
Standard solution Acetaminophen l;t> -......
~-~'-

RSand USP 4~Am op eno Acetaminophen dime!'" 1.57 0.15

Sample solution:' Nominally 1
acetaminophen in Dilu~nt pr Any
quantity equivalent to about unspeCified -
impurity 0.15.
from a volyrrie of Oral, Suspen
to a1 OO-mL volumetric flask.
shake by mechanical'meansfo
Totalimpurities -- 2.0

volume. Mix well. Pass a portio ',6' ~DlhYclroxy.[1/1'-biphenYI].3,3;~diyl)diacetainjde."

l-Aug-2018)
suitable filter.
Chromatograpl1/c system _, .'", _... _ _, , SPECIFIC TESTS
(See Chromatography (621 ),System Suitqbility.)
Mode: LC • pH (791): 4.0-6.9
Detector:UV 272 nm ADDITIONAL REQUIREMENTS
Column: 2.1-mm x l'S-ern; l~8-~mpacki!1g 11 • PACKAGING AND STORAGE: Preserve in tight containers, and
Column temperature: 40° store at controlled room temperature.
Flow rate: 0.5 mL/min • USP REFERENCE STANDARDS (11)
Injection volume: 2.SJJL USPAcetaminophen RS
System suitability USP 4-Aminophenol RS
Samples:, Sensltivltysolutlon atandCirdsoJution
[NoTE~See•Table 3 for, relativ eentio~ times:]
Suitability requirem~nts, '.._._ .. ' .. ,.'_
Tailing factor: NMT2.0 foracetaminophenand
4-amiilophenol, Standard solution, ,, "
Acetaminophen Suppositories
Relative standarddeviation:, NMT,S.O% for DEFINITION
acetamInophen and4-amirioph~nol,-Sta rnd(7rdso/iuiion
Acetaminophen Suppositories contain NLT 90.0% and NMT
Signal-to-nois~ ratio: NLT 10 fdracetaminop en and 110.0% of the labeled amount of acetaminophen
4-aminoplienol, Sensitivity solution (CsH9N02) ·

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USP 43 OfficialMonographs / Acetaminophen 45

IDENTIFICATION IMPURITIES
• A. The retention time of the major peak of the Sample • 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
solution corresponds to that of the Standardsolution, as PRODUCTS (227)
obtained in the Assay. Buffer: 4.0 giL of sodium citrate dihydrate and 1.5 giL of
• B. THIN-LAVER CHROMATOGRAPHIC IDENTIFICATION TEST anhydrous citric acid, in water
(201) Diluent: Bufferand acetonitrile (9:1)
Sample solution: Transfer the equivalent of 20 mg of Sample stock solution: Approximately 12-13 mg/mL of
acetaminophen from a portion of Suppositoriesto a beaker. acetaminophen prepared as follows. Transfer an
Add 20 mLof methanol and heat on a steam bath until appropriate number of whole Suppositories to a suitable
melted. Remove the beaker from the steam bath, allow to volumetric flask. Add Diluent until the flask is about half
cool with occasional stirring, and filter. Usethe clearfiltrate. filled and sonicate for 1 h with frequent swirling. Allow to
Chromatographic system cool and then dilute with Diluent to volume.
Developing solvent system: Methylene chloride and Sample solution: Approximately 4.8-5.2 mg/mL of
methanol (4:1) acetaminophen in Diluent from the Sample stocksolution
Acceptance criteria: Meet the requirements prepared as follows. Pipet 20.0 mLof the Sample stock
solution into a 50-mLvolumetricflask and dilute with
ASSAY Diluent to volume.
• PROCEDURE Standard stock solution: Prepare as indicated in the
Mobile phase: Methanol and water (1:3) chapter.
Standard solution: 0.01 mg/mL of USP Acetaminophen RS Standard solution: Add 20.0 mLof the Sample stock
in Mobile phase solution and 15.0 mLof the Standardstocksolution to a
Sample stock solution: Nominally0.5 mg/mL of 50-mL volumetric flask, and dilute with Diluent to volume.
acetaminophen prepared as follows. Tare a small dish and Acceptance criteria: Meet the requirements
a glass rod, place NLT 5 Suppositories in the dish, heat
gently on a steam bath until melted, stir, cool whilestirring, ADDITIONAL REQUIREMENTS
and weigh. Transfer a weighed portion of the mass, • PACKAGING AND STORAGE: Preserve in tight containers, and
equivalent to 100 mg of acetaminophen, to a separator, store at controlled room temperature or in a cool place.
add 30 mLof solvent hexane, and dissolve. Add 30 mLof • U-SP REFERENCE STANDARDS (11)
water, shake gently, and allow the phases to separate. If an USP Acetaminophen RS
emulsion forms, allow sufficienttime for it to separate.
Transfer the aqueous layer to a 200-mL volumetricflask,
and wash the solvent hexane in the separator with three
30-mL portions of water, adding the washings to the
volumetric flask. Dilute with Mobile phase to volume. Acetaminophen Tablets
Sample solution: Nominally 0.01 mg/mL of
acetaminophen in Mobile phase from the Sample stock DEFINITION
solution. Pass a portion of this solution through a filter of Acetaminophen Tablets contain NLT 90.0% and NMT 110.0%
0.5-l..Im or finer pore size, discarding the first 10 mL of the of the labeled amount of acetaminophen (CS H9N02) .
filtrate. Usethe clear filtrate. IDENTIFICATION
Chromatographic system • A. The retention time of the acetaminophen peak of the
(See Chromatography (621), System Suitability.) Sample solution corresponds to that of the Standard
Mode: LC solution, as obtained in the Assay.
Detector: UV 243 nm • B. The UV spectrum of the acetaminophen peak of the
Column: 3.9-mm x 30-cm; packing L1 Sample solution corresponds to that of the Standard
Flow rate: 1.5 mL/min solution, as obtained in the Assay.
Injection volume: 10 I..IL
System suitability ASSAY
Sample: Standardsolution • PROCEDURE
Suitability requirements Solution A: 1% (v/v) glacial acetic acid in water
Tailing factor: NMT 2 Solution B: Methanol
Relative standard deviation: NMT 2.0% Mobile phase: See Table 1. Return to original conditions
Analysis and re-equilibrate the system for 4 min.
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of Table 1
acetaminophen (CSH9N02) in the portion of Suppositories Time Solution A Solution B
taken: (min) (%) (%)
0.0 90 10
Result = (rulrs) x (CsICu) x 100
4.0 90 10
ru = peak response from the Sample solution
4.1 20 80
ts = peak response from the Standardsolution
Cs = concentration of USP Acetaminophen RS in the 6.0 20 80
Standardsolution (mg/mL)
Cu = nominal concentration of acetaminophen in the Diluent: Methanol and water (10:90)
Sample solution (mg/mL) Standard solution: 0.01 mg/mL of USP Acetaminophen RS
in Diluent
Acceptance criteria: 90.00/0-110.0% Sample stock solution: Nominally 0.1 mg/mL of
acetaminophen in Diluent prepared as follows. Transferan
appropriate amount of acetaminophen from NLT 10

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46 Acetaminophen / OfficialMonographs USP 43

Tablets to a suitable volumetric flask and dilute with Diluent • UNIFORMITY OF DOSAGE UNITS (905): Meet the
to volume. Centrifuge or pass a portion of this solution requirements
through a suitable filter. [NoTE-Sonication or shaking may
be necessary.] IMPURITIES
Sample solution: Nominally 0.01 mg/mL of • ORGANIC IMPURITIES
acetaminophen in Diluent from the Sample stocksolution. It is suggested to protect al! solutions contai.ning
Pass a portion of this solution through a suitable filter. acetaminophen or 4-amlnoph~nol from hg~t.
Chromatographic system Buffer: Dissolve 1.9 9 of ammonium formate In 1 Lof
(See Chromatography (621), System Suitability.) water. Add 1.0 mL of formic acid.
Mode: LC Solution A: Dissolve 3.1 9 of ammonium acetate in 1 L of
Detector: UV 243 nm. For Identification B, use a diode array water. Add 1.0 mL of trifluoroacetic acid.
detector in the range of 220-400 nm. Solution B: Acetonitrile, methanol, and water (10:75:15)
Column: 3.0-mm x 10-cm; 3.5-J.lm packing L1 Solution C: Dissolve 3.1 9 of ammonium acetate in 1000
Column temperature: 40° mL of Solution B. Add 1.0 mL of trifluoroacetic acid.
Flow rate: 0.5 mL/min Mobile phase: See Table 2. Return to original conditions
Injection volume: 10 J.lL and re-equilibrate the system for 4 min.
System suitability
Sample: Standardsolution Table 2
Suitability requirements Time Solution A Solution C
Tailing factor: NMT 2.0 (min) (%) (%)
Relative standard deviation: NMT 2.0% 0 97 3
Analysis
5 70 30
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of 10 10 90
acetaminophen (CaH9NOz) in the portion of Tablets taken:
11 10 90
Result = (r vir s) x (C siC v) x 100
Diluent: Methanol and Buffer (5:95)
=peak response of acetaminophen from the Sensitivity solution:. 0.0~0175 mg!mL of U~P .
Sample solution 4-Aminophenol RS In Diluent. SOnicate to dissolve, If
= peakresponse of acetaminophen from the necessary.
Standardsolution Standard solution: 0.00175 mg/mL of USP 4-Aminophenol
=concentration of USP Acetaminophen RS in the RS and 0.0035 mg/mL of USP Acetaminophen RS in
Standardsolution (mg/mL) Diluent. Sonicate to dissolve, if necessary.
= nominal concentration of acetaminophen in the Sample stock solution: Nominally 5 mg/mL of .
Sample solution (mg/mL) acetaminophen in Diluent from NLT 10 Tablets. [NOTE-It IS
recommended to shake on a flat bed at low speed (180
Acceptance criteria: 90.0%-110.0% oscillations/min) to dissolve, if necessary.]
Sample solution: Nominally 3.5 mg/mL of
PERFORMANCE TESTS acetaminophen in Diluent prepared as follows. Pass a
• DISSOLUTION (711) portion of the Sample stock solution through a suitable filter
Medium: pH 5.8 phosphate buffer (see Reaqents, Indicators, of 0.2-J.lm pore size. Discard the first 2 mL of the filtrate.
and Solutions-Buffer Solutions); 900 mL Dilute a suitable volume of the filtrate with Diluent to
Apparatus 2: 50 rpm volume.
Time: 30 min Chromatographic system
Standard solution: A known concentration of USP (See Chromatography (621), System Suitability.)
Acetaminophen RS in Medium Mode: LC
Sample solution: A filtered portion of the solution under Detector: UV 272 nm. For IdentificationB, use a diode array
test, suitably diluted with Medium to obtain a concentration detector in the range of 200-400 nm.
similar to that of the Standardsolution Column: 4.6-mm x 15-cm; 3-J.lm packing L1
Instrumental conditions Column temperature: 40°
Mode: UV Flow rate: 0.9 mL/min
Analytical wavelength: Maximum absorbance at about Injection volume: 25 J.lL
243 nm System suitability
Analysis Samples: Sensitivity solution and Standardsolution
Samples: Standardsolution and Sample solution Suitability requirements
Calculate the percentage of the labeled amount of Relative standard deviation: NMT 5.0% for
acetaminophen (CaH9NOz) dissolved. 4-aminophenol and acetaminophen, Standardsolution
Tolerances: NLT80% (Q) of the labeled amount of Signal-to-noise ratio: NLT 10 for 4-aminophenol,
acetaminophen (CaH9NOz) is dissolved. Sensitivity solution
For Tablets labeled as chewable Analysis
Medium: pH 5.8 phosphate buffer (see Reagents, Samples: Standardsolution and Sample solution
Indicators, and Solutions-Buffer Solutions); 900 mL Calculate the percentage of 4-aminophenol in the portion
Apparatus 2: 75 rpm of Tablets taken:
Time: 45 min
Standard solution, Sample solution, Instrumental Result = (r v/r s) x (C siC u) x 100
conditions, and Analysis: Proceed as directed above.
Tolerances: NLT 75% (Q) of the labeled amount of ru = peak response of 4-aminophenol from the
acetaminophen (CaH9NOi) is dissolved. Sample solution

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 USP43 OfficialMonographs / Acetaminophen 47

rs = peak response of 4-aminophenolfrom the Mobile phase: Methanol, Solution A, and water
Standardsolution (300:1:700)
Cs =concentration of USP 4-Aminophenol RS in the Standard solution: 0.65 mg/mL of USP Acetaminophen RS
Standardsolution (mg/mL) in Mobilephase. Prepare byfirst dissolving in methanol, and
Cu =nominal concentration of acetaminophen in the then diluting with Mobile phase to volume.
Sample solution (mg/mL) Sample stock solution: Transfer 10 Tablets to a 250-mL
volumetric flask containing50 mL of water and a magnetic
Calculate the percentage of any unspecified impurity in the stir bar. Stirfor at least 30 min or until the coating has
portion of Tablets taken: dissolved. Add 150 mL of methanol, and stir for 45 min.
Tablet cores should be disintegrated at least 15 min before
Result =(r vIr s) x (C sICv) x 100 ending the stirring. Remove the magnetic stir bar, and rinse
into the flask with methanol. Dilute with methanol to
ru =peak response of any unspecified impurity from volume, and centrifuge. Use the clear supernatant.
the Sample solution Sample solution: Dilute 5 mL of the Sample stock solution
rs =peak response of acetaminophen from the with Mobilephase to 200 mL.
Standardsolution Chromatographic system
Cs =concentration of USP Acetaminophen RS in the (See Chromatography (621), System Suitability.)
Standardsolution (mg/mL) . Mode: LC
Cu =nominal concentration of acetaminophen in the Detector: UV 295 nm
Sample solution (mg/mL) Column: 3.9-mm x 15-cm; packing L1
Flow rate: 2.0'mL/min
Acceptance criteria: See Table 3. Injection volume: 20 IJL
System suitability
Table 3 Sample: Standard solution
Relative Acceptance Suitability requirements
Retention Criteria, Tailing factor: NMT 3.0
Name Time NMT (%)
Relative standard deviation: NMT 2.0%
4-Aminophenol 0.53 0.15 Analysis
Samples: Standard solution and Sample solution
Acetaminophen 1.0 - Calculatethe percentage of the labeled amount of
Any unspecified impurity - 0.15 acetaminophen (CaH9N02) inthe portion ofTablets taken:
Total impurities - 0.60
Result = (ru/rs) x (CsfCu) x 100
ADDITIONAL REQUIREMENTS tu = peak responsefrom the Sample solution
• PACKAGING AND STORAGE: Preserve in tight containers,and rs = peak responsefrom the Standard solution
store at controlled room temperature. Cs = concentration of USP Acetaminophen RS in the
• LABELING: Label Tablets that must be chewed'to indicate Standard solution (mg/mL)
that they are to be chewed before swallowing. Cu = nominal concentration of acetaminophen in the
• USP REFERENCE STANDARDS (11) Sample solution (mg/mL)
USP Acetaminophen RS
USP 4-Aminophenol RS Acceptance criteria: 90.0%-110.0%
4-Aminophenol.
C6H7NO 109.13 PERFORMANCE TESTS
• DISSOLUTION (711)
Test 1
Medium: Simulated gastric fluid TS (without enzyme);900
mL
Acetaminophen Extended-Release Apparatus 2: 50 rpm
Tablets Times: 15 min, 1 h, and 3 h
Standard solution: A known concentration of USP
DEFINITION Acetaminophen RS in Medium
Acetaminophen Extended-Release Tablets contain NLT 90.0% Sample solution: Afiltered portion of the solution under
and NMT 110.0% of the labeled amount of acetaminophen test, suitablydiluted with Medium to obtain a
(CaH9 N0 2) · concentration similar to that of the Standard solution
Instrumental conditions
IDENTIFICATION Mode: UV
Analytical wavelength: 280 nm
Analysis
Samples: Standard solution and Sample solution
Calculatethe percentage of the labeled amount of
acetaminophen (CaH9N02 ) dissolved.
Sample: A porti~n()fpowdered Tablets Tolerances: See Table 7.
Acceptance criteria: Meet the requirements
• B. The retention time of the Sample solutioncorresponds to Table 1
that of the Standardsolution, as obtained in the Assay. Time Amount Dissolved
ASSAY 15 min 45%-65%
• PROCEDURE
1h 60%-85%
Solution A: Phosphoric acid and water (1 :9)

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48 Acetaminophen / OfficialMonographs USP 43

.....- T_able 1 (continued) IDENTIFICATION

_ _ _ _Time
_ _ _ _ _1 Amount Dlssolved • A. The retention times of the major peaks of the Sample
solution correspond to those of the Standard solution, as
3h . NLT85% obtained in the Assay.
The percentages of the labeled amount of acetaminophen ASSAY
(CSH9N02) dissolved at the times specified conform to • PROCEDURE
Acceptance Table 2 in (711). [NoTE-Use clean and dry glassware. Inject the
For gelatin-coated Tablets Standard solution and the Sample solution promptly
Medium, Apparatus, Standard solution, Sample after preparation.]
solution, Instrumental conditions, and Analysis: Solution A: Chloroform, methanol, and glacial acetic acid
Proceed as directed in Test 1. (78:20:2)
Mobile phase: Transfer 225 mg of tetramethylammonium
Times: 30 min, 90 min, and 4 h
hydroxide pentahydrate to a 1OOO-ml flask.Add 750 ml of
Tolerances: See Table 2.
water, 125 ml of methanol, 125 ml of acetonitrile, and 1.0
ml of glacial acetic acid. Stir for 3 min, and pass through a
Table 2
membrane filter of 0.5-~m or finer pore size.
Time Amount Dissolved Internal standard solution: 20 mg/ml of benzoic acid in
30 min 40%-60% Solution A
Standard solution: 3.25 mg/ml each of USP
90 min 55%-85% Acetaminophen RS and USP Aspirin RS, and 2.0 mg/ml of
4h NLT80% benzoic acid, from Internal standard solution, in Solution A
Sample solution: Nominally 3.25 mg/ml of
acetaminophen in Solution A, prepared as follows. Transfer
The percentages of the labeled amount of an equivalent of 325 mg of acetaminophen, from NlT 20
acetaminophen (CSH9N02) dissolved at the times finely powdered Tablets, to a 1OO-ml volumetric flask. Add
specified conform to Acceptance Table 2 in (711). 10.0 ml of Internal standard solution and 50 ml of Solution
Test 2: Ifthe product complies with this test, the labeling A, and sonicate for 3 min. Dilute with Solution A to volume.
indicates that it meets USP Dissolution Test 2. Pass a portion of this solution through a filter of 2.5-~m or
Medium, Apparatus, Standard solution, Sample finer pore size. Use the filtrate.
solution, Instrumental conditions, and Analysis: Chromatographic system
Proceed as directed in Test 1. (See Chromatography (621), System Suitability.)
Times: 15 min, 1 h, and 3 h Mode: lC
Tolerances: See Table 3. Detector: UV 280 nm
Column: 3.9-mm x 30-cm; packing l1
Table 3 Flow rate: 2 ml/min
Time Amount Dissolved Injection volume: 5 ~l
15 min 40%-60% System suitability
Sample: Standard solution
1h 55%-75% [NOTE-The retention times for acetaminophen,
3h NLT80% salicylic acid (if present), aspirin, and benzoic acid are
about 2, 3, 5, and 8 min, respectively.]
Suitability requirements
The percentages of the labeled amount of acetaminophen Relative standard deviation: NMT 3.0% for
(CSH9N02) dissolved at the times specified conform to acetaminophen or aspirin for four replicate injections
Acceptance Table 2 in (711). Analysis
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Samples: Standard solution and Sample solution
requirements Calculate individuallythe percentage of the labeled amount
IMPURITIES of acetaminophen (CSH9N02) and aspirin (C9 Hs04) in the
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG portion of Tablets taken:
PRODUCTS (227): Meet the requirements
Result = (Ru/R s) x (Cs/Cu) x 100
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers. = peak response ratio of acetaminophen or aspirin
• LABELING: Where the Tablets are gelatin-coated, the label to benzoic acid from the Sample solution
so states. When more than one Dissolution test is given, the = peak response ratio of acetaminophen or aspirin
labeling states the Dissolution test used only if Test 1 is not to benzoic acid from the Standard solution
used. = concentration of USPAcetaminophen RS or USP
• USP REFERENCE STANDARDS (11) Aspirin RS in the Standard solution (mg/ml)
USP Acetaminophen RS = nominal concentration of acetaminophen or
aspirin in the Sample solution (mg/ml)

Acceptance criteria: 90.0%-110.0% of the labeled amount

of acetaminophen (CSH9N02 ) and aspirin (C9H s0 4)
Acetaminophen and Aspirin Tablets
PERFORMANCE TESTS
DEFINITION • DISSOLUTION (711)
Acetaminophen and Aspirin Tablets contain NlT 90.0% and Procedure for a pooled sample
NMT 110.0% of the labeled amount of acetaminophen Medium: Water; 900 ml
(CSH9N02) and aspirin (C9H s04) . Apparatus 2: 50 rpm

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 USP 43 Official Monographs / Acetaminophen 49

Time: 45 min IMPURITIES

Solution A, Mobile phase, and Chromatographic system • LIMIT OF SALICYLIC ACID
(except for Injection volume): Proceedas directed in the Solution A, Mobile phase, Internal standard solution,
Assay. Sample solution, Chromatographic system, and System
Injection volume: 20 J,JL suitability: Proceed as directed in the Assay.
Internal standard solution: 1 mg/mL of benzoicacid in Standard stock solution: 1.0 mg/mL of USP Salicylic Acid
methanol RS in Solution A
Standard stock solution A: 70 J,Jg/mL of USP Salicylic Acid Standard solutions: Transfer 1.0-, 5.0-, and 10.0-mL
RS in Solution A portions of Standard stock solution to separate 100-mL
Standard solution A: Combine 4.0 mL of Standard stock volumetricflasks. Add 10.0 mL of Internal standard solution
solution A and 1.0 mL of Internal standard solution. to each flask, and dilute with Solution A to volume.
Standard stock solution B: 360 J,Jg/mL each of USP Analysis
Acetaminophen RS and USP Aspirin RS in Solution A Samples: Sample solution and Standard solutions
Standard solution B: Combine 4.0 mL of Standard stock Plot the ratios of the peak responses for salicylic acid and
solution Band 1.0 mL of Internal standard solution. benzoic acid for each of the Standard solutions versus
Sample stock solution: Filtered portions of sample concentrations, in mg/mL, ofsalicylic acid, and draw the
suitablydiluted with Medium to a concentration that is straight line best fitting the three plotted points. From
similar to that of Standard stock solution A or Standard stock the graph so obtained, and from the ratio of the peak
solution B responsesfor salicylic acid and benzoic acid from the
Sample solution: Combine 4.0 mLof the Sample stock Sample solution as obtained in the Assay, determine the
solution and 1.0 mL of Internal standard solution. concentration, in mg/mL,ofsalicylic acid (C7H6 0 3) inthe
Analysis Sample solution.
Samples: Standard solution A, Standard solution B, and Calculatethe percentage of salicylic acid in relationto the
Sample solution concentration of aspirin in the Sample solution, as
[NoTE-The relative retention times for obtained in the Assay.
acetaminophen, salicylic acid, aspirin, and benzoic Acceptance criteria: NMT 3.0%
acid are about 0.3, 004, 0.6, and 1.0, respectively.] • 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
Calculate the percentage of the labeled amount of 'PRODUCTS (227): Meet the requirements
acetaminophen (CSH9NO z) dissolved:
ADDITIONAL REQUIREMENTS
Result = (Ru/R s) x (C/W) x 90 • PACKAGING AND STORAGE: Preserve in tight containers, and
store at controlled room temperature.
Ru =relative peak response ratio of acetaminophen to • USP REFERENCE STANDARDS (11)
the internal standard from the Sample solution USP Acetaminophen RS
Rs = relative peak response ratio of acetaminophen to USP Aspirin RS
the internal standard from Standard solution B USP Salicylic Acid RS
C =concentration of USP Acetaminophen RS in
Standard solution B (lJg/mL) .
W = labeled amount of acetaminophen (mg)

Calculatethe percentage of the labeled amount of aspirin Acetaminophen, Aspirin, and Caffeine
(C9H s0 4) dissolved: Tablets
Result = {[90C1 x (RutlRs1) ] + [90Cz x (Ruz/Rsz) x l..3044]} DEFINITION
/W Acetaminophen, Aspirin, and Caffeine Tablets contain NLT
90.0% and NMT 110.0% of the labeled amount of
C1 = concentration of USP Aspirin RS in Standard acetaminophen (CSH 9NO z), aspirin (C9H s0 4) , and caffeine
solution B (lJg/mL) (CSH lON 40Z) '
RU1 = relative peak response ratio of aspirin to the
internal standard from the Sample solution IDENTIFICATION
RS1 = relative peak response ratio of aspirin to the • A. The retention times of the major peaks of the Sample
internal standard from Standard solution B solution correspond to those of the Standard solution, as
Cz =concentration of USP Salicylic Acid RS in Standard obtained in the Assay.
solution A (lJg/mL) ASSAY
Ruz =relative peak response ratio of salicylic acid to the • PROCEDURE
internal standard from the Sample solution Injectthe Standard solution and the Sample solution within 8
RS2 = relative peak response ratio ofsalicylic acid to the h of preparation.
internal standard from Standard solution A Mobile phase: Methanol, glacial acetic acid, and water
W = labeled amount of aspirin (mg) (28:3:69)
Tolerances: NLT 75% (Q) of the labeled amount of Diluent: Methanol and glacial acetic acid (95:5)
acetaminophen (CSH9NO z) and aspirin (C9H s0 4) is Internal standard solution: 6 mg/mL of benzoic acid in
methanol
dissolved. Standard stock solution: 0.25 mg/mL of USP
• UNIFORMITY OF DOSAGE UNITS (905), Content Uniformity: Acetaminophen RS, 0.25}mg/mL of USP Aspirin RS, and
Meet the requirements with respect to acetaminophen and 0.25}'mg/mLof USP Caffeine RS in Diluentt] being the ratio
to aspirin of the labeled amount, in milligrams, of aspirin to the
labeled amount, in,milligrams, of acetaminophen per
Tablet; and l' being the ratio of the labeled amount, in

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 50 Acetaminophen / Official Monographs USP 43

milligrams, of caffeine to the labeled amount, in milligrams, Standard solution: Transfer 20.0 mL of Standard stock
of acetaminophen per Tablet) solution, 3.0 mL of Internalstandardsolution, and 20 mL of
Standard solution: 0.1 mg/mL of USP Acetaminophen RS, water to a 50-mL volumetric flask, and allow to stand for 30
0.1} mg/mLof USP Aspirin RS, and 0.1}' mg/mL of USP s. Dilute with Diluent to volume. Use within 8 h.
Caffeine RS in Diluent, prepared as follows. Transfer 20.0 Sample solution: Transfer 20.0 mL of a filtered portion of
mL of the Standard stock solution and 3.0 mL of Internal the solution under test to a 50-mL volumetric flask. Add 3.0
standard solution to a 50-mL volumetric flask, and dilute mL of Internal standardsolution and 20 mL of Diluent, mix,
with Diluent to volume. and allow to stand for 30 s. Dilute with Diluent to volume.
Sample stock solution: Nominally 2.5 mg/mL of Analysis: Proceed as directed for Analysis in the Assay.
acetaminophen in Diluent prepared as follows. Transfer an Calculate individually the percentage of the labeled amount
equivalent of 250 mg of acetaminophen, from NLT 20 of acetaminophen (CaHgNOz), aspirin (CgH a0 4) , and
finely powdered Tablets, to a 1OO-mL volumetric flask. Add caffeine (CaH lON4 0 z) dissolved:
75 mL of Diluent, and shake by mechanical means for 30
min. Dilute with Diluent to volume. Result =(RvIR s) x (CslCv) x 100
Sample solution: Nominally 0.1 mg/mL of
acetaminophen in Diluent prepared as follows. Transfer 2.0 Rv = peak response ratio of acetaminophen, aspirin, or
mL of the Sample stock solution and 3.0 mL of Internal caffeine to the internal standard from the Sample
standard solution to a 50-mL volumetric flask, and dilute solution
with Diluent to volume. Rs =peak response ratio of acetaminophen, aspirin, or
Chromatographic system caffeine to the internal standard from the
(See Chromatography (621), System Suitability.) Standard solution
Mode: LC Cs =concentration of the corresponding USP
Detector: UV 275 nm Reference Standard in the Standard solution(mgl
Column: 4.6-mm x 10-cm; 5-lJm packing L1 mL)
Column temperature: 45 ± 1 0 Cv =nominal concentration of acetaminophen,
Flow rate: 2 mL/min aspirin, or caffeine in the Sample solution
Injection volume: 10 IJL (mg/mL)
System suitability
Sample: Standard solution Tolerances: NLT 75% (Q) of the labeled amount of
[NOTE-The relative retention times for acetaminophen (CaHgNOz), aspirin (CgH a0 4) , and caffeine
acetaminophen, caffeine, aspirin, benzoic acid, and (CaHlON 40 z) is dissolved.
salicylic acid are about 0.3, 0.5, 0.8, 1.0, and 1.2, • UNIFORMITY OF DOSAGE UNITS (905), ContentUniformity:
respectively.] Meet the requirements with respect to acetaminophen,
Suitability requirements aspirin, and caffeine'
Resolution: NLT 1.4 between any of the analyte and
internal standard peaks IMPURITIES
Tailing factor: NMT 1.2 for each analyte peak • LIMIT OF SALICYLIC ACID
Relative standard deviation: NMT 2.0% Mobile phase and Diluent: Prepare as directed in the Assay.
Analysis Standard solution: 0.02 mg/mL of USPSalicylicAcid RS in
Samples: Standard solution and Sample solution Diluent
Calculate individually the percentage of the labeled amount Sample solution: Nominally 2.5 mg/mL of aspirin in
of acetaminophen (CaHgNO z), aspirin (CgHa0 4) , and Diluent, prepared as follows. Transfer an equivalent of 250
caffeine (CaHlON40 z) in the portion of Tablets taken: mg of aspirin, from NLT 20 finely powdered Tablets, to a
1OO-mL volumetric flask. Add 75 mL of Diluent, and shake
Result = (RvIRs) x (CsICv) x 100 by mechanical means for 30 min. Dilute with Diluent to
volume.
Rv = peak response ratio of acetaminophen, aspirin, or Chromatographic system
caffeine to the internal standard from the Sample (See Chromatography (621), System Suitability.)
solution Mode: LC
Rs =peak response ratio of acetaminophen, aspirin, or Detector: UV 302 nm
Column: 4.6-mm x 10-cm; 5-lJm packing L1
caffeine to the internal standard from the
Standard solution Column temperature: 45 ± 1 0
Cs = concentration of the corresponding USP Flow rate: 2 mL/min
Reference Standard in the Standard solution(mgl Injection volume: 10 IJL
mL) System suitability
Cv = nominal concentration of acetaminophen, Sample: Standard solution
aspirin, or caffeine in the Sample solution Suitability requirements
(mg/mL) Tailing factor: NMT 1.6
Relative standard deviation: NMT 3.0%
Acceptance criteria: 90.0%-110.0% of the labeled amount Analysis
of acetaminophen, aspirin, and caffeine Samples: Standard solution and Sample solution
Calculate the percentage of salicylic acid (C 7H 60 3) relative
PERFORMANCE TESTS to the labeled amount of aspirin in the portion of Tablets
• DISSOLUTION (711) taken:
Medium: Water; 900 mL
Apparatus 2: 100 rpm Result = (rvlrs) x (CslCv) x 100
Time: 60 min
Mobile phase, Diluent, Internal standard solution, tu = peak response of salicylic acid from the Sample
Standard stock solution, and Chromatographic system: 'solution
Proceed as directed in the Assay.

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USP 43 OfficialMonographs / Acetaminophen 51

ts = peak response of salicylic acid from the Standard System suitability

solution Sample: Standard solution
Cs = concentration of USP Salicylic Acid RS in the [NOTE-The relative retention times for
Standard solution (mg/mL) acetaminophen, caffeine, and benzoic acid are about
Cu = nominal concentration of aspirin in the Sample 0.3, 0.5, and 1.0, respectively.]
solution (mg/mL) Suitability requirements
Resolution: NLT 1.4 between any of the analyteand
Acceptance criteria: NMT 3.0% internal standard peaks
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG Tailing factor: NMT 1.2 for each analyte peak
PRODUCTS (227): Meet the requirements Relative standard deviation: NMT 2.0%
Analysis
ADDITIONAL REQUIREMENTS Samples: Standard solution and Sample solution
• PACKAGING AND STORAGE: Preserve in tight containers, and
Calculate individually the percentages of the labeled
store at controlled room temperature. amounts of acetaminophen (CSH 9NO z) and caffeine
• USP REFERENCE STANDARDS (11)
(CSH lON 40Z) in the portion of Tablets taken:
USP Acetaminophen RS
USP Aspirin RS
Result =(Ru/R s) x (Cs/C u) x 100
USP Caffeine RS
USP Salicylic Acid RS = peak response ratio of acetaminophen or caffeine
to the internal standard from the Sample solution
= peak response ratio of acetaminophen or caffeine
to the internal standard from the Standard
Acetaminophen and Caffeine Tablets solution
= concentration of USP Acetaminophen RS or USP
DEfiNITION Caffeine RS in the Standard solution (mg/mL)
Acetaminophen and Caffeine Tablets contain NLT 90.0% and = nominal concentration of acetaminophen or
NMT 110.0% of the labeled amounts of acetaminophen caffeine in the Sample solution (mg/mL)
(CSH 9NO z) and caffeine (CSH10N40Z)'
Acceptance criteria: 90.0%-110.0% of acetaminophen
IDENTifiCATION (CSH 9NOz) and caffeine (CSH10N40Z) -
• A. The retention times of the major peaks of the Sample
solution correspond to those of the Standard solution, PERfORMANCE TESTS
relative to the internal standard, as obtained in the Assay. • DISSOLUTION (711)
Medium: Water; 900 mL
ASSAY Apparatus 2: 100 rpm
• PROCEDURE Time: 60 min
Solution A: Methanol and glacial acetic acid (95:5) Solution A,' Mobile phase, Internal standard solution,
Mobile phase: Methanol, glacial acetic acid, and water Standard stock solution, Chromatographic system, and
(28:3:69) System suitability: Proceed as directed in the Assay.
Internal standard solution: 6 mg/mL of benzoic acid in Standard solution: Transfer 20.0 mL of the Standard stock
methanol solution, 3.0 mL of Internal standard solution, and 20 mL of
Standard stock solution: 0.25 mg/mL of US~ water to a 50-mL volumetric flask, and allow to stand for 30
Acetaminophen RS and 0.25/ mg/mL of USP Caffeine RS in s. Dilute with Solution A to volume. Use within 8 h.
Solution A; / being the ratio of the labeled amount, in mg, Sample solution: Transfer an aliquot of a filtered portion of
of caffeine to the labeled amount, in mg, of acetaminophen the solution under test to a 50-mL volumetric flask to obtain
per Tablet an expected concentration of 0.1 mg/mL of
Standard solution: 0.1 mg/mL of USP Acetaminophen RS acetaminophen and 0.1/ mg/mL of caffeine, where / is
and 0.1/ mg/mL of USP Caffeine RS, prepared by defined for the Standard stock solution. Add 3.0 mL of
transferring 20.0 mL of Standard stock solution and 3.0 mL Internal standard solution and 20 mL of Solution A, and allow
of Internal standard solution to a 50-mL volumetric flask, and to stand for 30 s. Dilute with Solution A to volume.
diluting with Solution A to volume Analysis: Proceed as directed in the Assay, using the
Sample stock solution: Nominally 2.5 mg/mL of Standard solution and Sample solution prepared within the
acetaminophen in Solution A, prepared asfollows. Transfer Dissolution test.
a portion of the powder equivalent to 250 mg of Calculate the percentages of the labeled amounts of
acetaminophen, from NLT 20 finely powdered Tablets, to acetaminophen (CsHgNO z) and caffeine (CSH10N40Z)
a 1OO-mL volumetric flask. Add 75 mL of Solution A, and dissolved:
shake by mechanical means for 30 min. Dilute with Solution
A to volume. Result = (Ru/R s) x (Cs/Cu) x 100
Sample solution: Transfer 2.0 mL of the Sample stock
solution and 3.0 mL of Internal standard solution to a 50-mL = peak response ratio of acetaminophen or caffeine
volumetric flask, and dilute with Solution A to volume. to the internal standard from the Sample solution
Chromatographic system =peak response ratio of acetaminophen or caffeine
(See Chromatography (621), System Suitability.) to the internal standard from the Standard
Mode: LC solution
Detector: UV 275 nm = concentration of USP Acetaminophen RS or USP
Column: 4.6-mm x 10-cm; 5-lJm packing L1 Caffeine RS in the Standard solution (mg/mL)
Column temperature: 45 ± 10 = nominal concentration of acetaminophen or
Flow raterz {TIL/min caffeine in the Sample solution (mg/mL)
Injection volume: 10 IJL

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 52 Acetaminophen / Official Monographs USP 43

Tolerances: NLT 75% (Q) of the labeled amounts of major peak for pseudoephedrine in the chromatogram of the
acetaminophen (C SH9N02) and caffeine (CSH10N402) is Assay preparation corresponds to that in the chromatogram of
dissolved. the Standard preparation, as obtained in the Assay for
• UNIFORMITY OF DOSAGE UNITS (90S): Meet the pseudoephedrine hydrochloride or the Assay forpseudoephedrine
requirements sulfate.
8: If acetaminophen is claimed in the labeling to be
IMPURITIES present, the retention time of the major peak for
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG acetaminophen in the chromatogram of the Assay preparation
PRODUCTS (227): Meet the requirements corresponds to that in the chromatogram of the Standard
ADDITIONAL REQUIREMENTS preparation, as obtained in the Assay for acetaminophen.
• PACKAGING ANDSTORAGE: Preserve in tight containers, and C: If chlorpheniramine maleate is claimed in the labeling to
store at controlled room temperature. be present, the retention time of the major peak for
• USP REFERENCE STANDARDS (11) chlorpheniramine in the chromatogram of the Assay
USPAcetaminophen RS preparation corresponds to that in the chromatogram of the
USP Caffeine RS Standard preparation, as obtained in the Assay for
chlorpheniramine maleate.
D: If dextromethorphan hydrobromide is claimed in the
labeling to be present, the retention time of the major peak
for dextromethorphan in the chromatogram of the Assay
Capsules Containing at Least Three of preparation corresponds to that in the chromatogram of the
Standard preparation, as obtained in the Assay for
the Following-Acetaminophen and dextromethorphan hydrobromide.
Salts of Chlorpheniramine, Dissolution, Procedure for a Pooled Sample (711)-
Dextromethorphan, and Medium: water; 900 mL.
Apparatus 7: 100 rpm.
Pseudoephedrine Time: 45 minutes.
Test preparation-Mix 9.0 mL of a filtered portion of the
» Capsules Containing at Least Three of the solution under test with 1.0 mL of 1% phosphoric acid
solution.
FollOWing-Acetaminophen and Salts of Procedure-Determine the amounts of pseudoephedrine
Chlorpheniramine, Dextromethorphan, and, hydrochloride or pseudoephedrine sulfate (as appropriate),
Pseudoephedrine contain not less than 90.0 acetaminophen, chlorpheniramine maleate, and
percent and not more than 110.0 percent of the dextromethorphan hydrobromide dissolved, employing the
procedures set forth in the Assay for pseudoephedrine
labeled amounts of acetaminophen (C SH 9N02), hydrochloride or Assay for pseudoephedrine sulfate, Assay for
chlorpheniramine maleate (C16H19CIN2 . C4H404) , acetaminophen, Assay for chlorpheniramine maleate, and Assay
dextrornethorphan hydrobromide (C 18H2SNO . for dextromethorphan hydrobromide, respectively, making any
HBr· H20), and pseudoephedrine hydrochloride necessary volumetric adjustments.
Tolerances-Not less than 75% (Q) of the labeled amounts
(ClOH 1SNO· HCI) or pseudoephedrine sulfate of pseudoephedrine hydrochloride (C10H1SNO . HC!) or
[(C,oH1SNO)2' H2S04] , . pseudoephedrine sulfate [(C lOH 1SNO)2 . H2S04],
[NOTE-The headin9 of this monograph does not acetaminophen (C sH9N02), chlorpheniramine maleate
constitute the offlcial title. It is nor-intended that (C16H19CIN2' C4H404), and dextromethorphan hydrobromide
the name described herein be recognized as the (C1sH2SNO· HBr· H20) are dissolved in 45 minutes.
official title or the common or usual name. The Uniformity of dosage units (90S): meet the requirements.
name for each article encompassed by this
monograph shall be composed of the names of the Assay for pseudoephedrine hydrochloride (where
active Ingredients contained therein, as well as the pseudoephedrine hydrochloride is the salt form used, if
quantitative amount of each active ingredient, and present in the formulation)-
a statement of the function (or purpose) of die Mobilephase, Standard preparation, and Chromatographic
ingredient in the article.] , system-Proceed as directed in the Assay for pseudoephedrine
hydrochloride under Tablets Containing at Least Three of the
Packaging and storage-Preserve in tight containers, and Following-Acetaminophen and Salts of Chlorpheniramine,
store at controlled room temperature. Dextromethorphan, and Pseudoephedrine.
USP Reference standards (11)- Chlorpheniramine standardpreparation-Prepare as
USP Acetaminophen RS directed for Standard preparation in the Assay for
USP Chlorpheniramine Maleate RS chlorpheniramine maleate.
USP Dextromethorphan Hydrobromide RS Dextromethorphan standardpreparation-Prepare as
USP Pseudoephedrine Hydrochloride RS directed for Standard preparation in the Assay for
USP Pseudoephedrine Sulfate RS dextromethorphan hydrobromide.
Labeling-The label for each article encompassed by this System suitabilitysolution 7 (for Capsules that contain
monograph bears a name composed of the active ingredients either all four ingredients or a combination of three containing
contained in the article. The label states the name and quantity chlorpheniramine salt)-Mix equal volumes of the Standard
of each active ingredient and indicates its function (or preparation and the Chlorpheniramine standardpreparation.
purpose) in the article. System suitability solution 2 (for Capsules that contain no
cnlorphenlramlnej-c-Mix equal volumes of the Standard
Identification- preparation and the Dextromethorphan standardpreparation.
A: If pseudoephedrine hydrochloride or pseudoephedrine Assay preparation-Transfer not fewer than 10 Capsules,
sulfate is purported to be present, the retention time of the accurately counted, to a 500-mL volumetric flask. Add about

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 USP 43 OfficialMonographs / Acetaminophen 53

100 mLof water and 10 mL of5% phosphoricacid, and gently iscomplete. Add 0.2 mL of phosphoricacid, dilute with water
heat until the Capsules are fully dispersed. Cool the solution to volume, and mixto obtain a solution having a known
to room temperature, dilute with water to volume, mix, and concentration of about 0.25 mg per mL.
filter. Quantitatively dilute a portion of this solution, if Assay preparation-Transfer not fewer than 10 Capsules,
necessary, with water to obtain a solution having a accuratelycounted, to a 500-mL volumetric flask. Add about
concentration of about 0.12 mg of pseudoephedrine 100 mL ofwater and 10 mL of5% phosphoricacid, and gently
hydrochloride per mL. heat until the Capsules are fully dispersed. Cool the solution
Procedure-Separately inject equal volumes(about 10 JJL) to room temperature, dilute with water to volume, and mix.
of the Standardpreparation and the Assay preparation into the Quantitatively dilute a portion of this solution, if necessary,
chromatograph, record the chromatograms, and measurethe with 0.1% phosphoric acid to obtain a solution having a
responses for the pseudoephedrine peaks. Calculate the concentration of about 0.25 mg of acetaminophen per mL.
quantity, in mg, of pseudoephedrine hydrochloride Chromatographic system (see Chromatography (621 »- The
(C1oH1SNO· HCI) in the Capsules taken by the formula: liquid chromatograph is equipped with a 280-nm detector
and a 4.6-mm x 15-cm column that contains packing L7. The
(CL/D)(ru/rs) flow rate is about 1 mL per minute. Chromatograph the
Standardpreparation, and record the peak responses as
in which C isthe concentration, in mg per mL, of USP directed for Procedure: the tailing factor for the
Pseudoephedrine Hydrochloride RS in the Standard acetaminophen peak is not greater than 2.0; and the relative
preparation; L is the labeled quantity, in mg, of standard deviationfor replicate injections is not more than
pseudoephedrine hydrochloride in each Capsule; D isthe 2.0%.
concentration, in mg per mL, of pseudoephedrine Procedure-Separately inject equal volumes (about 10 JJL)
hydrochloride in the Assay preparation, based on the number of the Standard preparation and the Assay preparation into the
of Capsules taken, the labeled quantity, in mg, of chromatograph, record the chromatograms, and measure the
pseudoephedrine hydrochloride in each Capsuleand the responsesfor the acetaminophen peaks. Calculate the
extent of dilution; and ru and rs are the pseudoephedrine peak quantity, in mg, of acetaminophen (CSH 9N02) in each Capsule
responses obtained from the Assay preparation and the taken by the formula:
Standardpreparation, respectively.
(CL/D)(ru/rs)
Assay for pseudoephedrine sulfate (where
pseudoephedrine sulfateisthe saltform used, ifpresent in the in which C is the concentration, in mg per mL, of USP
formulation)- Acetaminophen RS in the Standard preparation; L isthe labeled
Mobile phase, System suitability solutions, and quantity, in mg, of acetaminophen in each Capsule; 0 is the
Chromatographic system-Proceed as directed in the Assay for concentration, in mg per mL, of acetaminophen in each mL
pseudoephedrine hydrochloride under Tablets Containing at of the Assay preparation, based on the number of Capsules
Least Three of the Following-Acetaminophen and Salts of taken, the labeled quantity, in mg, of acetaminophen in each
Chlorpheniramine, Dextromethorphan, and Pseudoephedrine. Capsule, and the extent of dilution; and ru and ts are the
Chlorpheniramine standardpreparation-Prepare as acetaminophen peak responses obtained from the Assay
directed for Standard preparation in the Assay for preparation and the Standard preparation, respectively.
chlorpheniramine maleate.
Dextromethorphan standardpreparation-Prepare as Assay for chlorpheniramine maleate (if present)-
directed for Standardpreparation in the Assay for Mobilephase and Chromatographic system-Proceed as
dextromethorphan hydrobromide. . directed in the Assay for pseudoephedrine hydrochloride under
Standardpreparation-Dissolve an accuratelyweighed Tablets Containing at Least Three of the Fo/lowing-
quantity of USP PseudoephedrineSulfate RS inwater to obtain Acetaminophen and Salts of Chlorpheniramine,
a solution having a known concentration of about 3.0'l11g per Dextromethorphan, and Pseudoephedrine.
mL. Transfer 2.0 mL of this solution to a 25-mL volumetric Standardpreparation-Dissolve an accuratelyweighed
flask, add 2.5 mLof methanol, dilute with 0.1% phosphoric quantity of USP Chlorpheniramine Maleate RS in water to
acid to volume, and mix. obtain a solution having a known concentration of about 0.8
Assay preparation-Proceed as directed for the Assay mg per mL. Quantitatively dilutea portion ofthis solutionwith
preparation in the Assay for pseudoephedrine hydrochloride to 0.1% phosphoric acid to obtain a solution having a known
obtain a solution having a concentration of about 0.24 mg of concentration of about 8 JJg per mL.
pseudoephedrine sulfate per mL. Assay preparation-Transfer not fewer than 10 Capsules,
Procedure-Proceed as directed for Procedure in the Assay accuratelycounted, to a 500-mL volumetric flask. Add about
for pseudoephedrine hydrochloride. Calculatethe quantity, in 100 mL of water and 10 mL of5% phosphoricacid, and gently
mg, of pseudoephedrine sulfate[(C1oH 15NO)2 . H2S04] in each heat until the Capsules are fully dispersed. Cool the solution
Capsule taken by the formula: to room temperature, dilute with water to volume, mix, and
filter. Quantitatively dilute a portion of this solution, if
(CL/D)(ru/rs) necessary, with 0.1% phosphoric acid to obtain a solution
having a concentration of about 8 JJg of chlorpheniramine
in which the terms are as defined therein, pseudoephedrine maleate per mL.
sulfate being substituted for pseudoephedrine hydrochloride. Procedure-Separately inject equal volumes(about 10 JJL)
of the Standardpreparation and the Assay preparation into the
Assay for acetaminophen (if present)-- chromatograph, record the chromatograms and measure the
Mobile phase-Prepare a filtered and degassed mixtureof responsesfor the chlorpheniramine peaks. Calculate the
water, methanol, and glacial acetic acid (79:20:1). Make quantity, in mg, of chlorpheniramine maleate (C16H19CIN2 .
adjustments, if necessary (see System Suitability under C4H 404) in each Capsuletaken by the formula:
Chromatography (621 »,
Standardpreparation-Transfer about 25 mg of USP
(CL/D)(ru/rs)
Acetaminophen RS, accurately weighed, to a 100-mL
volumetricflask. Add4 mL of methanol, and mixuntilsolution

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54 Acetaminophen / Official Monographs USP 43

in which C is the concentration, in mg per mL, of USP Chlorpheniramine, Dextromethorphan, and

Chlorpheniramine Maleate RS in the Standardpreparation; L is Pseudoephedrine contains not less than 90.0
the labeled quantity, in mg, of chlorpheniramine maleate in
each Capsule; 0 is the concentration, in mg per mL, of percent and not more than 110.0 percent of the
chlorpheniraminemaleate in each mL ofthe Assaypreparation, labeled amounts of acetaminophen (C8H9N02),
based on the number of Capsules taken, the labeled quantity, chlorpheniramine maleate (C16H19CIN2 ·C4H404 ) ,
in mg, of chlorpheniramine maleate in each Capsule, and the dextromethorphan hydrobromide (C18H2SNO .
extent ofdilution; and ru and rs are the chlorpheniramine peak.
responses obtained from the Assay preparation and the H~r· H20), and pseudoephedrine hydrochloride
Standard preparation, respectively. (C10H1SNO· HC!) or pseudoephedrine sulfate
Assay for dextromethorphan hydrobromide (if [(ClOH 1SNO)2· H2S04] .
present)- [NOTE-The headin9 of this monograph does not
Mobile phase and Chromatographicsystem-Proceed as constitute the official title. It is nofInfended that
directed in the Assay for pseudoephedrine hydrochloride under the name described herein be recognized as the
Tablets Containing at Least Three of the Following- official title or the common or usual name. The
Acetaminophen and Salts of Chlorpheniramine, name for each article encompassed by this
Dextromethorphan, and Pseudoephedrine. monograph shall be composed of the names of the
Standard preparation-Dissolve an accurately weighed active Ingredients contained therein, as well as the
quantity of USP Dextromethorphan Hydrobromide RS inwater quantitative amount of each active ingredient, and
to obtain a solution having a known concentration of about a statement of the function (or purpose) of the
0.4 mg per mL. Quantitatively dilute a portion of this solution ingredient in the article.] .
with 0.1% phosphoric acid to obtain a solution havinga
known concentration of about 0.04 mg per mL. Packaging and storage-Preserve in tight containers, and
Assay preparation-Transfer not fewer than 10 Capsules, store at controlled room temperature.
accuratelycounted, to a 500-mLvolumetric flask. Add about USP Reference standards (11 )-
100 mL ofwater and 10 mL of 5% phosphoricacid,and gently USP Acetaminophen RS
heat until the Capsules are fully dispersed. Cool the solution USP Chlorpheniramine Maleate RS
to room temperature, dilute with water to volume, mix, and USP Dextromethorphan Hydrobromide RS
filter. Quantitatively dilute a portion of this solution, if USP Pseudoephedrine Hydrochloride RS
necessary, with 0.1% phosphoricacid to obtain a solution USP Pseudoephedrine Sulfate RS
having a concentration of about 0.04 mg of Labeling-The labelfor each article encompassed by this
dextromethorphan hydrobromide per mL. monograph bearsa name composed of the activeingredients.
Procedure-Separately inject equal volumes (about 10 ~L) The label states the name and quantity of each active
of the Standardpreparation and the Assay preparation into the ingredient and indicates itsfunction (or purpose) in the article.
chromatograph, record the chromatograms, and measurethe Identification-
peak responsesforthe dextromethorphan peaks. Calculate the A: Ifpseudoephedrine hydrochloride or pseudoephedrine
quantity, in mg, of dextromethorphan hydrobromide sulfate is claimed in the labeling to be present, the retention
(C1sH 2s NO · HBr· H20 ) in each Capsuletaken by the formula: time of the major peak for pseudoephedrine in the
chromatogram of the Assay preparation corresponds to that in
(370.33/352. 32)(CL/D)(ru/rs) the chromatogram of the Standardpreparation, as obtained in
the Assay for pseudoephedrine hydrochloride or the Assay for
in which 370.33 and 352.32 are the molecular weights of pseudoephedrine sulfate.
dextromethorphan hydrobromide monohydrate and B: Ifacetaminophen is claimed in the labeling to be
anhydrous dextromethorphan hydrobromide, respectively; C present, the retention time of the major peak for
isthe concentration, in mg per mL, of USP Dextromethorphan acetaminophen inthe chromatogram of the Assay preparation
Hydrobromide RS in the Standard preparation; L isthe labeled corresponds to that in the chromatogram of the Standard
quantity, in mg, of dextromethorphan hydrobromide in each preparation, as obtained in the Assay for acetaminophen.
Capsule; D is the concentration, in mg per mL, of C: Ifchlorpheniramine maleate isclaimed in the labelingto
dextromethorphan hydrobromide in each mL of the Assay be present, the retention time of the major peak for
preparation, based on the number of Capsules taken, the chlorpheniramine in the chromatogram of the Assay
labeled quantity, in mg, of dextromethorphan hydrobromide preparation. corresponds to that in the chromatogram of the
in each Capsule, and the extent of dilution; and ru and rs are Standard preparation, as obtained in the Assay for
the dextromethorphan peak responses obtained from the chlorpheniraminemaleate.
Assay preparation and the Standard preparation, respectively. D: Ifdextromethorphan hydrobromide is claimed in the
labelingto be present, the retention time of the major peak
for dextromethorphan in the chromatogram of the Assay
preparation corresponds to that in the chromatogram of the
Standard preparation, as obtained in the Assay for
Oral Powder Containing at Least Three dextromethorphan hydrobromide.
of the following-Acetaminophen and Minimum fill (755): meets the requirements.
Salts of Chlorpheniramine, Uniformity of dosage units (905)-
FOR ORAL POWDER PACKAGED IN SINGLE-UNIT CONTAINERS: meets
Dextromethorphan, and the requirements.
Pseudoephedrine
»Oral Powder Containing at Least Three of the
FolloWing-Acetaminophen and Salts of

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 USP 43 Official Monographs / Acetaminophen 55

Assay for pseudoephedrine hydrochloride (where Dextromethorphan standardpreparation-Prepare as

pseudoephedrine hydrochloride is the salt form used, if directed for Standard preparation in the Assay for
present in the formulation)- dextromethorphan hydrobromide.
Mobilephase and Chromatographic system-Proceed as Standard preparation-Dissolve an accurately weighed
directed in the Assay for pseudoephedrine hydrochloride under quantity of USP Pseudoephedrine Sulfate RS in water to obtain
Tablets Containingat Least Three of the Following- a solution having a known concentration of about 6.0 mgper
Acetaminophen and Salts of Chlorpheniramine, mL. Transfer 2.0 mL of this solution to a 25-mL volumetric
Dextromethorphan, and Pseudoephedrine. flask, dilute with 0.1 % phosphoric acid to volume, and mix.
Chlorpheniramine standardpreparation-Prepare as System suitability solution 1 (for Oral Powder that contains
directed for Standard preparation in the Assay for either all four ingredients or a combination of three containing
chlorpheniramine maleate. chlorpheniramine salt)-Mix equal volumes of the Standard
Dextromethorphan standardpreparation-Prepare as preparation and the Chlorpheniramine standardpreparation.
directed for Standard preparation In the Assay for System suitability solution 2 (for Oral Powder that contains
dextromethorphan hydrobromide. no chlorpheniramine salt)-Mix equal volumes of the
Standardpreparation--Dissolve an accurately weighed Standard preparation and the Dextromethorphan standard
quantity of USP Pseudoephedrine Hydrochloride RS in water preparation.
to obtain a solution having a known concentration of about Assay preparation-Proceed as directed for the Assay
3.0 mg per mL. Transfer 2.0 mL of this solution to a 25-mL preparation in the Assay for pseudoephedrine hydrochloride to
volumetric flask, dilute with 0.1 % phosphoric acid to volume, obtain a solution having a concentration of about 0.48 mg of
and mix. pseudoephedrine sulfate per mL.
System suitability solution 1 (for Oral Powder that contains Procedure-Proceed as directed for Procedure in the Assay
either all four ingredients or a combination of three containing for pseudoephedrine hydrochloride. Calculate the quantity, in
chlorpheniramine salt)-Mix equal volumes of the Standard mg, of pseudoephedrine sulfate [(C 10H1SNO)2 . H2S04] in each
preparation and the Chlorpheniramine standardpreparation. unit-dose container of Oral Powder taken by the formula:
System suitability solution 2 (for Oral Powder that contains
no chlorpheniramine salt)-Mix equal volumes of the (CLID)(rulr s)
Standardpreparation and the Dextromethorphan standard
preparation. in which the terms are as defined therein, pseudoephedrine
Assay preparation-Transfer the contents of 10 unit-dose sulfate being substituted for pseudoephedrine hydrochloride.
containers of the Oral Powder to a 2000-mL volumetric flask.
Add 1000 mL of water and 2.0 mL of phosphoric acid. Gently Assay for acetaminophen (if present)-
heat to about 60° until the powder is fully dispersed. Cool the Mobilephase, Standardpreparation, and Chromatographic
flask to room temperature, add 40 mL of methanol, dilute with system-Proceed as directed in the Assay for pseudoephedrine
water to volume, and mix. Quantitatively dilute a portion of hydrochloride under Tablets Containing at Least. Three of the
this solution, if necessary, with 0.1 % phosphoric acid to obtain Following-Acetaminophen and Salts of Chlorpheniramine,
a solution having a concentration of about 0.24 mg of Dextromethorphan, and Pseudoephedrine.
pseudoephedrine hydrochloride per mL.
Assay preparation-Transfer the contents of 10 unit-dose
Procedure-Separately inject equal volumes (about 10 IJL) containers of the Oral Powder to a 2000-mL volumetric flask.
of the Standardpreparation and the Assay preparation into the Add 1000 mL of water and 2 mL of phosphoric acid. Gently
chromatograph, record the chromatograms, and measure the heat to about 60° until the powder is fully dispersed. Cool the
responses for the pseudoephedrine peaks. Calculate the flask to room temperature, add 40 mL of methanol, dilute with
quantity, in mg, of pseudoephedrine hydrochloride water to volume, and mix. Quantitatively dilute a portion of
this solution, if necessary, with 0.1 % phosphoric acid to obtain
(C 1oH1SNO· HCI), in each unit-dose container of Oral Powder
a solution having a concentration of about 0.50 mg of
taken by the formula:
acetaminophen per mL.
(CLID)(rulrs) Procedure-Separately inject equal volumes (about 10 IJL)
of the Standard preparation and the Assay preparation into the
in which C is the concentration, in mg per mL, of USP chromatograph, record the chromatograms, and measure the
Pseudoephedrine Hydrochloride RS in the Standard responses for the acetaminophen peaks. Calculate the
preparation; L is the labeled quantity, in mg, of quantity, in mg, of acetaminophen (CsH 9N02) in each
pseudoephedrine hydrochloride in each unit-dose container; unit-dose container of Oral Powder taken by the formula:
o is the concentration, in mg per mL, of pseudoephedrine (CLID)(ruI rs)
hydrochloride in each mL of the Assay preparation, based on
the number of unit-dose containers taken, the labeled
quantity, in mg, of pseudoephedrine hydrochloride in each in which C is the concentration, in mg per mL, of USP
unit-dose container, and the extent of dilution; and ruand rs Acetaminophen RS in the Standardpreparation;L is the labeled
quantity, in mg, of acetaminophen in each unit-dose
are the pseudoephedrine peak responses obtained from the container; 0 is the concentration, in mg per mL, of
Assay preparation and the Standard preparation, respectively. acetaminophen in the Assay preparation, based on the number
Assay for pseudoephedrine sulfate (where of unit-dose containers taken, the labeled quantity, in mg, of
pseudoephedrine sulfate is the salt form used, if present in the acetaminophen in each unit-dose container, and the extent of
formulation)- dilution; and tu and ts are the acetaminophen peak responses
Mobilephase and Chromatographic system-Proceed as obtained from the Assay preparation and the Standard
directed in the Assay for pseudoephedrine hydrochloride under preparation, respectively.
Tablets Containing at Least Three of the Following-
Acetaminophen and Salts of Chlorpheniramine, Assay for chlorpheniramine maleate (if present)-
Dextromethorphan, and Pseudoephedrine. Mobile phase and Chromatographic system-Proceed as
directed in the Assay for pseudoephedrine hydrochloride under
Chlorpheniramine standardpreparation-Prepare as
directed for Standardpreparation in the Assay for
Tablets Containing at Least Three of the Following-
chlorpheniramine maleate.

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 56 Acetaminophen / OfficialMonographs USP 43

Acetaminophen and Salts of Chlorpheniramine, (370. 33/352. 32)(CL/D)(rulrs)

Dextromethorphan, and Pseudoephedrine.
Standardpreparation-Dissolve an accurately weighed in which 370.33 and 352.32 are the molecular weights of
quantity of USP Chlorpheniramine Maleate RS in water to dextromethorphan hydrobromide monohydrate and
obtain a solution having a known concentration of about 0.8 anhydrous dextromethorphan hydrobromide, respectively; C
mg per mL. Quantitatively dilute a portion of this solution with is the concentration, in mg per mL, of USP Dextromethorphan
0.1 % phosphoric acid to obtain a solution having a known Hydrobromide RS in the Standardpreparation; L is the labeled
concentration of about 8 J..Ig per mL. quantity, in mg, of dextromethorphan hydrobromide in each
Assay preparation-Transfer the contents of 10 unit-dose unit-dose container; D is the concentration, in mg per mL, of
containers of Oral Powder to a 2000-mL volumetric flask. Add dextromethorphan hydrobromide in each mL of the Assay
1000 mL of water and 2 mL of phosphoric acid. Gently heat preparation, based on the number of unit-dose containers
to about 60° until the powder is fully dispersed. Cool the flask taken, the labeled quantity, in mg, of dextromethorphan
to room temperature, add 40 mL of methanol, dilute with hydrobromide in each unit-dose container, and the extent of
water to volume, and mix. Quantitatively dilute a portion of dilution; and tu and rs are the dextromethorphan peak
this solution, if necessary, with 0.1 % phosphoric acid to obtain responses obtained from the A$say preparation and the
a solution having a concentration of 8 J..Ig of chlorpheniramine Standardpreparation, respectively.
maleate per mL.
Procedure-Separately inject equal volumes (about 10 J..IL)
of the Standardpreparation and the Assay preparation into the
chromatograph, record the chromatograms, and measure the
responsesfor the chlorpheniramine peaks. Calculate the Oral Solution Containing at Least Three
quantity, in mg, of chlorpheniramine maleate (C16H19CIN2 .
C4H4 0 4) in each unit-dose container of Oral Powder taken by
of the Following-Acetaminophen and
the formula: Salts of Chlorpheniramine,
Dextromethorphan, and
. Pseudoephedrine
in which C is the concentration, in mg per mL, of USP
Chlorpheniramine Maleate RS in the Standardpreparation;L is
the labeled quantity, in mg, of chlorpheniramine maleate in » Oral Solution Containing at LeastThree of the
each unit-dose container; D is the concentration, in mg per Following-Acetaminophen and Salts of
mL, of chlorpheniramine maleate in each mL of the Assay Chlorpheniramine, Dextromethorphan, and
preparation, based on the number of unit-dose containers Pseudoephedrine contains not less than 90.0
taken, the labeled quantity, in mg, of chlorpheniramine
maleate in each unit-dose container, and the extent of percent and not more than 110.0 percent .of the
dilution; and rv and rs are the chlorpheniramine peak labeled amounts of acetaminophen (CgH 9N02),
responses obtained from the Assay preparation and the chlorpheniramine maleate (C16H19CIN2 . C4H404) ,
Standardpreparation, respectively. dextromethorphan hydrobromide (C1gH 2SNO .
Assay for dextromethorphan hydrobromide (if HBr· H20), and pseudoephedrine hydrochloride
present)- (C lO H1SNO· HCI) or pseudoephedrine sulfate
Mobilephase and Chromatographic system-Proceed as
directed in the Assay for pseudoephedrine hydrochloride under [(C lOH 1SNO)2' H2S04] ,
Tablets Containing at Least Three of the Following- [NOTE-The headin9 of this monoqraph does not
Acetaminophen and Salts of Chlorpheniramine, constitute the offlcial title. It is nofintended that
Dextromethorphan, and Pseudoephedrine. the name described herein be recognized as the
Standardpreparation-Dissolve an accurately weighed official title or the common or usual name. The
quantity of USP Dextromethorphan Hydrobromide RS in water name for each article encompassed by this
to obtain a solution having a known concentration of about monograph shall be composed of the names of the
0.8 mg per mL. Quantitatively dilute a portion of this solution active Inqredients contained therein, as well as the
with 0.1 % phosphoric acid to obtain a solution having a quantitatlve amount of each active ingredient, and
known concentration of 0.08 mg per mL. a statement of the function (or purpose) of the
Assay preparation-Transfer the contents of 10 unit-dose ingredient in the article.]
containers of Oral Powder to a 2000-mL volumetric flask. Add
1000 mL of water and 2 mL of phosphoric acid. Gently heat Packaging and storage-Preserve in tight containers, and
to about 60° until the powder is fully dispersed. Cool the flask store at controlled room temperature.
to room temperature, add 40 mL of methanol, dilute with USPReference standards (11)-
water to volume, and mix. If necessary, quantitatively dilute a USP Acetaminophen RS
portion of this solution with 0.1 % phosphoric acid to obtain a USP Chlorpheniramine Maleate RS
solution having a concentration of 0.08 mg of USP Dextromethorphan Hydrobromide RS
dextromethorphan hydrobromide per mL. USP Pseudoephedrine Hydrochloride RS
Procedure-Separately. inject equal volumes (about 10 J..IL) USP Pseudoephedrine Sulfate RS
of the Standardpreparation and the Assay preparation into the Labeling-The label for each article encompassed by this
chromatograph, record the chromatograms, and measure the monograph bears a name composed of the active ingredients.
responsesfor the dextromethorphan peaks. Calculate the The label states the name and quantity of each active
quantity, in mg, of dextromethorphan hydrobromide ingredient and indicates its function (or purpose) in the article.
(C1sH2SNO· HBr· H20 ) in each unit-dose container of Oral Identification-
Powder taken by the formula: A: If pseudoephedrine hydrochloride or pseudoephedrine
sulfate is claimed in the labeling to be present, the retention

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 USP 43 Official Monographs / Acetaminophen 57

time of the major peak for pseudoephedrine in the Standardpreparation and the Dextromethorphan standard
chromatogram of the Assay preparation corresponds to that in preparation.
the chromatogram of the Standardpreparation, as obtained in Assay preparation-Transfer an accurately measured
the Assay for pseudoephedrine hydrochloride or the Assay for . volume of the Oral Solution, equivalent to 15 mg of
pseudoephedrine sulfate. pseudoephedrine hydrochloride, to a 1OO-mL volumetric flask,
8: If acetaminophen is claimed in the labeling to be add 80.0 mL of Mobilephase, dilute with water to volume,
present, the retention time of the major peak for and mix.
acetaminophen in the chromatogram of the Assay preparation Chromatographic system (see Chromatography (621 »- The
corresponds to that in the chromatogram of the Standard liquid chromatograph is equipped with a 214-nm detector
preparation, as obtained in the Assay for acetaminophen. and a 4.6-mm x 15-cm column that contains packing L11. The
C: If chlorpheniramine maleate is claimed in the labeling to flow rate is about 2 mL per minute. Chromatograph the
be present, the retention time of the major peak for Standardpreparation, and record the peak responses as
chlorpheniramine in the chromatogram of the Assay directed for Procedure: the tailing factor for the
preparation corresponds to that in the chromatogram of the pseudoephedrine peak is not greater than 2.5; and the relative
Standardpreparation, as obtained in the Assay for standard deviation for replicate injections is not more than
chlorpheniramine maleate. 2.0%. Separately inject about 10 ~L of System suitability
0: If dextromethorphan hydrobromide is claimed in the solution 1 or System suitability solution 2, as appropriate. The
labeling to be present, the retention time of the major peak resolution, R, between pseudoephedrine and
for dextromethorphan in the chromatogram of the Assay chlorpheniramine or between pseudoephedrine and
preparation corresponds to that in the chromatogram of the dextromethorphan is not less than 2.0.
Standardpreparation, as obtained in the Assay for Procedure-Separately inject equal volumes (about 10 ~L)
dextromethorphan hydrobromide. of the Standardpreparation and the Assay preparation into the
chromatograph, record the chromatograms, and measure the
Uniformity of dosage units (905)- responses for the pseudoephedrine peaks. Calculate the
FOR ORAL SOLUTION PACKAGED IN SINGLE-UNIT CONTAINERS:
quantity, in mg, of pseudoephedrine hydrochloride
meets the requirements. (C1oH1SNO· HCI) in each mL of the Oral Solution taken by the
Deliverable volume (698)- formula:
FOR ORAL SOLUTION PACKAGED IN MULTIPLE-UNIT CONTAINERS:
meets the requirements. 100(C/V)(r u/rs)
pH (791): between 3.7 and 7.5.
Alcohol Determination, Method /I (611) (if present) : in which C is the concentration, in mg per mL, of USP
between 90.0% and 110.0% of the labeled amount of Pseudoephedrine Hydrochloride RS in the Standard
C2HsOH. preparation; V is the volume, in mL, of the Oral Solution
Microbial enumeration tests (61 ) and Absence of specified taken; and ru and rs are the pseudoephedrine peak responses
microorganisms (62)-The total bacterial count does not obtained from the Assay preparation and the Standard
exceed 100 cfu per g, the total combined molds and yeasts preparation, respectively.
count does not exceed 10 cfu per g, and it meets the Assay for pseudoephedrine sulfate (where
requirements of the tests for absence of Salmonella speciesand pseudoephedrine sulfate is the salt form used, if present in the
Escherichia coli. . formulation)-
Assay for pseudoephedrine hydrochloride (where Mobile phase, System SUitability solutions, and
pseudoephedrine hydrochloride is the salt form used, if Chromatographic system-Proceed as directed in the Assay for
present in the formulation)- pseudoephedrine hydrochloride.
Mobile phase-Prepare a filtered and degassed mixture of Chlorpheniramine standardpreparation-Prepare as
methanol and water (60:40) containing 0.34 g of monobasic directed for Standard preparation in the Assay for
potassium phosphate, 0.15 g of triethylamine hydrochloride, chlorpheniramine maleate.
0.25 g of sodium lauryl sulfate, and 0.1 mL of phosphoric acid Dextromethorphan standardpreparation-Prepare as
in each 100 mL of solution. Make adjustments if necessary(see directed for Standard preparation in the Assay for
System Suitability under Chromatography (621 ». dextromethorphan hydrobromide.
Standardpreparation-Dissolve an accurately weighed
Standardpreparation-Dissolve an accurately weighed
quantity of USP Pseudoephedrine Hydrochloride RS in water quantity of USP Pseudoephedrine Sulfate RS in water to obtain
to obtain a solution having a known concentration of about a solution having a known concentration of about 3.0 mg per
1.5 mg per mL. Transfer 1.0 mL of this solution to a 10-mL mL. Transfer 1.0 mL of this solution to a 1O-mL volumetric
volumetric flask, add 8.0 mL of Mobilephase, dilute with water flask, add 4.0 mL of Mobilephase, dilute with water to volume,
to volume, and mix. and mix.
Chlorpheniramine standardpreparation-Prepare as Assay preparation-Transfer an accurately measured
directed for Standardpreparation in the Assay for volume of Oral Solution, equivalent to 30 mg of
chlorpheniramine maleate. pseudoephedrine sulfate, to a 1OO-mL volumetric flask, add
Dextromethorphan standardprepardtion-Prepare as 80.0 mL of Mobilephase, dilute with water to volume, and mix.
directed for Standardpreparation in the Assay for Procedure-Proceed as directed for Procedure in the Assay
dextromethorphan hydrobromide. for pseudoephedrine hydrochloride. Calculate the .quantity, in
System suitability solution 1 (for Oral Solution that contains mg, of pseudoephedrine sulfate [(C 1oH1SNO)2 . H2S04] in each
either all the four ingredients or a combination of three mL of the Oral Solution taken by the formula:
containing chlorpheniramine salt)-Mix equal volumes of the
Standard preparation and the Chlorpheniramine standard 100(C/V)(ru/rs)
preparation.
System suitability solution 2 (for Oral Solution that contains in which the terms are as defined therein, pseudoephedrine
no chlorpheniramine salt)-Mix equal volumes of the sulfate being substituted for pseudoephedrine hydrochloride.

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58 Acetaminophen / Official Monographs USP 43

Assay for acetaminophen (if present)- Standardpreparation-Dissolve an accurately weighed

Mobile phase-Prepare a suitable degassed and filtered quantity of USP Dextromethorphan Hydrobromide RS in water
mixture of water, methanol, and glacial acetic acid (79:20:1). to obtain a solution having a known concentration of about
Make any necessary adjustments (see System SUitability under 1.5 mg per mL. Transfer 5.0 mL of this solution to a 100-mL
Chromatography (621 »). volumetric flask, add 80 mL of Mobilephase, dilute with water
Standardpreparation-Transfer about 16.5 mg of USP to volume, and mix.
Acetaminophen RS, accurately weighed, to a 100-mL Assay preparation-Transfer an accurately measured
volumetric flask. Add 2.5 mL of methanol, and mix until volume of Oral Solution, equivalent to about 7.5 mg of
solution is complete. Dilute with water to volume, and mix to dextromethorphan hydrobromide, to a 1OO-mL volumetric
obtain a solution having a known concentration of about flask, add 80 mL of Mobilephase, dilute with water to volume,
0.165 mg per mL. and mix.
Assay preparation-Transfer an accurately measured Procedure-Separately inject equal volumes (about 10 IJL)
volume of Oral Solution, equivalent to about 33 mg of of the Standardpreparation and the Assay preparation into the
acetaminophen, to a 200-mL volumetric flask, add 5 mL of chromatograph, record the chromatograms, and measure the
methanol, and mix. Dilute with water to volume, and mix. responsesfor the dextromethorphan peaks. Calculate the
Chromatographic system (see Chromatography (621»)-The quantity, in mg, of dextromethorphan hydrobromide
liquid chromatograph is equipped with a 280-nm detector (C,sH2SNO· HBr· H20) in each mL of the Oral Solution taken
and a 4.6-mm x 15-cm column that contains packing L7. The by the formula:
flow rate is about 1 mL per minute. Chromatograph the
Standardpreparation, and record the peak responses as (370.33/352.32)(100C/V)(rvlrs)
directed for Procedure: the tailing factorfor the acetaminophen
peak is not greater than 2.0; and the relative standard in which 370.33 and 352.32 are the molecular weights of
deviation for replicate injections is not more than 2.0%. dextromethorphan hydrobromide monohydrate and
Procedure-Separately inject equal volumes (about 10 IJL) anhydrous dextromethorphan hydrobromide, respectively; C
of the Standardpreparation and the Assay preparation into the is the concentration, in mg per mL, of USP Dextromethorphan
chromatograph, record the chromatograms, and measure the Hydrobromide RS in the Standardpreparation; Vis the volume,
responses for the acetaminophen peaks. Calculate the in mL, of the Oral Solution taken; and to and ts are the
quantity, in mg, of acetaminophen (CSH 9N02) in each mL of .dextromethorphan peak responses obtained from the Assay
the Oral Solution taken by the formula: preparation and the Standardpreparation, respectively.

200( C/V)(rvirs)

in which C is the concentration, in mg per mL, of USP

Acetaminophen RS in the Standardpreparation; V is the Tablets Containing at Least Three of the
volume, in mL, ofthe Oral Solution taken; and rv and rs are the
acetaminophen peak responses obtained from the Assay
Following-Acetaminophen and Salts of
preparation and the Standardpreparation, respectively. Chlorpheniramine, Dextromethorphan,
Assay for chlorpheniramine maleate (if present)- and Pseudoephedrine
Mobilephase and Chromatographic system-Proceed as
directed in the Assay for pseudoephedrine hydrochloride.
Standardpreparation-Dissolve an accurately weighed » Tablets Containing at Least Three of the
quantity of USP Chlorpheniramine Maleate RS in water to Following-Acetaminophen and Salts of
obtain a solution having a known concentration of about 1 mg Chlorpheniramine, Dextromethorphan, and
per mL. Transfer 1.0 mL ofthis solution to a 1OO-mL volumetric Pseudoephedrine contain not less than 90.0
flask, add 80 mL of Mobilephase, dilute with water to volume, percent and not more than 110.0 percent of the
and mix.
Assay preparation-Transfer an accurately measured labeled amounts of acetaminophen (CsH9N02),
volume of Oral Solution, equivalent to about 1 mg of chlorpheniramine maleate (C16H19CIN2 . C4H404) ,
chlorpheniramine maleate, to a 1OO-mL volumetric flask. Add dextromethorphan hydrobromide (C1sH2SNO .
80 mL of Mobile phase, dilute with water to volume, and rnlx.,
Procedure-Separately inject equal volumes (about 10 IJL) HBr· H20), and pseudoephedrine hydrochloride
of the Standardpreparation and the Assay preparation into the (ClOH 1SNO· HCI) or pseudoephedrine sulfate
chromatograph, record the chromatograms, and measure the [(ClOH 1SNO)2' H2S04] ,
responses for the chlorpheniramine peaks. Calculate the
quantity, in mg, of chlorpheniramine maleate (C16H19CIN2 .
[NOTE-The headin9 of this rnonoqraph does not
C4H 404) in the Oral Solution taken by the formula:
constitute the official title. It is notintended that
the name described herein be recognized as the
official title or the common or usual name. The
100(C/V)(rv/ rs) name for each article encompassed by this
in which C is the concentration, in mg per mL, of USP
monograph shall be composed of the names of the
Chlorpheniramine Maleate RS in the Standardpreparation; V
active inqredlents contained therein, as well as the
is the volume, in mL, of the Oral Solution taken; and rv and ts
quantitative amount of each active ingredient, and
a statement of the function (or purpose) of the
are the chlorpheniramine peak responses obtained from the ingredient in the article.]
Assay preparation and the Standardpreparation, respectively.
Assay for dextromethorphan hydrobromide (if Packaging and storage-Preserve in tight containers, and
present)- store at controlled room temperature.
Mobile phase and Chromatographic system-Proceed as USP Referencestandards (11)-
directed in the Assay for pseudoephedrine hydrochloride. USP Acetaminophen RS

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 USP 43 OfficialMonographs / Acetaminophen 59

USP Chlorpheniramine Maleate RS Assay for pseudoephedrine hydrochloride (where

USP Dextromethorphan Hydrobromide RS pseudoephedrine hydrochloride is the salt form used, if
USP Pseudoephedrine Hydrochloride RS present in the formulation)-
USP Pseudoephedrine Sulfate RS Mobile phase-Prepare a filtered and degassed mixture of
Labeling-The label for each article encompassed by this methanol and water (60:40) containing 0.34 g of monobasic
monograph bears a name composed of the active ingredients. potassium phosphate, 0.3 g of triethylamine hydrochloride,
The label states the name and quantity of each active 0.15 g of sodium lauryl sulfate, and 0.1 mL of phosphoric acid
ingredient and indicates its function (or purpose) in the article. in each 100 mL of solution. Make adjustments if necessary(see
When more than one Dissolution test is given, the labeling System Suitability under Chromatography (621 )).
states the Dissolution test used only if Test 7 is not used. Standard preparation-Dissolve an accurately weighed
quantity of USP Pseudoephedrine Hydrochloride RS in water
Identification-
to obtain a solution having a known concentration of about
A: If pseudoephedrine hydrochloride or pseudoephedrine 3.0 mg per mL. Transfer 1.0 mL of this solution to a 25-mL
sulfate is claimed in the labeling to be present, the volumetric flask, add 2.5 mL of methanol, dilute with 0.1 %
chromatogram of the Assay preparation, obtained as directed phosphoric acid to volume, and mix.
in the Assay for pseudoephedrine hydrochloride or the Assay for Chlorpheniramine standardpreparation-Prepare as
pseudoephedrine sulfate, exhibits a major peak for directed for the Standardpreparation in the Assay for
pseudoephedrine, the retention time of which corresponds to chlorpheniramine maleate.
that exhibited by the Standardpreparation. Dextromethorphan standardpreparation-Prepare as
B: If acetaminophen is claimed in the labeling to be directed for the Standardpreparation in the Assay for
present, the chromatogram of the Assay preparation, obtained dextromethorphan hydrobromide.
as directed in the Assay for acetaminophen, exhibits a major System suitability solution 7 (for Tablets that contain either
peak for acetaminophen, the retention time of which all the four ingredients or a combination of three containing
corresponds to that exhibited by the Standardpreparation. chlorpheniramine salt)-Mix equal volumes of the Standard
C: If chlorpheniramine maleate is claimed in the labeling to preparation and the Chlorpheniramine standard preparation.
be present, the chromatogram of the Assay preparation, System suitability solution 2 (for Tablets that contain no
obtained as directed in the Assay for chlorpheniramine maleate, chlorpheniramine salt)-Mix equal volumes of the Standard
exhibits a major peak for chlorpheniramine, the retention time preparation and the Dextromethorphan standard preparation.
of which corresponds to that exhibited by the Standard Assay preparation-Weigh and finely powder not fewer
preparation. than 20 Tablets. Transfer an accurately weighed quantity of
D: If dextromethorphan hydrobromide is claimed in the the powder, equivalent to about 6 mg of pseudoephedrine
labeling to be present, the chromatogram of the Assay hydrochloride, to a 50-mL volumetric flask. Add 5 mL of
preparation, obtained as directed in the Assay for methanol, and sonicate to disperse the powder. Dilute with
dextromethorphan hydrobromide, exhibits a major peak for 0.1 % phosphoric acid to volume, mix, and filter.
dextromethorphan, the retention time of which corresponds Chromatographic system (see Chromatography(621 ))-The
to that exhibited by the Standardpreparation. liquid chromatograph is equipped with a 214-nm detector
Dissolution, Procedure for a Pooled Sample (711 )-:- and a 4.6-mm x 15-cm column that contains packing L11. The
TEST 1- flow rate is about 2 mL per minute. Chromatograph the
Medium: pH 5.8 phosphate buffer (see Buffer Solutions in Standard preparation, and record the peak responses as
the section Reagents, Indicators, and Solutions); 900 mL. directed for Procedure: the tailing factor for the
Apparatus 2: 50 rpm. pseudoephedrine peak is not greater than 2.5, and the relative
Time: 45 minutes. standard deviation for replicate injections is not more than
Test preparation-Mix 9.0 mL of a filtered portion of the 2.0%. Separately inject about 10 IJLof System SUitability
solution under test with 1.0 mL of 1% phosphoric acid solution 7 or System suitability solution 2, as appropriate. The
solution. resolution, R, between pseudoephedrine and
Procedure-Determine the amounts of pseudoephedrine chlorpheniramine or between pseudoephedrine and
hydrochloride or pseudoephedrine sulfate (as appropriate), dextromethorphan is not less than 2.0.
acetaminophen, chlorpheniramine maleate, and Procedure-Separately inject equal volumes (about 10 IJL)
dextromethorphan hydrobromide dissolved, employing the of the Standardpreparation and the Assay preparation into the
procedures set forth in the Assay for pseudoephedrine chromatograph, record the chromatograms, and measure the
hydrochloride or Assay for pseudoephedrine sulfate, Assay for responses for the pseudoephedrine peaks. Calculate the
acetaminophen, Assay for chlorpheniramine maleate, and Assay quantity, in mg, of pseudoephedrine hydrochloride
for dextromethorphan hydrobromide, respectively, making any (ClOH 1SNO . HC!) in the portion of Tablets taken by the
necessaryvolumetric adjustments. formula:
Tolerances-Not less than 75% (Q) of the labeled amounts
of pseudoephedrine hydrochloride (C lOH15NO . HCI) or
pseudoephedrine sulfate [(C lOH 1SNO)2' H2S04] ,
acetaminophen (CSH 9N02), chlorpheniramine maleate in which C is the concentration, in mg per mL, of USP
Pseudoephedrine Hydrochloride RS in the Standard
(C16H19CIN2' C4H 404) , and dextromethorphan hydrobromide
preparation, and to and rs are the pseudoephedrine peak
(C1sH2SNO· HBr· H20) are dissolved in 45 minutes.
responses obtained from the Assay preparation and the
TEST 2-lf the product complies with this test, the labeling Standard preparation, respectively.
indicates that it meets USP Dissolution Test 2.
Medium: water; 900 mL. Assay for pseudoephedrine sulfate (where
Apparatus, Time, Test preparation, Procedure, and Tolerances- pseudoephedrine sulfate is the salt form used, if present in the
Proceed as directed for Test 7. formulation)-
Uniformity of dosage units (905): meet the requirements. Mobile phase, Chlorpheniramine standard preparation,
Dextromethorphanstandard preparation, System suitability
solutions, and Chromatographic system-Proceed as directed
in the Assay for pseudoephedrine hydrochloride.

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 60 Acetaminophen / OfficialMonographs USP 43

Standard preparation-Dissolve an accurately weighed 0.1% phosphoricacid to obtain a solution having a known
quantity of USP Pseudoephedrine Sulfate RS in water to obtain concentration of about 8 lJg per mL.
a solution havinga known concentration of about 3.0 mg per Assaypreparation-Weigh and finely powder not fewer
mL. Transfer 2.0 mL of this solution to a 25-mL volumetric than 20 Tablets. Transfer an accurately weighed portion ofthe
flask, add 2.5 mL of methanol, dilute with 0.1% phosphoric powder, equivalentto about 2 mg of chlorpheniramine
acid to volume, and mix. maleate, to a 250-mL volumetric flask. Add 25 mL of
Assay preparation-Weigh and finely powder not fewer methanol, and sonicate to dispersethe powder. Add 1 mL of
than 20 Tablets. Transfer an accuratelyweighed quantity of phosphoric acid, dilute with water to volume, mix, and filter.
the powder, equivalentto about 12 mg of pseudoephedrine Procedure-Separately inject equal volumes (about 10 lJL)
sulfate, to a 50-mL volumetric flask. Add 5 mL of methanol, of the Standard preparation and the Assay preparation into the
and sonicate to dispersethe powder. Dilute with 0.1% chromatograph, record the chromatograms, and measurethe
phosphoricacid to volume, mix, and filter. responsesfor the chlorpheniramine peaks. Calculate the
Procedure-Proceed as directed for Procedure in the Assay quantity, in mg, of chlorpheniramine maleate (C16H19CIN2 .
for pseudoephedrine hydrochloride. Calculate the quantity, in C4H 404) in the portion of Tablets taken by the formula:
mg, of pseudoephedrine sulfate [(C1oH1SNO)2 . H2S04] in the
portion of Tablets taken by the formula: 250qrv/ rs)

50qrulrs) in which C is the concentration, in mg per mL, of USP

Chlorpheniramine MaleateRS inthe Standard preparation; and
in which the terms are as defined therein, pseudoephedrine ruand rs are the peakresponsesforchlorpheniramine obtained
sulfate being substituted for pseudoephedrine hydrochloride. from the Assay preparation and the Standard preparation,
Assay for acetaminophen (if present)- respectively.
Mobile phase-Prepare a filtered and degassed mixtureof Assay for dextromethorphan hydrobromide (if
water, methanol, and glacial acetic acid (79:20:1). Make present)-
adjustments, if necessary (see System Suitability under Mobile phaseand Chromatographic system-Proceed as
Chromatography (621». directed in the Assay for pseudoephedrine hydrochloride.
Standard preparation-Transfer about 50 mg of USP . Standard preparation-Dissolve an accuratelyweighed
Acetaminophen RS, accuratelyweighed, to a 100-ml quantity of USP DextromethorphanHydrobromide RS inwater
volumetric flask. Add4 mL of methanol, and mix untilsolution to obtain a solution having a known concentration of about
iscomplete. Dilute with 0.1 % phosphoricacid to volume,and 0.6 mg per mL. Quantitatively dilute a portion of this solution
mix. with 0.1% phosphoric acid to obtain a solution having a
Assay preparation-Weigh and powder not fewer than 20 known concentration of about 0.06 mg per mL.
Tablets. Transfer an accuratelyweighed portion of the Assay preparation-Weigh and finely powder not fewer
powder, equivalentto about 100 mg of acetaminophen, to a than 20 Tablets. Transfer an accurately weighed portion of the
50-mL volumetric flask. Add about 7.5 mL of methanol, and powder, equivalentto about 6 mg of dextromethorphan
sonicate to dispersethe powder. Add 0.5 mL of phosphoric hydrobromide, to a 1OO-mL volumetric flask. Add 10 mL of
acid, dilutewith water to volume,mix,and filter. Transfer 25.0 methanol, and sonicate to dispersethe powder. Add 0.4 mL
mL of the filtered solutionto a 1OO-mL volumetric flask, dilute of phosphoric acid, dilute with water to volume, mix, and
with water to volume, and mix. filter.
Chromatographic system (see Chromatography (621 »-The Procedure-Separately inject equal volumes (about 10 lJL)
liquid chromatograph is equipped with a 2S0-nm detector of the Standard preparation and the Assay preparation into the
and a 4.6-mm x 15-cm column that contains packing L7. The chromatograph, record the chromatograms, and measurethe
flow rate is about 1 mL per minute. Chromatograph the peak responsesforthe dextromethorphan peaks.Calculate the
Standard preparation, and record the peak responses as quantity, in mg, of dextromethorphan hydrobromide
directedfor Procedure: the tailingfactorforthe acetaminophen (C1sH2SNO· HBr· H20) in the portion of Tablets taken by the
peak is not greater than 2.0, and the relative standard formula:
deviation for replicate injections is not more than 2.0%.
Procedure-Separately inject equal volumes(about 10 lJL) (370.33/352.32)(100C)(rv/rs)
of the Standard preparation and the Assay preparation into the
chromatograph, record the chromatograms, and measurethe in which 370.33 and 352.32 are the molecularweights of
responsesfor the acetaminophen peaks. Calculate the dextromethorphan hydrobromide monohydrate and
quantity, in mg, of acetaminophen (CSH 9N02) in the portion anhydrous dextromethorphan hydrobromide, respectively; C
of Tablets taken by the formula: isthe concentration, in mg per mL, of USP Dextromethorphan
Hydrobromide RS in the Standard preparation; and tu and ts
200qrv/rs) are the dextromethorphan peak responses obtained from the
Assay preparation and the Standard preparation, respectively.
in which C is the concentration, in mg per mL, of USP
Acetaminophen RS in the Standard preparation; and tu and rs
are the acetaminophen peak responses obtained from the
Assay preparation and the Standard preparation, respectively.
Assay for chlorpheniramine maleate (if present)-
Acetaminophen, Chlorpheniramine
Mobile phaseand Chromatographic system-Proceed as Maleate, and Dextromethorphan
directed in the Assay for pseudoephedrine hvdrochiorlde. Hydrobromide Tablets
Standard preparation-Dissolve an accuratelyweighed
quantity of USP Chlorpheniramine Maleate RS in water to DEFINITION
obtain a solution having a known concentration of about 0.8 Acetaminophen, Chlorpheniramine Maleate, and
mg per mL. Quantitatively dilute a portion ofthis solutionwith Dextromethorphan Hydrobromide Tablets contain NLT
90.0% and NMT 110.0% of the labeled amount of

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 USP 43 Official Monographs / Acetaminophen 61

acetaminophen (CSH 9N0 2), chlorpheniramine maleate sulfate, and 0.1 mL of phosphoric acid in each 100 mL of
(C16H19CIN2' C4H 404) , and dextromethorphan solution
hydrobromide monohydrate (C1sH2SNO . HBr· H20). Standard stock solution: 0.8 mg/mL of USP
Chlorpheniramine Maleate RS in water ..
IDIENTIFICATlON Standard solution: 8 IJg/mL of USP Chlorphenlrarnine
• A. The retention time of the major peak for acetaminophen Maleate RS in 0.1% phosphoric acid from Standard stock
of the Samplesolution corresponds to that of.the Standard solution
solution, as obtained in the Assayfor Acetaminophen. Sample solution: Nominally 8 IJg/mL of chlorpheniramine
• B. The retention time of the major peak for maleate prepared asfollows. Transfer a portion of
chlorpheniramine of the Sample solution corresponds to powdered Tablets (NLT 20), equivalent to 2 mg ~f
that of the Standard solution, as obtained in the Assayfor chlorpheniramine maleate, to a 250-mL volumetric flask.
ChlorpheniramineMaleate. Add 25 mL of methanol, and sonicate to disperse the
• C. The retention time of the major peak for powder. Add 1 mL of phosphoric acid, dilute with water to
dextromethorphan of the Sample solution corresponds to volume, and filter.
that of the Standard solution, as obtained in the Assayfor Chromatographic system
Dextromethorphan Hydrobromide. (See Chromatography(621), System SUitability.)
ASSAY Mode: LC
• ACETAMINOPHEN
Detector: UV 214 nm
Mobile phase: Methanol, glacial acetic acid, and water Column: 4.6-mm x 15-cm; 5-lJm packing L11
(20:1 :79) . Flow rate: 2 mL/min
Standard solution: 0.5 mg/mL of USP Acetaminophen RS Injection volume: 10 IJL
prepared asfollows. Transferan appropriate amount of USP System suitability
Acetaminophen RS to a suitable volumetric flask and add Samples: Standard solution
methanol using 4% of the final volume. Mi~ unt.i1 solution Suitability requirements
is complete and dilute with 0.1 % phosphoric acid to Tailing factor: NMT 2.5
volume. Relative standard deviation: NMT 2.0%
Sample stock solution: Nominally 2 mg/mL of . Analysis
acetaminophen prepared asfollows. Transfer a portion of Samples: Standard solution and Sample solution
powdered Tablets (NLT 20), equivalent to 100 mg of Calculate the percentage of the labeled amount of.
acetaminophen, to a 50-mL volumetric flask. Add 7.5 mL chlorpheniramine maleate (C,6H,9CIN2 . C4H404) In the
of methanol, and sonicate to disperse the powder. Add 0.5 portion of Tablets taken:
mL of phosphoric acid, dilute with water to volume, and
filter. Result =(ru/rs) x (Cs/Cu) x 100
Sample solution: Nominally 0.5 mg/mL of acetaminophen
from Sample stock solution in water
= peak responseof chlorpheniramine from the
Sample solution
Chromatographic system
(See Chromatography (621), System SUitability.)
= peak response of chlorpheniramine from the
Standard solution
Mode: LC
Detector: UV 280 nm =concentration of USP Chlorpheniramine Maleate
RS in the Standard solution (mg/mL)
Column: 4.6-mm x 15-cm; 5-lJm packing L7
Flow rate: 1 mL/min
=nominal concentration of chlorpheniramine
maleate in the Sample solution (mg/mL)
Injection volume: 10 IJL
System suitability
Acceptance criteria: 90.0%-110.0% of the labeled amount
Sample: Standard solution
of chlorpheniramine maleate (C,6H,9C1N2' C4H404)
Suitability requirements
• DEXTROMETHORPHAN HVDROBROMIDE
Tailing factor: NMT 2.0
Mobile phase, Chromatographic system, and System
Relative standard deviation: NMT 2.0%
Analysis suitability: Proceed as directed in the Assay for
Chlorpheniramine Maleate.
Samples: Standard solution and Sample solution
Standard stock solution: 0.6 mg/mL of USP
Calculate the percentage of the labeled amount of
Dextromethorphan Hydrobromide RS in water
acetaminophen (CSH 9N02) in the portion of Tablets taken:
Standard solution: 0.06 mg/mL of USP Dextromethorphan
Hydrobromide RS in 0.1% phosphoric acid, from Standard
Result = (rufrs) x (Cs/Cu) x 100
stock solution
to =peak response of acetaminophen from the Sample solution: Nominally 0.06 mg/mL of
Samplesolution dextromethorphan hydrobromide prepared as follows.
rs =peak response of acetaminophen from the Transfer a portion of powdered Tablets (NLT 20), .
Standard solution equivalent to 6 mg of dextromethorphan hydrobrornide,
Cs =concentration of USP Acetaminophen RS in the to a 1OO-mL volumetric flask. Add 10 mL of methanol, and
Standard solution (mg/mL) sonicate to dispersethe powder. Add 0.1 mL of phosphoric
Cu =nominal concentration of acetaminophen in the acid, dilute with water to volume, and filter.
Samplesolution (mg/mL) System suitability
Samples: Standard solution
Acceptance criteria: 90.0%-110.0% of the labeled amount Suitability requirements
of acetaminophen (CSH 9N02) Tailing factor: NMT 2.5
• CHLORPHENIRAMINE MALEATE
Relative standard deviation: NMT 2.0%
Mobile phase: Methanol and water (60:40) containing 0.34 Analysis .
9 of monobasic potassium phosphate, 0.3 9 of Samples: Standard solution and Sample solution
triethylamine hydrochloride, 0.15 9 of sodium lauryl

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62 Acetaminophen / Official Monographs USP 43

Calculate the percentage of the labeled amount of the labeling states the Dissolution Test used only if Test 7 is
dextromethorphan hydrobromide monohydrate not used.
(C1sH2SNO· HBr· H20) in the portion of Tablets taken: • USP REFERENCE STANDARDS (11)
USP Acetaminophen RS
Result = (rulrs) x (Cs/Cu) x (M,dM'2) x 100 USP Chlorpheniramine Maleate RS
USP Dextromethorphan Hydrobromide RS
= peak response of dextromethorphan from the
Sample solution .
= peak response of dextromethorphan from the
Standardsolution
= concentration of USP Dextromethorphan Acetaminophen and Codeine
Hydrobromide RS in the Standardsolution Phosphate Capsules
(mg/mL)
= nominal concentration of dextromethorphan DEfiNITION
hydrobromide in the Sample solution (mg/mL) Acetaminophen and Codeine Phosphate Capsules contain
= molecular weight of dextromethorphan NLT 90.0% and NMT 110.0% of the labeled amount of
hydrobromide monohydrate, 370.32 acetaminophen (CsHgN02) and codeine phosphate
= molecular weight of anhydrous (C1sH21N0 3 • H3P04 • VzHzO).
dextromethorphan hydrobromide, 352.32
IDENTIfICATION
Acceptance criteria: 90.0%-110.0% of the labeled amount • A. The retention times of the major peaks of the Sample
of dextromethorphan hydrobromide monohydrate solution correspond to those of the Standardsolution, as
(C1sH2SNO· HBr. H20) obtained in the Assay.
• B. THIN-LAVER CHROMATOGRAPHY
PERfORMANCE TESTS
Standard solution: 12 mg/mL each of USPAcetaminophen
• DISSOLUTION (711), Procedure, Apparatus 7 and Apparatus 2, RS and USP Codeine Phosphate RS in methanol
Immediate-Release Dosage Forms, Procedure for a pooled Sample solution: Transfer a portion of Capsule contents,
sample for immediate-release dosage forms equivalent to 12 mg of codeine phosphate, to a separator.
Test 1 Add 5 mL of water, 1 mL of ammonium hydroxide, and 5
Medium: Water; 900 mL mL of methylene chloride. Shake for 1 min, and allow the
Apparatus 2: 50 rpm layers to separate. Use the clear lower layer.
Time: 45 min Chromatographic system
Sample solution: Mix 9.0 mL of a filtered portion of the (See Chromatography (621), General Procedures, Thin-Layer
solution with 1.0 mL of 1% phosphoric acid solution. Chromatography.)
Analysis: Determine the percentage of the labeled amount Adsorbent: 0.25-mm layer of chromatographic silica gel
of acetaminophen, chlorpheniramine maleate, and mixture
dextromethorphan hydrobromide dissolved, using the Application volume: 10 I..IL .
Analysis set forth in the AssayforAcetaminophen, the Assay Developing solvent system: Methanol and ammonium
for Chlorpheniramine Maleate, and the Assay for hydroxide (49:1)
Dextromethorphan Hydrobromide, respectively, making Analysis .
any necessary volumetric adjustments. Samples: Standardsolution and Sample solution
Tolerances: NLT75% (Q) of the labeled amount of Allow the spots to dry after applying each sample to the
acetaminophen (CsHgN02), chlorpheniramine maleate adsorbent. Develop the chromatogram in the Developing
(C16H19CIN2' C4H404) , and dextromethorphan solvent system until the solvent front has moved
hydrobromide monohydrate (C1sH2SNO· HBr·· H20) is three-fourths of the length of the plate. Remove the plate
dissolved. from the developing chamber, mark the solvent front, and
Test 2: If the product complies with this test, the labeling allow the solvent to evaporate. Locate the spots on the
indicates that it meets USP Dissolution Test 2. plate by examination under short-wavelength UV light.
Medium: 0.1 M hydrochloric acid; 900 mL Acceptance criteria: The R F values of the two principal
Apparatus 2, Time, Sample solution, Analysis, and spots of the Sample solution correspond to those of the
Tolerances: Proceed as directed in Test 7. Standardsolution.
Test 3: If the product complies with this test, the labeling
indicates that it meets USP Dissolution Test 3. ASSAY
Medium: pH 5.8 phosphate buffer (see Reagents, • PROCEDURE
Indicators, and Solutions-Buffer Solutions); 900 mL Solution A: Dissolve 2.04 g of monobasic potassium
Apparatus 2, Time, Sample solution, Analysis, and phosphate in 950 mL of water. Add 2 mL of
Tolerances: Proceed as directed in Test 7. triethylamine, adjust with phosphoric acid to a pH of 2.35,
• UNIFORMITY OF DOSAGE UNITS (905): Meet the and dilute with water to 1000 mL.
requirements Mobile phase: Methanol and Solution A (8:92)
Codeine phosphate standard stock solution: 0.3 mg/mL
IMPURITIES of USP Codeine Phosphate RS in Mobilephase
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG Standard solution: 0.3 mg/mL of USP Acetaminophen RS
PRODUCTS (227): Meet the requirements and 0.3j mg/mL of codeine phosphate in Mobilephase,
ADDITIONAL REQUIREMENTS prepared as follows. Transfer,an appropriate am~u~t of USP
• PACKAGING AND STORAGE: Preserve in tight containers, and Acetaminophen RS and a SUItable volume (rnultlplled by J)
store at controlled room temperature. of Codeine phosphate standardstock solution Ubeing the
• LABELING: The label states the name and quantity of each
ratio of the labeled amount, in mg, of codeine phosphate
active ingredient and indicates its function (or purpose) in to that of acetaminophen) to a suitable volumetric flask.
the article. When more than one Dissolution Test is given, Dilute with Mobile phase to volume.

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USP 43 Official Monographs / Acetaminophen 63

Sample stock solution: Nominally 3.0 mg/mL of Analysis: Determine the percentage of the labeled amount
acetaminophen and 3.0j mg/ml of codeine phosphate of acetaminophen (CSH9NO z) and codeine phosphate
(equivalent to 2.93J mg/mL of anhydrous codeine (ClsHzlN03' H3P04 • V2H zO) dissolved by using the method
phosphate) in Mobile phase, prepared as follows. Transfer a set forth in the Assay, except use 0.01 N hydrochloric acid
portion of the combined contents, equivalent to 300 mg of to prepare the Codeine phosphate standard stocksolution,
acetaminophen, from NLT 20 Capsules, to a 100-mL and make any other necessary volumetric adjustments.
volumetric flask. Add 75 ml of Mobile phase, and sonicate Tolerances: NLT 75% (Q) of the labeled amount of
for 10 min. Dilute with Mobile phase to volume. acetaminophen (CSH9NOz) andcodeine phosphate
Sample solution: Dilute 5.0 mL of the Sample stock solution (ClsHzlN03' H3P04 • V2H zO) is dissolved.
with Mobile phase to 50 mL, and pass a portion through a • UNIFORMITY OF DOSAGE UNITS (905)
filter of 1-JJm pore size. Procedure for content uniformity
Chromatographic system Solution A, Mobile phase, Codeine phosphate standard
(See Chromatography(621), System SUitability.) stock solution, Standard solution, Chromatographic
Mode: lC system, and System suitability: Proceed as directed in
Detector: UV 214 nm the Assay.
Column: 4.6-mm x 25-cm; 5-JJm packing L1 Sample stock solution: Transfer the contents of 1 Capsule
Flow rate: 1.5 mL/min to a 1OO-mL volumetric flask. Add 75 mL of Mobile
Injection volume: 30 JJl phase, and sonicate for 10 min. Dilute with Mobile phase
System suitability to volume.
Sample: Standard solution Sample solution: Dilute 5.0 mL of the Sample stock
Suitability requirements solution with Mobile phase to 50 mL, and pass a portion
Resolution: NlT 2.0 between acetaminophen and through a suitable filter of 1-JJm pore size.
codeine Analysis
Relative standard deviation: NMT 2.0% for Samples: Standardsolution and Sample solution
acetaminophen; NMT 3.0% for codeine Calculate the quantity, in mg, of acetaminophen
Analysis (CSH9NO z) in the Capsule taken:
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of Result = (r vir s) x C s x F
acetaminophen (CSH9NO z) in the portion of Capsules
taken: = peak response of acetaminophen from the
Sample solution
Result = (r vir s) x (C siC v) x 100 = peak response of acetaminophen from the
Standardsolution
= peak response of acetaminophen from the = concentration of USP Acetaminophen RS in the
Sample solution Standardsolution (mg/mL)
= peak response of acetaminophen from the F = dilution volume, 1000 mL
Standardsolution
= concentration of USPAcetaminophen RS in the Calculate the quantity, in mg, of codeine phosphate
Standardsolution (mg/mL) (ClsHzlN03' H3P0 4 • V2H zO) in the Capsule taken:
= nominal concentration of acetaminophen in the
Sample solution (mg/ml) Result = (r vir s) x C s x (M rrlM r2) X F
Calculate the percentage of the labeled amount of codeine = peak response of codeine from the Sample
phosphate (ClsHzlN03 . H3P0 4 • V2H zO) in the portion of solution
Capsules taken: = peak response of codeine from the Standard
solution
Result = (r vir s) x (C siC v) x (M ,tiM (2) x 100 = concentration of USP Codeine Phosphate RS in
the Standardsolution (mg/mL)
= peak response of codeine from the Sample M r1 = molecular weight of codeine phosphate, 406.37
solution = molecular weight of anhydrous codeine
= peak response of codeine from the Standard Mr2
phosphate, 397.37
solution F = dilution volume, 1000 mL
= concentration of USP Codeine Phosphate RS in
the Standardsolution (mg/mL) Acceptance criteria: Meet the requirements
= nominal concentration of codeine phosphate in
the Sample solution (mg/mL) IMPURITIES
M'1 = molecular weight of codeine phosphate, 406.37 • 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
M,2 = molecular weight of anhydrous codeine PRODUCTS (227): Meet the requirements
phosphate, 397.37 ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
Acceptance criteria
containers, and store at controlled room temperature.
Acetaminophen: 90.0%-110.0%
• USP REFERENCE STANDARDS (11)
Codeine phosphate: 90.0%-110.0% USPAcetaminophen RS
PERFORMANCE TESTS USP Codeine Phosphate RS
• DISSOLUTION (711)
Medium: 0.01 N hydrochloric acid; 900 ml
Apparatus 2: 50 rpm
Time: 30 min

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64 Acetaminophen / OfficialMonographs USP43

= peak response of acetaminophen from the

Acetaminophen and Codeine Sample solution
Phosphate Oral Solution =peak response of acetaminophen from the
Standard solution
DEFINITION = concentration of USP Acetaminophen RS in the
Acetaminophen and Codeine Phosphate Oral Solution Standard solution (mgfmL)
contains NLT90.0% and NMT 110.0% of the labeled =nominal concentration of acetaminophen in the
amount of acetaminophen (CSH9NO~ and codeine Sample solution (mg/mL)
phosphate hemihydrate (ClsHzlN03' H3P04 • Y2H zO).
Acceptance criteria: 90.0%-110.0%
IDENTIFICATION • CODEINE PHOSPHATE
• A. The retention times of the major peaks of the Sample Mobile phase: Dissolve 4.44 g of docusate sodium in 1000
solutions correspond to those of the Standard solutions, as mL of a mixture of methanol, tetrahydrofuran, phosphoric
obtained in the Assays for Acetaminophen and Codeine acid, and water (600:40:1 :360) with stirring, and pass
Phosphate. through a membrane filter of 0.45-lJm or finer pore size.
• B. THIN-LAYER CHROMATOGRAPHY Diluent: Methanol and water (3:7)
Standard solution: 12 mgfmL each of USP Acetaminophen Standard solution: 0.12 mg/mL of USP Codeine Phosphate
RS and USP Codeine Phosphate RS in methanol RS in Diluent
Sample solution: Transfer a volume of Oral Solution, Sample solution: Nominally 0.12 mg/mL of codeine
equivalent to 12 mg of codeine phosphate, to a separator. phosphate hemihydrate in Diluent, prepared by adding a
Add 1 mL of ammonium hydroxide and 5 mL of methylene volume of Oral Solution, equivalent to 12 mg of codeine
chloride. Shake for 1 min, and allow the layers to separate. phosphate hemihydrate, to a 1OO-mL volumetric flask.
Use the clear lower layer. Dilute with Diluent to volume.
Developing solvent system: Methanol and ammonium Chromatographic system
hydroxide (49:1) (See Chromatography (621), System Suitability.)
Chromatographic system Mode: LC
(See Chromatography (621), Thin-Layer Chromatography.) Detector: UV 280 nm
Mode: TLC Column: 3.9-mm x 30-cm; packing L1
Adsorbent: 0.25-mm layer of chromatographic silica gel Flow rate: 1.5 mLfmin
mixture Injection volume: 10 IJL
Application volume: 10 IJL System suitability
Analysis Sample: Standard solution
Samples: Standard solution and Sample solution Suitability requirements
Develop the chromatogram in the Developing solvent Tailing factor: NMT 2.0
system until the solvent front has moved three-fourths of Relative standard deviation: NMT 3.0%
the length of the plate. Locate the spots on the plate by Analysis
examination under short-wavelength UV light. Samples: Standard solution and Sample solution
Acceptance criteria: The RF values of the two principal spots Calculate the percentage of the labeled amount of codeine
of the Sample solution correspond to those of the Standard phosphate hemihydrate (ClsHzlN03' H3P0 4 • VzHzO) in
solution. the portion of Oral Solution taken:
ASSAY
Result =(rufrs) x (CslC u) x (M,tlM'2) X 100
• ACETAMINOPHEN
Mobile phase: Methanol and water (3:7) = peak response of codeine phosphate from the
Standard solution: 0.48 mgfmL of USP Acetaminophen RS Sample solution
in Mobile phase = peak response of codeine phosphate from the
Sample solution: Nominally 0.48 mq/rnl, of Standard solution
acetaminophen in Mobile phase, prepared by adding a = concentration of USP Codeine Phosphate RS in
volume of Oral Solution, equivalent to 120 mg of the Standard solution (mgfmL)
acetaminophen, to a 250-mL volumetric flask. Dilute with = nominal concentration of codeine phosphate in
Mobile phase to volume. the Sample solution (mg/mL)
Chromatographic system = molecular weight of codeine phosphate
(See Chromatography (621), System Suitability.) hemihydrate, 406.37
Mode: LC = molecular weight of anhydrous codeine
Detector: UV 280 nm phosphate, 397.37
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 2 mt/rnln Acceptance criteria: 90.0%-110.0%
Injection volume: 10 IJL
System suitability PERFORMANCE TESTS
Sample: Standard solution • UNIFORMITY OF DOSAGE UNITS (905)
Suitability requirements For single-unit containers
Tailing factor: NMT 1.5 Acceptance criteria: Meets the requirements
Relative standard deviation: NMT 2.0% • DELIVERABLE VOLUME (698)
Analysis For multiple-unit containers :
Samples: Standard solution and Sample solution Acceptance criteria: Meets the requirements
Calculate the percentage of the labeled amount of
acetaminophen (CSH9NOz) in the portion of Oral Solution IMPURITIES
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
taken: PRODUCTS (227): Meets the requirements

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 USP 43 OfficialMonographs / Acetaminophen 65

SPECIFICTESTS volumetric flask. Dissolve in and dilute with Diluent to

• pH (791): 4.0-6.1 volume.
• ALCOHOL DETERMINATION (611), Method /I (if present): System suitability stock solution: 0.02 mg/mL of sodium
90.0%-120.0% of the labeled quantity of alcohol benzoate and 0.03 mg/mL of methylparaben in Diluent
(CzHsOH), acetone being used as the internal standard System suitability solution: Transfer10.0 mLof the System
suitability stock solution to a 50-mLvolumetric flask, add
ADDITIONAL REQUIREMENTS 10.0 mLof Standard stock solution, and dilute with Mobile
• PACKAGING. AND STORAGE: Preserve in tight, .light-resistant phase to volume.
containers, and store at controlled room temperature. Standard solution: 0.01 mg/mL of USP Codeine
• USP REFERENCE STANDARDS (11) Phosphate RS and 0.01} mg/ml of USP Acetaminophen RS
USP Acetaminophen RS in Mobile phase. Prepare by diluting 10.0 mLof the Standard
USP Codeine Phosphate RS stock solution with Mobile phase to 50 mL in a volumetric
flask.
Sample stock solution: Nominally 0.5 mg/mL of
acetaminophen and 0.5 mg/mL of codeine phosphate
Acetaminophen and Codeine hemihydrate in Diluent prepared as follows. Transfera
measured volume of well-mixed Oral Suspension,
Phosphate Oral Suspension equivalent to 50 mg of acetaminophen, to a 100-mL
volumetric flask. Add 50 mLof Diluent, and mix by
DEFINITION mechanical means for 30 min. Dilutewith Diluent to
Acetaminophen and Codeine Phosphate Oral Suspension is a volume. Foaming may be minimized by adding a few drops
suspension of Acetaminophen and Codeine Phosphate in a of acetonitrile before diluting with Diluent to volume.
suitable aqueous vehicle. It contains NlT 90.0% and NMT Centrifuge a portion of this mixture.
110.0% of the labeled amount of acetaminophen (CSH9NOz) Sample solution: Dilute 10.0 mLof the clear supernatant
and codeine phosphatehemihydrate (C1sH z,N0 3 • H3P04 • from the Sample stock solution with Mobile phase to 50 ml
V2H zO). in a volumetric flask.
Chromatographic system
IDENTIFICATION (See Chromatography (621), System SUitability.)
• A. The retention times of the major peaks of the Sample Mode: LC
solution correspond to those of the Standard solution, as Detector: UV 220 nm
obtained in the Assay. Column: 4.6-mm x 15-cm; 5-J.lm packing L11
• B. THIN-LAYER CHROMATOGRAPHY Flow rate: 2 mL/min
Standard solution: 12 mg/ml each of USP Acetaminophen Injection volume: 20 J.ll
RS and USP Codeine Phosphate RS in methanol System suitability
Sample solution: Transfera volume of Oral Suspension, Samples: System suitability solution and Standard solution
equivalent to 12 mg of codeine phosphate, to a separator. [NOTE-The relative retention times for
Add 1 ml of ammonium hydroxide and 5 ml of methylene acetaminophen, benzoate, codeine, and
chloride, shake for 1 min, and allow the layersto separate. methylparaben are about 0.25, 0.5, 1.0, and 1.3,
Use the clear lower layer. respectively.]
Chromatographic system Suitability requirements
(See Chromatography (621), Thin-Layer Chromatography.) Resolution: .NLT 2 between each pair of adjacent peaks,
Adsorbent: 0.25-mm layer of chromatographic silica gel System suitability solution
mixture Tailing factor: NMT2 for each analyte peak, Standard
Developing solvent system: Methanol and ammonium solution
hydroxide (49:1) Relative standard deviation: NMT 2.0%, Standard
Application volume: 10 J.ll solution
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standard solution and Sample solution
Develop the chromatogram in the Developing solvent Calculate the percentage of the labeled amount of
system until the solvent front has moved three-fourths of acetaminophen (CSH9NO z) in the portion of Oral
the length of the plate. locate the spots on the plate by Suspension taken:
examination under short-wavelength UV light.
Acceptance criteria: The RF values of the two principal spots Result =(ru/rs) x (CslCu) x 100
of the Sample solution correspond to those of the Standard
solution. =peak response of acetaminophen from the
Sample solution
ASSAY = peak response of acetaminophen from the
• PROCEDURE Standard solution
Diluent: Methanol and 0.01 N sodium hydroxide (30:70) = concentration of USP Acetaminophen RS in the
Mobile phase: Dissolve 4.9 g of monobasic potassium Standard solution (mg/ml)
phosphate in 900 ml of water, adjust with phosphoric acid = nominal concentration of acetaminophen in the
to a pH of 3.9, and add 216 mg of sodium Sample solution (mg/ml)
l-octanesulfonate. Add 100 ml of acetonitrile, and filter.
Codeine phosphate standard stock solution: 0.5 mg/ml Acceptance criteria: 90.0%-110.0%
of USP Codeine Phosphate RS in Diluent Calculate the percentage of the labeled amount of codeine
Standard stock solution: Transfer a quantity of 5}mg of phosphate hemihydrate (C,sH z,N0 3 • H3P04 ·VzHzO) in
USP Acetaminophen RS Ubeing the ratio of the labeled the portion of Oral Suspension taken:
amount, in mg, of acetaminophen to the labeled amount,
in mg, of codeine phosphate hemihydrate) and 10.0 ml of
Codeine phosphate standard stock solution to a 100-ml

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66 Acetaminophen / OfficialMonographs USP43

=peak response of codeine from the Sample Analysis

solution Samples: Standard solution and Sample solution
= peak response of codeine from the Standard Allow the spots to dry after applying each sample to the
solution adsorbent. Develop the chromatogram in the Developing
=concentration of USP Codeine Phosphate RS in solvent system until the solvent front has moved
the Standard solution(mg/mL) three-fourths of the length of the plate. Remove the plate
= nominal concentration of codeine phosphate from the developing chamber, mark the solvent front, and
hemihydrate in the Sample solution(mg/mL) allow the solvent to evaporate. Locate the spots on the
= molecular weight of codeine phosphate plate by examination under short-wavelength UV light.
hemihydrate, 406.37 Acceptance criteria: The R F values of the two principal
= molecular weight of anhydrous codeine spots of the Sample solution correspond to those of the
phosphate, 397.37 Standard solution.
Acceptance criteria: 90.00/0-110.0% ASSAY
• PROCEDURE
PERFORMANCE TESTS Solution A: Dissolve 2.04 g of monobasic potassium
• UNIFORMITY OF DOSAGE UNITS (905) phosphate in about 950 mL of water. Add 2 mL of
For single-unit containers triethylamine, adjust with phosphoric acid to a pH of 2.35,
Acceptance criteria: Meets the requirements and dilute with water to 1000 mL.
• DELIVERABLE VOLUME (698) Mobile phase: Methanol and Solution A (8:92)
For multiple-unit containers Codeine phosphate standard stock solution: 0.3 mg/mL
Acceptance criteria: Meets the requirements of USPCodeine Phosphate RS in Mobile phase
IMPURITIES
Standard solution: 0.3 mg/mL of USP Acetaminophen RS
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
and 0.3} mg/mL of codeine phosphate in Mobilephase,
PRODUCTS (227): Meets the requirements
prepared as follows. Transfer an appropriate amount of USP
Acetaminophen RS and a suitable volume (multiplied by J)
SPECIFIC TESTS of Codeine phosphate standardstock solution U being the
• pH (791): 4.0-6.1 ratio of the labeled amount, in mg, of codeine phosphate
to that of acetaminophen) to a 1OO-mL volumetric flask.
ADDITIONAL REQUIREMENTS Dilute with Mobilephase to volume.
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Sample stock solution: Nominally 3.0 mg/mL of
containers, and store at controlled room temperature. acetaminophen and 3.0} mg/mL of codeine phosphate
• USP REFERENCE STANDARDS (11) (equivalent to 2.93} mg/mL of anhydrous codeine
USP Acetaminophen RS phosphate) in Mobilephase, prepared as follows. Transfer a
USP Codeine Phosphate RS portion of the powder (equivalent to 300 mg of
acetaminophen, from NLT 20 finely powdered Tablets) to
a 1OO-mL volumetric flask. Add 75 mL of Mobilephase, and
sonicate for 10 min. Dilute with Mobilephase to volume.
Acetaminophen and Codeine Sample solution: Dilute 5.0 mL of the Sample stock solution
with Mobilephase to 50 mL, and pass a portion of the
Phosphate Tablets solution through a suitable filter of t-urn pore size.
Chromatographic system
DEFINITION (See Chromatography (621), System Suitability.)
Acetaminophen and Codeine Phosphate Tablets contain NLT Mode: LC
90.0% and NMT 110.0% of the labeled amountof Detector: UV214 nm
acetaminophen (CsHgN0 2) and codeine phosphate Column: 4.6-mm x 25-cm; 5-J.Jm packing L1
(ClsH21N03' H3P0 4 • V2H 2 0 ). Flow rate: 1.5 mL/min
Injection volume: 30 J.JL
IDENTIFICATION System suitability
• A. The retention times of the major peaks of the Sample Sample: Standard solution
solution correspond to those of the Standard solution, as Suitability requirements
obtained in the Assay. Resolution: NLT 2.0 between acetaminophen and
• B. THIN-LAYER CHROMATOGRAPHY codeine
Standard solution: 12 mg/mL each of USP Acetaminophen Relative standard deviation: NMT 2.0% for
RS and USP Codeine Phosphate RS in methanol acetaminophen; NMT 3.0% for codeine
Sample solution: Transfer a quantity of finely powdered Analysis
Tablets, equivalent to 12 mg of codeine phosphate, to a Samples: Standard solution and Sample solution
separator. Add 5 mL of water, 1 mL of ammonium Calculate the percentage of the labeled amount of
hydroxide, and 5 mL of methylene chloride. Shake for 1 acetaminophen (CsHgN02) in the portion of Tablets taken:
min, and allow the layers to separate. Use the clear lower
layer. Result =(r vir s) x (C siC v) x 100
Chromatographic system
(See Chromatography (621), General Procedures, Thin-Layer = peak response of acetaminophen from the
C;hromatography.) Sample solution
Adsorbent: 0.25-mm layer of chromatographic silica gel = peak response of acetaminophen from. the
mixture Standard solution
Application volume: 10 J.JL = concentration of USPAcetaminophen RS in the
Developing solvent system: Methanol and ammonium Standard solution (mg/mL)
hydroxide (49:1) Cu = nominal concentration of acetaminophen in the
Sample solution (mg/mL)

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USP 43 OfficialMonographs / Acetaminophen 67

Calculate the percentage of the labeled amount of codeine rs = peak response of codeine from the Standard
phosphate (ClsH21N03 . H3P04 • lhH20) in the portion of solution
Tablets taken: Cs = concentration of USP Codeine Phosphate RS in
the Standard solution(mg/mL)
Result = (r vir s) x (C siC v) x (M ,dM '2) x 100 M'I = molecularweight of codeine phosphate, 406.37
M ,2 = molecular weight of anhydrous codeine
ru = peak response of codeine from the Sample phosphate, 397.37
solution F =dilution volume, 1000 mL
rs = peak response of codeine from the Standard
solution Acceptance criteria: Meet the requirements
Cs = concentration of USP Codeine Phosphate RS in
the Standardsolution (mg/mL) IMPURITIES
Cu = nominal concentration of codeine phosphate in • 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
PRODUCTS (227): Meet the requirements
the Sample solution(mg/mL)
M'I = molecular weight of codeine phosphate, 406.37 ADDITIONAL REQUIREMENTS
M ,2 = molecular weight of anhydrous codeine • PACKAGING AND STORAGE: Preserve in tight, light-resistant
phosphate, 397.37 containers, and store at controlled room temperature.
• USP REFERENCE STANDARDS (11)
Acceptance criteria USP Acetaminophen RS
Acetaminophen: 90.00/0-110.0% USP Codeine Phosphate RS
Codeine phosphate: 90.0%-110.0%
PERFORMANCE TESTS
• DISSOLUTION (711)
Medium: 0.01 N hydrochloric acid; 900 mL Acetaminophen, Dextromethorphan
Apparatus 2: 50 rpm
Time: 30 min Hydrobromide, Doxylamine Succinate,
Analysis: Determine the percentage of the labeled amount and Pseudoephedrine Hydrochloride
of acetaminophen (CSH9N0 2) and codeine phosphate
(ClsH21N03' H3P04 • V2H 20) dissolved by usingthe method Oral Solution
set forth in the Assay, except use 0.01 N hydrochloric acid
to prepare the Codeine phosphate standardstock solution DEFINITION
and to make any other necessary volumetric adjustments. Acetaminophen, Dextromethorphan Hydrobromide,
Tolerances: NLT 75% (Q) of the labeled amount of Doxylamine Succinate, and PseudoephedrineHydrochloride
acetaminophen (CSH9N02) and codeine phosphate Oral Solution contains NLT 90.0% and NMT 110.0% of the
(ClsH21N03' H3P04 ·lhH 20) is dissolved. labeled amount of acetaminophen (CSH9N0 2),
• UNIFORMITV OF DOSAGE UNITS (905) dextromethorphan hydrobromide (C1sH2SNO . HBr· H20),
Procedure for content uniformity doxylamine succinate (C17H22N 20 . C4H604) , and
Solution A, Mobile phase, Codeine phosphate standard pseudoephedrine hydrochloride (C10H1SNO . HCI).
stock solution, Standard solution, Chromatographic IDENTIFICATION
system, and System suitability: Proceed as directed in • A. The retention time of the acetaminophen peak of the
the Assay. .
Sample solution corresponds to that of the Standard
Sample stock solution: Transfer 1 Tablet to a 100-mL solution, as obtained in the Assay for Acetaminophen.
volumetric flask. Add 75 mL of Mobilephase, and sonicate • B. The retention time of the dextromethorphan peak of the
for 10 min. Dilute with Mobilephase to volume. Sample solution correspondsto that of the Standard
Sample solution: Dilute 5.0 mL of the Sample stock solution, as obtained in the Assay for Dextromethorphan
solution with Mobile phase to 50 mL, and pass a portion Hydrobromide.
through a suitable filter of t-urn pore size. • C. The retention time of the doxylamine peak of the
Analysis Sample solution corresponds to that of the Standard
Samples: Standardsolution and Sample solution solution, as obtained in the Assay for Doxylamine Succinate.
Calculatethe quantity, in mg, of acetaminophen • D. The retention time of the pseudoephedrine peak of the
(CSH9N0 2) in the Tablet taken: Sample solution corresponds to that of the Standard
solution, as obtained in the Assay for Pseudoephedrine
Result = (r ulr s) x C s x F Hydrochloride.
ru = peak response of acetaminophen from the ASSAY
Sample solution • ACETAMINOPHEN
rs = peak response of acetaminophen from the Mobile phase: Methanol and water (45:55)
Standard solution Standard solution: 0.2 mg/mL of USP Acetaminophen RS
Cs =concentration of USP Acetaminophen RS in the in Mobilephase
Standard solution (mg/mL) Sample solution: Nominally 0.2 mg/mL of acetaminophen
F = dilution volume, 1000 mL from a volume of Oral Solution in Mobilephase prepared as
follows. Dilute a volume of Oral Solution, equivalentto
Calculate the quantity, in mg, of codeine phosphate about 200 mg of acetaminophen, in Mobilephase.
(ClsH21N03' H3P04 • V2H 20) in the Tablet taken: Chromatographic system
(See Chromatography (621), System Suitability.)
Result =(r ulr s) x C s x (M ,dM '2) x F Mode: LC
Detector: UV 254 nm
ru = peak response of codeine from the Sample Column: 4.6-mm x 25-cm; packing L1
solution

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68 Acetaminophen / OfficialMonographs USP 43

Flow rate: 1 mL/min =concentration of USP Dextromethorphan

Injection volume: 10 J.JL Hydrobromide RS in the Standard solution
System suitability (mg/mL)
Sample: Standard solution Cv =nominal concentration of dextromethorphan
Suitability requirements hydrobrornide-ln the Sample solution (mg/mL)
Tailing factor: NMT 2.0 for the acetaminophen peak M" = molecular weight of dextromethorphan
Relative standard deviation: NMT 2.0% hydrobromide monohydrate, 370.32
Analysis = molecular weight of anhydrous
Samples: Standard solution and Sample solution dextromethorphan hydrobromide, 352.32
Calculate the percentage of the labeled amount of
acetaminophen (CSH9N02) in the portion of Oral Solution Acceptance criteria: 90.0%-110.0% of the labeled amount
taken: of dextromethorphan hydrobromide (C 1sH2SNO· HBr·
H20)
Result = (rv/r s) x (CsICv) x 100 • DOXYLAMINE SUCCINATE
Solution A, Mobile phase, Standard solution,
rv = peak response of acetaminophen from the Chromatographic system, and System suitability:
Sample solution Proceed as directed in the Assay for Dextromethorphan
r, = peak response of acetaminophen from the Hydrobromide.
Standard solution Sample solution: Nominally 0.04 mg/mL of doxylamine
Cs = concentration of USP Acetaminophen RS in the succinate from a volume of Oral Solution in Mobilephase
Standard solution (mg/mL) prepared as follows. Dilute a volume of Oral Solution,
Cv = nominal concentration of acetaminophen in the equivalent to about 2 mg of doxylamine succinate, in
Sample solution (mg/mL) Mobile phase.
Analysis
Acceptance criteria: 90.0%-110.0% of the labeled amount Samples: Standard solution and Sample solution
of acetaminophen (CSH9NO~ Calculate the percentage of the labeled amount of
• DEXTROMETHORPHAN HYDROBROMIDE doxylamine succinate (C17H 22N20 . C4H604 ) in the portion
Solution A: 6.8 gIL of monobasic potassium phosphate in of Oral Solution taken:
water
Mobile phase: Acetonitrile and Solution A (45:55) Result = (rvlrs) x (Cs/Cv) x 100
Standard solution: 0.1 mg/mL of USP Dextromethorphan
Hydrobromide RS, 0.04 mg/mL of USP Doxylamine tu =peak response of doxylamine from the Sample
Succinate RS, and 0.2 mg/mL of USP Pseudoephedrine solution
Hydrochloride RS in Mobilephase rs =peak response of doxylamine from the Standard
Sample solution: Nominally 0.1 mg/mL of solution
dextromethorphan hydrobromide from a volume of Oral Cs = concentration of USP Doxylamine Succinate RS in
Solution in Mobilephase prepared as follows. Dilute a the Standard solution(mg/mL)
volume of Oral Solution, equivalent to about 5 mg of Cv =nominal concentration of doxylamine succinate
dextromethorphan hydrobromide, in Mobile phase. in the Sample solution (mg/mL)
Chromatographic system
(See Chromatography (621), System Suitability.) Acceptance criteria: 90.00/0-110.0% of the labeled amount
Mode: LC of doxylamine succinate (C17H22N20 . C4H604)
Detector: UV 220 nm • PSEUDOEPHEDRINE HYDROCHLORIDE
Column: 4.6-mm x 25-cm; packing L9 Solution A, Mobile phase, Standard solution,
Flow rate: 2.5 mL/min Chromatographic system, and System suitability:
Injection volume: 10 J.JL Proceed as directed in the Assay for Dextromethorphan
System suitability Hydrobromide.
Sample: Standard solution Sample solution: Nominally 0.2 mg/mL of
[NOTE-The relative retention times for pseudoephedrine hydrochloride from a volume of Oral
pseudoephedrine, dextromethorphan, and Solution in Mobilephase prepared as follows. Dilute a
doxylamine are 0.38, 0.65, and 1.0, respectively.] volume of Oral Solution, equivalent to about 10 mg of
Suitability requirements pseudoephedrine hydrochloride, in Mobilephase.
Tailing factor: NMT 2.5 for the dextromethorphan, Analysis
doxylamine, and pseudoephedrine peaks Samples: Standard solution and Sample solution
Relative standard deviation: NMT 2.0% for Calculate the percentage of the labeled amount of
dextromethorphan, doxylamine, and pseudoephedrine pseudoephedrine hydrochloride (C1oH1SNO· HCI) in the
Analysis portion of Oral Solution taken:
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of Result = (rvlr s) x (CsICv) x 100
dextromethorphan hydrobromide (C1sH2SNO· HBr· H20)
in the portion of Oral Solution taken: . = peak response of pseudoephedrine from the
Sample solution
Result = (rvlr s) x (CsICv) x (M,,/M'2) x 100 = peak responseof pseudoephedrine from the
Standard solution
ru = peak response of dextromethorphan from the = concentration of USP Pseudoephedrine
Sample solution Hydrochloride RS in the Standardsolution
ts = peak response of dextromethorphan from the (mg/mL) ,
Standard solution Cv = nominal concentration of pseudoephedrine
hydrochloride in the Sample solution (mg/mL)

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USP 43 OfficialMonographs / Acetaminophen 69

Acceptance criteria: 90.0%-110.0% of the labeled amount the powder from NLT 20 finely powdered Tablets,
of pseudoephedrine hydrochloride (C 10H1SNO . HCI) nominally equivalent to an appropriate amount of
acetaminophen, to a suitable volumetric flask. Add 25% of
PERFORMANCE TESTS the total volume of methanol, and shake by mechanical
• UNIFORMITY OF DOSAGE UNITS (905) means for 10 min. Dilute with water to volume.
For single-unit containers Sample solution: Nominally 0.02 mg/mL of
Acceptance criteria: Meets the requirements acetaminophen prepared as follows. Transfer 2.0 mL of
• DELIVERABLEVOLUME (698) Sample stocksolution to a 50-mL volumetric flask, add 5.0
For multiple-unit containers mL of Internal standard solution, and dilute with Mobile
Acceptance criteria: Meets the requirements phase to volume.
IMPURITIES Chromatographic system
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG (See Chromatography (621), System Suitability.)
PRODUCTS (227): Meets the requirements Mode: LC
Detector: UV 254 nm
SPECIFIC TESTS Column: 4.6-mm x 15-cm; 5-~m packing L1
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR Column temperature: 35 ± 0.5 0
SPECIFIED MICROORGANISMS (62): The total bacterial Flow rate: 1 mL/min
count does not exceed 100 du/g, the total combined Injection volume: 10 ~L
molds and yeasts count does not exceed 10 du/g, and it System suitability
meets the requirements of the tests for absence of Sample: Standardsolution
Salmonella species and Escherichia coli. [NoTE-The relative retention times for acetaminophen
• pH (791): 4.5-6.3 and guaifenesin are 0.5 and 1.0, respectively.]
• ALCOHOL DETERMINATION (611), Method" (if present): Suitability requirements
90.00/0-110.0% of the labeled amount of alcohol Resolution: NLT 6.0 between the analyte and internal
(C 2H sOH) standard peaks
Tailing factor: NMT 2 for the analyte peak
ADDITIONAL REQUIREMENTS
Relative standard deviation: NMT 2.5% for the peak
• PACKAGING AND STORAGE: Preserve in tight containers, and
response ratios
store at controlled room temperature. Analysis
• USP REFERENCE STANDARDS (11)
Samples: Standardsolution and Sample solution
USPAcetaminophen RS Calculate the percentage of the labeled amount of
USP Dextromethorphan Hydrobromide RS acetaminophen (C 8H9N02) in the portion of Tablets taken:
USP Doxylamine Succinate RS
USP Pseudoephedrine Hydrochloride RS Result = (Ru/R s) x (Cs/Cu) x 100

Ru = peak response ratio of acetaminophen to the

internal standard from the Sample solution
Acetaminophen and Diphenhydramine Rs =peak response ratio of acetaminophen to the
internal standard from the Standardsolution
Citrate Tablets Cs =concentration of USP Acetaminophen RS in the
Standardsolution (mg/ml)
DEFINITION Cu = nominal concentration of acetaminophen in the
Acetaminophen and Diphenhydramine Citrate Tablets contain Sample solution (mg/mL)
NlT 90.0% and NMT 110.0% of the labeled amount of
acetaminophen (C8H9N02) and diphenhydramine citrate Acceptance criteria: 90.0%-110.0% of the labeled amount
(C17H 21 NO· C6H80 7) . of acetaminophen (C 8H9N02 )
• DIPHENHVDRAMINE CITRATE
IDENTIFICATION Diluent: Methanol and water (1:1)
• A. The retention times of the major peaks of the Sample. Mobile phase: Methanol, water, and glacial acetic acid
solution, obtained in the AssayforAcetaminophen and in the (61:38:1) containing 1.0813 9 of sodium
Assayfor Diphenhydramine Citrate, relative to the retention l-octanesulfonate in each 1000 mL of solution
times of the respective internal standards, correspond to Internal standard solution: 8 mg/mL of xylometazoline
those of the respective Standardsolution. hydrochloride in water
ASSAY Standard solution: 0.38 mg/mL of USP Diphenhydramine
• ACETAMINOPHEN Citrate RS and 0.8 mg/mL of xylometazoline hydrochloride
Mobile phase: Methanol and water (40:60) from Internal standard solution in Diluent
Diluent: Methanol and water (1:4) Sample solution: Nominally 0.38 mg/mL of
Internal standard solution: 8.0 mg/ml of guaifenesin in diphenhydramine citrate prepared as follows. Transfer a
Diluent portion of the powder from NLT 20 finely powdered
Standard stock solution: 0.5 mg/ml of USP Tablets, nominally equivalent to 38 mg of
Acetaminophen RS, prepared asfollows. Transfer 50 mg of diphenhydramine citrate, to a 1OO-mL volumetric flask. Add
USPAcetaminophen RS to a 1OO-ml volumetric flask. 65 mL of Diluent, and shake by mechanical means for 15
Dissolve in 2.5 ml of methanol, and dilute with water to min. Add 5.0 mL of Internal standard solution, and dilute
volume. with Diluent to volume.
Standard solution: 0.02 mg/mL of acetaminophen from Chromatographic system
Standardstocksolution and 0.8 mg/mL of guaifenesin from (See Chromatography (621), System SUitability.)
Internal standard solution, in Mobilephase Mode: LC
Sample stock solution: Nominally 0.5 mg/mL of Detector: UV 265 nm
acetaminophen prepared as follows. Transfer a portion of Column: 3.9-mm x 30-cm; packing L1

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70 Acetaminophen / Official Monographs USP 43

Column temperature: 35 ± 0.5 0

Flow rate: ·1.5 ml/min
Acetaminophen, Diphenhydramine
Injection volume: 50 IJl Hydrochloride, and Pseudoephedrine
System suitability
Sample: Standardsolution Hydrochloride Tablets
[NoTE-The relative retention times for DEFINITION
diphenhydramine citrate and xylometazoline Acetaminophen, Diphenhydramine Hydrochloride, and
hydrochloride are 0.7 and 1.0, respectively.] Pseudoephedrine Hydrochloride Tablets contain NlT 90.0%
Suitability requirements and NMT 110;0% of the labeled amount of acetaminophen
Resolution: NlT 2.5 between the analyte and internal (CsH9N02 ) , diphenhydramine hydrochloride (C17H21NO .
standard peaks
HCI), and pseudoephedrine hydrochloride (C10H 1SNO . HCI).
Tailing factor: NMT 1.7 for the analyte peak
Relative standard deviation: NMT 2.0% for the peak IDENTIFICATION
response ratios • A. The retention time of the acetaminophen peak of the
Analysis Sample solution corresponds to that of the Standard
Samples: Standardsolution and Sample solution solution, as obtained in the Assay for Acetaminophen.
Calculate the percentage of the labeled amount of • B. The retention time of the diphenhydramine peak of the
diphenhydramine citrate (C17 H21NO . C6Hs0 7) in the Sample solution corresponds to that of the Standard
portion of Tablets taken: solution, as obtained in the Assay for Diphenhydramine
Hydrochloride.
Result = (RulR s) x (Cs/Cu) x 100 • C. The retention time of the pseudoephedrine peak of the
Sample solution corresponds to that of the Standard
= peak response ratio of diphenhydramine citrate solution, as obtained in the Assay for Pseudoephedrine
to the internal standard from the Sample solution Hydrochloride.
= peak response ratio of diphenhydramine citrate
to the internal standard from the Standard ASSAY
solution • ACETAMINOPHEN
= concentration of USP Diphenhydramine Citrate Solution A: Transfer 6.8 g of monobasic potassium
RS in the Standardsolution (mg/ml) phosphate to a 1OOO-ml volumetric flask, and add water to
= nominal concentration of diphenhydramine dissolve. Add 2.0 ml of triethylamine, and dilute with water
citrate in the Sample solution (mg/ml) to volume. Adjust with phosphoric acid to a pH of 4.0.
Diluent: Acetonitrile and Solution A (11 :89)
Acceptance criteria: 90.0%-110.0% of the labeled amount Mobile phase: Acetonitrile and Solution A (6:94)
of diphenhydramine citrate (C17H 21NO . C6H s0 7) Standard solution: 25 IJg/ml of USP Acetaminophen RS,
12.5 pq/rnl, of USP Diphenhydramine Hydrochloride RS,
PERFORMANCE TESTS and 30 IJg/ml of USP Pseudoephedrine Hydrochloride RS
• DISSOLUTION (711), Procedure, Apparatus 7 and Apparatus 2, in Diluent
Immediate-Release Dosage Forms, Procedure for a pooled Sample stock solution: Nominally 5 mg/ml of
sample for immediate-release dosage forms acetaminophen in Diluent prepared as follows. Transfer an
Medium: Water; 900 ml amount nominally equivalent to 500 mg of acetaminophen
Apparatus 2: 50 rpm from NlT 20 finely powdered Tablets to a 100-ml
Time: 45 min . volumetric flask, add 75 ml of Diluent, shake, and sonicate
Analysis: Calculate the percentage of the labeled amount of for 15 min. Dilute with Diluent to volume.
acetaminophen (CSH 9N02) and diphenhydramine citrate Sample solution: Nominally 25 IJg/ml of acetaminophen
(C17H 21 NO· C6 Hs0 7) dissolved using the procedures set from the Sample stock solution in Diluent
forth in the Assay for Acetaminophen and the Assay for Chromatographic system
Diphenhydramine Citrate, respectively, and make any (See Chromatography (621), System Suitability.)
necessary volumetric adjustments. Mode: lC
Tolerances: NlT 75% (Q) of the labeled amount of Detector: UV 220 nm
acetaminophen (CSH9N02 ) and diphenhydramine citrate Column: 4.6-mm x 15-cm; packing l10
(C17H21 NO· C6 Hs0 7) is dissolved. Flow rate: 2 ml/min
• UNIFORMITY OF DOSAGE UNITS (905), ContentUniformity: lnlectlon volume: 20 IJl
Meet the requirements with respect to acetaminophen and System suitability
diphenhydramine citrate Sample: Standardsolution
Suitability requirements
IMPURITIES Tailing factor: NMT 2.0 for the acetaminophen,
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG diphenhydramine, and pseudoephedrine peaks
PRODUCTS (227): Meet the requirements Relative standard deviation: NMT 2.0% determined
ADDITIONAL REQUIREMENTS from the acetaminophen, diphenhydramine
• PACKAGING AND STORAGE: Preserve in tight containers, and hydrochloride, and pseudoephedrine hydrochloride
store at controlled room temperature. responses for replicate injections
• USP REFERENCE STANDARDS (11) Analysis
USP Acetaminophen RS Samples: Standardsolution and Sample solution
USP Diphenhydramine Citrate RS Calculate the percentage of the labeled amount of
acetaminophen (CsH9N02) in the portion of Tablets taken:

Result = (ru/rs) x (CslCu) x 100

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USP 43 Official Monographs / Acetaminophen 71

=peak response of acetaminophen from the = peak response of pseudoephedrine from the
Sample solution Standardsolution
= peak response of acetaminophen from the = concentration of USP Pseudoephedrine
Standardsolution Hydrochloride RS in the Standardsolution
= concentration of USP Acetaminophen RS in the (lJg/ mL)
Standardsolution (lJg/mL) = nominal concentration of pseudoephedrine
=nominal concentration of acetaminophen in the hydrochloride in the Sample solution (lJg/mL)
Sample solution (lJg/mL)
Acceptance criteria: 90.0%-110.0% of the labeled amount
Acceptance criteria: 90.00/0-110.0% of the labeled amount of pseudoephedrine hydrochloride (C10H 1SNO . HCI)
of acetaminophen (CsH 9N0 2)
PERFORMANCE TESTS
• DIPHENHYDRAMINE HYDROCHLORIDE
Solution A, Diluent, Mobile phase, Standard solution, • DISSOLUTION (711), Procedure, Apparatus 1 and Apparatus 2,
Chromatographic system, and System suitability: Immediate-Release Dosage Forms, Procedure for a pooled
Proceed as directed in the Assay for Acetaminophen. sample for immediate-release dosage forms
Sample stock solution: Nominally 0.125 mg/mL of Medium: pH 5.8 phosphate buffer (see Reagents, Indicators,
diphenhydramine hydrochloride in Diluent prepared as and Solutions-Buffer Solutions); 900 mL
follows. Transfer an amount nominally equivalent to 12.5 Apparatus 2: 50 rpm
mg of diphenhydramine hydrochloride from a portion of Time: 45 min
finely powdered Tablets (NLT 20) to a 1OO-mL volumetric Solution A, Diluent, Mobile phase, Standard solution, and
flask, add 75 mL of Diluent, and sonicate for 15 min. Chromatographic system: Proceed as directed in the
Dilute with Diluent to volume. Assay for Acetaminophen.
Sample solution: Nominally 12.5 IJg/mL of Sample solution A: Combine equal volumes of the filtered
diphenhydramine hydrochloride from the Sample stock solutions, and use the pooled sample.
solution in Diluent Sample solution B: Transfer 5.0 mL of Sample solutionA to
Analysis a 1OO-mL volumetric flask. Dilute with Mobilephase to
Samples: Standardsolution and Sample solution volume.
Calculate the percentage of the labeled amount of Analysis: Using Sample solutionA and the Standardsolution,
diphenhydramine hydrochloride (C 17H 21 NO . HCI) in the and making any necessary volumetric adjustments,
proceed as directed in the Assay for Diphenhydramine
portion of Tablets taken:
Hydrochloride and the Assay for Pseudoephedrine
Result = (ru/rs) x (Cs/Cu) x 100 Hydrochloride, and determine the percentage of the labeled
amount of diphenhydramine hydrochloride (C17H 21NO·
ru = peak response of diphenhydramine from the HCI) and pseudoephedrine hydrochloride (C1oH 1SNO . HCI)
Sample solution dissolved. Using Sample solution B and the Standard
ts = peak response of diphenhydramine from the solution, and making any necessary volumetric
Standardsolution . adjustments, proceed as directed in the Assay for
Cs = concentration of USP Diphenhydramine Acetaminophen, and determine the percentage of the
Hydrochloride RS in the Standardsolution labeled amount of acetaminophen (CsH9N02) dissolved.
(uq/rnl) Tolerances: NLT 75% (Q) of the labeled amount of
Cv = nominal concentration of diphenhydramine acetaminophen (CsH9N02) , diphenhydramine
hydrochloride in the Sample solution (lJg/mL) hydrochloride (C17H 21NO· HCI), and pseudoephedrine
hydrochloride (C10H1SNO . HCI) is dissolved.
Acceptance criteria: 90.0%-110.0% of the labeled.amount For Tablets labeled as chewable
of diphenhydramine hydrochloride (C17H21NO . HCI) Medium: pH 5.8 phosphate buffer (see Reagents,
• PSEUDOEPHEDRINE HYDROCHLORIDE Indicators, and Solutions-Buffer Solutions); 900 mL
Solution A, Diluent, Mobile phase, Standard solution, Apparatus 2: 75 rpm
Chromatographic system, and System suitability: Time: 45 min
Proceed as directed in the Assay for Acetaminophen. Tolerances: NLT 75% (Q) of the labeled amount of
Sample stock solution: Nominally 0.3 mg/mL of acetaminophen (CsH9N02 ) , diphenhydramine
pseudoephedrine hydrochloride in Diluent prepared as hydrochloride (C17H 21NO· HCI), and pseudoephedrine
follows. Transfer an amount nominally equivalent to 30 mg hydrochloride (ClOH 1SNO . HCI) is dissolved.
of pseudoephedrine hydrochloride from a portion of finely
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
powdered Tablets (NLT 20) to a 100-mL volumetric flask,
requirements
add 75 mL of Diluent, and sonicate for 15 min. Dilute with
Diluent to volume. IMPURITIES
Sample solution: Nominally 30 IJg/mL of pseudoephedrine • 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
hydrochloride from the Sample stock solution in Diluent PRODUCTS (227): Meet the requirements
Analysis
Samples: Standardsolution and Sample solution ADDITIONAL REQUIREMENTS
Calculate the percentage of the labeled amount of • PACKAGING AND STORAGE: Preserve in tight containers, and
pseudoephedrine hydrochloride (C lOH 1SNO . HCI) in the store at controlled room temperature.
• USP REFERENCE STANDARDS (11)
portion of Tablets taken:
USP Acetaminophen RS
Result = (ru/rs) x (Cs/Cu) x 100 USP Diphenhydramine Hydrochloride RS
USP Pseudoephedrine Hydrochloride RS
rv =peak response of pseudoephedrine from the
Sample solution

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72 Acetaminophen / OfficialMonographs USP 43

Acetaminophen and Pseudoephedrine Result = (rvlrs) x (CslCv) x 100

Hydrochloride Tablets tu = peak response of the corresponding analyte

from the Sample solution
DEFINITION rs = peak response of the corresponding analyte
Acetaminophen and Pseudoephedrine Hydrochloride Tablets from the Standardsolution
contain NLT 90.0% and NMT 110.0% of the labeled amount Cs =concentration of the appropriate USP Reference
of acetaminophen (CaH9N02 ) and pseudoephedrine Standard in the Standard solution (mg/mL)
hydrochloride (C 1oH 1SNO . HCI). Cu = nominal concentration of the appropriate analyte
in the Sample solution (mg/mL)
IDENTIFICATION
• A. The retention times of the acetaminophen and Acceptance criteria: 90.0%-110.0% of the labele~ amount
pseudoephedrine peaks of the Sample solution correspond of acetaminophen (CaH9N02) and pseudoephedrine
to those of the Standard solution, as obtained in the Assay. hydrochloride (C 10H 1SNO . HCI)
ASSAY PERFORMANCE TESTS
• PROCEDURE • DISSOLUTION (711), Procedure, Apparatus 1 and Apparatus2,
Diluent: Acetonitrile and water (10:90) Immediate-Release Dosage Forms, Procedure for a pooled
Solution A: 0.005 M ethanesulfonic acid and 0.05 M sample for immediate-release dosage forms
monobasic potassium phosphate . Medium: pH 5.8 phosphate buffer (see Reagents, Indicators,
Mobile phase: Acetonitrile and SolutionA (1OO:?OO): Adjust and Solutions-Buffer Solutions); 900 mL
with 5 N sodium hydroxide or 1 N hydrochloric acid to a Apparatus 2: 50 rpm
pH of 4.6. .• Time: 45 min
Pseudoephedrine hydrochloride standard stock solution: Determine the percentage of the labeled amou~t of
0.6 mg/mL of USP Pseudoephedrine Hydrochloride RS in acetaminophen (CaH9N02) and pseudoephedrine
Diluent
hydrochloride (ClOH 15NO . HCI) dissolved by using the
Standard solution: Transfer 61mg of USP Acetaminophen
RS to a 1OO-mL volumetric flask, 1 being the ratio of the following method.
labeled quantity (mg) of aceta":linophen to th~ la~)eled Mobile phase: Proceed as directed in the Assay.
quantity (mg) of pseudoephedrine hydrochloride In each Standard solution: (L/900) mg/mL of USP
Tablet. Add 2.0 mL of 1 N hydrochloric acid and 20 mL of Pseudoephedrine Hydro~hlorid~ RS and (LI19~0) mg/mL of
Diluent, and mix to dissolve. Add 10.0 mL of USP Acetaminophen RS In MedlU'!1' [NOTE-L IS ~he !abeled
Pseudoephedrine hydrochloridesta!1dard ~tock solut!on and quantity, in mg, of pseudoephedrine hydroc~lorlge In each
dilute with Diluent to volume. This solution contains 0.061 Tablet; and 1 is the ratio of the labele.d q~antlty, In mg, of
mg/mL of USP Acetaminophen RS and 0.06 mg/mL of USP acetaminophen to the labeled quantity, In mg, of
Pseudoephedrine Hydrochloride RS. pseudoephedrine hydrochloride in each Tablet.]
Sample solution: Nominally 0.06 mg/mL of Sample solution: Filtered portion of the solution under test,
pseudoephedrine hydrochloride prepared as follows. SUitably diluted with Medium, if necessary ..
Transfer a portion of finely powdered .Tablets (NLT ~O), Chromatographic system and Syst~~ sultablllty: Proceed
equivalent to 30 mg of pseudoephedrine hydrochloride, to as directed in the Assay, except to Inject the Standard
a 500-mL volumetric flask, add 10.0 mL of 1 N solution.
hydrochloric acid and 100 mL of Diluent, and sonicat~ for Analysis
30 min with occasional shaking. Allow to cool, and dilute Samples: Standardsolution and Sample solution
with DHuent to volume. Pass a portion of this solution [NoTE-Inject 20 J.JL of the Samples, and measure the
through a glassfiber filter, and use the filtrate. . responses for the acetaminophen and
Chromatographic system pseudoephedrine peaks.]
(See Chromatography (621), System Suitability.) Calculate the percentage of the labeled amount. of
Mode: LC acetaminophen (CaH9N02) and pseudoephedrine
Detector: UV 214 nm hydrochloride (ClOH 15NO . HCI) dissolved:
Column: 4.6-mm x 25-cm; base-deactivated or
end-capped packing L1 Result = (rufrs) x V x (CsIL) x 100
Flow rate: 3 mL/min
Injection volume: 10 J.JL = peak response of the corresponding analyte
System suitability from the Sample solution
Sample: Standard solution. . . = peak response of the corresponding analyte
[NOTE-The relative retention times for acetaminophen from the Standardsolution
and pseudoephedrine are about 0.55 and 1.0, V =volume of Medium, 900 mL
respectively.] Cs =concentration of the appropriate USP Reference
Suitability requirements . Standard in the Standard solution (mg/mL)
Resolution: NLT 3.5 between acetaminophen and L = label amount of the corresponding analyte in a
pseudoephedrine Tablet (mg)
Tailing factor: NMT 2 for the pseudoephedrine peak
Tolerances: NLT 75% (Q) of the labeled amount of
Relative standard deviation: NMT 2.0% for replicate
injections acetaminophen (CaH9N02 ) and pseudoephedrine
Analysis hydrochloride (ClOH 1SNO . Hel) is dissolved.
Samples: Standard solution and Sample solution . For Tablets labeled as chewable
Calculate the percentage of the labeled amount. of Medium: pH 5.8 phosphate buffer (s~e Reagents,
acetaminophen (CaH9N02) and pseudoephedrine Indicators, and Solutions-Buffer Solutions); 900 mL
hydrochloride (C lOH 1SNO . HCI) in the portion of Tablets Apparatus 2: 75 rpm
Time: 45 min
taken:

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USP 43 OfficialMonographs / Acetazolamide 73

Standard solution, Sample solution, Chromatographic System suitability

system, System suitability, and Analysis: Proceed as Sample: Standard solution
directed above in Procedure for a pooledsample for Suitability requirements
immediate-release dosage forms. Tailing factor: NMT 1.5
Tolerances: NLT 75% (Q) of the labeled amount of Relative standard deviation: NMT 0.73%
acetaminophen (CgH 9N02) and pseudoephedrine Analysis
hydrochloride (ClOH 1SNO . HCI) is dissolved. Samples: Standard solution and Sample solution
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Calculate the percentage of acetazolamide (C4H6N403S2) in
requirements the portion of Acetazolamide taken:
IMPURITIES Result =(rulrs) x (CsICu) x 100
• 4-AMINOPHENOL IN ACETAMINOPHEN-CONTAINING DRUG
PRODUCTS (227): Meet the requirements = peak response of acetazolamide from the
ADDITIONAL REQUIREMENTS
Sample solution
• PACKAGING AND STORAGE: Preserve in tight containers, and = peak response of acetazolamide from the
store at controlled room temperature. Standard solution
• USP REFERENCE STANDARDS (11)
= concentration of USP Acetazolamide RS in the
USP Acetaminophen RS Standard solution(mg/mL)
USP Pseudoephedrine Hydrochloride RS =concentration of Acetazolamide in the Sample
solution(mg/mL)

Acceptance criteria: 98.0%-102.0% on the anhydrous

basis
Acetazolamide IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1%
• CHLORIDE AND SULFATE (221), Chloride
Sample solution: Digest 1.5 g with 75 mL of water at about
70° for 5 min. Cool to room temperature, and filter.
C4H6N403S2 222.25 Acceptance criteria: A 25-mL portion of the filtrate shows
Acetamide, N-[5-(aminosulfonyl)-1,3,4-thiadiazol-2-yl]-; no more chloride than corresponds to 0.10 mL of 0.020 N
N-(5-Sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide [59-66-5]. hydrochloric acid (0.014%).
• CHLORIDE AND SULFATE (221), Sulfate
DEFINITION Sample solution: A 25-mL portion of the filtrate prepared
Acetazolamide contains NLT 98.0% and NMT 102.0% of in the test for Chloride and Sulfate, Chloride
acetazolamide (C4H6N403S2), calculated on the anhydrous Acceptance criteria: It shows no more sulfate than
basis. corresponds to 0.20 mL of 0.020 N sulfuric acid (0.04%).
• SELENIUM (291)
IDENTIFICATION
Sample: 200 mg
Acceptance criteria: NMT 30 ppm

•. S:'The retention time of the maJor peak of the Sample

solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
• PROCEDURE
Mobile phase: Dissolve 4.1 g of anhydrous sodium acetate
in 950 mL of water, add 20 mL of methanol and 30 mL of
acetonitrile, and mix. Adjust with glacial acetic acid to a pH
of 4.0.
Standard solution: 0.1 mg/mL of USP Acetazolamide RS
prepared as follows. Transfer USP Acetazolamide RS into a
suitable volumetric flask, add 0.5 N sodium hydroxide
equivalent to 10% of the finalvolume, and dilute with water
to volume.
Sample solution: 0.1 mg/mL of Acetazolamide prepared as
follows. Transfer Acetazolamide into a suitable volumetric
flask, add 0.5 N sodium hydroxide equivalent to 10% of the
final volume, and dilute with water to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing L1
Flow rate: 2 mL/min
Injection volume: 20 IJL

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74 Acetazolamide / Official Monographs USP 43

Stanctar,d stock§olution:' 0.4 mgl' Table .. (continued)

Acetazolamide RS in'Mob71eph Rehithie RelatiVE! Accepfance
Transfer a welghedall19unt oa Retention Response Criteria,
suitablevolumetric fla of Name Time Factor NMT(%)
the finalvolume.·Soni Anyunspecified ImpuritY - 1.0 OJ
shaking'todissolv
Standard solution' Tofalimpurities -- - 1.0
in Mobile ph r
a 5-Amino-l ,3,4-t/liadiazole-2-sulfonal11ide.
Sensitivity, so n b S-Acetamido-l ,3,4:thiadiazole~2-sulfonic acid.
RS from Standard eN_ 1 34-Thiadiazol-2-yl)acetaroide.
Sample solution:' d capto-1,3,4-th I)acetamide.
phaseprepared ro:f,3,4-thiad .' cetamide., . . ,. ....' ..
Acetazolamid f N,N'-{5,5'-[(Hydrosulfonylamino)sulfonyl]bis(1,3,4-thii'i~iazole-5,2­
methanol to diyl)}diacetamide.,\ (USP 1·Aug-2019)
min with occas
phaseto volume. SPECIFIC TESTS
ChrortHltographic sys'tem • WATER DETERMINATION (921), Method I: NMT 0.5%
(See Chromatography (62 f); SystemStiitability.j
ADDITIONAL REQUIREMENTS
Mode: LC
• PACKAGING AND STORAGE: Preserve in tight containers, and
Detector: UV 2 nm store at room temperature.
Column: .4.6~m 15-cm; 3.5-J.lrh packing'L10 • USP REFERENCE STANDARDS (11)
Colu' re: 30 9 USPAcetazolamide RS
Flow in
Inje
Run time: NLT 9.5 ti the retention time of
acetazolamide
System suitability .Acetazolamide for Injection
Samples:" Standard solution and Sensitivity solupon
Suitability requirements' . DEFINITION
Tailing factor: N T Acetazolamide for Injection is prepared from Acetazolamide
~elative standar gndard with the aid of sodium hydroxide. It is suitable for parenteral
solution . use. The contents of each container, when constituted as
Signal.,to~l1oiser~tio: ,r\I~T f4;~~nsi~/vlty solytion directed in the labeling, yield a solution containing NLT
Analysis 95.0% and NMT 110.0% of the labeled amount of
Samples:' Standard soltition . ufion acetazolamide (C4H6N403S2)'
Calculate the percentage of e c inthep9rtion of IDENTIFICATION
Acetazolamide taken:
(hange tq read:
Result= (ru/ts) x,(~slCurx (1/F) x100
• A.",SPECTROSCOPIC I
ru = peak area of eachi(1lpurity from the Sample Spectroscopy: 191K,,( 20)
solution . Sample: Dissolve 500 mg in 5 mL of water, add 2 drops of
ts =peak area of acetazola'mideTrom the Standard hydrochloric acid, and allow the mixture to stand for about
sol . 15 min. Filter through a fine sintered-glass funnel, wash
C!s = with several small portions of water, and dry under vacuum
over silica gel for 3 h.
Cu the Sample Acceptance criteria: Meets the requirements
• B. The retention time of the major peak of the Sample
F solution corresponds to that of the Standard solution, as
obtained in the Assay.
• C. IDENTIFICATION TESTS-GENERAL (191), Chemical
Acceptance criteria.: . See Table t. Identification Tests, Sodium: Meets the requirements
Tablet ASSAY
• PROCEDURE
Relative Mobile phase: Dissolve 4.1 9 of anhydrous sodium acetate
Retention
t\lairie Time in 950 mL of water, add 20 mL of methanol and 30 mL of
acetonitrile, and mix. Adjust with glacial acetic acid to a pH
Desacetyl acetazolamidea 0.45 of 4.0.
Acetazolamide acid analogb 0.54 1.0 0.5 Standard solution: 0.1 mg/mL of USP Acetazolamide RS
prepared as follows. Transfer USP Acetazolamide RS to a
Acetamidothiadiazole c 0.82 0.5
1.5 suitable volumetric flask, add 0.5 N sodium hydroxide
Acetazolamide 1;0 1.0 equivalent to 10% of the final volume, and dilute with water
to volume.
Mercaptothiadiazoleanalog d 1.6 0.46 0.5 Sample solution: Nominally 0.1 mg/mL of acetazolamide
Chlorothiadiazole analoge 1.9 1.4 0.5
from Acetazolamide for Injection prepared as follows.
Dissolve the contents of one container of Acetazolamide for
Acetazolamide dimerf 6.3, 1.0 0.5 Injection in a volume of water corresponding to the volume

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USP 43 Official Monographs / Acetazolamide 75

of solvent specified in the labeling. Dilute with water as Suitability requirements

needed. Resolution: NLT 1.5 between acetazolamide related
Chromatographic system compound E and acetazolamide related compound 0,
(See Chromatography (621), System Suitability.) System suitability solution
Mode: LC Relative standard deviation: NMT 5.0%, Standard
Detector: UV 254 nm solution
Column: 4.6-mm x 25-cm; 10-l..Im packing L1 Analysis
Flow rate: 2 mL/min Samples: Standardsolution and Sample solution
Injection volume: 20 I..IL Calculate the percentage of each impurity in the portion of
System suitability Acetazolamide for Injection taken:
Sample: Standardsolution
Suitability requirements Result = (r ul r s) x (C sf C u) x (1 IF) x 100
Tailing factor: NMT 1.5
Relative standard deviation: NMT 1.0% ru = peak area of each impurity from the Sample
Analysis solution
Samples: Standardsolution and Sample solution rs = peak area of acetazolamide from the Standard
Calculate the percentage of the labeled amount of solution
acetazolamide (C4H6N403S2) in the portion of Cs = concentration of USP Acetazolamide RS in the
Acetazolamide for Injection taken: Standardsolution (mg/mL)
Cu = nominal concentration of acetazolamide in the
Result = (r vir s) x (C siC u) x 100 Sample solution (mg/mL)
F = relative response factor (see Table 1)
= peak response of acetazolamide from the
Sample solution Acceptance criteria: See Table 1. Disregard any impurity
= peak response of acetazolamide from the peak less than 0.05%.
Standardsolution
= concentration of USP Acetazolamide RS in the Table 1
Standardsolution (mg/mL) Relative Relative Acceptance
= nominal concentration of acetazolamide in the Retention Response Criteria,
Sample solution (mg/mL) Name Time Factor NMT(%)
Acetazolamide
Acceptance criteria: 95.0%-110.0% related compound E" 0.38 - -
PERFORMANCE TESTS Acetazolamide
• UNIFORMITY OF DOSAGE UNITS (905): Meets the related compound D 0.43 0.70 2.0
requirements Aminothiadiazole
mercaptan- b 0.55 - -
IMPURITIES
• ORGANIC IMPURITIES Acetamldothiadiazolev c 0.77 - -
Buffer: 6.8 giL of monobasic potassium phosphate in water Acetazolamide 1.0 - -
Mobile phase: Acetonitrile and Buffer (10:90)
System suitability solution: 0.16 I..Ig/mL each of USP Mercaptothiadiazole ana-
loga.d 1.4 - -
Acetazolamide Related Compound D RS and USP
Acetazolamide Related Compound E RS in Mobilephase Chlorothiadiazole
Standard stock solution: 0.1 mg/mL of USPAcetazolamide analoga,e 2.2 - -
RS prepared as follows. Transfer a weighed amount of USP Acetazolamidedimer'" f 2.8 - -
Acetazolamide RS to a suitable volumetric flask and add
acetonitrile equivalent to 10% of the final volume and Buffer Any unspecified -
equivalent to 20% of the final volume. Sonicate to dissolve degradation product 1.0 0.20
and dilute with Bufferto volume. Total degradation products - - 3.0
Standard solution: 0.8 I..Ig/mL of USP Acetazolamide RS
from Standardstock solution in Mobilephase a This process impurityis controlled in the drug substance. It is included in the
Sample solution: Nominally 0.4 mg/mL of acetazolamide table for identification only and is not to be reported in the total degradation
products.
from Acetazolamide for Injection prepared as follows. b 5-Amino-l,3,4-thiadiazole-2-thiol.
Dissolve the contents of one container of Acetazolamide for c N-(l ,3,4-Thiadiazol-2-yl)acetamide.
Injection in water corresponding to the volume of solvent d N-(5-Mercapto-l ,3,4-thiadiazol-2-yl)acetamide.
specified in the labeling. Dilute with Mobile phase as e N-(5-Chloro-l ,3,4-thiadiazol-2-yl)acetamide.
needed. f N,N'-{5,5'-[(Hydrosulfonylamino)sulfonyl]bis(1 ,3,4-thiadiazole-5,2-diyl)}
Chromatographic system diacetamide.
(See Chromatography (621), System Suitability.)
Mode: LC SPECIFIC TESTS
Detector: 265 nm • pH (791)
Column: 4.6-mm x 15-cm; 3.5-l..Im packing L11 Sample solution: Freshly prepared solution (1 in 10)
Flow rate: 1.0 mL/min Acceptance criteria: 9.0-10.0
Injection volume: 25 I..IL • BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.5 USP
Run time: NLT 3.5 times the retention time of Endotoxin Units/mg of acetazolamide.
acetazolamide • INJECTIONS AND IMPLANTED DRUG PRODUCTS (1), Product
System suitability Quality Tests Common to Parenteral Dosage Forms, Specific
Samples: System suitability solution and Standardsolution Tests, Completeness and Clarity of Solutions: Meets the
requirements at the time of use

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76 Acetazolamide / Official Monographs USP 43

• STERILITY TESTS (71): Meets the requirements Detector: UV 254 nm

Column: 4.6-mm x 25-cm; 5-lJm packing L1
ADDITIONAL REQUIREMENTS Flow rate: 2 mL/min
• PACKAGING AND STORAGE: Preserve as described in
Injection volume: 20 IJL
Packag~ng and Storage Requirements (659), Injection
System suitability
Packagmg, Packaging for constitution, preferablyin Sample: Standard solution
containers of Type III glass, and store at room temperature. [NOTE-The retention time for the acetazolamide peak
• LABELING (7), Labels and Labeling for Injectable Products: is about 3 min.]
Meets the requirements Suitability requirements
• USP REFERENCE STANDARDS (11) R~I~ti~e standard deviation: NMT 1.1% for replicate
USP Acetazolamide RS injections
USP Acetazolamide Related Compound D RS Analysis
5-Amino-1,3,4-thiadiazole-2-sulfonamide. Samples: Standard solution and Sample solution
C2H4N402S2 180.21 Calculatethe percentage of the labeled amount of
USP Acetazolamide Related Compound ERS acetazolamide (C4H6N403S2) in the portion of Oral
5-Acetamido-1 ,3,4-thiadiazole-2-sulfonic acid potassium Suspension taken:
salt.
C4H4KN304S2 261.32 Result = (rulrs) x (CsICu) x 100
= peak response from the Sample solution
= peak response from the Standard solution
= concentration of USP Acetazolamide RS in the
Acetazolamide Compounded Oral Standard solution (lJg/mL)
Suspension Cu =nominal concentration of acetazolamide in the
Sample solution (lJg/mL)
DEFINITION
AcetazolamideCompounded Oral Suspension contains NLT Acceptance criteria: 90.0%-110.0%
90.0% and NMT 110.0% of the labeled amount of
acetazolamide (C4H6N403S2)' SPECIFIC TESTS
Prepare Acetazolamide Compounded Oral Suspension, 25 • pH (791): 4.0-5.0 (Vehicle for Oral Solution and Vehicle for
mg/mL, as follows (see Pharmaceutical Compounding- Oral Suspension), 3.1-3.9 (CherrySyrup)
Nonsterile Preparations (795». ADDITIONAL REQUIREMENTS
• PACKA.GING AND STORAGE: Package in tight, light-resistant
Acetazolamide 2.5 9 containers. Store at controlled room temperature or in a
refrigerator. '
Vehicle: a 1:1 mixture of Vehicle for Oral Solution NF(regular
or sugar-free), and Vehicle for Oral '
• BEYOND-USE DATE: NMT 60 days after the day on which it
Suspe~sion, NF, or Cherry Syrup, NF, a sufficient was compounded when stored at controlled room
quantity to make 100 mL temperature, or in a refrigerator
• LABELING: Label it to state that it isto be wellshaken before
If using tablets, place in a mortar and comminute to a fine use, and to state the Beyond-Use Date.
powder, or add Acetazolamide powder. Add about 20 mL of • USP REFERENCE STANDARDS (11) .
the Vehicl~, and mix to a uniform paste. Add the Vehicle in USP Acetazolamide RS
small por~l?ns almost to volume, and mix thoroughly after
each addlt.lon: Transferthe contents of the mortar, stepwise
and .quantlt~tlvely, ~o a calibrated bottle. Add enough liquid
vehicle to bnng to final volume, and mixwell.
Acetazolamide Tablets
ASSAY
• PROCEDURE DEFINITION
Mobile phase: Dissolve 4.1 g of anhydrous sodium acetate Acetazolamide Tablets contain NLT 95.0% and NMT 105.0%
in 950 mLof water, and add 20 mLof methanol and 30 mL of the labeled amount of acetazolamide (C4H6N403S2)'
of acetonitrile. Adjustwith glacial acetic acid to a pH of 4.0. IDENTIFICATION
Standard stock solution: Transfer about 25 mg of USP
Acetazol~mide RS, accurately weighed, to a 50-mL
volum~tnc fl~sk, add ~.O mLof 0.5 N sodium hydroxide,
and mixto dissolve. Dilute with water to volume, and mix.
Standard solution: 250 IJg/mL of USP AcetazolamideRS • A.
from the Standardstock solution in water ;~fJl.i'i\..."'/{
Sam~le: Extracta quantity of finely powdered Tablets,
Sample solution: 250 IJg/mL of acetazolamide from Oral equivalent to.about 500 mg of acetazolamide, with 50 mL
Suspension in Mobilephase. Agitate the container of Oral of acetone. Filter, and add sufficient solvent hexane to the
Suspension for 30.min on a rotating mixer, remove a 5-mL filtrate to cause formation of a heavy, white precipitate.
sample, and store In a clear glassvialat -70 0 until analyzed. C;0llect the precipitate on a medium-porosity,
Atthe time of analysis, remove the sample from the freezer, sintered-qlassfunnel, and dry with suction.
all.ow to reach room temperature, and mix with a vortex Acceptance criteria: Meet the requirements
mixer for 30 s. Pipet 1.0 mL of this solution to a 100-mL • B. Th~ retention time of the major peak of the Sample
volumetric flask, and dilute with Mobilephase to volume. solution corresponds to that of the Standard solution as
Chromatographic system . obtained in the Assay. ' .
(See Chromatography (621), System Suitability.)
Mode: LC

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USP 43 Official Monographs / Acetic 77

ASSAY (A viA s) x C s x D x (VIL) x 100

• PROCEDURE
Mobile phase: Dissolve 4.1 g of anhydrous sodium acetate A u= absorbance of the Sample solutioh
in 950 mL of water, add 20 mL of methanol and 30 mL of A s= absorbance of the Standard solution
acetonitrile, and mix. Adjust with glacial acetic acid to a pH C s= concentration of the Standard solution (mg/mL)
of 4.0. D= dilution factor of the Sample solution, if needed
Standard solution: 0.1 mg/mL of USP Acetazolamide RS V= volume of Medium, 900 mL
prepared as follows. Transfer USP Acetazolamide RS into a L= label claim (mg/Tablet) .
suitable volumetric flask, add 0.5 N sodium hydroxide
equivalent to 10% of the final volume, and dilute with water Tolerances: NLT 75% (Q) of the labeled amount of
to volume. acetazolamide (C4H6N403S2) is dissolved.
Sample stock solution: Nominally equivalent to 1.0 mg/mL • UNIFORMITY OF DOSAGE UNITS (905): Meet the
of acetazolamide prepared as follows. Transfer a portion of requirements
the powder, from NLT 20 Tablets, equivalent to 100 mg
acetazolamide into a 1OO-mL volumetric flask. Add 10 mL ADDITIONAL REQUIREMENTS
of 0.5 N sodium hydroxide, sonicate for 5 min, cool to room • PACKAGING AND STORAGE: Preserve in tight containers, and
temperature, and dilute with water to volume. Filter a store at controlled room temperature.
portion of this solution, discarding the first 20 mL of the • USP REFERENCE STANDARDS (11 )
filtrate. USP Acetazolamide RS
Sample solution: Nominally equivalent to 0.1 mg/mL of
acetazolamide prepared as follows. Transfer 10.0 mL of
Sample stocksolution and 10 mL of 0.5 N sodium hydroxide
to a 1OO-mL volumetric flask, and dilute with water to
volume. Glacial Acetic Acid
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing L1 C2H402 60.05
Flow rate: 2 mL/min Acetic acid [64-19-7].
Injection volume: 20 IJL DEFINITION
System suitability Glacial Acetic Acid contains NLT 99.5% and NMT 100.5%, by
Sample: Standardsolution weight, of C2H402.
Suitability requirements
Tailing factor: NMT 1.5 IDENTIFICATION
Relative standard deviation: NMT 1.0% • IDENTIFICATION TESTS-CENERAL (191), Acetate: Meets the
Analysis requirements
Samples: Standardsolution and Sample solution Sample solution (for test A): Glacial Acetic Acid and water
Calculate the percentage of the labeled amount of (1:100)
acetazolamide (C4H6N403S2) in the portion of Tablets ASSAY
taken: • PROCEDURE
Sample solution: Measure 2 mL of Glacial Acetic Acid into
Result = (r vir s) x (C siC v) x 100 a glass-stoppered flask, previously tared while containing
= acetazolamide peak response from the Sample about 20 mL of water, and weigh again to obtain the
solution weight of the substance under assay.
=acetazolamide peak response from the Standard Analysis: Add 20 mLofwater, then add phenolphthalein TS.
solution Titratewith 1 N sodium hydroxide VS. Each mL of 1 N
= concentration of USP Acetazolamide RS in the sodium hydroxide is equivalent to 60.05 mg of C2H40 2 •
Standardsolution (mg/mL) Acceptance criteria: 99.5%-100.5%
= nominal concentration of acetazolamide in the IMPURITIES
Sample solution (mg/mL) INORGANIC IMPURITIES
• limit of Nonvolatile Residue: Evaporate 20 rnl, in a tared
Acceptance criteria: 95.0%-105.0% dish, and dry at 105° for 1 h: the weight of the residue does
PERFORMANCE TESTS not exceed 1.0 mg.
• DISSOLUTION (711) • CHLORIDE AND SULFATE, Chloride (221)
Medium: 0.01 N hydrochloric acid; 900 mL Sample solution: Dilute 1.0 mL with 20 mL of water.
Apparatus 1: 100 rpm Analysis: Add 5 drops of silver nitrate TS.
Time: 60 min Acceptance criteria: No opalescence is produced.
Standard solution: USP Acetazolamide RS in Medium • CHLORIDE AND SULFATE, Sulfate (221)
Sample solution: Dilute with Medium if necessary. Sample solution: Dilute 1.0 mL with 10 mL of water.
Instrumental conditions Analysis: Add 1 mL of barium chloride TS.
(See Ultraviolet-Visible Spectroscopy (857).) Acceptance criteria: No turbidity is produced.
Mode: UV ORGANIC IMPURITIES
Analytical wavelength: 265 nm • Procedure: Readily Oxidizable Substances
Analysis Sample solution: Dilute 2.0 mL in a glass-stoppered vessel
Samples: Standardsolution and Sample solution with 10 mL of water.
Determine the percentage of the labeled amount of Analysis: Add 0.10 mL of 0.10 N potassium permanganate.
acetazolamide (C4H6N403S2) dissolved:

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78 Acetic / OfficialMonographs USP 43

Acceptance criteria: The pink color is not changed to brown Acceptance criteria: Ethyl acetate, recognizable by its
within 2 h. characteristic odor, is evolved.
SPECIFIC TESTS ASSAY
• CONGEALING TEMPERATURE (651): NLT 15.6 0 • PROCEDURE
Sample: A quantity of Acetic Acid Otic Solution containing
ADDITIONAL REQUIREMENTS 100 mg of glacial acetic acid
• PACKAGING AND STORAGE: Preserve in tight containers, and Analysis: Transfer the Sample to a 250-mL conical flask, and
store at room temperature. add 5 mL of saturated sodium chloride solution, 40 mL of
water, and 3 drops of phenolphthalein TS.Titrate with 0.1
N sodium hydroxide VS to a faint pink endpoint. Each mL
of 0.1 N sodium hydroxide is equivalent to 6.005 mg of
Acetic Acid Irrigation acetic acid (C2H402) .
Acceptance criteria: 85.0%-130.0%
DEFINITION SPECIFIC TESTS
Acetic Acid Irrigation is a sterile solution of Glacial Acetic Acid • pH (791)
in Water for Injection. It contains, in each 100 mL, NLT 237.5 Sample solution: Acetic Acid Otic Solution and water
mg and NMT 262.5 mg of C2H402 • (1:1)
IDENTIFICATION Acceptance criteria: 2.0-4.0
• A. IDENTIFICATION TESTS-GENERAL, Acetate (191) ADDITIONAL REQUIREMENTS
Sample: 100 mL of Acetic Acid Irrigation • PACKAGING AND STORAGE: Preserve in tight containers, and
Analysis: Evaporate the Sample to about 10 mL. store at controlled room temperature.
Acceptance criteria: The resulting solution meets the
requirements.
ASSAY
• PROCEDURE
Sample: 50 mL of Acetic Acid Irrigation
Acetohydroxamic Acid
Analysis: Pipet the Sample into a 150-mL conical flask, add
2 drops of phenolphthalein TS, and titrate with 0.1 N
sodium hydroxide VS. Each mL of 0.1 N sodium hydroxide
is equivalent to 6.005 mg of acetic acid (C2H402) .
Acceptance criteria: 237.5-262.5 mg of C2H402 in each C2H sN0 2 75.07
100 mL of Acetic Acid Irrigation N-Acetyl hydroxyacetamide;
Acetohydroxamic acid [546-88-3].
SPECIFIC TESTS
• pH (791): 2.8-3.4 DEFINITION
• BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.5 USP Acetohydroxamic Acid, dried over phosphorus pentoxide for
Endotoxin Unit/mL. 16 h, contains NLT 98.0% and NMT 101.0% of
• OTHER REQUIREMENTS: It meets the requirements under acetohydroxamic acid (C2H sN02) .
Injections and Implanted Drug Products (1)" except that the
container in which it is packaged may be designed to empty IDENTIFICATION
rapidly and may exceed 1000 mL in capacity.
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in single-dose
containers, preferably of Type I or Type II glass, and store
at controlled room temperature. It may be packaged in
suitable plastic containers. Sample solution: 20 mg/mL in water
Analysis: To 10 mL of the Sample solution add 2 drops of
potassium permanganate TS.
Acceptance criteria: The pink color of the permanganate
disappears.
Acetic Acid Otic Solution
ASSAY
DEFINITION • PROCEDURE
Acetic Acid Otic Solution is a solution of Glacial Acetic Acid in Ferric chloride solution: 20 mg/mL of ferric chloride in 0.1
a suitable nonaqueous solvent. It contains NLT 85.0% and N hydrochloric acid
NMT 130.0% of the labeled amount of C2H402 • Standard solution: 500 IJg/mL of USP Acetohydroxamic
Acid RS in 0.1 N hydrochloric acid
IDENTIFICATION Sample solution: 500 IJg/mL of Acetohydroxamic Acid,
• A. previously dried, in 0.1 N hydrochloric acid
Sample solution: Dilute 5 mL of Acetic Acid Otic Solution Blank: 0.1 N hydrochloric acid
with 10 mL of water. Analysis
Analysis: Adjust'the Sample solution with 1 N sodium Samples: Standardsolutions, Sample solution, and Blank
hydroxide to a pH of 7. Add ferric chloride TS. Transfer 10.0 mL each of the Standardsolution, Sample
Acceptance criteria: A deep red color is produced, and it is solution, and Blankto separate 1OO-mL volumetric flasks.
destroyed by the addition of hydrochloric acid. To each flask add 50 mL of 0.1 N hydrochloric acid and
• B. 10.0 mL of Ferric chloride solution, and dilute with 0.1 N
Analysis: Warm it with sulfuric acid and alcohol. hydrochloric acid to volume. Without delay,

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USP 43 Official Monographs / Acetohydroxamic 79

concomitantly determine the absorbances of the M r2 = molecular weight of hydroxylamine

solutions at the wavelength of maximum absorbance at hydrochloride, 69.50
about 502 nm using the Blank to set the instrument.
Calculate the percentage of acetohydroxamic.acid. Acceptance criteria: NMT 0.5%
(C2H sN02 ) in the portion of Acetohydroxarnlc ACid
SPECIFIC TESTS
taken:
• Loss ON DRYING (731)
Analysis: Dry a sample over phosphorus pentoxide for 16 h.
Result =(A ulA s) x (C siC u) x 100
Acceptance criteria: NMT 1.0% .
Au = absorbance of the Sample solution • COMPLETENESS OF SOLUTION (641): A 1.0-g portion
dissolves in 10 mL of water to yield a clear solution.
As = absorbance of the Standardsolution
• COLOR OF SOLUTION
Cs =concentration of USP Acetohydroxamic Acid RS Sample solution: 200 mg/mL in water
in the Standardsolution (Jjg/mL) Blank: Water
Cu = concentration of Acetohydroxamic Acid in the Instrumental conditions
Sample solution (Jjg/mL) (See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis
Acceptance criteria: 98.0%-101.0% on the previously
Analytical wavelength: Between 400 and 750 nm
dried basis
Cell: 1 cm
IMPURITIES Acceptance criteria: The absorbance is NMT 0.050.
• RESIDUE ON IGNITION (281): NMT 0.1 % ADDITIONAL REQUIREMENTS
• LIMIT OF HYDROXYLAMINE • PACKAGING AND STORAGE: Preserve in tight containers, and
Buffer: 1.36 giL of monobasic potassium phosphate in
store in a cool, dry place.
water, adjusted with 1 M potassium hydroxide to a pH of
• USP REFERENCE STANDARDS (11)
7.4 USP Acetohydroxamic Acid RS
Solution A: 1 mg/ml of pyridoxal. 5-phospha~e.
monohydrate in Buffer, prepared In a low-actinic flask fresh
before use
Standard stock solution: 2.0 mg/ml of hydroxylamine
hydrochloride in water Acetohydroxamic Acid Tablets .
Standard solutions: Transfer 5.0, 10.0, and 15.0 ml of the
Standard stock solution to separate 1OO-mL volumetric DEFINITION
flasks, and dilute with water to volume. . Acetohydroxamic Acid Tablets contain NLT 90.0% and NMT
Sample solution: Transfer 1500 mg of Acetohydr~xamlc . 110.0% of the labeled amount of acetohydroxamic acid
Acid, previously dried, to a 1OO-ml beaker, and dissolve In (C2H sN02) ·
a sufficient amount of water to cover the electrode of a
calibrated pH meter (about 60 ml). While stirring, adjust IDENTIFICATION
with 0.05 M potassium hydroxide to a pH of 7.4. Transfer • A. Tablets produce a purple color when mixed with an
the contents of the beaker, with the aid of small portions of acidic solution of ferric chloride.
water, to a 1OO-mL volumetric flask, and dilute with water ASSAY
to volume.
• PROCEDURE
Blank: Water Ferric chloride solution: 20 mg/ml of ferric chloride in 0.1
Analysis N hydrochloric acid . .
Samples: Standard solutions, Sample solution, and Blank Standard solution: 500 Jjg/mL of USP Acetohydroxarnlc
Transfer 2.0 mL of each Standard solution, the Sample Acid RS in 0.1 N hydrochloric acid
solution and Blank into separate 1OO-mL volumetric Sample solution: Weigh, and finely powder NLT 20 Tablets.
flasks. T~ each flask add 4.0 mL of Solution A. After 8 n:'in, Transfer an accurately weighed portion of the r:?0w~er,
accurately timed, dilute the contents of each flask With equivalent to about 500 mg of acetohydroxarnlc acid, to a
Buffer to volume. 1OOO-mL volumetric flask, add about 500 mL of 0.1 N
Immediately determine the fluorescence intensities of the
hydrochloric acid, and shake for 1 ~in ..Dilute. with ~.1 N
solutions from the Standard solutions and the Sample hydrochloric acid to volume, and mix. Filter, discarding the
solution in a fluorometer at an excitation wavelength of first 40 ml of the filtrate. Use the clear filtrate.
350 nm and an emission wavelength of 450 nm, setting Blank: 0.1 N hydrochloric acid
the instrument to zero with the Blank. Determine the
Analysis
best-fit straight line from the fluorescence intensiti~s of Samples: Standard solutions, Sample solution, and Blank
the three Standardsolutions versus the hydroxylamine Transfer 10.0 ml each of the Standard solution, Sample
hydrochloride concentrations, in Jjg/mL. Fr0n:' th~ solution and Blankto separate 1OO-ml volumetric flasks.
best-fit straight line, determine the concentration, In Jjgl To each flask add 50 mL of 0.1 N hydrochloric acid and
mL, of hydroxylamine hydrochloride in the Sample 10.0 ml of Ferric chloride solution, and dilute with 0.1 N
solution. hydrochloric acid to volume. Without delay,
Calculate the percentage of hydroxylamine in the portion concomitantly determine the absorbances of the
of Acetohydroxamic Acid taken: solutions at the wavelength of maximum absorbance at
about 502 nm, using the Blank to set the instrument.
Result = (C ulC) x (M r1IM r2) X 100
Calculate the percentage of labeled amount of
Cu = concentration of hydroxylamine hydrochloride acetohydroxamic acid (C2H sN0 2) in the portion of
in the Sample solution (mg/mL). .. Tablets taken:
C =concentration of Acetohydroxarnic ACid In the Result =(AulAs) x (CsICu) x 100
Sample solution (mg/mL)
M rl = molecular weight of hydroxylamine, 33.03

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80 Acetohydroxamic / OfficialMonographs USP 43

Au = absorbance of the Sample solution Result = (CulC) x (M,tlM'2) x 100

As = absorbance of the Standardsolution
Cs = concentration of USP Acetohydroxamic Acid RS = concentration of hydroxylamine hydrochloride
in the Standardsolution (J.lg/mL) in the Sample solution (J.lg/mL)
Cu = nominal concentration of acetohydroxamic acid C = nominal concentration of acetohydroxamic acid
in the Sample solution (1J9/mL) in the Sample solution (lJg/mL)
= molecular weight of hydroxylamine, 33.03
Acceptance criteria: 90.0%-110.0% = molecular weight of hydroxylamine
hydrochloride, 69.50
PERfORMANCE TESTS
• DISSOLUTION, Procedure for a Pooled Sample (711) Acceptance criteria: NMT 0.5%
Medium: 0.01 N hydrochloric acid; 900 mL
Apparatus 1: 100 rpm ADDITIONAL REQUIREMENTS
Time: 30 min • PACKACING AND STORAGE: Preserve in tight containers.
Analysis: Calculate the percentage of the labeled amount of • USP REFERENCE STANDARDS (11)
acetohydroxamic acid (C2H sN02 ) dissolved, using the USP Acetohydroxamic Acid RS
procedure in the Assay, using a filtered portion of the
solution under test as Sample solution in comparison with a
Standardsolution having a known concentration of USP
Acetohydroxamic Acid RS in Medium.
Tolerances: NLT 85% (Q) of the labeled amount of
Acetylcholine Chloride
acetohydroxamic acid (C2H sN0 2 ) is dissolved.
• UNIFORMITY OF DOSACE UNITS (905): Meet the CI

requirements
IMPURITIES C7H 16CIN02 181.66
• LIMIT OF HYDROXYLAMINE Ethanaminium, 2-(acetyloxy)-N,N,N-trimethyl-, chloride;
Buffer: 1.36 giL of monobasic potassium phosphate in Choline acetate (ester) chloride [60-31-1].
water, adjusted with 1 M potassium hydroxide to a pH of DEfiNITION
7.4 Acetylcholine Chloride contains NLT 98.0% and NMT 102.0%
Solution A: 1 mg/mL of pyridoxal5-phosphate of acetylcholine chloride (C7H 16CIN02 ) , calculated on the
monohydrate in Buffer, prepared in a low-actinic flask fresh dried basis.
before use
Standard stock solution: 2.0 mg/mL of hydroxylamine IDENTifiCATION
hydrochloride in water-
Standard solutions: Transfer 5.0, 10.0, and 15.0 mL of the
Standardstocksolution to separate 1OO-mL volumetric
flasks, and dilute with water to volume.
Sample solution: Weigh, and finely powder NLT 20 Tablets.
Transfer a portion of the powder, equivalent to about 1500
mg of acetohydroxamic acid to a 50-mL stoppered Sample solution: 100 mg/mL in water
centrifuge tube. Add 30.0 mL of water, shake for about 2 Analysis: To 5 mL of Sample solution add 5 mL of silver
min, and centrifuge. Pipet 15.0 mL of the clear solution into nitrate TS.
a 50-mL beaker, add just enough water to cover the Acceptance criteria: A white, curdy precipitate, which is
electrode of a calibrated pH meter, and while stirring, adjust soluble in ammonium hydroxide but insoluble in nitric acid,
with 0.5 M potassium hydroxide to a pH of 7.4. is formed.
Quantitatively transfer the contents of the beaker with the
aid of small portions of water to a 50-mL volumetric flask, ASSAY
dilute with water to volume, and mix. • PROCEDURE
Blank: Water Sample: 400 mg of Acetylcholine Chloride
Analysis Analysis: Dissolve in 15 mL of water in a glass-stoppered
conical flask, add 40.0 mL of 0.1 N sodium hydroxide VS,
Samples: Standardsolutions, Sample solution, and Blank
and heat on a steam bath for 30 min. Insert the stopper,
Transfer 2.0 mL of each Standardsolution, the Sample
allow to cool, add phenolphthalein TS, and titrate the
solution, and Blank into separate 1OO-mL volumetric
excess alkali with 0.1 N sulfuric acid VS. Perform a blank
flasks.To each flask add 4.0 mL of Solution A. After 8 min,
determination (see Titrimetry(541), Residual Titrations).
accurately timed, dilute the contents of each flask with
Bufferto volume. Each mL of 0.1 N sodium hydroxide is equivalent to 18.17
mg of C7H 16CIN02 •
Immediately determine the fluorescence intensities of the
solutions from the Standardsolutions and the Sample Acceptance criteria: 98.0%-102.0% on the dried basis
solution in a fluorometer at an excitation wavelength of OTHER COMPONENTS
350 nm and an emission wavelength of 450 nm, setting • CONTENT OF CHLORIDE
the instrument to zero with the Blank. Determine the Sample: 280 mg of Acetylcholine Chloride
best-fit straight line from the fluorescence intensities of Analysis: Dissolvethe Sample in 140 mL of water, and add
the three Standardsolutions versus the hydroxylamine 1 mL of dichlorofluorescein TS. Titrate with 0.1 N silver
hydrochloride concentrations, in IJg/mL. From the nitrate VS until the silver chloride flocculates and the
best-fit straight line, determine the concentration, in IJgI mixture acquires a faint pink color. Each mL of 0.1 N silver
mL, of hydroxylamine hydrochloride in the Sample nitrate is equivalent to 3.545 mg of CI.
solution. Acceptance criteria: 19.3%-19.8% of Cion the dried basis
Calculate the percentage of hydroxylamine in the portion
of Tablets taken:

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USP 43 Official Monographs / Acetylcholine 81

IMPURITIES • B.
• RESIDUE ON IGNITION (281): NMT 0.2% Sample solution: Nominally 10 mg/mL of acetylcholine
chloride
SPECIFIC TESTS Analysis: To 2 mL of Sample solution add 1 drop of nitric acid
• MELTING RANGE OR TEMPERATURE, Class 1(741): 149°-152° and 1 mL of silver nitrate TS.
• ACIDITY Acceptance criteria: A curdy, white precipitate, soluble in
Sample: 100 mg of Acetylcholine Chloride an excess of 6 N ammonium hydroxide, is formed.
Analysis: Dissolvethe Sample in 10 mL of recently boiled
water, and add at once 1 drop of bromothymol blue TS. ASSAY
Acceptance criteria: NMT 0.50 mL of 0.010 N sodium • PROCEDURE
hydroxide is required to produce a color change. Mobile phase: Add 1.03 g of sodium 1-heptanesulfonate to
• Loss ON DRYING (731) a mixture of 900 mL of water and 10 mL of methanol.
Analysis: Dry a sample at 105° for 3 h. Adjust with ammonium hydroxide or glacial acetic acid to
Acceptance criteria: NMT 1.0% a pH of 4.0. Add 50 mL of acetonitrile. Dilute with water to
1 L. [NOTE-A slight variation of the amount of acetonitrile
ADDITIONAL REQ.UIREMENTS may be required to improve resolution or adjust retention
• PACKAGING AND STORAGE: Preserve in a tight container time.]
and store at controlled room temperature. ' Standard solution: A quantity of USP Acetylcholine
• USP REFERENCE STANDARDS (11) Chloride RS in Mobile phase, to obtain a solution having a
USP Acetylcholine Chloride RS known concentration equal to that of the acetylcholine
chloride in the Sample solution
Sample solution: Transfer the contents of 1 container of
Acetylcholine Chloride for Ophthalmic Solution to a 10-mL
Acetylcholine Chloride for Ophthalmic volumetric flask with the aid of Mobilephase and dilute
with Mobile phase to volume. '
Solution System suitability solution: 0.2% each of acetylcholine
chloride and choline chloride
DEFINITION Chromatographic system
Ace~ylcholine Chloride for Ophthalmic Solution is a sterile (See Chromatography (621), System SUitability.)
mixture of Acetylcholine Chloride with Mannitol or other Mode: LC
suitable diluent, prepared by freeze-drying. Each container Detector: Refractive index
contains NLT 90.0% and NMT 115.0% of the labeled Column: 3.9-mm x 30-cm; packing L1
amount of acetylcholine chloride (C7H16CIN02) . Flow rate: 2 mL/min
Injection volume: 50 ~L
IDENTIFICATION
System suitability
• A. Samples: Standardsolution and System suitability solution
Standard solution: 10 mg/mL of USP Acetylcholine
Suitability requirements
Chloride RS
Resolution: NLT 2.0 between acetylcholine chloride and
Sample solution: 10 mg/mL of acetylcholine chloride
choline chloride, System suitabilitysolution
Chromatographic system
Relative standard deviation: NMT 3.5%, Standard
(See Chromatography (621), Thin-Layer Chromatography.)
solution
Adsorbent: 0.25-mm layer of aluminum oxide
Analysis
Application volume: 2 ~L '.
Samples: Standardsolution and Sample solution
Devel.opi~g solvent system: Mix butyl alcohol, glacial
Calculate the percentage of acetylcholine chloride
acetic acid, and water (40:10:50). Allow the layers to
(C7H 16CIN02) in the container taken:
separate completely. Usethe upper layer.
Spray reagent A: Freshlyprepared solution of 5 mg/mL of Result = (rulrs) x Cs x V x (lIL) x 100
cobaltous chloride prepared asfollows. Dissolve the
required amount of cobaltous chloride in 50% of the final = peak response from the Sample solution
volume of water, and dilute with 50% alcohol.
= peak responsefrom the Standardsolution
[NoTE-This solution is freshly prepared.]
Spray reagent B: Freshlyprepared potassium ferrocyanide =concentration of USP Acetylcholine Chloride RS
solution prepared as follows. Dissolve 1.0 g of potassium in the Standardsolution (mg/mL)
ferrocyanide in 100 mL of water, and dilute with 50 mL of V = volume of the Sample solution, 10 mL
alcohol. . L = label claim (mg/vial)
Analysis
Acceptance criteria: 90.00/0-115.0%
Samples: Standardsolution and Sample solution
Develop the chromatogram, without delay, in a PERFORMANCE TESTS
vapor-saturated chamber containing the Developing • UNIFORMITY OF DOSAGE UNITS (905): Meets the
sotvent system. Allow the solvent front to move about 10 requirements
cm beyond the initial spotting line. Dry the plate with a
current of warm air. Immediately spray the plate with SPECIFIC TESTS
Spray reagent A. Dry the plate as before, and • STERILITY TESTS (71): Meets the requirements
immediately spray the plate with Spray reagent B. Dry • ACIDITY
the plate with a current of warm air. Analysis: Dissolve an amount of Acetylcholine Chloride for
Acceptance criteria: The RF value and color of the principal Ophthalmic Solution equivalent to 100 mg of acetylcholine
spot from the Sample solution correspond to those from the chloride in 10 mL of recently boiled water. Add at once 1
Standard solution. drop of bromothymol blue T5.
Acceptance criteria: NMT 0.50 mL of 0.. 010 N sodium
hydroxide is required to produce a color change.

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82 Acetylcholine / Official Monographs USP 43

• WATER DETERMINATION, Method 1(921) Injection size: 5 J.lL

Analysis: Perform the titration in the original container, System suitability
observing precautions against contact with water or moist Sample: Standard solution
atmosphere. Adjust the concentration of the reagent so [NOTE""7"The relative retention times for acetylcysteine
that the titration volume approaches but does not exceed and l-phenylalanine are about 0.5 and 1.0,
the capacity of the container. Titrate to an amber color that respectively.]
persists for 15 s after mixing. Suitability requirements
Acceptance criteria: NMT 1.0% Resolution: NLT 6 between acetylcysteine and
• CONSTITUTED SOLUTION: At the time of use, it meets the l-phenylalanine
requirements for Injections and ImplantedDrug Products (1), Relative standard deviation: NMT 2.0%
Specific Tests, Completeness and clarity of solutions. Analysis
Samples: Standardsolution and Sample solution
ADDITIONAL REQUIREMENTS Calculate the percentage of acetylcysteine (CsH9N03S) in
• PACKAGING AND STORAGE: Preserve in tight containers as the portion of Acetylcysteine taken:
described in Packaging and Storage Requirements (659),
Injection Packaging, Packaging for constitution, and store at Result = (R viR s) x (C siC u) x 100
controlled room temperature.
• USP REFERENCE STANDARDS (11) Ru = peak response ratio of acetylcysteine to
USP Acetylcholine Chloride RS l-phenylalanine from the Sample solution
Rs = peak response ratio of acetylcysteine to
l-phenylalanine from the Standardsolution
Cs =concentration of USP Acetylcysteine RS in the
Acetylcysteine Standardsolution (mg/mL)
Cu =concentration of acetylcysteine in the Sample
solution (mg/mL)

Acceptance criteria: 98.0~102.0% on the dried basis

IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.5%
CsH9N0 3S 163.19
l-Cysteine, N-acetyl-; SPECIFIC TESTS
N-Acetyl-l-cysteine [616-91-1]. • OPTICAL ROTATION, Specific Rotation (781 S)
Buffer: Mix 29.5 mL of 1 N sodium hydroxide, 50 mL of 1
DEFINITION M monobasic potassium phosphate, and sufficient water to
Acetylcysteine contains NLT 98.0% and NMT 102.0% of make 100 mL. Adjust to a pH of 7.0 ± 0.1 by adding more
CsH 9N03S, calculated on the dried basis. of either solution, as necessary.
Sample solution: In a 25-mL volumetric flask, mix 1.25 g
IDENTIFICATION with 1 mL of edetate disodium solution (1 in 100), add 7.5
mL of sodium hydroxide solution (1 in 25), and mix to
dissolve. Dilute with Bufferto volume.
Acceptance criteria: +21 0 to +2r
• pH (791): 2.0-2.8 in a solution (1 in 100)
• Loss ON DRYING (731): Dry a sample at a pressure of about
ASSAY 50 mm of mercury at 70 0 for 4 h: it loses NMT 1.0% of its
• PROCEDURE weight.
Mobile phase: 6.8 giL of monobasic potassium phosphate. ADDITIONAL REQUIREMENTS
Adjust with phosphoric acid to a pH of 3.0. • PACKAGING AND STORAGE: Preserve in tight containers, and
Sodium metabisulfite solution: 0.5 mg/mL of sodium store at controlled room temperature.
metabisulfite in water, freshly prepared • USP REFERENCE STANDARDS (11)
Internal standard solution: 5 mg/mL of USP USP Acetylcysteine RS
l-Phenylalanine RS in Sodium metabisulfite solution USP l-Phenylalanine RS
Standard stock solution: 10 mg/mL of USP Acetylcysteine
RS in Sodium metabisulfite solution
Standard solution: 0.5 mg/mL of USP Acetylcysteine RS
and 0.25 mg/mL of USP l-Phenylalanine RS in Sodium
metabisulfite solution from Standardstock solution and Acetylcysteine Solution
Internal standard solution
Sample stock solution: 10 mg/mL of Acetylcysteine in DEFINITION
Sodium metabisulfite solution Acetylcysteine Solution is a sterile solution of Acetylcysteine in
Sample solution: 0.5 mg/mL of Acetylcysteine and 0.25 water, prepared with the aid of Sodium Hydroxide, It
mg/mL of USP l-Phenylalanine RS in Sodium metabisulfite contains NLT90.0% and NMT 110.0% of the labeled
solution from Sample stock solution and Internal standard amount of acetylcysteine (CsH9N03S).
solution
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 214 nm
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 1.5 mt/rnln

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USP43 Official Monographs / Acetylcysteine 83

IDENTIFICAnON Cu =nominal concentration of acetylcysteine in the

Sample solution (mg/mL)

Acceptance criteria: 90.0%-110.0%

SPECIFIC TESTS
Sample solution: Place 10 mCin a suitable beaker, and • pH (791): 6.0-7.5
• STERILITY TESTS (71): Meets the requirements
adjust to a pH of 2 (pH indicator paper), using 3 N
hydrochloric acid. Add up to 2 g of finely powdered sodium ADDITIONAL REQUIREMENTS
chloride, in two portions of 200 mg each initially and then • PACKAGING AND STORAGE: Preserve in single-unit or
in smaller portions of 25 mg, stirring after each addition multiple-unit tight containers that effectively exclude
until the sodium chloride dissolvesand a precipitate is oxygen, and store at controlled room temperature.
formed. The precipitate appears asa very fine powder, and • USP REFERENCE STANDARDS (11)
the solution turns cloudy. If no precipitate forms, add an USP Acetylcysteine RS
additional drop of 3 N hydrochloric acid, and stir until the USP L-Phenylalanine RS
precipitate forms. Allow to stand at room temperature for
15 min, and collect the residue by suction filtration. Usethe
acetylcysteine so obtained after being dried at a pressureof
50 mm of mercury at 70° for 4 h.
Acceptance criteria: Meets the requirements Acetylcysteine Compounded Solution
ASSAY DEFINITION
• PROCEDURE Acetylcysteine Compounded Solution contains NLT 90.0%
Solution A: 0.5 mg/mL of sodium metabisulfite solution in and NMT 110.0% of the labeled amount of acetylcysteine
water, freshly prepared (CsH 9N03S). Prepare Acetylcysteine Compounded Solution
Solution B: 0.5 mg/mL of sodium bisulfite solution 20% as follows (see Pharmaceutical Compounding-Sterile
Mobile phase: 6.8 gIL of monobasic potassium phosphate. Preparations (797».
Adjust with phosphoric acid to a pH of 3.0.
Internal standard solution: 5 mg/mL of USP
L-Phenylalanine RS in Solution A Acetylcysteine 2g
Standard stock solution: 10 mg/mL of USP Acetylcysteine Edetate Disodium Dihydrate 5.5 mg
RS in Solution A
Sodium Hydroxide 10% Solution To adjust pHto 6.5-7.5
Standard solution: 0.5 mg/mL of USP Acetylcysteine RS
and 0.25 mg/mL of USP L-Phenylalanine RS in Solution A SterileWater for Injection,
from Standardstock solution and Internal standardsolution a sufficient quantity to make 10 mLa
Sample stock solution: Equivalent to 10 mg/mL of
acetylcysteine from the volume of Solution in Solution B a It is necessaryto adjust the formula and compound an additionalamount to
completelyfill each single-unitcontainer to minimize exposure to oxygen
Sample solution: 0.5 mg/mL of acetylcysteine "and 0.25 because the preparation issusceptibleto oxidation.
mg/mL of USP L-PhenylalanineRS in Solution A from Sample
stock solution and Internal standard solution Dissolve Edetate Disodium Dihydrate in 7 mL of Sterile Waterfor
Chromatographic system Injection. Slight heating may be necessary. Allow to cool.
(See Chromatography (621), System Suitability.) Dissolve Acetylcysteine in the edetate disodium solution. Add
Mode: LC Sodium Hydroxide 70% Solution dropwise with mixing to
Detector: UV 214 nm adjust the pH to between 6.5 and 7.5. Bring to final volume
Column: 3.9-mm x 30-cm; packing L1 with Sterile Water for Injection and mix well. Pass through a
Flow rate: 1.5 mL/min sterile filter of 0.22-JJm pore size into single-unit sterile
Injection volume: 5 JJL containers. It is necessaryto completely fill the container to
System suitability minimize the amount of oxygen present because the
Sample: Standardsolution preparation is susceptible to oxidation.
Suitability requirements
Resolution: NLT 6 between acetylcysteine and ASSAY
L-phenylalanine • PROCEDURE
Relative standard deviation: NMT 2.0% Mobile phase: Acetonitrile, phosphoric acid, and water (3:
Analysis 0.5: 96.5)
Samples: Standardsolution and Sample solution Standard solution: 0.4 mg/mL of acetylcysteine prepared
[NOTE-The relative retention times for acetylcysteine from USP Acetylcysteine RS in Mobilephase
and L-phenylalanine are about 0.5 and 1.0, Sample solution: Transfer 0.4 mL of Solution to a 200-mL
respectively.] volumetric flask, dilute with Mobile phase to volume, and
Calculate the percentage of the labeled amount of mix well.
acetylcysteine (CsH9N03S) in the portion of Solution Chromatographic system
taken: (See Chromatography (621), System SUitability.)
Mode: LC
Result = (R viR s) x (C siC v) x 100 Detector: UV 200 nm
Column: 4.6-mm x 25-cm; 5-JJm packing L96
= peak response ratio of acetylcysteine to Column temperature: 15°
L-phenylalanine from the Sample solution Flow rate: 2.0 mL/min
= peak response ratio of acetylcysteine to Injection volume: 10 JJL
L-phenylalanine from the Standardsolution System suitability
= concentration of USP Acetylcysteine RS in the Sample: Standardsolution
Standardsolution (mg/mL)

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 84 Acetylcysteine / OfficialMonographs USP 43

[NoTE-The retention time for acetylcysteine is about percent of the labeled amount of isoproterenol
3.8 min.]
Suitability requirements
hydrochloride (C ll H17N03 • HCI).
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% for replicate Packaging and storage-Preserve in single-dose ~r
injections multiple-dose containers, preferably of Type I glass, tlqhtly
Analysis closed with a glass or polyethylene closure, and store at
Samples: Standard solution and Sample solution controlled room temperature. " .
Calculate the percentage of the labeled amount of Labeling-The label indicates that the Inhalation S?lutlon IS
acetylcysteine (CsH9N0 3S) in the portion of Solution not to be used if its color is pinkish or darker than slightly
yellow or if it contains a precipitate.
taken:
USP Reference standards (11)-
USP Acetylcysteine RS
Result = (rvlrs) x (CsICv) x 100
USP Isoproterenol Hydrochloride RS
=peak response of acetylcysteine from the Sample USP L-Phenylalanine RS
solution Color and darity-Using the Inhalation Solution as the Test
= peak responseof acetylcysteinefrom the Standard solution, proceed as directed for Colorand clarity under
solution Isoproterenol Inhalation Solution.
= concentration of USP Acetylcysteine RS in the Identification-
Standard solution (mg/mL) A: Place 2 mL in a 1O-mL beaker, and adjust with 3 N
Cv = nominal concentration of acetylcysteine in the hydrochloric acid to a pH of about 3 (p~ indicat~rpaper). Add
Sample solution (mg/mL) 500 mg to 1 g of finely powder~d. ~odlum chlond~, 10 two
portions of about 200 mg ~a~h Initially, and th~~ In sm~lIer
Acceptance criteria: 90.0%-110.0% portions (about 25 mg), stirring after each addition, until a
SPECIFIC TESTS precipitate is formed. Allow to stand at room temperature for
• pH (791): 6.5-7.5 15 minutes and collect the residue by suction filtration: the
• STERILITY TESTS (71), Test for Sterilityof the Product to Be .acetylcystei~e so obtained, after being dried as directed in the
Examined, Membrane Filtration: Meets the requirements test for Loss on drying under Acetylcysteine, responds to the
Identification test under Acetylcysteine.
ADDITIONAL REQUIREMENTS B: Ferro-Citrate Solution and Buffer Solution-Prepare as
• PACKAGING AND STORAGE: Package in single-unit sterile directed under Epinephrine Assay (391).
glass containers and store at controlled room temperature. Procedure-Place a volume of Inhalation Solution,
equivalent to about 0.26 mg of isoproter.enol h~drochlorid~,
in a test tube with 3 mL of 0.1 M mercuric chloride, and mix.
Add 100 ~L of Ferro-Citrate Solution and 1.0 mL of Buffer
• B~Y()(Id~:.tJSE I)A!E:ln t~~~b~~n~~()! Solution, and mix: the presenceof isoproterenol hydrochloride
t~st,~.~~k~l.J[)s<im>(7~7)i~BB1~(,IEPf7P~r is confirmed by the development of a purple color.
<:~R~Dtf.·i9.~te~il.it)lit~~tj.~p~s~~.~,.fN>ty1~i9. Sterility Tests (71): meets the requirements.
8~.\f'i~.i£Q.• ii..~. .;.-y.~~S8.mB.g~;~~~gi~m.~n ,.•sJclr~c;t pH (791): between 6.0 and 7.0.
r(2)(2)rTl.t~rnp~rCltlJr~~(CNl ..tylaY:,2Q.zQ)·
• LABELING: Label it to state the Beyond-Use Date. The label Assay for acetylcysteine-
indicates that the Solution is not to be used if it contains a Mobile phase, Internal standardsolution, Standard
precipitate. Label it to state that it is a single-unit c~ntainer, preparation, and Chromatographic system-Proceed as
that it is overfilled with an excess that should be discarded directed in the Assay under Acetylcysteine.
after a measured single dose is used, and to store at Assay preparation-Pipet a volume of In~alat!on Solution,
controlled room temperature. Label it for inhalation o~ oral equivalent to about 1000 mg of acetylcystelne, lnto a 100-mL
administration only. Label it to state that the preparation volumetric flask, dilute with sodium meta bisulfite solution (1
may have a disagreeable odor and light purple color that is in 2000) to volume, and mix. Pipet 10 mL of this solution and
a result of a chemical reaction that does not affect the 10 mL of Internal standard solution into a 200-mL volumetric
strength of the preparation. flask, dilute with sodium metabisulfite solution (1 in 2000) to
• USP REFERENCE STANDARDS (11) volume, and mix. .
USP Acetylcysteine RS Procedure-Proceed as directed for Procedure in the Assay
under Acetylcysteine. Calculate the quantity, in mg, of
CSH9N0 3S in each mL of the Inhalation Solution taken by the
formula:

Acetylcysteine and Isoproterenol 2000( CI V)(Rvl Rs)

Hydrochloride Inhalation Solution in which C is the concentration, in mg per mL, of USP

Acetylcysteine RS in the Standardpreparation; Vis the volume,
in mL, of Inhalation Solution taken; and Rvand Rs are the ratios
» Acetylcysteine and Isoproterenol Hydrochloride
of the peak response of acetylcysteine to that of
Inhalation Solution is a sterile solution of DL-phenylalanine obtained from the Assay preparation and the
Acetylcysteine and Isoproterenol Hydrochloride in Standardpreparation, respectively.
water. It contains not less than 90.0 percent and Assay for isoproterenol hydrochloride-. .
not more than 110.0 percent of the labeled Mobile phase-Dissolve 13.6 g of monobasic potassium
amount of acetylcysteine (CsH9N03S), and not less phosphate in 1000 mL of water, and passthrough a
than 90.0 percent and not more than 115.0 membrane filter having a 0.45-~m porosity. Add 20.0 mL of
methanol, mix, and degas.

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 USP 43 Official Monographs / Acitretin 85

Inter'!al stand~rd solution-Place about 150 mg of IIDENTIFICATION

acetaminophen In a 500-mL volumetric flask add 5 mL of
glacial acetic acid, dilute with water to volu~e, and mix.
Sta'!dard preparation-Dissolve an accurately weighed
qua.ntlty of US~ Isoproterenol Hydrochloride RS in 0.05 M
sodium metabisulfite to obtain a solution having a known
concentration of 0.15 mg per mL. Transfer 10.0 ml of this peak of the Sample
solution to a 25-ml volumetric flask.add 10.0 mL of Internal solution corresponds to that of Standardsolution, as
stCfndard solution, dilute with 0.2 M acetic acid to volume, and obtained in the Assay.
mix.
Assay preparation-Transfer an accurately measured ASSAY
volume of Inhalation Solution, equivalent to about 1 5 mg of • PROCEDURE
isoproterenol hydrochloride, and 10 mL of Internal standard [NOTE-Store the solutions at 4° before injection.]
so~ution. to a 25-mL volumetric flask, add dilute glacial acetic
Mobile phase: Alcohol, glacial acetic acid, and water (92:
acid (1 In 100) to volume, and mix. 0.3: 8)
. ~hromatographic sy~tem (~ee Chromatography (621 »- The
System suitability stock solution: 0.01 mg/mL each of USP
Acitretin RS and USP Tretinoin RS in alcohol.
llquld chromatograph IS equipped with a 280-nm detector
and a 3.9-mm x 40-cm column that contains packing L1. The [NOTE-Dissolve in tetrahydrofuran before diluting with
flow rate is about 2 mL per minute. Chromatograph the alcohol.]
S~andard preparation, and record the peak responses as
System suitability solution: 0.25 J,Jg/mleach of USP .
Ac!tre~i~ RS and USP Tretinoin RS in alcohol, from System
directed for Procedure: the relative retention times are about
0.5 for isoproterenol hydrochloride and 1.0 for SUItability stock solution
acetaminophen; the resolution, R, between isoproterenol Standard solution: 0.1 mg/ml of USp· Acitretin RS in
hydr~)Chloride and acetaminophen is not less than 6; and the
alcohol. [NOTE-Dissolve in tetrahydrofuran before diluting
relative standard deviation for replicate injections is not more with alcohol. The final concentration of tetrahydrofuran in
than 2.0%. the preparation will be 2%.]
Procedure-Separately inject equal volumes (about 25 J.Jl) Sample stock solution: 0.25 mg/ml of Acitretin in
of the Standardpreparation and the Assay preparation into the tetrahydrofuran and alcohol (1:19). [NOTE-Dissolve in
chromatograph, record the chromatograms, and measure the tetrahydrofuran before diluting with alcohol.]
responses for the major peaks. Calculate the quantity, in mg, Sample solution: 0.1 mg/ml of Acitretin in alcohol from
of Isoproterenol hydrochloride (C ll H17N0 3. HCI) in each ml Sample stock solution '
Chromatographic system
of the Inhalation Solution taken by the formula:
(See Chromatography (621), System Suitability.)
(25C/V)(Ru/Rs) Mode: LC
Detector: UV 360 nm
in which C is the concentration, in mg per mL, of USP Column: 4-mm x 25-cm; packing II
!soproterenol ~ydrochloride RS in the Standardpreparation; V Flow rate: 0.6 ml/min
IS the volume, In ml, of Inhalation Solution taken; and Ru and Injection size: 10 J.Jl
System suitability
Rs are the ratios of the peak responses of isoproterenol
Samples: System suitability solution and Standardsolution
hydrochloride to those of acetaminophen obtained from the [NoTE-The relative retention times for tretinoin and
Assay preparation and the Standardpreparation! respectively. acitretin are 0.84 and 1.0, respectively.]
Suitability requirements
Resolution: NlT 2.0 between tretinoin and acitretin
System SUitability solution '
Acitretin Relative standard deviation: NMT 1.0% of acitretin,
Standardsolution
CH, CH, CH, 0 Analysis
H'C~~~~
I ""
Samples: Standardsolution and Sample solution
OH Calculate the percentage of C21H2603 in the portion of
H,CO ~ CH, Acitretin taken:
C2l H2603 326.43 Result =(ru/rs) x (Cs/Cu) x 100
2,4!6,8-Nonatetraenoic acid, 9-(4-methoxy-2,3,6-
tnmethylphenyl)-3,7-dimethyl-, (all~£)-; =peak response from the Sample solution
(all-£)-9-( 4-Methoxy-2, 3,6-trimethylphenyl)-3, 7-dimethyl- =peak responsefrom the Standardsolution
2,4,6,8-nonatetraenoic acid [55079-83-9]. . = concentration of USP Acitretin RS in the Standard
DEFINITION solution (mg/mL)
Acitretin contains NlT 98.0% and NMT 102.0% of C21H2603, =concentration of Acitretin in the Sample solution
calculated on the dried basis. (mg/mL)
[CAUTION-Acitretin is a teratogen. Great care should be
taken when handling to avoid inhalation of dust or Acceptance criteria: 98.0%-102.0% on the dried basis
contact with skin.] IMPURITIES
[NoTE-Use low-actinic glassware and perform all tests INORGANIC IMPURITIES
under yellow and subdued light.] • Residue on Ignition (281): NMT 0.1%
ORGANIC IMPURITIES
[NOTE-Store the solutions at 4° before injection.]

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86 Acitretin / Official Monographs USP 43

• Procedure SPECIFIC TESTS

Mobile phase and Chromatographic system: Proceed as • Loss ON DRYING (731): Dry a sample in a vacuum at a
directed in the Assay. pressure not exceeding 19 mm of mercury at 100° for 4 h:
Standard solution: 0.8 ~g/ml each of USP Acitretin RS, USP it loses NMT 0.2% of its weight.
Acitretin Related Compound A RS, and USP Acitretin Related
Compound B RS in alcohol. [NOTE-Dissolve in ADDITIONAL REQUIREMENTS
tetrahydrofuran before diluting with alcohol.] • PACKAGING AND STORAGE: Preserve in tight containers,
Sample solution: 0.25 mg/ml of Acitretin in tetrahydrofuran protected from light. Store at controlled room temperature.
and alcohol (1:19). [NOTE-Dissolve in tetrahydrofuran • USP REFERENCE STANDARDS (11)
before diluting with alcohol.] USPAcitretin RS
System suitability USPAcitretin Related Compound A RS
(See Chromatography (621), System Suitability.) (2Z,4E,6E,8 £)-9-(4-Methoxy-2,3,6-trimethylphenyl)-3, 7-
Sample: Standard solution dimethylnona-2,4,6,8-tetraenoic acid.
Suitability requirements C21H2603 326.43
Resolution: NlT 1.5 between acitretin related compound USPAcitretin Related Compound B RS
A and acitretin; NlT 1.5 between acitretin related Ethyl (all-£)-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-
compound Band acitretin dimethylnona-2,4,6,8-tetraenoate.
Relative standard deviation: NMT 10.0% for acitretin C23H3003 354.48
related compound A and NMT 10.0% for acitretin related USPTretinoin RS
compound B
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of acitretin related compound A
and acitretin related compound B in the portion of Acitretin Capsules
Acitretin taken: DEFINITION
Acitretin Capsules contain NlT 90.0% and NMT 110.0% of
Result =(ru/rs) x (Cs/Cu) x 100
the labeled amount of acitretin (C21H2603)'
ru = peak response from the relevant impurity from [CAUTION-Acitretin is a teratogen. Great care should be
the Sample solution taken when handling to avoid inhalation of dust or
rs = peak response from the relevant impurity from contact with skin.]
the Standardsolution [NoTE-Use low-actinic glasswareand perform all tests
C, = concentration of USP Acitretin Related under yellow and subdued light. Make all injections within
Compound A RS or USP Acitretin Related 1 h of the Sample solution preparation.]
Compound B RS in the Standard solution IDENTIFICATION
(Ilg/ ml ) • A. The retention time of the major peak of the Sample
Cu = concentration of Acitretin in the Sample solution solution corresponds to that of the Standard solution, as
(Ilg/ml ) . obtained in the Assay.
• B. The UV spectrum of the major peak of the Sample
Calculate the percentage of impurities other than acitretin solution corresponds to that of the Standardsolution, as
related compounds A and B in the portion of Acitretin obtained in the Assay.
taken:
ASSAY
Result =(ru/r s) x (Cs/Cu) x 100 • PROCEDURE
Diluent: Methanol and tetrahydrofuran (13:10)
ru =peak response of each individual unspecified Mobile phase: Methanol, alcohol, glacial acetic acid, and
impurity from the Sample solution water (74: 5: 0.5: 21)
rs = peak response of USP Acitretin RS in the Standard Standard solution: 0.1 mg/ml of USP Acitretin RS in a
solution mixture of Diluent and water (23:2). Dissolve USP Acitretin
C, = concentration of USP Acitretin RS in the Standard RS in Diluent equivalent to 80% of the final volume, sonicate
solution (uq/rnl.) for 5 min, add water equivalent to 8% of the final volume,
Cu = concentration of Acitretin in the Sample solution and dilute with Diluent to volume.
(~g/ml) System suitability solution: Transfer 2 mL of the Standard
solution to a clear 4-ml glassvial. After sealing the vial with
Acceptance criteria a Teflon-lined silicone septum and cap, place the vial on its
Individual impurities: See Impurity Table 1. side in a light chamber, expose it to 400 foot-candles of
Total impurities: NMT 1.0% fluorescent light for 5 min, and then completely wrap the
vial with aluminum foil.
Impurity Table 1 [NOTE-Exposure to the fluorescent light allows for the
Relative Acceptance formation of two degradation products: acitretin
Retention Criteria, related compound A and 6Z-isomer «2E,4E,6Z,8£)-
Name Time NMT(%) 9-(4-methoxy-2, 3,6-trimethylphenyl)-
Acitretin related compound A 0.78 0.3 3,7-dimethylnona-2,4,6,8-tetraenoic acid).]
Sample solution: Nominally 0.1 mg/ml of acitretin in a
Acitretin 1.0 - mixture of Diluent and water (23:2). Open NlT 20
Acitretin related compound B 1.61 0.3 Capsules, composite the Capsulefill, and mix well. Transfer
the Capsule fill to a volumetric flask, add water equivalent
Anyunspecified impurity - 0.1
to 8% of the final volume to wet the sample, and sonicate
Total unspecified impurities - 0.4 for 5 min. Dilute with Diluentto volume, and sonicate for 5
min. Cool to room temperature, pass the suspension

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USP 43 Official Monographs / Acitretin 87

through a suitable filter of 0.5-l..Im pore size, and use the Result = [(A u- A dl A s] x (C sl L) x Vx 100
clear filtrate. [NoTE-Inject the Sample solution within 1 h of
preparation.] Au =absorbance of the Sample solution
Chromatographic system A cs =Capsule shell correction, calculated as shown
(See Chromatography (621), System Suitability.) below
Mode: LC As =absorbance of the Standard solution
Detector: UV 365 nm. For Identification B, use a diode array C5 =concentration of the appropriate Standard
detector in the range of 200-400 nm. solution (mg/mL) .
Column: 4.6-mm x 15-cm; 5-l..Im packing L1 L = label claim (mg/Capsule)
Flow rate: 1 mL/min V = volume of Medium, 900 mL
Injection volume: 25 I..IL
System suitability The Capsule shell correction, A cs, is calculated:
Samples: Standard solution and System suitability solution
[NOTE-The relative retention times for acitretin related A C5 = A css/ N
compound A (2Z-isomer), acitretin, and the
6Z-isomer are 0.84, 1.0, and 1.09, respectively.] A C55 =absorbance of the Capsule shellsolution
Suitability requirements N = number of Capsule shells used to prepare the
Resolution: NLT 3.0 between acitretin related Capsule shellsolution
compound A and acitretin; NLT 1.8 between the
6Z-isomer and acitretin, System suitabilitysolution Tolerances: NLT85% (Q) of the labeled amount of
Relative standard deviation: NMT 2.0%, Standard acitretin (CzlHz603) is dissolved.
solution Test 2: If the product complies with this test, the labeling
Analysis indicates that the product meets USP Dissolution Test 2.
Samples: Standard solution and Sample solution Tier 1
Calculate the percentage of the labeled amount of acitretin Medium: 3% sodium lauryl sulfate in deaerated water,
(CZl Hz60 3) in the portion of Capsules taken: pH 9.6-10.0 (adjusted with 1 N sodium hydroxide);
900 mL
Result = (r vIr s) x (C sICv) x 100 Apparatus 1: 100 rpm
Time: 30 min
ru = peak response of acitretin from the Sample Tier 2
solution Medium A: Prepare a solution containing pancreatin
rs = peak response of acitretin from the Standard with NMT 2000 USP Units/L of protease activity in
solution deaerated water, pH 8.0 (adjusted with 1% sodium
Cs = concentration of USP Acitretin RS in the Standard hydroxide); 450 mL. Use immediately.
solution (mg/mL) Medium B: 6% sodium lauryl sulfate in deaerated water,
Cu = nominal concentration of acitretin in the Sample pH 10.5 (adjusted with 1% sodium hydroxide); 450 mL
solution (mg/mL) . Apparatus 1: 100 rpm
Time: 15 min, MediumA; 15 min, MediumA with the
Acceptance criteria: 90.0%-110.0% addition of Medium B
PERFORMANCE TESTS Determine the amount of acitretin (CZl Hz603) dissolved
• DISSOLUTION (711) using the following method.
Test 1 Mobile phase: Methanol, water, and glacial acetic acid
Medium: 3% sodium lauryl sulfate in deaerated water, pH (750:250:1)
9.6-10.0; 900 mL Standard stock solution: 280 I..Ig/mL of USP Acitretin RS
Apparatus 1: 100 rpm in absolute alcohol. Use sonication to dissolve.
Time: 30 min Standard solution: 20 I..Ig/mL of USP Acitretin RS in
Determine the amount of acitretin (CZl Hz60 3) dissolved Medium under Tier 1, from Standard stock solution
using the following method. Sample solution: Pass a portion of the solution under test
Standard solution: Transfer about 14 mg of USPAcitretin through a suitable glass filter with l-urn pore size, discard
RS to a 500-mL volumetric flask. Dissolve in 50 mL of the first few mL, and use the filtrate for analysis.
alcohol, and dilute with Medium to volume. Dissolution procedure: Perform the test using the
For Capsules labeled to contain 10 mg: Transfer 20 mL conditions under Tier 1. In the presence of cross-linking
of this solution to a 50-mL volumetric flask, and dilute repeat the test with new Capsules using the conditions
with Medium to volume. under Tier2 as follows. After 15 min, stop the dissolution
Sample solution: Use portions of the solution under test bath and timer (do not lift the baskets), and add 450 mL
passed through a suitable filter of 0,45-l..Im pore size. of Medium B pre-equilibrated at 37 ± 0.5°. Restart the
Capsule shell solution: Dissolve 6 clean empty-shell timer and bath, and after 5 min check the pH of the
Capsules in 900 mL of Medium. medium and adjust with 1% sodium hydroxide to a range
Instrumental conditions of 9.6-10.0. Continue dissolution for an additional 10
Mode: UV min.
Analytical wavelength: 347 nm Chromatographic system .
Cell: 2 mm (See Chromatography (621), System Suitability.)
Blank: Medium Mode: LC
Analysis Detector: 360 nm
Samples: Standard solution, Sample solution, and Capsule Columns
shellsolution Guard: 4-mm xl-em; 5-l..Im packing L1
Calculate the percentage of the labeled amount of Analytical: 4.6-mm x 5-cm; 5-l..Im packing L1
acitretin (CZl Hz603) dissolved: Temperatures
Autosampler: 40°

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88 Acitretin / Official Monographs USP43

Column: 35° • USP REFERENCE STANDARDS (11)

Flow rate: 2.0 mL/min USP Acitretin RS
Injection volume: 10 J.lL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0 Acyclovir
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
acitretin (C21H2603) dissolved:

Result = (r vir s) x (C slL) x V x 100

CSH llN s0 3 225.20
= peak response of acitretin from the Sample 6H-Purin-6-one, 2-amino-1,9-dihydro-9-[(2-hydroxyeth oxy)
solution methyl]-.
= peak response of acitretin from the Standard 9-[(2-Hydroxyethoxy)methyl]guanine [59277-89-3].
solution
=concentration of USP Acitretin RS in the Standard » Acyclovir containsnot less than 98.0 percent and
solution(mg/mL) not more than 101.0 percent of CgH" NS0 31
L =label claim (mg/Capsule) calculated on the anhydrous basis.
V =volume of Medium, 900 mL
Tolerances: NLT 85% (Q) of the labeled amount of Packaging and storage-Preserve in tight containers. Store
acitretin (C21H2603) is dissolved. at room temperature. Protect from light and moisture.
USP Reference standards (11)-
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
·USP Acyclovir RS
requirements
Identification-
IMPURITIES
• ORGANIC IMPURITIES: LIMIT OF DEGRADATION PRODUCTS
Diluent, Mobile phase, System suitability solution,
Sample solution, Chromatographic system, and System
suitability: Proceed as directed in the Assay.
Analysis -
retention time of the major peak in the
Sample: Sample solution chromatogram of the Assay preparation corresponds to that in
Calculate the percentage of each degradation product in the chromatogram of the Standard preparation, asobtained in
the portion of Capsulestaken: . the Assay and limit for guanine.
Water Determination, MethodI (921): not more than 6.0%.
Result =(r vir r) x 100
Ordinary impurities (466)-
ru = peak response of each individual.impurity from Test solution: dimethyl sulfoxide.
the Sample solution Standardsolution: dimethyl sulfoxide.
rT = sum of the responses of all the peaks from the Eluant: a mixture of chloroform, methanol, and
Sample solution . ammonium hydroxide (80:20:2).
Visualization: 1.
Acceptance criteria: See Table 7. Application volume: 5 J.lL.
Limit: 1%.
Table 1
Relative Acceptance
Assay and limit for guanine-
Retention Criteria, Mobile phase-Prepare a filtered and degassed solution of
Name Time NMT(%) glacial acetic acid in water (1 in 1000). Make adjustments if
necessary (see System Suitability under Chromatography
Acitretin related
compound A (621».
(2Z-isomer)a 0.84 0.5 System suitability solution 7-Dissolve accurately weighed
quantities of USP Acyclovir RS and guanine in 0.1 N sodium
Acitretin 1.0 - hydroxide, and dilute quantitatively, and stepwise if necessary,
Any unspecified with water to obtain a solution havlnq known concentrations
impurity - 0.4 of about 0.1 rnq of each per mL.
Total unspecified System suitability solution 2-Dissolve an accurately
impurities - 0.8 weighed quantity of guanine in 0.1 N sodium hydroxide, and
dilute quantitatively, and stepwise if necessary, with water to
a (2Z,4 £,6£, 8E)-9-( 4-Methoxy-2,3,6-trimethylphenyl)-3,7-dimethylnona- obtain a solution having a known concentration of about 0.7
2,4,6,8-tetraenoic acid (C21H 2P 3326.43). J.lg per mL.
Guanine standardpreparation-Transfer about 8.75 mg of
ADDITIONAL REQUIREMENTS guanine, accurately weighed, to a 500-mL volumetric flask.
• PACKAGING AND STORAGE: Preserve in well-closed, Dissolve in 50 mL of 0.1 N sodium hydroxide, dilute with water
light-resistant containers. to volume, and mix. Transfer 2.0 mL of this solution to a 50-mL
• LABELING: When more than one Dissolution test is given, the volumetric flask, dilute with 0.01 N sodium hydroxide to
labeling states the test used only if Test 7 is not used.

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 USP 43 OfficialMonographs / Acyclovir 89

volume, and mix to obtain a solution having a concentration System suitability solution B: 2.0 IJg/ml of guanine.
of about 0.7 IJg per mL. Dissolve in 0.1 N sodium hydroxide, and dilute with water.
Standard preparation-Dissolve about 25 mg of USP Standard solution: 0.1 mg/mL of USP Acyclovir RS. Dissolve
Acyclovir RS, accurately weighed, in 5 mL of 0.1 N sodium in 0.1 N sodium hydroxide, and dilute with water.
hydroxide in a 50-ml volumetric flask, dilute with water to Sample solution: Nominally 0.1 mg/mL of acyclovir
volume, and mix. Transfer 10.0 ml of this solution to a 50-mL prepared asfollows. Transfer the contents of Capsules
volumetric flask, dilute with 0.01 N sodium hydroxide to equivalent to 10 mg of acyclovir (NLT 10 Capsules) to a
volume, and mix to obtain a solution having a known 1OO-ml volumetric flask. Dissolvein 10 mL of 0.1 N sodium
concentration of about 0.1 mg of USP Acyclovir RS per ml. hydroxide, dilute to volume with water, and filter.
Assay preparation-Dissolve about 100 mg of Acyclovir, Chromatographic system
accurately weighed, in 20 ml of 0.1 N sodium hydroxide in a (See Chromatography (621), System Suitability.)
200-mL volumetric flask, dilute with water to volume, and mix. Mode: LC
Transfer 10.0 mL of this solution to a 50-ml volumetric flask, Detector: UV 254 nm
dilute with 0.01 N sodium hydroxide to volume, and mix. Column: 4.2-mm x 25-cm; packing II
Chromatographic system (see Chromatography (621 »)-The Flow rate: 1.5 mL/min
liquid chromatograph is equipped with a 254-nm detector Injection volume: 20 IJl
and a 4.6-mm x 25-cm column that contains packing ll. The System suitability
flow rate is about 3 ml per minute. Chromatograph System Samples: System SUitability solution A and System suitability
suitability solution 7, and record the peak responses as directed solution B
for Procedure: the resolution, R, between acyclovir and guanine [NoTE-The relative retention times for guanine and
is not less than 2.0; the tailing factor for the analyte peak is not acyclovir are about 0.6 and 1.0, respectively, in
more than 2; and the relative standard deviation for replicate System suitability solution A.]
injections for the acyclovir peak is not more than 2.0%. Suitability requirements
Chromatograph System suitability solution 2, and record the Resolution: NlT 2.0 between guanine and acyclovir,
peak responses as directed for Procedure: the relative standard System suitability solution A
deviation for replicate injections is not more than 2.0%. Relative standard deviation: NMT 2.0% for the
Procedure-Separately inject equal volumes (about 20 IJl) acyclovir peak, System suitability solution A
of the Standard preparation, the Guanine standard Relative standard deviation: NMT 2.0%, System
preparation, and the Assay preparation into the suitability solution B
chromatograph, record the chromatograms, and measure the Analysis: Standard solution and Sample solution
responses for all the peaks. Calculate the quantity, in IJg, of Calculate the percentage of the labeled amount of acyclovir
guanine in the portion of Acyclovir taken by the formula: (CSH ll N S0 3) in the portion of Capsules taken:

1000qrvlrs) Result = (rvlrs) x (CsICv) x 100

in which C is the concentration, in IJg per ml, of guanine in = peak response of the Sample solution
the Guanine standard preparation; and rv and rs are the peak = peak response of the Standard solution
responses due to guanine in the Assay preparation and the =concentration of USP Acyclovir RS in the Standard
Guanine standard preparation, respectively: not more than solution (mg/ml)
0.7% of guanine is found. Calculate the quantity, in mg, of Cv = nominal concentration of acyclovir in the Sample
CSH11Ns03 in the portion of Acyclovir taken by the formula: solution (mg/ml)
1000qrvlrs) Acceptance criteria: 93.0%-107.0%

in which C is the concentration, in mg per mL, of USP Acyclovir PERFORMANCE TESTS

RS in the Standard preparation; and rvand rs are the peak • DISSOLUTION (711 )
responses due to acyclovir in the Assay preparation and the Medium: 0.1 N hydrochloric acid; 900 ml
Standard preparation, respectively. Apparntusl: 100 rpm
Time: 45 min
Detector: UV 254 nm
Standard solution: USP Acyclovir RS in Medium
Sample solutions: Dilute with Medium to a concentration
Acyclovir Capsules that is similar to the Standard solution.
Analysis: Determine the amount of acyclovir (CSH ll N S0 3)
DEFINITION dissolved from UV absorption at the wavelength of
Acyclovir Capsules contain NLT 93.0% and NMT 107.0% of maximum absorption on filtered portions of the solution
the labeled amount of acyclovir (CSH11Ns03)' under test.
Tolerances: NLT 75% (Q) of the labeled amount of
IDENTIFICATION acyclovir (CSH ll N S0 3) is dissolved.
• A. The retention time of the major peak of the Sample • UNIFORMITY OF DOSAGE UNITS (905): Meet the
solution corresponds to that of the Standard solution, as requirements for Content Uniformity .
obtained in the Assay.
IMPURITIES
ASSAY • PROCEDURE
• PRO(EDURE Mobile phase, System suitability solution A, System
Mobile phase: 0.02 M acetic acid suitability solution B, Sample solution,
System suitability solution A: 0.1 mg/ml each of USP Chromatographic system, and System suitability:
Acyclovir RS and guanine. Dissolve in 0.1 N sodium Proceed as directed in the Assay.
hydroxide, and dilute with water. Analysis: Sample solution

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90 Acyclovir / OfficialMonographs USP 43

Calculate the percentage of each impurity in the portion of Result = (r vir s) x (C siCv) x 100
Capsules taken:
= peak response of the Sample solution
Result = (rvlrr) x 100 = peak response of the Standard solution
= concentration of USP Acyclovir RS in the Standard
ru = peak response for each impurity solution (mg/mL)
rr = sum of the responses for all of the peaks = concentration of the Sample solution (mg/mL)
Acceptance criteria Acceptance criteria: 90.0%-110.0%
Guanine: NMT 2.0%
Any individual impurity: NMT 0.5% IMPURITIES
• PROCEDURE
ADDITIONAL REQUIREMENTS Solution A: 0.17 M acetic acid and methanol (125:8)
• PACKAGING AND STORAGE: Preserve in tight containers. Solution B: Methanol
Store between 15° and 25°. Protect from light and Mobile phase: See Table 1.
moisture.
• USP REFERENCE STANDARDS (11) Table 1
USP Acyclovir RS
Time Solution A Solution B
(min) (%) (%)
0 100 0
15 100 0
Acyclovir for Injection
45 65 35
DEFINITION 46 100 0
Acyclovir for Injection contains NLT 90.0% and NMT 110.0%
-
of the labeled amount of acyclovir (CsH"Ns0 3) . 56 100 0

IDENTIFICATION
• A. The retention time of the major peak of the Sample System suitability solution: 0.5 IJg/mL each of purine and
solution corresponds to that of the Standard solution, as USP Acyclovir RS in Solution A
obtained in the Assay. Acyclovir standard solution: 5 IJg/mL of USP Acyclovir RS
in Solution A
ASSAY Guanine solution: 0.05 mg/mL of guanine prepared as
• PROCEDURE follows. Dissolve 25 mg of guanine in 50 mL of 0.1 N
Mobile phase: 0.02 M acetic acid sodium hydroxide in a 500-mL volumetric flask, and bring
System suitability solution A: 0.1 mg/mL each of USP the solution to volume with water.
Acyclovir RS and guanine in 0.1 N sodium hydroxide Standard solution A: 0.5 IJg/mL of Acyclovir standard
System suitability solution B: 2.0 IJg/mL of guanine in 0.1 solution in Solution A
N sodium hydroxide Standard solution B: 5 IJg/mL of Guanine solution in
Standard solution: 0.1 mg/mL of USP Acyclovir RS in 0.1 N Solution A
sodium hydroxide Sample solution: Equivalent to 0.5 mg/mL of acyclovir from
Sample solution: Nominally 0.1 mg/mL of acyclovir a mixture of NLT 10 reconstituted vials of Acyclovir for
prepared asfollows. Constitute 1 vial of Acyclovir for Injection in Solution A
Injection with water. Transfer an amount, equivalent to 10 Chromatographic system
mg of acyclovir, to a 1OO-mL volumetric flask, and dilute (See Chromatography (621), System Suitability.)
with water to volume. Mode: LC
Chromatographic system Detector: UV 254 nm
(See Chromatography (621), System Suitability.) Column: 4.6-mm x 25-cm; packing L1
Mode: LC Flow rate: 1 mL/min
Detector: UV 254 nm Injection volume: 50 IJL
Column: 4.2-mm x 25-cm; packing L1 System suitability
Flow rate: 1.5 mL/min Samples: System suitability solution, Standard solution A,
Injection volume: 20 IJL and Standard solution B
System suitability . . [NOTE-Typical retention times for guanine and
Samples: System sUitability solution A and System suitability acyclovir of Standard solution A and Standard solution
solution B Bare 5.8 and 14 min, respectively.]
[NoTE-The relative retention times for guanine and Suitability requirements
acyclovir are 0.6 and 1.0, respectively, in System Resolution: NLT 2.0 between purine and acyclovir,
suitability solution A.] System suitability solution
Suitability requirements Relative standard deviation: NMT 1% for the acyclovir
Resolution: NLT 2.0 between guanine and acyclovir, and the guanine peaks, Standard solution A and Standard
System suitability solution A solution B
Relative standard deviation: NMT 2.0% for the Analysis 1
acyclovir peak, System suitability solution A Calculate the percentage of guanine in the Acyclovir for
Relative standard deviation: NMT 2.0%, System Injection taken:
suitability solution B
Analysis Result = (r vir s) x (C siCv) x 100
Calculate the percentage of acyclovir (CsH"N s 0 3) in the
portion of Acyclovir for Injection taken: ru = peak response for guanine, if present, in the
Sample solution

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 USP 43 Official Monographs / Acyclovir 91

rs = peak responseof guanine in the Standard solution flask. Dissolve in and dilute with 0.1 N sodium hydroxide
Cs =concentration of guanine in the Standard solution to volume.
(mg/mL) Chromatographic system
Cu = nominal concentration of acyclovir in the Sample (See Chromatography (621), System Suitability.)
solution (mg/mL) Mode: LC
Detector: UV 254 nm
Acceptance criteria 1: NMT 1.0% guanine Column: 4.6-mm x 25-cm; packing L1
Analysis 2 Flow rate: 3 mL/min
Calculate the percentage of each other impurity in the Injection volume: 20 IJL
portion of Acyclovir for Injection taken: System suitability
Samples: System suitabilitysolution A and System suitability
Result = (r u/r s) x (C s/Cu ) x 100 solution 8
[NoTE-The relative retention times for guanine and
ru = peak response for each impurity acyclovir are about 0.6 and 1.0, respectively, in
rs = peak responseof acyclovir in the Standardsolution System sUitability solution A.]
Cs = concentration of USP Acyclovir RS in the Standard Suitability requirements
solution (mg/mL) Resolution: NLT 2.0 between guanine and acyclovir,
Cu = nominal concentration of acyclovir in the Sample System sUitability solution A
solution (mg/mL) Relative standard deviation: NMT 2.0% for the
acyclovir peak, System suitability solution A; NMT 2.0%,
Acceptance criteria 2: NMT 0.15% for any peak having a System sUitability solution 8
relative retention time of about 0.7 compared to the Analysis
acyclovir peak; NMT 0.5% for any other individual Samples: Standardsolution and Sample solution
impurity; and NMT 1.0% for the total of all other impurities Calculate the percentage of the labeled amount of acyclovir
(CaH'lNS03) in the portion of Ointment taken:
SPECifiC TESTS
• pH (791): 11.0-12.5, 50 mg/mL of acyclovir Result = (rulrs) x (CsICu) x 100
-WATER DETERMINATION, Method 1(921): NMT 5.5%
• STERILITY TESTS (71): Meets the requirements t» = peak response from the Sample solution
• UNIFORMITY OF DOSAGE UNITS (905): Meets the
requirements
rs =peak responsefrom the Standardsolution
Cs = concentration of USP Acyclovir RS in the Standard
• BACTERIAL ENDOTOXINS TEST (85): NMT 0.174 USP
solution (mg/mL)
Endotoxin Unit/mg of acyclovir
• OTHER REQUIREMENTS: Meets the requirements for
Cu =nominal concentration of acyclovir in the Sample
solution (mg/mL)
labeling in Labeling (7), Labels and Labeling for Injectable
Products Acceptance criteria: 90.0%-110.0%
ADDITIONAL REQUIREMENTS
PERfORMANCE TESTS
• PACKAGING AND STORAGE: Preserve in tight containers.
• MINIMUM fiLL (755): Meets the requirements
Store between 15° and 25°. Protect from light.
• USP REFERENCE STANDARDS (11) IMPURITIES
USP Acyclovir RS • LIMIT OF GUANINE
Mobile phase, Sample solution, and Chromatographic
system: Proceed as directed in the Assay.
Standard solution: 2.0 IJg/mL of guanine in 0.1 M sodium
hydroxide
Acyclovir Ointment Analysis
Samples: Standardsolution and Sample solution
DEfiNITION Calculate the percentage of guanine in the portion of
Acyclovir Ointment contains NLT 90.0% and NMT 110.0% of Ointment taken:
the labeled amount of acyclovir (CaH ll Ns03),in a suitable
ointment base. Result =(rulrs) x (CsICu) x 100
IDENTifiCATION
ru = peak response of guanine from the Sample
• A. The retention time of the major peak of the Sample solution
solution corresponds to that of the Standardsolution, as rs = peak response of guanine from the Standard
obtained in the Assay. solution
ASSAY Cs = concentration of guanine in the Standardsolution
• PROCEDURE (mg/mL)
Mobile phase: 0.02 M acetic acid Cu = nominal concentration of acyclovir in the Sample
System suitability solution A: 0.1 mg/mL each of USP solution (mg/mL)
Acyclovir RS and guanine in 0.1 N sodium hydroxide
System suitability solution B: 2.0 IJg/mL of guanine in 0.1 Acceptance criteria: NMT 2.0%
N sodium hydroxide SPECifiC TESTS
Standard solution: 0.1 mg/mL of USP Acyclovir RS in 0.1 N • MICROBIAL ENUMERATION TESTS (61) and TESTS FO.R
sodium hydroxide SPECIFIED MICROORGANISMS' (62): It meets the
Sample solution: Nominally 0.1 mg/mL of acyclovir requirements. of the tests for the absence of Staphylococcus
prepared as follows. Transfer an amount of Ointment, aureus and Pseudomonas aeruginosa.
equivalent to 10 mg of acyclovir, to a 1OO-mL volumetric

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92 Acyclovir / OfficialMonographs USP43

ADDITIONAL REQUIREMENTS = concentration of USP Acyclovir RS in the Standard

··PACKAGBNG AND STORAGE: Preserve in tight containers. solution (mg/mL)
Store between 15° and 25° in a dry place. = nominal concentration of acyclovir in the Sample
• USP REFERENCE STANDARDS (11) solution (mg/ml)
USP Acyclovir RS
Acceptance criteria: 90.0%-110.0%
IMPURITIES
• LIMIT OF GUANINE
Acyclovir Oral Suspension Mobile phase, System suitability solution A, System
suitability solution B, Sample solution,
DEFINITION Chromatographic system, and System suitability:
Acyclovir Oral Suspension contains NlT 90.0% and NMT Proceed as directed in the Assay.
110.0% of the labeled amount of acyclovir (CSH ll NS0 3)· Standard solution: 2.0 IJg/ml of guanine in 0.1 M sodium
hydroxide
IDENTIFICATION Analysis
• A. The retention time of the major peak of the Sample Samples: Standard solution and Sample solution
solution corresponds to that of the Standard solution, as Calculatethe percentage of guanine in the portion of Oral
obtained in the Assay. Suspension taken:
ASSAY Result =(ru/rs) x (CslCu) x 100
• PROCEDURE
Mobile phase: 0.02 M acetic acid tu = peak responsefor guanine from the Sample
System suitability solution A: 0.1 mg/ml each of USP solution
Acyclovir RS and guanine in 0.1 N sodium hydroxide rs = peak responsefor guanine from the Standard
System suitability solution B: 2.0 IJg/ml of guanine in 0.1 solution ,
N sodium hydroxide Cs =concentration of guanine in the Standard solution
Standard solution: 0.1 mg/mL of USP Acyclovir RS in 0.1 N (mg/mL)
sodium hydroxide Cu = nominal concentration of acyclovir in the Sample
Sample stock solution: Nominally 1 mg/ml of acyclovir solution (mg/ml)
prepared asfollows. Transfer an amount ofwell-shaken Oral
Suspension equivalent to 200 mg of acyclovir to a 200-ml Acceptance criteria: NMT 2.0%
volumetric flask. Add 100 ml of 0.1 N sodium hydroxide,
shake by mechanical means for 15 min, and sonicate, if SPECIFICTESTS
necessary, to dissolve the Oral Suspension completely. • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Dilute with 0.1 N sodium hydroxide to volume. SPECIFIED MICROORGANISMS (62): Itstotal count does not
Sample solution: Transfer 10.0 ml of the Sample stock exceed 101 cfu/ml, and it meets the requirementsof the
solution to a 1OO-ml volumetric flask, and dilutewith water tests for absence of Salmonella species and Escherichia coli.
to volume. • pH (791): 4.5-7.0
Chromatographic system PERFORMANCE TESTS
(See Chromatography (621), System Suitability). • UNIFORMITY OF DOSAGE UNITS (905): Meetsthe
Mode: lC requirementsfor Oral Suspension packaged in single-unit
Detector: UV 254 nm containers
Column: 4.6-mm x 25-cm; packing II • DELIVERABLE VOLUME (698): Meetsthe requirementsfor
Flowrate: 3 ml/min Oral Suspension packaged in multiple-unit containers
Injection volume: 20 IJl
System suitability ADDITIONAL REQUIREMENTS
Samples: System SUitability solution A and System suitability • PACKAGING AND STORAGE: Preserve in tight containers.
solution B Store between 15° and 25°. Protectfrom light.
[NOTE-The relative retention times for guanine and • USP REFERENCE STANDARDS (11)
acyclovir are about 0.6 and 1.0, respectively, in . USP Acyclovir RS
System suitability solution A.]
Suitability requirements
Resolution: NLT 2.0 between guanine and acyclovir,
System suitability solution A
Relative standard deviation: NMT 2.0% for replicate Acyclovir Tablets
injections for the acyclovir peak, System suitability
solution A DEFINITION
Relative standard deviation: NMT 2.0% for replicate Acyclovir Tablets contain NLT 90.0% and NMT 110.0% of the
injections, System suitability solution B labeled amount of acyclovir (CSH11NS0 3)·
Analysis IDENTIFICATION
Samples: Standard solution and Sample solution • A. The retention time of the major peak of the Sample
Calculate the percentage ofthe labeledamount of acyclovir solution corresponds to that of the Standard solution, as
(CSH11Ns03) inthe portion of Oral Suspension taken: obtained in the Assay~
Result =(rulrs) x (CsICu) x 100 ASSAY
• PROCEDURE
ru = peak response from the Sample solution Mobile phase: 0.02 M acetic acid
ts = peak response from the Standard solution

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USP 43 Official Monographs / Adapalene 93

System sultablllty solution A: 0.1 mg/mL each of USP IMPURITIES

Acyclovir RS and guanine. Dissolve in 0.1 N sodium • PROCEDURE
hydroxide, and dilute with water. Mobile phase, System suitability solution A, System
System sultablllty solution B: 2.0 ~g/mL of guanine. suitability solution B, Sample
Dissolve in 0.1 N sodium hydroxide, and dilute with water. solution,Chromatographic system, and System
Standard solution: 0.1 mg/mL of USP Acyclovir RS. Dissolve suitability: Proceed as directed in the Assay.
in 0.1 N sodium hydroxide, and dilute with water. Analysis
Sample solution: Nominally 0.1 mg/mL of acyclovir Sample: Sample solution
prepared as follows. Transferan amount offinelypowdered Calculate the percentage of each impurity in the portion of
Tablets equivalent to 10 mg of acyclovir (NLT 10 Tablets) Tablets taken:
to a 1OO-mL volumetric flask. Dissolve in 10 mLof 0.1 N
sodium hydroxide, dilute with water to volume, and filter. Result = (ru/rr) x 100
Chromatographic system
(See Chromatography (621), System SUitability.) ru = peak response for each impurity
Mode: LC rr = sum of the responses for all of the peaks
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing L1 Acceptance criteria
Column temperature: 40° Guanine: NMT2.0%
Flow rate: 1.5 mL/min Any other impurity: NMT 0.5%
Injection volume: 20 IJL
System suitability ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers.
Samples: System suitability solution A and System suitability
solution B Store between 15° and 25°. Protect from light and
[NoTE-The relative retention times for guanine and moisture.
• USP REFERENCE STANDARDS (11)
acyclovir are about 0.6 and 1.0, respectively, in
System suitability solution A.] USP Acyclovir RS
Suitability requirements
Resolution: NLT 2.0 between guanine and acyclovir,
System suitability solution A
Relative standard deviation: NMT2.0% for the Adapalene
acyclovir peak, System suitability solution A; NMT 2.0%,
System suitability solution B o
Analysis OH
Samples: Standard solution and Sample solution
Calculatethe percentage of the labeled amount of acyclovir
(CSH ll NS03) in the portion of Tablets taken:
Result = (ru/rs) x (Cs/Cu) x 100
ru = peak response from the Sample solution
r5 = peak response from the Standard solution C2sH2S03 412.52
C5 = concentration of USP Acyclovir RS in the Standard 2-Naphthalenecarboxylic acid, 6-(4-methoxy-3-tricyclo
solution (mg/mL) [3.3.1.1 3,7]dec-l-ylphenyl)-;
Cu = nominal concentration of acyclovir in the Sample 6-[3-(1-Adamantyl)-4-methoxyphenyl]-2-naphthoic acid.
solution (mg/mL) [106685-40-9].
DEFINITION
Acceptance criteria: 90.0%-110.0% Adapalene contains NLT 98.0% and NMT 102.0% of
PERFORMANCE TESTS adapalene (C2sH2S03), calculated on the dried basis.
• DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL IDENTIFICATION
Apparatus 2: 50 rpm
Time: 45 min
Instrumental conditions
Mode: UV _ J
Wavelength: 254 nm • B. The retention time of the major peak of the Sample
Standard solution: USP Acyclovir RS in Medium solution corresponds to that of the Standard solution, as
Sample solutions: Dilute with Medium to a concentration obtained in the Assay.
that is similarto the Standard solution.
Analysis: Determine the amount of acyclovir (CSH ll NS0 3) ASSAY
dissolvedfrom UV absorption at the wavelength of • PROCEDURE
maximum absorbance on filtered portions of the solution Mobile phase: Acetonitrile, tetrahydrofuran, trifluoroacetic
under test. acid, and water (21: 16: 0.01 : 13)
Tolerances: NLT 80% (Q) of the labeled amount of Standard stock solution: 0.2 mg/mL of USP Adapalene RS
acyclovir (CSHllNs03) is dissolved. in Mobile phase. Dissolve USP Adapalene RS in a minimal
• UNIFORMITY OF DOSAGE UNITS (905): Meet the amount of tetrahydrofuran (about 1%-5% of the final
requirements for Weight Variation volume), using sonication as needed, and dilute with Mobile
phase to volume.

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94 Adapalene / OfficialMonographs USP43

Standard solution: 40 I-lg/mL of USPAdapalene RS in Suitability requirements

Mobile phase from the Standard stocksolution Relative standard deviation: NMT 3.0% for the
Sample stock solution: 0.2 mg/mL of Adapalene in Mobile adapalene peak
phase. Dissolve Adapalene in a minimal amount of Column efficiency: NLT 3000 theoretical plates for the
tetrahydrofuran (about 1%-5% of the final volume), using adapalene peak
sonication as needed, and dilute with Mobile phase to Analysis
volume. Samples: Standardsolution and Sample solution
Sample solution: 40 IJg/mL of Adapalene in Mobile phase Calculate the percentage of adapalene related compounds
from the Sample stock solution A and B in the portion of Adapalene taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result =(r vir s) x (C siC v) x 100
Mode: LC
Detector: UV 235 nm ru = peak area of each impurity from the Sample
Column: 4.6-mm x 25-cm; 5-lJm packing L1 solution
Flow rate: 1 mL/min rs = peak area of corresponding adapalene related
Injection volume: 20 IJL compound A or adapalene related compound B
System suitability from the Standardsolution
Sample: Standardsolution Cs = concentration of corresponding USPAdapalene
Suitability requirements Related Compound A RS or USP Adapalene
Relative standard deviation: NMT 1.0% Related Compound B RS in the Standardsolution
Analysis (mg/mL)
Samples: Standardsolution and Sample solution Cu = concentration of Adapalene in the Sample
Calculate the 'percentage of adapalene (C2sH2S03) in the solution (mg/mL)
portion of Adapalene taken:
Calculate the percentage of each unspecified impurity in
Result = (r vir s) x (C siC v) x 100 the portion of Adapalene taken:

= peak response from the Sample solution Result = (r vir s) x (C siC v) x 100
= peak response from the Standardsolution
ru = peak area of each unspecified impurity from the
= concentration of USPAdapalene RS in the
Sample solution
Standardsolution (lJg/mL)
rs = peak area of adapalene from the Standardsolution
= concentration of Adapalene in the Sample
solution (lJg/mL) Cs = concentration of USPAdapalene RS in the
Standardsolution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis Cu = concentration of Adapalene in the Sample
solution (mg/mL)
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.20% Acceptance criteria: See Table 1. Disregard any impurity
[NoTE-On the basis of the synthetic route, perform either peaks less than 0.05%.
Organic Impurities, Procedure 1 or OrganicImpurities,
Procedure 2.] Table 1
• ORGANIC IMPURITIES, PROCEDURE 1 Relative Acceptance
Procedure 1 is recommended if adapalene related ,
Retention Criteria,
compounds A and B may be present. , Name TIme NMT (%)
Mobile phase: Proceed as directed in the Assay. Adapalene related compound A- 0.52 0.10
Standard stock solution: 0.2 mg/mL of USPAdapalene RS,
0.3 mg/mL of USP Adapalene Related Compound A RS, and Adapalene 1.0 -
0.2 mg/mL of USP Adapalene Related Compound B RS in Adapalene related compound Bb 1.57 0.10
Mobile phase. Dissolve USP Adapalene RS, USP Adapalene
Related Compound A RS, and USP Adapalene Related Any individual unspecified impurity - 0.10
Compound B RS in a minimal amount of tetrahydrofuran Total impurities - 0.50
(about 1%-5% of the final volume), using sonication as
needed, and dilute with Mobilephase to volume. a Methyl 6-bromo-2-naphthoate.
Standard solution: 0.21Jg/mL of USP Adapalene RS, 0.3 IJgI b Methyl 6-[3-(1-Adamantyl)-4-methoxyphenyl]-2-naphthoate.
mL of USP Adapalene Related Compound A RS, and 0.2
IJg/mL of USPAdapalene Related Compound B RS in Mobile • ORGANIC IMPURITIES, PROCEDURE 2
phase from the Standardstocksolution ' Procedure 2 is recommended if adapalene related
Sample solution: 0.2 mg/mL of Adapalene in Mobile compounds E, C, and D may be present.
phase. Dissolve Adapalene in a minimal amount of Solution A: Glacial acetic acid and water (0.1 : 100)
tetrahydrofuran (about 1%-5% of the final volume), using Solution B: Acetonitrile and tetrahydrofuran (65:35)
sonication as needed, and dilute with Mobile phase to Mobile phase: See Table 2.
volume. '
Chromatographic system: Proceed as directed in the Table 2
Assay, except use a run time of NLT two times the retention Time Solution A Solution B
time of adapalene peak for Standardsolution and NLT six (min) (%) (%)
times the retention time of adapalene peak for Sample 0 50 50
solution.
System suitability 2.5 50 50
Sample: Standardsolution 40 72
28

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USP 43 OfficialMonographs / Adapalene 95

Table 2 (continued) Table 3 (continued)

Time Solution A Solution B Relative Relative Acceptance
(min) (%) (%) Retention Response Criteria,
Name Time Factor NMT (%)
42 28 72
Adapalene related com-
42.1 50 50 pound CC 0.9 0.14 0.1
50 50 50 Adapalene 1.0 - -
Adapalene related com-
Diluent: Acetonitrile, tetrahydrofuran, and water pound Dd 1.9 0.71 0.2
(37:20:43)
Any individual unspeci-
Standard stock solution: 0.2 mg/mL of USP Adapalene RS fied impurity - 1.0 0.1
in tetrahydrofuran .
Standard solution: 2.0 ~g/mL of USP Adapalene RS In Total impurities - - 0.5
Diluent from the Standardstock solution
a 2 2'-Binaphthyl-6,6'-dicarboxylie acid.
System suitability solution: 0.2 mg/mL of USP Adapalene
RS and 1.2 ~g/mL each of USP Adapalene Related b 6~[3-(3-Hydroxyadamant-1-yl)-4-methoxyphenyl]-2-naphthoie acid.
c 2-(Adamant-1-yl)methoxybenzene.
Compound C RS, USP Adapalene Related Compound D d 4,4'-Dimethoxy-3,3'-di(adamant-l-yl)biphenyl.
RS, and USP Adapalene Related Compound E RS I?repared
by dissolving the standards in te~ra~ydro~uran .equlvalent to II RESIDUAL SOLVENT: LIMIT OF TRIETHYLAMINE
50% of the final volume, and diluting with DIluent to [NOTE-This test should be performed if triethylamine is
volume used in the manufacturing process.]
Sample solution: 2.0 mg/mL of Adapalene prepared by Diluent: Dimethyl sulfoxide . . .
dissolving in tetrahydrofuran equivalent to 50% of the final Standard solution: 4.0 ~g/mL of USP Triethylamine RS In
volume, and diluting with Diluent to volume Diluent. Transfer 4.0 mL of this solution to a 20-mL
Chromatographic system headspace vial, and add 1.0 mL of 1 N Na~H ~olution.
(See Chromatography (621), System Suitability.) Sample solution: 50 mg/mL of Adapalene In DIluent. .
Mode: LC Transfer 4.0 mL of this solution to a 20-mL headspace vial,
Detector: UV 270 nm and add 1.0 mL of 1 N NaOH solution.
Column: 4.6-mm x 25-cm; 5-~m packing Lll with 7.5% Chromatographic system
carbon loading (See Chromatography (621), System Suitability.)
Column temperature: 30 0 Mode: GC
Flow rate: 1.2 mL/min Detector: Flame ionization
Injection volume: 25 ~L Column: 30-m x 0.53-mm; 3.0-~m coating of G27
System suitability Temperatures
Sample: System suitability solution Injection port: 250 0
Suitability requirements Detector: 300 0
Resolution: NLT4.5 between the adapaleneand Column: See Table 4.
adapalene related compound C peaks
Signal-to-noise ratio: NLT 10 for the adapalene related Table 4
compound C peak
Hold Time at Fi-
Analysis . Initial Temperature Final nal
Samples: Standard solution and Sample solution Temperature Ramp Temperature Temperature
Calculate the percentage of each impurity in the portion of CO) CO/min) CO) (min)
Adapalene taken:
40 0 40 5
Result = (r vir s) x (C siC v) x (1 IF) x 100 40 40 240 5

ru = peak response of each impurity from the Sample

solution . Headspace operating parameters
[NOTE-Headspace operating parameters can be
rs = peak response of adapalene from the Standard modified in order to optimize the performance.]
solution
Equilibration temperature: 95 0
Cs = concentration of adapalene in the Standard
Equilibration time: 15 min
solution (mg/mL)
Transfer line temperature: 125 0
Cu = concentration of Adapalene in the Sample
Pressurization time: 3 min
solution (mg/mL) . .. Carrier gas: Nitrogen
F = relative response factor for each indivldual
Flow rate: 4.8 mL/min
impurity (see Table 3)
Injection volume: 1 mL
Acceptance criteria: See Table 3. Disregard any impurity System suitability
peaks less than 0.05%. Sample: Standard solution
Suitability requirements
Table 3 Relative standard deviation: NMT 15%
Analysis
Relative Relative Acceptance Samples: Standard solution and Sample solution
Retention Response Criteria,
Name Time Factor NMT (%) Calculate the content, in ppm, of triethylamine in the
portion of Adapalene taken:
Adapalene related com-
pound Ea 0.3 1.4 0.3
Result = (r vir s) x (C siC u) x 10 6
Hydroxyadapalene" 0.5 0.91 0.1

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96 Adapalene / OfficialMonographs USP 43

ru = peak response of triethylamine from the Sample • B. The retention time of the major peak of the Sample
solution solution corresponds to that of the Standardsolution, as
rs = peak response of triethylamine from the Standard obtained in the Assay.
solution
Cs = concentration of triethylamine in the Standard ASSAY
solution (mg/mL) • PROCEDURE
Cu = concentration of Adapalene in the Sample Mobile phase: Acetonitrile, tetrahydrofuran, trifluoroacetic
solution (mg/mL) acid, and water (43: 36: 0.02: 21)
Standard stock solution: 0.25 mg/mL of USP Adapalene
Acceptance criteria: NMT 80 ppm RS, prepared as follows. Transfer USP Adapalene ~S to a
suitable volumetric flask, add tetrahydrofuran equivalent to
SPECIFIC TESTS 1% ofthe final volume, and sonicate to dissolve. Dilutewith
• Loss ON DRYING (731) Mobilephase to volume.
Analysis: Drya sample at 105° for 4 h. Standard solution: 20 ~g/mL of USP Adapalene RS in
Acceptance criteria: NMT 0.6% Mobilephase, from Standardstock solution
Sample stock solution: Nominally equivalent to 20 ~g/mL
ADDITIONAL REQUIREMENTS of adapalene, prepared as follows. Transfer 2.0 9 of Gel to
• PACKAGING AND STORAGE: Preserve in tight, light-resistant a 1OO-mL volumetric flask, add 25 mL of tetrahydrofuran,
containers, and store at room temperature. and sonicate to dissolve. Add 25 mLof acetonitrile and
• LABELING: Ifa test for OrganicImpurities other than sonicate for 20 min. Cool to room temperature and dilute
Procedure 7 is used, the labelingstates the test with which with Mobilephase to volum~. .
the article complies. Sample solution: Pass a portion of Sample stock solution
• USP REFERENCE STANDARDS (11) through a Teflon filterof 0045-~m pore size and use the
USP Adapalene RS filtrate.
USP Adapalene Related Compound A RS Chromatographic system
Methyl 6-bromo-2-naphthoate. (See Chromatography (621), System Suitability.)
C12H9Br02 265.10 Mode: LC
USP Adapalene Related Compound BRS Detector: UV 235 nm
Methyl 6-[3-(1-adamantyl)-4-methoxyphenyl]-2- Column: 4.6-mm x 25-cm; 5-~m packing L1
naphthoate. Flow rate: 1 mL/min
C29H3003 426.55 Injection volume: 20 ~L
USP Adapalene Related Compound C RS System suitability
2-(Adamant-1-yl)methoxybenzene. Sample: Standard solution
C17H 220 242.36 Suitability requirements
USP Adapalene Related Compound D RS Tailing factor: NMT 2.0
4,4' -Dimethoxy-3,3'-di(adamant-1-yl)biphenyl. Relative standard deviation: NMT 2.0%
C34H4202 482.70 Analysis
USP Adapalene Related Compound ERS Samples: Standard solution and Sample solution
2,2' -Binaphthyl-6,6'-dicarboxylic acid. Calculatethe percentage of the labeled amount of
C22H1404 342.34 adapalene (C2sH2S03) in the portion of Gel taken:
USP Triethylamine RS
Triethylamine. Result = (rulrs) x (CsICu) xl 00
C6H,sN 101.19'
= peak response from the Sample solution
= peak response from the Standardsolution
=concentration of USP Adapalene RS in the
Standard solution (mg/mL)
Adapalene Gel Cu = nominal concentration of adapalene in the
Sample solution (mg/mL)
DEFINITION
Adapalene Gel contains NLT 90.0% and NMT 110.0% of the Acceptance criteria: 90.0%-110.0%
labeled amount of adapalene (C2sH2S03);
IMPURITIES
IDENTIFICATION • ORGANIC IMPURITIES
Buffer: 6.8 giL of monobasic potassium phosphate in water.
Adjustwith phosphoric acid to a pH of 3.5.
Solution A: Use Mobilephase in the Assay.
• A•. ~.~~EC"I"~OSCOf>I<:il~~N·IJ~I~~"I",~~>[~~~~~,f~l~~<~ Solution B: Buffer and Solution A (50:50)
Ultraviolet-Visible Spect"oscoPY:J~?Q.t(CN14v1ay".z020) Mobile phase: See Table 1.
Diluent: Use Mobile phase in the Assay.
Sample stock solution: Use Sample stock solution in the Table 1
Assay. Time Solution A Solution B
Sample solution: Nominally equivalent to 004 ~g/mL of (min) (%) (%)
adapalene, prepared as follows. Dilute 2.0 mL of SC!mple
stocksolution with Diluent to 100.0 mL. Pass a portion 0 0 100
through a Teflon filter of 0045-~m pore size and use the 4 0 100
filtrate.
Acceptance criteria: Meets the requirements 30 55 45
65 55 45

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USP 43 OfficialMonographs / Adenine 97

Table 1 (continued) Table 2 (continued)

Time Solution A Solution B Relative Acceptance
(min) (%) (%) Retention Criteria,
Name Time NMT (%)
68 0 100
Total impurities - 1.0
80 0 100
a Methyl 6-bromo-2-naphthoate.
Diluent: Acetonitrile and tetrahydrofuran (3:2) b This process impurity is controlled in the drug substance monograph. It is
included in the table for identification only and it is not to be reported in the
System suitability stock solution: 0.5 mg/mL of USP total impurities.
Adapalene RS, prepared as follows. Transfer USP c Methyl 6-[3-(adamant-1-yl)-4-methoxyphenyl]-Z.naphthoate.
Adapalene RS to a suitable volumetric flask, add
tetrahydrofuran equivalent to 40% of the final volume, and SPECIFIC TESTS
sonicate to dissolve. Dilute with acetonitrile to volume. • pH (791): 4.0-6.0
System suitability solution: 0.2 mg/mL of USP Adapalene • MINIMUM FILL (755): Meets the requirements
RS in Diluent, from System suitability stocksolution • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Standard solution: 1.0 IJg/mL of USPAdapalene RS in SPECIFIED MICROORGANISMS (62): The total aerobic
Diluent, from System suitability solution microbial count is NMT 10 2 du/g. The total yeasts and
Sample solution: Nominally equivalent to 0.2 mg/mL of molds count is NMT 10' du/g. It meets the requirements
adapalene, prepared as follows. Transfer 5.0 9 of Gel to a of the tests for the absence of Escherichia coli, Salmonella
25-mL volumetric flask. Add 10 mL of tetrahydrofuran and species, Staphylococcus aureus, and Pseudomonas
sonicate to disperse for 10 min. Add 10 mL of acetonitrile aeruginosa species.
and sonicate for 10 min. Cool to room temperature and
dilute with acetonitrile to volume. Pass a portion through a ADDITIONAL REQUIREMENTS
Teflon filter of 0.45-lJm pore size and use the filtrate. • PACKAGING AND STORAGE: Preserve in tight containers.
Chromatographic system Store at controlled room temperature and protect from
(See Chromatography (621), System Suitability.) freezing.
Mode: LC • ~SP REFERENCE STANDARDS (11)
Detector: UV 235 nm USPAdapalene RS
Column: 4.6-mm x 25-cm; 5-lJm packing L1
Column temperature: 40°
Flow rate: 1 mL/min
Injection volume: 20 IJL
System suitability Adenine
Samples: System suitability solution and Standard solution NH,

Cr~
Suitability requirements
Tailing factor: NMT 2.0, System suitability solution
Relative standard deviation: NMT 5.0%, Standard N ~
solution
Analysis CsHsNs 135.13
Samples: Standardsolution and Sample solution 9 H-Purin-6-amine;
Calculate the percentage of each individual impurity in the 1,6-Dihydro-6-iminopurine [73-24-5].
portion of Gel taken:
DEFINITION
Result = (rufr s) x (Cs/Cu) x 100 Adenine contains NLT98.0% and NMT 102.0% of adenine
(CsHsNs), calculated on the dried basis.
tu = peak area of each impurity from the Sample IDENTIFICATION
solution
ts =peak area of adapalene from the Standard solution
Cs = concentration of USPAdapalene RS in the
Standardsolution (mg/mL) • A. A SPECTROSCOPIC IDENTIFICATION TESTS (191) ~ Infrcired
Cu = nominal concentration of adapalene in the Spectroscopy: 197K A (CN1.May-2020) .
Sample solution (mg/mL) • B. The retention time of the major peak of the Sample
solution corresponds to that of the Standardsolution, as
Acceptance criteria: See Table 2. Disregard any peak less obtained in the Assay.
than 0.1%.
ASSAY
Table 2 • PROCEDURE
Relative Acceptance Buffer solution: Dissolve 6.90 9 of monobasic ammonium
Retention Criteria, phosphate in about 800 mL of water. Adjust with
Name Time NMT (%) ammonium hydroxide to a pH of 6.2, and dilute with water
Adapalene related com-
to 1 L.
pound N·b 0.5 - Mobile phase: See Table 7.
Adapalene 1.0 - Table 1
Adapalene related com- Buffer
pound Bb.c 1.3
- Time Solution Acetonitrile Water
(min) (%) (%) (%)
Any unspecified
impurity - 0.2 0 5 5 90

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98 Adenine / OfficialMonographs USP 43

Table 1 (continued) Analysis

Buffer Sample: Sample solution
Time Solution Acetonitrile Water Calculate the percentage of each impurity in the portion of
(min) (%) (%) (%) Adenine taken:
20 5 5 90
Result = (r vir T) x 100
20.1 10 10 80
30 10 10 80 rv = peak response of each impurity from the Sample
solution
30.1 5 5 90 r T = sum of all the peak responses from the Sample
40 5 5 90 solution

Acceptance criteria
System suitability solution: 50 ~g/mL each of USPAdenine Individual impurity: NMT 0.1 %
RS and 7-methyladenine in water Total impurities: NMT 2.0%
Standard solution: 0.1 mg/mL of USP Adenine RS in water.
If necessary, sonicate the solution at 30° until the substance SPECIFIC TESTS
is completely dissolved. • Loss ON DRYING (731): Dry a sample at 110° for 4 h: it loses
Sample solution: 0.1 mg/mL of Adenine in water. If NMT 1.0% of its weight.
necessary, sonicate the solution at 30° until the substance ADDITIONAL REQUIREMENTS
is completely dissolved. • PACKAGING AND STORAGE: Preserve in well-closed
Chromatographic system containers.
(See Chromatography (621), System Suitability.) • USP REFERENCE STANDARDS (11)
Mode: LC USPAdenine RS
Detector: UV 260 nm
Column: 4.6-mm x 25-cm; 5-~m packing L85
Flow rate: 1.0 mL/min
Injection volume: 10 ~L
System suitability Adenosine
Sample: System sUitability solution
[NoTE-The relative retention times for
7-methyladenine and adenine are 0.88 and 1.0,
respectively.]
SUitability requirements
Resolution: NLT2.0 between the 7-methyladenine and CloH13Ns04 267.24
adenine peaks e-Arnlno-s-f-o-rlbofuranosyt-s H-purine;
Analysis 9-~-D-Ribofuranosyladenine [58-61-7].
Samples: Standardsolution and Sample solution
Calculate the percentage of adenine (CsHsN s) in the DEFINITION
portion of Adenine taken: Adenosine contains NLT 98.0% and NMT 102.0% of
adenosine (CloH13Ns04)' calculated on the dried basis.
Result = (r vir s) x (C siC u) x .100
IDENTIFICATION
= peak response from the Sample solution
= peak response from the Standard solution
= concentration of USP Adenine RS in the Standard
solution (mg/mL)
= concentration of Adenine in the Sample solution • B. The retention times of the major peaks of the Sample
(mg/mL)
solution correspond to those of the Standard solution, as
Acceptance criteria: 98.00/0-102.0% on the dried basis obtained in the Assay.

IMPURITIES ASSAY
• RESIDUE ON IGNITION (281): NMT 0.1% • PROCEDURE
• RELATED COMPOUNDS Buffer: 6.8 giL of potassium hydrogen sulfate and 3.4 giL
Buffer solution, Mobile phase, System suitability solution, of tetrabutylammonium hydrogen sulfate in a solution
Standard solution, and System suitability: Proceed as prepared as follows. Transfer suitable quantities of
directed in the Assay. potassium hydrogen sulfate and tetrabutyl ammonium
Sample solution: Dissolve 25 mg of Adenine in hydrogen sulfate to an appropriate volumetric flask, and
approximately 15 mL of boiling water. Cool, quantitatively dissolve in 90% of the flask volume of water. Adjust with 2
transfer to a 25-mL volumetric flask, and dilute with water N potassium hydroxide to a pH of 6.5, and dilute with water
to volume. to volume.
Chromatographic system Mobile phase: Buffer and water (60:40)
(See Chromatography (621), System Suitability.) System suitability solution: 4 ~g/mL each of USP Adenine
Mode: LC RS and inosine in Mobilephase
Detector: UV 240 nm Standard solution: 0.2 mg/mL of USP Adenosine RS in
Column: 4.6-mm x 25-cm; 5-~m packing L85 Mobilephase
Flow rate: 1.0 mL/min Sample solution: 0.2 mg/mL of Adenosine in Mobilephase
Injection volume: 20 ~L Chromatographic system
(See Chromatography (621), System Suitability.)

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USP 43 Official Monographs / Adenosine 99

Mode: LC Table 1
Detector: UV 254 nm Relative Relative Acceptance
Column: 4.6-mm x 25-cm; 5-!Jm packing L1 Retention Response Criteria,
Flow rate: 1.5 mL/min Name Time Factor NMT(%)
Injection volume: 20!JL Urldlne" 0.29 0.73 0.10
Run time: NLT 1.5 times the retention time of the
adenosine peak Adenine 0.34 1.6 0.2
System suitability lnoslne" 0.42 0.73 0.1
Samples: System suitability solution and Standardsolution
[NoTE-See Table 7 for the relative retention times.] Guanosine' 0.51 0.86 0.10
Suitability requirements Adenosine 1.0 - -
Resolution: NLT 1.5 between adenine and inosine,
Any individual unspe-
System suitability solution cified impurity - 1.0 0.10
Tailing factor: NMT 2.0, Standardsolution
Relative standard deviation: NMT 0.7%, Standard Total impurities - - 0.5
solution
Analysis a 1-~-D-Ribofuranosylpyrimidine-2,4(1 H,3H)-dione.
b 9-~-D-Ribofuranosylpurine-6(1 H)-one.
Samples: Standard solution and Sample solution
c 2-Amino-9.~-D-ribofuranosylpurine-6(1 H)-one.
Calculate the percentage of adenosine (CloH13Ns04) in the
portion of Adenosine taken:
SPECIFICTESTS
Result = (r vir s) x (C siC v) x 100 • OPTICAL ROTATION, Specific Rotation (781 S): -68° to -72°
Test solution: 20 mg/mL in sodium hydroxide solution (1
ru = peak response from the Sample solution in 20), determined on a sample previously dried at 105° for
rs = peak response from the Standardsolution 2h
Cs =concentration of USPAdenosine RS in the • Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Standardsolution (mg/mL)
Cu = concentration of Adenosine in the Sample
Acceptance criteria: NMT 0.5%
solution (mg/mL) ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
Acceptance criteria: 98.0%-102.0% on the dried basis containers, and store at controlled room temperature.
IMPURITIES • USP REFERENCE STANDARDS (11)
• RESIDUE ON IGNITION (281): NMT 0.1 % USP Adenine RS
USP Adenosine RS
• ORGANIC IMPURITIES
Buffer, Mobile phase, System suitability solution, and
Chromatographic system: Proceed as directed in the
Assay.
Standard solution: 0.001 mg/mL of USP Adenosine RS in Adenosine Injection
Mobile phase
Sample solution: 1 mg/ml of Adenosine in Mobilephase DEFINITION
System suitability . Adenosine Injection is a sterile solution of Adenosine in Water
Samples: System suitability solution and Standardsolution for Injection. It may contain Sodium Chloride. It contains NLT
[NoTE-See Table 7 for the relative retention times.]. 90.0% and NMT 110.0% of the labeled amount of adenosine
Suitability requirements (C lOH 13 Ns 0 4 ) ·
Resolution: NLT 1.5 between adenine and inosine,
System suitability solution IDENTIFICATION
Relative standard deviation: NMT 5%, Standard • The retention time of the adenosine peak of the Sample
solution solution corresponds to that of the Standardsolution, as
Analysis obtained in the Assay.
Samples: Standard solution and Sample solution
ASSAY
Calculate the percentage of each impurity in the portion of
Adenosine taken: • PROCEDURE
Mobile phase: Dissolve 2.0 9 of monobasic potassium
Result = (r vir s) x (C siC v) x (l/A x 100 phosphate in 800 mL of water. Add 5 mL of 1.0 M
tetrabutylammonium dihydrogen phosphate, dilute with
ru = peak response from the Sample solution water to 980 mL, and mix. Add 20 mL of acetonitrile.
rs = peak response from the Standardsolution System suitability solution: 0.03 mg/mL each of USP
Adenosine RS and inosine dissolved in warm water (50° to
Cs = concentration of USP Adenosine RS in the
55°), and diluted with water
Standardsolution (mg/mL)
Standard solution: 0.03 mg/mL of USP Adenosine RS
Cu = concentration of Adenosine in the Sample
dissolved in warm water (50° to 55°), and diluted with
solution (mg/mL) water to volume. Before addition of the warm water, if
F = relative response factor (see Table 7)
sodium chloride is present in the Injection, add 0.01 mlof
Acceptance criteria: See Table 7. Disregard peaks that are a solution of sodium chloride (0.9 in 100) per ml of the
less than 0.05% of the adenosine peak. anticipated final volume of the Standard solution.
Sample solution: Nominally 0.03 mg/ml of adenosine,
from a suitable volume of Injection in water
Chromatographic system
(See Chromatography (621), System SUitability.)

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 100 Adenosine / OfficialMonographs USP 43

Mode: LC • liSP REFERENCE STANDARDS (11)

Detector: UV 254 nm USP Adenosine RS
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 2.5 mL/min
Injection volume: 10 IJL
Run time: 2.5 times the retention time of adenosine
System suitability Medical Air .
Samples: System suitability solution and Standard solution
[NOTE-The relative retention times of inosine and DEFINITION
adenosine are 0.43 and 1.0, respectively.] Medical Air isa natural or syntheticmixtureof gases consisting
Suitability requirements largelyof nitrogen and oxygen. It contains NLT 19.5% and
Resolution: NLT 6.0 between adenosine and inosine, NMT 23.5%, by volume, of oxygen (0 2) ,
System suitability solution IDENTIFICATION
Tailing factor: NMT 2.0 for the adenosine peak, System • A. The paramagnetic signal exhibited by the Sample gas in
suitability solution the Assay confirms the presence of oxygen.
Relative standard deviation: NMT 1.5%, Standard • B. The Sample gas in the Assay meets the assay Acceptance
solution criteria.
Analysis
Samples: Standardsolution and Sample solution ASSAY
Calculatethe percentage of the labeled amount of • PROCEDURE
adenosine (ClO H13Ns0 4) in the portion of Injection taken: The certified standards called for in the followlnq test are
listed in Reagents, Indicators, and Solutions.
Result =(r vir s) x (C siC v) x 100 Zero gas: Nitrogen certified standard
Span gas: 21% Oxygen certified standard. [NOTE-See
ru =peak responsefrom the Sample solution Reagents, Indicators, and Solutions.]
rs = peak response from the Standard solution Sample gas: Medical Air
Cs = concentration of USP Adenosine RS in the Mode: Paramagnetic oxygen measurement (see Medical
Standardsolution (mg/mL) Gases Assay (41 5»
Cu = nominal concentration of adenosine in the Analysis: Determine the concentration of oxygen in
Sample solution (mg/mL) percentage by volume of Medical Air using a suitable
paramagnetic analyzer.
Acceptance criteria: 90.00/0-110.0% Acceptance criteria: 19.50/0-23.5% of oxygen by volume
IMPURITIES IMPURITIES
• ORGANIC IMPURITIES See Impurities Testing in Medical Gases Assay (413). The
Mobile phase, System suitability solution, Standard detector tubes called for in the following tests are listed in
solution, Chromatographic system, and System Reagents, Indicators, and Solutions.
suitability: Proceed as directed in the Assay. If the label indicates that Medical Air is a synthetic mixture of
Sample solution: Nominally 0.3 mg/mL of adenosine from oxygen and nitrogen, and where oxygen complies to
a volume of Injection, in water Oxygen USP and Nitrogen complies to Nitrogen NF, then
Analysis the Impurities tests are not required.
Sample: Sample solution . • LIMIT OF CARBON DIOXIDE
Calculate the percentage of each impurity in the volumeof Sample: Detector tube manufacturer's recommended
Injection taken: volume ±5910 of Medical Air
Analysis: Pass the Sample through a carbon dioxidedetector
Result = (r vir r) x 100 tube at the rate specified for the tube by the detector tube
manufacturer.
ru = peak responsefor each impurity Acceptance criteria: NMT 500 ppm
rt =sum of the responsesof all of the peaks • LIMIT OF CARBON MONOXIDE
Sample: Detector tube manufacturer's recommended
Acceptance criteria volume ±5% of Medical Air
Any individual impurity: NMT 1.0% Analysis: Pass the Sample through a carbon monoxide
Total impurities: NMT 1.5% detector tube at the rate specified for the tube by the
detector tube manufacturer.
SPECIFIC TESTS
Acceptance criteria: NMT 10 ppm
• pH (791): 4.5-7.5 • LIMIT OF SULFUR DIOXIDE
• PARTICULATE MATTER IN INJECTIONS (788): It meets the Sample: Detector tube manufacturer's recommended
requirements for small-volume injections. volume ±5% of Medical Air
• BACTERIAL ENDOTOXINS TEST (85): When the product is Analysis: Pass the Sample through a sulfurdioxide detector
used for rapid intravenousinjection, it contains NMT 11.62 tube at the rate specified for the tube by the detector tube
USP Endotoxin Units/mg of adenosine. When the product manufacturer.
is used for continuous peripheral intravenous infusion, it Acceptance criteria: NMT 5 ppm
contains NMT 5.95 USP Endotoxin Units/mg of adenosine. • LIMIT OF NITRIC OXIDE AND NITROGEN DIOXIDE
• OTHER REQUIREMENTS: It meets the requirements under Sample: Detector tube manufacturer's recommended
Injections and ImplantedDrug Products (1). volume ±5910 of Medical Air
ADDITIONAL REQUIREMENTS Analysis: Pass the Sample through a nitric oxide-nitrogen
• PACKAGING AND STORAGE: Preserve in tight, single-dose dioxide detector tube at the rate specified for the tube by
containers, preferably of Type I glass, and store at the detector tube manufacturer.
controlled room temperature. Acceptance criteria: NMT 2.5 ppm

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USP43 Official Monographs / Alanine 101

CD LIMIT OF WATER AND OIL CDClHlORIDIE AND SULFATE, Sulfate (221)

Analysis: Support one container in an inverted position Standard solution: 0.30 mL of 0.020 N sulfuric acid
(with the valve at the bottom) for 5 min. Cautiously open Sample: 1.0 g of Alanine
the valve slightly, maintaining the container in an inverted Acceptance criteria: NMT 0.03%
position. Vent the gas with a barely audible flow against a • IRON (241): NMT 30 ppm
stainless steel mirror for a few seconds. • RELATED COMPOUNDS
Acceptance criteria: No liquid is discernible on the mirror. Mobile phase: 0.008 N sulfuric acid solution
System suitability solution: A mixture of 0.05 mg/mL of
ADDITIONAL REQUIREMENTS USP Fumaric Acid RS, 0.05 rnq/m], of USP Maleic Acid RS,
CD PACKAGING AND STORAGE: Preserve in pressurized
and 3 mg/mL of USP Malic Acid RS in water
containers. Container connections shall be appropriate for Maleic acid standard solution: 0.05 mg/mL of USP Maleic
air. Adaptors shall not be used to connect containers to Acid RS in water
patient use supply system piping or equipment. Malic acid standard solution: 3 mg/mL of USP Malic Acid
CD LABELING: Label states if Medical Air is a synthetic mixture
RS in water
of Oxygen USP and Nitrogen NF. Where it is piped directly Fumaric acid standard solution: 0.05 mg/mL of USP
from the collecting tank to the patient point of use, label Fumaric Acid RS in water
each outlet "Medical Air". Sample solution: 100 mg/mL of Alanine in water
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Alanine Detector: UV 214 nm
Column: 7.8-mm x 30-cm; 9-J.Im packing L17
Column temperature: 30°
Flow rate: 0.6 mL/min
Injection volume: 10 J.IL
System suitability
C3H 7N02 89.09 Sample: System SUitability solution
L-Alanine [56-41-7]. [NoTE-See Table 1 for the relative retention times.]
Suitability requirements
DEFINITION Resolution: NLT 1.5 between maleic acid and malic acid
Alanine contains NLT 98.5% and NMT 101.5% of L-alanine Relative standard deviation: NMT 5.0% for each of
(C3H 7N02) , calculated on the dried basis. fumaric acid, maleic acid, and malic acid
Analysis
IDENTIFICAnON
Samples: Standard solutions and Sample solution
Calculate the percentage of each specified acid in the
portion of Alanine taken:

Result = (r vir s) x (C siC v) x 100

ASSAY =peak response of maleic acid, malic acid, or

CD PROCEDURE fumaric acid from the Sample solution
Sample: 80 mg of Alanine = peak response of maleic acid, malic acid, or
Titrimetric system fumaric acid from the corresponding Standard
(See Titrimetry (541).) solution
Mode: Direct titration =concentration of USP Maleic Acid RS, USP Malic
Titrant: 0.1 N perchloric acid VS Acid RS, or USP Fumaric Acid RS in the
Endpoint detection: Potentiometric corresponding Standard solution (mg/mL)
Blank: 3 mL of formic acid in 50 mL of glacial acetic acid =concentration of Alanine in the Sample solution
Analysis: Dissolve the Sample in a mixture of 3 mL of formic (mg/mL)
acid and 50 mL of glacial acetic acid, and titrate with
Titrant. Calculate the percentage of any unspecified impurity in the
Calculate the percentage of L-alanine (C3H7N02) in the portion of Alanine taken:
portion of Alanine taken:
Result = (r vir s) x (C siC v) x 100
Result = [(V s" V B) x N x Fx 100]/W
ru = peak response of any unspecified impurity from
VS = Titrant volume consumed by the Sample (mL) the Sample solution
V8 = Titrant volume consumed by the Blank (mL)
rs = peak response of fumaric acid from the Fumaric
acid standardsolution
N = actual normality of the Titrant (mEq/mL) Cs = concentration of USP Fumaric Acid RS in the
F = equivalency factor, 89.09 mg/mEq Fumaric acid standardsolution(mg/mL)
W = Sample weight (mg) Cu = concentration of Alanine in the Sample solution
(mg/mL)
Acceptance criteria: 98.5%-101.5% on the dried basis
IMPURITIES Acceptance criteria: See Table 1.
CD RESIDUE ON IGNITION (281): NMT 0.15%

CD CHLORIDE AND SULFATE, Chloride (221)

Standard solution: 0.70 mL of 0.020 N hydrochloric acid

Sample: 1.0 g of Alanine
Acceptance criteria: NMT 0.05%

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102 Alanine / Official Monographs USP 43

Table 1 a blank determination. Each ml of 0.1 N perchloric acid is

Relative Acceptance equivalent to 26.53 mg of C12H,sN 3025.
Retention Criteria, Acceptance criteria: 98.0%-102.0% on the dried basis
Name Time NMT (%)
IMPURITIES
Maleicacid 0.5 0.05
• RESIDUE ON IGNITION (281): NMT 0.2%
Malicacid 0.6 0.05 • ORGANIC IMPURITIES
Standard stock solution: 5 mg/ml of USP Albendazole RS
Fumaricacid 1.0 0.05
.in glacial acetic acid
Alanine Not observed - Standard solution: 0.05 mg/ml of USP Albendazole RS in
Anyunspecified
glacial acetic acid from Standardstock solution
impurity - 0.05 Sample solution: 10 mg/ml in glacial acetic acid
Chromatographic system
Total unspecified im- (See Chromatography (621), Thin-Layer Chromatography.)
purities - 0.20
Mode: TlC
Adsorbant: 0.25-mm layer of silica gel mixture
SPECIFIC TESTS Application volume: 10 IJl
• OPTICAL ROTATION, Specific Rotation (7815) Developing solvent system: Chloroform, ether, and
Sample solution: 100 mg/ml in 6 N hydrochloric acid glacial acetic acid (60:10:10)
Acceptance criteria: +13.7 to +15.1 0
0
Analysis: Proceed as directed for Chromatography (621),
• pH (791): 5.5-7.0, in a solution (1 in 20) Thin-Layer Chromatography.
• Loss ON DRYING (731) Samples: Standardstock solution, Standardsolution, and
Analysis: Dry at 105 0 for 3 h. Sample solution
Acceptance criteria: NMT 0.2% Develop the chromatogram in the Developing solvent
ADDITIONAL REQUIREMENTS system until the solvent front has moved about
three-fourths of the length of the plate. Remove the plate
• PACKAGING AND STORAGE: Preserve in tight containers, and
from the developing chamber, mark the solvent front,
store at controlled room temperature.
allow the solvent to evaporate from the plate, and
• USP REFERENCE STANDARDS (11)
USP L-Alanine RS examine the plate under short-wavelength UV light.
U5P Fumaric Acid RS Acceptance criteria: 0.5%; no spot, other than the principal
USP Maleic Acid RS spot of the Sample solution, is larger or more intense than
USP Malic Acid RS the principal spot of the Standard solution.
SPECIFIC TESTS
• Loss ON DRVING (731)
Analysis: Dry at 105 0 for 4 h.
Acceptance criteria: NMT 0.5%
Albendazole
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers, and
store at controlled room temperature.
• USP REFERENCE STANDARDS (11)
USPAlbendazole RS
C12H,sN 302S 265.33
Carbamic acid, [5-(propylthio)-1 H-benzimidazol-2:,yl]-,
methyl ester;
Methyl 5-(propylthio)-2-benzimidazolecarbamate [54965-
21-8]. Albendazole Oral Suspension
DEFINITION DEFINITION
Albendazole contains NlT 98.0% and NMT 102.0% of Albendazole Oral Suspension is Albendazole in an aqueous
albendazole (C'2H,sN302S), calculated on the dried basis. vehicle. It contains one or more preservatives and dispersing
or suspending agents. It contains NlT 90.0% and NMT
IDENTIFICATION 110.0% of the labeled amount of albendazole
(C'2H,sN30 2S).
IDENTIFICATION
• A•. ~SfE~TR()SSOPI(:I~E~."'lfIS~II~~'t~$r~!<j~t)ilnffi:lr;~a
SpgcJrqs~qpy:.]l ~i;M.{C:N.l"Ma),-2Q20)
• B. The R F value of the principal spot of the Sample solution
corresponds to that of the principal spot of the Standard • A... SPECTROSCOPIC IDENTIFICATION
solution, as obtained in the test for Organic Impurities. Ultraviolet-Visible Spectroscopy:. 197U" 020)
Sample stock solution: 1 mg/ml of albendazole from a
ASSAY quantity of Suspension, in a mixture of methanol and
• PROCEDURE hydrochloric acid (99:1). Filter the mixture, if necessary, to
Sample: 250 mg of Albendazole obtain a clear solution.
Analysis: Transfer the Sample to a suitable flask, and dissolve Sample solution: 0.01 mg/ml of albendazole in 0.1 N
in 100 ml of glacial acetic acid, warming gently if sodium hydroxide from Sample stock solution
necessary. Cool, and titrate with 0.1 N perchloric acid VS Acceptance criteria: Meets the requirements
to a potentiometric endpoint (see Titrimetry (541». Perform

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 USP 43 Official Monographs / Albendazole 103

ASSAY USP Reference standards (11)-

• PROCEDURE USP Albendazole RS
Solution A: Methanol and hydrochloric acid (99:1) USP Parbendazole RS
Solution B: 13.75 gIL of monobasic sodium phosphate
Mobile phase: Methanol and Solution B (60:40) Identification-
Standard stock solution: 1 mg/mL of USP Albendazole RS
in Solution A
Standard solution: 100 ~g/mL of USP Albendazole RS from
Standard stocksolution in Mobilephase .~.~~"~B~fl.~~~.€9P{fi~~~~~{t{~g~{g~in~~f~(1QZ);!Qlt(qYi91~t2Yisit>1~
Sample stock solution: Equivalent to 1 mg/mL of SP~ctt;Q~qoPY:lQZl.)Jr.(CN.j~MaY;2o"20)-
albendazole from a volume of Oral Suspension in Solution
Solution: Dilute a portion of the clear filtrate used to prepare
the Assay preparation and a portion of the stock solution used
A
to prepare the Standard preparationprepared in the Assaywith
Sample solution: Nominally 100 ~g/mL of albendazole
from Sample stock solution in Mobilephase. [NOTE-Filter, if
Acidified methanol, prepared as directed for Dissolution, to
obtain solutions containing about 10 ~g of albendazole per
necessary, to obtain a clear solution.]
mL.
Chromatographic system
B: The retention time of the major peak for albendazolein
(See Chromatography (621), System SUitability.)
the chromatogram of the Assay preparation corresponds to
Mode: LC
that in the chromatogram of the Standard preparation, as
Detector: UV 308 nm
obtained in the Assay.
Column: 4-mm x 25-cm; packing L1
Flow rate: 2 mL/min Dissolution (711)-
Injection volume: 20 ~L Medium: 0.1 N hydrochloric acid; 900 mL.
System sultablllty Apparatus 2: 50 rpm.
Sample: Standardsolution Time: 30 minutes.
Suitability requirements Determine the amount of C12H1SN302S dissolved using the
Column efficiency: NLT 2000 theoretical plates following procedure.
Tailing factor: NMT 2.0 Acidified methanol-To about 50 mL of methanol in a
Relative standard deviation: NMT 2.0% 1OO-mLvolumetric flask add 2 mL of hydrochloric acid, dilute
Analysis with methanol to volume, and mix.
Samples: Standard solution and Sample solution Standardsolution-Transfer about 90 mg of USP
Calculate the percentage of albendazole (C12H1SN302S) in Albendazole RS, accurately weighed, to a 250-mL volumetric
the portion of Oral Suspension taken: flask, add 10 mL of Acidified methanol, and shake to dissolve.
Dilute with 0.1 N hydrochloric acid to volume, and mix.
Result = (rulrs) x (CsICu) x 100 Transfer 5.0 mL of this solution to a 200-mL volumetric flask,
dilute with 0.1 N sodium hydroxide to volume, and mix.
ru = peak response from the Sample solution Procedure-Transfer 10.0 mL of a filtered portion of the
ts = peak response from the Standard solution solution under test to a 250-mL volumetric flask, dilute with
Cs = concentration of USP Albendazole RS in the 0.1 N sodium hydroxide to volume, and mix. Concomitantly
Standard solution (uq/rnt) determine the absorbances of this solution and the Standard
Cu = nominal concentration of albendazole in the solution at the wavelengths of maximum and minimum
Sample solution (~g/mL) absorbance at about 308 nm and 350 nm, using 0.1 N sodium
hydroxide as the blank. Calculate the quantity, in mg, of
Acceptance criteria: 90.0%-110.0% C12H1SN302S dissolved by the formula:
SPECIFIC TESTS
22.5C(A ulA s)
• pH (791): 4.5-5.5
ADDITIONAL REQUIREMENTS in which C is the concentration, in ~g per mL, of USP
• PACKAGING AND STORAGE: Preservein tight containers, and Albendazole RS in the Standard solution; and A u and A s are
store at controlled room temperature. the differences in absorbance between 308 nm and 350 nm
• LABELING: Label it to indicate that it is for veterinary use obtained from the solution under test and the Standard
only. solution, respectively.
• USP REFERENCE STANDARDS (11) Tolerances-Not less than 80% (Q) of the labeled amount
USPAlbendazole RS of C12H1SN302S is dissolved in 30 minutes.
Uniformity of dosage units (905): meet the requirements.
Procedure for content uniformity-
Acidified methanol and Standard solution-Prepare as
directed under Dissolution.
Albendazole Tablets Test solution-Place 1 Tablet in a 500-mL volumetric flask,
add about 300 mL of Acidified methanol, and shake by
mechanical means for about 30 minutes. Dilute with Acidified
» Albendazole Tablets contain not less than 90.0 methanol to volume, and mix. Filter a portion of this solution,
percent and not more than 110.0 percent of the discarding the first 20 mL of the filtrate. Transfer 4.0 mL of the
labeled amount of albendazole (C12H1SN30ZS). clear filtrate to a 200-mL volumetric flask, dilute with 0.1 N
sodium hydroxide to volume, and mix.
Packaging and storage-Preserve in tight containers, and Procedure-Concomitantly determine the absorbances of
store at controlled room temperature. the Standardsolution and the Test solution at the wavelengths
Labeling-Tablets intended for veterinary use only are so of maximum and minimum absorbance at about 308 nm and
labeled. 350 nm, using 0.1 N sodium hydroxide astheblank. Calculate

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 104 Albendazole / OfficialMonographs USP 43

the quantity, in mg, of C12H15N 30 2S in the Tablet taken by the

formula: Albumin Human
DEFINITION
25qA ulA s) Albumin Human conforms to the regulations of the federal.
Food and Drug Administration con~erning ~iologics (640.8.0
in which C is the concentration, in ~g per mL, of USP
Albendazole RS in the Standard preparation; and A u and A s to 640.86) (see Biologics (1011». It !s a sterile, n<?npy~ogenlc
preparation of serum albumin obtained by fractionating
are the differences in absorbance between 308 nm and 350 material (source blood, plasma, serum, or placentas) from
nm obtained from the Test solution and the Standard solution, healthy human donors, the source material being tested for
respectively.
the absence of hepatitis B surface antigen. It is made by a
Assay- process that yields a product that is safe for intravenous use.
Mobilephase-Dissolve 0.50 g of monobasic ammonium NLT 96% of its total protein is albumin. It is a solution .
phosphate in 400 mL of water. Add 600 mL of methanol, mix, containing, in each 100 mL, either 25 g of serum albumin
and filter, discarding the first 15 mL of the filtrate. Degas the osmotically equivalent to 500 mL of normal human plasma,
clear filtrate before use. Make adjustments if necessary (see or 20 g equivalent to 400 mL, or 5 g equivalent to 100 mL,
System Suitability under Chromatography (621». or 4 g equivalent to 80 mL, and contains NLT 93.75% and
Sulfuric acid in methanol-Prepare a mixture of 1 mL of NMT 106.25% of the labeled amount in the case of the
sulfuric acid and 99 mL of methanol. solution containing 4 g in each 100 mL, and NLT 94.0% and
Internalstandardsolution-Transfer about 150 mg of USP NMT 106.0% of the labeled amount in the other cases..It
Parbendazole RS to a 50-mL volumetric flask. Add 5 mL of contains no added antimicrobial agent, but may contain
Sulfuric acid in methanol, 25 mL of methanol, and shake to sodium acetyltryptophanate with or with0L!t sodium
dissolve. Dilute with methanol to volume, and mix. caprylate as a stabilizing agent. It has a sodium content of
Standard preparation-Transfer about 100 mg of USP NLT 130 mEq/L and NMT 160 mEq/L. It has a heme content
Albendazole RS, accurately weighed, to a 50-mL volumetric such that the absorbance of a solution, diluted to contain 1%
flask. Add 5 mL of Sulfuric acid in methanol and 25 mL of of protein in a 1-cm holding cell, measured at a wavelength
methanol, and shake to dissolve. Dilute with methanol to of 403 n~, is NMT 0.25. It meets the requirements of the
volume, and mix. Transfer 5.0 mL of this stock solution and test for heat stability and for pH.
5.0 mL of Internal standardsolution to a second 50-mL ADDITIONAL REQUIREMENTS
volumetric flask, dilute with methanol to volume, and mix. • PACKAGING AND STORAGE: Preserve in tight containers, and
Assay preparation-Weigh and finely powder not fewer store at the temperature recommended by the
than 20 Tablets. Transfer an accurately weighed portion of the manufacturer or indicated on the label.
powder, equivalent to about 100 mg of albendazole, to a • EXPIRATION DATE: The expiration date is not later than 5
50-mL volumetric flask. Add 5 mL of Sulfuric acid in methanol years after issuefrom manufacturer's cold storage (5°, 3
and 20 mL of methanol, and shake by mechanical means for years) if labeling recommends storage between 2° and 10°;
about 15 minutes. Dilute with methanol to volume, mix, and not later than 3 years after issue from manufacturer's cold
filter, discarding the first 15 mL of the filtrate. Transfer 5.0 mL storage (5°, 3 years) if labeling recommends storage at
of the clear filtrate and 5.0 mL of Internal standardsolution to temperatures not higher than 3r; and not later than 10
a second 50-mL volumetric flask,dilute with methanol to years after date of manufacture if in a hermetically sealed
volume, and mix. metal container and labeling recommends storage
Chromatographic system (see Chromatography (621»-The between 2° and 10°.
liquid chromatograph is equipped with a 254-nm detector • LABELING: Label it to state that it is not to be used if it is
and a 4.6-mm x 25-cm column that contains 5-~m packing turbid and that it is to be used within 4 h after the container
L1. The flow rate is about 2 mL per minute. Chromatograph is entered. Label it also to state the osmotic equivalent in
the Standard preparation, and record the peak responses as terms of plasma, the sodium content, and the type of
directed for Procedure: the tailing factor is not more than 2.0; source material (venous plasma, placental plasma, or both)
the column efficiency is not less than 1000 theoretical plates; from which it was prepared. Label it also to indicate that
the resolution between the albendazole peak and the additional fluids are needed when the 20-g/1 OO-mL or
'parbendazole peak is not less than 2.0; and the relative 25-g/1 OO-mL product is administered to a markedly
standard deviation for replicate injections is not more than dehydrated patient.
2.0%.
Procedure- [NoTE-Use peak heights where peak responses
are indicated.] Separately inject equal volumes (about 20 ~L)
of the Standard preparation and the Assay preparation into the
chromatograph, record the chromatograms, and measure the Albuterol
responses for the major peaks. Calculate the quantity, in mg,
of C12H1SN302S in the portion of Tablets taken by the formula:

~
OH ~~CH3
HO ~ 1\
500QR ulR s) I h H,C CH3
HO

in which C is the concentration, in mg per mL, of USP C13H 21N03 239.31

Albendazole RS in the Standard preparation;and Ru and Rs are 1,3-Benzenedimethanol, a1-[[(1, 1-dimethylethyl)amino]
the peak response ratios of the albendazole peak to the methyl]-4-hydroxy-;
parbendazole peak obtained from the Assay preparation and a1_[(tert-Butylamino)methyl]-4-hydroxy-m-xylene-a,a'-diol '
the Standard preparation, respectively. [18559-94-9].
DEFINITION
Albuterol contains NLT 98.5% and NMT 101.0% of albuterol
(C13H 21N03) , calculated on the anhydrous basis.

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USP 43 Official Monographs / A/buteral 105

IDENTIFICATION Albuterol Tablets

DEFINITION
Albuterol Tablets contain an amount of albuterol sulfate
[(C 13Hz1N03) z ' HzS04] equivalent to NLT 90.0% and NMT
110.0% of the labeled amount of albuterol (C 13Hz1 N03) .
IDENTIFICATION
• A. The RF value of the principal spot of the Sample solution
• B. corresponds to that of Standard solution A, obtained as
1.Ilt>~i~1 directed in the Procedure for Organic Impurities.
Sample so ution: 80 I-Ig ml in 0.1 N hydroc • B. IDENTIFICATION TESTS-GENERAL, Sulfate (191)
Acceptance criteria: Meets the requirements Sample solution: Shake a quantity of powdered Tablets
ASSAY equivalent to 4 mg of albuterol with 10 mL of water and
• PROCEDURE filter. Use the filtrate. '
Sample solution: 8 mg/ml of Albuterol in glacial acetic acid Acceptance criteria: Meet the requirements
Analysis: To 50 mL of the Sample solution add 2 drops of ASSAY
crystal violet TS, and titrate with 0.1 N perchloric acid VS. • PROCEDURE
Perform a blank determination, and make any necessary Solution A: 10 ml/l of glacial acetic acid in water
correction. Each ml of 0.1 N perchloric acid is equivalent Solution B: 1.13 g of sodium 1-hexanesulfonate in 1200 mL
to 23.93 mg of C nH z1N0 3 • of water. Add 12 mL of glacial acetic acid.
Acceptance criteria: 98.5%-101 .0% on the anhydrous Diluent: Methanol and water (40:60)
basis Mobile phase: Methanol and Solution B (40:60)
IMPURITIES Standard stock solution: 0.12 mg/mL of USPAlbuterol
• RESIDUE ON IGNITION (281): NMT 0.1 % Sulfate RS, prepared as follows. Transfer USPAlbuterol
• ORGANIC IMPURITIES .Sulfate RS to a suitable volumetric flask, and add a volume
Standard solution: 0.10 mg/ml of USP Albuterol RS in of Solution A corresponding to 60% of the flask volume.
methanol Sonicate for 5 min, and dilute with methanol to volume.
Sample solution: 20 mg/ml of Albuterol in methanol Standard solution: 0.03 mg/mL of USP Albuterol Sulfate RS
Chromatographic system in Diluent, from Standard stock solution
(See Chromatography (621), Thin-Layer Chromatography.) Sample solution: Nominally 0.025 mg/ml of albuterol,
Mode: TLC prepared as follows. Transfer a number of whole Tablets
Adsorbent: 0.25-mm layer of chromatographic silica gel equivalent to 50 mg of albuterol, to a suitable volumetri~
Application volume: 10 I-Il flask. Add 60% of the flask volume of Solution A, shake by
Developing solvent system: Methyl isobutyl ketone, mechanical means for 45 min, sonicate for 10 min, allow to
isopropyl alcohol, ethyl acetate, ammonium hydroxide, cool to room temperature, and dilute with methanol to
and water (50:45:35:3:18) volume. Pass through a suitable filter of 0.45-l-Im or finer
Visualization: Iodine vapor pore size.
Analysis Chromatographic system
Samples: Standardsolution and Sample solution (See Chromatography (621), System Suitability.)
Proceed as directed in the chapter, applying aliquots of Mode: LC
the Standardsolution and the Sample solution. Develop Detector: UV 276 nm
in the Developing solvent system until the solvent front Column: 4.6-mm x 15-cm; packing L1
has moved three-fourths the length of the plate. Remove Flow rate: 1.5 mL/min
the plate from the developing chamber, air-dry, and Injection volume: 25 I-Il
expose it to iodine vapor. System suitability
Acceptance criteria: Any spot, other than the principal Sample: Standard solution
spot, obtained from the Sample solutionis not greater in size Suitability requirements
and intensity than the spot produced by the Standard Column efficiency: NLT 800 theoretical plates
solution (0.5%), and the sum of the impurities is not greater Tailing factor: NMT 2.5
than 2.0%. Relative standard deviation: NMT 2.0%
Analysis
SPECIFIC TESTS I Samples: Standard solution and Sample solution
• WATER DETERMINATION, Method I (921): NMT 0.5% Calculate the percentage of the labeled amount of albuterol
(C 13Hz1N03) in the Tablets taken:
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed, Result =(rulrs) x (CsICu) x M x (MrtlMr2 ) x 100
light-resistant containers.
• USP REFERENCE STANDARDS (11) = peak response from the Sample solution
USPAlbuterol RS = peak response from the Standard solution
= concentration of USP Albuterol Sulfate RS in the
Standard solution (mg/mL)
= nominal concentration of albuterol in the Sample
solution (mg/mL)
M = number of moles of albuterol per mole of
albuterol sulfate, 2
= molecular weight of albuterol, 239.31
= molecular weight of albuterol sulfate, 576.70

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106 Albuteral / Official Monographs USP43

Acceptance criteria: 90.0%-110.0% Analysis

Samples: StandardsolutionA, StandardsolutionB, Standard
PERFOR.MANCE TESTS solution C, and Sample solution
• DISSOLUTION, Procedure for a Pooled Sample (711) Air-dry the plate. Develop the chromatograms until the
Medium: Water; 500 mL solventfront has moved about 17 cm. Spraythe plate
Apparatus 2: 50 rpm first with Spray reagentA, and then Spray reagent B, and
Time: 30 min finally again with Spray reagent A. Examine the plate and
Diluent, Mobile phase, and Standard stock solution: estimate the responses of any secondaryspots observed
Proceed as directed in the Assay. in the lane of the Sample solution by comparison with
Standard solution: 0.03 mg/mL of USP Albuterol Sulfate RS those of Standardsolutions A, B, and C.
in Diluent, from Standard stocksolution. If necessary, dilute Acceptance criteria
with Diluent to a concentration corresponding to the 1. 2.0%; no major secondaryspot is greater in size or
Sample solution.
Sample solution: Pass a portion of the solution under test intensity than the principal spot produced by
through a nylon filterof 0.45-lJm pore size. Standardsolution A.
Chromatographic system 2. 0.75%; no other secondaryspot is greater in size or
(See Chromatography (621), System Suitability.) intensity than the principal spot produced by
Mode:. LC Standardsolution B.
Detector: UV 276 nm 3. 0.25%; no more than two other secondaryspots are
Column: 4.6-mm x 15-cm; packing L1 equal in size or intensity to the principal spot
Flow rate: 1.5 mL/min produced by Standardsolution C.
Injection volume: 100 IJL 4. The sum of the intensities of all secondaryspots
System suitability obtained from the Sample solution corresponds to
Sample: Standardsolution NMT 3.5%.
Suitability requirements
Column efficiency: NLT 800 theoretical plates ADDITIONAL REQUIREMENTS
Tailing factor: NMT 2.5 • PACKAGING AND STORAGE: Preserve-in tight, light-resistant
Relative standard deviation: NMT 2.0% containers, and store at controlled room temperature.
Analysis • USP REFERENCE STANDARDS (11)
Samples: Standardsolution and Sample solution USP Albuterol Sulfate RS
Calculate the percentage ofthe labeledamount ofalbuterol
(C13H 21N03) dissolved by comparing the major peak
response from the Sample solution to that from the
Standardsolution. Albuterol Extended-Release Tablets
Acceptance criteria: NLT 80% (Q) of the labeledamount of
albuterol (C13H21N03) isdissolved. DEFINITION
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Albuterol Extended-Release Tablets contain albuterol sulfate
requirements . equivalentto NLT 90.0% and NMT 110.0% of the labeled
IMPURITIES amount of albuterol (C13H 21N03).
• ORGANIC IMPURITIES IDENTIFICATION
Standard solution A: 0.580 mg/mL of U~P Albuterol • A. The retention time of the major peak of the Sample
Sulfate RS in water, equivalentto 0.483 mg/mLofalbuterol solution corresponds to that of the Standardsolution, as
Standard solution B: 0.218 mg/mL of USP Albuterol obtained in the Assay.
Sulfate RS in water, equivalentto 0.183 mg/mL of albuterol
Standard solution C: 0.073 mg/mL of USP Albuterol
Sulfate RS in water, equivalentto 0.061 mg/mL ofalbuterol
Sample solution: Place a quantity of finely powdered
Tablets, equivalent to 48 mg of albuterol, into a suitable
container. Add 60 mL of diluted alcohol (1 in 2), and shake Standard solution: 80 IJg/mL of albuterolfrom USP
by mechanical means for 30 min. Filter the mixture, and Albuterol Sulfate RS in methanol
wash the filter with small portions of alcohol, combining Sample solution: 80 IJg/mL of albuterol in methanol,
this with the filtrate. Evaporate the filtrate to drynessunder prepared as follows. Transfer a suitable number of Tablets
reduced pressure below 40°. Dissolve the residueas to a volumetric flask, and dilute with methanol to volume;
completely as possible in 2 mL of water. Stir for 30 min, and centrifuge.
Chromatographic system Wavelength range: 210-350 nm
(See Chromatography (621), Thin-Layer Chromatography.) Cell path: 0.2 cm
Mode: TLC Acceptance criteria: The Sample solution exhibits maxima
Adsorbent: 0.25-mm layer of chromatographic silica gel and minima only at the same wavelengths as the Standard
Application volume: 10 IJL. Apply two successive 5-IJL solution.
aliquots, allowing the solvent to evaporate between
applications. ASSAY
Developing solvent system: Methyl isobutyl ketone, • PROCEDURE
isopropyl alcohol, ethyl acetate, ammonium hydroxide, Buffer: 0.65 giL of sodium l-octane sulfonate and 21.7 giL
and water (50:45:35:3:18) of ammonium acetate in water
Spray reagent A: 3-Methyl-2-benzothiazolinone Mobile phase: Glacial acetic acid, 2-propanol, methanol,
hydrazone hydrochloride TS and Buffer (4:3:1 :92)
Spray reagent B: Ammoniacal potassium ferricyanide TS Diluent: 10 mL/L of triethylamine in water
Standard stock soiution: 0.2 mg/mL of USP Albuterol
Sulfate RS in Diluent

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 USP 43 Official Monographs / Albuteroll 07

Standard solution: 0.02 mg/mL of USP Albuterol Sulfate RS Analysis

in Diluent, from the Standardstock solution. Transfer an Samples: Standard solution and Sample solution
aliquot of the Standardstocksolution to a suitable Calculate the concentration (C/) of albuterol (C13 Hz1N03) in
volumetric flask, and add 4% of the flask volume of the sample withdrawn from the vessel at each time point
methanol. Allow to cool to room temperature, and dilute (I):
with Diluent to volume.
Sample solution: Nominally 0;016 mg/mL of albuterol,' Result; = (rulrs) x Cs
prepared as follows. Transfer Tablets (NLT 10) to a suitable
volumetric flask, add 10% of the flask volume of methanol, ru =peak response from the Sample solution
and sonicate for 30 min with regular swirling. Add 60% of Is = peak response from the Standardsolution
the flask volume of Diluent, and sonicate for 30 min with Cs =concentration of albuterol in the Standard
swirling. Stir for 60 min, allow the solution to cool to room solution (mg/mL)
temperature, and dilute with Diluent to volume. Centrifuge
a portion of this solution at 2500 rpm for 15 min. Transfer Calculate the percentage of the labeled amount of albuterol
10 mL of the supernatant into a 50-mL volumetric flask, add (C 13Hz1N03) released at each time point (I):
1 mL of methanol, cool to room temperature, and dilute
with Diluent to volume. Pass the solution though a T-um Result, = C1 x V X (1I L) x 1 00
glass fiber or equivalent filter, and discard the first 3 mL of
the filtrate. Result, = {[C z x (V- Vs)] + [C, x Vsn x (l1L) x 100
Chromatographic system
(See Chromatography (621), System Suitability.) Result, ={[ C3 X (V - (2 x Vs))] + [( Cz + C,) x Vsn x (1I L) x
Mode: LC 100
Detector: UV 276 nm
Column: 4.6-mm x 15-cm; 5-l.lm packing L1 Result, = {[C4 x (V - (3 x Vs))] + [(C3 + Cz + C,) x Vsn x (1 I
Column temperature: 30° L) x 100
Flow rate: 1 mL/min
Injection volume: 40 ~L = concentration of albuterol in the portion of
System suitability sample withdrawn at time point i (mg/mL)
Sample: Standardsolution V =volume of the Medium (900 mL)
Suitability requirements L = label claim (mg/Tablet)
Column efficiency: NLT 2000 theoretical plates Vs =volume of the Sample solution withdrawn from
Tailing factor: NMT 2.0 the Medium (mL)
Relative standard deviation: NMT 2.0%
Analysis Tolerances: See Table 7.
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of albuterol Table 1
(C 13H z1 N0 3) in the portion of Tablets taken: Time point Time Amount released
(i) (h) (%)
Result =(rulrs) x (CsICu) x (M x M,,/M(2) x 100 1 1 25-45
= peak response from the Sample solution 2 2 45-65
= peak response from the Standardsolution 3 4 65-85
= concentration of USP Albuterol Sulfate RS in the
Standardsolution (mg/mL) . 4 9 NLT80
= nominal concentration of albuterol in the Sample
solution (mg/mL) The cumulative percentages of the labeled amount of
=moles of albuterol per mole of albuterol sulfate, 2 albuterol (C13Hz1 N03) , released at the times specified,
= molecular weight of albuterol, 239.31 conform to Acceptance Table 2 in Dissolution (711).
= molecular weight of albuterol sulfate, 576.70 • UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
Acceptance criteria: 90.0%-110.0%
IMPURITIES
PERFORMANCE TESTS • ORGANIC IMPURITIES, PROCEDURE 1
• DISSOLUTION (711) Buffer: 6.8 gIL of monobasic potassium phosphate in water.
Medium: 0.1 N hydrochloric acid; 900 mL Adjust with hydrochloric acid to a pH of 3.0.
Apparatus 2: 50 rpm, with helix sinker Mobile phase: Methanol and Buffer(5:95)
Time: 1, 2,4, and 9 h System suitability solution: 2 I.lg/mL each of USP Albuterol
Buffer, Mobile phase, Chromatographic system, and Sulfate RS and USP Albuterol Related Compound B RS in
System suitability: Proceed as directed in the Assay, Mobile phase
except to use an Injection volume of 50 1.lL. Standard solution: 2.4 I.lg/mL of USP Albuterol Sulfate RS,
Standard stock solution: 0.2 mg/mL of albuterol, using 0.80 I.lg/mL of USP Levalbuterol Related Compound C RS,
USPAlbuterol Sulfate RS, in Medium and 0.25 I.lg/mL of USP Levalbuterol Related Compound
Standard solution: Dilute the Standardstocksolution with D RS (equivalent to 0.20 I.lg/mL free base) in Mobilephase
Medium to obtain a final concentration of (L/1 000) mg/mL Sensitivity solution: 0.06 I.lg/mL of USP Albuterol Sulfate RS
of albuterol, where L is the label claim in mg/Tablet of in Mobile phase
albuterol. Sample solution: Transfer an amount of powder from NLT
Sample solution: Pass a portion of the solution under test 20 Tablets to a suitable volumetric flask to obtain a solution
through a suitable filter. with a final nominal concentration of 0.2 mq/rn], of
albuterol. Add 70% of the flask volume of Mobilephase, and

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108 A/butera/ / OfficialMonographs USP 43

sonicate for 10 min. Stir for 30 min, and dilute with Mobile Cu =nominal concentration of albuterol in the Sample
phase to volume. Pass the solution though a I-prn glass solution (mg/mL)
fiber or equivalent filter, and discard the first 3 mL of the M = moles of albuterol per mole of albuterol sulfate, 2
filtrate. Mr1 = molecular weight of albuterol, 239.31
Chromatographic system Mr2 = molecular weight of albuterol sulfate, 576.70
(See Chromatography (621), System Suitability.)
Mode: LC Acceptance criteria: See Table 2. Disregard peaks eluting
Detector: UV 225 nm after levalbuterol related compound C or with areas less
Column: 4.6-mm x 25-cm; 5-lJm packing L11 than that of the Sensitivity solution.
Column temperature: 30°
Flow rate: 1.5 mL/min Table 2
Injection volume: 80 IJL Relative Acceptance
Run time: 5 times the retention time of albuterol Retention Criteria,
System suitability Name Time NMT (%)
Samples: System sUitability solution and Standard solution Albuterol relatedcom-
[NoTE-Identify the impurities using the relative . pound Ba 0.88 _b
retention times shown in Table 2.]
Suitability requirements: Albuterol 1.0 -
Tailing factor: NMT 2.0 for each compound, Standard Chloroalbuterone" 1.7 _b
solution _b
Resolution: NLT 2 between albuterol and albuterol Chloroalbuterol" 2.5
related compound B, System suitability solution Albuteral related com-
Relative standard deviation: NMT 10.0% for each pound Ae 2.7 _b

com pound, Standard solution

Levalbuterol related com-
Analysis pound Of 3.2 0.1
Samples: Standardsolution, Sensitivity solution, and Sample
solution Levalbuterol related com-
pound (9,h 3.5 0.4
[NoTE-Identify the impurities using the relative
retention times shown in Table 2.] Any other individual un-
Calculate the percentage of levalbuterol related compound specified impurity - 0.2
C in the portion of Tablets taken:
a 2-(tert-Butylamino)-1-[4-hydroxy-3-(hydroxymethyl)phenyl]ethanone.
Result = (rulrs) x (Cs/Cu) x 100 b Process impurityincludedin the table foridentification only.Process impurities
are controlled in the drug substance, and are not to be reported or included in
the total impurities for the drug product.
ru =peak response of levalbuterol related compound c 2-(tert-Butylamino)-1-[3-chloro-4-hydroxy-5-(hydroxymethyl)phenylJ
C from the Sample solution ethanone.
rs = peak response of levalbuterol related compound d4-[2-(tert-Butylamino)-l-hyd roxyethylJ-2-chloro-6-(hydroxymethyl)phenol.
C from the Standard solution . e 4-{2-[(1,1-0imethylethyl)aminoJ-1-hydroxyethyl}-2-methylphenol.
Cs =concentration of USP Levalbuterol Related f 5-[2-{(1, 1-0imethylethyl)amino}-1-hydroxyethyl]-2-hydroxy-benzaIdehyde.
Compound C RS in the Standardsolution 9 a-[{(l, 1-0imethylethyl)amino}methyl]-4-hydroxy-3-(methoxymethyl)-
(mg/mL) benzenemethanol.
h Disregard peakseluting after levalbuterol related compound C.
Cu = nominal concentration of albuterol in the Sample
solution (mg/mL)
• ORGANIC IMPURITIES, PROCEDURE 2
Calculate the percentage of levalbuterol related Buffer: 6.8 giL of monobasic potassium phosphate in water.
compound D in the portion of Tablets taken: Adjust with hydrochloric acid to a pH of 3.0.
Mobile phase: Methanol and Buffer (30:70)
Result =(rulrs) x (CslCu) x 100 Diluent: Methanol and Buffer(5:95)
Peak identification solution: 2.4 IJg/mL of USP Albuterol
=peak response of levalbuterol related compound Sulfate RS, 1.0 IJg/mL of USP Levalbuterol Related
D from the Sample solution Compound C RS, and 1.2 IJg/mL of USP Levalbuterol
= peak response of the levalbuterol related Related Compound D RS in Diluent
compound D from the Standard solution Standard solution: 2.4 IJg/mL of USP Albuteral Sulfate RS
=concentration of USP Levalbuterol Related and 1.0 IJg/mL of USP Albuterol RelatedCompound E RS in
Compound D RS (as the free base) in the Diluent
Standardsolution(mg/mL) Sensitivity solution: 0.06 IJg/mL of USP Albuteral Sulfate RS
= nominal concentration of albuterol in the Sample in Diluent from Standard solution
solution (mg/mL) Sample solution: Transfer an amount of powder from NLT
20 Tablets to a suitable volumetric flask to obtain a solution
Calculate the percentage of any other impurity in the with a final concentration of 0.2 mg/mL of albuterol. Add
portion of Tablets taken: 70% of the flask volume of Diluent, and sonicate for 10 min.
Stir for 30 min, and dilute with Diluent to volume. Pass the
Result = (rulrs) x (Cs/Cu) x (M x MrtlMr2 ) x 100 solution though a glassfiber or equivalent filter of t-urn
pore size, and discard the first 3 mL of the filtrate.
ru = peak response of the impurity from the Sample Chromatographic system
solution (See Chromatography (621), System Suitability.)
rs = peak response of albuterol from the Standard Mode: LC
solution Detector: ·UV 225 nm
Cs = concentration of USP Albuterol Sulfate RS in the Column: 4.6-mm x 25-cm; 5-lJm packing L11
Standardsolution (mg/mL) Flow rate: 0.7 mL/min

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 USP 43 OfficialMonographs / Albuterol 109

Injection volume: 80 ~L ADDITIONAL REQUIREMENTS

Run time: 5 times the retention time of albuterol • PACKAGING AND STORAGE: Preserve in tight, light-resistant
System suitability containers. Store at controlled room temperature.
Sample: Standardsolution • USP REfERENCE STANDARDS (11)
Suitability requirements USP Albuterol Sulfate RS
Tailing factor: NMT 2.0 for each compound USP Albuterol Related Compound BRS
Relative standard deviation: NMT 10.0% for each 2-(tert-Butylamino)-1-[4-hydroxy-3-(hydroxymethyl)
compound phenyl]ethanone.
Analysis C13H,9N03 237.29
Samples: Standardsolution, Peak identification solution, USP Albuterol Related Compound ERS
Sensitivity solution, and Sample solution . 2,2'-Oxybis(methylene)bis{4-[2-( tert-butylamino)-1-
Calculate the percentage of albuterol related compound E hydroxyethyl]phenol}.
in the portion of Tablets taken: C26H40N20S 460.61
USP Levalbuterol Related Compound C RS
Result = (rulr s) x (CsICu) x 100 a-[{(l,l-Dimethylethyl)amino}methyl]-4-hydroxy-3-
(methoxymethyl)-benzenemethanol.
ru = peak response of albuterol related compound E C14H23N03 253.34
from the Sample solution USP Levalbuterol Related Compound D RS
ts = peak response of albuterol related compound E 5-[2-{(l,l-Dimethylethyl)amino}-1-hydroxyethyl]-2-
from the Standardsolution hydroxy-benzaldehyde sulfatesalt.
Cs = concentration of USP AlbuterolRelated (C13H,9N03h· H2S04 572.67
Compound E RS in the Standard solution
(mg/mL)
Cu = nominal concentration of albuterol in the Sample
solution (mg/mL)

Calculate the percentage of any other impurity in the Albuterol Sulfate

portion of Tablets taken:
(C13H21N03)2' H2S04 576.70
Result =(rulrs) x (CsIC u) x (M x Mr,/Mr2) x 100 l,3-Benzenedimethanol, a'-[[(l,l-dimethylethyl)amino]
methyl]-4-hydroxy-, sulfate(2:1) (salt).
ru = peak response of the impurityfrom the Sample a'-[(tert -Butylamino)methyl]-4-hydroxy-m-xylene-a,a'-diol
solution sulfate (2:1) (salt) [51022-70-9].
ts =peak response of albuterol from the Standard
solution » Albuterol Sulfate contains not less than 98.5
Cs = concentration of USP Albuterol Sulfate RS in the percent and not more than 101.0 percent of
Standardsolution(mg/mL)
Cu = nominal concentration of albuterol in the Sample (C13H21 N0 3) 2 • H2S04, calculated on the anhydrous
solution(mg/mL) basis.
M =moles of albuterol per mole of albuterolsulfate, 2
Mr1 = molecularweight of albuterol, 239.31 Packaging and storage-Preserve in well-closed,
Mr2 = molecularweight of albuterol sulfate, 576.70 light-resistant containers.
USP Reference standards (11 )-
Acceptance criteria: See Table 3. Disregard the levalbuterol USP Albuterol Related Compound A RS
related compound C peak and any peak eluting before it. 4-[2-[(l,l-Dimethylethyl)amino]-1-hydroxyethyl]-2-
Disregard peaks with areas lessthan that of the Sensitivity methylphenol sulfate.
solution. USP Albuterol Sulfate RS
Table 3 Identification-
Relative Acceptance
Retention Criteria,
Compound Time NMT (0/0)
Albuterol 1.0 -
Levalbuterol related com-
pound C 1.79 -
Levalbuterol related com-
pound D 1.83 -
Albuterol related ution: ~g per
compound EO 3.67 0.5
Medium: 0.1 N hydrochloric acid.
Anyother individual C: Shake a quantity of it, equivalentto 4 mg of albuterol,
unspecified impurity - 0.2 with 10 mLof water, and filter: the filtrate so obtained meets
Total impurities - 1.5b the requirements of the tests for Sulfate (191 ).
D: The retention time of the major peak in the
a 2,2'-Oxybis(methylene)bis{4-[2-(tert-butylamino)-1-hydroxyethyl]phenol}. chromatogram of the Assay preparation corresponds to that in
b From the sum of the impuritiesin Procedure 1 and Procedure 2. the chromatogram of the Standard preparation, as obtained in
the Assay. .
Water Determination, MethodI (921): not more than 0.5%.
Residue on ignition (281): not more than 0.1%.

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 110 Albuterol / Official Monographs USP 43

Chromatographic purity-It meets the requirements of IDENTIFICATION

the test for Organic Impurities under Albuterol, except to read
Albuterol Sulfate in place of Albuterol and to use water instead
of methanol as the solvent to prepare the Standard solution
and the Sample solution. • A·.. /~i
Assay-
Spe;gtCQ
• B. the ~retention time of t e major peak of the Sample
0.05 ± 0.07 M Ammonium acetate solution-Dissolve 3.85 g solution corresponds to that of the Standard solution, both
of ammonium acetate in 1000 ml of water, and mix. relative to the Internal standard solution, as obtained in the
Mobile phase-Prepare a degassed mixture of water, 0.05 Assay.
± 0.07 M Ammonium acetate solution, and isopropanol [65: 30:
(5 ± 1)], and adjust dropwise with acetic acid to a pH of 4.5 ASSAY
± 0.3. • PROCEDURE
Resolution solution-Dissolve accurately weighed quantities Solution A: 6.80 mg/ml of monobasic potassium
of USP Albuterol Sulfate RS and USP Albuterol Related phosphate (0.05 M)
Compound A RS in water, and dilute quantitatively, and Mobile phase: Methanol and Solution A (2:1)
stepwise if necessary, with Mobile phaseto obtain a solution Internal standard solution: 2 mg/ml of betamethasone
having a known concentration of about 0.140 mg per ml and dipropionate in methanol
0.030 mg per ml, respectively. Standard stock solution: 1.2 mg/ml of USP Alclometasone
Standard preparation-Dissolve an accurately weighed Dipropionate RS in methanol
quantity of USP Albuterol Sulfate RS in water, and dilute Standard solution: 4.0 ml of Standard stock solution and
quantitatively with water to obtain a solution having a known 4.0 mL of Internal standard solution. Dilute with methanol
concentration of about 0.6 mg per ml. to 25 ml. [NOTE-This solution contains approximately 0.2
Assay preparation-Transfer about 60 mg of Albuterol mg/mL of USP Alclometasone Dipropionate RS.]
Sulfate, accurately weighed, to a 1OO-ml volumetric flask, Sample stock solution: 1.2 mg/ml of Alclometasone
dissolve in and dilute with water to volume, and mix. Dipropionate in methanol
Chromatographic system (see Chromatography (621 »-The Sample solution: 4 ml of Sample stock solution and 4 ml of
liquid chromatograph is equipped with a 276-nm detector Internal standard solution. Dilute with methanol to 25 ml.
and a 4.6-mm x 20-cm column that contains packing L10. The Chromatographic system
flow rate is about 2.0 ml per minute. Chromatograph the (See Chromatography (621), System Suitability.)
Resolution solution, and record the peak responses as directed Mode: lC
for Procedure: the resolution, R, between albuterol and Detector: UV 254 nm
albuterol related compound A is not less than 1.5; and the Column: 4-mm x 30-cm; packing II
relative standard deviation for replicate injections is not more Flow rate: 1.2 mL/min
than 1.5%. Injection size: 10 ul,
Procedure-Separately inject equal volumes (about 10 ut) System suitability
of the Standard preparation and the Assay preparation into the Sample: Standard solution
chromatograph, record the chromatograms, and measure the [NoTE-The relative retention times for alclometasone
responses for the major peaks. Calculate the quantity, in mg, dipropionate and betamethasone dipropionate are
of (C13H 21N0 3)2 . H2S04 in the portion of Albuterol Sulfate about 0.7 and 1.0, respectively.]
taken by the formula: Suitability requirements
Resolution: NlT 3.0 between the analyte and the
100C(rulrs) internal standard peaks
Relative standard deviation: NMT 2%
in which C isthe concentration, in mg per ml, of USP Albuterol Analysis
Sulfate RS in the Standard preparation; and ru and rs are the Samples: Standard solution and Sample solution
peak responses obtained from the Assay preparation and the Calculate the percentage of C2sH37C107 in the portion of
Standard preparation, respectively. Alclometasone Dipropionate taken:
Result = (Ru/Rs) x (Cs/Cu) x 100
= peak height ratio from the Sample solution
Alclometasone Dipropionate '7peak height ratio from the Standard solution
= concentration of USP Alclometasone
Dipropionate RSin the Standard solution
(mg/ml)
= concentration of the Sample solution (mg/ml)

Acceptance criteria: 97.0%-102.0% on the dried basis

IMPURITIES
C2sH37CI07 521.04 INORGANIC IMPURITIES
Pregna-l,4-diene-3,20-dione, 7-chloro-ll-hydroxy-16- • Residue on Ignition (281): NMT 0.1 %
ORGANIC IMPURITIES
methyl-17,21-bis(1-oxopropoxy)-, (7a, 11~, 16a)-;
Ze-Chloro-l lp, 17,21-trihydroxy-16a-methylpregna-l ,4- • Procedure
diene-3,20-dione 17,21-dipropionate[66734-13-2]. Mobile phase: Acetonitrile and water (3:2)
Diluent: Acetonitrile and water (2:1)
DEFINITION System suitability solution: 1.5 mg/mL of USP
Alclometasone Dipropionate contains NlT 97.0% and NMT Alclometasone Dipropionate RS and 0.015 mg/mL of USP
102.0% of C2sH37CI07, calculated on the dried basis.

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 USP 43 OfficialMonographs / Alclometasone 111

Alclometasone Dipropionate Related Compound A RS in USP Alclometasone Dipropionate Related Compound A RS

Diluent 11 p, 17,21-Trihydroxy-16a.-methylpregna-l ,4-diene-
Sample solution: 1.5 mg/mL of Alclometasone 3,20-dione 17,21-dipropionate.
Dipropionate in Diluent C2sH3S07 486.60
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 15-cm; 5-l-Im packing L1 Alclometasone Dipropionate Cream
Flow rate: 1 mL/min
Injection size: 5 I-IL DEFINITION
Run time: Three times the retention time of alclometasone Alclometasone Dipropionate Cream contains NLT 90.0% and
System suitability NMT 110.0% of the labeled amount of alclometasone
Sample: System suitability solution dipropionate (C2sH37C107) in a suitable cream base.
Suitability requirements IDENTIFICATION
Tailing factor: NMT 1.5 for alclometasone dipropionate • A. The retention time of the major peak of the Sample
Relative standard deviation: NMT 2.0% for solution corresponds to that of the Standardsolution, both
alclometasone dipropionate relative to the internal standard, as obtained in the Assay.
Resolution: NLT 2.0 between alclometasone dipropionate • B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
and alclometasone dipropionate related compound A (201 )
Analysis Standard solution: 0.08 mg/mL of USP Alclometasone
Sample: Sample solution Dipropionate RS in methanol
Calculate the percentage of each impurity in the portion of Sample solution: Plate a quantity of Cream, equivalent to
Alclometasone Dipropionate taken: 1.25 mg of alclometasone dipropionate, in a 50-mL
Result = (rU/rT) x (1 IF) x 100 centrifuge tube, and add 15 mL of methanol. Insert a
stopper securely into the tube, and place the tube in a water
ru = peak area for each impurity from the Sample bath maintained at 60 0 until the semisolid components
solution melt. Remove the tube from the bath, shake vigorously until
rr = sum of all the peaks from the Sample solution the specimen components resolidify, and place the tube in
an ice-methanol bath for 15 min. Remove the tube from
F = relative response factor (see Impurity Table 1)
the bath, and centrifuge at 2500 rpm for 5 min. Transfer
Acceptance criteria the clear supernatant to a vial, and allow to equilibrate to
Individual impurities: See Impurity Table 1. room temperature.
Total impurities: NMT 2.0% Chromatographic system
(See Chromatography(621), Thin-Layer Chromatography.)
Impurity Table 1 Adsorbent: 0.25-mm layer of chromatographic silica gel
mixture
Relative Relative Acceptance Application volume: 20I-lL
Retention Response Criteria,
Name Time Factor NMT(%) Developing solvent system: Chloroform and acetone
(7:1 )
Alclometasone dipropionate 1.0 - - Analysis
Alclometasone dipropionate Samples: Standardsolution and Sample solution
related compound A" 1.2 0.93 1.0 Dry the applications with the aid of a stream of nitrogen,
and develop the chromatograms in a saturated, unlined
2-Bromo aclometasone di-
proplonate" 1.7 0.91 0.5 chromatographic chamber. When the solvent front has
moved three-fourths of the length of the plate, remove
Any individual, unspecified the plate from the chamber, mark the solvent front, and
impurity - 1.0 0.10
allow the solvent to evaporate. Observe the plate under
a 11~, 17,21-Trihydroxy-16a-methylpregna-1 ,4-diene-3,20-dione 17,21-
short-wavelength UV light.
dipropionate. Acceptance criteria: The RF value of the principal spot
b 2-Bromo-7a-chloro-11~, 17,21-trihydroxy-16a-methylpregna-1 ,4-diene-3,20- obtained from the Sample solution corresponds to that of
dione 17,21-dipropionate. the Standardsolution. .,
SPECIFIC TESTS ASSAY
• OPTICAL R.OTATION, Specific Rotation (781 S) • PROCEDURE
Sample solution: 30 mg/mL in dioxane Buffer: 6.80 giL of monobasic potassium phosphate (0.05
Acceptance criteria: +21 0 to +25 0 M)
• Loss ON DRYING (731): Dry a sample in a vacuum at a Mobile phase: Methanol and Buffer (2:1)
pressure not exceeding 5 mm of mercury at 105 0 for 3 h: it Internal standard solution: 0.4 mg/mL of betarnethasone
loses NMT 0.5% of its weight. dipropionate in methanol
Standard stock solution: 0.25 mg/mL of USP
ADDITIONAL REQUIREMENTS Alclometasone Dipropionate RS in methanol
• PACKAGING AND STORAGE: Preserve in tight containers, and Standard solution: 0.08 mg/mL of USP Alclometasone
store at controlled room temperature. Dipropionate RS obtained by combining, in a small
• USP REFERENCE STANDARDS (11) stoppered flask, 5.0 mL of Standardstock solution, 5.0 mL
USPAlclometasone Dipropionate RS of methanol, and 5.0 mL of Internal standard solution
Sample solution: Transfer a quantity of Cream, equivalent
to 1.25 mg of alclometasone dipropionate, to a 50-mL
centrifuge tube. Add 5.0 mL of Internal standard solution

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 112 Alclometasone / OfficialMonographs USP 43

and 10.0 mL of methanol. Inserta stopper securelyinto the

tube, and place it in a water bath maintained at 60° until Alclometasone Dipropionate Ointment
the semisolidcomponents melt. Remove the tube from the DEFINITION
bath, shake vigorously untilthe specimen components Alclometasone Dipropionate Ointment contains NLT 90.0%
resolidify, and return the tube to the 60° water bath until and NMT110.0% of the labeled amount of alclometasone
the semisolidcomponents melt. Remove the tube from the dipropionate (C2sH37CI07) in a suitable ointment base.
bath, shake vigorously untilthe specimen components
resolidify, and place the tube in an ice-methanol bath for IDENTIFICATION
15 min. Remove the tube from the bath, and centrifuge at • A. The retention time of the major peak of the Sample
2500 rpm for 5 min. Transfer the clear supernatant to a solution corresponds to that of the Standardsolution, both
small stoppered flask, and allow to equlllbrate to room relative to the internal standard, as obtained in the Assay.
temperature. • B. THIN-LAVER CHROMATOGRAPHIC IDENTIFICATION TEST
Chromatographic system (201 )
(See Chromatography (621), System Suitability.) Standard solution: 0.25 mg/mL USP Alclometasone
Mode: LC Dipropionate RS in methanol
Detector: UV 254 nm Sample solution: Place a quantity of Ointment, equivalent
Column: 4-mm x 30-cm; packing L1 to 1.25 mg of alclometasone dipropionate, in a 50-mL
Flow rate: 1.2 mL/min centrifuge tube, add 10 mL of 2,2,4-trimethylpentane,
Injection volume: 20 IJL insert a stopper securely into the tube, and disperse the
System suitability specimen using a vortex mixer. Add5.0 mL of a solution of
Sample: Standardsolution methanol in water (45 in 50), insert the stopper securely,
[NoTE-The relative retention times for alclometasone shake vigorously for 2 min, and centrifuge at 2500 rpm for
dipropionate and betamethasone dipropionate are 3 min. Remove the lower, aqueous alcohol phase, .end
about 0.7 and 1.0, respectively.] transfer to a stoppered vial.
SUitabilityrequirements Chromatographic system
Resolution: NLT 3.0 between the analyte and internal (See Chromatography (621), Thin-Layer Chromatography.)
standard peaks Adsorbent: 0.25-mm layer of chromatographic silica gel
Relative standard deviation: NMT 2% mixture
Analysis Application volume: 20 IJL
Samples: Standardsolution and Sample solution Developing solvent system: Chloroform and acetone
Calculate the percentage of the labeled amount of (7:1 )
alclometasone dipropionate (C2sH37C107) inthe portion of Analysis
Cream taken: Samples: Standardsolution and Sample solution
Drythe applicationswith the aid of a stream of nitrogen,
Result = (RulRs) x (CslCu) x 100 and develop the chromatograms in a saturated, unlined
chromatographic chamber. When the solvent front has
Ru = peak height ratio of alclometasone dipropionate moved three-fourths of the length of the plate, remove
to the internal standard from the Sample solution the plate from the chamber, mark the solvent front, and
Rs = peak height ratio of alclometasone dipropionate allow the solvent to evaporate. Observe the plate under
to the internal standard from the Standard short-wavelength UV light.
solution Acceptance criteria: The RF value of the principal spot
Cs =concentration of USP Alclometasone obtained from the Sample solution corresponds to that of
Dipropionate RS in the Standardsolution the Standardsolution.
(mg/mL) .
Cu = nominal concentration of aldometasone ASSAY
dipropionate in the Sample solution (mg/mL) • PROCEDURE
Buffer: 6.80 gIL of monobasic potassium phosphate (0.05
Acceptance criteria: 90.00/0-110.0% M)
Solution A: Dilute450 mL of methanol with water to 500
PERFORMANCE TESTS mL.
• MINIMUM FILL (755): Meets the requirements Mobile phase: Methanol and Buffer (2:1)
SPECIFIC TESTS Internal standard solution: 0.15 mg/mL of betamethasone
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR dipropionate in Solution A
SPECIFIED MICROORGANISMS (62): Meets the requirements Standard stock solution: 0.1 mg/mL of USP Alclometasone
of the tests for absence of Staphylococcus aureus and Dipropionate RS in Solution A
Pseudomonas aeruginosa Standard solution: 0.05 mg/mL of USP Alclometasone
Dipropionate RS obtained by combining, in a small
ADDITIONAL REQUIREMENTS stoppered flask, 5.0 mLof Standard stocksolution and 5.0
• PACKAGING AND STORAGE: Preserve in collapsible tubes or mL of Internal standard solution
tight containers, and store at controlled room temperature. Sample solution: Transfer a quantity of Ointment,
• USP REFERENCE STANDARDS (11) equivalent to 0.5 mg of alclometasone dipropionate, to a
USP Alclometasone Dipropionate RS 50-mL centrifuge tube, add 10 mL of
2,2,4-trimethylpentane, insert a stopper securely into the
tube, and disperse the specimen using a vortex mixer. Add
5.0 mL of Internal standardsolution and 5.0 mLof Solution
A, insert the stopper securely, shake vigorously for 2 min,
and centrifuge at 2500 rpm for 3 min. Remove the lower,
aqueous alcohol phase, and transfer this Sample solution to
a stoppered vial.

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 USP 43 OfficialMonographs / Alcohol 113

Chromatographic system IDENTIFICATION

(See Chromatography (621), System Suitability.) • A. It meets the requirements of the test for Specific Gravity
Mode: LC (841).
Detector: UV 254 nm
Column: 4-mm x 30-cm; packing L1
Flow rate: 1.2 mL/min
Injection volume: 20 ~L
System suitability
Sample: Standard solution
IMPURiTIES
[NoTE-The relative retention times for alclometasone
dipropionate and betamethasone dipropionate are • LIMIT OF NONVOLATILE RESIDUE
about 0.7 and 1.0, respectively.] Sample: 100 mL of Alcohol
Suitability requirements Analysis: Evaporate the Sample in a tared dish on a water
Resolution: NLT 3.0 between the analyte and internal bath, and dry at 100°-105° for 1 h. .
standard peaks Acceptance criteria: The weight of the residue is NMT 2.5
Relative standard deviation: NMT 2% mg.
Analysis • ORGANIC IMPURITIES
Samples: Standard solution and Sample solution Sample solution A: Alcohol (substance under test)
Calculate the percentage of the labeled amount of Sample solution B: 300 IJL/L of 4-methylpentan-2-ol in
alclometasone dipropionate (C2sH37CI07) in the portion of Sample solution A
Standard solution A: 200 ~L/L of methanol in Sample
Ointment taken: solution A
Result = (Ru/R s) x (Cs/Cu) x 100 Standard solution B: 10 ~L/L of methanol and 10 ~L/L of
acetaldehyde in Sample solution A
= peak height ratio of alclometasone dipropionate Standard solution C: 30 IJL/L of acetal in Sample solution A
to the internal standard from the Sample solution Standard solution D: 2 ~L/L of benzene in Sample solution
= peak height ratio of alclometasone dipropionate A
to the internal standard from the Standard Chromatographic system
solution (See Chromatography (621), System Suitability.)
= concentration of USP Alclometasone Mode: GC
Dipropionate RS in the Standard solution Detector: Flame ionization
(mg/mL) Column: 0.32-mm x 30-m fused-silica capillary; bonded
= nominal concentration of alclometasone with a 1.8-~m layer of phase G43
dipropionate in the Sample solution (mg/mL) Split ratio: 20:1
Temperatures
Acceptance criteria: 90.0%-110.0% Injection port: 200°
Detector: 280°
PERFORMANCE TESTS Column: See Table 1.
• MINIMUM FILL (755): Meets the requirements
Table 1
SPECIFIC TESTS
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR Mold Time
Initial Temperature Final at Final
SPECIFIED MICROORGANISMS (62): Meets the requirements Temperature Ramp Temperature Temperature
of the tests for absence of Staphylococcus aureus and e) e/min) e) (min)
Pseudomonas aeruginosa .
40 0 40 12
ADDITIONAL REQUIREMENTS
40 10 240 10
• PACKAGING AND STORAGE: Preserve in collapsible tubes or
tight containers, and store at controlled room temperature.
• USP REFERENCE STANDARDS (11) Linear velocity: 35 cmls
USP Alclometasone Dipropionate RS Carrier gas: Helium
Injection volume: 1.0 ~L
System suitability
Sample: Standard solution 8
Suitability requirements
Alcohol Resolution: NLT 1.5 between the first major peak
(acetaldehyde) and the second major peak (methanol)
Portions of this monograph that are national USP text, and are Analysis
not part of the harmonized text, are marked with symbols Samples: Sample solutionA, Sample solution 8, Standard
(+.) to specify this fact. solution A, Standard solution 8, Standard solution C, and
Standard solution 0
Methanol calculation
C2H 60 46.07
Ethanol; Result =(rulr s)
Ethyl alcohol ,[64-17-5].
= peak area of methanol from Sample solution A
DEFINITION = peak area of methanol from Standard solution A
·Alcohol contains NLT 92.3% and NMT 93.8%, by weight,
corresponding to NLT 94.9% and NMT 96.0%, by volume,
at 15.56°, of C2HsOH.•

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 114 Alcohol/Official Monographs USP 43

Acetaldehyde calculation (sum of acetaldehyde and Acceptance criteria

acetal) Absorbance: NMT 0.40 at 240 nm; NMT 0.30 between
250 nm and 260 nm; NMT 0.10 between 270 nm and
Result = ([AE/(A r - AE)] x CAl + {[DE/(D r - DE)] x Co x (Mrtl 340 nm
Mr2 ) } Curve: The spectrum shows a steadily descending curve
with no observable peaks or shoulders.
= peak area of acetaldehyde from Sample solutionA
= peak area of acetaldehyde from Standard
solution B
=concentration of acetaldehyde in Standard • ·CLARITY OF SOLUTION
solution B (~L/L) [NoTE-The Sample solution is to be compared to
= peak area of acetal from Sample solutionA Standardsuspension A and to water in diffused
= peak area of acetal from Standardsolution C daylight 5 min after preparation of Standard
=concentration of acetal in Standardsolution C (~L/ suspension A] .
L) Hydrazine solution: 10 mg/mL of hydrazine sulfate in
= molecular weight of acetaldehyde, 44.05 water. Allow to stand for 4-6 h.
= molecular weight of acetal, 118.2 Methenamine solution: Transfer 2.5 g of methenamine to
a 1OO-mL glass-stoppered flask, add 25.0 mL of water,
Benzene calculation insert the glass stopper, and mix to dissolve.
Primary opalescent suspension: Transfer 25.0 mL of
Result = [BE/(B T - BE)] X C8 Hydrazine solution to the Methenamine solution in the
1OO-mL glass-stoppered flask. Mix, and allow to stand for
BE = peak area of benzene from Sample solution A 24 h. This suspension is stable for 2 months, provided it is
BT =peak area of benzene from Standardsolution D stored in a glass container free from surface defects. The
suspension must not adhere to the glass and must be well
C8 =concentration of benzene in Standardsolution 0 mixed before use.
(~L/L)
Opalescence standard: Transfer 15.0 mL of the Primary
[NOTE-Ifnecessary, the identity of benzene can be opalescent suspension to a 1OOO-mL volumetric flask and
. confirmed using another suitable chromatographic dilute with water to volume. This suspension should 'not be
system (stationary phase with a different polarity).] used beyond 24 h after preparation.
Any other impurity calculation Standard suspension A: Opalescence standard and water (1
in 20)
Result = (rV/rM ) x CM Standard suspension B: Opalescence standard and water (1
in 10)
rv = peak area of each impurity in Sample solution B Sample solution A: Substance to be examined
rM = peak area of 4-methylpentan-2-01 in Sample Sample solution B: Dilute 1.0 mL of Sample solutionA with
solution B °
water to 20 mL,and allow to stand for 5 min before testing.
CM =concentration of 4-methylpentan-2-01 in Sample Blank: Water
solution B (~L/L) Analysis: Transfer a sufficient portion of Sample solution A
and Sample solution B to separate test tubes of colorless,
Acceptance criteria: See Table 2. transparent, neutral glass with a flat base and an internal
diameter of 15-25 mm to obtain a depth of 40 mm.
Table 2 Similarly transfer portions of Standardsuspension A,
Acceptance Standardsuspension B, and Blank to separate matching test
Name Criteria tubes. Compare Sample solution A, Sample solution B,
Standardsuspension A, Standardsuspension B, and Blank in
NMT 0.5, correspondingto
Methanol 200 IJl/L diffused daylight,.. ~.i,~~iQ~~.;,~!.;~I,!X . .~.Q.~i~~; . .: . . ~I~.~.~
backgrou nd (see~.V1iS;YC11.~gtrJPIJ.~lS;ga/(~~. g)~:.. (qlil·1+M~~~~qj'~}).
Acetaldehyde NMT 10 IJL/L, expressedas The diffusion of light must be such that Standardsuspension
and acetal acetaldehyde
A can readily be distinguished from water, and Standard
Benzene NMT 21JL/L suspension B can readily be distinguished from Standard
Sum of allother suspension A.
impurities' NMT 300 IJL/L Acceptance criteria: Sample solution A and Sample solution
~ show the same clarity as that of water or their opalescence
a Disregard .any peaks of less than 9 IJL/L (0.03 times the area of the peak IS not more pronounced than that of Standardsuspension A..
correspondlnq to 4-methylpentan-2-olin the chromatogram obtained with • ACIDITY OR ALKALINITY
Sample solutionB).
Phenolphthalein solution: Dissolve 0.1 g of
SPECIFIC TESTS phenolphthalein in 80 mL of alcohol, and dilute with water
•• SPECIFIC GRAVITY (841): 0.812-0.816 at
015.56°

to 100 mL.
indicating 92.30/0-93.8%, by weight, or 94.90/0-96.0%, by Sample: 20 mL of Alcohol
volume, of C2HsO H. Analysis: To the Sample add 20 mL of freshly boiled and
cooled water and 0.1 mL of Phenolphthalein solution. The
• ULTRAVIOLET ABSORPTION solution is colorless. Add 1.0 mL of 0.01 N sodium
Analytical wavelength: 235-340 nm hydroxide.
Cell: 5 cm
Acceptance criteri.a: The solution is pink (30 ~L/L,
Reference: Water
expressed.as acetic acid).

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 USP 43 Official Monographs / Alcohol 115

Standard solution A: 200 ~L/L of methanol in Sample

solutionA
• ·COLOR OF SOLUTION Standard solution B: 10 ~L/L of methanol and 10 ~L/L of
Standard stock solution: Combine 3.0 mLof ferric chloride acetaldehyde in Sample solution A
CS, 3.0 mL of cobaltous chloride CS, 2.4 mL of cupric Standard solution C: 30 ~L/L of acetal in Sample solution A
sulfate CS, and 1.6 mL of dilute hydrochloric acid (10 gIL). Standard solution D: 2 ~L/L of benzene in Sample solution
Standard solution: Transfer 1.0 mL of Standard stock A
solution to a 1OO-mL volumetric flask, and dilute with dilute Chromatographic system
hydrochloric acid (10 gIL). Prepare the Standard solution (See Chromatography (621), System SUitability.)
immediately before use. Mode: GC
Sample solution: Substance to be examined Detector: Flame ionization
Blank: Water Column: 0.32-mm x 30-m fused-silica capillary; bonded
Analysis: Transfer a sufficient portion of the Sample solution with a 1.8·~m layer of phase G43
to a test tube of colorless, transparent, neutral glass with a Split ratio: 20:1
flat base and an internal diameter of 15-25 mm to obtain Temperatures
a depth of 40 mm. Similarly transfer portions of the Injection port: 200°
Standardsolution and Blank to separate, matching test Detector: 280 0
tubes. Compare the Sample solution, Standard solution, and Column: See Table 1.
Blank in diffused d~~lig~tr~i;i~i~.g~~j~i:~II~<:~:j~.~~~):)~;~ite Table 1
background (see~\1i$ygl;i~Qt1JJi{iei~Qb:~(§j~fi»ii(<+~i'l::fit~Y£iQi?».
Acceptance criteria: The Sample solution has the Hold Time
appearance of water or is not more intensely colored than Initial Temperature Final at Final
Temperature Ramp Temperature Temperature
the Standard solution.• (") ("/min) (") (min)
ADDITIONAL REQUIREMENTS 40 0 40 12
• PACKAGING AND STORAGE: Preserve in tight containers,
40 10 240 10
protected from light.
• USP REFERENCE STANDARDS (11)
USP Alcohol RS Flow rate: 35 cm/s
Carrier gas: Helium
Injection volume: 1.0 ~L
System suitability
Sample: Standard solution B
Dehydrated Alcohol Suitability requirements
Resolution: NLT 1.5 between the first major peak
Portions of this monograph that are national USP text, and are (acetaldehyde) and the second major peak (methanol)
not part of the harmonized text, are marked with symbols Analysis
(+.) to specify this fact. Samples: Sample solution A, Sample solutionB, Standard
solutionA, Standard solution B, Standard solution C, and
Standardsolution 0
C2H60 46.07 Methanol calculation
Ethanol;
Ethyl alcohol [64-17-5]. Result = rulrs
DEFINITION ru = peak area of methanol from Sample solution A
·Dehydrated Alcohol contains NLT 99.2% by weight, rs =peak area of methanol from Standard solution A
corresponding to NLT 99.5% by volume, at 15.56°, of
C2H sOH .• Acetaldehyde calculation (sum of acetaldehyde and
IDENTIFICATION acetal)
• A. It meets the requirements of the test for Specific Gravity Result = {[AEI(A r - AE)] x C.J + {[DEI(D r - DE)] x CD x (M,d
(841 ).
M(2)}

AE = peak area of acetaldehyde from Sample solution A

Ar = peak area of acetaldehyde from Standard
solution B
CA =concentration of acetaldehyde in Standard
IMPURITIES solution B (~L/L)
• LBMIT OF NONVOLATILE RESIDUE oE = peak area of acetal from Sample solutionA
Sample: 100 mL of Dehydrated Alcohol DT = peak area of acetal from Standard solution C
Analysis: Evaporate the Sample in a tared dish on a water CD =concentration of acetal in Standard solution C (~LI
bath, and dry at 100°-105° for 1 h. L)
Acceptance criteria: The weight of the residue is NMT 2.5 M ,1 = molecular weight of acetaldehyde, 44.05
mg. M ,2 = molecular weight of acetal, 118.2
• ORGANIC IMPURITIES
Sample solution A: Substance to be examined Benzene calculation
Sample solution B: 300 ~L/L of 4-methylpentan-2-ol in
Sample solutionA Result = [Bi(B r - BJ] x Cs

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116 Alcohol/Official Monographs USP 43

BE = peak area of benzene from Sample solution A Opalescence standard: Transfer 15.0 mL of the Primary
BT = peak area of benzene from Standard solution D opalescent suspension to a 1OOO-mL volumetric flask, and
CB = concentration of benzene in Standard solution 0 dilute with water to volume. This suspensionshould not be
(IJL/L) used beyond 24 h after preparation.
Standard suspension A: Dilute 5.0 mL of the Opalescence
[NOTE-If necessary, the identity of benzene can be standard with water to 100.0.mL.
confirmed using another suitable chromatographic Standard suspension B: Dilute 10.0 mL of the Opalescence
system (stationaryphase with a different polarity).] standard with water to 100.0 mL.
Any other impurity calculation Sample solution A: Substanceto be examined
Sample solution B: 1.0 mL of Sample solution A diluted with
Result =(rU/rM) x CM water to 20 mL. Allow to stand for 5 min before testing.
Blank: Water
ru = peak area of each impurityfrom Sample solution Analysis
B Samples: Standard suspension A, Standard suspension B,
rM = peak area of 4-methylpentan-2-01 from Sample Sample solution A, Sample solution B) and Blank
solution B Transfer a sufficient portion of Sample solution A and
CM = concentration of 4-methylpentan-2-ol in Sample Sample solution B to separate test tubes-of colorless,
solution B (IJL/L) transparent, neutralglasswith a flat base and an internal
diameter of 15-25 mm to obtain a depth of 40 mm.
Acceptance criteria: See Table 2. Similarly transfer portions of Standardsuspension A,
Standard suspension B, and Blank to separate matching
Table 2 test tubes. Compare samples in diffused daylight,
Name
Acceptance
Criteria \,!~~in~.~~~!~~.!I.~>/~gai~st;~;~!.~;~l;!~;~.;'~~ round (see
~i\{t~qgl(f.Qmpgcl~QI'1.i(g~.Q);+i£(~.~£1.~M~Yfg9,lg». The diffusion of
NMT 0.5, corresponding to light must be such that Standard suspension A can be
Methanol 200 J.IL/L readily distinguished from water, and Standard
Acetaldehyde NMT 10 J.Il/L, expressedas suspension B can be readily distinguished from Standard
and acetal acetaldehyde suspension A
Benzene
Acceptance criteria: Sample solution A and Sample solution
NMT 2 J.IL/L
B show the same clarity as that of water, or their
Sum of allother opalescence isnot more pronounced than that of Standard
impurities' NMT 300 J.IL/L suspension A..
• ACIDITY OR ALKALINITY
a Disregard any peaks of lessthan 9 J.Il/L (0.03 times the area of the peak
correspondingto 4-methylpentan-2-o1 in the chromatogram obtained with Phenolphthalein solution: Dissolve 0.1 g of
Sample solution B). phenolphthalein in 80 mL of alcohol, and dilute with water
to 100 mL.
SPECIFIC TESTS Sample: 20 mL of DehydratedAlcohol
• • SPECIFIC GRAVITY (841): NMT 0.7962 at 15.56°, Analysis: To the Sample add 20 mL of freshly boiled and
indicating NLT 99.2% of C2 HsOH by weight. cooled water and 0.1 mL of Phenolphthalein solution. The
• ULTRAVIOLET ABSORPTION solution is colorless. Add 1.0 mL of 0.01 N sodium
Analytical wavelength: 235-340 nm hydroxide.
Cell: 5 cm Acceptance criteria: The solution is pink (30 IJg/g,
Reference: Water expressed as acetic acid).
Acceptance criteria
Absorbance: NMT 0.40 at 240 nm; NMT 0.30 between
250 and 260 nm; NMT 0.10 between 270 and 340 nm
Curve: The spectrum shows a steadilydescending curve • ·COLOR OF SOLUTION
with no observable peaks or shoulders. Standard stock solution: Combine 3.0 mLof ferric chloride
CS, 3.0 mLof cobaltous chlorideCS, 2.4 mL of cupric
sulfate CS, and 1.6 mL of dilute hydrochloric acid (10 mg/
mL).
• ·CLARITY OF SOLUTION Standard solution: 1.0 mL of Standard stock solution,
[NoTE-The Sample solution isto be compared to diluted with dilute hydrochloric acid (10 mg/mL) to 100
Standard suspension A and to water in diffused mL. Prepare the Standard solution immediatelybefore use.
daylight 5 min after preparation of Standard Sample solution: Substanceto be examined
suspension A] Blank: Water
Hydrazine solution: 10 mg/mLof hydrazine sulfate in Analysis . .
water. Allow to stand for 4-6 h. Samples: Standard solution, Sample solution, and Blank
Methenamine solution: Transfer 2.5 g of methenamine to Transfer a sufficient portion of each of the Samples to
a 1OO-mL glass-stoppered flask, add 25.0 mL of water, individual test tubes of colorless, transparent, neutral
insert the glass stopper, and mix to dissolve. glass with a flat base and an internal diameter of 15-25
Primary opalescent suspension: Transfer 25.0 mL of mm to.obtain a of 40 mm. Compare the Samples
Hydrazine solution to the Methenamine solution in the in diffused a white
1OO-mL glass-stopperedflask. Mix, and allow to stand for background
24 h. This suspension isstable for 2 months, provided it is ).
stored in a glasscontainer free from surface defects.The Acceptance criteria: The Sample solution has the
suspension must not adhere to the glass and must be well appearance of water or is not more intenselycolored than
mixed before use. . the Standard solution.•

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 USP 43 Official Monographs / Alcohol 117

ADDITIONAL REQUIREMENTS stand until the permanganate color is discharged. Add 3 mL

• PACKAGING AND STORAGE: Preserve in tight containers, of 2.5 N sodium hydroxide, and pass, without washing,
protected from light. through a sintered-glass filter. Prepare a control containing 1
• liSP REFERENCE STANDARDS (11) mL of the saturated solution of dibasic sodium phosphate, 3
USP Dehydrated Alcohol RS mL of 2.5 N sodium hydroxide, and 80 IJgof acetone in 9 mL.
To each solution add 1 mL of furfural solution (1 in 100), and
allow to stand for 10 minutes, then to 1.0 mL of each solution
add 3 mL of hydrochloric acid: any pink color produced in the
test solution is not more intense than that in the control.
Dehydrated Alcohol Injection Methanol-To 1 drop add 1 drop of water, 1 drop of dilute
phosphoric acid (1 in 20), and 1 drop of potassium
permanganate solution (1 in 20). Mix, allow to stand for 1
» Dehydrated Alcohol Injection is Dehydrated minute, and add sodium metabisulfite solution (1 in 20),
Alcohol suitable for parenteral use. dropwise, until the permanganate color is discharged. If a
brown color remains, add 1 drop of the dilute phosphoric acid.
Packaging and storage-Preserve in tight, single-dose To the colorless solution add 5 mL of freshly prepared
containers, preferably of Type I glass, and store at controlled chromotropic acid TS, and heat on a water bath at 60° for 10
room temperature. The container may contain an inert gas in minutes: no violet color appears.
the headspace. Other requirements-It meets the requirements under
Identification- Injections and Implanted Drug Products (1).
A: Mix 5 drops in a small beaker with 1 mL of potassium
permanganate solution (1 in 100) and 5 drops of 2 N sulfuric
acid, and cover the beaker immediately with a filter paper
moistened with a solution recently prepared by dissolving 0.1 Alcohol in Dextrose Injection
g of sodium nitroferricyanide and 0.25 g of piperazine in 5 mL
of water: an intense blue color is produced on the filter paper, DEFINITION
the color becoming paler after a few minutes. Alcohol in Dextrose Injection is a sterile solution of Alcohol and
B: To 5 mL of a solution (1 in 10) add 1 mL of 1.0 N sodium Dextrose in Water for Injection. It contains NlT 90.0% and
hydroxide, then slowly (over a period of 3 minutes) add 2 rnL NMT 110.0% of the labeled amount of alcohol (C2H sOH),
of 0.1 N iodine: the odor of iodoform develops, and a yellow and NLT 95.0% and NMT 105.0% of the labeled amount of
precipitate is formed within 30'minutes. dextrose (C6 H120 6 • H20).
Spedfic gravity (841): not more than 0.8035 at 15.56°,
indicating not less than 96.8%, by weight, of C2H sOH. IDENTIFICATION
Acidity-To 50 mL, in a glass-stoppered flask, add 50 mL of • A.
recently boiled water. Add phenolphthalein TS, and titrate Sample solution: A few drops of Injection (1 in 20) in water
with 0.020 N sodium hydroxide to a pink color that persists Analysis: Add the Sample solution to 5 mL of hot alkaline
for 30 seconds: not more than 10.0 mL of 0.020 N sodium cupric tartrate TS.
hydroxide is required for neutralization. Acceptance criteria: A copious red precipitate of cuprous
Limit of nonvolatile residue-Evaporate 40 mL in a tared oxide is formed.
dish on a water bath, and dry at 105° for 1 hour: the weight • B. SPECIFIC GRAVITY (841): NMT 0.7962 at 15.56°,
of the residue does not exceed 1 mg. . indicating NLT 99.2% of alcohol (C 2H sOH) by weight
Water-insoluble substances-Dilute it with an equal
volume of water: the mixture is clear and remains clear for 30
minutes after cooling to 10°.
Aldehydes and other foreign organic substances- ij~~~(;)~I~I~~N"'IFI~~r~~~j~~~JS(1~7);.lfiffq[~i:J
Place 20 mL in a glass-stoppered cylinder that has been .sP~§t PY;1?7;'~·c>r·1.?7'F.(CNJ-MaY':2020):Meets the
thoroughly cleaned with hydrochloric acid, then rinsed with requirements
water and finally with the dehydrated alcohol to be tested.
Cool the contents to approximately 15°, and add, by means ASSAY
of a carefully cleaned pipet, 0.10 mL of 0.10 N potassium • ALCOHOL DETERMINATION, Method 7-Distillation Method
permanganate, noting accurately the time of addition. Mix at (611 )
once by inverting the stoppered cylinder, and allow it to stand Sample solution: 50.0 mL
at 15° for 5 minutes: the pink color does not entirely disappear. Acceptance criteria: 90.00/0-110.0%
Amyl alcohol and nonvolatile, carbonizable • DEXTROSE
substances-Allow 25 mL to evaporate spontaneously from Sample: Equivalent to 2-5 g of dextrose from a suitable
a porcelain dish, carefully protected from dust, until the volume of injection
surface of the dish is barely moist: no red or brown color is Analysis: Transfer the Sample solution to a 100-mL
produced immediately upon the addition of a few drops of volumetric flask, add 0.2 mL of 6 N ammonium hydroxide,
sulfuric acid. and dilute with water to volume. Determine the angular
Ultraviolet absorbance-Record the UV absorption rotation in a suitable polarimeter tube (see Optical Rotation
spectrum between 340 nm and 235 nm in a t-ern cell, with (781 »).
water in a matched cell in the reference beam: the absorbance Calculate the percentage of dextrose (C6H 12 0 6 • H2 0 ) in the
is not more than 0.08 at 240 nm, and 0.02 between 270 nm portion of Injection taken:
and 340 nm, and the curve drawn through these points is
smooth. Result = [(M ,d M (2)1 R mid]A R x 100
Limit of acetone and isopropyl alcohol-To 1.0 mL add
1 mL of water, 1 mL of a saturated solution of dibasic sodium M'I = molecular weightof dextrose monohydrate,
phosphate, and 3 mL of a saturated solution of potassium 198.17
permanganate. Warm the mixture to 4SO to 50°, and allow to M ,2 =molecular weight of anhydrous dextrose, 180.16

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118 Alcohol/Official Monographs USP 43

R mid = midpoint of the specific rotation range for Standard solution: 50 J-lg/mL of USP Denatonium Benzoate
anhydrous dextrose, 52.9° RS in water
A = 100 mm divided by the length of the polarimeter Sample solution: Dissolvethe residue obtained in the test
tube (mm) for Limit of Nonvolatile Residue in 50.0 mL of water, and
R = observed rotation CO) transfer to a suitable flask.
Instrumental conditions
Acceptance criteria: 95.00/0-105.0% Analytical wavelength: Maximum absorbance at about
410 nm
IMPURITIES
Cell: 1 em
• LIMIT OF 5-HvDROXVMETHVLFURFURAL AND RELATED
Analysis
SUBSTANCES
Samples: Buffer, Standard solution, and Sample solution
Sample solution: Equivalent to 2 mg/mL of dextrose in Transfer 10.0 mL each of Buffer, Standardsolution, and
water from a suitable volume of Injection Sample solution to individual 250-mL separators. Add to
Instrumental conditions each 40 mL of Buffer 10 mL of a 1-in-1 000 solution of
Mode: UV bromophenol blue in chloroform and 60 mL of
Analytical wavelength: 284 nm chloroform. Shake the separators vigorously for 2 min,
Cell: 1 em allow to stand for 15 min, then withdraw the chloroform
Blank: Water layers through chloroform-washed cotton into 100-mL
Acceptance criteria: The absorbance is NMT 0.25. volumetric flasks. Repeat the extraction with 20 mL of
SPECIFIC TESTS chloroform, adding the filtered chloroform extracts to the
• pH (791): 3.5-6.5 respective volumetric flasks, and dilute with chloroform to
Sample solution: A portion of Injection to which 0.30 mL volume. Without delay, concomitantly determine the
of saturated potassium chloride solution has been added for absorbances of the solutions, using the blank to set a
each 100 mL and which previously has been diluted with suitable spectrophotometer.
water, if necessary,to a concentration of NMT 5% of Calculate the quantity, in mg, of denatonium benzoate
dextrose (C2sH34N203 . H20) in 100 mL of Rubbing Alcohol:
• BACTERIAL ENDOTOXINS TEST (85): NMT 0.5 USP Endotoxin
unit/mL Result = (Au/As) x Cs x 0.025
• OTHER REQUIREMENTS: Meets the requirements in Injections
and Implanted Drug Products (1) = absorbance of the Sample solution
= absorbance of the Standard solution
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, single-dose
= concentration of USP Denatonium Benzoate RS in
the Standard solution (J-lg/mL)
containers, preferably of Type I or Type II glass, and store
at controlled room temperature. Acceptance criteria: NLT 1.40 mg
• LABELING: The label states the total osmolarity of the • SUCROSE OCTAACETATE
solution expressed in mOsmol/L. Sample solution: Using about 50 mL of 70% alcohol,
transfer the residue obtained in the test for Limit of
Nonvolatile Residue to a 500-mL conical flask.
Analysis: Neutralize the Sample solution with 0.1 N sodium
hydroxide VS, using phenolphthalein TS as the indicator.
Rubbing Alcohol Add 25.0 mL of 0.1 N sodium hydroxide, attach an air
DEFINITION
condenser to the flask, and reflux on a steam bath for 1 h.
Rubbing Alcohol and all preparations under the classification Remove from the steam bath, cool quickly, and titrate the
of Rubbing Alcohols are manufactured in accordance with excess alkali with 0.1 N sulfuric acid VS, using
the requirements of the U.S.Treasury Department, Bureau of phenolphthalein TS as the indicator. Perform a blank
Alcohol, Tobacco, and Firearms, Formula 23-H (8 parts by determination (see Titrimetry(541), Residual Titrations).
volume of acetone, 1.5 parts by volume of methyl isobutyl Each mL of 0.1 N sodium hydroxide is equivalent to 8.483
ketone, and 100 parts by volume of ethyl alcohol) being mg of sucrose octaacetate (C2SH3S019)'
used. It contains NLT 68.5% and NMT71.5% by volume of Acceptance criteria: NLT 355 mg of sucrose octaacetate
dehydrated alcohol, the remainder consisting of water and per 100 mL of Rubbing Alcohol
the denaturants, with or without color additives, and IMPURITIES
perfume oils. Rubbing Alcohol contains, in each 100 mL, NLT • METHANOL
355 mg of sucrose octaacetate or NLT 1.40 mg of Sample solution: Dilute 0.50 mL of Rubbing Alcohol with
denatonium benzoate. The preparation may be colored with water to 1.0 mL.
one or more color additives, listed by the FDA for use in Analysis: To 0.50 mL of the Sample solution add 1 drop of
drugs. A suitable stabilizer may be added. Rubbing Alcohol dilute phosphoric acid (1 in 20) and 1 drop of potassium
complies with the requirements of the Bureau of Alcohol, permanganate solution (1 in 20). Mix, allow to stand for 1
Tobacco, and Firearms of the U.S. Treasury Department. min, and add dropwise sodium metabisulfite solution (1 in
[NOTE-Rubbing Alcohol is packaged, labeled, and sold in 20) until the permanganate color is discharged. If a brown
accordance with the regulations issued by the U.S. color remains, add 1 drop of dilute phosphoric acid (1 in
Treasury Department, Bureau of Alcohol, Tobacco, and 20). To the colorless solution add 5 mL of freshly prepared
Firearms.] chromotropic acid TS, and heat in a water bath at 60° for
ASSAY 10 min.
• DENATONIUM BENZOATE Acceptance criteria: No violet color appears.
Buffer: 9.23 g of anhydrous dibasic sodium phosphate in SPECIFIC TESTS
800 mL of water. Adjust with saturated citric acid solution • SPECIFIC GRAVITY (841): 0.8691-0.8771 at 15.56° (the U.S.
to a pH of 4 ± 0.1, dilute with water to 1000 mL, and mix. government standard temperature for alcohol

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 USP 43 OfficialMonographs / Alendronate 119

determination) for Rubbing Alcohol manufactured with Standard solution: Transfer 5.0 mL of the Standardstock
specially denatured alcohol Formula 23-H solution to a 50-mL polypropylene, screw-cap centrifuge
• LIMIT OF NONVOLATILE RESIDUE tube containing 5 mL of Borate solution. Add 5 mL of
Where the denaturant is denatonium benzoate Solution A, and shakefor 30 s. Allow to stand at room
Sample: 200.0 mL of Rubbing Alcohol temperature for 25 min. Add 25 mL of methylene chloride,
Analysis: Evaporatethe Sample, transferred in convenient and shakevigorously for 1 min. Centrifuge for 5-10 min.
portions, in a suitable tared dish on a steam bath, and dry Usea portion of the clear upper aqueous layer.
the residue at 105° for 1 h. Retain the residuefor the Assay Reagent blank: Transfer 5.0 mL of Diluent to a 50-mL
for Denatonium Benzoate. polypropylene, screw-cap centrifuge tube containing 5
Acceptance criteria: The weight of the residue is NLT 2.8 mL of Borate solution. Add 5 mL of Solution A, and shakefor
mg. 30 s. Allow to stand at room temperature for 25 min. Add
Where the denaturant is sucrose octaacetate 25 mL of methylene chloride, and shake vigorously for 1
Sample: 25.0 mL of Rubbing Alcohol min. Centrifuge for 5-10 min. Use a portion of the clear
Analysis: Evaporate the Sample in a suitable tared dish on upper aqueous layer.
a steam bath, and dry the residue at 105° for 1 h. Retain Sample stock solution: 0.1 mg/mL of Alendronate Sodium
the residue for the Assay for Sucrose Octaacetate. in Diluent
Acceptance criteria: The weight of the residue is NLT 89 Sample solution: Transfer 5.0 mL of the Sample stock
mg. solution to a 50-mL polypropylene, screw-cap centrifuge
tube containing 5 mL of Borate solution. Add 5 mL of
ADDITIONAL REQUIREMENTS Solution A, and shakefor 30 s. Allow to stand at room
• PACKAGING AND STORAGE: Preserve in tight containers, temperature for 25 min. Add 25 mL of methylene chloride,
remote from fire, and store at controlled room temperature. and shake vigorously for 1 min. Centrifuge for 5-10 min.
• LABELING: Label it to indicate that it is flammable. Usea portion of the clear upper aqueous layer.
• USP REFERENCE STANDARDS (11) Chromatographic system
USP Denatonium Benzoate RS (See Chromatography (621), System SUitability.)
Mode: LC
Detector: UV 266 nm
Column: 4.1-mm x 25-cm; packing L21
Alendronate Sodium Column temperature: 35°
Flow rate: 1.2 mL/min
Injection volume: 10 ~L
System suitability
Sample: Standardsolution
SUitability requirements
Tail~ng factor: NMT 1.5
Relative standard deviation: NMT 2.0% for replicate
injections
C4H12NNa07P2·3H20 325.12
Analysis
Phosphonic acid, (4-amino-l-hydroxybutylidene) bis-, Samples: Standardsolution, Reagent blank, and Sample
monosodium salt, trihydrate; solution
Sodium trihydrogen (4-amino-l-hydroxybutylidene) Calculate the percentage of alendronate sodium
diphosphonate, trihydrate [121268-17-5]. . (C4H12NNa07P2) in the portion of Alendronate Sodium
DEFINITION taken:
Alendronate Sodium contains NLT 98.0% and NMT 102.0%
of alendronate sodium (C4H12NNa07P2), calculated on the Result = (r vir s) x (C siC v) x 100
dried basis.
ru = peak area from the Sample solution
IDENTIFICATION r5 = peak area from the Standardsolution
Cs = concentration of USP Alendronate Sodium RS in
the Standardstock solution (mg/mL)
Cu =concentration of Alendronate Sodium in the
• A. Sample stocksolution (mg/mL)
~R~. )
• B. IDENTIFICATION TESTs-GENERAL (191), Sodium: Meets Acceptance criteria: 98.0%-102.0% on the dried basis
the requirements of test A
IMPURITIES
ASSAY • ORGANIC IMPURITIES
• PROCEDURE Buffer solution: 2.94 giL of sodium citrate dihydrate and
Buffer solution: 14.7 giL of sodium citrate dihydrate and 1.42 giL of anhydrous dibasic sodium phosphate. Adjust
7.05 giL of anhydrous dibasic sodium phosphate. Adjust with phosphoric acid to a pH of 8 and pass through a filter
with phosphoric acid to a pH of 8. of 0.5-~m or finer pore size.
Mobile phase: Acetonitrile, methanol, and Buffer solution Solution A: Acetonitrile and Buffer solution (3:17)
(25:5:70) Solution B: Acetonitrile and Buffer solution (7:3)
Diluent: 29.4 giL of sodium citrate dihydrate Mobile phase: See Table 7.
Borate solution: 19.1 giL of sodium borate
Solution A: 0.5 mg/mL of 9-fluorenylmethyl chloroformate Table 1
in acetonitrile. [Note-Prepare this solution fresh just before
Time Solution A Solution B
use.] (min) (%) (%)
Standard stock solution: 0.1 mg/mL of USP Alendronate
Sodium RS in Diluent 0 100 0

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120 Alendronate / Official Monographs USP 43

Table 1 (continued) Calculate the percentage of each impurity in the portion of

Time Solution A Solution B Alendronate Sodium taken:
(min) (%) (%)
Result = (r vir r) x 100
15 50 50
25 0 100 ru = peak area of each impurity
27 100 0
rr = sum of all impurity peaksand the main peak

32 100 0 Acceptance criteria

Individual impurities: NMT 0.1%
Diluent and Borate solution: Proceed as directed in the Total impurities: NMT 0.5%
Assay. SPECIFIC TESTS
Solution C: 4 mg/mL of 9-fluorenylmethyl chloroformate in • Loss ON DRYING (731)
acetonitrile. [Nora-Prepare this solution fresh just before Sample: Dry at a pressureof NMT 5 mm of mercury at 140 0
use.] to constant weight.
Standard stock solution: 0.6 mg/mL of USP Alendronate Acceptance criteria: 16.1%-1 7.1%
Sodium RS in Diluent
Standard solution A: Transfer 5.0 mL of the Standard stock ADDITIONAL REQUIREMENTS
solution to a 50-mL polypropylene, screw-cap centrifuge • PACKAGING AND STORAGE: Preserve in well-closed
tube containing 5 mL of Borate solution. Add 5 mL of containers, and store at room temperature.
acetonitrile and 5 mL of Solution C, and shakefor 45 s.Allow • USP REFERENCE STANDARDS (11)
to stand at room temperature for 30 min. Add 20 mL of USP Alendronate Sodium RS
methylene chloride, and shake vigorously for 1 min.
Centrifuge for 5-10 min, and use a portion of the clear
upper aqueous layer.
Standard solution B: 0.6 JJg/mLof USP Alendronate
Sodium RS in Diluent from Standard stock solution. Transfer Alendronate Sodium Tablets
5 mL of this diluted solution (0.6 JJg/mL) to a 50-mL
polypropylene, screw-cap centrifuge tube containing 5 DEFINITION
mL of Borate solution. Add 5 mL of acetonitrile and 5 mL of Alendronate Sodium Tablets contain an amount of
Solution C, and shake for 45 s. Allow to stand at room Alendronate Sodium equivalent to NLT 90.0% and NMT
temperature for 30 min. Add 20 mL of methylene chloride, 110.0% of the labeled amount of alendronic acid
and shake vigorously for 1 min. Centrifuge for 5-10 min, (C4H 13N07P 2) ·
and use a portion of the clear upper aqueous layer. IDENTIFICATION
Reagent blank: Transfer 5.0 mL of Diluent to a 50-mL • The retention time of the major peak of the Sample solution
polypropylene, screw-cap centrifuge tube containing 5 corresponds to that of the Standard solution, as obtained in
mL of Borate solution. Add 5 mL of acetonitrile and 5 mL of the Assay.
Solution C, and shake for 45 s. Allow to stand at room
temperature for 30 min. Add 20 mL of methylene chloride, ASSAY
and shake vigorously for 1 min. Centrifuge for 5-10 min, • PROCEDURE
and use a portion of the clear upper aqueous layer. Solution A: 14.7 giL of sodium citrate dihydrate and 7.05
Sample stock solution: 0.6 mg/mL of Aleridronate Sodium giL of anhydrous dibasic sodium phosphate in water.
in Diluent [NOTE-Adjust with phosphoric acid to a pH of 8.0 before
Sample solution: Transfer 5.0 mL of Sample stock-solution to bringing the solution to volume.]
a 50-mL polypropylene, screw-cap centrifuge tube Solution B: 38.1 giL of sodium borate in water
containing 5 mL of Borate solution. Add 5 mL of acetonitrile Solution C: 1 mg/mL of 9-fluorenylmethyl chloroformate in
and 5 mL of Solution C, and shake for 45s. Allow to stand acetonitrile. [NOTE-Prepare this solution fresh just before
at room temperature for 30 min. Add 20 mL of methylene use.]
chloride, and shakevigorously for 1 min. Centrifuge for 5- Mobile phase: Acetonitrile, methanol, and Solution A
10 min, and use a portion of the clear upper aqueous layer. (20:5:75)
Chromatographic system Diluent: 29.4 giL of sodium citrate dihydrate in water
(See Chromatography (621), System Suitability.) Standard stock solution: 0.03 mg/mL of anhydrous
Mode: LC alendronate sodium in Diluent, from USP Alendronate
Detector: UV 266 nm Sodium RS
Column: 4.1-mm x 25-cm; packing L21 Standard solution: Transfer 5.0 mL of the Standardstock
Column temperature: 45 0 solution to a 50-mL polypropylene screw-cap centrifuge
Flow rate: 1.8mL/min tube containing 5 mL of Solution B, and mix for 3 min. Add
Injection volume: 20 JJL 4 mL of Solution C, and agitate for 30 s. Allow the solution
System suitability to.stand at room temperature for 25 min. Add 25 mL of
Samples: Standardsolution A and Standard solution B methylene chloride, and agitate for 40 s. Centrifuge the
Suitability requirements .mixture for 10 min. Use the clear upper aqueous layer.
Tailing factor: NMT 2.0 for the main peak, Standard Sample stock solution: Transfer NLT 10 Tablets to a
solution A 1OOO-mL volumetric flask. Add 500 mL of Diluent, shake by
Signal-to-noise ratio: NLT 3 for the main peak, Standard mechanical means for 30 min, and sonicate for 5 min.
solution B Dilute with Diluent to volume, and centrifuge a portion of
. Analysis this solution. Quantitatively dilute a portion of the clear
Samples: Reagent blank and Sample solution supernatant to a concentration of 0.02-0.03 mg/mL of
[NoTE-Disregard any peak corresponding to those alendronic acid.
obtained from the Reagent blank.]

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USP 43 Official Monographs / Alendronate 121

Sample solution: Transfer 5.0 mL of the Sample stock concentration, C (mg/mL), of anhydrous alendronate
solution to a 50-mL polypropylene screw-cap centrifuge sodium in this solution.
tube containing 5 mL of Solution B, and mix for 3 min. Add Standard solution: Transfer 5.0 mL of the Standard stock
4 mL of Solution C, and agitate for 30 s, Allow the solution solution to a 50-mL polypropylene screw-cap centrifuge
to stand at room temperature for 25 min. Add 25 mL of tube containing 1.0 mL of Diluent and 5.0 mL of Solution
methylene chloride, and agitate for 40 s. Centrifuge the B, and mix for 3 min. Add 4.0 mL of Solution C, and agitate
mixture for 10 min. Usethe clear upper aqueous layer. for 30 s. Allow the solution to stand at room temperature
Blank: Transfer 5 mL of Diluent to a 50-mL polypropylene for 25 min. Add 25 mL of methylene chloride, and agitate
screw-cap centrifuge tube containing 5 mL of Solution B, for 40 s. Centrifuge the mixture for 5 min. Use a portion
and mix for 3 min. Add 4 mL of Solution C, and agitate for of the clear, upper aqueous layer.
30 s. Allow the solution to stand at room temperature for Blank: Transfer 5 mL of water to a 50-mL polypropylene
25 min. Add 25 mL of methylene chloride, and agitate for screw-cap centrifuge tube containing 1.0 mL of Diluent
40 s. Centrifuge the mixture for 10 min. Usethe clear upper and 5.0 mL of Solution B, and mix for 3 min. Add 4.0 mL
aqueous layer. of Solution C, and agitate for 30 s. Allow the solution to
Chromatographic system stand at room temperature for 25 min. Add 25 mL of
(See Chromatography (621), System Suitability.) methylene chloride, and agitate for 40 s. Centrifuge the
Mode: LC mixture for 5 min. Use a portion of the clear, upper
Detector: UV 266 nm aqueous layer. .
Column: 4.1-mm x 25-cm; packing L21 Sample solution: Withdraw a portion of the solution
Column temperature: 35° under test, and centrifuge immediately. Transfer 5.0 mL
Flow rate: 1 mL/min of the supernatant to a 50-mL polypropylene screw-cap
Injection size: 50 tJL centrifuge tube containing 1.0 mL of Diluent and 5.0 mL
System suitability of Solution B, and mix for 3 min. Add 4.0 mL of Solution
Sample: Standardsolution C, and agitate for 30 s. Allow the solution to stand at room
Suitability requirements temperature for 25 min. Add 25 mL of methylene
Capacity factor: NLT 2.0 chloride, and agitate for 40 s. Centrifuge the mixture for
Relative standard deviation: NMT 2.0% for replicate , 5 min. Use a portion of the clear, upper aqueous layer.
injections Chromatographic system and System suitabilitity:
Analysis Proceed as directed in the Assay.
Samples: Standardsolution, Sample solution, and Blank Analysis
Calculate the percentage of the label claim in the portion Samples: Standard solution and Sample solution
of C4HnN0 7P2 taken: Calculate the percentage of C4HnN07P2 dissolved:

Result = (ru/rs) x (Cs/Cu) x (Mr1/Mr2) x 100 Result = (ru/r s) x C x (M r1/Mr2) x V x (100/L)

ru = peak area from the Sample solution ru =peak area from the Sample solution
rs = peak area from the Standard solution . rs =peak area from the Standard solution
Cs =concentration of anhydrous USP Alendronate C = defined under the Standard stock solution
Sodium RS in the Standard stock solution M r1 = molecular weight of alendronic acid, 249.10
(mg/mL) M r2 =molecular weight of alendronate sodium, 271.09
Cu = nominal concentration of alendronic acid in the V =volume of the Medium, 900 mL
Sample stocksolution (mg/mL) . L =Tablet label claim (mg)
M r1 = molecular weight of alendronic acid, 249.10
M r2 = molecular weight of anhydrous alendronate Tolerances: NLT 80% (Q) of the labeled amount of
sodium, 271.09 C4H13N07P2 is dissolved; for tablets labeled for weekly
dosing, NLT 75% (Q) of the labeled amount of
Acceptance criteria: C4H 12NNa0 7P2 equivalent to 90.0%- C4H nN0 7P2 is dissolved.
110.0% of the labeled amount of C4H nN0 7P2 Test 2
If the product complies with this test, the labeling indicates
PERFORMANCE TESTS that the product meets USP Dissolution Test 2.
• DISSOLUTION (711) Medium: Water; 900 mL
Test 1 Apparatus 2: 50 rpm
Medium: Water; 900 mL Time: 30 min
Apparatus 2: 50 rpm Determine the amount of C4H 12NNa0 7P2 . 3H20 dissolved
Time: 15 min
Determine the amount of C4H nN0 7P2 dissolved by using using the following method.
Solution B and Solution C: Proceed as directed in the
the following method. Assay.
Solution A and Mobile phase: Proceed as directed in the 0.6 M citrate buffer: 176.4 gIL of sodium citrate dihydrate
Assay. in water
Diluent: 176.4 gIL of sodium citrate in Medium 0.05 M buffer: Transfer 14.7 g of sodium citrate dihydrate
Solution B: Dissolve 6.2 g of boric acid in approximately and 7.05 g of anhydrous dibasic sodium phosphate to a
950 mL of water. Adjust with 1 N sodium hydroxide to a 1OOO-mL volumetric flask, dissolve in about 900 mL of
pH of 9.0, and dilute with water to 1 L. water, adjust with phosphoric acid to a pH of 8.0, and
Solution C: 0.5 mg/mL of 9-fluorenylmethyl dilute with water to volume.
thloroformate in acetonitrile. [Nora-Prepare this solution Mobile phase: 0.05 M buffer, acetonitrile, and methanol
fresh.] (76:19:5)
Standard stock solution: USP Alendronate Sodium RS in Standard stock solution: Prepare a solution of USP
Medium to make a concentration equivalent to dissolving Alendronate Sodium RS in Medium with a final
1 Tablet in 900 mL of the same Medium. Calculate the concentration corresponding to the concentration

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122 Alendronate / Official Monographs USP 43

obtained by dissolving 1 tablet in 900 mL of Medium. DEFINITION

Calculate the concentration, C (mg/mL), of anhydrous Alfentanil Hydrochloride contains NLT 98.0% and NMT
alendronate sodium in this solution. 102.0% of alfentanil hydrochloride (C21H32N603. HCI),
Standard solution: Transfer 5.0 mL of the Standardstock calculated on the anhydrous basis.
solution to a 50-mL screw-cap polypropylene centrifuge [CAUTION-Handle Alfentanil Hydrochloride with great
tube containing 1.0 mL of 0.6 M citrate buffer and 5.0 mL care because it is a potent opioid analgesic. Great care
of Solution B, and mix for about 3 min. Add 4.0 mL of should be taken to prevent inhaling particles of
Solution C, and agitate for about 30 s. Allow the solution Alfentanil Hydrochloride and exposing the skin to it.]
to stand at room temperature for about 30 min. Add 25
mL of methylene chloride, and agitate vigorously for IDENTIFICATION
about 40 s. Centrifuge the mixture for 10 min. Use a
portion of the clear upper aqueous layer.
Blank: Using 5 mL of water, proceed as directed for the
Standardsolution, beginning with "to a 50-mL screw-cap ·A
polypropylene centrifuge tube". $pg ZQ)

Sample solution • B. The retention time of the major peak of the Sample
For Tablets labeled to contain S mg, 10 mg, 3S mg, or solution corresponds to that of the Standardsolution, as
40 mg: After 30 min, withdraw 30 rnl, of the solution obtained in the Assay.
under test, and passthrough a suitable OAS-J.lm filter, ASSAY
discarding the first 10 mL. Using 5.0 mL of the filtrate, • PROCEDURE
proceed as directed for the Standardsolution, beginning Mobile phase: Acetonitrile and 0.01 M
with "to a 50-mL screw-cap polypropylene centrifuge tetrabutylammonium hydrogen sulfate (14:86)
tube". Standard solution: 0.54 mg/mL of USP Alfentanil
For Tablets labeled to contain 70 mg: After 30 min, Hydrochloride RS in Mobile phase
withdraw 30 mL of the solution under test, and pass Sample solution: 0.54 mg/mL of Alfentanil Hydrochloride
through a suitable 0.45-J.lm filter, discarding the first 10 in Mobile phase
mL. Transfer 6.0 mL of the filtrate to a 1O-mLvolumetric Chromatographic system
flask, and dilute with water to volume. Using 5.0 mL of (See Chromatography (621), System Suitability.)
this dilution, proceed as directed for the Standard Mode: LC
solution, beginning with "to a 50-mL screw-cap Detector: UV 235 nm
polypropylene centrifuge tube". Column: 4.0-mm x 25-cm; 5-J.lm packing L1
Chromatographic system and System suitability: Flow rate: 2 mL/min
Proceed as directed in the Assay. Injection volume: 25 J.lL
Analysis: Proceed as directed in Test 7. System suitability
Tolerances: NLT 80% (Q) of the labeled amount of Sample: Standardsolution
alendronate sodium (C4H12NNa07P2 . 3H20) is dissolved. Suitability requirements
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Column efficiency: NLT 5400 theoretical plates
requirements Tailing factor: NMT 1.3
ADDITIONAL REQUIREMENTS Relative standard deviation: NMT 0.73%
• PACKAGING AND STORAGE: Preserve in tight containers. Analysis
Store between 15° and 30°. . Samples: Standardsolution and Sample solution
Calculate the percentage of alfentanil hydrochloride
• LABELING: The labeling indicates weekly dosing where
(C21H32N603 . HCI) in the portion of Alfentanil
appropriate. When more than one Dissolution test is given,
the labeling states the test used only if Test 7 is riot used. Hydrochloride taken:
• USP REFERENCE STANDARDS (11)
USP Alendronate Sodium RS Result =(rufrs) x (Cs/Cu) x 100

ru =peak response from the Sample solution

rs = peak response from the Standardsolution
Cs =concentration of USP Alfentanil Hydrochloride RS
Alfentanil Hydrochloride in the Standardsolution (mg/mL)
Cu =concentration of Alfentanil Hydrochloride in the
N=N Sample solution (mg/mL)
,Y
HC 0 I
P~N[(N'../CH'
\

• HCI • H20 Acceptance criteria: 98.00/0-102.0% on the anhydrous

d
N 0
basis
OCH,
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1%
C21H32N603' HCI· H20 470.99
• ORGANIC IMPURITIES
C21H32N603. HCI 452.98 Mobile phase: Acetonitrile and 0.01 M
Propanamide, N-[1-[2-(4-ethyl-4,5-dihydro-5-oxo-1 H- tetrabutylammonium hydrogen sulfate (14:86)
tetrazol-1--yl)ethyl]-4-(methoxymethyl)-4-piperidinyl]- N- Standard solution: 0.54 mg/mL of USP Alfentanil
phenyl, monohydrochloride, monohydrate; Hydrochloride RS in Mobile phase
N-[1-[2-( 4-Ethyl-5-oxo-2-tetrazolin-1-yl)-ethyl]-4- Sample solution: 0.54 mg/mL of Alfentanil Hydrochloride
(methoxymethyl)-4-piperidyl]propionanilide in Mobile phase
monohydrochloride monohydrate [70879-28-6]. Chromatographic system
Anhydrous [69049-06-5]. (See'Chromatography (621), System Suitability.)
Mode: LC

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 USP 43 OfficialMonographs / Alfentanil 123

Detector: UV 235 nm Standard solution: 0.54 mg/mL of USP Alfentanil

Column: 4.6-mm x 25-cm; 5-~m packing L1 Hydrochloride RS in saline TS
Flow rate: 2 mL/min Sample solution: Equivalent to 0.50 mg/mL of alfentanil
lnlectlon volume: 25 ~L from a suitable volume of Injection in saline TS
System suitability Chromatographic system
Sample: Standardsolution (See Chromatography (621), System Suitability.)
Suitability requirements Mode: LC
Column efficiency: NLT 5400 theoretical plates Detector: UV 235 nm
Tailing factor: NMT 1.3 Column: 4.6-mm x 25-cm; 5-~m packing L1
Relative standard deviation: NMT 0.73% Flow rate: 2 mL/min
Analysis Injection volume: 25 ~L
Sample: Sample solution System suitability
Calculate the percentage of each impurity in the portion of Sample: Standardsolution
Alfentanil Hydrochloride taken: Suitability requirements
Column efficiency: NLT 5400 theoretical plates
Result = (rUlrT) x 100 Tailing factor: NMT 1.3
Relative standard deviation: NMT 1.0%
ru = peak responsefor each impurity Analysis
rr = sum of all the peak responses Samples: Standardsolution and Sample solution
Calculate the percentage of alfentanil (C2l H32N6 0 3) in the
Acceptance criteria portion of Injection taken:
Any single impurity: NMT 0.5%
Total impurities: NMT 1.0% Result =(r ulr s) x (C siC u) x (M rllM r2) X 100
SPECifiC TESTS = peak response from the Sample solution
• WATER DETERMINATION, Method I (921): NMT 4.0%
= peak response from the Standardsolution
ADDITIONAL REQUIREMENTS = concentration of USP Alfentanil Hydrochloride RS
• PACKAGING AND STORAGE: Preserve in tight containers, and in the Standardsolution(mg/mL)
store at controlled room temperature. = nominal concentration of alfentanil in the Sample
• USP REFERENCE STANDARDS (11) solution (mg/mL)
USP Alfentanil Hydrochloride RS M rl = molecular weight of alfentanil, 416.52
M r2 = molecular weight of alfentanil hydrochloride,
452.98

Acceptance criteria: 90.0%-110.0%

Alfentanillnjection
IMPURITIES
DEfiNITION • ORGANIC IMPURITIES
Alfentanil Injection is a sterile solution of Alfentanil Mobile phase: Acetonitrile and 0.01 M
Hydrochloride in Water for Injection. It contains an amount tetrabutylammonium hydrogen sulfate (14:86)
of Alfentanil Hydrochloride equivalent to NLT 90.0% and Standard solution: 0.54 mg/mL of USP Alfentanil
NMT 110.0% of the labeled amount of alfentanil Hydrochloride RS in saline TS
(C2l H32N603) · Sample solution: Equivalent to 0.50 mg/mL of alfentanil
[CAUTIoN-Handle Alfentanil Injection with great care from a suitable volume of Injection in saline TS
because it is a potent opioid analgesic.] Chromatographic system
(See Chromatography (621), System Suitability.)
IDENTIFICATION Mode: LC
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Detector: UV 235 nm
(201 ) Column: 4.6-mm x 25-cm; 5-~m packing L1
Standard solution: 0.54 mg/mL of USP Alfentanil Flow rate: 2 mL/min
Hydrochloride RS Injection volume: 25 ~L
Sample solution: 0.5 mg/mL of alfentanil in water System suitability
Chromatographic system Sample: Standardsolution
Application volume: 200 ~L Suitability requirements
Developing solvent system: Chloroform, methanol, and Column efficiency: NLT 5400 theoretical plates
formic acid (85:10:5) Tailing factor: NMT 1.3
Visualizing agent: Dragendorff's reagent Relative standard deviation: NMT 1.0%
Analysis Analysis
Samples: Standardsolution and Sample solution Sample: Sample solution
Proceed as directed in the chapter. Calculate the percentage of each impurity in the portion of
Acceptance criteria: Meets the requirements Injection taken:
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standardsolution, as Result = (r ulr T) x 100
obtained in the Assay.
ASSAY
ru = peak response of each impurity
rT = sum of all of the peaks
• PROCEDURE
Mobile phase: Acetonitrile and 0;01 M
tetrabutylammonium hydroqen sulfate (14:86) Acceptance criteria: The sum of all impurities is NMT 2.0%.

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124 Alfentanil / OfficialMonographs USP 43

SPECIFIC TESTS Mode: LC

- pH (791): 4.0-6.0 Detector: UV 254 nm
CD PARTICULATE MATTER IN INJECTIONS (788): Meets the Column: 4.6-mm x 25-cm; 5-/..Im packing II
requirements for small-volume injections Column temperature: 30°
CD BACTERIAL ENDOTOXINS TEST (85): NMT 10 USP Endotoxin Autosampler temperature: 5°
Units/mL Flow rate: 1.5 mL/min
- OTHER REQUIREMENTS: Meets the requirements in Injections Injection volume: 10 /..IL
and Implanted Drug Products (1) System suitability
Sample: Standard solution
ADDITIONAL REQUIREMENTS
Suitability requirements
CD PACKAGING AND STORAGE: Preserve in tight, single-dose or
Relative standard deviation: NMT 1.0%
multiple-dose containers, preferably of Type I glass, and Tailing factor: NMT 1.5
store at controlled room temperature.
Analysis . .
CD USP REFERENCE STANDARDS (11)
Samples: Standard solution and Sample solution
USP Alfentanil Hydrochloride RS Calculate the percentage of alfuzosin hydrochloride
(C19H27Ns04 . HCI) in the portion of Alfuzosin
Hydrochloride taken:

Alfuzosin Hydrochloride Result = (ru/rs) x (Cs/Cu) x 100

= peak response from the Sample solution
H'CO:ce;Ny~~~yO . HCI = peak response from the Standardsolution
=
I",;; ~N
H,CO 0
concentration of USP Alfuzosin Hydrochloride RS
in the Standardsolution (mg/mL)
NH, = concentration of Alfuzosin Hydrochloride in the
Sample solution (mg/mL)
C19H27Ns04 . HCI 425.91
2-Furancarboxamide, N-[3-[(4-amino":6,7-dimethoxy-2- Acceptance criteria: 98.0%-102.0% on the anhydrous and
quinazolinyl)methylamino]propyl]tetrahydro-, solvent-free basis
monohydrochloride (±);
(±)-N-[3-[(4-Amino-6, 7-dimethoxy-2-quinazolinyl) IMPURITIES
methylamino] propyl]tetrahydro-2-furamide - RESIDUE ON IGNITION (281): NMT 0.1 %
monohydrochloride [81403-68-1]. - ORGANIC IMPURITIES
Buffer: Dilute 5 mL of perchloric acid in 900 mL of water.
DEFINITION
Adjust with 2 M sodium hydroxide to a pH of 3.5, and dilute
Alfuzosin Hydrochloride contains NLT 98.0% and NMT with water to 1 L.
102.0% of alfuzosin hydrochloride (C19H27Ns04 . HCI), Mobile phase: Acetonitrile, tetrahydrofuran, and Buffer
calculated on the anhydrous and solvent-free.basis. (20:1 :80)
IDENTIFICATION System suitability solution: 0.4 mg/mL of USP Alfuzosin
System Suitability Mixture RS in Mobilephase
Sample solution: 0.40 mg/mL of Alfuzosin Hydrochloride in
Mobilephase
- A. Reference solution: 0.40 /..Ig/mL of Alfuzosin
~j:J Hydrochloride in Mobilephase from the Sample solution
-B. IDENTIFICAnON TESTs-GENERAL, Chloride (191): Meets Chromatographic system
the requirements (See Chromatography (621), System Suitability.)
- C. The retention time of the Sample solution corresponds to Mode: LC
that of the Standardsolution, as obtained in the Assay. Detector: UV 254 nm
Column: 4.6-mm x 15-cm; 5-/..Im packing L1
ASSAY Flow rate: 1.5 mL/min
CD PROCEDURE
Injection volume: 10 /..IL
[NOTE-Use low-actinic glassware.] System suitability
Solution A: 2 M sodium hydroxide Sample: System suitability solution
Solution B: 5.0 mL of perchloric acid in 900 mL of water. Suitability requirements
Adjust with Solution A to a pH of 3.5, and dilute with water Peak-to-valley ratio: The ratio of the height of the
to 1000 mL. furamide analog peak to the height of the valley between
Mobile phase; Acetonitrile and Solution B (30:70) the furamide analog peak and alfuzosin is NLT 5.
Diluent: Methanol and Solution A (500:3) Analysis
Standard stock solution: 0.5 mg/mL of USP Alfuzosin Samples: Sample solution and Reference solution
Hydrochloride RS in Diluent Calculate the percentage of each impurity in the portion of
Standard solution: 0.04 mg/ml of USP Alfuzosin Alfuzosin Hydrochloride taken:
Hydrochloride RS in Mobile phasefrom the Standardstock
solution; the solution is stable for 14 h at 5°. Result = (rulrs) x (1/0) x 100
Sample stock solution: 0.5 mg/mL of Alfuzosin
Hydrochloride in Diluent rv = peak response of each impurity from the Sample
Sample solution: 0.04 mg/mL of Alfuzosin Hydrochloride in solution
Mobile phase from the Sample stock solution; the solution is ts = peak response of alfuzosin from the Reference
stable for 14 h at 5°. solution
Chromatographic system . D =dilution factor between the Sample solution and
(See Chromatography (621), System Suitability.) the Reference solution, 1000

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 USP 43 Official Monographs / Alfuzosin 125

Acceptance criteria: See Table 7. [NOTE-Disregard any a phase separation filter. Take 2.0 mL of the dried organic
peak with an area less than 0.05%.] solution, and mix with 200 mg of finely ground potassium
bromide. Evaporate the methylene chloride at 60°, then at
Table 1 105° for 30 min. Make a disk. Alternatively, evaporate
Relative Acceptance methylene chloride from the dried organic solution at 60°,
Retention Criteria, then at 105° for 30 min. Perform the IR spectrum.
Name Time NMT(%) Acceptance criteria: The maxima of the spectrum obtained
Deacylatedalfuzosln" 0.5 0.20 from the Sample correspond in position and relative
intensity to those obtained from USP Alfuzosin
Alfuzosin 1.0 - Hydrochloride RS, treated in the same manner as the
Furamide ana10gb 1.2 _c Sample, beginning with "add 20 mL of water."
• B. The retention time of the major peak of the Sample
Anyother individual, uniden- solution corresponds to that of the Standardsolution, as
tifiedimpurity - 0.10 obtained in the Assay.
Totalimpurities - 0.30
ASSAY
a Nl-(3-Aminopropyl)-6,7.dimethoxy-N2-methylquinazoline-2,4.diamine. • PROCEDURE
b N-{3-[( 4-Amino-6,7-dimethoxyquinazolin-2.yl)(methyl)amino]propyl}furan- Solution A: 5.0 mL of perchloric acid in 900 ml of water.
2-carboxamide. Adjust with 2 M sodium hydroxide to a pH of 3.5, and dilute
C Furamide analog, a component of USP Alfuzosin SystemSuitability Mixture with water to 1000 mL.
RS, is not a specified impurity. Mobile phase: Acetonitrile, tetrahydrofuran, and Solution A
(20:1 :80)
SPECIFIC TESTS Diluent: 0.01 N hydrochloric acid
• OPTICAL ROTATION (781) Standard stock solution: 0.15 mg/mL of USP Alfuzosin
Sample solution: 20 mg/mL in carbon dioxide-free water Hydrochloride RS in methanol .
Acceptance criteria: -0.10° to +0.10° Standard solution: 0.03 mg/mL of USP Alfuzosin
• WATER DETERMINATION, Method 1(921): NMT2.0% Hydrochloride RS in Diluentfrom the Standardstock solution
• pH (791) Sample stock solution: Place a suitable number of Tablets
Sample solution: 20 mg/mL in carbon dioxide-free water into a suitable volumetric flask to obtain a solution having
Acceptance criteria: 4.0-5.5 a concentration of 0.16 mg/mL of alfuzosin hydrochloride.
ADDITIONAL REQUIREMENTS Add 80% of the flask volume of methanol, and stir for at
• PACKAGING AND STORAGE: Preserve in tight containers. least 1 h using a magnetic stirrer. Add 10% of the flask
Protect from light and humidity, and store at room volume of Diluent, mix, and allow it to cool to room
temperature. temperature. Dilute the resulting suspensionwith methanol
• USP REFERENCE STANDARDS (11) to volume, stir, and allow to settle for 30 min.
USP Alfuzosin Hydrochloride RS Sample solution: 0.03 mg/mL of alfuzosin hydrochloride in
USP Alfuzosin System Suitability Mixture RS Diluent from the Sample stock solution supernatant. Pass
Alfuzosin Hydrochloride containing approximately 0.4% through a suitable filter.
furamide analog (N-{3-[(4-amino-6,7- Chromatographic system
dimethoxyquinazolin-2-yl)(methyl)amino]propyl}furan- (See Chromatography (621), System Suitability.)
2-carboxamide), and about 0.4% deacylated alfuzosin Mode: LC
(N2-(3-aminopropyl)-6,7-dimethoxy-N2- . Detector: UV 254 nm
methylquinazoline-2,4-diamine). Column: 4.6-mm x 15-cm; 5-l.lm packing L1
Flow rate: 1.5 mL/min
Injection volume: 20 I.lL
System suitability
Sample: Standardsolution
Alfuzosin Hydrochloride Suitability requirements
Tailing factor: 0.8-1.5 for alfuzosin
Extended-Release Tablets Relative standard deviation: NMT 2.0%
Analysis
DEFINITION Samples: Standardsolution and Sample solution
Alfuzosin Hydrochloride Extended-Release Tablets contain Calculate the percentage of the labeled amount of alfuzosin
NLT 90.0% and NMT 110.0% of the labeled amount of hydrochloride (C19H27Ns04' HCI) in the portion of Tablets
alfuzosin hydrochloride (C19H27Ns04 . HCI).
taken:
IDENTIFICATION
Result =(rulrs) x (CsICu) x 100

ru = peak responsefrom the Sample solution

.~~ ~'.~.~.~
§P~(i~'i9§g;
.......•

Sample: Grind 4 Tablets, and add 20 mL of water.

c
ts
Cs
=peak responsefrom the Standardsolution
= concentration of USP Alfuzosin Hydrochloride RS
in the Standardsolution(mg/mL)
[NoTE-When analyzing multi-layer Tablets, isolate the layer Cu =nominal concentration of the Sample solution
containing alfuzosin hydrochloride using a suitable tool.] (mg/mL)
Add 20 mL of strong ammonia solution. Extract with 20 mL
of methylene chloride, and separate the organic layer. Acceptance criteria: 90.00/0-110.0%
Repeat the extraction successively with 20 mL, then with
10 mL of methylene chloride. Wash the combined organic
layers with 20 mL of water. Dry th~ organic solution using

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126 Alfuzosin / OfficialMonographs USP 43

PERFORMANCE TESTS Table 1 (continued)

• DISSOLUTION (711) Time Amount Dissolved
Test 1 Level (h) (%)
Medium: 0.01 N hydrochloric acid; 500 mL Average of 24 Tablets complies with L1, NMT
Apparatus 2: 100 rpm, with Tablet holder (see Figure 7. 2 Tablets outside L2, and all Tablets within:
37.5-mm (I) x 20-mm (d) stainless steel cylinders are used
as sample holders. The cylinders contain screw caps drilled. 1 8-24
with seven 4.5-mm holes. Seven 4.5-mm holes are drtlled In 6 32-66
the bottom, and 72 longitudinalseries of five s-mm holes are
drilledon the cylinders, alternatively starting and endingwith 12 52-102
one 7.7-mm hole.) L3 20 NLT68

Test 2: If the product complies with this test, the labeling

indicates that it meets USP Dissolution Test 2.
Medium: 0.01 N hydrochloric acid; 900 mL
Apparatus 2: 100 rpm
Times: 1, 3, 12, and 24 h
Buffer: Dilute 5.0 mL of perchloric acid in 900 mL of
water, adjust with diluted sodium hydroxide (0.1 g/mL)
to a pH of 3.5 ± 0.5, and dilute with water to 1 L.
Mobile phase: Acetonitrile and Buffer (25:75) .
Standard stock solution: 0.28 mg/mL of USP Alfuzosln
Hydrochloride RS, prepared as follows. In a 200-!T1L
volumetric flask dissolve 55.5 mg of USP Alfuzosln
Hydrochloride RS in 5 mL of methanol, sonicate to-
dissolve, and dilute with Medium to volume.
Standard solution: 0.011 mg/mL of USPAlfuzosin
Hydrochloride RS in Medium from the Standardstock
solution
Sample solution: Pass a portion of the sol~tion unde~ test
through a suitable filter. Replace the port.lon of solution
Figure 1. 37.5-mm (I) x 20-mm (d) stainless steel cylinders withdrawn with an equal volume of Medium.
are used as sample holders. The cylinders contain screw Chromatographic system
caps drilled with seven 4.5-mm holes. Seven 4.5-mm (See Chromatography (621), System Suitability.)
holes are drilled in the bottom, and 12 longitudinal series Mode: LC
of five 5-mm holes are drilled on the cylinders, Detector: UV 244 nm
alternatively starting and ending with one 1.7 -rnrn hole. Column: 4.6-mm x 15-cm; 5-lJm packing L7
Column temperature: 30°
Times: 1, 6, 12, and 20 h Flow rate: 1.2 mL/min
Sample solution: Pass a portion of the solution under test Injection volume: 20 IJL
through a suitable filter. System suitability
Standard solution: (LI500) mg/mL of USPAlfuzosin Sample: Standardsolution
Hydrochloride RS in Medium, where L is the Tablet label Suitability requirements
claim in mg Tailing factor: NMT 2.0
Detector: UV 330 nm Column efficiency: NLT 3000 theoretical plates
Blank: Medium Relative standard deviation: NMT 2.0%
Path length: 1 cm Analysis
Tolerances: See Table 7. Samples: Standardsolution and Sample ~olution .
Calculate the concentration (C/) of alfuzosln hydrochloride
Table 1
(C,9H27Ns04' HCI) in the sample withdrawn from the
Time Amount Dissolved vessel at each time point (I):
Level (h) (%)
Each Tablet: Result, =(rvirs) x Cs
1 10-20
= peak response from the Sample solution
6 40-55 = peak response from the Standardsolution
12 65-85 =concentration of USP Alfuzosin Hydrochloride RS
in the Standardsolution (mg/mL)
L1 20 NLT85
Average of 12 Tablets complies with L1 and
Calculate the percentage of the labeled amount of
each Tablet: alfuzosin hydrochloride (C19H27Ns04' HCI)dissolved at
each time point (I):
1 9-22
6 36-61 Result, = C7 x Vx(1IL) x 100
12 59-94 Result, = [(C2 x V) + (C7 x Vs)] x (lIL) x 100
L2 20 NLT77
Result, = {(C3 x V) + [(C2 + C7) x Vs]} x (lIL) x 100

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USP 43 OfficialMonographs / Alfuzosin 127

Result, = {(C4 X V) + [(C3 + C2 + C1) x Vs]} x (lIL) x 100 Table 3

Amount
= concentration of alfuzosin hydrochloride in the Time Point Time Dissolved
portion of sample withdrawn at the specified (i) (h) (%)
time point (mg/mL) 1 1 NMT15
V = volume of Medium, 900 mL
L = label claim (mg/Tablet) 2 6 20-40
Vs = volume of the Sample solution withdrawn at each 3 12 45-70
time point and replaced with Medium (mL)
4 24 NLT80
Tolerances: See Table 2.
The percentages of the labeled amount of alfuzosin
Table 2 hydrochloride dissolved at the times specified conform
Amount to Dissolution (711), Acceptance Table 2.
Time Point Time Dissolved Test 4: If the product complies with this test, the labeling
(I) (h) (%) indicates that it meets USP Dissolution Test 4.
1 1 NMT20 Medium: 0.01 N hydrochloric acid; 900 mL
Apparatus 2: 100 rpm
2 3 15-35 Times: 1, 6, 12, and 20 h
3 12 50-70 Standard solution: 0.01 mg/mL of USP Alfuzosin
Hydrochloride RS in Medium
4 24 NLT80
Sample solution: Centrifuge a portion of the solution
under test.
The percentages of the labeled amount of alfuzosin Instrumental conditions
hydrochloride dissolved at the times specifiedconform (See Ultraviolet-Visible Spectroscopy (857).)
to Dissolution (711), Acceptance Table 2. Mode: UV
Test 3: Ifthe product complies with this test, the labeling Analytical wavelength: 245 nrn
indicates that it meets USP Dissolution Test 3. Blank: Medium
Medium: 0.25% sodium dodecyl sulfatein 0.05 M sodium Path length: 0.2 cm
phosphate buffer, pH 6.8 (2.5 gIL of sodium dodecyl Analysis
sulfate, 6.9 gIL of monobasic sodium phosphate Samples: Standard solution and Sample solution
monohydrate, and 0.83 gIL of sodium hydroxidein water Calculatethe concentration (Cj) of alfuzosin hydrochloride
previously degassed with helium. Adjustwith either (C'9H27Ns04 . HCI) in the sample withdrawn from the
phosphoric acid or 1 N sodium hydroxide to a pH of 6.8 vessel at each time point (I):
± 0.05); 900 mL
Apparatus 1: 100 rpm Result, = (AulAs) x Cs
Times: 1,6, 12, and 24 h .
Standard stock solution: 1.1 mg/mL of USP Alfuzosin Au = absorbance of the Sample solution
Hydrochloride RS in methanol As = absorbance of the Standard solution
Standard solution: 0.011 mg/mL of USP Alfuzosin Cs = concentration of USP Alfuzosin Hydrochloride RS
Hydrochlorlde RS in Medium from the Standard stock in the Standard solution(mg/mL)
solution .
Sample solution: Passa portion of the solution under test Calculate the percentage of the labeledamount of
through a suitable filter. alfuzosin hydrochloride (C,9H27Ns04· HC!) dissolved at
Instrumental conditions each time point (I):
(See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis Result, = C1 x Vx (lIL) x 100
Analytical wavelength: 331 nm, background correction
at 490 nm Result, = {[C2 x (V - Vs)] + (C1 x Vs)} x (lIL) x 100
Blank: Medium
Cell: 1.0 cm Result, = ({C3 x [V - (2 x Vs)]} + [(C2 + C1) x Vs]) x (1I L) x
Analysis 100
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of Result, = ({C4 x [V - (3 x Vs)]} +[(C3 + C2 + C1) x Vs]) x (1I
alfuzosin hydrochloride (C'9H27Ns04 . HCI) dissolved at L) x 100
each time point (I):
c, = concentration of alfuzosin hydrochloride in the
Result, = (AulAs) x Cs x V x (1I L) x 100 portion of sample withdrawn at the specified
time point (mg/mL)
= absorbance of the Sample solution V = volume of Medium, 900 mL
= absorbance of the Standard solution L = label claim (mg/Tablet)
= concentration of USP Alfuzosin Hydrochloride RS Vs = volume of the Sample solution withdrawn at each
in the Standard solution (mg/mL) time point (mL)
V = volume of Medium (mL)
L = label claim (mg/Tablet) Tolerances: See Table 4.

Tolerances: See Table 3.

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128 Alfuzosin / OfficialMonographs USP 43

Table 4 Result, =({C3 x [V - (2 x Vs)]} + [(C2 + C1) x Vs]) x (lIL) x

Amount 100
Time Point Time Dissolved
(I) (h) (%) Result, = ({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C1) x Vs]) ~ (1I
1 1 NMT30 L) x 100
2 6 40-60 Results =({Cs x [V - (4 x Vs)]} + [(C4 + C3 + C2 + C1) x Vs]
3 12 65-85 ) x (1I L) x 100
4 20 NLT 80 c, = concentration of alfuzosin hydrochloride in the
portion of sample withdrawn at the specified
The percentages of the labeled amount of alfuzosin time point (mg/mL)
hydrochloride dissolved at the times specified conform V = volume of Medium, 900 mL
to Dissolution (711), Acceptance Table 2. L = label claim (mg/Tablet)
TestS: If the product complies with this test, the labeling v, =volume of the Sample solution withdrawn at each
indicates that it meets USP Dissolution Test 5. time point (mL)
Medium: 0.01 N hydrochloric acid; 900 mL
Apparatus 2: 100 rpm Tolerances: See Table 5.
Times: 1, 3, 6, 12, and 20 h
Buffer: Add 1 mL of triethylamine in 1000 mL of water. Table 5
Adjust with phosphoric acid to a pH of 2.5 ± 0.05. Pass Amount
through a suitable filter of 0.45-~m pore size. TIme Point Time Dissolved
Mobile phase: Methanol and Buffer (40:60) (I) (h) (%)
Standard stock solution: 0.55 mg/mL of USP Alfuzosin 1 1 NMT20
Hydrochloride RS. Prepare by transferring a portion of USP
Alfuzosin Hydrochloride RS to a suitable flask. Add 2 3 20-40
methanol to 20% of the flask volume, and sonicate at 3 6 35-55
room temperature to dissolve. Dilute with Medium to
volume. 4 12 60-80
Standard solution: 0.011 mg/mL of USP Alfuzosin 5 20 NLT80
Hydrochloride RS in Medium from the Standardstock
solution. Pass through a suitable filter of 0.45-~m pore size. The percentages of the labeled amount of alfuzosin
Sample solution: Pass a portion of the solution under test hydrochloride dissolved at the times specified conform
through a suitable filter of O.4S-~m pore size. to Dissolution (711), Acceptance Table 2.
Chromatographic system Test 6: If the product complies with this test, the labeling
(See Chromatography (621), System Suitability.) indicates that it meets USP Dissolution Test 6.
Mode: LC Medium: 0.01 N hydrochloric acid; 900 mL
Detector: UV 245 nm Apparatus 2: 100 rpm. Adjust the paddle height to 4.5 cm
Column: 4.6-mm x 15-cm; 5-~m packing L1 above the bottom of the vessel and use a 10# mesh basket
Flow rate: 1 mL/min as sinker.
Injection volume: 10 ~L Times: 1, 6, 12, and 20 h
System suitability Buffer: 2.3 giL of anhydrous dibasic sodium phosphate
Sample: Standard solution and 1.75 giL of monobasic potassium phosphate
Suitability requirements Mobile phase: Acetonitrile and Buffer (50:50)
Tailing factor: NMT 2.0 Standard stock solution: 0.45 mg/mL of USP Alfuzosin
Column efficiency: NLT 2000 theoretical plates Hydrochloride RS. Prepare by transferring a portion of USP
Relative standard deviation: NMT 2.0% Alfuzosin Hydrochloride RS to a suitable flask. Add 20% of
Analysis the flask volume of water. Sonicate to dissolve, and-
Samples: Standard solution and Sample solution dilute with Medium to volume.
Calculate the concentration (Cj) of alfuzosin hydrochloride Standard solution: 0.011 mg/mL of USPAlfuzosin
(C'9H27Ns04' HCI) in the sample withdrawn from the Hydrochloride RS in Medium from the Standard stock
vessel at each time point (I): solution
Sample solution: Pass a portion of the solution under test
Result, = (rulrs) x Cs through a suitable filter. Replace with the same volume of
Medium.
= peak response from the Sample solution Chromatographic system
= peak response from the Standard solution (See Chromatography (621), System Suitability.)
=concentration of USP Alfuzosin Hydrochloride RS Mode: LC
in the Standard solution(mg/mL) Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-~m packing L1
Calculate the percentage of the labeled amount of Column temperature: 30°
alfuzosin hydrochloride (C'9H27Ns04' HCI) dissolved at Flow rate: 1 mL/min
each time point (I): Injection volume: 20 ~L
System SUitability
Result, = C1 x VX (lIL) x 100 Sample: Standard solution
Suitablllty requirements
Result2'= {[C2 x (V- Vs)] + (C1 x Vs)} x (lIL) x 100 Tailing factor: NMT 2.0
Column efficiency: NLT 3500 theoretical plates
Relative standard deviation: NMT 2.0%

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USP 43 Official Monographs / Alfuzosin 129

Analysis Detector: UV 245 nm

Samples: Standard solution and Sample solution Column: 4.6-mm x 5-cm; 3-lJm packing L7
Calculate the concentration (C;) of alfuzosin hydrochloride Flow rate: 1.5 mL/min
(C19H27Ns04' HCI) in the sample withdrawn from the Injection volume: 10 I.lL
vessel at each time point (I): Run time: NLT 1.5 times the retention time of alfuzosin
System suitability
Result; = (rulr s) x Cs Sample: Standard solution
Suitability requirements
ru =peak response from the Sample solution Tailing factor: NMT 1 .8
rs = peak response from the Standard solution Relative standard deviation: NMT 2.0%
Cs = concentration of USP Alfuzosin Hydrochloride RS Analysis
in the Standard solution (mg/mL) Samples: Standard solution and Sample solution
Calculate the concentration (C;) of alfuzosin hydrochloride
Calculate the percentage of the labeled amount of (C19H27Ns04' HCI) in the sample withdrawn from the
alfuzosin hydrochloride (C19H27Ns04 . HCI) dissolved at vessel at each time point (I):
each time point (I):
Result; = (rulrs) x Cs
Result, = C7 x VX OIL) x 100
= peak responsefrom the Sample solution
Result, = [(C2 x V) + (C7 x Vs)] x (lIL) x 100 =peak response from the Standard solution
= concentration of USP Alfuzosin Hydrochloride RS
Result, = {(C3 x V) + [(C2 + C7) x Vs]} x (lIL) x 100 in the Standard solution (mg/mL)

Result, = {(C4 x V) + [(C3 + C2 + C7) x Vs]} x (lIL) x 100 Calculate the percentage of the labeled amount of
alfuzosin hydrochloride (C19H27Ns04 . HCI) dissolved at
C; = concentration of alfuzosin hydrochloride in the each time point (I):
portion of sample withdrawn at the specified
time point (mg/mL) Result, = C7 x V x (1 I L) x 100
V = volume of Medium, 900 mL
L = label claim (mg/Tablet) Result, = {[C2 x (V - Vs)] + (C7 x Vs)} x (lIL) x 100
Vs =volume of the Sample solution withdrawn at each
time point and replaced with Medium (mL) Result, = ({C3 x [V - (2 x Vs)]} + [(C2 + C7) x Vs]) x (1I L) x
" 100
Tolerances: See Table 6.
Result, =({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C7) x Vs]) x (1I
Table 6 L) x 100
Amount
Time Point Time Dissolved C; = concentration of alfuzosin hydrochloride in the
(I) (h) (%) portion of sample withdrawn at the specified
1 1 NMT30 time point (mg/mL)
= volume of Medium, 900 mL
2 6 " 45-65 = label claim (mg/Tablet)
3 12 70-90 = volume of the Sample solution withdrawn at each
time point (mL)
4 20 NLT85
Tolerances: See Table 7.
The percentages of the labeled amount of alfuzosin
hydrochloride dissolved at the times specified conform Table 7
to Dissolution (711), Acceptance Table 2. Amount
Test 7: If the product complies with this test, the labeling Time Point Time Dissolved
indicates that it meets USP Dissolution Test 7. (i) (h) (%)
Medium: 0.01 N hydrochloric acid; 900 mL 1 1 NMT25
Apparatus 2: 100 rpm with sinker; see Dissolution (711),
Figure 20. 2 6 40-60
Times: 1, 6, 12, and 20 h 3 12 65-85
Solution A: 500 glL of sodium hydroxide
4 20 NLT 85
Solution B: 100 giL of sodium hydroxide
Buffer: Add 5 mL of perchloric acid in 1000 mL of water.
Adjust with Solution A to a pH of 2.5, then adjust with The percentages of the labeled amount of alfuzosin
Solution B to a pH of3.50 ± 0.05. Pass through a suitable hydrochloride dissolved at the times specified conform
filter of 0.45-l.lm pore size. . to Dissolution "(711), Acceptance Table 2.
Mobile phase: Acetonitrile and Buffer (24: 76) • UNIFORMITY OF DOSAGE UNITS (90S): Meet the
Standard solution: 0.01 mg/mL of USP Alfuzosin requirements
Hydrochloride RS in Medium
IMPURITIES
Sample solution: Centrifuge or pass a portion of the
• ORGANIC IMPURITIES
solution under test through a suitable filter.
Chromatographic system Solution A, Mobile phase, Diluent, Sample solution, and
(See Chromatography (621), System Suitability.) Chromatographic system: Proceed as directed in the
Mode: LC Assay.

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 130 Alfuzosin / OfficialMonographs USP 43

System suitability stock solution: 0.4 mg/mL of USP Deacylated alfuzosin: N2-(3-Aminopropyl)-6, 7-
Alfuzosin System Suitability Mixture A RS in methanol dimethoxy-N2-methylquinazoline-2,4-diamine.
System suitability solution: 0.03 mg/mL of USP Alfuzosin C14H21N S0 2 291.35
System Suitability Mixture A RS in Diluent from the System N-Formyl analog: N-[3-[(4-Amino-6,7-
suitability stock solution dimethoxyquinazolin-2-yl)(methyl)amino]propyl]
Standard stock solution: 0.15 mg/mL of USP Alfuzosin formamide.
Hydrochloride RS in methanol ClsH21Ns03 319.36
Standard solution: 0.03 mg/mL of USP Alfuzosin
Hydrochloride RS in Diluentfrom the Standard stocksolution
System sultablllty
Samples: System suitability solution and Standard solution
Suitability requirements Anantoin
Resolution: NLT 1.0 between alfuzosin and the furamide
analog; NLT 1.0 between deacylated alfuzosin and the o O~NH
N-formyl analog, System suitability solution HN~NA)::::O
Relative standard deviation: NMT 2.0%, Standard 2 H H

solution
Analysis C4H6N 403 158.12
Samples: Standard solution and Sample solution Urea, (2,5-d ioxo-4-i midazolid inyl)-;
Calculate the percentage of each impurity in the portion of Allantoin [97-59-6].
Tablets taken: DEFINITION
Allantoin contains NLT 98.5% and NMT 101.0% of C4H 6N 40 3.
Result = (ru/rs) x (CslCu) x 100
IDENTIFICATION
ru = peak response of each impurity from the Sample
solution
rs = peak response of alfuzosin from the Sample
solution '. A·~~~~~"'~P~~~~I~<I~
Cs =concentration of USP Alfuzosin Hydrochloride RS .spi\'c;tr0.sc;Opy:rl?lKA(CN>lt 2Q'
in the Standard solution (mg/mL) • B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Cu =nominal concentration of alfuzosin hydrochloride (201): The RF value of the principal spot from Sample
in the Sample solution (mg/mL) solution B corresponds to that from Standard solution A, as
described in the test for Organic Impurities.
Acceptance criteria: See Table 8
ASSAY
Table 8 • PROCEDURE
Relative Acceptance
Sample: 120 mg
Retention . Criteria, Analysis: Transfer the Sample to a 1OO-mL beaker, dissolve
Name Time NMT (%) by stirring in 40 mL of water, and titrate with 0.1 M sodium
Deacylated alfuzosln- 0.40
hydroxide. Use a suitable electrode system (see Titrimetry
0.46
(541». Each mL of 0.1 M sodium hydroxide is equivalent
N-Formyl analoq" 0.50 0.30 to 15.81 mg of C4H 6N 403.
Alfuzosin 1.0 - Acceptance criteria: 98.50/0-101.0%
Furamide analoq- 1.18 _d IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1 %
Any individual unspecified impur-
ity - 0.20
• ORGANIC IMPURITIES
Adsorbent: Cellulose
Total impurities - 0.80 Diluent: Methanol and water (1:1)
Urea stock solution: 1 _mg/mL of USP Urea RS in water
a NZ-(3-Aminopropyl)-6,7-dimethoxy-N2-methylquinazoline-2,4-diamine. Standard solution A: 1 mg/mL of USP AllantoinRS in
b N-[3-[( 4-Amino-6,7-dimethoxyquinazolin-2-yl)(methyl)amino]propyl] Diluent
formamide.
c N-{3-[( 4-Ami no-6,7 -dimethoxyqu inazolin-2-yl)(methyl)amino]propyl}furan-
Standard solution B: 0.1 mg/mL of USP Urea RS in ,/
2-carboxamide. methanol, from Urea stock solution
d Furamide analog, a component of USP Alfuzosin System Suitability Mixture A Standard solution C: Standard solution A and Standard
RS, is not a specified impurity. solution B (1:1)
Sample solution A: Transfer 0.10 9 of Allantoin to a 10-mL
ADDITIONAL REQUIREMENTS volumetric flask,add 5 mL of water, dissolve by heating,
• PACKAGING AND STORAGE: Protect from light and moisture. and allow to cool. Dilute with methanol to volume.
Store at controlled room temperature. - [NOTE-Use immediately after preparation.]
• LABELING: When more than one Dissolution test is given, the Sample solutionB: Transfer 1 mL of Sample solution A to a -
labeling states the Dissolution test used only if Test 1 is not 1O-mL volumetric flask, and dilute with Diluent to volume.
used. . Spray reagent: 5 mg/mL of p-dimethylaminobenzaldehyde
• USP REFERENCE STANDARDS (11) in a mixture of methanol and hydrochloric acid (3:1)
USP Alfuzosin Hydrochloride RS Application volume
USP Alfuzosin System Suitability Mixture A RS Standard solution A: 5 IJL
Furamide analog: N-{3-[(4-Amino-6,7- Standard solution B: 5 IJL
dimethoxyquinazolin-2-yl)(methyl)amino]propyl}furan- Standard solution C: 5 IJL
2-carboxamide. Sample solution A: 10 IJL
C19H23Ns04 385.42 Sample solution B: 5 IJL

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 USP 43 Official Monographs / Allopurinol 131

Developing solvent system: Butyl alcohol, glacial acetic and USP Allopurinol Related Compound C RS, prepared as
acid, and water (60:15:25) follows. Transfer weighed quantities of USP Allopurinol RS,
Analysis: Proceed as directed for Chromatography (621), USPAllopurinol Related Compound B RS, and USP
Thin-Layer Chromatography. Develop the chromatogram Allopurinol Related Compound C RS to three separate
until the solvent front has moved about 10 em. Spray the suitable volumetric flasks, dissolve in a small volume of 0.1
plate with Spray reagent, dry in a current of hot air, and after N sodium hydroxide, and immediately dilute with Mobile
30 min examine under visible light. phase to volume to obtain solutions containing 0.05
Acceptance criteria: Any spot from Sample solution A, mg/ml each. Transfer 1.0 ml of each of these three
except for the principal spot, is not more intense than the solutions to a 1OO-mL volumetric flask and dilute with
spot from Standardsolution B (NMT 0.5%). The test is'not Mobile phase to volume.
valid unless the principal spots from Standardsolution Care Standard stock solution: 0.5 mg/ml of USP Allopurinol
clearly separated. RS, prepared asfollows. Transfer a weighed quantity of USP
Allopurinol RS to a suitable volumetric flask, dissolve in a
SPECIFIC TESTS small volume of 0.1 N sodium hydroxide, and immediately
• ACIDITY OR ALKALINITY dilute with Mobilephase to volume.
Sample solution: 5 mg/mL in carbon dioxide-free water Standard solution: 0.08 mg/ml of USP Allopurinol RS in
Analysis: To 5 mL of the Sample solution add 5 mL of water, Mobile phase from the Standardstock solution
0.1 mL of methyl red TS, and 0.2 mL of 0.01 M sodium Sample stock solution: 0.5 mg/ml of Allopurinol, prepared
hydroxide. asfollows. Transfer 50 mg of Allopurinol to a 100-mL
Acceptance criteria: A yellow color is observed. The volumetric flask, dissolve in 5.0 ml of 0.1 N sodium
solution turns red upon the addition of 0.4 mL of 0.01 M hydroxide, and immediately dilute with Mobilephase to
hydrochloric acid. volume.
• loss ON DRYING (731): Dry a sample at 105° to constant Sample solution: 0.08 mg/mL of Allopurinol in Mobile
weight: it loses NMT 0.1% of its weight. phase from the Sample stock solution
• REDUCING SUBSTANCES Chromatographic system
Sample solution: 1.0 g of Allantoin in 10 mL of water. Shake (See Chromatography (621), System Suitability.)
for 2 min, and filter. Mode: LC
Analysis: To the Sample solution add 1.5 mL of 0.02 M Detector: UV 230 nm
potassium permanganate. Column: 4.6-mm x 25-cm; packing II
Acceptance criteria: The solution remains violet for at least Flow rate: 1.8 mL/min
10 min. Injection volume: 20 ~L
ADDITIONAL REQUIREMENTS System suitability
• USP REFERENCE STANDARDS (11) Samples: System suitability solution and Standardsolution
USP Allantoin RS [NOTE-The relative retention times for allopurinol
USP Urea RS related compound B, allopurinol related compound
C, and allopurinol are about 0.7, 0.8, and 1.0,
respectively.]
Suitability requirements
Resolution: NlT 1.1 between allopurinol related
Allopurinol compound B and allopurinol related compound C; NLT
6.0 between allopurinol related compound C and
o allopurinol, System suitability solution
HN~N
~. Jl.~
Relative standard deviation: NMT 2.0% for replicate
injections, Standardsolution
N N
H
Analysis
Samples: Standardsolution and Sample solution
CSH4N 40 136.11 Calculate the percentage of allopurinol (CSH4N4 0 ) in the
4H-Pyrazolo[3,4-d]pyrimidin-4-one, 1,5-dihydro-; portion of Allopurinol taken:
1,5-Dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one;
1H-Pyrazolo[3,4-d]pyrimidin-4-01 [315-30-0]. Result = (ru/rs) x (Cs/Cu) x 100
DEFINITION
Allopurinol contains NLT 98.0% and NMT 102.0% of ru =peak response from the Sample solution
allopurinol (CS H4N 40 ), calculated on the dried basis. rs =peak response from the Standardsolution
Cs =concentration of USP Allopurinol RS in the
IDENTIFICATION Standardsolution (mg/mL)
Cu = concentration of Allopurinol in the Sample
solution (mg/mL)

Acceptance criteria: 98.0%-102.0% on the dried basis

IMPURITIES
ASSAY • ORGANIC IMPURITIES
• PROCEDURE [NOTE-Store and inject the Standard solution and the
[NOTE-Store and inject the System suitability solution, Sample solution at 8°, using a cooled autosampler.]
Standardsolution, and Sample solution at 8°, using a Solution A: 1.25-g/L solution of monobasic potassium
cooled autosampler.] phosphate in water, filtered and degassed
Mobile phase: 1.25-g/L solution of monobasic potassium Solution B: Methanol
phosphate in water, filtered and degassed Mobile phase: See Table 7.
System suitability solution: 0.5 ~g/mL each of USP
Allopurinol RS, USP Allopurinol Related Compound B RS,

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132 Allopurinol/Official Monographs USP 43

Table 1 =concentration of Allopurinol in the Sample

Time Solution A Solution B solution (mg/mL)
(min) (%) (%)
0
Calculate the percentage of any other individual impurity
90 10
in the portion of Allopurinol taken:
30 70 30
35
Result = (rv/rs) x (Cs/Cv) x 100
70 30
36 90 10 = peak response of each impurity from the Sample
solution
46 90 10
=peak response of allopurinol from t~tandard
solution
Diluent: Solution A and Solution B (90: 10) =concentration of USP Allopurinol RS in the
Standard stock solution: 0.05 mg/mL each of USP . Standard solution (mg/mL)
Allopurinol RS, USP Allopurinol Related Compound A RS =concentration of Allopurinol in the Sample
USP Allopurinol Related Compound B RS, USP Allopurinb, solution (mg/mL)
Related Compound C RS, USP Allopurinol Related '
Compound DRS, and USP Allopurinol Related Compound Acceptance criteria: See Table 2.
E RS, prepared as follows. Transfer 5 mg each of USP
Allopurinol RS, USP Allopurinol Related Compound A RS Table 2
USP Allopurinol Related Compound B RS, USP Allopurinb, Relative Acceptance
Related Compound C RS, USP Allopurinol Related Retention Criteria,
Compound DRS, and USP Allopurinol Related Compound Name Time NMT (%)
E RS to a 1OO-mL volumetric flask. Add 2.0 mL of 0.1 N Allopurinol related com-
sodium hydroxide, and promptly sonicate with swirling for pound A 0.62 0.2
NMT 1 min to dissolve. Add 80 mL of Diluent, and sonicate
Allopurinol related com-
for an additional 5 min. Dilute with Diluent to volume. pound C 0.79 0.2
[NOTE-This solution is stable for 48 h when stored at 8°.]
Standard solution: 0.5 J.Ig/mL each of USP Allopurinol RS Allopurinol related com-
USP Allopurinol Related Compound A RS, USP Allopurinol pound B 0.81 0.2
Related Compound B RS, USP Allopurinol Related Allopurinol 1.0 -
Compound C RS, USP Allopurinol Related Compound D
Allopurinol related com-
RS, and USP Allopurinol Related Compound E RS in Diluent pound 0 4.4 0.2
from the Standard stock solution
Sample solution: 0.25 mg/mL of Allopurinol, prepared as Allopurinol related com-
follows. Transfer 25 mg of Allopurinol to a 100-mL pound E 4.8 0.2
v?'umetric flask. Add 5.0 mL of 0.1 N sodium hydroxide to Ethyl-(E/Z)-3-(2-carbe-
dissolve, promp~ly sonicate with swirling for NMT 1 min, thoxy-2-cyanoethenyl)
add 80 mL of Diluent, and sonicate for an additional 5 min. amino-l H-pyrazole-
4-carboxylate 6.5 0.2
Dilute with Diluent to volume.
Chromatographic system Unspecified
impurity - 0.1
(See Chromatography (621), System Suitabmty.)
Mode: LC Total impurities - 1.0
Detector: UV 220 nm
Column: 4.6-mm x 25-cm; 5-J.Im packing L1
Column temperature: 30° • LIMIT OF HVDRAZINE
Flow rate: 1.0 mL/min [NOTE-Under the following conditions, any hydrazine
Injection volume: 40 J.IL present in the sample will react with benzaldehyde to
System suitability form benzalazine.]
Sample: Standard solution Mobile phase: Hexane and isopropyl alcohol (95:5)
[NoTE-See Table 2 for relative retention times.] 2 N sodium hydroxide solution: Dissolve 8.5 9 of sodium
Suitability requirements hydroxide in water, and dilute with the same solvent to 100
Resolution: NLT 0.8 between allopurinol related mL. Alternatively, a commercially available 2 N sodium
compound C and allopurinol related compound B hydroxide solution can be used.
Tailing factor: NMT 1.5 for the allopurinol peak Diluent: Methanol and 2 N sodium hydroxide solution (1:1)
Analysis Benzaldehyde solution: 40 mg/mL of benzaldehyde in
Samples: Standardsolution and Sample solution Diluent. [NOTE-Prepare immediately before use.]
Calculate the percentages of allopurinol related Hydrazine solutlon: 2.0 I-'g/mL of hydrazine sulfate in
compounds A, B, C, D, and E in the portion of Allopurinol Diluent. Use sonication if necessary.
taken: . Standard solution: Transfer 5.0 mL of Hydrazine solution to
a suitable flask and add 4 mL of Benzaldebyde solution. Mix
Result = (rv/r s) x (CslCv) x 100 and allow to stand for 2.5 h at room temperature. Add 5.0
mL of hexane, and shake for 1 min. Allow the layers to
rv = peak response of each individual impurity from separate, and use the upper (hexane) layer.
the Sample solution Allopurinol solution: Dissolve 250 mg of Allopurinol in 5
rs = peak response of each individual impurity from mL of Diluent.
the Standard solution Sample solution: Transfer the Allopurinol solution to a
Cs = concentration of each individual impurity in the suitable flask; and add 4 mL of Benzaldehyde solution. Mix,
Standardsolution (mg/mL) and allow to stand for 2.5 h at room temperature. Add 5.0

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USP 43 OfficialMonographs / Allopurinol 133

mL of hexane, and shakefor 1 min. Allow the layers to USP Allopurinol Related Compound D RS
separate, and use the upper (hexane) layer. Ethyl 5-amino-1 H-pyrazole-4-carboxylate.
Blank solution: Mix 5.0 mL of Diluent and 4 mL of C6 H9N30 2 155.15
Benzaldehyde solution, and allow to stand for 2.5 h at room USP Allopurinol Related Compound E RS
temperature. Add 5.0 mL of hexane, and shakefor 1 min. Ethyl 5-(formylamino)-1H-pyrazole-4-carboxylate.
Allow the layers to separate, and use the upper (hexane) C7H9N 30 3 183.16
layer.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 310 nm Allopurinol Compounded Oral
Column: 4.0-mm x 25-cm; 5-\.Im packing L10
Column temperature: 30°
Suspension
Flow rate: 1.5 mL/min DEFINITION
Injection volume: 20 \.IL
System suitability
Sample: Standard solution
[NoTE-The relative retention times for benzalazine and Allopurinol Compounded Oral Suspension contains NLT
benzaldehyde are about 0.8 and 1.0, respectively.] 90.0% and NMT 110.0% of the labeled amount of
Suitability requirements allopurinol (CsH4N40 ).
Resolution: NLT 2.0 between benzalazine and PrepareAlloplJrinol Compounded Oral S,:!spel'}sion _20 mg/mL
benzaldehyde ~i . defor Oral Suspension andVe~ide fQrpral
Relative standard deviation: NMT 15.0% for the Sol . A (USP 1.M;y.2020) as follows (see Pharmaceutical
benzalazine peak
Compounding-Nonsterile Preparations (795».
Analysis
Samples: Standard solution and Sample solution
Calculate the amount, in ppm, of hydrazine in the portion Allopurinol tablets equivalent to 2 9 of allopurinol
of Allopurinol taken: Glycerin 5 ml

Result = (ru/rs) x (Cs/Cu) x (M rdMr2 ) x F Vehicle for Oral Suspension

(1J~l'il:M·}i'2Q2Q) 45 ml
= peak response of benzalazine from the Sample Vehicle for Solution,
solution 'i,," !i·..··.>",,·>!.i!. a sufficient quantity to
= peak response of benzalazine from the Standard ma"l<e 100mL
solution
= concentration of hydrazine sulfate in the Selectthe number of tablets that contain the specified amount
Hydrazine solution (\.Ig/mL) . of allopurinol, and calculate the quantity of each ingredient
= concentration of Allopurinol in the Allopurinol
required for the total amount to be prepared. Count,
solution (mg/mL)
weigh, or measure each ingredient. Thoroughly pulverize
= molecular weight of hydrazine, 32.05
the tablets. Mix the powdered Allopurinol tablets and
= molecular weight of hydrazine sulfate, 130.12
9'ys~rin to forr:'asmooth paste. Incorporat;,j"<'
= unit conversion factor (from \.Ig/mg to ppm),
1000 .~i~'Ve~'!;!;"~~~'iOral Suspension;~gmeJi\(ehic/~IQr
(p(g!;Qy .trv1~y.f4()?Q)Add sufficient Vehicle for Oral
Acceptance criteria: NMT 10 ppm of hydrazine Solution to volume, and mix well. Adjust the pH, if
e
SPECIFIC TESTS
• Loss ON DRYING (731)
Analysis: Dry under vacuum at 105° for 5 h.
Acceptance criteria: NMT 0.5%
ADDITIONAL REQUIREMENTS 29
• PACKAGING AND STORAGE: Preserve in well-closed
containers. Store at room temperature. 100mL

• USP REFERENCE STANDARDS (11)

USP Allopurinol RS
USP Allopurinol Related Compound A RS
3-Amino.-4-carboxamidopyrazQlehemisulfate.
A(C4H6N40 )2' H2S04 . 350.31 i.. (ERRi.Dec:~018)
USP Aflopurinol Related Compound B RS
5-(Formylamino)-1 H-pyrazole-4-carboxamide.
CSH6N 402 154.13
USP Allopurinol Related Compound C RS
5-(4H-1,2,4-Triazol-4-yl)-1 H-pyrazole-4-carboxamide.
C6H6N60 178.15

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134 Allopurinol/Official Monographs USP 43

ASSAY

• BEVOND-USE DATE

NMT 60 days
after the date on which it was compounded when stored
at r,......"tl",...lIor!

f!S (»)
• LABELING: Label it to state that it is to be shaken well before
use, and to state the Beyond-Use Date.

Allopurinol Tablets
» Allopurinol Tablets contain not less than 93.0
percent and not more than 107.0 percent of the
labeled amount of allopurinol (CSH4N4 0 ).
Packaging and storage-Preserve in well-closed
containers.
USP Reference standards (11)-
USP Allopurinol RS
Identification-Extract a quantity of finely powdered
Tablets, equivalent to about 50 mg of allopurinol, by
trituration with 10 mL of 0.1 N sodium hydroxide. Filter,
acidify the filtrate with 1 N acetic acid, collect the precipitated
allopurinol (allow 10 to 15 minutes for sufficient precipitation
to occur), wash the precipitate with 3 mL of dehydrated
alcohol, in portions, and finally wash with 4 mL of anhydrous
ethyl ether. Allow to dry in air for 15 minutes, then dry at 105°
for 3 hours: the residue so obtained meets the requirements
for the Identification test under Allopurinol.
Dissolution (711)-
Medium: 0.01 N hydrochloric acid; 900 mL.
Apparatus 2: 75 rpm.
Time: 45 minutes.
Standard stock solution-Prepare a stock solution by
SPECIFIC TESTS
transferring about 40 mg of USP Allopurinol RS, accurately
weighed, to a 200-mL volumetric flask. Add 10 ml of 0.1 N
sodium hydroxide, sonicate for about 2 minutes, shake by
mechanical means for about 10 minutes, dilute with
Dissolution Medium to volume, and mix.
Standard solution-Dilute the Standard stock solution with
Dissolution Medium to obtain a solution having a
concentration similar to that expected in the solution under
_J test.
ADDITIONAL REQUIREMENTS Procedure-Determine the amount of CSH 4N 40 dissolved
by employing UV absorption at the wavelength of maximum
absorbance at about 250 nm on filtered portions of the
solution under test, suitably diluted with Dissolution Medium,
in comparison with the Standard solution.
Tolerances-Not lessthan 75% (Q) of the labeled amount
of CSH 4N4 0 is dissolved in 45 minutes.
Uniformity of dosage units (905): meet the requirements.
Assay- [NOTE-Do not allow the Mobile phase to remain in
the column overnight. After performing the procedure, flush

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 USP 43 OfficialMonographs / Almotriptan 1 35

the system with water for not less than 20 minutes and then IDENTIFICATION
flush with methanol for 20 minutes.] . '.
Mobile phase-Prepare a filtered and degassed 0.05 M
solution of monobasic ammonium phosphate.
Internal standard solution-On the day of use, dissolve • A. .:@~J~;?(~·~~)~lk)lfJrizQ~~CI
about 50 mg of hypoxanthine in 10 mL of 0.1 N sodium }.< ..0): The spectrum -exhibits
hydroxide, shake by mechanical means until dissolved (about pronounced peaks at about 700, 950, 980, 1300, 1340,
10 minutes), dilute with water to 50 mL, and mix. 1350, 141 0, 1420~ 1650, 2100, and 2200 crrr".
Standard preparation-On the day of use, transfer about 50
mg of USP Allopurinol RS, accurately weighed, to a 50-mL ASSAY
volumetric !Iask, add 10 mL of 0.1 N sodium hydroxide, shake • PROCEDURE
by mechanical means for 10 minutes, dilute with water to Sample solution: Transfer 4 mL into a 1OO-mL volumetric
volume, and mix. Transfer 4.0 mL of this solution and 2.0 flask, and dilute with alcohol to volume.
mL of Internal standard solution to a 200-mL volumetric flask Analysis: Transfer 5.0 mL of the Sample solution to a 100-mL
dilute with Mobile phase to volume, and mix. ' conical flask, and add 50.0 mL of 0.1 N silver nitrate VS and
Assay preparation-Weigh and finely powder not fewer 5 mL of ammonia TS. Connect the flask to a reflux
than 20 Tablets. Transfer an accurately weighed portion of the condenser; heat on a water bath for 1 h, and allow to cool
powder, ~quivalent to about 50 mg of allopurinol, to a 50-mL to room temperature. Disconnect the flask from the
volumetric !Iask, add 10 mL of 0.1 N sodium hydroxide, shake condenser, transfer the contents of the conical flask to a
by mechanical means for 10 minutes, add water to volume, 1OO-mL volumetric flask with the aid of water, and dilute
and mix. [Nora-Prom this point, conduct the remainder of with water to volume. Pass through a dry filter, discarding
the Assaywithout delay.] Filter, rejecting the first 10 mL of the t~e first 10 mL of the filtrate. To 50.0 mL of the subsequent
filtrate. Transfer 4.0 mL of the filtrate and 2.0 mL of Internal filtrate add 5 mL of nitric acid and 2 mL of ferric ammonium
standard solution to a 200-mL volumetric flask, dilute with sulfate TS, and titrate the excess silver nitrate with 0.1 N
Mobile phase to volume, and mix. ammonium thiocyanate VS. Perform a blank determination,
Chromatographic system (see Chromatography (621 »- The using 5 mL of alcohol in place of the Sample solution, and
make any necessary correction. Each mL of 0.1 N silver
liquid chromatograph is equipped with a 254-nm detector
and a 4-mm x 30-cm column that contains packing L1. The nitrate is equivalent to 4.958 mg of allyl isothiocyanate
flow rate is about 1.5 mL per minute. Chromatograph the (C4H sNS).
S~andard preparation, and record the peak responses as Acceptance criteria: 93.0%-105.0%
directed for Procedure: the relative retention times are about IMPURITIES
0.6 for hypoxanthine and 1.0 for allopurinol; the resolution, • LIMIT OF PHENOLS
R, between the analyte and internal standard is not less than Sample solution: Dilute a 1-mL sample with 5 mL of
5; and the relative standard deviation for replicate injections is alcohol.
not more than 3.0%. Analysis: Add 1 drop of ferric chloride TS to the Sample
Procedure-Separately inject equal volumes (aboutl Sjrl.) solution.
of the Standard preparation and the Assay preparation into the Acceptance criteria: A blue color is not produced
chromatograph, record the chromatograms, and measure the immediately.
responses for the major peaks. Calculate the quantity, in mg,
of allopurinol (CSH4N40) in the portion of Tablets taken by the SPECIFIC TESTS
formula: • SPECIFIC GRAVITY (841): 1.013-1.020
• REFRACTIVE INDEX (831): 1.527-1.531, determined at 20 0
2.5C(Ru/Rs) • DISTILLING RANGE, Method I (721): 148 0 - 1 5 4 0
ADDITIONAL REQUIREMENTS
in which C is the concentration, in IJg per mL, of USP
• PACKAGING AND STORAGE: Preserve in tight containers.
Allopurinol RS in the Standard preparation; and Ru and Rs are
the peak response ratios of allopurinol to hypoxanthine
obtained from the Assay preparation and the Standard
preparation, respectively.
Almotriptan Malate

Allyl Isothiocyanate HO~If y 1

»<:
'OH
o OH

C4H sNS 99.15 C17H2SN302S, C4H60S 469.55

3-lsothiocyanato-l-propene; Pyrrolidine, 1-[[[3-[2-(dimethylamino)ethyl]-1 H-indol-5-yl]
Isothiocyanic acid allyl ester [57-06-7]. methyl]sulfonyl]-, hydroxybutanedioate (1:1);
1-[({3-[2-(Dimethylamino)ethyl]indol-5-yl}methyl)sulfonyl]
DEFINITION pyrrolidine malate (1:1) [181183-52-8].
Allyllsothiocyanate contains NLT 93.0% and NMT 105.0% of
allyl isothiocyanate (C4H sNS). DEFINITION
[CAUTION-Allyl Isothiocyanate is a potent lachrymator, Almotriptan Malate contains NLT 98.0% and NMT 102.0% of
with a pungent, irritating odor. Care should be taken to almotriptan malate (C17H2SN302S . C4H60 S), calculated on
protect the eyes, to prevent inhalation of fumes, and to the anhydrous and solvent-free basis.
avoid tasting.]

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136 Almotriptan / Official Monographs USP 43

IDENTIFICATION • LIMIT OF ALMOTRIPTAN RELATED COMPOUND D AND

ALMOTRIPTAN N-DIMER
Run buffer: 23.5 giL of phosphoricacid in water. Adjust
with triethanolamine to a pH of 3.0 and pass through a
• A. suitable filter of 0,45-l..Im pore size.
'$. Diluent: Methanol and water (50:50)
• B. the retention time of the major peak of the Sample Internal standard solution: 0.01 mg/mL of 4-hydroxy-
solution corresponds to that of the Standard solution, as 4-phenylpiperidine in Diluent
obtained in the Assay. System suitability solution: 0.005 mg/mL each of. USP
ASSAY
Almotriptan Related Compound BRS, USP Almotrrptan
Related Compound DRS, and USP Almotriptan Malate RS
• PROCEDURE
in the Internal standardsolution. Pass through a suitable
Buffer: 2.72 gIL of monobasic potassium phosphate in filterof 0,45-l..Im pore size.
water. Adjust with phosphoricacid to a pH of 3.5. Standard stock solution: 0.5 mg/mL of USP Almotriptan
Mobile phase: Methanol and Buffer (40:60) Malate RS in Diluent
System suitability solution: 0.14 mg/mL each of USP Standard solution: 0.005 mg/mL of USP Almotriptan
Almotriptan Malate RS and USP Almotriptan Related Malate RS from the Standard stock solution in the Internal
Compound BRS in Mobilephase. Sonication may be used standard solution. Pass through a suitable filterof 0,45-l..Im
to promote dissolution. pore size.
Standard solution: 0.14 mg/mL of USP Almotriptan Malate Sample solution: 2.5 mg/mL of Almotriptan Malate in the
RS in Mobilephase. Sonication may be used to promote Internal standardsolution. Sonication may be used to
dissolution. promote dissolution. Pass the solution through a suitable
Sample solution: 0.14 mg/mL of Almotriptan Malatein filter of 0,45-l..Im pore size. .
Mobilephase. Sonication may be used to promote Capillary rinsing t:?rocedure: Use separa~e Run b~,!er Vials
dissolution. for the capillary nnse and sample analysis. Condition the
Chromatographic system capillary by rinsing with water, 0.1 N sodium hydroxide,
(See Chromatography (621), System Suitability.) water, and the Run buffer before each injection. [NOTE-It
Mode: LC may be suitable to rinsewith water, 0.1 N sodium
Detector: UV 230 nm hydroxide, and water using a pressure of 20 psifor Nl,T 2
Column: 4.6-mm x 15-cm; 5-l..Im packing L10 min each and then to rinse with the Run buffer using a
Flowrate: 1 mL/min pressure of 20 psifor NLT 5 min.]
Injection volume: 10 I..IL Instrumental conditions
Run time: NLT 2 times the retention time of almotriptan Mode: CE
System suitability Detector: UV 214 nm
Samples: System suitability solution and Standard solution Capillary, Capillaryeffective length, Capillary .
[NOTE-The relative retention times for almotriptan temperature, and Voltage: Use parameters described
relatedcompound Band almotriptanare 0.7 and 1.0, under A or B as indicated in Table 7.
respectively.]
Suitability requirements Table 1
Resolution: NLT 2.0 between almotriptan and
almotriptan related compound B, System suitability Parameter A B
solution . 75-lJm x 48.5-cm; 75-lJm x 60-cm;
Tailing factor: NMT 2.0, Standard solution Capillary uncoated fused silica uncoated fused silica
Relative standard deviation: NMT 0.85% for six Capillary
injections, Standard solution effective length 40cm 47cm
Analysis
Samples: Standard solution and Sample solution Capillary
temperature 15° 25°
Calculate the percentage of almotriptan malate
(C17H2SN302S, C4H60 S) in the portion of Almotriptan Avoltage of 15.5 kV may Avoltage of 15.0 kV may
Malatetaken: Voltage be suitable. be suitable.

Result = (r ulr s) x (C sIC u) x 100 Injection sequence: 0.5 psifor 8 s for the Sample
solution, followed by 0.5 psifor 1 s for the Run buffer
= peak response of almotriptan from the Sample Runtime: NLT 2.5 timesthe migration time of almotriptan
solution System suitability
= peak response of almotriptanfrom the Standard Samples: System suitabilitysolution and Standard solution
solution [NOTE-See Table 2 for the relative migration times.]
=concentration of USP Almotriptan Malate RS in Suitability requirements
the Standard solution (mg/mL) Resolution: NLT 2.0 between almotriptan related
Cu = concentration of Almotriptan Malate in the compound Band almotriptan; NLT 2.0 between
Sample solution(mg/mL) almotriptan and almotriptan related compound D,
System suitability solution
Acceptance criteria: 98.0%-102.0% on the anhydrousand Relative standard deviation: NMT 5.0% for the ratio of
solvent-free basis the peak response of almotriptan to the peak response
IMPURITIES of the internalstandard, Standard solution
• RESIDUE ON IGNITION (281): NMT 0.10% Analysis .
Samples: Standardsolution and Sample solution
Calculate the corrected peak response:
Result = (rim)

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 USP 43 Official Monographs / Almotriptan 137

r = peak response System suitability

m = migration time of the peak (min) Samples: System sUitability solution and Standardsolution
[NoTE-See Table 3 for the relative retention times.]
Calculate the percentage of almotriptan related compound Suitability requirements,
D, almotriptan N-dimer, and other impurities in the Resolution: NLT 1.0 between almotriptan related
portion of Almotriptan Malate taken: compound C and almotriptan related compound D,
System suitability solution
Result = (R vIR s) x (C sIC v) x 100 Relative standard deviation: NMT 5.0% for six replicate
injections, Standard solution
Ru =corrected peak response ratio of the impurity to Analysis
the internal standard from the Sample solution Samples: System suitabilitysolution, Standardsolution, and
Rs = corrected peak response ratio of almotriptan to Sample solution
the internal standard from the Standardsolution Chromatograph the System suitability solution and identify
Cs = concentration of USPAlmotriptan Malate RS in the components on the basis of their relative retention
the Standardsolution (mg/mL) times, as shown in Table 3.
Cu = concentration of Almotriptan Malate in the Calculate the percentage of each impurity in the portion of
Sample solution (mg/mL) Almotriptan Malate taken:
Acceptance criteria: See Table 2. Result =(r vIr s) x (C sIC v) x 100
Table 2 = peak response of each impurity from the Sample
Relative Acceptance solution
Migration Criteria, = peak response of almotriptan from the Standard
Name Time NMT(%) solution
Almotriptan N·dimer" 0.71 0.3 = concentration of USPAlmotriptan Malate RS in
the Standard solution(mg/mL)
Internalstandard" 0.78 - = concentration of Almotriptan Malate in the
Almotriptan related Sample solution(mg/mL)
compound Be 0.92 -
Acceptance criteria: See Table 3.
Almotriptan 1.0 -
Almotriptan related Table 3
compound o 1.02 -
Relative Acceptance
Almotriptan related Retention Criteria,
compound D 1.22 0.1 Name Time NMT(%)

Anyindividual unspecified impur-

-
Malic acid" 0.10 -
ities 0.1
Almotriptan related com-
pound B 0.62 0.1
a 2-{1·({3-[2-(Dimethylamino)ethyl]-1 H.indol-5-yl}methyl)-5-[(pyrrolidin-l-
ylsulfonyl)methyl]-l H-indol-3-yl}-N,N-dimethylethan-l-amine. Almotriptan related com-
b 4-Hydroxy-4-phenylpiperidine. pound C 0.77 0.5
e Ifpresent, this impuritymay not be fully resolved from almotriptan.It is
quantified using the test for OrganicImpurities. Almotriptan related com-
.
pound Db 0.92 -
• ORGANIC IMPURITIES Almotriptan 1.00 -
Buffer: Add 10 mLof triethylamine to every 1000 mLof 0.01 Anyother individual
M phosphoric acid. Adjust with phosphoric acid to a pH of impurity - 0.1
6.5.
Mobile phase: Acetonitrile and Buffer (15:85) Total irnpurltles- - 1.0
System suitability stock solution: 0.5 mg/mL each of USP
aThispeak is due to the malate counterion;hence it is not an impurity. It is not
Almotriptan Related Compound B RS, USPAlmotriptan to be reported or included in the total impurities.
Related Compound C RS, and USP Almotriptan Related bThisimpurityis quantifiedusingthe Limit of Almotriptan Related Compound D
Compound D RS in methanol and AlmotriptanN-Dimer test.
System suitability solution: 0.005 mg/mL each of USP eThe sum of allimpurities from the test for Organic Impurities and the Limit of
Almotriptan Related Compound B RS, USP Almotriptan AlmotriptanRelated Compound D andAlmotriptan N-Dimertest.
Related Compound C RS, and USP Almotriptan Related
Compound D RS from the System suitability stock solution in • LIMIT OF FUMARIC ACID
water Buffer: 6.8 giL of monobasic potassium phosphate in
Standard solution: 0.007 mg/mL of USPAlmotriptan water. Adjust with phosphoric acid to a pH of 2.8.
Malate RS in water Mobile phase: Methanol and Buffer (5:95)
Sample solution: 3.5 mg/mL of Almotriptan Malate in Standard solution: 0.0085 mg/mL of USP Fumaric Acid RS
water. Sonication may be used to promote dissolution. and 0.0017 mg/mL of USP Maleic Acid RS in water.
Chromatographic system Sonication may be used to promote dissolution.
(See Chromatography (621), System Suitability.) Sample solution: 2.8 mg/mL of Almotriptan Malate in
Mode: LC water. Sonication may be used to promote dissolution.
Detector: UV 210 nm Chromatographic system
Column: 4.6-mm x 3D-em; 5-lJm packing L1 (See Chromatography (621), System Suitability.)
Flow rate: 1 mL/min Mode: LC
Injection volume: 20 IJL Detector: UV220 nm
Run time: NLT 3 times the retention time of almotriptan Column: 4.6-mm x 25-cm; 5-l.Im packing L7

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1 38 Almotriptan / OfficialMonographs USP 43

Flow rate: 0.7 ml/min

Injection volume: 10 IJl
Almotriptan Tablets
Run time: NlT 1.6 times the retention time of fumaric acid DEFINITION
System suitability Almotriptan Tablets contain an amount of almotriptan malate
Sample: Standardsolution (C17H2SN302S . C4H 60s) equivalent to NlT 90.0% and NMT
[NoTE-See Table 4 for the relative retention times.]
110.0% of the labeled amount of almotriptan (C17H2sN302S),
Suitability requirements
Resolution: NlT 2.0 between fumaric acid and maleic r: IDENTIFICATION
acid
Relative standard deviation: NMT 5.0% forfumaric acid
from six injections
Analysis )~JqN,itE~[~i.(l'gZ);lfjrri:lr;eq
Samples: Standardsolution and Sample solution .s../.. ..' .»>: • ..20)
Calculate the percentage of fumaric acid (C4H404) in the Sample: Sonicate 5 powdered Tablets in 25 ml of water.
portion of Almotriptan Malate taken: Extract the suspension with 25 ml of methylene chloride,
and discard the organic phase. Add another 25 ml of
Result = (r vir 5) x (C siC v) x 100 methylene chloride and 3 ml of 1 N sodium hydroxide.
Extract the precipitated base into the organic layer. Dry
ru = peak response of fumaric acid from the Sample with anhydrous sodium sulfate, and evaporate the organic
solution solvent. Prepare the residue oil as a film on a sodium
rs =peak response of fumaric acid from the Standard chloride pellet.
solution Acceptance criteria: The IRspectrum obtained from the
Cs =concentration of USP Fumaric Acid RS in the Sample is consistent with that of similarly prepared USP
Standardsolution (mg/ml) Almotriptan Malate RS.
Cu = concentration of Almotriptan Malate in the • B. The retention time of the major peak of the Sample
Sample solution (mg/ml) solution corresponds to that of the Standardsolution, as
obtained in the Assay.
Acceptance criteria: See Table 4.
ASSAY
Table 4 • PROCEDURE
Relative Acceptance Protect samples, the Reference Standards, and solutions
Retention Criteria, containing them from light.
Name Time NMT (%) Buffer: Add 10 ml of triethylamine to every 1000 mL of 0.01
M phosphoric acid. Adjust with phosphoric acid to a pH of
Malicacid" 0.60 - 6.0.
Maleicacid" 0.80 - Mobile phase: Acetonitrile and Buffer(10:90)
Fumaricacid 0.2
Standard solution: 0.5 mg/mL of USP Almotriptan Malate
1.0
RS in Mobile phase. Sonication may be used to aid in
a Includedfor identification purposes only. dissolution.
System suitability stock solution: 0.1 mg/mL each of USP
SPECIFIC TESTS Almotriptan Related Compound B RS, USP Almotriptan
• WATER DETERMINATION (921), Method I, Method la: NMT Related Compound C RS, and USP Almotriptan Related
0.5% Compound D RS in methanol. Sonication may be used to
aid in dissolution.
ADDITIONAL REQUIREMENTS System suitability solution: 0.001 mg/mL each of USP
• PACKAGING AND STORAGE: Preserve in well-closed Almotriptan Related Compound B RS, USP Almotriptan
containers. Store at controlled room temperature. Related Compound C RS, and USP Almotriptan Related
• USP REFERENCE STANDARDS (11) Compound D RS prepared from the System suitability
USP Almotriptan Malate RS stocksolution in Standardsolution
USP Almotriptan Related Compound B RS Sample solution: Nominally 0.5 mg/mL of almotriptan
2-{5-[(Pyrrolidin-l-ylsulfonyl)methyl]-1 H-indol-3-yl} from Tablets prepared as follows. Transfer NLT 8 Tablets
ethanamine hemifumarate. into a suitable volumetric flask, and add 80% of the flask
C1sH21N 302S . V2C 4H404 365.46 volume of Mobilephase. Sonicate for NLT 10 min, and
USP Almotriptan Related Compound C RS dilute with Mobilephase to volume. Stir for 30 min, and
N-Methyl-2-{5-[(pyrrolidin-1-ylsulfonyl)methyl]-1 H- centrifuge. Pass a portion of the supernatant through a
indol-3-yl}ethanamine. suitable filter of 0.45-lJm pore size. Use the filtrate.
C16H23N302S 321.44 Chromatographic system
USP Almotriptan Related Compound DRS (See Chromatography (621), System Suitability.)
1-[( {3-[2-(Dimethylamino)ethyl]indol-5-yl}methyl) Mode: LC
sulfonyl]pyrrolidine N-oxide hydrochloride. Detector: UV 210 nm
C17H2SN303S, HCI 387.92 Column: 2.1-mm x 10-cm; 1.8-lJm packing L1
USP Fumaric Acid RS Column temperature: 40°
USP Maleic Acid RS Flow rate: 0.55 mL/min
Injection volume: 3 IJL
System suitability
Samples: Standardsolution and System suitability solution
[NOTE-See Table 7 for the relative retention tlrnes.]

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USP 43 Official Monographs / Almotriptan 139

Suitability requirements Standard stock solution: 0.425 mg/mL of USP

Resolution: NLT 1.5 between almotriptan related Almotriptan Malate RS, equivalent to 0.30 mg/mL of
compound C and almotriptan peaks, System suitability almotriptan, in water
solution Standard solution: 0.021 mg/mL of USP Almotriptan
Tailing factor: NMT 3.0, Standard solution Malate RS, equivalent to 0.015 mg/mL of almotriptan,
Relative standard deviation: NMT 2.0%, Standard from the Standard stock solution in Medium
solution Sample solution: Pass a portion of the solution under test
Analysis through a suitable filter of 0.45-J.Jm pore size, and discard
Samples: Standard solution and Sample solution the first 5 mL. Use the remaining filtrate.
Calculate the percentage of the labeled amount of Instrumental conditions
almotriptan (C17HzsN30ZS) in the portion of Tablets taken: (See Ultraviolet- Visible Spectroscopy (857).)
Mode: UV
Result = (r vir s) x (C siC v) x (M ,dM (2) x 100 Analytical wavelength: 283 nm
Blank: Medium
ru = peak response of almotriptan from the Sample Analysis
solution Samples: Standard solution and Sample solution
rs = peak response of almotriptan from the Standard Calculate the percentage of the labeled amount of
solution almotriptan (C17HzsN30ZS) dissolved:
Cs = concentration of USP Almotriptan Malate RS in
the Standard solution (mg/mL) Result =(A viA s) x (C slL) x V x (M ,dM (2) x 100
Cu = nominal concentration of almotriptan in the
Sample solution (mg/mL) Au =absorbance of the Sample solution
M ,1 = molecular weight of almotriptan, 335.46 As =absorbance of the Standard solution
M ,2 = molecular weight of almotriptan malate, 469.55 Cs = concentration of USPAlmotriptan Malate RS in
the Standard solution(mg/mL)
Acceptance criteria: 90.00/0-110.0% of the labeled amount L = label claim (mg/Tablet)
of almotriptan V =volume of Medium, 900 mL
PERFORMANCE TESTS
.M ,1 = molecular weight of almotriptan, 335.46
• DISSOLUTION (711)
M ,2 = molecular weight of almotriptan malate, 469.55
Test 1
Medium: 0.1 N hydrochloric acid; 900 mL Tolerances: NLT 80% (Q) of the labeled amount of
Apparatus 2: 50 rpm almotriptan (C17HzsN30ZS) is dissolved.
Time: 15 min • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Standard solution: (L/600) mg/mL of USP Almotriptan requirements
Malate RS in Medium, where L is the label claim in mgl IMPURITIES
Tablet • ORGANIC IMPURITIES
Sample solution: Pass a portion of the solution under test Mobile phase, Standard solution, System suitability
through a suitable filter of 0.45-J.Jm pore size. solution, Sample solution, Chromatographic system,
Instrumental conditions and System suitability: Proceed as directed in the Assay.
Mode: UV Analysis
Analytical wavelength Samples: Standardsolution, System suitability solution, and
For Tablets labeled to contain 6.25 mg: 228 nm Sample solution
For Tablets labeled to contain 12.5 mg: 284 nm Chromatograph the System suitabilitysolution and identify
Blank: Medium . the components on the basis of their relative retention
Analysis times, as shown in Table 1.
Samples: Standardsolution and Sample solution Calculate the percentage of each degradation product in
Calculate the percentage of the labeled amount of the portion of Tablets taken:
almotriptan (C17HzsN30ZS) dissolved:
Result =(r vir s) x (C siC v) x (M r1IM (2) x 100
Result = (A viA s) x (C slL) x V x (M ,dM (2) x 100
ru = peak response of each degradation product
Au = absorbance of the Sample solution from the Sample solution
As = absorbance of the Standard solution rs = peak response of almotriptan from the Standard
Cs = concentration of USP Almotriptan Malate RS in solution
_the Standardsolution (mg/mL) Cs =concentration of USP Almotriptan Malate RS in
L = label claim (mg/Tablet) the Standardsolution(mg/mL)
V =volume of Medium, 900 mL Cu = nominal concentration of almotriptan in the
M ,1 = molecular weight of almotriptan, 335.46 Sample solution (mg/mL)
M ,2 = molecular weight of almotriptan malate, 469.55 M" = molecular weight of almotriptan, 335.46
M '2 = molecular weight of alrnotrlptan malate, 469.55
Tolerances: NLT 80% (Q) of the labeled amount of
almotriptan (C17HzsN30ZS) is dissolved. Acceptance criteria: See Table 1.
Test 2: If the product complies with this test, the labeling
indicates that it meets USP Dissolution Test 2. Table 1
Medium: 0.1 N hydrochloric acid; 900 mL Relative Acceptance
Apparatus 2: 50 rpm Retention Criteria,
Time: 30 min Name Time NMT(%)
Splroalmotriptan" 0.32 0.4

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140 Almotriptan / Official Monographs USP 43

Table 1 (continued) Acceptance criteria: The color of the filtrate, viewed in the
Relative Acceptance bulb of a 1OO..:mL volumetric flask, is dark orange with
Retention Criteria, Curacao aloe and greenish-yellow with Cape aloe. The
Name Time NMT(%) filtrate darkens on standing. [NOTE-Reserve the filtrate for
2-Hydroxyalmotriptan b 0.47 0.2 Identification B.]
Almotriptan related com- • lB.
- Sample: 5 mL of the filtrate obtained in Identification A
pound BC 0.82 Analysis: Add 2 mL of nitric acid to the Sample, and mix.
Almotriptan related com- Acceptance criteria: The mixture exhibits a reddish-orange
pound CC 0.93 - color with aloe vera and a reddish-brown color that
Almotriptan 1.0 - changes rapidly to green with Cape aloe.
• C. THIN-LAYER CHROMATOGRAPHY
Almotriptan related com- Standard solution: 1.0 mg/mL of USP Aloin RS in methanol
pound D 1.39 0.2 Sample solution: 0.5 g of finely powdered Aloe in 10 mL of
Anyindividual methanol. Sonicate for 15 min, centrifuge or filter, and use
unspecified - the supernatant or the filtrate.
degradation product 0.2 Chromatographic system
Totaldegradation (See Chromatography (621), General Procedures, Thin-Layer
products - 1.0 Chromatography.)
Adsorbent: Chromatographic silica gel mixture with an
a l'-Methyl-S-[(pyrrolidin-l-ylsulfonyl)methyl]spiro[indoline-3,3'-pyrrolidin]-2- average particle size of 5 IJm (HPTLCplates)
01.
Application volume: 2 IJLof the Standardsolution and 5
b 3-[2-(Dimethylamino)ethyl]-S-[(pyrrolidin-l-ylsulfonyl)methyl]-l H-indol-2-01.
C This is a process impurity that isincludedin this table for identification only.
IJLof the Sample solution as 8-mm bands
Thisimpurityis controlledin the drug substance. Thisimpurityis not to be Relative humidity: Condition the plate to a relative
reported for the drug product and isnot to be included inthe total degradation humidity of about 33% using a suitable device.
products. Developing solvent system: Ethyl acetate, methanol, and
water (100:17:13)
ADDITIONAL REQUIREMENTS Developing distance: 6 em
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Derivatization reagent: 10% potassium hydroxide
containers. Store at controlled room temperature. solution in methanol (prepare in an ice bath)
• LABELING: The labeling states the Dissolution test used only Analysis
if Test 1 is not used. Samples: Standardsolution and Sample solution
• USP REFERENCE STANDARDS (11) Apply the Samples as bands to a suitable HPTLCplate. Use
USP Almotriptan Malate RS a saturated chamber. Develop the chromatograms, dry in
USP Almotriptan Related Compound B RS air, derivatize with Derivatization reagent, and heat at 110°
2-{5-[(Pyrrolidin-1-ylsulfonyl)methyl]-1 H-indol-3-yl} for 5 min. Examine under visible light and UV light at 365
ethanamine hemifumarate. nm.
ClsH21N302S, V2C 4H 40 4 365.46 Acceptance criteria: Under visible light, the Sample solution
USP Almotriptan Related Compound C RS exhibits a brown band due to aloin at about the middle of
N-Methyl-2-{5-[(pyrrolidin-1-ylsulfonyl)methyl]-1 H- the chromatogram, corresponding in color and R F to the
indol-3-yl}ethanamine. band exhibited by the Standardsolution. The Sample
C16H23N302S 321.44 solution containing aloe vera exhibits an additional violet
USP Almotriptan Related Compound D RS band due to 7-hydroxyaloin right below the aloin band.
1-[ ({3-[2-(Dimethylamino)ethyl]indol-5-yl} methyl) The Sample solution containing Cape aloe lacks the violet
sulfonyl]pyrrolidine N-oxide hydrochloride. band due to 7-hydroxyaloin. Under UV light at 365 nm, the
C17H2SN303S . HCI 387.92 Sample solution exhibits a yellow fluorescence band due to
aloin, corresponding in color and R F to the band exhibited
by the Standardsolution, and a light blue fluorescence band
due to aloesine at about one-third of the chromatogram.
Aloe COMPOSITION
• CONTENT OF ALOIN
DEFINITION Mobile phase: A mixture of acetonitrile and water (3:7)
Aloe is the dried latex of the leaves of Aloe vera (L.) Burm. f. Standard solution: 0.1 mg/mL of USP Aloin RS in methanol
(syn. Aloebarbadensis Mill.), known in commerce as aloe and water (1 :1)
vera, Curacao aloe, or Barbados aloe; or of Aloeferox Mill., or Sample solution: Transfer about 0.1 g of aloe vera or 0.2 g
of hybrids of Aloe ferox Mill. with Aloe africana Mill. and Aloe of Cape aloe, finely powdered and accurately weighed, to
spicata L.f., known in commerce as Cape aloe (Fam. a 1OO-mL volumetric flask, and add about 75 mL of
Liliaceae). Aloe vera contains NLT 16.0% of aloin, and Cape methanol. Sonicate for 30 min, cool to room temperature,
aloe and its hybrids contain NLT 6.0% of aloin, both adjust with methanol to volume, and mix. Before injection,
calculated on the dried basis. pass through a PTFE membrane filter of 0.45-lJm pore size,
discarding the first few mL of the filtrate.
IDENTIFICATION Chromatographic system
• A. (See Chromatography (621), System Suitability.)
Sample: 1 g, finely powdered Mode: LC
Analysis: Mix the Sample with 25 mL of cold water. Shake Detector: UV 295 nm
the mixture occasionally for 2 h, filter, and wash the filter Column: 4.6-mm x 25-cm; end-capped 5-lJm packing L1
and residue with sufficient cold water to make the filtrate Column temperature: 43 ± 10
measure 100 mL. Flow rate: 1.0 mL/min

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 USP 43 Official Monographs / Alosetron 141

Injection volume: 20 J..lL Acceptance criteria: The weight of the residue is NMT
System suitability 10.0% of the weight of Aloe taken.
Sample: Standard solution • BOTANICAL CHARACTERISTICS
Suitability requirements Curacao aloe: Brownish-black, opaque masses. Itsfractured
Column efficiency: NLT 2000 theoretical plates for the surface is uneven, waxy, and somewhat resinous.
aloin peak Cape aloe: Dusky to dark brown irregular masses, the
Tailing factor: NMT 2.0 for the aloin peak surfaces of which are often covered with a yellowish
Relative standard deviation: NMT 2.0% determined powder. Its fracture is smooth and glassy.
from the aloin peak in repeated injections Powdered Aloe: Yellow, yellowish brown to olive-brown in
Analysis color. When mounted in olive oil, it appears as
Samples: Standard solution and Sample solution greenish-yellow to reddish-brown irregular fragments, the
[NoTE-The Standard solution and Sample solution are hues of which depend to some extent upon the thickness
stable for 8 h at room temperature.] of the fragments.
Using the chromatogram of the Standard solution, identify
ADDITIONAL REQUIREMENTS
the retention time of the peak corresponding to aloin in
• USP REFERENCE STANDARDS (11)
the Sample solution.
Calculate the percentage of aloin in the portion of Aloe USP Aloin RS
taken:

Result = (r vir s) xes x (VIW) x 100

ru = peak area of aloin from the Sample solution
rs = peak area of aloin from the Standard solution
Cs = concentration of USP Aloin RS in the Standard
solution (mg/mL)
V =final volume of the Sample solution (mL)
W =weight of Aloe taken to prepare the Sample
solution (mg)
Acceptance criteria: Aloe vera contains NLT 16.0% of aloin,
and Cape aloe and its hybrids contain NLT 6.0% of aloin,
both on the dried basis.
• WATER-SOLUBLE EXTRACTIVE
Sample: 2 g of powdered Aloe
Analysis: Macerate the Sample in 70 mL of water in a
suitable flask. Shake the mixture for 8 h at 30-min intervals,
and allow it to stand for 16 h without shaking -. Filter, and
wash the flask and residue with small portions of water,
passing the washings through the filter until the filtrate
measures 100.0 mL. Evaporate a 50-mL aliquot of the'
filtrate in a tared dish on a steam bath to dryness, and dry
at 110° to constant weight.
Acceptance criteria: The weight of water-soluble extractive
is NLT50% of the weight of Aloe taken.
CONTAMINANTS
• ARTICLES OF BOTANICAL ORIGIN (561), Pesticide Residue
Analysis: Meets the requirements
SPECIFIC TESTS
• Loss ON DRYING (731)
Sample: Use a powdered sample. If the Aloe is not
powdered, crush it in a mortar until it p.asses through a no.
40 sieve, and mix the ground material before weighing the
sample. -
Analysis: Dry the Sample at 105° for 5 h.
Acceptance criteria: NMT 12.0%
• ARTICLES OF BOTANICAL ORIGIN (561), Methods of
Analysis, Total Ash
Acceptance criteria: NMT 4.0%
• ALCOHOL-INSOLUBLE SUBSTANCES
Sample: 1 g of powdered Aloe
Analysis: Add the Sample to 50 mLof alcohol in a flask. Heat
the mixture to boiling, and maintain at incipient boiling for
15 min, replacing any loss due to evaporation. Remove
from the heat, and shake the mixture at intervals for 1 h.
Pass through a small dried and tared filter paper or a dried
and tared filtering crucible, and wash the residue on the
filter with alcohol until the last washing is colorless. Dry the
residue at 105° to constant weight.

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142 Alosetron / Official Monographs USP 43

Ari~dysi~ .. , ..
~amples' Stein n
Calc , ri~}/in the
PC)
~es~lt :i::(r~/ rs)'x}CiCu) ~ ,(1 / f)~1 00

p6rise of' e~ch imp4rjtyfrorllJ!le:Sattlpl~

()nse'ofalosetroqJrom th~ Stanaqr9

.rochloride' in)h~
Result ~ (fu!is)x(GslC~) ~.~ 1;90
F
=··peak.respoTls~.frorn .the§9mple~C!'ati.Orj
= peak response f[QI1J the,StalJdarcJ solutiOb Acceptance criteriif: See .Table 2.
=c' . ride RS
Table 2
ce
Name
Afbsetron 1';0
baSIS Alosefron related
compoundA . 1.3 0.15
'ther individual
ity . 1~0 0.10
ining 'alos.etron hydrocf:llodd~~fr9,rli

n1ato9r~phic$yster:n:pr()c~~~t:a::s~gif~~~.d 'TS
MIN~TIO'" <9215~ Metf;1odf;' Metnodla::NMT
l1ifrfle
'. e Table 'J. Return tooriginit~()flQ]f1c>ris
uilibrate the system.' . rners~

Table 1
Time
(min)
SblUfior{A
(%)
S()I!~lfi?J~
,Q ~o 10
'etrahydro-lH-pyrido[4,3-bJindol,;,l:';
is 90 'lQ
2~ ?~ .~~

25 $0 ~O,

IDENTIFICATION

o ,
J..to~noise· rat~o:' NlT·1 O~Se~sigvjtY~ioJfiflcjf1

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USP 43 Official Monographs / Alosetron 143

M:rl =molecular weight ofalosetro_ 94;36

Mil =ri1orecular'weight()[alQse~ro ,'(Q.ch!Qrlde,'
330.82

Acceptanc~ 'criteria: 90.0%':'1 J 9.0%

TESTS
11>-
'hs cohlainln~falosef..oriJlyqr6~liforfde~trQffi

OOrriL'of
5 '{Tig!mL o(O-Sp'.f\losetron

Result == (rvI rs)x (tslL) x: V x(M;ll Mr2f >r. 'fQ(j

fu =peak, response of alosetron from tn~ $alfikle
sol .
rs F= pe p6nseofalosetrorjfrCim tH(SJiindqrd
Cs ochlorfde'RS

ES
rde:'fioi11
ntaining'a.fosetl'on~h'yarochr()-..

as di~ectedJrrthe Ass:ay:
er
F'pe(lJ('response trom tne_5(JrnplfsoluIion : ceonitrHe c

~ peak'response ff()~tn.~ Stcfnc[aiq' 'jo7i.itNrj

ase: See Table "1. Ret
~eq uJlibrate the systel1}'f

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144 Alosetron / OfficialMonographs USP 43

Table 1 Table 2 (continued)

Time SoliltiollA SolutionB Relative Relative
(infn) (%) (0/6) Retention Response
Name_ Tim~ Factor
0 90 10
er inc;lividual
5 90 Hi 1.0 0,5
20 5Q 5Q i.oS,
25 50 So

Alprazolam
H3C"r

~
~ N:fN
CI ....-:: )
-N

~ Ii
•fty solution :alia1tanaajdJc>{~ti9Yl
C17 H13CIN4 308.76
4H-[1 ,2,4]Triazolo[4,3-a][1 ,4]benzodiazepine, 8-chloro-1-
methyl-6-phenyl-;
8-Chloro-1-methyl-6-phenyl-4H-s-triazolo[4, 3-a][1,4]
benzodiazepine [28981-97-7].
DEFINITION
Alprazolam contains NLT 98.0% and NMT 102.0% of
rttyinthe
C17 H13CIN4 •
[CAUTIoN-Care should be taken to prevent inhaling
Result = (rulrs)x (c:.lCu) x (l/F) x (MrrlMr2) _X 109 particles of Alprazolam and exposing the skin to it.]
IDENTIFICAnON
ru = -peak re~ponse of e_achimpuritYfrqm~h~ ~ample
solution CfltllJge-~orea
rs == peak resp_onse ofalos~tronf[orn. tt1e_$tandard
S' • Ii • A.A-SPECTROSCOPICJDE
Cs == c loride',RS Specttoscdpy; 197M A (CN1~J\.1 )
i • B. The retention time of the major peak from the Sample
Cu solution corresponds to that from the Standardsolution, as
obtained in the Assay.
F
ASSAY
Mrl • PROCEDURE
M~2 =_molecularweight ofaI6setron-hya(ochlori(je; Diluent: Acetonitrile and water (1:1)
330;82 Buffer: 1.4 giL of monobaslc potassium phosphate in water
Mobile phase: Acetonitrile and Buffer (1:1)
Acceptance criteria:-See Table 2. Standard solution: 25 J,Jg/mL of USP Alprazolam RS.in
Diluent. [NoTE-The solution is stable for 48 h at room
Ti~ble 2 temperature when stored in closed containers.]
Relative Relative Sample solution: 25 J,Jg/mL of Alprazolam in Diluent.
Retention Response Sonicate for about 1 min. [NOTE-The solution is stable for
Name Time Facto" 48 h at room temperature when stored in closed
Alosetron 1.0 containers.] .'
Chromatographic system
Alosetron related com-
pound A 1.1 0.3 (See Chromatography (621), System Suitability.)
Mode: LC .
Detector: UV 231 nm

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USP 43 Official Monographs / Alprazolam 145

Column: 4.6-mm x 25-cm; packing L1 Table 1

Column temperature: 40° Relative Relative Acceptance
Flow rate: 1 mL/min Retention Response Criteria,
Injection size: 20 IJL Name Time Factor NMT (%)
System suitability Alprazolam related com-
Sample: Standard solution pound A 0.8 0.76 0.15
Suitability requirements
Alprazolam 1.0 1.0 -
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% 2-Amino-5-chloro-
Analysis benzophenone 4.0 1.0 0.15
Samples: Standardsolution and Sample solution Individual unspecified im-
Calculate the percentage of alprazolam (C17H13CIN 4) in the purity - 1.0 0.10
portion of Alprazolam taken: Total impurities - - 1.0

Result = (r vir s) x (C siC v) x 100

SPECIFICTESTS
ru =peak area from the Sample solution • Loss ONDRYING (731): Dry a sample at 105° for 1 h: it loses
rs =peak area from the Standard solution NMT 0.5% of its weight.
Cs = concentration of USP Alprazolam RS in the ADDITIONAL REQUIREMENTS
Standard solution (mg/mL) • PACKAGING ANDSTORAGE: Preserve in tight containers, and
Cu = concentration of Alprazolam in the Sample store at controlled room temperature.
solution (mg/mL) " USP REFERENCE STANDARDS (11)
USP Alprazolam RS
Acceptance criteria: 98.0%-102.0% USP Alprazolam Related Compound A RS
IMPURITIES 2-(2-Acetylhydrazino)-7 -chloro-5-phenyl-3H-l,4-
• RESIDUE ON IGNITION (281): NMT 0.5% benzodiazepine.
• ORGANIC IMPURITIES USP 2-Amino-5-chlorobenzophenone RS
Diluent, Buffer, Mobile phase, and Chromatographic 2-Amino-5-chlorobenzophenone.
system: Proceed as directed in the Assay. C13H lOCINO 231 .68
System suitability solution: 20 IJg/mL each of USP
Alprazolam RS, USP Alprazolam Related Compound A RS,
and USP 2-Amino-5-chlorobenzophenone RS in Diluent
Standard solution: 0.25 IJg/mL of USP Alprazolam RS in
Diluent. [NoTE-When stored in closed containers, the Alprazolam Compounded Oral
solution is stable for 48 h at room ternperature.] Suspension
Sample solution: 250 IJg/mL of Alprazolam in Diluent.
Sonicate for about 1 min. [NoTE-When stored in closed DEFINITION
containers, the Sample solution is stable for 24 h at room Alprazolam Compounded Oral Suspension contains NLT
temperature.] 90.0% and NMT 110.0% of the labeled amount of
System suitability alprazolam (C17H13CIN4)'
Samples: Standardsolution and System suitabilitysolution Prepare Alprazolam Compounded Oral Suspension 1 mg/mL
[NoTE-For relative retention times, see Table 7.] asfollows (see Pharmaceutical Compounding-Nonsterile
Suitability requirements Preparations (795) ).
Resolution: NLT 2.0 between alprazolam related
compound A and alprazolam, System suitabilitysolution Alprazolam 100 mg
Relative standard deviation: NMT 5.0%, Standard
solution Vehicle: a 1:1 mixture of Vehicle tor Oral Solution
(regular or sugar-tree), NF, and Vehicle tor Oral Sus-
Analysis pension, NF, a sufficient quantity to make 100 mL
Samples: Standardsolution and Sample solution
Calculate the percentage of each impurity in the portion of
Alprazolam taken: Comminute tablets in a suitable mortar to a fine powder, or
add Alprazolampowder. Add about 20 mL of the Vehicle, and
Result = (r vir s) x (C siC v) x (liP> x 100 mix to a uniform paste. Add the Vehicle in small portions
almost to volume, and mix thoroughly after each addition.
ru = peak response for each impurity in the Sample Transfer the contents of the mortar, stepwise and
solution quantitatively, to a calibrated bottle. Add sufficient Vehicle to
rs =peak response for alprazolam from the Standard bring to final volume, and mix well.
solution
ASSAY
Cs = concentration of USP Alprazolam RS in the
Standardsolution (lJg/mL) • PROCEDURE
Buffer: 0.04 M sodium acetate solution. Adjust with glacial
Cu = concentration of Alprazolam in the Sample
acetic acid to a pH of 2.4.
solution (uq/rnt)
Mobile phase: Methanol, acetonitrile, and Buffer (45:8:47)
F = relative response factor (see Table 7)
Standard solution: 20 1J9/mL of USP Alprazolam RS in
Acceptance criteria: See Table 7. Mobilephase
Sample solution: .Agitate the container of Oral Suspension
for 30 min on arotating mixer, remove a 5-mL sample, and
store in a clear glass vial at -70° until analyzed. At the time
of analysis, remove the sample from the freezer,'allow it to

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146 Alprazolam / Official Monographs USP 43

reach room temperature, and mix with a vortex mixer for

30 s. Dilute a suitable volume of the Oral Suspension in
Mobile phase to obtain a nominal concentration of 20 ~g/
mL.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-~m packing L1
Flow rate: 0.6 mL/min
Injection volume: 20 ~L
System suitability
Sample: Standardsolution
[NOTE-The retention time of alprazolam is about 10
min.]
Suitability requirements
Relative standard deviation: NMT 1.4% for replicate
injections
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of
alprazolam (C 17H13CIN4) in the portion of Oral Suspension
taken:
Result = (rulrs) x (CslCu) x 100
= peak response from the Sample solution
= peak response from the Standardsolution
=concentration of USP Alprazolam RS in the
Standardsolution (~g/mL) ASSAY
= nominal concentration of alprazolam in the
Sample solution (~g/mL)

Acceptance criteria: 90.0%-110.0% • PROCEDURE

SPECIFIC TESTS
• pH (791): 4.0-5.0
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store at controlled room temperature, or in a
refrigerator.
• BEYOND-USE DATE: NMT 60 days after theday on which it
was compounded when stored at controlled room
temperature or in a refrigerator " o 60 40
• LABELING: Label it to state that it is to be well-shaken before
use, and to state the Beyond-Use Date. 2.5 60
• USP REFERENCE STANDARDS (11)
9.0
USP Alprazolam RS

Alprazolam Tablets
DEFINITION
Alprazolam Tablets contain NLT 90.0% and NMT 110.0% of
the labeled amount of alprazolam (C17H13CIN4) .
IDENTIFICATION

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USP 43 OfficialMonographs / Alprazolam 147

Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
alprazolam (C17H13CIN4) dissolved.
Tolerances: NLT 80% (Q) of the labeled amount of
alprazolam (C17H13CIN4) is dissolved.

>' _',c3")
Internal standard solution: 0.032mg/mL of triazolam in
acetonitrile
Standard solution: 0.025 mg/mL of USP Alprazolam RS in
Internalstandardsolution
Sample solution: Transfer 1 Tablet to a container. Add 0.4
mL of water directly onto the Tablet, allow the Tablet to
stand for 2 min, and then swirl the container to disperse the
Tablet. For every 0.25 mg of alprazolam contained in the
add 10.0 mL of Internal standardsolution to the
if necessary.

Cu

Acceptance criteria: 90.00/0-110.0%

PERFORMANCE TESTS
• DISSOLUTION (711 >, Procedure, Apparatus 1 and Apparatus 2,
Immediate-release dosage forms, Procedure for a pooled
sample for immediate-release dosage forms
Buffer stock solution: Dissolve 80 9 of monobasic I
potassium phosphate and 20 9 of dibasic potassium Analysis
phosphate in 1 Lof water. Add, with mixing, phosphoric Samples: Standard solution and Sample solution
acid or potassium hydroxide solution (45 in 100), as Calculate the percentage of the labeled amount of
necessary to adjust the solution, such that the resulting alprazolam (C17H 13CIN4) in the Tablet taken:
solution has a pH of 6.0 ± 0.1. .
Buffer: Prepare a 1-in-10 dilution of the Buffer stock solution Result =(Rul Rs) x C x V x (1001L)
to obtain a solution that has a pH of 6.0 ± 0.1.
Medium: Buffer, 500 mL Ru = peak area response ratio of alprazolam relative to
Apparatus 1: 100 rpm the internal standard from the Sample solution
Time: 30 min Rs = peak area response ratio of alprazolam relative to
Mobile phase: Acetonitrile, tetrahydrofuran, and Buffer the internal standard from the Standard solution
(35:5:60) C = concentration of USP Alprazolam RS in the
Standard stock solution: 0.05 mg/mL of USPAlprazolam Standard solution (mg/mL)
RS in methanol V = volume of the Internal standardsolution usedto
Standard solution: Add 50 mL of Buffer stock solution and prepare the Sample solution (mL)
250 mL of water to a 500-mL flask. Add to the flask 5.0 mL L = label claim (mg/Tablet)
of Standardstock solution for every 0.25 mg of alprazolam
contained in the Tablet being assayed. Dilute with water to Acceptance criteria: Meet the requirements
volume.
Sample solution: Pass a portion of the solution under test IMPURITIES
through a suitable filter.
Chromatographic system
(See Chromatography (621 >, System Suitability.)
Mode: LC '
Detector: UV 254 nm
Column: 4.6-mm x 10-cm; packing L7
Flow rate: 1 mL/min
System suitability
Sample: Standardsolution
Suitability requirements
Column efficiency: NLT 500 theoretical plates
Relative standard deviation: NMT 3.0% for replicate
injections

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148 Alprazolam / Official Monographs USP 43

Also ~nown inydro-5~phenyl-1H~1~4~

benzodia e;
C15HnCIN 2

Alprazolam Extended-Release Tablets

DEFINITION
Alprazolam Extended-Release Tablets contain NLT90.0% and
NMT 110.0% of the labeled amount of alprazolam
(C17 H13CIN4) .
IDENTIFICAnON
• A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
• B. The UV spectrum of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
• PROCEDURE
Mobile phase: Acetonitrile, water, and phosphoric acid
(350:650:1)
Standard solution: 0.05 mg/mL of USPAlprazolam RS in
Table 2 methanol
Sample solution: Nominally 0.05 mg/mL of alprazolam
Relative prepared as follows. Transfer an appropriate number of
Rete-ntion
Name Tiin~ ~ Tablets to a suitable volumetric flask. Sonicate in 80% of the
flask volume of methanol for 15 min, and shake
mechanically for 30 min. Dilute with methanol to final
volume, filter a portion of the solution, and discard the first
3 mL offiltrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mod~ LC .
Detector: UV 254 nm. For Identification B, use a diode array
detector in the range of 200-400 nm.
Column: 4.6-mm x 15-cm; 5-l.Im packing L7
Column temperature: 30°
Flow rate: 1 mL/min
Injection volume: 10 I.IL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0
Column efficiency: NLT 3000 theoretical plates
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
ADDITIONAL REQUIREMENTS alprazolam (C17H13CIN4) in the portion of Tablets taken:
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at controlled room temperature. Result = (rulrs) x (CslCu) x 100
= peak response from the Sample solution
= peak response from the Standard solution
• USP REFERENCE STANDARDS (11 ) = concentration of USP Alprazolam RS in the
USPAI razolam RS Standard solution (mg/mL)
~U = nominal concentration of alprazolam in the
:2 Sample solution (mg/mL)
Acceptance criteria: 90.0%-110.0%

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USP 43 Official Monographs / Alprazolam 149

PERFORMANCE TESTS Times: 1,4,8,and16h

• DISSOLUTION (711) Mobile phase: Acetonitrile, tetrahydrofuran, and Medium
Test 1 (35:5:60)
Medium: pH 6.0 phosphate buffer (8.0 giL of monobasic Standard stock solution: 0.05 mg/mL of USP Alprazolam
potassium phosphate and 2.0 giL of dibasic potassium RS in methanol
phosphate in water. Adjust with phosphoric acid or Standard solution: (LI500) mg/mL of USP Alprazolam RS
potassium hydroxide to a pH of 6.0 ± 0.1); 500 mL in Medium from the Standardstocksolution, where L isthe
Apparatus 1: 100 rpm label claim in mg/Tablet
Times: 1,4,8, and 12 h Sample solution: Pass a portion of the solution under test
Mobile phase: Acetonitrile, tetrahydrofuran, and Medium through a suitable filter.
(7:1 :12) Chromatographic system
Standard stock solution: 0.5 mg/mL of USP Alprazolam (See Chromatography (621), System Suitability.)
RS in acetonitrile Mode: LC
Standard solution: (LI500) mg/mL of USP Alprazolam RS Detector: UV 254 nm
in Medium from the Standardstocksolution, where L is the Column: 4.6-mm x 7.5-cm; 5-l-Im packing L7
label claim in mg/Tablet Flow rate: 1.3 mL/min
Sample solution: Pass a portion of the solution under test Injection volume: 80 I-IL -
through a suitable filter. System suitability
Chromatographic system Sample: Standard solution
(See Chromatography (621), System Suitability.) Suitability requirements
Mode: LC Tailing factor: NMT 1.5
Detector: UV 254 nm Relative standard deviation: NMT 2.0%
Column: 4.6-mm x 10-cm; 5-l-Im packing L7 Analysis
Flow rate: 1 mL/min Samples: Standard solution and Sample solution
Injection volume: 100 I-IL Calculate the concentration (Ci) of alprazolam
System suitability (C17H13CIN4) in the sample withdrawn from the vessel at
Sample: Standard solution each time point (I):
Suitability requirements
Tailing factor: NMT 2.0 Result, = (rulrs) x Cs
Column efficiency: NLT 3000 theoretical plates
Relative standard deviation: NMT 2.0% ru = peak response of alprazolam from the Sample
Analysis solution at each time point
Samples: Standard solution and Sample solution ts = peak response of alprazolam from the Standard
Calculate the percentage of the labeled amount of solution
alprazolam (C17H 13CIN4 ) dissolved: Cs = concentration of USP Alprazolam RS in the
Standard solution (mg/mL)
Result =(rulrs) x (CsiL) x V x 100
Calculate the percentage of the labeled amount of
ru = peak response from the Sample solution alprazolam (C17H13CIN4) dissolved at each time point (I):
rs = peak response from the Standard solution
Cs = concentration of USP Alprazolam RS in the Result, = C1 x V x (1I L) x 100
Standard solution (mg/mL)
L = label claim (mg/Tablet) Result, = {[C2 x (V - Vs)] + (C1 x Vs)} x (lIL) x 100
V = volume of Medium, 500 mL
Result, = ({C3 x [V - (2 x Vs)]} + [(C2 + C1) x Vs]) x (lIL) x
Tolerances: See Table 1. 100

Table 1 Result, = ({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C1) x Vs]) x (1I

Amount Dissolved L) x 100
Time 2-mg Tablet 3-mg Tablet Ci =concentration of alprazolam in the Sample
(h) O.5-mgTablet (%) (%) (%) solution at the specified time point (mg/mL)
1 NMT25 NMT20 NMT20 V =volume of Medium, 500 mL
L = label claim (mg/Tablet)
4 40-60 30-55 30-55 Vs =volume of the Sample solution withdrawn at each
8 70-90 65-90 65-90 time point (mL)
12 NLT 85 NLT 85 NLT 85 Tolerances: See Table 2.

The percentages of the labeled amount of alprazolam Table 2

(C17H13C1N4) released at the times specified conform to Amount Dissolved
Dissolution (711), Acceptance Table 2. Time
Point Time O.5-mg 1-mg 2-mg 3-mg
Test 2: Ifthe product complies with this test, the labeling (i) (h) Tablet (%) Tablet (%) Tablet (%) Tablet (%)
indicates that it meets USP Dissolution Test 2.
Medium: pH 6.0 phosphate buffer (8.0 giL of monobasic 1 1 NMT25 NMT25 NMT20 NMT20
potassium phosphate and 2.0 giL of dibasic potassium 2 4 45-60 40-55 30-50 25-45
phosphate in water. Adjust with phosphoric acid or
potassium hydroxide to a pH of 6.0 ± 0.1); 500 mL 3 8 70-90 65-85 55-75 50-70
Apparatus 1: 100 rpm . 4 16 NLT 85 NLT85 NLT 85 NLT80

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150 Alprazolam / OfficialMonographs USP 43

The percentages of the labeled amount of alprazolam Table 3

(C17H 13C1N4) released at the times specified conform to Amount Dissolved
Dissolution (711), Acceptance. Table 2. Time
Point Time O.5-mg I-mq 2-mg 3-mg
T~st .3: If the p~oduct complies with this test, the labeling (i) Tablet (%) Tablet (%) Tablet (%)
(h) Tablet (%)
indicates that It meets USP Dissolution Test 3.
Mediun;': pH 6.0 phosphate buffer (8.0 gIL of monobasic 1 1 15-35 10-30 10-30 5-25
potassium phosphate and 2.0 gIL of dibasic potassium 2 4 50-75 45-65 30-55 25-50
phosphate in water. Adjust with phosphoric acid or
potassium hydroxide to a pH of 6.0 ± 0.1); 500 mL, 3 8 NlT 75 NlT70 60-80 50-75
deaerated 4 16 - - NlT 85 NlT 80
Apparatus 1: 100 rpm
Times: 1, 4, and 8 h for Tablets labeled to 'contain 0.5 mg
or 1 mg; 1, 4, 8, and 16 'h for Tablets labeled to contain The percentages of the labeled amount of alprazolam
2 mg or 3 mg (C17H13C1N4) released at the times specified conform to
Mobile phase: Acetonitrile and Medium (40:60) Dissolution (711), Acceptance Table 2.
Standard stock solution: 0.5 mg/mL of USP Alprazolam Test 4: If the product complies with this test, the labeling
RS in methanol indicates that it meets USP Dissolution Test 4.
S~andar~ solution: (L/500) mg/mL of USPAlprazolam RS Medium: pH 6.0 phosphate buffer (8.0 gIL of monobasic
In Medium from the Standardstock solution, where L is the potassium phosphate and 2.0 gIL of dibasic potassium
label claim in mg/Tablet phosphate in water. Adjust with phosphoric acid or
Sample solution: Pass a portion of the solution under test potassium hydroxide to a pH of 6.0); 500 mL
through a suitable filter of t-urn pore size. Apparatus 1 (20-mesh basket): 100 rpm
Chromatographic system Times: 1,4,8, and 16 h
(See Chromatography (621), System SUitability.) Mobile phase: Acetonitrile and Medium (32:68)
Mode: LC Standard stock solution: 0,4 mg/mL of USPAlprazolam
Detector: UV 254 nm RS in methanol
Column: 4.6-mm x 10-cm; 3-l-Im or 5-l-Im packing L7 Standard solution: (L/500) mg/mL of USP Alprazolam RS
Flow rate: 1 mL/min in Medium from the Standardstocksolution, where L is the
Injection volume: 100 I-IL label claim in mg/Tablet. Pass through a suitable filter of
System suitability 0,45-l-Im pore size, and use the filtrate.
Sample: Standardsolution Sample solution: At the end of specified time intervals,
Suitability requirements withdraw a known volume (Vs) of the solution from the
Relative standard deviation: NMT 5.0% dissolution vessel, and replace an equal volume of fresh
Analysis Medium into the dissolution vessel. Pass the withdrawn
Samples: Standard solution and Sample solution sample through a suitable filter of 0,45-l-Im pore size, and
Calculate the concentration (C,) of alprazolam use the filtrate.
(C'7H13CIN4) in the sample withdrawn from the vessel at Chromatographic system
each time point (I): . (See Chromatography (621), System Suitability.)
Mode: LC
Result, = (rvirs) x Cs Detector: UV254 nm
Column: 4.6-mm x 15-cm; 5-l-Im packing L1
t» = peak response of alprazolam from the Sample Flow rate: 1.5 mL/min
solution at each time point Injection volume: 100 I-IL
rs = peak response of alprazolam from the Standard System suitability
solution . Sample: Standardsolution
Cs = concentration of USPAlprazolam RS in the Suitability requirements
Standard solution (mg/mL) Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0%
Calculate the percentage of the labeled amount of Analysis
alprazolam (C17H13CIN 4) dissolved at each time point (I): Samples: Standard solution and Sample solution
Calculate the concentration (C,) of alprazolam
Result, =C 1 x Vx (lIL) x 100 (C,7H,3C1N4) in the sample withdrawn from the vessel at
each time point (I):
Result, = {[C2 x (V - Vs)] + (C 1 x Vs)} x (1I L) x 100
Result, = (rvlrs) x Cs
Result, = ({C3 x [V - (2 x Vs)]} + [(C2 + C1) x Vs]) x (lIL) x
100 tu = peak response of alprazolam from the Sample
solution at each time point
Result, = ({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C1) x VsD x (1 I rs = peak response of alprazolam from the Standard
L) x 100 solution
Cs = concentration of USP Alprazolam RS in the
C; = concentration of alprazolam in the Sample Standard solution (mg/mL)
solution at the specified time point (mg/mL)
=volume of Medium, 500 mL Calculate the percentage of the labeled amount of
= label claim (mg/Tablet) alprazolam (C17H13C1N4) dissolved at each time point (I):
= volume of the Samplesolution withdrawn at each
time point (mL) Result, = C1 x VX (lIL) x 100

Tolerances: See Table 3. Result, = [(C2 x \I) + (C1 x Vs)] x (lIL) x 100

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USP 43 Official Monographs / Alprazolam 151

Result, = HC3 X VJ + [(C2 + C7) x Vs]} x (1 I L) x 100 rs = peak response of alprazolam from the Standard
solution
Result, = {[C4 X VJ + [(C3 + C2 + C7) x Vs]} x (lIL) x 100 Cs = concentration of USPAlprazolam RS in the
Standard solution(mg/mL)
C, =concentration of alprazolam in the Sample
solution at the specified time point (mg/mL) Calculate the percentage of the labeled amount of
V =volume of Medium, 500 mL alprazolam (C17H nCIN4) dissolved at each time point (I):
L = label claim (mg/Tablet)
Vs =volume of the Sample solutionwithdrawn at each Result, = C7 x V x (1I L) x 100
time point and replaced with Medium (mL)
Result, ={[C2 x (V - Vs)] + (C7 x Vs)} x (lIL) x 100
Tolerances: See Table 4.
Result, = ({C3 x [V- (2 x Vs)]} + [(C2 + C7) x VsD x (1I L) x
Table 4 100
Amount Dissolved
Time Result, = ({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C7) x Vs]) x (1I
Point Time O.5-mg Tab- 1-mg Tablet 2-mg Tablet 3-mg Tablet
(I) (h) let (%) (%) (%) (%) L) x 100
1 1 NMT40 NMT35 NMT35 NMT35 C; =concentration of alprazolam in the Sample
4
solution at the specified time point (mg/mL)
2 50-75 45-65 35-55 30-55
V = volume of Medium, 500 mL
3 8 NLT 75 70-90 55-75 50-70 L = label claim (mg/Tablet)
4 16 NlT 85 NlT 85 NlT 85 NlT75
Vs =volume of the Sample solutionwithdrawn at each
time point (mL)
The percentages of the labeled amount of alprazolam Tolerances: See Table 5.
(C'7H13C1N4) released at the times specified conform to
Dissolution (711), Acceptance Table 2. Table 5
Test 5: If the product complies with this test, the labeling Time Point Time
indicates that it meets USP Dissolution Test 5. (i) (h) Amount Dissolved (%)
Medium: pH 6.0 phosphate buffer (8.0 gIL of monobasic
1 1 NMT25
potassium phosphate and 2.0 gIL of dibasic potassium
phosphate in water. Adjust with phosphoric acid to a pH 2 4 40-65
of 6.0); 500 mL
3 8 65-95
Apparatus 1: 100 rpm
Times: 1,4,8, and 16 h 4 16 NlT 85
Mobile phase: Acetonitrile, water, and phosphoric acid
(350:650:1 ) The percentages of the labeled amount of alprazolam
Standard stock solution: 0.5 mg/mL of USPAlprazolam (C17H nCIN4) released at the times specified conform to
RS in methanol Dissolution (711), Acceptance Table 2.
Standard solution: (LI500) mg/mL of USP Alprazolam RS • UNIFORMITY OF DOSAGE UNITS (905): Meet the
in Medium from the Standard stock soiutlon, where L is the requirements
label claim in mg/Tablet . .
Sample solution: Pass a portion of the solution under test IMPURITIES
through a suitable filter of 0.45-lJm pore size, arid use the • ORGANIC IMPURITIES
filtrate. Buffer: 5.4 gIL of monobasic potassium phosphate
Chromatographic system (KH zP0 4) in water. Adjust with phosphoric acid to a pH of
(See Chromatography (621), System Suitability.) 3.4.
Mode: LC Solution A: Acetonitrile, methanol, and Buffer (27:1 0:63)
Detector: UV 254 nm Solution B: Acetonitrile, methanol, and Buffer (7:3:10)
Column: 4.6-mm x 15-cm; 5-lJm packing L7 Mobile phase: See Table 6.
Column temperature: 30°
Flow rate: 1 mL/min Table 6
Injection volume: 50 IJL Time Solution A Solution B
System suitability (min) (%) (%)
Sample: Standardsolution
0 95 5
Suitability requirements
Tailing factor: NMT 2.0 22 95 5
Relative standard deviation: NMT 2.0%
25 15 85
Analysis
Samples: Standardsolutionand Sample solution 60 15 85
Calculate the concentration (Ci) of alprazolam 5
60.1 95
(C'7HnCIN4) in the sample withdrawn from the vessel at
each tlrne point (I): 70 95 5

Result; = (rvIrs) x Cs System suitability solution: 1 IJg/mL each of USP

Chlordiazepoxide Related Compound A RS, USP
= peak response of alprazolam from the Sample Alprazolam Related Compound A RS, and USP
solution at each time point

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152 Alprazolam / Official Monographs USP43

Nordazepam RS; and 0.4 IJg/mL of USP Alprazolam RS in Table 7 (continued)

methanol Relative Relative Acceptance
Standard solution: 0.4 IJg/ml of USP Alprazolam RS in Retention Response Criteria,
methanol Name Time Factor NMT (%)
Sample solution: From NlT 20 Tablets ground to a fine Total impurities - - 2.0
powder, transfer an amount of powder to a suitable flask to
obtain a nominal concentration of 0.2 mg/ml of a Ifpossible from the manufacturingprocess.
alprazolam in methanol. [NoTE-Sonicatefor 15 min to b 7-Chloro-5-phenyl-l, 3-dihydro-2H-l ,4.benzodiazepin-2-one.
dissolve the contents.] Filter a portion, and discard the first c 7-Chloro-l-methyl-5-phenyl[1 ,2,4]triazolo[4,3-a]quinolin-4-amine.
1 ml of filtrate.
Chromatographic system ADDITIONAL REQUIREMENTS
(See Chromatography (621), System Suitability.) • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Mode: lC containers, and store at room temperature.
Detector: UV 230 nm • LABELING: The labeling states the Dissolution test used only
Column: 4.6-mm x 25-cm; 5-lJm packing l7 if Test 1 is not used.
Flow rate: 1.5 ml/min • USP REFERENCE STANDARDS (11)
Injection volume: 10 IJL USP Alprazolam RS
System suitability USP Alprazolam Related Compound A RS
"Samples: System suitability solution and Standard solution 2-(2-Acetylhydrazino)-7-chloro-5-phenyl-3H-l,4-
[NOTE-The relative retention times are listed in Table benzodiazepine.
7.] C17H1SCIN40 326.78
Suitability requirements USP Chlordiazepoxide Related Compound A RS
Resolution: NLT 1.5 between nordazepam and 7-Chloro-l,3-dihydro-5-phenyl-2H-l,4-benzodiazepin-2-
alprazolam; NLT 1.5 between chlordiazepoxide related one 4-oxide.
compound A and alprazolam related compound A, ClsHllCIN202 286.71
System suitability solution USP Nordazepam RS
Tailing factor: NMT 2.0 for the alprazolam peak, System
suitability solution
Relative standard deviation: NMT 5%, Standard
solution
Analysis Alprazolam Orally Disintegrating
Samples: Standard solution and Sample solution Tablets
Calculate the percentage of each impurity in the portion of
Tablets taken: DEFINITION
Alprazolam Orally Disintegrating Tablets contain NlT 90.0%
Result = (ru/rs) x (Cs/Cu) x (1/F) x 100 and NMT 110.0% of the labeled amount of alprazolam
tu = peak response of the impurity from the Sample (C17H13CIN4) ·
solution IDENTIFICATION
rs = peak response from the Standard solution • A. The retention time of the major peak of the Sample
Cs = concentration of USP Alprazolam RS in the solution corresponds to that of the Standardsolution, as
Standard solution (mg/mL) obtained in the Assay.
Cu = nominal concentration of alprazolam in the • B. The UV spectrum of the major peak of the Sample
Sample solution (mg/mL) solution corresponds to that of the Standardsolution, as
F = relative response factor (see Table 7) obtained in the Assay.
Acceptance criteria: See Table 7. ASSAY
• PROCEDURE
Table 7 Buffer: 6.8 gILof monobasic potassium phosphate in water.
Acceptance
Adjustwith phosphoric acid to a pH of 3.5.
Relative Relative
Retention Response Criteria, Diluent: Acetonitrileand water (60:40) .
Name Time Factor NMT (%) Mobile phase: Acetonitrile, methanol, and Buffer
(35:10:55)
Chlordiazepoxide Standard solution: 10 IJg/ml of USP Alprazolam RS in
related compound N 0.36 1.0 0.2
Diluent
Alprazolam related com- Sample solution: Nominally10 IJg/ml of alprazolam from
pound A 0.45 0.7 0.5 Tablets prepared as follows. Transfer 10 Tabletsto a suitable
Nordazepam- b 0.8 1.0 0.2 volumetric flask. Add Diluent to volume and pass through
a suitable filter. (NOTE-Sonicate with intermittent shaking
Alprazolam 1.0 - - to help dissolve, if necessary.]
2-Amino-5-chloro-benzo- Chromatographic system
phenone 1.8 0.9 0.5 (See Chromatography (621), System Suitability.)
Arnlno-derlvatlve- 2.2 1.2 0.5 Mode: lC
Detector: UV 221 nm. For Identification 8, use a diode array
Anyother individual degra- detector in the range of 200-400 nm.
dation - Column: 4.6-mm x 15-cm; 5-lJm packing l7
product 1.0 0.2
Column temperature: 30°
Flow rate: 1.5 ml/min
Injection volume: 30 IJL

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USP 43 Official Monographs / Alprazolam 153

System suitability phosphate in water. Adjust with phosphoric acid or

Sample: Standardsolution potassium hydroxide to a pH of 6.0 ± 0.1); 500 mL
Suitability requirements Apparatus 2: 50 rpm
Tailing factor: NMT1.5 Time: 10 min
Relative standard deviation: NMT 2.0% Buffer: 1.36 giL of monobasic potassium phosphate.
Analysis Adjust with dilute sodium hydroxide to a pH of 6.0.
Samples: Standardsolution and Sample solution Mobile phase: Acetonitrile and Buffer(35:65)
Calculate the percentage of the labeled amount of Standard stock solution: 0.05 mg/mL of USP Alprazolam
alprazolam (C17H nCIN 4) in the portion of Tabletstaken: RS in methanol. [NoTE-Sonicate to help dissolve, if
necessary.]
Result = (rulr s) x (CsICu) x 100 Standard solution: (LI500) mg/mL of USP Alprazolam RS
from the Standard stock solution in Medium, where L isthe
to = peak response from the Sample solution label claim in mg/Tablet
ts = peak response from the Standardsolution Sample solution: Pass a 5-mLaliquot of the solution under
Cs = concentration of USP Alprazolam RS in the test through a suitablefilter of 0.45-~m pore size,
Standardsolution (~g/mL) discarding the first 3 mL.
Cu = nominal concentration of alprazolam in the Chromatographic system
Sample solution (~g/mL) (See Chromatography (621), System Suitability.)
Mode: LC
Acceptance criteria: 90.0%-110.0% Detector: UV 230 nm
Column: 4.6-mm x 7.5-cm; 5-~m packing L7
PERFORMANCE TESTS Flow rate: 1.5 mL/min
• DISINTEGRATION (701) Injection volume: 40 ~L
Test 1 Run time: 3 times the retention time of alprazolam
Time: NMT 60 s System suitability
Test 2: If the product complies with this test, the labeling Sample: Standard solution
indicates that it meets USP Disintegration Test 2. Suitability requirements
Time: NMT 30 s Tailing factor: NMT 2.0
• DISSOLUTION (711) Relative standard deviation: NMT 2.0%
Test 1 Analysis
Medium: pH 6.0 phosphate buffer (8 giL of monobasic Samples: Standard solution and Sample solution
potassium phosphate and 2 giL of dibasic potassium Calculate the percentage of the labeled amount of
phosphate in water. Adjustwith phosphoric acid or alprazolam (C17H13CIN4) dissolved:
diluted potassium hydroxide to a pH of 6.0 ± 0.1); 900 mL
Apparatus 2: 50 rpm Result = (rulrs) x Cs x V x (1 I L) x 100
Time: 10 min
Mobile phase, Chromatographic system, and System ru = peak responsefrom the Sample solution
suitability: Proceed as directed in the Assay, except use ts = peak responsefrom the Standardsolution
an Injection volume of 100 ~L. Cs = concentration of USP Alprazolam RS in the
Standard stock solution: 0.05 mg/mL of USP Alprazolam Standard solution (mg/mL)
RS in methanol. [Nors-Sonlcate to help dissolve, if V = volume of Medium, 500 mL
necessary.] . L = label claim (mg/Tablet)
Standard solution: (Lll 000) mg/mL of USP Alprazolam RS
from the Standardstock solution in Medium, where L isthe Tolerances: NLT 70% (Q) of the labeled amount of
label claim in mg/Tablet . alprazolam (C17H nCIN4 ) is dissolved.
Sample solution: Passa portion of the solution under test • UNIFORMITY OF DOSAGE UNITS (905): Meet the
through a nylon membrane filter of 0.45-~m pore size, requirements
discarding the first few mL.
Analysis IMPURITIES
Samples: Standardsolution and Sample solution • ORGANIC IMPURITIES
Calculate the percentage of the labeled amount of Diluent: Prepare as directed in the Assay.
alprazolam (C17H13CIN4) dissolved: Buffer: 6.8 giL of monobasic potassium phosphate inwater.
Adjust with phosphoric acid to a pH of 3.0.
Result = (rulr s) x Cs x V x (lIL) x 100 Solution A: Acetonitrile, methanol, and Buffer(25:20:55)
Solution B: Acetonitrile, methanol, and Buffer(40:5:55)
ru = peak response from the Sample solution Mobile phase: See Table 1.
ts = peak response from the Standardsolution
Cs = concentration of USP Alprazolam RS in the Table 1
Standardsolution (mg/mL) Time Solution A Solution B
V = volume of Medium, 900 mL (min) (%) (%)
L = label claim (mg/Tablet)
0 100 a
Tolerances: NLT 80% (Q) of the labeled amount of 12 100 a
alprazolam (C17H nCIN4) is dissolved. 15 0 100
Test 2: Ifthe product complies with this test, the labeling
indicates that it meets USP Dissolution Test 2. 60 a ioo
Medium: pH 6.0 phosphate buffer (8 giL of monobasic 65 100 0
potassium phosphate and 2 giL of dibasic potassium
70 100 0

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154 Alprazolam / Official Monographs USP43

Standard solution: 0.6 ~g/ml of USP Alprazolam RS in

Diluent Alprostadil
Sa"!1ple solution: ~ominally 200 uq/ml, of alprazolam in
Diluent. Prepare usrnq 10 Tablets, and pass through a .
suitable filter. ~""~OH
Chromatographic system ~CH,
(See Chromatography (621), System Suitability.) Hl 6H
Mode: lC
Detector: UV 240 nm C2oH340S 354.48
Column: 4.6-mm x 15-cm; 5-~m packing l7 Prost-13-en-l-oic acid, 11, 15-dihydroxy-9-oxo-, (11 0.,13£,
Column temperature: 30° 155)-;
Flow rate: 1.2 ml/min (1 R,2R,3R)-3-Hydroxy-2-[(E)-(35)-3-hydroxy-l-octenyl]-5-
Injection volume: 25 ut, oxocyclopentaneheptanoic acid [745-65-3].
System suitability DEFINITION
Sample: Standard solution Alprostadil contains NlT 95.0% and NMT 105.0% of
Suitability requirements alprostadil (C2oH340S), calculated on the anhydrous basis.
Theoretical plates: NlT 2000 [CAUTION-Great care should be taken to prevent inhaling
Tailing factor: .NMT 1.5 particles of Alprostadil and exposing the skin to it.]
Relative standard deviation: NMT 6.0%
Analysis IDENTIFICATION
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of
Tablets taken:
Result = (rulrs) x (CslCu) x (1 IF) x 100
= peak response of each impurity from the Sample ASSAY
solution .• PROCEDURE
= peak response of alprazolam from the Standard Use freshly prepared solutions.
solution Mobile phase: Methanol, acetonitrile, and 0.1 M
= concentration of USP Alprazolam RS in the monobasic potassium phosphate (2:1:2). Adjust with
Standard solution (~g/ml) phosphoric acid to a pH of 3.0.
= nominal concentration of alprazolam in the Diluent: Methanol and water (90: 10)
Sample solution (~g/ml) Internal standard solution: 0.05 mg/ml of ethylparaben in
F = relative response factor (see Table 2) Diluent
Standard stock solution: 0.3 mg/ml of USP Alprostadil RS
Acceptance criteria: See Table 2. Disregard any peaks less in Diluent
than 0.05%. Standard solution: 0.2 mg/ml of USP Alprostadil RS
prepared by combining 2.0 ml of Standard stock solution
Table 2 with 1.0 ml of Internal standard solution
System suitability stock solution: 4.5 ~g/ml of USP
Relative Relative Acceptance
Retention Response Criteria, Prostaglandin A1 RS in Standard solution
Name Time Factor NMT (%) System suitability solution: Combine 2.0 ml of System
suitability stock solution with 1.0 ml of Internal standard
Alprazolam related com-
pound N,b 0.8 - - solution.
Sample stock solution: 0.3 mg/ml of Alprostadil in Diluent
Alprazolam 1.0 - - Sample solution: 0.2 mg/ml of Alprostadil prepared by
2-Amino-S-chlorobenzo- combining 2.0 ml of Sample stock solution and 1.0 ml of
phenone 2.9 1.9 0.5 Internal standard solution
Chromatographic system
Anyother unknown impuri- (See Chromatography (621), System Suitability.)
ty - 1.0 0.5
Mode: LC
Total impurities - - 2.0 Detector: Photodiode array detector or equivalent capable
of detecting UV wavelengths of 200-300 nm
: 2-.(2-Acetylhydrazino)-7-chloro-S-phenyl-3H-l ,4-benzodiazepine. Analytical wavelength: 200 nm
DIsregard the peak due to alprazolam related compound A, becauseit is a Column: 4.6-mm x 25-cm; packing II
processimpurityin alprazolam.
Column temperature: 40°
ADDITIONAL REQUIREMENTS Flow rate: 1 ml/min
• PACKAGING AND STORAGE: Preserve in tight containers, and
Injection volume: 20 ~l
store at controlled room temperature. System suitability
• LABELING: When more than one Disintegration test is given,
Sample: System suitability solution
~he labeling states the Disintegration test used only if Test 1
Suitability requirements
IS not us~d. When more than one Dissolution test is given,
Resolution: NlT 7.5 between prostaglandin A, and
the labeling states the Dissolution test used only if Test 1 is aJprostadil, and NlT 2.0 between prostaglandin A, and
not used. ~h~parnben . .
• USP REFERENCE STANDARDS (11) Relative standard deviation: NMT 2.0%, determined
USP Alprazolam RS fro.m the peak area ratio of alprostadil to ethylpara ben
Analysis
Samples: Standard solution and Sample solution

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USP 43 Official Monographs / Alprostadil 155

Calculate the percentage of alprostadil (C2oH340s) in the Temperatures

portion of Alprostadil taken: Drying: 100°
Ashing: 1000°
Result = (RuIR s) x (CsICu) x 100 Atomization: 2800°
Injection volume: 20 I-IL
= peak area ratio of alprostadil to the internal Analysis
standard from the Sample solution Samples: Standardsolution and Sample solution
= peak area ratio of alprostadil to the internal Calculate the percentage of rhodium (Rh) in the portion of
standard from the Standardsolution Afprostadil taken:
=concentration of USP Alprostadil RS in the
Standardsolution (mg/mL) Result = (AulAs) x (CsfCu) x 100
= concentration of Alprostadil in the Sample
solution (mg/mL) Au = absorbance of the Sample solution
As = absorbance of the Standard solution
Acceptance criteria: 95.0%-105.0% on the anhydrous Cs =concentration of rhodium in the Standard
basis solution (mg/mL)
IMPURITIES Cu =concentration of Alprostadil in the Sample
• RESIDUE ON IGNITION (281) solution (mg/mL)
Sample: 0.3 9
Acceptance criteria: NMT 0.002%
Acceptance criteria: NMT 0.5%
• LIMIT OF FOREIGN PROSTAGLANDINS, TEST 1
• LIMIT OF CHROMIUM
Use freshly prepared solutions.
Standard stock solution: 3.04 I-Ig/mL of chromium
trichloride in 0.05 M nitric acid Mobile phase: Methanol, acetonitrile, and 0.1 M
Standard solution: 20 ng/mL of chromium (Cr) in alcohol, monobasic potassium phosphate (2:1:2). Adjust with
phosphoric acid to a pH of 3.0.
prepared as follows. Transfer 2. mL of the St~ndard.stock
solution to a 1OO-mL volumetnc flask, and dilute with Diluent: Methanol and water (90:10)
alcohol to volume. Standard solution: 6 I-Ig/mL of USP Alprostadil RS,15
Sample solution: 1.0 mg/mL of Alprostadil in alcohol I-Ig/mL of USP Prostaglandin A1 RS, and 6 I-Ig/mL of USP
Prostaglandin B1 RS in Diluent
Blank: Alcohol
Instrumental conditions Sample solution: 3.0 mg/mL of Alprostadil in Diluent
Chromatographic system
(See Atomic Absorption Spectroscopy (852).)
Mode: Atomic absorption spectroscopy (See Chromatography (621), System Suitability.)
Mode: LC
lamp: Chromium hollow-cathode
Analytical wavelength: 357.9 nm Detector: Photodiode array detector or equivalent capable
of detecting UV wavelengths of 200-300 nm
Atomization type: Graphite furnace Analytical wavelengths
Temperatures Prostaglandin A1 : 224 nm
Drying: 100°
Ashing: 1000° Prostaglandin B1: 280 nm
Atomization: 2700° Other foreign prostaglandins: 200 nm
Injection volume: 20 I-IL Column: 4.6-mm x 25-cm; packing L1
Ana~ili . Column temperature: 40°
Samples: Standardsolution and Sample solution Flow rate: 1 mL/min
Calculate the percentage of chromium (Cr) in the portion Injection volume: 20 I-IL
of Alprostadil taken: System suitability
Sample: Standardsolution
Result = (AulAs) x (CslCu) x 100 Suitability requirements
Resolution: NLT 7.5 between prostaglandin A1 and
= absorbance of the Sample solution alprostadil
=absorbance of the Standardsolution Relative standard deviation: NMT 4.0%, determined
= concentration of chromium in the Standard from the peaks at their respective wavelength for
solution (mg/mL) replicate injections
=concentration of Alprostadil in the Sample Samples: Standardsolution and Sample solution
solution (mg/mL) Calculate the percentage of prostaglandin A1 and
prostaglandin B1 in the portion of Alprostadil taken:
Acceptance criteria: NMT 0.002%
• LIMIT OF RHODIUM Result = (rulr s) x (CsfCu) x 100
Standard stock solution: 100 I-Ig/mL of rhodium in 1.2 M
hydrochloric acid, prepared by diluting rhodium chloride = peak response of prostaglandin A 1 or
hydrate prostaglandin B1 from the Sample solution
Standard solution: 50 ng/mL of rhodium (Rh) in alcohol = peak response of prostaglandin A1 or
from Standardstock solution prostaglandin B1 from the Standardsolution
Sample solution: 2.0 mg/mL of Alprostadil in alcohol =concentration of USP Prostaglandin A1 RS or USP
Blank: Alcohol Prostaglandin Bl RS in the Standardsolution(mgl
Instrumental conditions mL)
(See Atomic Absorption Spectroscopy (852).) = concentration of Alprostadil in the Sample
Mode: Atomic absorption spectroscopy solution (mg/mL)
lamp: Rhodium hollow-cathode
Analytical wavelength: 343.5 nm
Atomization type: Graphite furnace

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156 Alprostadil / Official Monographs USP 43

Calculate the percentage of each impurity occurring at 200 Relative standard deviation: NMT 2.0%, determined
nm and eluting before prostaglandin A1 in the portion of from the main peak, Standardsolution
Alprostadil taken: Analysis
Samples: Standardsolution and Sample solution
Result = (rufrs) x (CslCv) x 100 Calculate the percentage of each impurity, excluding
prostaglandin B1, observed at 200 nm and eluting after
ro = peak response for each impurityfrom the prostaglandin A1 in the portion of Alprostadil taken:
Sample solution
rs = peak response for alprostadilfrom the Standard Result = (rvirs) x (CsiCv) x 100
solution
Cs = concentration of USP Alprostadil RS in the rv = peak response for each impurityfrom the
Standardsolution(mg/mL) Sample solution
Cv =concentration of AlprostadiJ in the Sample rs = peak response for alprostadil from the Standard
solution (mg/mL) solution
Cs = concentration of USP Alprostadil RS in the
Calculate the percentage of the impurity having a relative Standardsolution (mg/mL)
retention time of 0.6, relative to the prostaglandin A1 peak Cv = concentration of Alprostadil in the Sample
detected at 224 nm, in the portion of AlprostadiJ taken: solution (mg/mL)
Result = (rvlrs) x (CsICv) x 100 Acceptance criteria
Sum of the impurities at relative retention times 2.0 and
rv = peak response for any impurity having a relative 2.3: NMT 0.6%
retention time of 0.6, relativeto the Any other foreign prostaglandin impurity eluting after
prostaglandin A1 peak, from the Sample solution prostaglanin A,: NMT 0.9%
rs = peak response for prostaglandin A1 from the Total impurities: The sum of the impurities from Limit of
Standardsolution Foreign Prostaglandins, Test 7 and Test 2, is NMT 2.0%.
Cs = concentration of USP Prostaglandin A1 RS in the SPECIFIC TESTS
Standardsolution (mg/mL)
Cv = concentration of Alprostadil in the Sample • WATER DETERMINATION, Method I (921)
solution (mg/mL) Sample: 0.5 g
Acceptance criteria: NMT 0.5%
Acceptance criteria ADDITIONAL REQUIREMENTS
Prostaglandin A,: NMT 1.5% • PACKAGING AND STORAGE: Preserve in tight containers, and
Prostaglandin 8,: NMT 0.1% store in a refrigerator.
Any foreign prostaglandin impurity eluting before • USP REFERENCE STANDARDS (11)
prostaglandin A,: NMT 0.9% USP Alprostadil RS
Impurity at relative retention time 0.6, relative to USP Prostaglandin A1 RS
prostaglandin A,: NMT 0.9% USP Prostaglandin B1 RS
• LIMIT OF FOREIGN PROSTAGLANDINS, TEST 2 (13~,15S)-15-Hydroxy-9-oxoprosta-8(12),13-dien-l-oic
Mobile phase: Methanol, acetonitrile, and 0.02 M acid.
monobasic potassium phosphate (2:1:1).,Adjust with CZOH3Z04 336.47
phosphoric acid to a pH of 3.
System suitability solution: 6 ~g/mL of USP Alprostadil RS,
15 ~g/mL of USP Prostaglandin A1 RS, and 6 ~g/mL of USP
Prostaglandin Bl RS in methanol and water (9:1)
Standard solution: 10 ~g/mL of USP Alprostadil RS in Alprostadil Injection
acetonitrile and water (1 :1)
Sample solution: 5.0 mg/mL of Alprostadil in acetonitrile DEFINITION
and water (1 :1.). [NoTE-Sonicate if necessary.] AlprostadiUnjection is a sterile solution of Alprostadil in
Chromatographic system Dehydrated Alcohol. It contains NLT 90.0% and NMT
(See Chromatography (621), System Suitability.) 115.0% of the labeled amount of alprostadiJ (CZOH340S)'
Mode: LC IDENTIFICATION
Detector: Photodiode array detector or equivalent,
capable of detecting UV wavelengths of 200-300 nm
Analytical wavelengths
Prostaglandin A,: 224 nm
Prostaglandin 8,: 280 nm
Other foreign prostaglandins: 200 nm an amount equivalent to 2 mg of
Column: 4.6-mm x 25-cm; packing L1 atorostadu. on 500 mg of spectroscopic grade potassium
Flow rate: 1.2 mL/min bromide 40°-50° under vacuum. Prepare a pellet from
Injection volume: 20 ~L this mixture.
System suitability Standard: A solution of USP Alprostadil RS dissolved in
Samples: System suitability solution and Standardsolution dehydrated alcohol and processed as described in the
[NOTE-The relative retention times for alprostadiJ and Sample
prostaglandin A1 are 1.0 and 1.2, respectively, Acceptance criteria: Meets the requirements
System suitability solution.]
Suitability requirements
Resolution: NLT 4.0 between prostaglandin A1 ' and
alprostadil, System suitability solution

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USP 43 OfficialMonographs / Alteplase 157

ASSAY • STERILITY TESTS (71): It meets the requirements when

• PROCEDURE tested as directed in Test for Sterility of the Product to Be
Solution A: 40 mg/mL of a-bromo-2'-acetonaphthone in Examined, Membrane Filtration.
acetonitrile, freshly prepared • WATER DETERMINATION, Method I (921): NMT 0.4%
Solution B: 5 mg/mL of diisopropylethylamine in • OTHER REQUIREMENTS: It meets the requirements in
acetonitrile, freshly prepared Injections and ImplantedDrug Products (1).
Mobile phase: Methylene chloride, l,3-butanediol, and
water (1000: 6: 0.5) ADDITIONAL REQ.UIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, single-dose
Internal standard solution: 50 I-Ig/mL of ethylparaben in
methylene chloride containers, preferably of Type I glass. Store in a refrigerator.
• USP REFERENCE STANDARDS (11)
Standard stock solution: 0.5 mg/mL of USP Alprostadil RS
in dehydrated alcohol USP Alprostadil RS
Standard solution: 0.13 mg/mL of USP Alprostadil RS,
prepared as follows. Gently evaporate a 0.5-mL portion of
the Standardstock solution to dryness with a stream of
nitrogen. Add 150 I-IL of Solution A, rinse the inside of the Alteplase
container with this solution, and swirl. Add 150 I-IL of
Solution B to the container, rinse the inside of the container SYQVICROEK TQMIYQQHQS WLRPVLRSNR VEY<;WCNSGR AQCHSVPVKS
with this solution, and swirl. Cap and sonicate to dissolve. ttSEPRCFNGG TljQQALYFSO FVCQ~EIOTRA TCYEOQGISY

Heat the container at 45° for 45 min, swirling occasionally. RGTW~CTNWNSSA LAQKPYSGRR POAIRLGLGN~Y<rRNPORO
SKPWCYVFKA GKYSSEFCST PACSEGNSO YFGNGSAYRG THSLTESGAS
Sonicate again after heating is complete. Discard the CLPWNSMILI GKVYTAQNPS AQALGLGKHN Y RNPOGOAK PWCHVLKNRR
specimen if the entire sample does not dissolve. Evaporate LTWEYCOVPS CSTCGLRQYS QPQFR
the solution using a stream of nitrogen, add 2.0 mL of
Internal standard solution, and sonicate to dissolve. Discard IKGGLFAOIA SHPWQAAIFA KHRRSPGERF LCGGILISS WILSAAHCFQ
ERFPPHHLTV ILGRTYRWP GEEEQKFEVE KYIVHKEFOO OTYONOIALL
the specimen if the entire sample does not dissolve. QLKSOSSRCA QESSWRTVC LPPAOLQLPO WTE<;:ELSGYG KHEALSPFYS
Sample solution: Nominally 0.1 3 mg/mL of alprostadil, ERLKEAHV~ YPSSRljTSQH LLNRTVTONM LCAGOTRSGG PQA~Q
prepared as follows. Pool the contents of several containers GOSGGPLVCL NOGRMTLVGI ISWGLGCGQK OVPGVYTKVTrNYLoWIRONM

of the Injection. Gently evaporate a volume, equivalent to RP

0.25 mg of alprostadil, to dryness using a stream of

nitrogen. Proceed as directed for the Standardsolution, CZS69H3894N7460781S40 59,007.61
beginning with "Add 150 I-IL of Solution A". [105857-23-6].
Chromatographic system DEFINITION
(See Chromatography (621), System Suitability.) Alteplase is a highly purified glycosylated serine protease with
Mode: LC fibrin-binding properties and plasminogen-specific .
Detector: UV254 nm proteolytic activities. It is produced by recombinant DNA
Column: 4.4-mm x 25-cm; packing L18 synthesis in mammalian cell culture. It has a biological
Flow rate: 1.5 mL/min potency of NLT90.0% and NMT 115.0% of the potency
Injection volume: Equal volumes of Standardsolution and stated on the label, the potency being 580,000 USP Alteplase
Sample solution Units/mg of protein.
System suitability The presence of host cell DNAand host cell protein impurities
Sample: Standardsolution . in Alteplase is process specific; the limits of these impurities
[NOTE-The relative retention times for ethyl para ben are determined by validated methods.
and alprostadil are about 0.4 and 1.0,
respectively. ] IDENTIFICATION
Suitability requirements • A.
Resolution: NLT 9.0 between alprostadil and the internal Standard solution: 1.0-2.5 mg/mL of USP Alteplase RS
standard Sample solution: Prepare similarly to the Standardsolution.
Relative standard deviation: NMT 2.5% Analysis
Analysis Samples: Standardsolution and Sample solution
Samples: Standardsolution and Sample solution To each of three test tubes transfer 1 mL of 0.5-mg/mL
Calculate the percentage of the labeled amount of H-D-isoleucyl-prolyl-arginyl-p-nitroaniline
alprostadil (CzoH340s) in the portion of Injection taken: dihydrochloride. Separately transfer 200 I-IL of the
Standardsolution and 200 I-IL of the Sample solution to two
Result = (R v/R s) x (C sIC v) x 100 of the test tubes. To the third test tube add 200 I-IL of 0.2
M arginine solution that has been adjusted with
Ru = peak response ratio of alprostadil to the internal phosphoric acid to a pH of 7.3 (negative control). Mix the
standard from the Sample solution solutions in the three test tubes, and allow to stand for 1
R5 = peak response ratio of alprostadil to the internal min.
standard from the Standardsolution Acceptance criteria: A yellow color is produced in the
Cs = concentration of USPAlprostadil RS in the solutions from the Standardsolution and the Sample
Standardsolution (mg/mL) solution, while no yellow color is produced in the negative
Cu = nominal concentration of alprostadil the Sample control.
solution (mg/mL) • B. PEPTIDE MAPPING
Solution A: 6.9 mg/mL of monobasic sodium phosphate in
Acceptance criteria: 90.00/0-115.0% water, adjusted with phosphoric acid to a pH of 2.85. Filter,
and degas.
SPECIFIC TESTS Solution B: Acetonitrile
• BACTERIAL ENDOTOXINS TEST (85): It contains NMT 5 USP Mobile phase: See Table 7.
Endotoxin Units/100 I-Ig of alprostadil.

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158 Alteplase / OfficialMonographs USP 43

Table 1 Human fibrinogen solution: 2 mg/mL of human fibrinogen

Time Solution A Solution B in Buffer
(min) (%) (%) Human plasminogen solution: 1 mg/mL of human
0 100 0 plasminogen in Buffer
Standard stock solution: 1.0 mg/mL (580,000 USP
91 70 30 Alteplase Units) of USP Alteplase RS in water
121 40 60 Standard solutions: Dilute volumes of Standardstock
solution with water to obtain a series of five Standard
131 40 60 solutions having known concentrations ranging from 145
to 9.3 USPAlteplase Units/mL.
Dialysis solution: 480 mg/mL of urea, 44 mg/mL of Sample stock solution: 1.0 mg/mL of Alteplase in water
tris(hydroxymethyl)aminomethane, and 0.88 mg/mL of Sample solutions: Dilute a volume of Sample stock solution
edetic acid in water. Adjust with hydrochloric acid to a pH with Bufferto obtain a series of dilutions of about 1:20,000;
of 8.6. 1:10,000; and 1:5,000.
Standard solution: Prepare a solution containing 1.0 Analysis
mg/mL of USPAlteplase RS in water. Dialyze 2.0 mL of this Samples: Standardsolutions and Sample solutions
solution into the Dialysis solution at room temperature for To a set of labeled glass test tubes add 0.5 mL of Human
NLT 12 h. Measure the volume of the solution, and transfer thrombin solution. To separate test tubes add 0.5 mL of
it to a clean test tube. For each mL of solution in the tube, each Standardsolution or Sample solution, and store on ice.
add 10 f.JL ofl M dithiothreitol. Incubate at room To a second set of labeled glass tubes, add 20 f.JL of Human
temperature for 4 h, then add 25 lJL of 1 M iodoacetic acid plasminogen solution and 1 mL of Human fibrinogen
per mL of the solution, and incubate in the dark for 30 min. solution, and store on ice. Beginning with the thrombin-
Quench the reaction by adding 50 f.JL of 1 M dithiothreitol Standardsolution mixture containing the Standard
per mL of the solution. Dialyze the solution against 0.1 M solution with the lowest number of USP Units/mL, record
ammonium bicarbonate for 24 h, replacing the 0.1 M the time, and separately add 200 f.JL of each of the
ammonium bicarbonate twice during the dialysis period. thrombin-Standard solution mixtures to the test tubes
To 2:0 mL of the dialyzed solution, add 20 lJg of trypsin, " containing the plasminogen-fibrinogen mixture. Using a
and Incubate for 6-8 h at room temperature. Again add 20 vortex mixer, intermittently mix the contents of each tube
f.Jg of trypsin, and incubate for 16-18 h for a total of 24 h for a total of 15 s, and carefully place into a rack in a 3r
of incubation of the trypsin-treated solution. circulating water bath. A visually turbid clot forms within
[NOTE-Store the Standardsolution in a freezer.] 30 s, followed by the formation of bubbles within the clot.
Sample solution: Using an accurately weighed quantity of Record the clot lysis time (t c/) from the first addition of the
Alteplase, proceed as directed in the Standardsolution. Alteplase solution to the last bubble to rise to the surface.
Chromatographic system Using a least squares fit, determine the equation of the line
(See Chromatography (621), System Suitability.) using the log values of the standard concentration, in USP
Mode: LC Alteplase Units/mL, versus the log values of their clot lysis
Detector: UV 214 nm times in s taken:
Column: 4.6-mm x 10-cm; packing L1
Flow rate: 1 mL/min log t= m(log Us) + b
Injection volume: 100 f.JL
System suitability t = time to bubble release (s)
Sample: Standardsolution m = slope of the line
Suitability requirements Us =activity of the Standard solution (USP Alteplase
Resolution: NLT 1.5 between peaks 6 and 7 "as defined Units/mL)
by the USPAlteplase RS Data Sheet. The times for peaks b = y-intercept of the line
6 and 7 baseline widths are NMT 0.5 min.
Analysis The correlation coefficient is NLT -0.9900. From the line
Samples: Standardsolution, Sample solution, and a mixture equation and using the log of the clot lysis time for the
of the Standardsolution and the Sample solution (1:1) Sample solution, calculate the log of the activity (U .J:
Measure the responses for NLT 20 major peaks as defined
in the USPAlteplase RS Data Sheet. log U A = {[(log t) - b]/m}
Acceptance criteria: The retention times of the
corresponding peaks of the Standardsolution and the Calculate the alteplase activity in USP Alteplase Units/mL
Sample solution do not differ by more than 0.4 min, and the taken:
peak area ratios relative to peak 19 (as shown on the USP Result = 0(1 0 log U)
Alteplase RS Data Sheet) do not differ by more than 20%.
No additional significant peaks or shoulders are found, a o = dilution factor for the Sample solution
significant peak or shoulder being defined as one having a
peak response of NLT 5% of peak 19. Calculate the specific activity in the portion of Alteplase
ASSAY taken:
• BIOLOGICAL POTENCY
Buffer: 1.38 mg/mL of monobasic sodium phosphate, 7.10 Result =(U A/ P)
mg/mL of anhydrous dibasic sodium phosphate, 0.20 mg/
P = concentration of protein obtained in the test for
!TIL of sodium azide, and 0.10 mg/mL of polysorbate 80 Protein Content
In water
Human thrombin solution: 33 U.S. Units in terms of the Acceptance criteria: 90.00/0-115.0% of the potency stated
U.S. Standard Thrombin/mL in Buffer on the label, the potency being 580,000 USP Alteplase
Units/mg of protein

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USP 43 OfficialMonographs / Alteplase 159

OTHER COMPONENTS Molecular weight standard solution: Usea commercially

• PROTEIN CONTIENT available preparation of low molecular weight protein
Arginine solution: 34.8 mg/mL of arginine in water. Adjust standards (10,000-100,000 Da) at 2 mg/mL. Mix 990 ~L
with phosphoric acid to a pH of 7.3. of Diluted 505 bufferand 10 ~L of the molecular weight
Sample stock solution: 1 mg/mL of Alteplase in water standard mixture.
Sample solution: Dilute a volume of Sample stock solution Control solution: Preparea control solution of bovine
with a volume of Arginine solution to obtain a solution serum albumin containing 10 ~g/mL. For a 10 ng/25 ~L
having an absorbance value of 0.5-1.0 at the wavelength load, mix 600 ~L of Diluted505 buffer and 25 ~L of the
of maximum absorbance at about 280 nm. Determine the control solution, and heat at 90° for 2 min. For a 2.5 ng/25
dilution volume (V). ~L load, mix 594 ~L of Diluted505 buffer and 6 ~L of the
Instrumental conditions control solution, and heat at 90° for 2 min.
(See Ultraviolet-Visible Spectroscopy (857).) Standard stock solution: 1 mg/mL of USP Alteplase RS in
Mode: UV water
Wavelength range: 240-500 nm Standard solution: Dilute an accurately measured volume
Analytical wavelengths: 320 nm and maximum of Standardstock solution in Arginine solution to obtain a
absorbance at about 280 nm solution having a final concentration of 0.25 mg/mL. Heat
Cell: 1 cm 0.5 mL of this solution with 116 ~L of 505 buffer and 10 ~L
Blank: Arginine solution of 1 M dithiothreitol at 80° for 2 min.
Analysis Carboxymethylated standard solution: Dilute 1.0 mL of
Samples: Sample solution and Blank Standard stock solution with 1 mL of Carboxymethylation
Calculate the protein content in the portion of Alteplase buffer, and adjust with 1 M sodium hydroxide to a pH of
taken: 8.5. Add 20 ~L of 1 M dithiothreitol, and incubate at 37°
for 60 min. Add 100 ~L of 1 M iodoacetic acid, and incubate
Result = [(A max - A 320)/8] X V in the dark for 20 min. Desalt by passing the solution
through a chromatographic column containing fine gel
A max = absorbance value at the wavelength of chromatographic packing equilibrated with a buffer
maximum absorbance solution containing 20 mg/mL of sodium dodecyl sulfate,
A 320 = absorbance of the Sample solution at 320 nm 100 mg/mL of glycerol, 1.42 mg/mL of tris(hydroxymethyl)
8 = molar absorptivity of Alteplase, 1.9 aminomethane hydrochloride, and 0.85 mg/mL of
V = volume of Arginine solution required to prepare tris(hydroxymeth yl)aminomethane. Collect the protein
the Sample solution fraction of the preparation by elution with the same buffer,
and add 20 ~L of 1 M dithiothreitol. Adjust the protein
IMPURITIES concentration to about 0.2 mg/mL with a buffer solution
• CHROMATOGRAPHIC PURITY containing 20 mg/mL of sodium dodecyl sulfate, 100
SDS buffer: 400 mg/mL of glycerol, 5.52 mg/mL of mg/mL of glycerol, 1.42 mg/mL of tris(hydroxymeth yl)
tris(hydroxymethyl)aminomethane hydrochloride, 3.28 aminomethane hydrochloride, 0.85 mg/mL of
mg/mL of tris(hydroxymethyl)aminomethane, 0.20 tris(hydroxymethyl)aminomethane, 1.06 mg/mL of
mg/mL of bromophenol blue, and 0.20 mg/mL of xylene dithiothreitol, 0.05 mg/mL of bromophenol blue, and 0.05
cyanole FF in sodium dodecyl sulfate solution (8 in 100) mg/mL of xylene cyanole FF.
Diluted SDS buffer: Dilute 1 volume of 505 buffer with 4 Sample stock solution, Sample solution, and
volumes of water. Carboxymethylated sample solution: Using an
Running buffer: 3.03 mg/mL of tris(hydroxymeth yl) accurately weighed quantity of Alteplase, proceed as
aminomethane and 14.26 mg/mL of glycine in sodium directed for Standard stock solution, Standard solution, and
dodecyl sulfate (1 in 1000) . Carboxymethylated standardsolution.
Carboxymethylation buffer: 480 mg/mL of urea, 44 Blank: Mix 500 ~L of water, 126 ~L of 505 buffer, and 10 ~L
mg/mL of tris(hydroxymethyl)aminomethane, and 1.2 mg/ of 1 M dithiothreitol.
mL of edetic acid in water. Adjust with hydrochloric acid, if Analysis
necessary, to a pH of 8.6. Samples: Molecularweight standardsolution, Control
Ammoniacal silver nitrate solution: Transfer 105 mL of solution, Standard solution, Carboxymethylated standard
sodium hydroxide solution (0.36 in 100) and 7.0 mL of solution, Sample solution, Carboxymethylated sample
ammonium hydroxide to a 500-mL volumetric flask, and solution, and Blank
add slowly, with stirring, 20.0 mL of silver nitrate solution Separately apply equal volumes (about 25 ~L) of the
(20 in 100). Dilute with water to volume. Standardsolution, Carboxymethylated standardsolution,
[NoTE-Prepare this solution immediately before use, Sample solution, and Carboxymethylated sample solution at
and protect it from light. This amount of solution is the 5-~g load; apply equal volumes (about 38 ~L) of the
sufficient for two slab gels.] Standard solution and the Carboxymethylated standard
Citric acid-formaldehyde solution: To 500 mL of water solution at the 7.5-~g load; and apply the Control
add 25 mg of citric acid, 0.25 mL of formaldehyde, and solutions at the 10- and 2.5-ng load onto separate lanes
0.025 mL of methanol, omitting the methanol if the of the gel. Apply about 25 ~L of the Molecular weight
formaldehyde is preserved with methanol. standardsolution to each side of the gel, and apply about
[Nora-Prepare this solution fresh at the time of use.This 25 ~L of the Blank onto a separate lane. Apply the
amount of solution is sufficient for two slab gels.] Standard solution and the Sample solution on one half and
Gel: Preparea 10% T (total acrylamide)-0.25% C the Carboxymethylated standardsolution and
(cross-linked bisacrylamide) resolving gel containing 0.1% Carboxymethylated sample solution on the other half.
sodium dodecyl sulfate; 0.375 M tris(hydroxymethyl) Perform the electrophoresis using a constant current of
aminomethane hydrochloride, and 0.05 M 1.3-1.5 mAmp/cm of gel length and the Running buffer.
·tris(hydroxymethyl)aminomethane. Remove the gel from the apparatus 10-20 min after the
Arginine solution: 34.8 mg/mL of arginine in water. Adjust tracking dye starts to move. Place the gel in 250 mL of a
with phosphoric acid to a pH of 7.3. solution of 20% alcohol and 6% glacial acetic acid for NLT

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160 Alteplase / Official Monographs USP 43

1 h, and change the solution every 20 min, leavingthe gel Suitability requirements
to soak overnight following the last change. Resolution: NLT 1.1 between the single-chain and
Perform silver staining of the gel by placing the gel in 250 two-chain alteplase peaks
mLof a 10% glutaraldehyde solution (vlv) in a shallow Analysis
dish, and shake for about 30 min. Replace the Samples: Standard solution and Sample solution
glutaraldehyde solution with distilled water, allowthe gel [NOTE-The major peaks are from single-chain and
to soak for about 20 min, and then change the water. two-chain alteplase and from higher and lower
Repeat for a total of three washings. Transfer the gel to a molecular weight species.]
dish, and cover with 250 mLof Ammoniacal silver nitrate Calculate the percentage of single-chain alteplase in the
solution. Placethe dish on a shakerfor about 15min. Rinse portion of Alteplase taken:
four times with 250 mL of water, rocking the dish for 1
min between rinses. Continue rocking to prevent the gel Result = (r vir r) x 100
from sticking and to facilitate washing.
Transferthe gel to a clear dish containing 250 mLof Citric ru = peak response for single-chain alteplase
acid-formaldehyde solution, and rock the dish. Protein rr =sum of all the peak responses of alteplase
bands become visible. When the gel is visibly stained,
wash immediatelywith water, and rinseit repeatedly with Acceptance criteria: No peaks or shoulders inthe Sample
water to remove the Citric acid-formaldehyde solution. solution that are not present in the Standard solution are
Rinse the gel for NLT 1 h, and dry. Soakcellophane found; NLT 60%.
membranes in glycerol solution (2 in 100). Roll a
membrane onto a rigid sheet of plastic. Roll the gel onto ADDITIONAL REQUIREMENTS
the membrane, and coverwith another membrane. Lay a • PACKAGING AND STORAGE: Preserve in tight containers, and
frame on the edges of the membranes, and clamp it to store in the frozen state at a temperature of -20° or below.
the rigid plastic sheet. Dismantle the dryer, and cut off • USP REFERENCE STANDARDS (11)
excess cellophane when dry (about 24 h). Visually USP Alteplase RS
examine the gel under light.
System suitability: The 2.5- and 10-ng Control solutions
must be visible. The nonreduced Control solutions migrate
with an apparent molecularweight of slightly less than Alteplase for Injection
66,000 Da,as compared with the Molecular weight standard
solution. DEFINITION
Acceptance criteria: The Sample solution exhibits three Alteplase for Injection is a sterile lyophilized preparation of
major bands in the region between 66,000 Da and 31,000 Alteplase. Its biological activity is NLT 90% and NMT 115%
Da, corresponding to the major bands from the Standard of that stated on the label in USP Alteplase Units. It contains
solution. The Carboxymethylated sample solution exhibits six NLT 95% and NMT 111% of the total protein content stated
major bands in the region between 92,500 Da and 45,000 on the label.
Da, corresponding to the major bands from the
Carboxymethylated standard solution. IDENTIFICATION
SPECIFIC TESTS
• A.
Standard solution: 1.0-2.5 mg/mL of USP Alteplase RS in
• BACTERIAL ENDOTOXINS TEST (85): NMT 1 USP Endotoxin water
Unit/mg of Alteplase . Sample solution: Prepare similarly to the Standard solution.
• SINGLE-CHAIN CONTENT Analysis
Mobile phase: 27.6 9 of monobasic sodium phosphate in Samples: Standard solution and Sample solution
1000 mLof sodium dodecyl sulfate solution (1 in 1000). To each of three test tubes transfer 1 mLof 0.5-mg/mL
Adjustwith sodium hydroxide to a pH of 6.8. Filter, and H-o-isoleucyl-prolyl-arginyl-p-nitroaniline
degas. . dihydrochloride. Separatelytransfer 200 IJL of the
Dithiothreitol solution: 3.12 mg/mL of dithiothreitol in Standard solution and 200 IJL of the Sample solution to two
Mobile phase of the test tubes. To the third test tube add 200 IJL of 0.2
Standard stock solution: Using an accuratelyweighed M arginine solution that has been adjusted with
quantity of USP Alteplase RS, make a 1-mg/mL solution in phosphoric acid to a pHof 7.3 (negative control). Mix the
water. solutions in the three test tubes, and allow to stand for 1
Standard solution: Pipet 1 mL of the Standard stock min. I
solution into a glass tube, add 3 mL of Dithiothreitol Acceptance criteria: Ayellowcolor is produced in the
solution, cap the tube, and invert to mix. Heatfor 3-5 min solutions from the Standard solution and the Sample
at about 80°. solution, while no yellow color is produced in the negative
Sample stock solution: Using an accuratelyweighed control.
quantity of Alteplase, make a 1-mg/mL solution in water. • B. PEPTIDE MAPPING
Sample solution: Pipet 1 mL of the Sample stock solution Solution A: 6.9 mg/mL of monobasic sodium phosphate in
into a glasstube, add 3 mL of Dithiothreitol solution, cap the water, adjusted with phosphoric acid to a pHof 2.85. Filter,
tube, and invert to mix. Heat for 3-5 min at about 80°. and degas.
Chromatographic system Solution B: Acetonitrile
(See Chromatography (621), System Suitability.) Mobile phase: See Table 1.
Mode: LC
Detector: UV 214 nm Table 1
Column: 7.5-mm x 60-cm; packing L25 Time Solution A Solution B
Flow rate: 0.5 mL/min (min) (%) (%)
Injection volume: 50 IJL
System suitability 0 100 0
Sample: Standard solution

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 USP 43 Official Monographs / Alteplase 161

Table 1 (continued) Human plasminogen solution: 1 mg/mL of human

Time Solution A Solution B plasminogen in Buffer
(min) (%) (%) Standard stock solution: 1.0 mg/mL (580,000 USP
Alteplase Units) of USP Alteplase RS in water
91 70 30
Standard solutions: Dilute volumes of Standardstock
121 40 60 solution with water to obtain a series of five Standard
131 40 60
solutions having known concentrations ranging from 145
to 9.3 USP Alteplase Units/mL.
Sample stock solution: 1.0 mg/mL of Alteplase for
Dialysis solution: 480 mg/mL of urea, 44 mg/mL of Injection in water
tris(hydroxymethyl)aminomethane, and 0.88 mg/mL of Sample solutions: Dilute a volume of Sample stocksolution
edetic acid in water. Adjust with hydrochloric acid to a pH with Bufferto obtain a series of dilutions of about 1:20,000;
of 8.6. 1:10,000; and 1:5,000.
Standard solution: Prepare a solution containing 1.0 Analysis
mg/mL of USP Alteplase RS in water. Dialyze 2.0 mL of this Samples: Standardsolutions and Sample solutions
solution into the Dialysis solution at room temperature for To a set of labeled glasstest tubes add 0.5 mL of Human
NLT 12 h. Measure the volume of the solution, and transfer thrombin solution. To separate test tubes add 0.5 mL of
it to a clean test tube. For each mL of solution in the tube, each Standardsolution or Sample solution; and store on ice.
add 10 ~L of 1 M dithiothreitol. Incubate at room To a second set of labeled glass tubes, add 20 ~L of Human
temperature for 4 h, then add 25 ~L of 1 M iodoacetic acid plasminogen solution and 1 mL of Human fibrinogen
per mL of the solution, and incubate in the dark for 30 min. solution, and store on ice. Beginning with the thrombin-
Quench the reaction by adding 50 ~L of 1 M dithiothreitol Standardsolution mixture containing the Standard
per mL of the solution. Dialyze the solution against 0.1 M solution with the lowest number of USP Units/mL, record
ammonium bicarbonate for 24 h, replacing the 0.1 M the time, and separately add 200 ~L of each of the
ammonium bicarbonate twice during the dialysis period. thrombin-Standard solution mixtures to the test tubes
To 2.0 mL of the dialyzed solution, add 20 ~g of trypsin, containing the plasminogen-fibrinogen mixture. Using a
and incubate for 6-8 h at room temperature. Again add 20 vortex mixer, intermittently mix the contents of each tube
~g of trypsin, and incubate for 16-18 h for a total of 24 h for a total of 15 s, and carefully place into a rack in a 3r
of incubation of the trypsin-treated solution. [NOTE-Store circulating water bath. A visually turbid clot forms within
the Standardsolution in a freezer.] 30 s, followed by the formation of bubbles within the clot.
Sample solution: Using a quantity of Alteplase for Injection, Record the clot lysistime (t C/) from the first addition of the
proceed as directed in the Standardsolution. alteplase solution to the last bubble to rise to the surface.
Chromatographic system Using a least squaresfit, determine the equation of the line
(See Chromatography (621), System Suitability.) using the log valuesof the standard concentration, in USP
Mode: LC Alteplase Units/mL, versusthe log values of their clot lysis
Detector: UV 214 nm times in seconds taken:
Column: 4.6-mm x 10-cm; packing L1
Flow rate: 1 mL/min log t = m(log U s) + b
Injection volume: 100 ~L
System suitability t =time to bubble release (s)
Sample: Standardsolution m =slope of the line
Suitability requirements . Us =activity of the Standardsolution (USP Alteplase
Resolution: NLT 1.5 between peaks 6 and 7 as defined Units/mL)
by the USP Alteplase RS Data Sheet. The times for peaks b =y-intercept of the line
6 and 7 baseline widths are NMT 0.5 min.
Analysis The correlation coefficient is NLT -0.9900. From the line
Samples: Standardsolution, Sample Solution, and a mixture equation and using the log of the clot lysis time for the
of the Standardsolution and the Sample solution (1:1) Sample solution, calculate the log of the activity (U A):
Measure the responses for NLT 20 major peaks as defined
in the USP Alteplase RS Data Sheet. log U A = {[(log t) - b]1m}
Acceptance criteria: The retention times of corresponding
peaks of the Standardsolution.and the Sample solution .do Calculate the alteplase activity in USP Alteplase Units/mL
not differ by more than 0.4 min, and the peak area ratios taken:
relative to peak 19 (as shown on the USP Alteplase RS Data
Sheet) do not differ by more than 20%. No additional Result= 0(1 0 log U)
significant peaks or shoulders are found, a significant peak
or shoulder being defined asone having a peak responseof o = dilution factor for the Sample solution
NLT 5% of peak 19.
Calculate the specific activity in the portion of Alteplase for
ASSAY Injection taken:
• BIOLOGICAL POTENCY
Buffer: 1.38 mg/mL of monobasic sodium phosphate, 7.10 Result = (U AlP)
mg/mL of anhydrous dibasic sodium phosphate, 0.20 mgl
mL of sodium azide, and 0.10 mg/mL of polysorbate 80 P =concentration of protein obtained in the test for
in water Protein Content
Human thrombin solution: 33 U.S. Units in terms of the
U.S. Standard Thrombin/mL in Buffer Acceptance criteria: 90%-115% of the potency stated on
Human fibrinogen solution: 2 mg/mL of human fibrinogen the label in USP Alteplase Units
in Buffer

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162 Alteplase / Official Monographs USP 43

OTHER COMPONENTS Result = (r vir T) x 100

• PROTEIN CONTENT
Arginine solution: 34.8 mg/mL of arginine in water. Adjust ro = peak response of the alteplase monomer
with phosphoric acid to a pH of 7.3. rT =sum of all the peak responses related to alteplase
Sample stock solution: 1 mg/mL of Alteplase for Injection
in water Acceptance criteria: NLT 95.0%
Sample solution: Dilute a volume of Sample stock solution • SINGLE-CHAIN CONTENT
with a volume of Arginine solution to obtain a solution Mobile phase: 27.6 mg/mL of monobasic sodium
having an absorbance value of 0.5-1.0 at the wavelength phosphate in sodium dodecyl sulfate solution (1 in 1000).
of maximum absorbance at about 280 nm. Determine the Adjust with sodium hydroxide to a pH of 6.8. Filter, and
dilution volume (\I). degas.
Instrumental conditions Dithiothreitol solution: 3.12 mg/mL of dithiothreitol in
(See Ultraviolet- Visible Spectroscopy (857).) Mobile phase
Mode: UV Standard stock solution: Using an accurately weighed
Wavelength range: 240-500 nm quantity of USP Alteplase RS; make a 1-mg/mL solution in
Analytical wavelengths: 320 nm and maximum water.
absorbance at about 280 nm Standard solution: Pipet 1 mL of the Standard stock
Cell: 1 cm solution into a glass tube. Add 3 mLof Dithiothreitol
Blank: Arginine solution solution, cap the tube, and invert to mix. Heat for 3-5 min
Analysis at about 80°.
Samples: Sample solution and Blank Sample stock solution: Using an accurately weighed
Calculate the protein content in the portion of Alteplasefor quantity of Alteplase for Injection, make a 1-mg/mL
Injection taken: solution in water.
Sample solution: Pipet 1 mL of the Sample stock solution
Result = [(A max - A 320)18] X V into a glass tube. Add3 mLof Dithiothreitol solution, cap the
tube, and invert to mix. Heat for 3-5 min at about 80°.
A max =absorbance value at the wavelength of Chromatographic system
maximum absorbance (See Chromatography (621), System Suitability.)
A 320 = absorbance of the Sample solution at 320 nm Mode: LC
8 = molar absorptivity of alteplase, 1.9 Detector: UV 214 nm
V = volume of Arginine solution required to prepare Column: 7.5-mm x 60-cm; packing L25
the Sample solution Flow rate: 0.5 mL/min
Injection volume: 50 J..IL
Acceptance criteria: 950/0-111 % of the total protein System suitability
content stated on the label Sample: Standard solution
PER.FOR.MANCE TESTS Suitability requirements
• UNIFORMITY OF DOSAGE UNITS (905) Resolution: NLT 1.1 between the single-chain and
Acceptance criteria: Meets the requirements for Content two-chain alteplase peaks
Uniformity Analysis
Samples: Standard solution and Sample solution
SPECIFIC TESTS [NoTE-The major peaks are from single-chain and
• PERCENT MONOMER two-chain alteplase and from higher and lower molecular
Mobile phase: 34.84 mg/mL of arginine, 158.56 mg/mL of weight species.]
ammonium sulfate, and 100 mL/L of isopropyl alcohol in Calculate the percentage of single-chain alteplase in the
water. Adjust with phosphoric acid to a pH of 7.3, degas, portion of Alteplase for Injection taken:
and pass through a filter of OA5-J..Im pore size.
System suitability solution: 1 mg/mL each of chicken Result = (r vir T) x 100
ovalbumin and bovine gamma globulin
Standard solution: 1 mg/mL of USP Alteplase RS in water ru = peak response for single-chain alteplase
Sample solution: 1 mg/mL of Alteplasefor Injection in rT =sum of allthe peak responses of alteplase
water
Chromatographic system Acceptance criteria: No peaks or shoulders in the Sample
(See Chromatography (621), System Suitability.) solution that are not present in the Standard solution are
Mode: LC found; NLT 60%.
Detector: UV 280 nm • INJECTIONS AND IMPLANTED DRUG PRODUCTS (1): Meets the
Column: 7.5-mm x 30-cm; packing L25 requirements of constituted solutions at the time of use
Flow rate: 0.5-1.0 mL/min • pH (791)
Injection volume: 50 J..IL Sample solution: Constitute as directed in the labeling.
System suitability Acceptance criteria: 7.1-7.5
Samples: System suitability solution and Standard solution • WATER DETERMINATION (921), Method I: NMT4.0%
Suitability requirements • BACTERIAL ENDOTOXINS TEST (85): NMT 1 USP Endotoxin
Resolution: NLT 1.6 between gamma globulin and Unit/mg
ovalbumin, System suitability solution • STERILITY TESTS (71): Meets the requirements when tested
Column efficiency: NLT 1200 theoretical plates, as directed in Test for Sterility of the Product to Be Examined,
determined from the alteplase peak, Standard solution Membrane Filtration .
Analysis • BIOLOGICAL REACTIVITY TESTS, IN VIVO (88): Meets the
Sample: Sample solution requirements for Safety Tests-Biologicals
Calculate, as a percentage, the monomer in the portion of
Alteplase for Injection taken:

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USP 43 Official Monographs / Altretamine 163

ADDITIONAL REQUIREMENTS the solution to reach room temperature. Dilute with water
" PACKAGING AND STORAGE: Preserve in hermetic, to volume and mix.
light-resistant containers, and store in a refrigerator. Sample solution: 0.1 mg/mL of Altretamine in water from
" LAIBEUNG: Label it to state the biological activity in USP Sample stock solution
Alteplase Units/vial and the amount of protein/vial. Chromatographic system
" USP REFERENCE STANDARDS (11) (See Chromatography (621), System Suitability.)
USP Alteplase RS Mode: LC
Detector: UV 242 nm
Column: 4.6-mm x 25-cm; 5-J.lm packing L96
Flow rate: 1.5 ml/min
Injection volume: 20 J.lL. [NOTE-An in-line filter may be
Altl"etamine needed to reduce system pressure.]
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 1.5
Relative standard deviation: NMT 2.0%
Analysis
C9H,8N6 210.28 Samples: Standard solution and Sample solution
Calculate the percentage of altretamine (C9H18N6) in the
1,3,5-Triazine-2,4,6-triamine, N,N,N',N',N",N"-hexamethyl-;
Hexamethylmelamine [645-05-6]. portion of Altretamine taken:

DEFINITION Result = (ru/rs) x (Cs/C u) x 100

Altretamine contains NLT98.0% and NMT 102.0% of
altretamine (C9H18N6), calculated on the anhydrous basis. t» = peak response from the Sample solution
rs = peak response from the Standard solution
IDENTIFICATION (s = concentration of USP Altretamine RS in the
Standard solution (mg/mL)
Cu = concentration of Altretamine in the Sample
solution (mg/ml)
-A
:~ Acceptance criteria: 98.0%-102.0% on the anhydrous
requirements basis
" B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as IMPURITIES
obtained in the Assay. - RESIDUE ON IGNITION (281): NMT 0.1 %
- ORGANIC IMPURITIES
ASSAY Solution A, Solution B, Mobile phase, Standard solution,
- PROCEDURE and Sample solution: Prepare as directed in the Assay.
Solution A: 1.7 gil of dibasic potassium phosphate in Sensitivity solution: 0.06 J.lg/mL of USP Altretamine RS in
water. Adjust with phosphoric acid to a pH of 7.0 ± 0.1. water from Standard solution
Solution B: Acetonitrile Chromatographic system
Mobile phase: See Table 7. (See Chromatography (621), System Suitability.)
Mode: LC
Table 1 Detector: UV 215 and 242 nm (see Table 2)
Time Solution A Solution B Column: 4.6-mm x 25-cm; 5-J.lm packing L96
(min) (%) (%) Flow rate: 1.5 ml/min
0 100 0 Injection volume: 20 ul,
System suitability
3 100 0- Samples: Standard solution and Sensitivity solution
25 50 50 Suitability requirements
Tailing factor: NMT 1.5, Standard solution
28 50 50 Relative standard deviation: NMT 2.0%, Standard
29 100 0 solution
Signal-to-noise ratio: NlT 10 for the altretamine peak,
35 100 0 Sensitivity solution
Analysis
Standard stock solution: 1.0 mg/mL of USP Altretamine RS Samples: Standard solution and Sample solution
prepared as follows. Dissolve a suitable amount ofUSP Calculate the percentage of each impurity in the portion of
Altretamine RS in about 40% of the flask volume of Altretamine taken:
acetonitrile. Sonicate as needed to dissolve. Add water
almost to volume and allow the solution to reach room Result =(ru/rs) x (Cs/Cu) x (1/F) x 100
temperature. Dilute with water to volume and mix.
Standard solution: 0.1 mg/mL of USPAltretamine RS in to = peak response of each impurity from the Sample
water from Standard stock solution solution
Sample stock solution: 1.0 mg/mL of Altretamine prepared rs =peak response of altretamine from the Standard
as follows. Dissolve a suitable amount of Altretamine in solution
about 40% of the flask volume of acetonitrile. Sonicate as Cs = concentration of USPAltretamine RS in the
needed to dissolve. Add water almost to volume and allow Standard solution (mg/mL)

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164 Altretamine / Official Monographs USP 43

Cu = concentration of Altretamine in the Sample chloroform. Filter, and evaporate the chloroform solution
solution (mg/ml) to dryness.
F = relative response factor (see Table 2) Acceptance criteria: Meet the requirements
• B. The retention time of the major peak of the Sample
Acceptance criteria: See Table 2. The reporting threshold is solution corresponds to that of the Standard solution, as
NMTO.06%. obtained in the Assay.
Table 2 ASSAY
Relative Relative Acceptance
Retention Response Detector Criteria,
Name Time Factor (nm) NMT (%)
Altretamine
diketo
analog a 0.21 0.44 242 0.1
Altretamine
chloro keto
ana 10gb 0.35 0.60 215 0.1
Table 1
Altretamine
Time Soltit:iOIlA S()@iQO~
keto
(miD) (0/1) (0/0)
analog' 0.56 1.3 242 0.1
Altretamine 0 100 0
dichloro 1(')()
3 0
analoq" 0.87 0.87 242 0.1
Altretamine 25 50 SO
monochloro 28 50 50
analoq" 0.96 1.3 242 0.1
29 100 0
Altretamine 1.00 1.0 215,2421 -
35 10Q 0
Any other
individual
impurities
- - 0.1
242
Total
impurities - - - 0.3

a 6-(Dimethylamino)-1 ,3,5-triazine-2,4[1 H,3H)-dione.

b 4-Chloro-6-(dimethylamino).1 ,3,5-triazine·2[1 H)-one.
c 4,6-Bis(dimethylamino).1 ,3,5-triazine-2[1 H)-one.
d 4,6-Dichloro-N,N-dimethyl.l ,3,5-triazine.2-amine.
e 6-Chloro-Nl,Nl,N4,N4-tetramethyl-l ,3,5-triazine-2,4-diamine.
f Use the detector consistent with respective impurity.

SPECIFIC TESTS
• WATER DETERMINATION (921), Method I: NMT 1%
• TESTS FOR SPECIFIED MICROORGANISMS (62): The total
aerobic microbial count is NMT 10 3 du/g. The total
combined molds and yeasts count is NMT 10 2 du/g.
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers, and
store at controlled room temperature.
• USP REFERENCE STANDARDS (11)
USP Altretamine RS

I
Altretamine Capsules r'le~(j
System suitability
DEFINITION Sample: Standard solution
Altretamine Capsules contain NlT 90.0% and NMT 110.0% Suitability requirements
of the labeled amount of altretamine (C9H 1SN6) . Tailing factor: NMT 1.5
Relative standard deviation: NMT 2.0%
IDENTIFICATION Analysis .
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
altretamine (C9H1SN6) in the portion of Capsules taken:
• A....SP,ECTROSCO
Spectroscopy: 19 Result = (ru/rs) x (CslCu) x 100
Sample: Remove as rnl'TIn,ll:>tl:>h,
5 Capsules, and dissolve, ru = peak response from the Sample solution

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USP 43 OfficialMonographs / Alum 165

=peak responsefrom the Standard solution Table 2 (continued)

= concentration of USP Altretamine RS in the Relative Relative Acceptance
Standard solution (mg/mL) Retention Resp~nse Detector Criteril:l,
=nominal concentration of altretamine in the Name Time Factor (11m) NMT(%)
Sample solution (mg/mL) AltretaJ11me 1;00 1.0 215, 242 f ..:.;;,.;.;

Acceptance criteria: 90.00/0-110.0% Any other:

individual .,.- -
IMPURITIES IrnpUri,tY 242 0.4

A,cJd,the fol,lowing
"(otalJmpufifies - - '--' 1.0

~' IMPURITIES

PERFORMANCE TESTS
• DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 1: 100 rpm
Time: 30 min
Detector: UV 242 nm
Standard solution: USP Altretamine RS in Medium
Analysis: Determine the amount of altretamine (C g H,sN 6)
dissolved from UV absorbances on filtered portions of the
. solution under test, suitably diluted if necessary with
Medium, compared with the Standard solution.
Tolerances: NLT 80% (Q) of the labeled amount of
altretamine (C g H,sN 6) is dissolved.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at controlled room temperature.
• USP REFERENCE STANDARDS (11)
Res!:Jlt~(ru/r~),xICslturx(11f)x)qg USP Altretamine RS
ru=peak response of eac~ impuritYfrom~1lJ'~Jcit:riple
solution
rs =peak response of altretamine from theStiiqaciid
solution Ammonium Alum
Cs ;:: concentrati tretamine RSinthe,
Sta dard )., ' ", . AINH4(S04h· 12HzO 453.33
Cu =n Ic altretamineintbe AINH4(S04)2 237.15
S. '. ' solutio inL) ", ' Sulfuric acid, aluminum ammonium salt (2:1:1),
F = relative responsefactor' (seeTable) dodecahydrate;
Aluminum ammonium sulfate (1:1 :2), dodecahydrate
Acceptance criteria; .See Table 2. The reportirig·thle~noldi~
[7784-26-1 ].
NMTO.1%.
Anhydrous [7784-25-0].
Table 2 DEFINITION
Relative Relative Ammonium Alum contains NLT 99.0% and NMT 100.5% of
Retention Response [)eteCtor ammonium alum [AINH4(S04)2], calculated on the dried
Name Time Factor (hill)' basis.
Altretamine diketo IDENTIFICATION
analoga 0.21 0.44 242 0:4
• A.
Sample solution: 50 mg/mL
0.35 0.60 21? OA Analysis: Add 1 N sodium hydroxide dropwise to the Sample
solution.
0.5~ 1.3 ,242 ();4 Acceptance criteria: A precipitate isformed, and it dissolves
in an excess of the reagent with the evolution of ammonia,
recognizable by its alkaline effect upon moistened red
0.87 0.87 2:4~ 0;.1
litmus paper exposed to the vapor.
• B. IDENTIFICATION TESTS-GENERAL, Aluminum (191)
Sample solution: 50 mg/mL
0.96 1.3 '242 °A Acceptance criteria: Meets the requirements

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 166 Alum / OfficialMonographs USP 43

til C. IDENTIFICATION TESTS-GENERAL (191), Sulfate IIDENTIFICATION

Sample solution: 50 mg/mL • A.
Analysis: Proceed as directed in Identification Tests- Sample solution: 50 mg/mL in water
General, Sulfate (191), except centrifuge the neutral Analysis: Add 1 N sodium hydroxide dropwise to the Sample
solutions of sulfates and use the supernatants for test B. solution.
Acceptance criteria: Meets the requirements Acceptance criteria: A precipitate is formed that dissolves
in an excess of the reagent. Ammonia is not evolved
ASSAY (distinction from ammonium alum).
- PROCEDURE
Edetate disodium titrant: Prepare and standardize as - lB.
Analysis: Hold it in a non luminous flame.
directed in Reagents, Volumetric Solutions, Edetate Disodium, Acceptance criteria: A violet color is imparted to the flame.
Twentieth-Molar (0.05 M).
Sample: 800 mg of Ammonium Alum - C.
Sample solution: Saturated solution in water
Analysis: Transfer the Sample to a 400-mL beaker, moisten Analysis: Add 10 mL of sodium bitartrate TS to 5 mL of the
with 1 mL of glacial acetic acid, and add 50 mL of water, Sample solution.
50.0 mL of Edetate disodium titrant and 20 mL of acetic Acceptance criteria: A white, crystalline precipitate is
acid-ammonium acetate buffer TS. Warm on a steam bath formed within 30 min.
until the solution is complete, and boil gently for 5 min. • D. IDENTIFICATION TESTS-GENERAL, Aluminum (191) AND
Cool, add 50 mL of alcohol and 2 mL of dithizone TS, and Sulfate (191)
titrate the excess edetate disodium with 0.05 M zinc sulfate Sample solution: 50 mg/mL in water
VS to a bright rose-pink color. Perform a blank Acceptance criteria: Meets the requirements
determination, and make any necessary correction. Each
mL of 0.05 M Edetate disodium titrant is equivalent to 11.86 ASSAY
mg of AINH 4(S04)z, - PROCEDURE
Acceptance criteria: 99.0%-100.5% on the dried basis Edetate disodium titrant: Prepare and standardize as
directed in Reagents, Indicators, and Solutions-Volumetric
IMPURITIES Solutions, Edetate Disodium, Twentieth-Molar (0.05 M).
-IRON Sample: 800 mg
Sample solution: 6.7 mg/mL Analysis: Transfer the Sample to a 400-mL beaker, moisten
Analysis: Add 5 drops of potassium ferrocyanide TS to 20 with 1 mL of glacial acetic acid, and add 50 mL of water,
mL of the Sample solution. 50.0 mL of Edetate disodium titrant, and 20 mL of acetic
Acceptance criteria: No blue color is produced acid-ammonium acetate buffer TS. Warm on a steam bath
immediately. until solution is complete, and boil gently for 5 min. Cool,
SPECIFIC TESTS add 50 mL of alcohol and 2 mL of dithizone TS, and titrate
- loss ON DRYING (731) 0.05 M zinc sulfate VS to a bright rose-pink color. Perform
Sample: 2.0 g a blank determination, and make any necessary correction.
Analysis: Transfer the Sample, in a tared porcelain crucible, Each mL of 0.05 M Edetate disodium titrant is equivalent to
to a muffle furnace at 200°. Increase the temperature to 12.91 mg of potassium alum [AIK(S04)z].
300°, and dry at 300° to a constant weight. Cool in a Acceptance criteria: 99.00/0-100.5% on the dried basis
desiccator, and weigh. IMPURITIES
Acceptance criteria: 45.0%-48.0%
• IRON
- LIMIT OF ALKALIES AND ALKALINE EARTHS' Sample solution: Potassium alum in water (1 in 150)
Sample: 1 g Analysis: Add 5 drops of potassium ferrocyanide TS to 20
Analysis: Completely precipitate the aluminum from a mL of the Sample solution.
boiling solution of the Sample in 100 mL of water by the Acceptance criteria: No blue color is produced
addition of sufficient 6 N ammonium hydroxide to render immediately.
the solution distinctly alkaline to methyl red TS, and filter.
Evaporate the filtrate to dryness, and ignite. SPECIFICTESTS
Acceptance criteria: The weight of the residue is NMT 5 mg - loss ON DRYING (731)
(0.5%). Sample: 2.0 g
Analysis: Transfer the Sample in a tared porcelain crucible to
a muffle furnace at 200°. Increase the temperature to 400°,
and dry at 400° to constant weight. Cool in a desiccator,
and weigh.
Potassium Alum Acceptance criteria: 43.00/0-46.0%
AIK(S04)z·12H zO 474.39 ADDITIONAL REQUIREMENTS
- PACKAGING AND STORAGE: Preserve in tight containers, and
AIK(S04)z 258.21 store at room temperature.
Sulfuric acid, aluminum potassium salt (2:1:1),
dodecahydrate;
Aluminum potassium sulfate (1:1 :2) dodecahydrate [7784-
24-9].
Anhydrous [10043-67-1]. Alumina and Magnesia Oral Suspension
DEFINITION DEFINITION
Potassium Alum contains NLT 99.0% and NMT 100.5% of Alumina and Magnesia Oral Suspension is a mixture
potassium alum [AIK(S04)z], calculated on the dried basis. containing aluminum hydroxide [AI(OH)3] and Magnesium
Hydroxide [Mg(OH)z]. It contains the equivalent of NLT
90.0% and NMT 110.0% of the labeled amounts of

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 USP 43 Official Monographs / Alumina 167

aluminum hydroxide [AI(OH)3l and magnesium hydroxide Acceptance criteria: NMT 0.14%; a 1O-mL portion of the
[Mg(OH)2l. It may contain a flavoring agent, and may Sample solution shows no more chloride than corresponds
contain suitable antimicrobial agents. to 1.0 mL of 0.020 N hydrochloric acid.
• CHLORIDE AND SULFATE, Sulfate (221)
IDENTIFICATION Sample solution: Dissolve5.0 g in 5 mL of 3 N hydrochloric
• A. IIDENTIFICATION TESTS-GENERAL, Magnesium (191) acid, with gentle heating. Cool, add water to make 250 mL,
Sample solution: To a solution of 5 g in 10 mL of 3 N and filter.
hydrochloric acid add 5 drops of methyl red TS, heat to Acceptance criteria: NMT 0.1%; a 20-ml portion of the
boiling, add 6 N ammonium hydroxide until t~e color.?f Sample solution shows no more sulfate than corresponds to
the solution changes to deep yellow, then continue boilinq 0.40 ml of 0.020 N sulfuric acid.
for 2 min, and filter. • ARSENIC, Method I (211)
Acceptance criteria: The filtrate meets the requirements. Standard preparation: Prepareasdirected in Arsenic (211),
• B. IDENTIFICATION TESTS-GENERAL, Aluminum (191) except prepare it to contain 5 IJgof arsenic instead of 3 IJg.
Sample solution: Wash the precipitate obtained in Test preparation: Dissolve a portion of Oral Suspension,
Identification test A with a hot solution containing 20 equivalent to 0.5 g of aluminum hydroxide [AI(OH)3l, in 20
mg/mL of ammonium chloride, and dissolvethe precipitate ml of 7 N sulfuric acid.
in hydrochloric acid. Acceptance criteria: NMT 10 ppm, based on the aluminum
Acceptance criteria: The solution meets the requirements. hydroxide [AI(OH)3l content
ASSAY SPECIFIC TESTS
• ALUMINUM HYDROXIDE • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Edetate disodium titrant: Prepare and standardize as SPECIFIED MICROORGANISMS (62): Its total aerobic
directed in Reagents, Volumetric Solutions, Edetate Disodium, microbial count does not exceed 10 2 du/mL, and it meets
Twentieth-Molar (0.05 M). the requirements for the absence of Escherichia coli.
Sample solution: Transfer a volume of Oral Suspension, • pH (791): 7.3-8.5
previously well shaken in its original container, equivalent • ACID-NEUTRALIZING CAPACITY (301)
to 1200 mg of aluminum hydroxide, to a suitable beaker. Acceptance criteria: The acid consumed by the minimum
Add 20 mL of water, stir, and slowly add 10 mL of . single dose recommended in the labeling is NlT 5 mEq, and
hydrochloric acid. Heat gently, if necessary, to aid solution, NlT the number of mEq calculated by the formula:
cool, and filter into a 200-mL volumetric flask. Wash the
filter with water into the flask, and add water to volume. Result = 0.55 x (FAX A) + 0.8 x (F M x M)
Analysis: Pipet 10 mL of the Sample solution into a beaker,
add 20 mL of water, then add, in the order named and with =theoretical acid-neutralizing capacity of
continuous stirring, 25.0 mL of Edetate disodium titrant and aluminum hydroxide [AI(OHhl, 0.0385 mEq
20 mL of acetic acid-ammonium acetate buffer TS, and A =amount of aluminum hydroxide [AI(OH)3l in the
heat near the boiling point for 5 min. Cool, add 50 mL of specimen tested, based on the labeled quantity
alcohol and 2 mL of dithizone TS, and mix. Titrate the (mg)
excess edetate disodium with 0.05 M zinc sulfate VS until =theoretical acid-neutralizing capacity of
the color changes from green-violet to rose-pink. Perform magnesium hydroxide [Mg(OH)2]' 0.0343 mEq
a blank determination, substituting 10 mL of water for the
M = amount of magnesium hydroxide [Mg(OH)2l in
Sample solution, and make any necessary correction. Each
mL of Edetate disodium titrant consumed is equivalent to the specimen tested, based on the labeled
3.900 mg of aluminum hydroxide [AI(OH)3l. quantity (mg) <

Acceptance criteria: 90.0%-110.0% ADDITIONAL REQUIREMENTS

• MAGNESIUM HYDROXIDE
• PACKAGING AND STORAGE: Preserve in tight containers, and
Sample solution: Prepare as directed in the Assay for avoid freezing.
AluminumHydroxide. • LABELING: Oral Suspension may be labeled to state the
Analysis: Pipet a volume of the Sample solution, equivalent aluminum hydroxide content in terms of the equivalent
to 40 mg of magnesium hydroxide, into a 400-mL beaker. amount of dried aluminum hydroxide gel, on the basisthat
Add 200 mL of water and 20 mL of triethanolamine, and each mg of dried gel is equivalent to 0.765 mg of aluminum
stir. Add 10 mL of ammonia-ammonium chloride buffer TS hydroxide [AI(OH)3]'
and 3 drops of an eriochrome black indicator solution .
(prepared by dissolving 200 mg of eriochrome black T In a
mixture of 15 mL of triethanolamine and 5 mL of
dehydrated alcohol), and mix. Cool the solution to between
3° and 4° by immersion of the beaker in an ice bath, then Alumina and Magnesia Tablets
remove, and titrate with 0.05 M edetate disodium VS to a
blue endpoint. Perform a blank determination, substituting DEFINITION
10 mL of water for the Sample solution, and make any Alumina and Magnesia Tablets contain NlT 90.0% and NMT
necessary correction. Each mL of 0.05 M edetate disodium 110.0% of the labeled amounts of aluminum hydroxide
consumed is equivalent to 2.916 mg of magnesium [AI(OH)3] and magnesium hydroxide [Mg(OH)2l.
hydroxide [Mg(OH)2l.
Acceptance criteria: 90.0%-110.0% IDENTIFICATION
• A. IDENTIFICATION TESTS-GENERAL, Magnesium (191)
IMPURITIES Sample solution: To a O.7-g portion offinely powdered
• CHLORIDE AND SULFATE, Chloride (221) Tablets add 10 mL of 3 N hydrochloric acid and 5 drops of
Sample solution: Dissolve 5.0 g in the minimum volume of methyl red TS, heat to boiling, and add 6 N ammonium
nitric acid required to achieve complete solution, add ~ mL hydroxide until the color of the solution changes to deep
of acid in excess, then add water to make 100 mL, and filter. yellow. Continue boiling for 2 min, and filter.
Acceptance criteria: The filtrate meets the requirements.

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168 Alumina / OfficialMonographs USP 43

• B. IDENTIIFICATION TESTS-GENERAL, Aluminum (191) A = amount of aluminum hydroxide [AI(OH)3l in the

Sample solution: Wash the precipitate obtained in specimen tested, based on the labeled quantity
Identification test A with hot ammonium chloride solution (mg)
(1 in 50), and dissolve the precipitate in hydrochloric acid. FM =theoretical acid-neutralizing capacity of
Acceptance criteria: The solution meets the requirements. magnesium hydroxide [Mg(OH)2l, 0.0343 mEq
ASSAY M =amount of magnesium hydroxide [Mg(OH)2l in
• ALUMINUM HYDROXIDE the specimen tested, based on the labeled
Ed~tate d~sodium titrant: Prepare and standardize as quantity (mg)
directed In Reagents, Volumetric Solutions, Edetate Disodium,
Twentieth-Molar (0.05 M). ADDITIONAL REQUIREMENTS
Sample solution: Finely powder NLT 20 Tablets. Transfer a • PACKAGING AND STORAGE: Preserve in well-closed
portion. of the powder, equivalent to 1200 mg of aluminum containers.
hydroxide, to a 150-mL beaker. Add 20 mL of water, stir, • LABELING: Tablets prepared with the useof Dried Aluminum
~nd slowly add 3~ mL of.3 N hydrochlor!c acid. Heat gently,
Hydroxide Gel may be labeled to state the aluminum
If necessary, to aid solution, cool, and filter into a 200-mL hydroxide content in terms of the equivalent amount of
volumetric flask. Wash the filter with water into the flask, dried aluminum hydroxide gel, on the basis that each mg
and add water to volume. of dried gel is equivalent to 0.765 mg of aluminum
Analysis: Pipet 10 mL of the Sample solution into a 250-mL hydroxide [AI(OH)3l.
beaker, add 20 mL of water, then add, in the order named
and with continuous stirring, 25 mL of Edetate disodium
titrant and 20 mL of acetic acid-ammonium acetate buffer
TS, and heat near the boiling point for 5 min. Cool, add 50
mL of alcohol and2 mL of dithizone TS, and mix. Titrate
Alumina, Magnesia, and Calcium
the excess edetate disodium with 0.05 M zinc sulfate VS Carbonate Oral Suspension
until the color changes from green-violet to rose-pink.
Perform a blank determination, substituting 10 mL of water DEFINITION
for the Sample solution, and make any necessary correction. Alumina, Magnesia, and Calcium Carbonate Oral Suspension
Each mL of Edetate disodium titrant is equivalent to 3.900 contains NLT 90.0% and NMT 110.0% of the labeled
mg of aluminum hydroxide [AI(OH)3l. amounts of aluminum hydroxide [AI(OH)3l, magnesium
Acceptance criteria: 90.0%-110.0% hydroxide [Mg(OH)2l, and calcium carbonate (CaC0 3).
• MAGNESIUM HYDROXIDE
Sample solution: Prepare as directed in the Assay for IDENTIFICATION
Aluminum Hydroxide. • A. IDENTIFICATION TESTS-GENERAL, Calcium (191)
Analysis: Pipet a volume of the Sample solution, equivalent Sample solution: To 5 g of Oral Suspension add 25 mL of 2
to 40 mg of magnesium hydroxide, into a 400-mL beaker. N sulfuric acid, stir, and allow to stand for 5 min. Add 25
Add 200 mL of water and 20 mL of triethanolamine, and mL of alcohol, stir, and place in an ice bath for 30 min. Filter
stir. Add 10 mL of ammonia-ammonium chloride buffer TS while cold, retaining the filtrate for Identification test B.
and 3 drops of an eriochrome black indicator solution Wash the precipitate with 50 mL of 0.75 N sulfuric acid, and
(prepared by dissolving 200 mg of eriochrome black TS in discard the washings. Dissolvethe precipitate in 3 N
a mixture of 15 mL of triethanolamine and 5 mL of hydrochloric acid, filter, and use the filtrate.
dehydrated alcohol), and mix. Cool the solution to between Acceptance criteria: Meets the requirements .
3° and 4° by immersion of the beaker in an ice bath, then • B. IDENTIFICATION TESTS-GENERAL, Aluminum (191)
remove, and titrate with 0.05 M edetate disodium VSto a Sample solution: To the filtrate obtained in Identification
blue endpoint. Perform a blank determination, substituting test A, add 5 drops of methyl red TS, and heat to boiling.
10 mL of water for the Sample solution, and make any Add 6 N ammonium hydroxide until the color of the
necessary correction. Each mL of 0.05 M edetate disodium solution changes to deep yellow, continue boiling for 2
consumed is equivalent to 2.916 mg of magnesium min, and filter through hardened filter paper. (Retain the
hydroxide [Mg(OH)Zl. filtrate for Identification test C.) Wash the precipitate with
350 mL of a hot solution containing 20 mg/mL of
Acceptance criteria: 90.00/0-110.0%
ammonium chloride, discarding the washings. Dissolve the
PERFORMANCE TESTS precipitate so obtained in 3 N hydrochloric acid.
• DISINTEGRATION (701) Acceptance criteria: Meets the requirements
Time: 10 min, simulated gastric fluid TS being substituted • C. IDENTIFICATION TESTS-GENERAL, Magnesium (191)
for water in the test Sample solution: The filtrate obtained in Identification test
Acceptance criteria: Meet the requirements B.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Acceptance criteria: Meets the requirements
requirements for Weight Variation with respect to alumina
ASSAY
and to magnesia
• ALUMINUM HYDROXIDE
SPECIFIC TESTS Edetate disodium titrant: Prepareand standardize as
• ACID-NEUTRALIZING CAPACITY (301): The acid consumed directed in Reagents, Volumetric Solutions, Edetate Disodium,
by the minimum single dose recommended in the labeling Twentieth-Molar (0.05 M).
is NLT 5 mEq, and NLT the number of mEq calculated by Sample solution: Transfer an amount of Oral Suspension,
the formula: previously shaken in its original container, equivalent to 600
mg of aluminum' hydroxide, to a tared beaker, and weigh.
Result = 0.55 x (FAx A) + 0.8 X (FM x M) Add 20 mL of water, stir, and slowly add 40 mL of 3 N
hydrochloric acid. Heat gently, if necessary, to aid solution,
FA = theoretical acid-neutralizing capacity of cool, and transfer to a 200-mL volumetric flask. Wash the
aluminum hydroxide [AI(OH)3l, 0.0385 mEq

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 USP 43 Official Monographs / Alumina 169

beaker with water, adding the washings to the flask, and Test preparation: Dissolvea portion of Oral Suspension,
add water to volume. equivalent to 0.5 g of aluminum hydroxide [AI(OH)3]' in 20
Analysis: Pipet 10 mL of the Sample solution into a 250-mL mL of 7 N sulfuric acid.
beaker, add 20 mL of water, then add, in the order named Acceptance criteria: NMT 10 ppm, based on the aluminum
and with continuous stirring, 25.0 mL of Edetate disodium hydroxide [AI(OH)3] content
titrant and 20 mL of acetic acid-ammonium acetate buffer
TS, and heat the solution near the boiling temperature for SPECIFIC TESTS
5 min. Cool, and add 50 mL of alcohol and 2 mL of o MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
dithizone TS, and mix. Titrate the excess edetate disodium SPECIFIED MICROORGANISMS (62): Its total aerobic
with 0.05 M zinc sulfate VS until the color changes from microbial count does not exceed 102 cfu/mL, and it meets
green-violet to rose-pink. Perform a blank determination, the requirements of the test for the absence of Escherichia
substituting 10 mL of water for the Sample solution, and coli.
make any necessary correction. Each mL of Edetate disodium o pH (791): 7.5-8.5
titrant consumed is equivalent to 3.900 mg of aluminum • ACID-NEUTRALIZING CAPACITY (301)
hydroxide [AI(OH)3]' Acceptance criteria: The acid consumed by the minimum
Acceptance criteria: 90.0%-110.0% single dose recommended in the labeling is NLT 5 mEq, and
• MAGNESIUM HYDROXIDE NLT the number of mEq calculated by the formula:
Sample solution: Prepare as directed in the Assay for
Aluminum Hydroxide. Result = 0.55 x (FAX A) + 0.8 x (F M x M) + 0.9 x (F eX C)
Analysis: Pipet a volume of the Sample solution, equivalent
= theoretical acid-neutralizing capacity of
to 40 rnq'of magnesium hydroxide, into a 400-mL beaker,
add 200 mL of water and 20 mL oftrolamine, and mix. Add aluminum hydroxide [AI(OH)3]' 0.0385 mEq
50 mL of ammonia-ammonium chloride buffer TS and 2 A = amount of aluminum hydroxide [AI(OH)3] in the
drops of an eriochrome black indicator solution (prepared specimen tested, based on the labeled quantity
by dissolving 200 mg of eriochrome black T in a mixture of (mg)
15 mL of trolamine and 5 mL of dehydrated alcohol, and = theoretical acid-neutralizing capacity of
mixing). Cool the solution to between 3° and 4° by magnesium hydroxide [Mg(OH)2]' 0.0343 mEq
immersing the beaker in an ice bath, and titrate with 0.05 iv1 = amount of magnesium hydroxide [Mg(OH)z] in
M edetate disodium VSuntil the color changes to pure blue. the specimen tested, based on the labeled
Perform a blank determination, substituting 10 mL of water quantity (mg)
for the Sample solution, and make any necessary correction. Fe = theoretical acid-neutralizing capacity of calcium
From the volume of 0.05 M edetate disodium consumed, carbonate (CaC0 3), 0.02 mEq
subtract the volume of 0.05 M edetate disodium consumed c = amount of calcium carbonate (CaC0 3) in the
in the Assayfor Calcium Carbonate. Each mL of 0.05 M specimen tested, based on the labeled quantity
edetate disodium is equivalent to 2.916 mg of magnesium (mg)
hydroxide [Mg(OH)2]'
Acceptance criteria: 90.0%-110.0% ADDITIONAL REQUIREMENTS
• CALCIUM CARBONATE • PACKAGING AND STORAGE: Preserve in tight containers, and
Sample solution: Prepare as directed in the Assay for avoid freezing.
Aluminum Hydroxide. o LABELING: Oral Suspension may be labeled to state the
Analysis: Pipet a volume of the Sample solution, equivalent aluminum hydroxide content in terms of the equivalent
to 50 mg of calcium carbonate, into a 400-mL beaker, and amount of dried aluminum hydroxide gel, on the basisthat
add 200 mL of water,S mL of sodium hydroxide solution each mg of dried gel is equivalent to 0.765 mg of aluminum
(1 in 2), and 250 mg of hydroxy naphthol blue. Stir with a hydroxide [AI(OH)3]'
magnetic stirrer, and titrate immediately with 0.05 M
edetate disodium VS until the solution is distinctly blue.
Each mL of 0.05 M edetate disodium is equivalent to 5.004
mg of calcium carbonate (CaC0 3).
Acceptance criteria: 90.0%-110.0% Alumina, Magnesia, and Calcium
IMPURITIES Carbonate Chewable Tablets
• CHLORIDE AND SULFATE, Chloride (221)
DEFINITION
Sample solution: Dissolve 5.0 g in 3 mL of nitric acid, add
Alumina, Magnesia, and Calcium Carbonate Chewable Tablets
water to make 100 mL, and filter.
contain NLT 90.0% and NMT 110.0% of the labeled
Acceptance criteria: NMT 0.14%; a 1O.O-mL portion of the
amounts of aluminum hydroxide [AI(OH)~], magnesium
Sample solution shows no more chloride than corresponds
to 1.0 mL of 0.020 N hydrochloric acid. hydroxide [Mg(OH)2]' and calcium carbonate (CaC03).
• CHLORIDE AND SULFATE, Sulfate (221) IDENTIFICATION
Sample solution: Dissolve5.0 gin 7 mL of 3 N hydrochloric o A. IDENTIFICATION TESTS-GENERAL, Calcium (191)
acid, and gently heat. Cool, add water to make 250 mL, Sample solution: To 3 g of finely powdered Chewable
and filter. Tablets add 25 mL of water and 25 mL of 2 N sulfuric acid,
Acceptance criteria: NMT 0.1%; a 20.0-mL portion of the stir, and heat on a steam bath for 10 min. Cool, add 50 mL
Sample solution shows no more sulfate than corresponds to of alcohol, stir, and place in an ice bath for 30 min. Filter
0.40 mL of 0.020 N sulfuric acid. while cold, retaining the filtrate for. Identification test B.
• ARSENIC, Method I (211) Wash the precipitate with 50 mL of 0.75 N sulfuric add, and
Standard preparation: Prepareasdirected in Arsenic (211), discard the washings. Dissolvethe precipitate in 3 N
except prepare it to contain 5 ~g of arsenic instead of 3 ~g. hydrochloric acid, filter, and use the filtrate.
Acceptance criteria: Meet the requirements

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170 Alumina / OfficialMonographs USP 43

• B. IDENTIFICATION TESTS-GENERAL, Aluminum (191) add 200 mL of water, 5 mL of sodium hydroxide solution
Sample solution: To the filtrate obtained in Identification (1 in 2), and 250 mg of hydroxy naphthol blue. Stir with a
test A add 5 drops of methyl red TS, and heat to boiling. magnetic stirrer, and titrate immediately with 0.05 M
Add 6 N ammonium hydroxide until the color of the edetate disodium VS until the solution is distinctly blue.
solution changes to deep yellow, continue boiling for 2 Each mL of 0.05 M edetate disodium is equivalent to 5.004
min, and filter through hardened filter paper. (Retain the mg of calcium carbonate (CaC0 3).
filtrate for Identification test C.) Wash the precipitate with Acceptance criteria: 90.0%-110.0%
350 mL of a hot solution containing 20 mg/mL of
ammonium chloride, discarding the washings. Dissolve the PERFORMANCE TESTS
precipitate so obtained in 3 N hydrochloric acid. • DISINTEGRATION (701)
Acceptance criteria: Meet the requirements Time: 45 min
• C. IDENTIFICATION TESTS-GENERAL, Magnesium (191) Acceptance criteria: Meet the requirements
Sample solution: The filtrate obtained in Identification test • UNIFORMITV OF DOSAGE UNITS (905): Meet the
B requirements for Weight Variation with respect to
Acceptance criteria: Meet the requirements aluminum hydroxide, to magnesium hydroxide, and to
calcium carbonate.
ASSAY
• ALUMINUM HVDROXIDE SPECIFIC TESTS
Edetate disodium titrant: Prepare and standardize as • ACID-NEUTRALIZING CAPACITY (301)
directed in Reagents, Volumetric Solutions, Edetate Disodium, Acceptance criteria: The acid consumed by the minimum
Twentieth-Molar (0.05 M). single dose recommended in the labeling is NLT 5 mEq, and
Sample solution: Weigh and finely powder NLT 20 NLT the number of mEq calculated by the formula:
Chewable Tablets. Transfer a portion of the powder,
Result = 0.55 x (FAx A) + 0.8 X (FM x M) + 0.9 x (Fe x C)
equivalent to 600 mg of aluminum hydroxide, to a beaker,
add 20 mL of water, and slowly add 40 mL of 3 N
hydrochloric acid, with mixing. Heat the mixture to boiling,
FA =theoretical acid-neutralizing capacity of
aluminum hydroxide [AI(OH)3l, 0.0385 mEq
cool, and filter into a 200-mL volumetric flask. Wash the
beaker with water, adding the washings to the filter. Add A = amount of aluminum hydroxide [AI(OH)3l in the
water to volume. specimen tested, based on the labeled quantity
Analysis: Pipet 10 mL of the Sample solution into a 250-mL (mg)
beaker, add 20 mL of water, then add, in the order named FM = theoretical acid-neutralizing capacity of
and with continuous stirring, 25.0 mL of Edetate disodium magnesium hydroxide [Mg(OH)2l, 0.0343 mEq
titrant and 20 mL of acetic acid-ammonium acetate buffer M =amount of magnesium hydroxide [Mg(OH)2l in
TS, and heat the solution near the boiling temperature for the specimen tested, based on the labeled
5 min. Cool, add 50 mL of alcohol and 2 mL of dithizone quantity (mg)
TS, and mix. Titrate the excess edetate disodium with 0.05 Fe = theoretical acid-neutralizing capacity of calcium
M zinc sulfate VS until the color changes from green-violet carbonate (CaC0 3), 0.02 mEq
to rose-pink. Perform a blank determination, substituting C =amount of calcium carbonate (CaC0 3) in the
10 mL of water for the Sample solution, and make any specimen tested, based on the labeled quantity
necessary correction. Each mL of Edetate disodium titrant (mg)
consumed is equivalent to 3.900 mg of aluminum
hydroxide [ A I ( O H ) 3 l . · ADDITIONAL REQUIREMENTS
Acceptance criteria: 90.0%-110.0% • PACKAGING AND STORAGE: Preserve in well-closed
• MAGNESIUM HVDROXIDE containers.
Sample solution: Prepare as directed in the Assay for • LABELING: Label the Chewable Tablets to indicate that they
Aluminum Hydroxide. . are to be chewed before being swallowed. Chewable
Analysis: Pipet a volume of the Sample solution, equivalent Tablets prepared using dried aluminum hydroxide gel may
to 40 mg of magnesium hydroxide, into a 400-mL beaker, be labeled to state the aluminum hydroxide content in
add 200 mL of water and 20 mL of trolamine, and mix. Add terms of the equivalent amount of dried aluminum
50 mL of ammonia-ammonium chloride buffer TS and 2 hydroxide gel, on the basis that each mg of dried gel is
drops of an eriochrome black indicator solution (prepared equivalent to 0.765 mg of aluminum hydroxide [AI(OH)3l.
by dissolving 200 mg of eriochrome black T in a mixture of
15 mL of trolamine and 5 mL of dehydrated alcohol, and
mixing). Cool the solution to between 3° and 4° by
immersing the beaker in an ice bath, and titrate with 0.05
M edetate disodium VS until the color changes to pure blue. Alumina, Magnesia, Calcium Carbonate,
Perform a blank determination, substituting 10 mL of water and Simethicone Chewable Tablets
for the Sample solution, and make any necessary correction.
From the-volume of 0.05 M edetate disodium consumed, DEfiNITION
subtract the volume of 0.05 M edetate disodium consumed Alumina, Magnesia, Calcium Carbonate, and Simethicone
in the Assayfor Calcium Carbonate. Each mL of 0.05 M Chewable Tablets contain NLT 90.0% and NMT 110.0% of
edetate disodium is equivalent to 2.916 mg of magnesium the labeled amounts of aluminum hydroxide [AI(OH)3l,
hydroxide [Mg(OH)2l. magnesium hydroxide [Mg(OH)2l, and calcium carbonate
Acceptance criteria: 90.0%-110.0% (CaC0 3), and an amount of polydimethylsiloxane ([-(CH 3)2
• CALCIUM CARBONATE SiO-l n) that is NLT 85.0% and NMT 115.0% of the labeled
Sample solution: Prepare as directed in the Assayfor amount of simethicone.
Aluminum Hydroxide.
Analysis: Pipet a volume of the Sample solution, equivalent
to 50 mg of calcium carbonate, into a 400-mL beaker, and

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USP 43 Official Monographs / Alumina 171

IDENTlflCAnON water, slowly add 10 mL of hydrochloric acid, and swirl to

• A. IDENTIFICATION TESTS-GENERAL, Calcium (191) dissolve the metal. Dilute with water to volume. Transfer
Sample solution: Cut a Chewable Tablet into pieces, add 1.0 mL of this solution to a 1OO-mL volumetric flask to
50 mL of 1 N sulfuric acid, stir until the pieces disintegrate, obtain a solution containing 10 IJg/mL of magnesium (Mg).
and heat on a steam bath for 10 min. Cool, add 50 mL of Standard solutions: To three separate 1OO-mL volumetric
alcohol, and stir. Place in an ice bath for 30 min. Filter while flasks each containing 5.0 mL of Lanthanum chloride
cold, retaining the filtrate for Identification test B. Wash the solution, add 1.0, 2.0, and 3.0 mL, respectively, of the
precipitate with 50 mL of 0.75 N sulfuric acid, and discard Magnesium stock solution. Dilute each with water to
the washings. Dissolve the precipitate in 3 N hydrochloric volume. Thesesolutions contain 0.1, 0.2, and 0.3 IJg/mL of
acid, filter, and use the filtrate. magnesium (Mg), respectively.
Acceptance criteria: Meet the requirements Sample stock solution: Transfer a number of Chewable
• B. IDENTIFICATION TESTS-GENERAL, Aluminum (191) Tablets, equivalent of 250 mg of magnesium hydroxide
Sample solution: To the filtrate obtained in Identification (100 mg of magnesium), to a 1OOO-mL volumetric flask.
test A, add 5 drops of methyl red TS, and heat to boiling. Add 500 mL of Dilutehydrochloric acid, and swirl to dissolve
Add 6 N ammonium hydroxide until the color of the the Chewable Tablets. Add 100.0 mL of Potassium chloride
solution changes to deep yellow, continue boiling for 2 solution, and dilute with water to volume. Transfer 10.0 mL
min, and filter through hardened filter paper. (Retain the of this solution to a 1OO-mL volumetric flask, and dilute with
filtrate for Identification test C.) Wash the precipitate with water to volume.
350 mL of a hot solution containing 20 mg/mL of Sample solution: Transfer 2.0 mL of Sample stock solution to
ammonium chloride, discarding the washings. Dissolvethe a second 1OO-mL volumetric flask, add 5.0 mL of
precipitate so obtained in 3 N hydrochloric acid. Lanthanum chloride solution, and dilute with water to
Acceptance criteria: Meet the requirements volume.
• C. IDENTIFICATION TESTS-GENERAL, Magnesium (191) Blank: Add 50 mL of Dilute hydrochloric acid and 10.0 mL of
Sample solution: The filtrate obtained in Identification test Potassium chloride solution to a 1OO-mL volumetric flask,
B and dilute with water to volume. Transfer 10.0 mL of this
Acceptance criteria: Meet the requirements solution to a second 1OO-mL volumetric flask, and dilute
with water to volume. Transfer 2.0 mL of this solution to a
third 1OO-mL volumetric flask, add 5.0 mL of Lanthanum
chloride solution, and dilute with water to volume.
• D·i~~g~...·(~~~~il Analysis: Concomitantly determine the absorbances of the
$pg~M9.~~9PY;·l~7'$ Standard solutions and the Sample solution at the
Sample solution: Prepare as rli .."rt""rl magnesium emission line at 285.2 nm, with a suitable
Polydimethylsiloxane. atomic absorption spectrophotometer (see Atomic
Analysis: Proceed as directed using a 0.5-mm cell. Absorption Spectroscopy (852» equipped with a magnesium
Acceptance criteria: Meet the requirements hollow-cathode lamp and an air-acetylene flame, using the
Blank to set the instrument. Plot the absorbances of the
ASSAY Standard solutions versus concentration, in j.Jg/mL, of
• ALUMINUM HYDROXIDE magnesium, and draw the straight line best fitting the three
Edetate disodium titrant: Prepare and standardize as plotted points. From the graph so obtained, determine the
directed in Reagents, Volumetric Solutions, Edetate Disodium, concentration, C, in IJg/mL, of magnesium in the Sample
Twentieth-Molar (0.05 M). solution.
Sample solution: Transfer a number of Chewable Tablets, Calculate the percentage of the labeled amount of
equivalent of about 665 mg of aluminum hydroxide, to a magnesium hydroxide [Mg(OHhl in the portion of
suitable beaker. Add 15 mL of hydrochloric acid, and swirl
Tablets taken:
to dissolve the Chewable Tablets. Add 80 mL of water, and
filter into a 200-mL volumetric flask. Wash the filter with Result = (CICu) x (M,/Ar) x 100
water into the flask, and add water to volume.
Analysis: Pipet 20 mL of the Sample solution into a 250-mL C =concentration of magnesium in the Sample
beaker, then add, in the order named and with continuous solution, as determined above (lJg/mL)
stirring, 25.0 mL of Edetate disodium titrant and 20 mL of Cu = nominal concentration of magnesium hydroxide
acetic acid-ammonium acetate buffer TS, and heat the in the Sample solution (lJg/mL)
solution near the boiling temperature for 5 min. Cool, add Mr = molecular weight of magnesium hydroxide,
50 mL of alcohol and 2 mL of dithizone TS. Titrate the 58.34
excess edetate disodium with 0.05 M zinc sulfate VS until Ar = atomic weight of magnesium, 24.305
the color changes from green-violet to rose-pink. Perform
a blank determination, substituting 20 mL of water for the Acceptance criteria: 90.0%-110.0%
Sample solution, and make any necessary correction. Each • CALCIUM CARBONATE
mL of Edetate disodium titrant consumed is equivalent to Sample solution: Transfer a number of Chewable Tablets,
3.900 mg of AI(OH)3' equivalent of about 665 mg of aluminum hydroxide, to a
Acceptance criteria: 90.0%-110.0% suitable beaker. Add 15 mL of hydrochloric acid, and swirl
• MAGNESIUM HYDROXIDE to dissolve the Chewable Tablets. Add 80 mL of water, and
Lanthanum chloride solution: Transfer 17.6 g of filter into a 200-mL volumetric flask. Wash the filter with
lanthanum chloride to a 200-mL volumetric flask, add 100 water into the flask, and add water to volume.
mL of water, and carefully add 50 mL of hydrochloric acid. Analysis: Pipet a volume of the Sample solution, equivalent
Allow to cool, and dilute with water to volume. to 50 mg of calcium carbonate, into a 400-mL beaker, and
Dilute hydrochloric acid: Dilute 226 mL of hydrochloric add 200 mL of water, a volume of sodium hydroxide
acid to 1000 mL with water. solution (1 in 2) equivalent to the volume of the Sample
Potassium chloride solution: 30 mg/mL in water solution taken, and 250 mg of hydroxy naphthol blue. Stir
Magnesium stock solution: Transfer 1.000 g of magnesium with a magnetic stirrer, and titrate immediately with 0.05
metal to a 1OOO-mL volumetric flask containing 10 mL of

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172 Alumina / Official Monographs USP 43

M edetate disodium VS until the solution is distinctly blue. ChewableTablets meet the requirements of the test for the
Perform a blank determination, substituting a volume of absence of Salmonella species and Escherichia coli.
water equivalent to the volume of the Sample solution e ACID-NEUTRALIZING CAPACITY (301)
taken, and make any necessarycorrection. Each mL of 0.05 Sample solution: Dissolve an accuratelycounted number of
M edetate disodium is equivalent to 5.004 mg of calcium ChewableTablets, equivalent to about 120 mEq of
carbonate (CaC0 3). acid-neutralizing capacity, in 400 mLof water. Transferthe
Acceptance criteria: 90.0%-110.0% mixtureto a 500-mLvolumetricflask, and dilute with water
• POLYDIMETHYLSILOXANE to volume.
Dilute hydrochloric acid: Dilute 400 mL of hydrochloric Analysis: Proceed as directed 'in the section Procedure for
acid with sufficient water to make 1000 mL. Powders, Effervescent Solids, Suspensions and Other Liquids,
Standard solution: Transfer 60 mg of USP Lozenges Nonchewable Tablets, Chewable Tablets, and
Polydimethylsiloxane RS to a separator, add 30.0 mLof Capsules using 75.0 mLof the Sample solution.
chloroform and 60 mLof Dilute hydrochloric acid, shake for Acceptance criteria: The acid consumed 'by the minimum
30 s, and allow the phases to separate. Remove 10 rnl, of singledose recommended in the labelingisNLT5 mEq, and
the lower, organic layerto a screw-capped, 15-mLtest tube NLT the number of mEq calculated by the formula:
containing 0.5 g of anhydrous sodium sulfate. Close the
tube with a screw-cap having an inert liner, agitate Result = 0.55 x (FAX A) + 0.8 X (FM x M) + 0.9 x (Fe x C)
vigorously, and centrifuge the mixture to obtain a clear
supernatant. FA = theoretical acid-neutralizing capacity of
Sample solution: Weigh and finely powder NLT 20 aluminum hydroxide [AI(OH)3l, 0.0385 mEq
Chewable Tablets. Transfer a portion of the powder, A = amount of aluminum hydroxide [AI(OH)3l in the
equivalent of 60 mg of simethicone, to a suitable specimen tested, based on the labeled quantity
screw-capped bottle. Add 30.0 mL of chloroform and 60 (mg)
mL of Dilute hydrochloric acid, and allow to stand, with FM = theoretical acid-neutralizing capacity of
frequent shaking, until the ChewableTablets are dissolved. magnesium hydroxide [Mg(OH)2l, 0.0343 mEq
Transfer the contents of the bottle to a separator, shake, M = amount of magnesium hydroxide [Mg(OH)2l in
and allow the phases to separate. Remove 10 mLof the the specimen tested, based on the labeled
lower, organic layer to a screw-capped, 15-mLtest tube quantity (mg)
containing 0.5 g of anhydrous sodium sulfate. Close the Fe = theoretical acid-neutralizing capacity of calcium
tube with a screw-cap having an inert liner, agitate carbonate (CaC0 3), 0.02 mEq
vigorously, and centrifuge the mixture to obtain a clear C = amount of calcium carbonate (CaC0 3) in the
supernatant. specimen tested, based on the labeled quantity
Blank: Place 30.0 mL of chloroform and 60 mLof Dilute (mg)
hydrochloric acid in a separator, shake for 30 s, and allow
the phases to separate. Remove 10 mL ofthe lower,organic • SODIUM CONTENT
layerto a screw-capped, 15-mLtest tube containing 0.5 g Potassium chloride solution: 30 mg/mL of potassium
of anhydrous sodium sulfate. Closethe tube with a chloride
screw-cap that has an inert liner, agitate vigorously, and Dilute hydrochloric acid: Dilute226 mLof hydrochloric
centrifuge the mixture to obtain a clear supernatant. acid with sufficient water to make 1000 mL.
Analysis: Concomitantly determine the absorbances of the Standard stock solution: Transfer2.5420 g of sodium
Standard solution and the Sample solution in 0.5-mm cells chloride, previously dried at 105° for 2 h, to a 1000-mL
at the wavelength of maximum absorbance at about 7.9 volumetric flask, and dissolve in and dilute with water to
J.Im, with a suitable IR spectrophotometer, using the Blank volume. Transfer 10.0 mLof this solution to a 100-mL
to set the instrument. , volumetric flask, and dilute with water to volume. Transfer
Calculate the percentage of polydimethylsiloxane 10.0 mL of this solution to a second 1OO-mL volumetric
([-(CH3)2 SiO-ln) in the portion of Tablets taken: flask, and dilute with water to volume.
Standard solutions: To three separate 1OO-mL volumetric
Result =(Au/As) x (Ws/Wu) x 100 flasks, each containing 10.0 mLof Potassium chloride
solution and 3.0 mLof Dilute hydrochloric acid, add 10.0,
Au =absorbance of the Sample solution 20.0, and 30.0 mL, respectively, of the Standard stock
As =absorbance of the Standard solution solution. The resulting Standard solutions contain 1.0, 2.0,
Ws = weight of USP Polydimethylsiloxane RS taken to and 3.0 J.Ig/mL of sodium (Na), respectively.
prepare the Standard solution (mg) Sample stock solution: Weigh 10 Chewable Tablets, and
Wu =nominal amount of simethicone in the portion of determine the average weight, A, in mg. Cut 4 Chewable
the Tablets taken to prepare the Sample solution Tabletsinto pieces, combine the pieces, and weigh them.
(mg) Transfer the combined pieces to a 500-mLvolumetricflask,
add 150 mL of Dilute hydrochloric acid, and swirl gently to
Acceptance criteria: 85.00/0-115.0% dissolve the pieces. Dilute with water to volume.
PERfORMANCE TESTS
Sample solution: Transfer10.0 mL of the Sample stock
• UNIFORMITY OF DOSAGE UNITS (905): Meet the solution to a 1OO-mL volumetric flask, add 10.0 mL of
requirements for Weight Variation with respect to Potassium chloride solution, and dilute with water to volume.
aluminum hydroxide, to magnesium hydroxide, and to Blanksolution: Combine 3.0 mLof Dilute hydrochloric acid
calcium carbonate. and 10.0 mL of Potassium chloride solution in a 100-mL
volumetric flask, and dilute with water to volume.
SPECifiC TESTS Analysis
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR Samples: Standard solution and Sample solution
SPECIFIED MICROORGANISMS (62): The total aerobic Concomitantlydetermine the absorbances of the
microbial count is NMT 2 x 1.02 du/g, the total combined Standard solutions and the Sample solution at the sodium
molds and yeasts count is NMT 2 x 102 du/g, and the emission line at 589.0 nm with a suitable atomic

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 USP 43 OfficialMonographs / Alumina 173

absorption spectrophotometer (see AtomicAbsorption • C. IIDENTIFICATION TESTS-GENERAL, Aluminum (191)

Spectroscopy (852» equipped with a sodium hollow- Sample solution: Wash the precipitate from Identification
cathode lamp and an air-acetylene flame, using the test B with hot ammonium chloride solution (1 in 50), and
Blank solution as the blank. Plot the absorbances of the dissolve the precipitate in hydrochloric acid. Divide this
Standardsolutions versus concentration, in J,Jg/mL, of solution into two portions.
sodium, and draw the straight line best fitting the three Analysis 1: Add, dropwise, 6 N ammonium hydroxide to
plotted points. From the graph so obtained, determine one portion of the Sample solution.
the concentration, C, in J,Jg/mL, of sodium in the Sample Acceptance criteria 1: A gelatinous white precipitate, which
solution. does not dissolve in an excess of 6 N ammonium hydroxide,
Calculate the quantity, in mg, of sodium (Na) in each is obtained.
Chewable Tablet taken: Analysis 2: Add, dropwise, 1 N sodium hydroxide to the
second portion of the Sample solution.
Result = (AI W) x (x D x F Acceptance criteria 2: A gelatinous white precipitate, which
dissolves in an excess of 1 N sodium hydroxide, leaving
A = average weight of each Tablet (mg) some turbidity, is obtained.
W = weight of the portion of Chewable Tablets taken
to prepare the Sample solution (mg) ASSAY
C =concentration of sodium in the Sample solution • ALUMINUM HYDROXIDE
(J,Jg/mL) Edetate disodium titrant: Prepare and standardize as
D = dilution factor for the Sample solution, 5000 directed in Reagents, Volumetric Solutions, Edetate Disodium,
F = conversion factor, 0.001 mg/J,Jg Twentieth-Molar(0.05 M).
Sample solution: Transfer a measured amount of Oral
Acceptance criteria: Chewable Tablets contain NMT 5 mgl Suspension, previously well shaken in its original container,
Tablet of sodium, except when labeled as containing more equivalent to 800 mg of aluminum hydroxide, to a suitable
than 5 mg/Tablet of sodium; then they contain NMT 110% beaker. Add 20 mL of water, stir, and slowly add 10 mL of
of the labeled amount. hydrochloric acid. Heat gently, if necessary, to aid solution,
cool, and filter into a 200-mL volumetric flask. Wash the
ADDITIONAL REQUIREMENTS filter with water into the flask, and add water to volume.
• PACKAGING AND STORAGE: Preserve in well-closed Analysis: Pipet 10 mL of the Sample solution into a 250-mL
containers. beaker, add 20 mL of water, then add, in the order named
• LABELING: The labeling indicates that the Chewable Tablets and with continuous stirring, 25.0 mL of Edetate disodium
are to be chewed before swallowing. Label the Chewable titrant and 20 mL of acetic acid-ammonium acetate buffer
Tablets to state the sodium content, if it is greater than 5 TS, and heat the solution near the boiling temperature for
mg/Chewable Tablet. 5 min. Cool, add 50 mL of alcohol and 2 mL of dithizone
• USP REFERENCE STANDARDS (11) TS. Titrate the excess edetate disodium with 0.05 M zinc
USP Polydimethylsiloxane RS sulfate VS until the color changes from green-violet to
rose-pink. Perform a blank determination, substituting 10
mL of water for the Sample solution, and make any
necessary correction. Each mL of Edetate disodium titrant
consumed is equivalent to 3.900 mg of aluminum
Alumina, Magnesia, and Simethicone hydroxide [AI(OH)31.
Oral Suspension Acceptance criteria: 90.0%-115.0%
• MAGNESIUM HYDROXIDE
DEFINITION Sample solution: Prepare as directed in the Assay for
Alumina, Magnesia, and Simethicone Oral Suspension Aluminum Hydroxide.
contains the equivalent of NLT 90.0% and NMT 115.0% of Analysis: Pipet a volume of the Sample solution, equivalent
the labeled amounts of aluminum hydroxide [AI(OH)31 and to 40 mg of magnesium hydroxide, into a 400-mL beaker,
magnesium hydroxide [Mg(OH)21, and an amount of add 200 mL of water and 20 mL of triethanolamine, and
polydimethylsiloxane ([-(CH 3)2 SiO-l n) that is NLT 85.0% stir. Add 10 mL of ammonia-ammonium chloride buffer TS
and NMT 115.0% of the labeled amount of simethicone. and 3 drops of an eriochrome black indicator solution
(prepared by dissolving 200 mg of eriochrome black T in a
IDENTIFICATION mixture of 15 mL of triethanolamine and 5 mL of
dehydrated alcohol, and mixing). Cool the solution to
between 3° and 4° by immersion of the beaker in an ice
bath, then remove, and titrate with 0.05 Medetate
• A:•. ~~ij~~.·) .•. )\).~i~'!~~ disodium VS to a blue endpoint. Perform a blank
~PJ~~tf9§99PY;~~'l~.4.(q., . . <1 " .• 9) determination, substituting water for the Sample solution,
Sample solution: Prepare as directed in the Assay for and make any necessarycorrection. Each mL of 0.05 M
Polydimethylsiloxane. edetate disodium consumed is equivalent to 2.916 mg of
Analysis: Proceed as directed using a 0.5-mm cell. magnesium hydroxide [Mg(OH)21.
Acceptance criteria: Meets the requirements Acceptance criteria: 90.0%-115.0%
• B. IDENTIFICATION TESTS-GENERAL, Magnesium (191) • POLYDIMETHYLSILOXANE
Sample solution: Add 5 9 of Oral Suspension tol 0 mL of 3 Standard solution: Prepare similarly to the Sample solution
N hydrochloric acid, then add 5 drops of methyl red TS, below, except dissolve 50 mg of USP Polydimethylsiloxane
heat to boiling, add 6 N ammonium hydroxide until the RSin 25.0 mL of toluene, add 40 mL of 0.1 N sodium
color of the solution just changes to deep yellow, then hydroxide, and add a volume of water equal to that of the
continue boiling for 2 min, and filter. specimen of Oral Suspension taken for the Sample solution.
Acceptance criteria: Meets the requirements Sample solution: Transfer an amount of Oral Suspension,
equivalent to 50 mg of simethicone, to a suitable round,

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174 Alumina / Official Monographs USP 43

narrow-mouth, screw-capped, 120-mL bottle, add 40 mL Standard solutions contain 0.5 and 1.0 I-lg/mL of sodium
of 0.1 N sodium hydroxide, and swirl to disperse. Add 25.0 (Na), respectively.
mL of toluene, close the bottle securely with a cap having Sample solution: Transfer 5.0 mL of Oral Suspension,
an inert liner, and shake for 15 min on a reciprocating previously well shaken in its original container, to a 100-mL
shaker (e.g., about 200 oscillations/min and a stroke of 38 volumetric flask. Add 50 mL of 1 N hydrochloric acid, boil
± 2 mm). Transfer the mixture to a 125-mL separator. for 15 min, cool to room temperature, and dilute with water
Remove about 5 mL of the upper, organic layer to a to volume. Filter, discarding the first few mL of the filtrate.
screw-capped, 15-mL test tube containing 0.5 g of Transfer 5.0 mL of the filtrate to a 1OO-mL volumetric flask
anhydrous sodium sulfate. Close the tube with a screw-cap containing 10.0 mL of Potassium chloride solution, and
having an inert liner, agitate vigorously, and centrifuge the dilute with water to volume.
mixture until a clear supernatant is obtained. Blank solution: Combine 4.0 mL of 1 N hydrochloric acid
Blank: Mix 10 mL of toluene with 0.5 g of anhydrous sodium and 10.0 mL of Potassium chloride solution in a 100-mL
sulfate, and centrifuge to obtain a clear supernatant. volumetric flask, and dilute with water to volume.
Analysis: Concomitantly determine the absorbances of the Analysis
Standard solution and the Sample solution in 0.5-mm cells Samples: Standard solution and Sample solution
at the wavelength of maximum absorbance at about 7.9 Concomitantly determine the absorbances of the
urn, with a suitable IR spectrophotometer, using the Blank Standard solutions and the Sample solution at the sodium
to set the instrument. emission line at 589.0 nm with a suitable atomic
Calculate the percentage of polydimethylsiloxane absorption spectrophotometer (see Atomic Absorption
([-(CH 3)2 SiO-]n) in the portion of Oral Suspension taken: Spectroscopy (852» equipped with a sodium hollow-
cathode lamp and an air-acetylene flame, usi":!g the
Result = (Au/As) x (Ws/Wu) x 100 Blank solution asthe blank. Plot the absorbances of the
Standard solutions versus concentration, in J.Jg/mL, of
Au =absorbance of the Sample solution sodium, and draw a straight line between the plotted
As =absorbance of the Standard solution points. From the graph so obtained, determine the
Ws = weight of USP Polydimethylsiloxane RS taken to concentration, C, in J.Jg/mL, of sodium in the Sample
prepare the Standard solution (mg) solution.
Wu =nominal amount of slmethlcone in the portion of Calculate the quantity, in mg, of sodium (Na) in each mL
the Oral Suspension taken to prepare the Sample of Oral Suspension taken:
solution (mg)
Result = (1/ N) x C x 0 x F
Acceptance criteria: 85.0%-115.0%
N =volume of the Oral Suspension taken to prepare
SPECIFIC TESTS the Sample solution, 5.0 mL
o MICROBIAL ENUMERATION TESTS (61) and TESTS FOR C = concentration of sodium in the Sample solution
SPECIFIED MICROORGANISMS (62): The total aerobic (J.Jg/mL)
microbial count is NMT 102 cfu/g, and it meets the o = dilution factor for the Sample solution, 2000
requirements of the test for the absence of Escherichia coli. F = conversion factor, 0.001 mg/J.Jg
o ACID-NEUTRALIZING CAPACITY (301)
Acceptance criteria: The acid consumed by the minimum ADDITIONAL REQUIREMENTS
single dose recommended in the labeling is NLT 5 mEq, and o PACKAGING AND STORAGE: Preserve in tight containers, and
NLT the number of mEq calculated by the-formula: avoid freezing.
o LABELING: Oral Suspension may be labeled to state the
Result = 0.55 x (FAx A) + 0.8 X (FM x M) aluminum hydroxide content in terms of the equivalent
amount of dried aluminum hydroxide gel, on the basisthat
FA = theoretical acid-neutralizing capacity of each mg of dried gel is equivalent to 0.765 mg of aluminum
aluminum hydroxide [AI(OH)3]' 0.0385 mEq hydroxide (AI(OH)3)' Label it to state the sodium content if
A =amount of aluminum hydroxide [AI(OH)3] in the it is greater than.l mg/mL.
specimen tested, based on the labeled quantity o USP REFERENCE STANDARDS (11)
(mg) USP Polydimethylsiloxane RS
FM = theoretical acid-neutralizing capacity of
magnesium hydroxide [Mg(OH)2]' 0.0343 mEq
M =amount of magnesium hydroxide [Mg(OH)2l in
the specimen tested, based on the labeled
quantity (mg) Alumina, Magnesia, and Simethicone
Chewable Tablets
o pH (791): 7.0-8.6
o SODIUM CONTENT DEFINITION
Potassium chloride solution: 38 mg/mL of potassium Alumina, Magnesia, and Simethicone Chewable Tablets
chloride contain the equivalent of NLT 90.0% and NMT 115.0% of
Sodium chloride stock solution: 25.42 J.Jg/mL of sodium the labeled amounts of aluminum hydroxide [AI(OH)3] and
chloride (previously dried at 105° for 2 h) in water. The magnesium hydroxide [Mg(OH)2]' and an amount of
solution contains 10 J.Jg/mL of sodium. polydimethylsiloxane ([-(CH 3)2 SiO-]n) that is NLT 85.0%
Standard solutions: On the day of use, transfer 4.0 mL of 1 and NMT 115.0% of the labeled amount of simethicone.
N hydrochloric acid and 10.0 mL of Potassium chloride
solution to each of two 1OO-mL volumetric flasks. To the
respective flasks add 5.0 and 10.0 mL of Sodium chloride
stock solution. Dilute with water to volume. The resulting

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USP 43 OfficialMonographs / Alumina 175

IDENTlIFBCAnON (prepared by dissolving 200 mg of eriochrome black T in a

mixture of 15 mL of triethanolamine and 5 mL of
dehydrated alcohol, and mixing). Cool the solution to
between 3° and 4° by immersion of the beaker in an ice
• A. bath, then remove, and titrate with 0.05 M edetate
!~R~ disodium VSto a blue endpoint. Perform a blank
Sample solution: Prepareas in the Assay for determination, substituting water for the Sample solution,
Polydimethylsiloxane. and make any necessary correction. Each mL of 0.05 M
Analysis: Proceed as directed using a 0.5-mm cell. edetate disodium consumed is equivalent to 2.916 mg of
Acceptance criteria: Meet the requirements magnesium hydroxide [Mg(OH)2J.
• B. IDENTIFICATION TESTS-GENERAL, Magnesium (191) Acceptance criteria: 90.0%-115.0%
Sample solution: To a portion offinely powdered Chewable • POL VDIMETHVLSILOXANE
Tablets, equivalent to 600 mg of magnesium hydroxide, Standard solution: Prepare similarly to the Sample solution
add 25 mL of 3 N hydrochloric acid and 25 mL of water, below, using 33 mg of USP Polydimethylsiloxane RS.
and mix. Boil gently for 2 min. Allow to cool, and filter. Add Sample solution: Weigh and finely powder NLT 20
5 drops of methyl red TS, heat to boiling, and add 6 N Chewable Tablets. Transfer a portion of the powder,
ammonium hydroxide until the color of the solution just equivalent of 33 mg of simethicone, to a suitable round,
turns to deep yellow. Continue boiling for 2 min, and filter. narrow-mouth, screw-capped, 120-mL bottle. Add 40 mL
Acceptance criteria: Meet the requirements of 0.1 N sodium hydroxide, and swirl to disperse.Add 20.0
• C. IDENTIFICATION TESTS-GENERAL, Aluminum (191) mL of toluene, close the bottle securely with a cap having
Sample solution: Wash the precipitate from Identification an inert liner, and shake for 30 min on a reciprocating
test B with hot ammonium chloride solution (1 in 50), and shaker(e.g., 200 oscillations/min and a stroke of 38 ± 2
dissolve the precipitate in hydrochloric acid. Divide this rnm), Transferthe mixture to a 125-mL separator, and allow
solution into two portions. to separate. Remove the upper, organic layer to a
Analysis 1: Add, dropwise, 6 N ammonium hydroxide to screw-capped, centrifuge tube containing 2 g of anhydrous
one portion of the Sample solution. sodium sulfate. Close the tube with a screw-cap having an
Acceptance criteria 1: A gelatinous white precipitate, which inert liner, agitate vigorously, and centrifuge the mixture
does not dissolve in an excess of 6 N ammonium hydroxide, until a clear supernatant is obtained.
is obtained. Blank: Mix 10 mL of toluene with 1 g of anhydrous sodium
Analysis 2: Add, dropwise, 1 N sodium hydroxide to the sulfate, and centrifuge to obtain a clear supernatant.
second portion of the Sample solution. Analysis: Concomitantly determine the absorbances of the
Acceptance criteria 2: A gelatinous white precipitate, which Standard solution and Sample solution in 0.5-mm cellsat the
dissolves in an excess of 1 N sodium hydroxide, leaving wavelength of maximum absorbance at about 7.9 IJm
some turbidity, is obtained. (1265.8 crrr"), with a suitable IRspectrophotometer, using
the Blank to set the instrument.
ASSAY
Calculate the percentage of polydimethylsiloxane
• ALUMINUM HVDROXIDE
([-(CH 3)2 SiO-Jn) in the portion of Chewable Tablets
Edetate disodium titrant: Prepare and standardize as
directed in Reagents, Volumetric Solutions, Edetate Disodium, taken:
Twentieth-Molar (0.05 M). Result =(Au/As) x (Ws/Wu) x 100
Sample solution: Weigh and finely powder NLT 20 .
Chewable Tablets. Transfer a portion of the powder, Au = absorbance of the Sample solution
equivalent of 800 mg of aluminum hydroxide, to a 150-mL As =absorbance of the Standard solution
beaker. Add 20 mL of water, stir, and slowly add 30 mL of
3 N hydrochloric acid. Heat gently, if necessary, to aid
Ws =weight of USP Polydimethylsiloxane RS taken to
solution, cool to room temperature, and filter into a 200-mL prepare the Standard solution (mg)
volumetric flask. Wash the filter with water into the flask, Wu =nominal amount of simethicone in the portion of
the Chewable Tablets taken to prepare the
and add water to volume.
Analysis: Pipet 10 mL of the Sample solution into a 250-mL Sample solution (mg)
beaker, add 20 mL of water, then add, in the order named
Acceptance criteria: 85.0%-115.0%
and with continuous stirring, 25.0 mL of Edetate disodium
titrant and 20 mL of acetic acid-ammonium acetate buffer PERFORMANCE TESTS
TS, and heat the solution near the boiling temperature for • UNIFORMITY OF DOSAGE UNITS (905): Meet the
5 min. Cool, add 50 mL of alcohol and 2 mL of dithizone requirements for Weight Variation with respect to
TS. Titrate the excess edetate disodium with 0.05 M zinc aluminum hydroxide and to magnesium hydroxide
sulfate VS until the color changes from green-violet to
rose-pink. Perform a blank determination, substituting 10 SPECIFIC TESTS
mL of water for the Sample solution, and make any • ACID-NEUTRALIZING CAPACITV (301)
necessarycorrection. Each mL of Edetate disodium titrant Acceptance criteria: The acid consumed by the minimum
consumed is equivalent to 3.900 mg of aluminum single dose recommended in the labeling is NLT 5 mEq, and
hydroxide [AI(OH)3J. NLT the number of mEq calculated by the formula:
Acceptance criteria: 90.0%-115.0%
Result =0.55 x (FAX A) + 0.8 X (FM x M)
• MAGNESIUM HVDROXIDE
Sample solution: Prepare as directed in the Assay for FA = theoretical acid-neutralizing capacity of ..
Aluminum Hydroxide. aluminum hydroxide [AI(OH)3J, 0.0385 mEq
Analysis: Pipet a volume of the Sample solution, equivalent
to 40 mg of magnesium hydroxide, into a 400-mL beaker,
A =amount of aluminum hydroxide [AI(OH)3J in the
add 200 mL of water and 20 mL of triethanolamine, and specimen tested, based on the labeled quantity
stir. Add 10 mL of ammonia-ammonium chloride buffer TS (mg)
and 3 drops of an eriochrome black indicator solution

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176 Alumina / Official Monographs USP 43

FM =theoretical acid-neutralizing capacity of Alumina and Magnesium Carbonate

magnesium hydroxide [Mg(OH)2]' 0.0343 mEq
M =amount of magnesium hydroxide [Mg(OH)2] in Oral Suspension
the specimen tested, based on the labeled
quantity (mg)
» Alumina and Magnesium Carbonate Oral
• SODIUM CONTENT Suspension contains the equivalent of not less than
Potassium chloride solution: 38 mg/ml of potassium 90.0 percent and not more than 110.0 percent of
chloride the labeled amounts of aluminum hydroxide
Sodium chloride stock solution: 25.42 ~g/ml of sodium
chloride (previously dried at 105° for 2 h) in water. The [AI(OH)3J and magnesium carbonate (MgC0 3)·
solution contains 10 ~g/ml of sodium.
Standard solutions: On the day of use, transfer 4.0 ml of 1 Packaging and storage-Preserve in tight containers, and
N hydrochloric acid and 10.0 ml of Potassium chloride avoid freezing.
solution to each of two 1OO-ml volumetric flasks. To the Identification-
respective flasks add 5.0 and 10.0 ml of Sodium chloride A: Place about 1 g in a flask equipped with a stopper and
stock solution. Dilute with water to volume. The resulting glass tubing, the tip of which is immersed in calcium hydroxide
Standard solutions contain 0.5 and 1.0 ~g/ml of sodium TS in a test tube. Add 5 ml of 3 N hydrochloric acid to the
(Na), respectively. flask, and immediately insert the stopper: gas evolves in the
Sample solution: Weigh and finely powder NLT 20 flask and a precipitate is formed in the test tube.
Chewable Tablets. Transfer a portion of the powder, B: To a solution of 5 gin 10 ml of 3 N hydrochloric acid
equivalent to the average weight of 1 Chewable Tablet, to add 5 drops of methyl red TS, heat to boiling, add 6 N
a 1OO-ml volumetric flask. Add 50 ml of 1 N hydrochloric ammonium hydroxide until the color of the solution changes
acid, boil for 15 min, cool to room temperature, and dilute to deep yellow, then continue boiling for 2 minutes, and filter:
with water to volume. Filter, discarding the first few ml of the filtrate responds to the tests for Magnesium (191). .
the filtrate. Transfer 5.0 ml of the filtrate to a 100-ml C: Wash the precipitate obtained in Identification test Bwith
volumetric flask containing 10.0 ml of Potassium chloride a hot solution of ammonium chloride (1 in 50), and dissolve
solution, and dilute with water to volume. the precipitate in hydrochloric acid: the solution responds to
Blank solution: Combine 4.0 ml of 1 N hydrochloric acid the tests for Aluminum (191).
and 10.0 mL of Potassium chloride solution in a 100-ml Microbial enumeration tests (61) and Tests for specified
volumetric flask, and dilute with water to volume. microorganisms (62)-lts total aerobic microbial count does
Analysis not exceed 100 du per ml, and it meets the requirements of
Samples: Standard solution and Sample solution the test for absence of Escherichia coli,Salmonella species,
Concomitantly determine the absorbances of the Staphylococcus aureus, and Pseudomonas aeruginosa.
Standard solutions and the Sample solution at the sodium Acid-neutralizing capacity (301 )-Not less than 5 mEq of
emission line at 589.0 nm with a suitable atomic acid is consumed by the minimum single dose recommended
absorption spectrophotometer (see Atomic Absorption in the labeling, and not less than the number of mEq
Spectroscopy (852») equipped with a sodium hollow- calculated by the formula:
cathode lamp and an air-acetylene flame, using the
Blank solution as the blank. Plot the absorbances of the 0.55(0.0385A) + 0.8(0.024M)
Standard solutions versus concentration, in ~g/mL, of
sodium, and draw a straight line between the plotted in which 0.0385 and 0.024 are the theoretical
points. From the graph so obtained, determine the acid-neutralizing capaclties, in mEq, of AI(OH)3 and MgC0 3,
concentration, C, in ~g/ml, of sodium in the Sample respectively; and A and M are the respective quantities, in mg,
solution. of AI(OHh and MgC0 3 in the specimen tested, based on the
Calculate the quantity, in mg, of sodium (Na) in each labeled quantities.
Chewable Tablet taken: pH (791): between 7.5 and 9.5.
Result = C x D x F Assay for aluminum hydroxide-
Potassium chloride solution-Prepare a solution containing
C = concentration of sodium in the Sample solution 4.5 g of potassium chloride in each 100 ml.
(~g/ml) Aluminum stock solution-Transfer 1.000 g of aluminum
o = dilution factor for the Sample solution, 2000 wire to a 1OOO-ml volumetric flask, and add 50 ml of 6 N
F = conversion factor, 0.001 mg/~g hydrochloric acid. Swirl to ensure contact of the aluminum
and the acid, and allow the reaction to proceed until all of the
ADDITIONAL REQUIREMENTS aluminum has dissolved. Dilute with water to volume, and
• PACKAGING AND STORAGE: Preserve in well-closed mix.
containers. Standard preparations-To separate 1OO-ml volumetric
• LABELING: Label the Chewable Tablets to indicate that they flasks, each containing 10 ml of Potassium chloride solution,
are to be chewed before being swallowed. label the transfer 9.0, 10.0, and 11.0 ml, respectively, of Aluminum
Chewable Tablets to state the sodium content if it is greater stock solution, dilute with water to volume, and mix. These
than 5 mg/Tablet. The Chewable Tablets may be labeled to Standard preparations contain 90.0, 100.0, and 110.0 ~g of
state the aluminum hydroxide content in terms of the aluminum per ml, respectively.
equivalent amount of dried aluminum hydroxide gel, on Assay preparation-Transfer an accurately measured
the basis that each mg of dried gel is equivalent to 0.765 quantity of Or~1 Suspension, previously shak~n in its origi~al
mg of aluminum hydroxide [AI(OH)3]' container, equivalent to about 75 mg of aluminum hydroxide,
• USP REFERENCE STANDARDS (11) to a suitable beaker. Add 25 rnl, of 6 N hydrochloric acid,and
USP Polydimethylsiloxane RS heat on a steam bath for 30 minutes, with occasional SWirling.
Cool, and transfer with the aid of water to a 250-ml volumetric

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 USP 43 Official Monographs / Alumina 177

flask containing 25 mL of Potassium chloride solution. Dilute

with water to volume, mix, and filter. Alumina and Magnesium Carbonate
Procedure-Concomitantly determine the absorbances of Tablets
the Standardpreparations and the Assay preparation at the
aluminum emission line at 309.3 nm, with a suitable atomic
absorption spectrophotometer (see AtomicAbsorption » Alumina and Magnesium Carbonate Tablets
Spectroscopy (852» equipped with an aluminum contain the equivalent of not less than 90.0 percent
hollow-cathode lamp and a nitrous oxide-acetylene flame, and not more than 110.0 percent of the labeled
using water as the blank. Calculate the quantity, in mg, of
AI(OH)3 in the portion of Oral Suspension taken by the
amounts of aluminum hydroxide [AI(OH)3l and
formula: magnesium carbonate (MgC0 3).
(78.00/26.98)(0.25)(A u/ Rs) Packaging and storage-Preserve in tight containers.

in which 78.00 is the molecular weight of aluminum Identification-

hydroxide; 26.98 is the atomic weight of aluminum; Au is the A: Place about 1 g of finely powdered Tablets in a flask
equipped with a stopper and glass tubing, the tip of which is
absorbance of the Assay preparation; and Rs is the average of
immersed in calcium hydroxide TS in a test tube. Add 5 mL of
the ratios of the absorbances of the Standard preparations to 3 N hydrochloric acid to the flask, and immediately insert the
their respective concentrations, in IJg of aluminum per mL. stopper: gas evolves in the flask and a precipitate is formed in
Assay for magnesium carbonate- the test tube.
Lanthanumchloride solution-Prepare a solution of B: To a 7-g portion of finely powdered Tablets add 10 mL
lanthanum chloride in water containing 5 mg per mL. of 3 N hydrochloric acid and 5 drops of methyl red TS, heat
Magnesium stock solution-Transfer 1.000 g of magnesium to boiling, and add 6 N ammonium hydroxide until the color
metal to a 1OOO-mL volumetric flask containing 50 mL of of the solution changes to deep yellow. Continue boiling for
water, and slowly add 10 mL of hydrochloric acid: Dilut~ with 2 minutes, and filter: the filtrate responds to the tests for
water to volume, and mix. Transfer 10.0 mL of this solution to Magnesium (191).
a 1OO-mL volumetric flask, dilute with water to volume, and C: Wash the precipitate obtained in Identification test B
mix. with a hot solution of ammonium chloride (1 in 50), and
Standardpreparations-To separate 1OO-mL volumetri~ dissolvethe precipitate in hydrochloric acid: the solution
flasks each containing 10 mL of Lanthanum chloride solution, responds to the tests for Aluminum (191).
transfer 1.70 mL and 1.80 mL, respectively, of Magnesium Disintegration (701): 10 minutes, simulated gastric fluid TS
stock solution, dilute with water to volume, and mix. These being substituted for water in the test.
Standard preparations contain 1.7 IJg of magnesium per mL Uniformity of dosage units (905): meet the requirements
and 1.8 J.lg of magnesium per mL, respectively. for Weight Variation with respect to aluminum hydroxide and
Assay preparation-Quantitatively dilute an accurately to magnesium carbonate.
measured volume of the Assay preparation prepared as Acid-neutralizing capacity (301)-Not less than 5 mEq of
directed in the Assay for aluminum hydroxide with water to acid is consumed by the minimum single dose recommended
obtain a solution having a concentration of about 6 IJg of in the labeling.
magnesium carbonate per mL. Assay for aluminum hydroxide- . .
Procedure-Concomitantly determine the absorbances of Potassium chloride solution-Prepare a solution containing
the Standardpreparations and the Assay preparation at the . 38.1 g of potassium chloride in each 1000 mL.
magnesium emission line at 285.2 nm, w~th a suita!Jle atomic Digestion fluid-Mix 5 mL of hydrochloric acid, 10 mL of
absorption spectrophotometer (see AtomiC Absorption . nitric acid, and 10 mL of water, and use promptly.
Spectroscopy (852» equipped with a magnesium Aluminum stocksolution-Transfer 1.000 g of aluminum
hollow-cathode lamp and an air-acetylene flame, using water metal to a 1OOO-mL volumetric flask, and add 50 mL of 6 N
as the blank. Calculate the quantity, in mg, of magnesium hydrochloric acid. Swirl to ensur~ contact of the al~minum
carbonate (MgC0 3) in the portion of Oral Suspension taken and the acid, and allow the reaction to proceed until all of the
by the formula:) aluminum has dissolved. Dilute with water to volume, and
mix.
(84.31/24.31 )(L/D)(Au/Rs) Standard preparations-To separate 1OO-mL volumetric
flasks transfer 3.0 mL, 4.0 mL, and 5.0 mL of Aluminum stock
in which 84.31 is the molecular weight of magnesium solution, respectively. To each flask add 10 mL of Potassium
carbonate; 24.31 isthe atomic weight of magnesium; L is the chloride solution and 7.5 mL of Digestion fluid, dilute with water
labeled quantity, in mg,. of magnesiu.m carbonate in ~he . to volume, and mix. These Standard preparations contain 30,
portion of Oral Suspension taken; D IS the concentration, ~n 40, and 50 J.lg of aluminum per mL, respectively.
IJg of magnesium carbonate per mL, of the Assay preparation, Assay preparation-Weigh and finely powder not fewer
based on the labeled amount of magnesium carbonate in the than 20 Tablets. Transfer an accurately weighed portion of the
portion of Oral Suspension taken and the extent of dilution; powder, equivalent to about 30 mg of aluminum hydroxide,
Au is the absorbance of the Assay preparation; and Rs is the to a 1OO-mL volumetric flask, add 25-mL of Digestion fluid, and
average of the ratios of the absorbances of the Standard heat on a steam bath for 30 minutes or on a hot plate until the
preparations to their respective concentrations, in IJg of volume is reduced by about one-half. Cool, dilute with water
magnesium per mL. to volume, and mix. Filter, discarding the first 20 mL of the
filtrate. Transfer 15.0 mL of the filtrate to a 50-mL volumetric
flask, add 5.0 mL of Potassium chloride solution, dilute with
water to volume, and mix. [NoTE-Reserve a portion of the
filtrate for use in the Assay for magnesium carbonate.]
Procedure-ConcoiTIitantly determine the absorbances of
the Standard preparations and the Assay preparation at the

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178 Alumina / OfficialMonographs USP 43

aluminum emission line at 309.3 nm, with a suitable atomic

absorption spectrophotometer (see Atomic Absorption Alumina, Magnesium Carbonate, and
Spectroscopy (852» equipped with an aluminum Magnesium Oxide Tablets
h~lIow-cathode lamp and a nitrous oxide-acetylene flame,
usmq water as the blank. Plot the absorbances of the Standard
preparations versus concentration, in IJg per mL, of aluminum, » Alumina, Magnesium Carbonate, and
and draw the straight line best fitting the three plotted points. Magnesium Oxide Tablets contain the equivalent
From the graph so obtained, determine the concentration C of not less than 90.0 percent and not more than
in IJg pe~ mL, of aluminum in each mL of the Assay " 110.0 percent of the labeled amounts of aluminum
prepar~tlon. Calculate the quantity, in mg, of aluminum
hydroxide [AI(OH)3l in the portion of Tablets taken by the hydroxide [AI(OH)3J and magnesium carbonate
formula: (MgC0 3), and not less than 85.0 percent and not
more than 115.0 percent of the labeled amount of
(78.00/26.98)(C/3)
magnesium oxide (MgO).
in whic.h 78.00 is the molecular weight of aluminum
hydroxide, and 26.98 is the atomic weight of aluminum. Packaging and storage-Preserve in tight containers.

Assay for magnesium carbonate- Identification-

Lanthanum chloride solution-Transfer 17.6 g of lanthanum A: Place about 3 g of finely powdered Tablets in a flask
chloride to a 1OOO-mL volumetric flask, add 500 mL of water, equipped with a stopper and glass tubing, the tip of which is
and caref~lIy ad~ 50 mL of hydrochloric acid. Mix, and allow immersed in calcium hydroxide TS in a test tube. Add 5 mL of
to cool. Dilute with water to volume, and mix. 3 N hydrochloric acid to the flask, and immediately insert the
Digestion fluid-Mix 5 mL of hydrochloric acid 10 mL of stopper: gas evolves in the flask and a precipitate is formed in
nitric acid, .and 10 mL of .water, and use promptly. the test tube.
Maqnesium stock solution-Transfer 1.000 g of magnesium B: To the solution in the flask obtained in Identification test
metal to a 1OOO-mL volumetric flask containing 50 mL of A add 5 drops of methyl red TS, and heat to boiling. Add 6 N
water, and slowly add 10 mL of hydrochloric acid. Dilute with .ammonium hydroxide until the color of the solution changes
water to volume, and mix. Transfer 5.0 mL of this solution to to deep yellow, continue boiling for 2 minutes, and filter
a ?OO-mL volumetric flask, dilute with water to volume, and through hardened filter paper. (Retain the filtrate for
mix. Identification test C.) Wash the precipitate with 350 mL of a
Standard preparations-To separate 1OO-mL volumetric hot ammonium chloride solution (1 in 50), discarding the
flask~ transfer 4.?, 6.0, and 8.0 mL of Magnesium stock
washings: the precipitate so obtained, dissolved in 3 N
so/~tlOn, respectively. To each flask add 0.5 mL of Digestion
hydrochloric acid, responds to the tests for Aluminum (191).
ttuid and 10 mL of Lanthanum chloride solution, dilute with C: The filtrate obtained in Identification test B responds to
water. to volume,' and mix. These Standard preparations the tests for Magnesium (191).
contain, 0.40, 0.60, and 0.80 IJg of magnesium per mL, Disintegration (701): 10 minutes, simulated gastric fluid TS
respectively. being substituted for water in the test.
Assay prepar~tion-Transfer an accurately measured Uniformity of dosage units (905): meet the requirements
volume of the filtrate used to prepare the Assaypreparation in for Weight Variation with respect to alumina, to magnesium
the Assay f?r aluminum hydroxide, equivalent to about 0.4 mg carbonate, and to magnesium oxide.
of magnesium carbonate, to a 200-mL volumetric flask, add Acid-neutralizing capacity (301 )-Not less than 5 mEq of
20 mL of Lanthanum chloride solution, dilute with water to acid is consumed by the minimum single dose recommended
volume, and mix. in the labeling.
Procedure--Concomitantly determine the absorbances of Assay for aluminum hydroxide-
the Sta~dard pr~p~rati?ns and the Assaypreparation at the Edetate disodium titrant-Prepare and standardize as
magnesium errussron line at 285.2 nm, with a suitable atomic directed in the Assay under Ammonium Alum.
absorption spectrophotometer (see Atomic Absorption Assay preparation-Weigh and finely powder not fewer
Spectroscopy (852» equipped with a magnesium than 20 Tablets. Transfer an accurately weighed portion of the
hollow-cathode lamp and an air-acetylene flame, using water powder, equivalent to about 1200 mg of aluminum
as the blank. Plot the absorbances of the Standard hydroxide, to a 150-mL beaker, add 20 mL of water, stir, and
prepara~ions versus concentration, in IJg per mL, of slowly add 30 mL 3 N hydrochloric acid. Heat gently, if
magneslu':l' and draw the straight line best fitting the three necessary, to aid solution, cool, and filter into a 200-mL
plotted pOI.nts. Fr~m the graph so obtained, determine the volumetric flask. Wash the filter with water into the flask, add
concentration, C, In IJg per mL, of magnesium in each mL of water to volume, and mix. .
the Ass~y preparation. Calculate the quantity, in mg, of Procedure-Pipet 10 mL of the Assaypreparation into a
magnesium carbonate (MgC0 3) in the portion of Tablets 250-mL beaker, add 20 mL of water, then add, in the order
taken by the formula: named and with continuous stirring, 25.0 mL of Edetate
disodium titrant and 20 mL of acetic acid-ammonium acetate
(84.31/24.31 )(20C/V) buffer TS, and heat near the boiling point for 5 minutes. Cool,
add 50 mL of alcohol and 2 mL of dithizone TS, and mix.
in which 84.31 is the molecular weight of magnesium Titrate with 0.05 M zinc sulfate VS until the color changes from
~arbonate; 24.31 is t~e atomic weight of magnesium; and V green-violet to rose-pink. Perform a blank determination
IS th.e volu~e taken, In mL, of th,e Assaypreparation prepared substituting 10 mL of water for the Assaypreparation, an'd
as directed In the Assay for aluminum hydroxide. make any necessary correction. Each mL of 0.05 M Edetate
disodium titrant consumed is equivalent to 3.900 mg of
AI(OH)3' .
Assay for magnesium carbonate-Weigh and finely
powder not fewer than 20 Tablets. Transfer an accurately

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 USP 43 OfficialMonographs / Alumina 179

weighed portion of the powder, equivalent to about 750 mg in which 40.30 and 24.31 are the molecular weight of
of magnesium carbonate, to a 250-mL conical flask fitted with magnesium oxide and the atomic weight of magnesium,
a two-hole stopper. Fill the lower transverse section of a respectively; A isthe quantity, in mg, of magnesium equivalent
U-shaped drying tube of about 15-mm internal diameter and in each Tablet; and 8 is the quantity, in mg, of magnesium
15-cm height with loosely packed glasswool. Placein one arm carbonate in each Tablet, as determined in the Assay for
of the tube about 5 g of anhydrous calcium chloride, and magnesium carbonate.
accurately weigh the tube and the contents. Into the other arm
of the tube place 9.5 g to 10.5 g of soda lime, and again weigh
accurately. Insert stoppers in the open arms of the U-tube, and
connect the side tube of the arm filled with soda lime to a
calcium chloride drying tube, which in turn is connected to Alumina and Magnesium Trisilicate
one of the holes in the stopper of the 250-mL conical flask. Oral Suspension
Attach a dropping funnel to the other hole in the stopper of
the 250-mL conical flask. Add 100 mL of water and 10 mL of
a mixture of hydrochloric acid and nitric acid (4:1) to the » Alumina and Magnesium Trisilicate Oral
250-mL conical flask through the dropping funnel, and close Suspension contains the equivalent of not less than
the dropping funnel. Heat the 250-mL conical flask at 95° for
1 hour, and allow the evolved carbon dioxide to pass through 90.0 percent and not more than 110.0 percent of
the U-tube. Replace the dropping funnel with a source of the labeled amounts of aluminum hydroxide
carbon dioxide-free air, and passthe carbon dioxide-free air [AI(OH)3l and magnesium trisilicate (MgzSi 30 s).
through the apparatus at a rate of about 75 mL per minute for
30 minutes. Disconnect the U-tube, cool to room Packaging a...d storage-Preserve in tight containers.
temperature, remove the stoppers, and weigh. The increasein
weight corresponds to the quantity of carbon dioxide evolved. Identification-
Calculate the quantity, in mg, of magnesium carbonate in A: To a mixture of 5 mL in 10 mL of 3 N hydrochloric acid
each Tablet taken by the formula: add 5 drops of methyl red TS, heat to boiling, add 6 N
ammonium hydroxide until the color of the solution changes
(84.31/44.01 )(/)(WA/Wp) to deep yellow, then continue boiling for 2 minutes, and filter:
the filtrate responds to the tests for Magnesium (191).
in which 84.31 and 44.01 are the molecular weights of 8: Washthe solids on the filter obtained in Identification test
magnesium carbonate and carbon dioxide, respectively; I is A with hot ammonium chloride solution (1 in 50), add 10 mL
the quantity, in mg, of carbon dioxide evolved from the of 3 N hydrochloric acid, and filter: the filtrate responds tothe
portion of Tablets taken; WA is the average weight, in g, of 1 tests for Aluminum (191 ).
Tablet; and Wp is the weight, in g, of the portion of Tablets C: Transfer the filter paper and contents from Identification
taken. test 8 to a small platinum dish, ignite, cool in a desiccator, and
Assay for magnesium oxide-Weigh and finely powder weigh. Moisten the residue with water and add 6 mL of
not fewer than 20 Tablets. Transfer an accurately weighed hydrofluoric acid. Evaporate to dryness, ignite for 5 minutes,
portion of the powder, equivalent to about 1000 mg of cool in a desiccator, and weigh: a loss of more than 10% in
magnesium carbonate and magnesium oxide combined, to a relation to the weight of the residue from the initial ignition
beaker, add 20 mL of water, and slowly add 40 mL of 3 N indicates Si0 2 •
hydrochloric acid, with mixing. Heat the mixture to boiling, Acid-neutralizing capacity (301 )-Not less than 5 mEq of
cool, and filter into a 200-mL volumetric flask. Wash the beaker acid is consumed by the minimum single dose recommended
with water, adding the washings to the filter. Add water to in the labeling.
volume, and mix. Transfer 20.0 mL of this solution to a 400-mL pH (791): between 7.5 and 8.5.
beaker, add 180 mL of water and 20 mL of triethanolamine, Assay for aluminum hydroxide-
and stir. Add 10 mL of ammonia-ammonium chloride buffer Edetate disodium titrant-Prepare and standardize as
TS and 3 drops of an eriochrome black indicator solution directed in the Assay under Ammonium Alum.
prepared by dissolving 200 mg of eriochrome black T in a Assaypreparation-Transfer about 109 of well-shaken Oral
mixture of 15 mL of triethanolamine and 5 mL of dehydrated Suspension to a tared beaker, and weigh accurately. Add 50
alcohol, and mix. Cool the solution to between 3° and 4° by mL of water and 10 mL of hydrochloric acid, and digest on a
immersion of the beaker in an ice bath, then remove, and steam bath for 1 hour. Cool, and filter into a 200-mL
titrate with 0.05 M edetate disodium VS to a blue endpoint. volumetric flask, washing the filter with water into the flask.
Perform a blank determination, substituting 20 mL of water Dilute with water to volume, and mix.
for the assay solution, and make any necessary correction. Procedure-Pipet 20 mL of Assaypreparation into a 250-mL
Each mL of 0.05 M edetate disodium consumed is equivalent beaker, add 20 mL of water, then add, in the order named and
to 1.216 mg of Mg. Calculate the quantity, in mg, of with continuous stirring, 25.0 mL of Edetate disodium titrant
magnesium equivalent in each Tablet taken by the formula: and 20 mL of acetic acid-ammonium acetate buffer TS, and
heat near the boiling point for 5 minutes. Cool, add 50 mL of
10T(WA/Wp) alcohol and 2 mL of dithizone TS, and mix. Titrate with 0.05
M zinc sulfate VS until the color changes from green-violet to
in which T is the magnesium equivalent obtained in the rose-pink. Perform a blank determination, substituting 20 mL
titration; WA is the average weight, in g, of 1 Tablet; and Wp is of water for the Assay preparation, and make any necessary
the weight, in g, of the portion of Tablets taken. Calculate the correction. Each mL of 0.05 M Edetate disodium titrant
quantity, in mg, of magnesium oxide in each Tablet taken by consumed is equivalent to 3.900 mg of AI(OH)3'
the formula:
Assay for magnesium trisilicate-
(40.30/24.31 )(A - 0.28838) Assay preparation-Prepare as directed in the Assay for
aluminum hydroxide.

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 180 Alumina / Official Monographs USP43

Procedure-Pipet 20 mL of Assay preparation into a 400-mL test. [NoTE-Take care that the electrodes do not touch the
beaker, add 180 mL of water and 20 mL of triethanolamine, liquid beneath the foam.]
and stir. Add 10 mL of ammonia-ammonium chloride buffer
TS and 3 drops of an eriochrome black indicator solution Assay for aluminum hydroxide-
prepared by dissolving 200 mg of eriochrome black T in a Edetate disodium titrant-Prepare and standardize as
mixture of 15 mL of triethanolamine and 5 mL of dehydrated directed in the Assay under Ammonium Alum.
alcohol, and mix. Cool the solution to between 3° and 4° by Assay preparation-Weigh and finely powder not less than
immersion of the beaker in an ice bath, then remove and 20 Tablets. Transfer an accurately weighed portion of the
titrate with 0.05 M edetate disodium VS to a blue endpoint. powder, equivalent to about 600 mg of aluminum hydroxide,
Perform a blank determination, substituting 20 mL of water to a beaker, add 20 mL of water, stir, and slowly add 40 mL of
for the Assay preparation, and make any necessary correction. 3 N hydrochloric acid. Heat gently, if necessary, to aid
Each mL of 0.05 M edetate disodium consumed is equivalent solution, cool, and transfer to a 200-mL volumetric flask. Wash
to 6.521 mg of Mg 2Si 30 s . the beaker with water, adding the washings to the flask, add
water to volume, and mix.
Procedure-Pipet 10 mL of Assay preparation into a 250-mL
beaker, add 20 mL of water, then add, in the order named and
with continuous stirring, 25.0 mL of 0.05 M Edetate disodium
Alumina and Magnesium Trisilicate titrant and 20 mL of acetic acid-ammonium acetate buffer TS,
and heat the solution near the boiling temperature for 5
Tablets minutes. Cool, add 50 mL of alcohol and 2 mL of dithizone
TS, and mix. Titrate with 0.05 M zinc sulfate VS until the color
changes from green-violet to rose-pink. Perform a blank
» Alumina and Magnesium Trisilicate Tablets determination, substituting 10 mL of water for the Assay
contain not less than 90.0 percent and not more preparation, and make any necessary correction. Each mL of
than 110.0 percent of the labeled amounts of 0.05 M Edetate disodium titrant consumed is equivalent to
aluminum hydroxide [AI(OH)3l and magnesium 3.900 mg of AI(OH)3'
trisilicate (Mg 2Si 30 g) . Assay for magnesium trisilicate-
Potassium chloride solution-Prepare a solution in water
Packaging and storage-Preserve in well-closed containing 5 g of potassium chloride per 100 mL.
containers. Magnesium standard solution-Transfer 1.000 g of
Labeling-Tablets prepared with the use of Dried Aluminum magnesium metal to a 1OOO-mL volumetric flask containing
Hydroxide Gel may be labeled to state the aluminum hydroxide 50 mL of water, and slowly add 10 mL of hydrochloric acid.
content in terms of the equivalent amount of dried aluminum Dilute with water to volume, and mix. Transfer 5.0 mL of this
hydroxide gel, on the basis that each mg of dried gel is solution to a 500-mL volumetric flask, dilute with water to
equivalent to 0.765 mg of AI(OH)3' Tablets intended for the volume, and mix.
temporary relief of heartburn (acid indigestion) due to acid Standard preparations-Transfer 16.0 mL, 18.0 mL, and
reflux are so labeled. Tablets that must be chewed before 20.0 mL of Magnesium standard solution to separate 100-mL
swallowing are so labeled. volumetric flasks, add 2.0 mL of Potassium chloride solution to
Identification-One powdered Tablet responds to the each flask, dilute with water to volume, and mix. These
Identification tests under Alumina and Magnesium Trisilicate Standard preparations contain 1.6, 1.8, and 2.0 J.lg of
Oral Suspension. . magnesium per mL, respectively. [NOTE-Prepare these
Disintegration (701): 10 minutes, simulated gastric fluid TS solutions on the day of use.]
being substituted for water in the test. [NOTE-Tablets that Assay preparation-Weigh and finely powder not fewer
must be chewed before swallowing are exempt from this than 20 Tablets. Transfer an accurately weighed portion of the
requirement.] powder, equivalent to about 5 mg of magnesium trisilicate, to
Uniformity of dosage units (905): meet the requirements a 1OO-mL volumetric flask, and add 10 mL of 18 N sulfuric acid.
for Weight Variation with respect to aluminum hydroxide and Heat on a steam bath for 30 minutes with occasional swirling.
to magnesium trisilicate. Allow to cool, dilute with water to volume, and mix. Filter this
Acid-neutralizing capacity (301 )-Not less than 5 mEq of solution, discarding the first 20 mL of the filtrate. Transfer 20.0
acid is consumed by the minimum single dose recommended mL of the filtrate to a second 1OO-mL volumetric flask, add 2.0
in the labeling. [NoTE-Tablets labeled for the temporary relief mL of Potassium chloride solution, dilute with water to volume,
of heartburn (acid indigestion) due to acid reflux are exempt and mix.
from this requirement.] Procedure-Concomitantly determine the absorbance of
Foam [where Tablets are labeled for the temporary relief of the Standard preparations and the Assay preparation at the
heartburn (acid indigestion) due to acid reflux]-Finely magnesium emission line at 285.2 nm, with an atomic
powder a number of Tablets, accuratelycounted, equivalent absorption spectrophotometer (see Atomic Absorption
to the minimum single dose recommended in the labeling, Spectroscopy (852», equipped with a magnesium
and transfer the powder to a 1OO-mL beaker having an inside hollow-cathode lamp and a nitrous oxide-acetylene flame,
diameter of 45 mm. Add 5 mL of alcohol and sufficient water using water as the blank. Plot the absorbances of the Standard
to make 40 mL. Mix at 300 rpm for 60 seconds, using a preparations, in J.lg per mL, of magnesium, and draw the line
magnetic stirrer and a 9.5- x 38-mm polytef-coated stirring best fitting the three plotted points. From the graph so
bar. Stop the stirrer, and carefully add 10 mL of 0.5 N obtained determine the concentration, C, in J.lg per mL, of
hydrochloric acid down the side of the beaker. Stir for 30 magnesium in the Assay preparation. Calculate the quantity,
seconds at 300 rpm. Allow to stand for 10 minutes, and in mg, of magnesium trisilicate (Mg 2Si 30 s) in the portion of
measure the thickness of the foam layer above the liquid in the Tablets taken by the formula:
beaker: the thickness of the foam is not less than 10 mm.
pH (791) [where Tablets are labeled for the temporary relief 0.5 C(260.86/48.62)
of heartburn (acid indigestion) due to acid reflux]: not less
than 4.5, determined on the foam layer obtained in the Foam

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USP 43 OfficialMonographs / Aluminum 181

in which 260.86 is the molecular weight of anhydrous contained in a receiving flask. Distill about 160 mL, then
magnesium trisilicate and 48.62 is twice the atomic weight of remove the delivery tube from below the surface of the
magnesium. liquid. Allow the distilling flask to cool, add 50 mL of water,
and distill an additional 40-45 mL into the receiving flask.
Add phenolphthalein TS to the distillate, and titrate the
excess Titrant with Back-titrant. Each mL of Titrant is
equivalent to 30.03 mg of acetic acid (C2H402 ) .
Aluminum Acetate Topical Solution Acceptance criteria: 4.24-5.12 g of acetic acid (C2H 402) in
100 mL
DEFINITION
Aluminum Acetate Topical Solution yields NLT 1.20 g and OTHER COMPONENTS
NMT 1.45 g of aluminum oxide (AI203) and NLT 4.24 g and • LIMIT OF BORIC ACID
NMT 5.12 g of acetic acid (C2H 402) , corresponding to NLT Sample: 25 mL
4.8 g and NMT 5.8 g of aluminum acetate (C6H9AI06) in each Titrimetric system
100 mL. Aluminum Acetate Topical Solution may be Mode: Direct titration
stabilized by the addition of NMT 0.6% of boric acid (H3B03) . Titrant: 0.5 N sodium hydroxide VS
Endpoint detection: Visual
Analysis: Pipet the Sample into 75 mL of water in a conical
Aluminum Subacetate Topical Solution 545 mL
flask.Add 3 mL of phenolphthalein TS, and add Titrantfrom
Glacial Acetic Acid 15 mL a buret until a faint pink color is obtained. Heat to boiling,
and again neutralize. Add 150 mL of glycerin to the
Purified Water, a sufficient quantity to make 1000 mL
neutralized solution, and titrate with Titrant. Perform a
blank determination in a similar manner. Subtract the
Add the GlacialAcetic Acid to the AluminumSubacetate Topical volume of Titrant used in the blank from the volume of
Solution and sufficient Purified Waterto bring to final volume. Titrant used after the addition of the glycerin. Each mL of
Mix, and filter if necessary. Dispense only clear Aluminum Titrant is equivalent to 30.92 mg of boric acid (H 3B03) .
Acetate Topical Solution. Acceptance criteria: NMT 0.6% of boric acid (H 3B03)
IDENTIFICATION IMPURITIES
• A. IDENTIFICATION TESTS-GENERAL (191): It meets the
requirements of the test for Aluminum and for the test B SPECIFIC TESTS
under Acetate. Ferricchloride TS produces a deep red color • pH (791): 3.6-4.4
that is destroyed by the addition of a mineral acid.
ADDITIONAL REQUIREMENTS
ASSAY • PACKAGING AND STORAGE: Package in tight containers.
• ALUMINUM OXIDE
Edetate disodium titrant: Prepare and standardize 0.05 M
edetate disodium titrant asdirected in Reagents, Volumetric
Solutions, Edetate Disodium, Twentieth-Molar (0.05 M).
Sample: 25 mL Aluminum Chloride
Blank: 25 mL of water
Titrimetric system AICI3·6H 20 241.43
Mode: Residual titration AICI3 133.34
Back-titrant: 0.05 M zinc sulfate VS Aluminum chloride, hexahydrate;
Endpoint detection: Visual Aluminum chloride hexahydrate [7784-1 3-6].
Analysis: Pipet the Sample into a 250-mL volumetric flask, Anhydrous [7446-70-0].
add 5 mL of hydrochloric acid, and dilute with water to
volume. Pipet 25 mL of this solution into a 250-mL beaker, DEFINITION
and add, in the order named and with continuous stirring, Aluminum Chloride contains NLT 95.0% and NMT 102.0% of
25.0 mL of Edetate disodium titrant and 20 mL of acetic aluminum chloride (AICI3) , calculated on the anhydrous
acid-ammonium acetate buffer TS, then heat the solution basis.
near the boiling point for 5 min. Cool, and add 50 mL of
IDENTIFICATION
alcohol and 2 mL of dithizone TS. Titrate the solution with
• A. IDENTIFICATION TESTS-GENERAL, Aluminum (191) and
Back-titrant to a bright rose-pink color. Perform a blank Chloride (191)
determination, and make any necessary correction. Each
Sample solution: 100 mg/mL
mL of Edetate disodium titrant is equivalent to 2.549 mg of
Acceptance criteria: Meets the requirements
aluminum oxide (AI203) .
Acceptance criteria: 1.20-1.45 g of aluminum oxide ASSAY
(AI203) in 100 mL • PROCEDURE
• ACETIC ACID Edetate disodium titrant: Prepare and standardize as
Sample: 20 mL directed in Reagents, Volumetric Solutions, Edetate Disodium,
Titrimetric system Twentieth-Molar (0.05~M).
Mode: Residual titration Sample solution: 20 mg/mL of aluminum chloride in water
Titrant: 0.5 N sodium hydroxide VS Titrimetric system
Back-titrant: 0.5 N sulfuric acid VS Mode: Back-titration
Endpoint detection: Visual Titrant: 0.05 M zinc sulfate VS
Analysis: Pipet the Sample into a Kjeldahl flask containing. a Endpoint detection: Visual
mixture of 20 mL of phosphoric acid and 150 mL of water. Analysis: Transfer 10.0 mL of the Sample solution into a
Connect the flask to a condenser, the delivery tube from 250-mL beaker, and add, in the order named and with
which dips beneath the surface of 50.0 mL of Titrant continuous stirring, 25.0 mL of Edetate disodium titrant and

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182 Aluminum / Official Monographs USP 43

20 ml of acetic acid-ammonium acetate bufferTS, and boil Titrimetric system

gently for 5 min. Cool, and add 50 ml of alcohol and 2 ml Mode: Direct titration
of dithizone TS. Titrate the excess edetate disodium with Titrant: 0.1 N silver nitrate VS
Titrant to a bright rose-pink color. Perform a blank Electrode system: A glass silver-silver chloride electrode
determination, substituting 10 ml of water for the Sample and a silver billet electrode system
solution, and make any necessary correction. Each ml of Endpoint detection: Potentiometric
Edetate disodium titrant consumed is equivalent to 6.667 Analysis: Transfer the Sample to a 250-ml beaker and add
mg of aluminum chloride (AICI 3) . 100 ml of water and 10 ml of diluted nitric acid with
Acceptance criteria: 95.0%-102.0% on the anhydrous stirring. Titrate with Titrant and determine the endpoint
basis potentiometrically. Each ml of 0.1 N silver nitrate is
equivalent to 3.545 mg of chloride (CI). Use the chloride
IMPURITIES content thus obtained to calculate the aluminum:chloride
• LIMIT OF SULFATE atomic ratio.
Sample solution: 10 mg/ml • PROCEDURE 2: CONTENT OF ALUMINUM
Analysis: Add 0.2 ml of barium chloride TS to 10 ml of the Edetate disodium titrant: Prepare and standardize as
Sample solution. directed in Reagents, Volumetric Solutions, Edetate Disodium, '
Acceptance criteria: No turbidity is produced within 1 min. Twentieth-Molar(0.05 M), except use 37.2 g of edetate
• IRON (241) disodium.
Sample: 1.0 g Sample solution: Transfer 200 mg of Aluminum
Analysis: Dissolve the Sample in 45 ml of water, and add 2 Chlorohydrate to a 250-ml beaker, add 20 ml of water and
ml of hydrochloric acid. 5 ml of hydrochloric acid, boil on a hot plate for NlT 5 min,'
Acceptance criteria: NMT 10 ppm and allow to cool.
• LIMIT OF ALKALIES AND ALKALINE EARTHS Titrimetric system
Sample: 1.0 g Mode: Back-titration
Analysis: To a boiling solution of the Sample in 150 ml of Titrant: 0.1 M zinc sulfate VS
water add a few drops of methyl red TS, then add 6 N Endpoint detection: Visual
ammonium hydroxide until the color of the solution just -Analysls: To the Sample solution add 25.0 ml of Edetate
changes to a distinct yellow. Add hot water to restore the disodium titrant, and adjust with 2.5 N ammonium
volume to 150 ml, and filter while hot. Evaporate 75 rnl, of hydroxide or 1 N acetic acid to a pH of 4.7 ± 0.1 . Add 20
the filtrate to dryness, and ignite to a constant weight. mlof acetic acid-ammonium acetate buffer TS, 50 ml of
Acceptance criteria: 0.5%; the weight of the residue is alcohol, and 5 ml of dithizone TS. The pH of this solution
NMT 2.5 mg. should be 4.7 ± 0.1. Titrate the excess edetate disodium
SPECIFIC TESTS with Titrant until the color changes from green-violet to
• WATER DETERMINATION, Method I (921): 42.00/0-48.0% rose-pink. Perform a blank titration, and make any
necessary correction. Each ml of 0.1 M Edetate dlsodium
ADDITIONAL REQUIREMENTS titrant consumed is equivalent to 2.698 mg of aluminum
• PACKAGING AND STORAGE: Preserve in tight containers. (AI). Use the aluminum content thus obtained to calculate
the aluminum:chloride atomic ratio.
• PROCEDURE 3: ALUMINUM:CHLORIDE ATOMIC RATIO
Analysis: Use the percentage of aluminum found in the
test for Content of Aluminum and the percentage of chloride
Aluminum Chlorohydrate found in the test for Content of Chloride.
Calculate the aluminum:chloride atomic ratio (X) as follows:
AI y (OH) 3y-z CI z • H20
AI y (OH) 3y-z CI z • 2H20 210.48 Result = (p Alp d x (A oIA AI)
AI y (OH) 3y-z CI z 174.45 = percentage of aluminum found in Content of
Aluminum chlorohydroxide, dihydrate; Aluminum
Aluminum hydroxychloride, dihydrate; PCl = percentage of chloride found in Content of
Aluminum chlorohydroxide; Chloride
Aluminum hydroxychloride; = atomic weight of chlorine (CI), 35.453
Dihydrate [12359-72-7]. = atomic weight of aluminum (AI), 26.98
Anhydrous [12042-91-0].
DEFINITION Acceptance criteria: Between 1.91:1 and 2.10:1
Aluminum Chlorohydrate consists of complex basic aluminum • PROCEDURE 4
chloride that is polymeric and loosely hydrated and Analysis: Calculate the percentage of anhydrous aluminum
encompasses a range of aluminum-to-chloride atomic ratios chlorohydrate [AI y (OH) 3y-zCI z] in the portion of Aluminum
between 1.91:1 and 2.10:1. It contains the equivalent ofNlT Chlorohydrate taken:
90.0% and NMT 110.0% of the labeled amount of
anhydrous aluminum chlorohydrate [AI y (OH) 3y-z CI z]. Result = P AI ({A AI X + [M(3X - 1)] + A (I }/A AI X)

IDENTIFICATION P AI = percentage of aluminum as obtained in the test

• A. IDENTIFICATION TESTS-GENERAL, Aluminum (191) and for Content of Aluminum
Chloride (191) A AI = atomic weight of aluminum (AI), 26.98
Sample solution: 100 mg/ml X =aluminum:chloride atomic ratio, as determined in
Acceptance criteria: Meets the requirements the test for Aluminum:Chloride Atomic Ratio
ASSAY
M = molecular weight of the hydroxide anion (OH),
17.01
• PROCEDURE 1: CONTENT OF CHLORIDE A (I == atomic weight of chlorine (CI), 35.453
Sample: 700 mg

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 USP 43 Official Monographs / Aluminum 183

Acceptance criteria: 90.0%-110.0% on the anhydrous Sample solution: 2 g of Solution in 10 mL of isopropyl

basis alcohol. Mix, filter, and evaporate the filtrate to 1 mL on a
steam bath.
IMPURITIES
Acceptance criteria: The IRspectrum of a filmof the Sample
e ARSENIC, Method I (211): NMT 2 ppm solution on a silver chloride disk exhibits maxima only at the
• LIMIT OF IRON same wavelengths as that of a similar preparation of a film
Standard solution: Transfer 2.0 mL of Standard Iron of dipropylene glycol.
Solution, prepared as directed in Iron (241), to a 50-mL • D. IDENTIFICATION OF ALCOHOL
beaker. Perform this test where alcohol is stated on the label.
Sample solution: Transfer 2.7 g of Aluminum Analysis: Mix 5 drops of Solution in a small beaker with 1
Chlorohydrate to a 1OO-mL volumetric flask, dilute with mL of potassium permanganate solution (1 in 100) and 5
water to volume, and mix. Transfer 5.0 mL of this solution drops of 2 N sulfuric acid, and cover the beaker immedia~ely
to a 50-mL beaker. with filter paper moistened with a freshly prepared solution
Analysis: To each of the beakers containing the Standard of 0.1 g of sodium nitroferricyanide and 0.25 g of
solution and the Sample solution, add 5 mL of 6 N nitric acid, piperazine in 5 mL of water.
cover with a watch glass, and boil on a hot plate for 3-5 Acceptance criteria: An intense ~Iue color is produced ~n
min. Allow to cool. Add 5 mL of Ammonium Thiocyanate the filter paper, the color becoming paler after a few min.
Solution (prepared as directed in Iron (241», transfer to
separate 50-mL color comparison tubes, and dilute with ASSAY
water to volume. • PROCEDURE 1: CONTENT OF CHLORIDE
Acceptance criteria: 150 ppm; the color of the solution Sample: 1.4 g of Solution
from the Sample solution is not darker than that of the Titrimetric system
solution from the Standardsolution. Mode: Direct titration
Titrant: 0.1 N silver nitrate VS
SPECIFIC TESTS Electrode system: A silver-silver chloride glass electrode
epH(791) and a silver billet electrode system
Sample solution: 15 g of Aluminum Chlorohydrate in 100 Endpoint detection: Potentiometric
g of water Analysis: Transfer the Sample to a 250-mL beaker, and add
Acceptance criteria: 3.0-5.0 100 mL of water and 10 mL of diluted nitric acid with
ADDITIONAL REQUIREMENTS stirring. Titrate with Titrant, and determine the endpoint.
• PACKAGING AND STORAGE: Preserve in well-closed Acceptance criteria: Each mL of .0.1 N silver nitrate is .
containers. equivalent to 3.545 mg of chloride (CI). Use the chloride
• LABELING: The label states the content of anhydrous content thus obtained to calculate the aluminum/chloride
aluminum chlorohydrate. atomic ratio.
• PROCEDURE 2: CONTENT OF ALUMINUM
Edetate disodium titrant: Prepare and standardize as
directed in Reagents, Volumetric Solutions, Edetate Disodium,
Twentieth-Molar (0.05 M), except use 37.2 g of edetate
Aluminum Chlorohydrate Solution disodium.
Sample solution: Transfer 400 mg of Solution to a 250-mL
DEFINITION beaker, add 20 mL of water and 5 mL of hydrochloric acid,
Aluminum Chlorohydrate Solution consists of complex basic boil on a hot plate for NLT 5 min, and allow to cool.
aluminum chloride that is polymeric and encompasses a Titrimetric system
range of aluminum-to-chloride ratios between 1.91:J and Mode: Back titration
2.10:1. The following solvents may be used: water, Titrant: 0.1 M zinc sulfate VS
propylene glycol, dipropylene glycol, or alcohol. It contains Endpoint detection: Visual
the equivalent of NLT 90.0% and NMT 110.0% of the Analysis: To the Sample solution add 25.0 mL of Edetate
labeled concentration of anhydrous aluminum disodium titrant, and adjust with 2.5 N ammonium
chlorohydrate (AI y (OH) 3y.z CI z). hydroxide or 1 N acetic acid to a pH of 4.7 ± 0.1 . Add 20
mL of acetic acid-ammonium acetate buffer TS, 50 mL of
IDENTIFICATION alcohol and 5 mL of dithizone TS. The pH of this solution
• A. IDENTifiCATION TESTS-GENERAL, Aluminum (191) and should he 4.7 ± 0.1. Titrate excess edetate disodium with
Chloride (191) Titrant until the color changes from green-violet to
Sample solution: Nominally equivalent to 100 mg/mL of rose-pink. Perform a blank titration, and make any
anhydrous aluminum chlorohydrate necessary correction.
Acceptance criteria: Meets the requirements Acceptance criteria: Each mL of 0.1 M Edetate disodium
• B. IDENTifiCATION Of PROPYLENE GLYCOL titrant consumed is equivalent to 2.698 mg of aluminum
Perform this test where propylene glycol is stated on the (AI). Usethe aluminum content thus obtained to calculate
label. the aluminum/chloride atomic ratio.
Sample solution: 2 g of Solution in 10 mL of isopropyl • PROCEDURE 3: ALUMINUM/CHLORIDE ATOMIC RATIO
alcohol. Mix, filter, and evaporate the filtrate to 1 mL on a Analysis: Use the percentage of aluminum found in Content
steam bath. of Aluminum and the percentage of chloride found in
Acceptance criteria: The IRspectrum of a film of the Sample Contentof Chloride.
solution on a silver chloride disk exhibits maxima only at the Calculate the aluminum/chloride atomic ratio (X) asfollows:
same wavelengths as that of a similar preparation of a film
of propylene glycol. Result = (P Ai P C/) x (A of A AI)
• C. IDENTIFICATION Of DIPROPYLENE GLYCOL
Perform this test where dipropylene glycol is stated on the P AI = percentage of aluminum found in Contentof
label. Aluminum

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184 Aluminum / Official Monographs USP 43

P (I = percentage of chloride found in Contentof polyethylene glycol. It encompasses a range of

Chloride aluminum-to-chloride atomic ratios between
A (I = atomic weight of chlorine (CI), 35.453 1.91:1 and 2.10:1. It contains not less than 90.0
A AI =atomic weight of aluminum (AI), 26.98 percent and not more than 110.0 percent of the
Acceptance criteria: 1.91:1 to 2.10:1 labeled amount of anhydrous aluminum
• PROCEDURE 4 chlorohydrate.
Analysis: Calculate the percentage of the labeled
concentration of anhydrous aluminum chlorohydrate (AI y Packaging and storage-Preserve in well-closed
(OH) 3y.z CI z) in the portion of Solution taken: containers.
Labeling-The label states the content of anhydrous
Result= PAl X {[A AI X + (M(3X - 1» + A CI]IA AI X} aluminum chlorohydrate.

P AI = percentage of aluminum found in Contentof Identification-

Aluminum A: A solution (1 in 10) responds to the tests for Aluminum
A AI = atomic weight of aluminum (AI), 26.98 (191) and for Chloride (191).
X = aluminum/chloride atomic ratio, as determined
in Aluminum/Chloride AtomicRatio
M = molecular weight of the hydroxide anion (OH),
17.01
A (I = atomic weight of chlorine (CI), 35.453 g in about 40 mL of water, and
with 2.5 N sodium hydroxide to a pH of
Acceptance criteria: 90.00/0-110.0% 9.55 ± the suspension of precipitate thus obtained.
IMPURITIES Evaporate about 15 mL of the filtrate to about 1 mL on a hot
• ARSENIC, Method I (211): NMT 2 ppm plate. Deposit this solution on a silver chloride disk.
• LIMIT OF IRON Standard specimen: a similar preparation of polyethylene
Standard preparation: 2.0 mL of Standard Iron Solution, ,glycol.
prepared as directed in Iron (241) pH (791): between 3.0 and 5.0, in a solution [15 in 100 (wi
Test preparation: Transfer 5.3 g of Solution to a 100-mL w)].
volumetric flask, and dilute with water to volume. Arsenic, Method I (211): 2 ~g per g.
Analysis: Transfer 2.0 mL of the Standard preparation into a Limit of iron-Using Aluminum Chlorohydrex Polyethylene
50-mL beaker. Transfer 5.0 mL of the Test preparation into Glycol instead of Aluminum Chlorohydrate, proceed as
a second 50-mL beaker. To each of the beakers add 5 mL directed in the test for Limit of iron under Aluminum
of 6 N nitric acid, cover with a watch glass, and boil on a Chlorohydrate. The limit is 150 ~g per g.
hot plate for 3-5 min. Allow to cool. Add 5 mL of Content of aluminum-Using Aluminum Chlorohydrex
Ammonium Thiocyanate Solution, prepared as directed in Polyethylene Glycol instead of Aluminum Chlorohydrate,
Iron (241), transfer to separate 50-mL color-comparison proceed as directed in the test for Contentof aluminum under
tubes, and dilute with water to volume. Aluminum Chlorohydrate. Use the result obtained to calculate
Acceptance criteria: 75 ppm; the color of the solution from the Aluminum/chloride atomic ratio.
the Test preparation is not darker than that from the Content of chloride-Using Aluminum Chlorohydrex
Standard preparation. . Polyethylene Glycol instead of Aluminum Chlorohydrate,
proceed as directed in the test for Content of chloride under
SPECIFIC TESTS Aluminum Chlorohydrate. Use the result obtained to calculate
• pH (791) the Aluminum/chloride atomic ratio.
Sample solution: Dilute 3 g of Solution with water to 1,0 mL. Aluminum/chloride atomic ratio-Divide the
Acceptance criteria: 3.0-5.0 percentage of aluminum found in the test for Content of
ADDITIONAL REQUIREMENTS aluminum by the percentage of chloride found in the test for
• PACKAGING AND STORAGE: Preserve in well-closed Contentof chloride, and multiply by 35.453/26.98, in which
containers. 35.453 and 26.98 are the atomic weights of chlorine and
• LABELING: Label Solution to state the solvent used and the aluminum, respectively: the ratio is between 1.91:1 and
claimed concentration of anhydrous aluminum 2.10:1.
chlorohydrate contained therein. Assay-Calculate the percentage of anhydrous aluminum
chlorohydrate in the Aluminum Chlorohydrex Polyethylene
Glycol by the formula:

AI({26.98x + [17.01 (3x - 1)] + 35.453}/26.98x)

Aluminum Chlorohydrex Polyethylene in which AI is the percentage of aluminum found in the test
Glycol for Contentof aluminum, x is the aluminum/chloride atomic
ratio found in the test for Aluminum/chloride atomicratio,
26.98 is the atomic weight of aluminum, 17.01 is the
AliOH)3y-zClz' nHzO . mH(OCHzCHz)nOH molecular weight of the hydroxide anion (OH), and 35.453 is
Aluminum chlorohydroxide polyethylene glycol complex. the atomic weight of chlorine (CI).
Aluminum hydroxychloride polyethylene glycol complex.

» Aluminum Chlorohydrex Polyethylene Glycol

consistsof aluminum chlorohydrate in which some
of the waters of hydration have been replaced by

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 USP 43 OfficialMonographs / Aluminum 185

40 to 50 mL of alcohol, and titrate with 0.1 M zinc sulfate VS

Aluminum Chlorohydrex Propylene until the color changes from a green-violet to a rose-pink..
Glycol Perform a blank titration, and make any necessary correction.
Each mL of 0.1 M Edetate disodium titrant consumed is
equivalent to 2.698 mg of aluminum (AI). U~e the alu~inum
AI y(OH)3y_zCI z ' nH20 . mC 3H s02 content thus obtained to calculate the Aluminum/chloride
AliH20)y-z(OH)6_n(CI)n(C3Hs02)z atomic ratio.
Aluminum chlorohydroxide, hydrate: propylene glycol Content of chloride-Using Aluminum Chlorohydrex
complex (1:1). . Propylene Glycol instead of Aluminum Chlorohydrate,
Aluminum hydroxychloride, hydrate: propylene glycol proceed as directed in the test for Procedure 1: Conten.t of ,
complex (1:1) [53026-85-0]. Chloride under Aluminum Chlorohydrate. Usethe chloride
content thus obtained to calculate the Aluminum/chloride
» Aluminum Chlorohydrex Propylene Glycol is a atomic ratio.
Aluminum/chloride atomic ratio-Divide the
complex of aluminum chlorohydrate and percentage of aluminum found in the Assay by the percentage
propylene glycol in which some of the waters of of chloride found in the test for Content of chloride, and
hydration of the aluminum chlorohydrate have multiply by 35.453/26.98, in which 35.453 and 26:98 are the
been replaced by propylene glycol. It contains the atomic weights of chlorine and aluminum, respectively: the
ratio is between 1.91:1 and 2.1:1.
equivalent of not less than 90.0 percent and not Assay-Calculate the percentage of anhydrous aluminum
more than 110.0 percent of the labeled amount of chlorohydrate in the Aluminum Chlorohydrex Propylene
anhydrous aluminum chlorohydrate. Glycol by the formula:

Packaging and storage-Preserve in well-closed AI({26.98x + [17.01 (3x - 1)] + 35.453}/26.98x)

containers.
Labeling-The label states the content of anhydrous in which AI is the percentage of aluminum found in the te~t
aluminum chlorohydrate. for Content of aluminum, x is the aluminum/chloride atomic
ratio 26.98 is the atomic weight of aluminum, 17.01 is the
Identification- mol~cular weight of the hydroxide ion (OH), and 35.453 is the
A: A solution (1 in 10) responds to the tests for Aluminum atomic weight of chlorine (CI).
(191) and for Chloride (191).

.~: ...~~ Aluminum Dichlorohydrate

..

$pgqtrq~L'.. c. ..J-
Test specimen-Dissolve 0.5 g in about 40 mL of water, and AIy(OH)3y_zCl z • nH20
while mixing adjust with 2.5 N sodium hydroxide to a p~ of Aluminum chlorohydroxide;
9.55 ± 0.05. Filter the suspension of precipitate thus obtained. Aluminum hydroxychloride.
Evaporate about 15 mL.of the filt~ate to ab?ut 1.mL on a hot
plate. Deposit this solution on a Silverchloride disk. DEFINITION
Standard specimen: a similar preparation of propylene Aluminum Dichlorohydrate consists of complex basic
glycol. aluminum chloride that is polymeric and loosely hydrated
pH (791): between 3.0 and 5.0, in a solution [15 in 10,0(w/ and encompasses a range of aluminum-to-chloride atomic
w)]. ratios between 0.90:1 and 1.25:1. It contains the equivalent
Arsen;c, Method I (211): 2 J,lg per g. of NLT 90.0% and NMT 110.0% of the labeled amount of
Limit of iron-Using Aluminum Chlorohydrex Propylene anhydrous aluminum dichlorohydrate [Al y(OH)3y.zCl z]'
Glycol instead of Aluminum Chlorohydrate, proceed as IDENTIFICATION
directed in the test for Limit of iron under Aluminum • A. IDENTIFICATION TESTS-GENERAL, Aluminum (191) and
Chloro~ydrate. The limit is 150 J,lg per g. Chloride (191)
Content of aluminum- Sample solution: 100 mg/mL
Edetate disodium titrant-Prepare and standardize as Acceptance criteria: Meets the requirements
directed in the ASSA Y under AmmoniumAlum, except to use ASSAY
37.2 g of edetate disodium instead of 18.6 g. • PROCEDURE 1: CONTENT OF CHLORIDE
Test solution-Transfer about 1.6 g of Aluminum Sample: 700 mg
Chlorohydrex Propylene Glycol, accurately weighed, to a Titrimetric system
1OO-mL beaker, add 15 to 20 mL of water and 5 to 6 ~L of Mode: Direct titration
hydrochloric acid, and boil on a hot plate for 15 to 20 minutes. Titrant: 0.1 N silver nitrate VS
Cool the solution, and with the aid of water transfer to a Electrode system: A glass silver-silver chloride electrode
1OO-mL volumetric flask. Dilute with water to volume, and and a silver billet electrode system
mix. Endpoint detection: Potentiometric
Procedure-Transfer 5.0 mL of the Test solutionto a 250-mL Analysis: Transfer the Sample to a 250-mL beaker, and add
beaker, add 10 to 15 mL of water, and adjust with 1 N sodium 100 mL of water and 10 mL of diluted nitric acid with
hydroxide to a pH of 1.5 ± 0.5. Add 10.0 mL of Edetate stirring. Titrate with Titrant, and deterr:nine t~e en~point
disodium titrant, and heat to boiling. Cool the solution and potentiometrically. Each mL of ~.1 N Silver nitrate IS .
carefullyintroduce a magnetic stirring bar into the beaker.Add equivalent to 3.545 mg of chloride (CI). Use. the chlon~e
10 to 15 mL of acetic acid-ammonium acetate buffer TS, 40 content thus obtained to calculate the alurnlnurn.chlorlde
to 50 mL of alcohol, and while stirring adjust with glacial acetic atomic ratio.
acid to a pH of 4.6 ± 0.1. Add 1 to 2 mL of dithizone TS and

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186 Aluminum / Official Monographs USP 43

• PROCEDURE 2: CONTENT Of ALUMINUM Analysis: To each of the beakers containing the Standard
Edetate disodium titrant: Prepare and standardize as solution and the Sample solution, add 5 mL of 6 N nitric acid,
directed in Reagents, Volumetric Solutions, Edetate Disodium, cover with a watch glass, and boil on a hot plate for 3-5
Twentieth-Molar (0.05 M), except use 37.2 g of edetate min. Allow to cool. Add 5 mL of Ammonium Thiocyanate
disodium. Solution (prepared as directed in Iron (241 »), transfer to
Sample solution: Transfer 200 mg of Aluminum separate 50-mL color comparison tubes, and dilute with
Dichlorohydrate to a 250-mL beaker, add 20 mL of water water to volume. ,
and 5 mL of hydrochloric acid, boil on a hot plate for NLT Acceptance criteria: 150 ppm; the color of the solution
5 min, and allow to cool. from the Sample solution is not darker than that of the
Titrimetric system solution from the Standardsolution.
Mode: Back-titration
Titrant: 0.1 M zinc sulfate VS SPECIFIC TESTS
Endpoint detection: Visual • pH (791)
Analysis: To the Sample solution add 25.0 mL of Edetate Sample solution: 15 g of Aluminum Dichlorohydrate in 100
disodium titrant, and adjust with 2.5 N ammonium g of water
hydroxide or 1 N acetic acid to a pH of 4.7 ± 0.1. Add 20 Acceptance criteria: 3.0-5.0
mL of acetic acid-ammonium acetate buffer TS, 50 mL of ADDITIONAL REQUIREMENTS
alcohol, and 5 mL of dithizone TS. The pH of this solution • PACKAGING AND STORAGE: Preserve in well-closed
should be 4.7 ± 0.1. Titrate the excess edetate disodium containers.
with Titrant until the color changes from green-violet to • LABELING: The label states the content of anhydrous
rose-pink. Perform a blank titration, and make any aluminum dichlorohydrate.
necessary correction. Each mL of 0.1 M Edetate disodium
titrant consumed is equivalent to 2.698 mg of aluminum
(AI). Usethe aluminum content thus obtained to calculate
the aluminum:chloride atomic ratio.
• PROCEDURE 3: ALUMINUM:CHLORIDE ATOMIC RATIO Aluminum Dichlorohydrate Solution
Analysis: Use the percentage of aluminum found in the
test for Contentof Aluminum and the percentage of chloride , DEFINITION
found in the test for Contentof Chloride. Aluminum Dichlorohydrate Solution consists of complex basic
Calculate the aluminum:chloride atomic ratio (X) as follows: aluminum chloride that is polymeric and encompasses a
range of aluminum-to-chloride atomic ratios between 0.90:1
Result = (PA/PC!) x (Ac/AA/) and 1.25:1. The following solvents may be used: water,
propylene glycol, dipropylene glycol, or alcohol. It contains
PAl =percentage of aluminum found in Contentof the equivalent of NLT 90.0% and NMT 110.0% of the
Aluminum labeled concentration of anhydrous aluminum
PC! = percentage of chloride found in Contentof dichlorohydrate (AIy(OH)3y.zCl z)'
Chloride
AC! = atomic weight of chlorine (CI), 35.453 IDENTIFICATION
AA/ = atomic weight of aluminum (AI), 26.98 • A. IDENTIFICATION TESTs-GENERAL, Aluminum (191) and
Chloride (191)
Acceptance criteria: Between 0.90:1 and 1.25:1 Sample solution: Nominally equivalent to 100 mg/mL of
• PROCEDURE 4
anhydrous aluminum dichlorohydrate
Analysis: Calculate the percentage of anhydrous aluminum Acceptance criteria: Meets the requirements
dichlorohydrate [Al y(OH)3Y'zCl z] in the portion of Aluminum
Dichlorohydrate taken:
• B. ... SI'ECTR()SCOPIIC I ..u:........r.,. A .....I'I....
Result = PA/({AA/X + [M(3X - 1)] + AC!}/AA/X)

=percentage of aluminum as obtained in the test is on

for Contentof Aluminum Sample solution: Add 1 mL of isopropyl alcohol to 2 g of
= atomic weight of aluminum (AI), 26.98 Solution, and filter. Evaporatethe filtrate to 1 mL on a steam
=aluminum:chloride atomic ratio, asdetermined in bath. Deposit this solution on a silver chloride disk.
the test for Aluminum:Chloride Atomic Ratio Standard solution: A similar preparation of propylene
=molecular weight of the hydroxide anion (OH), glycol
17.01 Acceptance criteria: Meets the requirements
= atomic weight of chlorine (CI), 35.453
Acceptance criteria: 90.0%-110.0% on the anhydrous • C.... SPECTROSC()PIC IDENTIFICATION TEsTS (197);' Infrared
basis
Spectroscopy: 197F... (eN l-May.2020): (where "dipropylene
IMPURITIES glycol is indicated on the label)
• ARSENIC, Method I (211): NMT 2 ppm Sample solution: Add 10 mL of isopropyl alcohol to 2 g of
• LIMIT OF IRON Solution, and filter. Evaporatethe filtrate to 1 mL on a steam
Standard solution: Transfer 2.0 mL of Standard Iron bath. Deposit this solution on a silver chloride disk.
Solution, prepared as directed in Iron (241), to a 50-mL Standard solution: A similar preparation of dipropylene
beaker. glycol
Sample solution: Transfer 2.7 g of Aluminum Acceptance criteria: Meets the requirements
Dichlorohydrate to a 1OO-mL volumetric flask, dilute with • D. IDENTIFICATION OF ALCOHOL
water to volume, and mix. Transfer 5.0 mL of this solution Perform this test where alcohol is stated on the label.
to a 50-mL beaker.

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USP 43 Official Monographs / Aluminum 187

Analysis: Mix 5 drops of Solution in a small beaker with 1 Result = PAl x {[AAI X + (M(3X - 1» + Ad/AAI X}
mL of potassium permanganate solution (1 in 100) and 5
drops of 2 N sulfuric acid, and cover the beakerimmediately PAl = percentage of aluminum found in Content of
with filter paper moistened with a freshly prepared solution Aluminum
of 0.1 g of sodium nitroferricyanide and 0.25 g of AAI = atomic weight of aluminum (AI), 26.98
piperazine in 5 mL of water. X = aluminum/chloride atomic ratio, as determined
Acceptance criteria: An intense blue color is produced on in Aluminum/Chloride Atomic Ratio
the filter paper, the color becoming paler after a few min. M = molecular weight of the hydroxide anion (OH),
17.01
ASSAY ACI == atomic weight of chlorine (CI), 35.453
• PROCEDURE 1: CONTENT OF CHLORIDE
Sample: 1.4 g of Solution Acceptance criteria: 90.0%-110.0%
Titrimetric system
Mode: Direct titration IMPURITIES
Titrant: 0.1 N silver nitrate VS • ARSENIC, Method I (211): NMT 2 ppm
Electrode system: A silver-silver chloride glass electrode • LIMIT OF IRON
and a silver billet electrode system Standard preparation: 2.0 ml of StandardIron Solution,
Endpoint detection: Potentiometric . prepared as directed in Iron (241)
Analysis: Transfer the Sample to a 250-ml beaker, and add Test preparation: Transfer 5.3 g of Solution to a 100-ml
100 mL of water and 10 mL of diluted nitric acid with volumetric flask, and dilute with water to volume.
stirring. Titrate with Titrant, and determine the endpoint. Analysis: Transfer 2.0 ml of the Standard preparation into a
Acceptance criteria: Each ml of 0.1 N silver nitrate is 50-ml beaker. Transfer 5.0 ml of the Test preparation into
equivalent to 3.545 mg of chloride (CI). Use the chloride a second 50-mL beaker. To each of the beakers add 5 mL
content thus obtained to calculate the aluminum/chloride of 6 N nitric acid, cover with a watch glass, and boil on a
atomic ratio. hot plate for 3-5 min. Allow to cool. Add 5 mL of
• PROCEDURE 2: CONTENT OF ALUMINUM Ammonium Thiocyanate Solution, prepared as directed in
Edetate disodium titrant: Prepare and standardize as Iron (241), transfer to separate 50-mL color-comparison
directed in Reagents, Volumetric Solutions, Edetate Disodium, tubes, and dilute with water to volume.
Twentieth-Molar (0.05 M), except use 37.2 g of edetate Acceptance criteria: 75 ppm; the color of the solution from
disodium. the Test preparation is not darker than that from the
Sample solution: Transfer 400 mg of Solution to a 250-mL Standardpreparation.
beaker, add 20 mL of water and 5 mL of hydrochloric acid,
boil on a hot plate for NlT 5 min, and allow to cool. SPECIFIC TESTS
Titrimetric system • pH (791)
Mode: Backtitration Sample solution: Dilute 3 g of Solution with water to 10 mL.
Titrant: 0.1 M zinc sulfate VS Acceptance criteria: 3.0-5.0
Endpoint detection: Visual . ADDITIONAL REQUIREMENTS
Analysis: To the Sample solution add 25.0 mL of Edetate • PACKAGING AND STORAGE: Preserve in well-closed
disodium titrant, and adjust with 2.5 N ammonium containers.
hydroxide or 1 N acetic acid to a pH of 4.7 ± 0.1. Add 20 • LABELING: Label Solution to state the solvent used and the
mL of acetic acid-ammonium acetate buffer TS, 50 mL of claimed concentration of anhydrous aluminum
alcohol, and 5 mL of dithizone TS. The pH of this solution dichlorohydrate contained therein.
should be 4.7 ± 0.1. Titrate excess edetate disodium with
Titrant until the color changes from green-violet to
rose-pink. Perform a blank titration, and make any
necessary correction.
Acceptance criteria: Each ml of 0.1 M Edetate disodium Aluminum Dichlorohydrex
titrant consumed is equivalent to 2.698 mg of aluminum
(AI). Use the aluminum content thus obtained to calculate Polyethylene Glycol
the aluminum/chloride atomic ratio.
• PROCEDURE 3: ALUMINUM/CHLORIDE ATOMIC RATIO Aly(OHhy.zClz· nHzO. mH(OCHzCHz)n OH
Analysis: Usethe percentage of aluminum found in Content Aluminum chlorohydroxide polyethylene glycol complex.
of Aluminum and the percentage of chloride found in Aluminum hydroxychloride polyethylene glycol complex.
Content of Chloride.
Calculate the aluminum/chloride atomic ratio (X) asfollows: » Aluminum Dichlorohydrex Polyethylene Glycol
Result = (PA,/PC/) x (Ac/AA/) consists of aluminum dichlorohydrate in which
some of the waters of hydration have been
PAl =percentage of aluminum found in Content of replaced by polyethylene glycol. It encompasses a
Aluminum range of aluminum-to-chloride atomic ratios
PCI = percentage of chloride found in Content of
Chloride between 0.90:1 and 1.25:1. It contains not less
ACI =atomic weight of chlorine (CI), 35.453 than 90.0 percent and not more than 110.0
AAI = atomic weight of aluminum (AI), 26.98 percent of the labeled amount of anhydrous
aluminum dichlorohydrate.
Acceptance criteria: 0.90:1 to 1.25:1
•. PROCEDURE 4 Packaging and storage-Preserve in well-closed
Analysis: Calculate the percentage of the labeled containers.
concentration of anhydrous aluminum dichlorohydrate Labeling-The label states the content of anhydrous
(Aly(O H)3Y.zClz) in the portion of Solution taken: , aluminum dichlorohydrate.

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188 Aluminum / OfficialMonographs USP43

Identification- than 90.0 percent and not more than 110.0

A: A solution (1 in 10) responds to the tests for Aluminum percent of the labeled amount of anhydrous
(191) and for Chloride (191).
aluminum dichlorohydrate.
Packaging and storage-Preserve in well-closed
containers.
~~ i~~e~£t~~~£~Pl~ Labeling-The label states the content of anhydrous
$p¢gtr.q~q.gpY;;l~Zc&"'t(C
Test specimen-Dissolve 0.5 g in about 40 mL of water, and aluminum dichlorohydrate.
while mixing adjust with 2.5 N sodium hydroxide to a pH of Identification-
9.55 ± 0.05. Filter the suspensionof precipitate thus obtained. A: A solution (1 in 10) responds to the tests for Aluminum
Evaporate about 15 mL of the filtrate to about 1 mL on a hot (191) and for Chloride (191). . ..
plate. Deposit this solution on a silver chloride disk. B: Dissolve0.5 g in about 40 mL of water, and while rruxmq
Standardspecimen: a similar preparation of polyethylene adjust with 2.5 N sodium hydroxide to a pH of 9.55 ± 0.05.
glycol. Filter the suspension of precipitate thus obtained. Evaporate
pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/ about 15 mL of the filtrate to about 1 mL on a hot plate: the
w)]. IRabsorption spectrum of a film of this solution on a silver
Arsenic, Method I (211): 2 ~g per g. chloride disk exhibits maxima only at the same wavelengths
Limit of iron-Using Aluminum Dichlorohydrex as that of a similar preparation of a film of propylene glycol.
Polyethylene Glycol instead of Aluminum Chlorohydrate, pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/
proceed asdirected in the test for Limit of iron under Aluminum ~)].
Chiorohvdrate. The limit is 150 ~g per g. Arsenic, Method I (211): 2 ~g per g.
Content of aluminum-Using Aluminum Dichlorohydrex Limit of iron-Using Aluminum Dichlorohydrex Propylene
Polyethylene Glycol instead of Aluminum Chlorohydrate, Glycol instead of Aluminum Chlorohydrate, proceed as
proceed as directed in the test for Contentof aluminum under directed in the test for Limit of iron under Aluminum
Aluminum Chtorohvdrate. Usethe result obtained to calculate Chlorohydrate. The limit is 150 ~g per g.
the Aluminum/chlorideatomic ratio. Content of aluminum-Using Aluminum Dichlorohydrex
Content of chloride-Using Aluminum Dichlorohydrex Propylene Glycol instead of Aluminum Chlorohydrate,
Polyethylene Glycol instead of Aluminum Chlorohydrate, proceed as directed in the test for Contentof aluminum under
proceed as directed in the test for Contentof chloride under Aluminum Chlorohydrate. Usethe result obtained to calculate
Aluminum Chlorohydrate. Use the result obtained to calculate the Aluminum/chloride atomic ratio.
the Aluminum/chlorideatomic ratio. Content of chloride-Using Aluminum Dichlorohydrex
Aluminum/chloride atomic ratio-Divide the Propylene Glycol instead of Aluminum Chlorohydrate,
percentage of aluminum found in the test for Contentof proceed as directed in the test for Contentof chloride under
aluminum by the percentage of chloride found in the test for Aluminum Chlorohydrate. Usethe result obtained to calculate
Content of chloride, and multiply by 35.453/26.98, in which the Aluminum/chloride atomic ratio.
35.453 and 26.98 are the atomic weights of chlorine and Aluminum/chloride atomic ratio-Divide the
aluminum, respectively: the ratio is between 0.90:1 and percentage of aluminum found in the test for Contentof
1.25:1. aluminum by the percentage of chloride found in the test for
Assay-Calculate the percentage of anhydrous aluminum Contentof chloride, and multiply by 35.453/26.98, in which
dichlorohydrate in the Aluminum Dichlorohydrex 35.453 and 26.98 are the atomic weights of chlorine and
Polyethylene Glycol by the formula: . aluminum, respectively: the ratio is between 0.90:1 and
1.25:1.
AI({26.98x + [17.01 (3x - 1)] + 35.453}/26.98x) Assay-Calculate the percentage of anhydrous aluminum
dichlorohydrate in the Aluminum Dichlorohydrex Propylene
in which AI is the percentage of aluminum found in the test Glycol by the formula:
for Contentof aluminum, x is the aluminum-to-chloride atomic
ratio, 26.98 is the atomic weight of aluminum, 17.01 is the AI({26.98x + [17.01 (3x - 1)] + 35,453}/26.98x)
molecular weight of the hydroxide anion (OH), and 35.453 is
the atomic weight of chlorine (CI). in which AI is the percentage of aluminum found in the test
for Contentof aluminum, x is the aluminum-to-chloride atomic
ratio, 26.98 is the atomic weight of aluminum, 17.01 is the
molecular weight of the hydroxide anion (OH), and 35.453 is
the atomic weight of chlorine (CI).
Aluminum Dichlorohydrex Propylene
Glycol
Aly(OH)3y_zCl z ' nHzO . mC3H aOz Aluminum Hydroxide Gel
Aluminum chlorohydroxide propylene glycol complex.
Aluminum hydroxychloride propylene glycol complex.
AI(OH)3 78.00
Aluminum hydroxide.
» Aluminum Dichlorohydrex Propylene Glycol Aluminum hydroxide [21645-51-2].
consists of aluminum dichlorohydrate in which
some of the waters of hydration have been » Aluminum Hydroxide Gel is a suspension of
replaced by propylene glycol. .It encompasses a amorphous aluminum hydroxide in which there is
range of aluminum-to-chloride atomic ratios a partial substitution of carbonate for hydroxide. It
between 0.90:1 and 1.25:1. It contains not less contains the equivalent of not less than 90.0

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 USP 43 Official Monographs / Aluminum 189

percent and not more than 110.0 percent of the Dried Aluminum Hydroxide Gel
labeled amount of aluminum hydroxide [AI(OH)3]'
It may contain Peppermint Oil, Glycerin, Sorbitol, AI(OH)3 78.00
Sucrose, Saccharin, or other suitable flavors, and it Aluminum hydroxide [21645-51-2].
may contain suitable antimicrobial agents.
» Dried Aluminum Hydroxide Gel is an amorphous
Packaging and storage-Preserve in tight containers, and
avoid freezing.
form of aluminum hydroxide in which there is a .
partial substitution of carbonate for hydroxide. It
Identification- contains the equivalent of not less than 76.5
A: Placeabout 1 g in a flask equipped with a stopper and
glasstubing, the tip of which is immersed in calcium hydroxide
percent of AI(OHh, and it may contain varying
TS in a test tube. Add 5 mL of 3 N hydrochloric acid to the quantities of basic aluminum carbonate and
flask, and immediately insert the stopper: gas evolves in the bicarbonate.
flask and a precipitate is formed in the test tube.
B: The solution remaining in the flask responds to the tests Packaging and storage-Preserve in tight containers.
for Aluminum (191). Labeling-Where the quantity of dried aluminum hydroxide
Microbial enumeration tests (61) and Tests for specified gel equivalent is stated in the labeling of any preparation, this
microorganisms (62)-lts total aerobic microbial count does shall be understood to be on the basis that each mg of dried
not exceed 100 cfu per mL, and it meets the requirements of gel is equivalent to 0.765 mg of AI(OH)3'
the test for the absence of Escherichia coli. USP Reference standards (11)-
Acid-neutralizing capacity (301 )-Not less than 65.0% of USP Dried Aluminum Hydroxide Gel RS
the expected mEq value, calculated from the results of the
Assay, is obtained. Each mg of AI(OH)3 has an expected Identification-
acid-neutralizing capacity value of 0.0385 mEq.
pH (791): between 5.5 and 8.0, determined
potentiometrically.
Chloride-Transfer an accurately measured quantity of the
Gel, equivalent to 0.6 g of AI(OH)3' to a porcelain dish. Add
mg of 3 N hydrochloric acid, with
0.1 mL of potassium chromate TS and 25 mL of water. Stir,
gentle warming: the solution responds to the tests for
and add 0.10 N silver nitrate until a faint, persistent pink color
Aluminum (191).
is obtained: not more than 8.0 mL of 0.10 N silver nitrate is
Acid-neutralizing capacity (301): not less than 25.0 mEq
required [4.7%, based on the AI(OH)3 content].
per g, 400 mg being tested as directed for Powders under Test
Sulfate (221)-Add 5.0 mL of 3 N hydrochloric acid to an Preparation.
accurately measured quantity of the Gel, equivalent to 0.3 g pH (791): not higher than 10.0, in an aqueous dispersion (1
of AI(OH)3' and heat to dissolve the specimen under.test. Cool, in 25).
dilute with water to 250 mL, and filter if necessary: a 20-mL Chloride (221 )-Dissolve 1.0 g in 30 mL of 2 N nitric acid,
portion of the filtrate shows no more sulfate than corresponds heat to boiling, add water to make 100 mL, and filter: a 5.0-mL
to 0.20 mL of 0.020 N sulfuric acid [0.8%, based on the portion of the filtrate, diluted with an equal volume of water,
AI(OH)3 content]. shows no more chloride than corresponds to 0.60 mL of 0.020
Arsenic, Method 1(211)-Prepare a StandardPreparation as N hydrochloric acid (0.85%).
directed in the test for Arsenic (211), except to prepare it to Sulfate (221)-Dissolve 330 mg in 15 mL of 3 N hydrochloric
contain 5 ~g of arsenic instead of 3 I-Ig. Prepare the Test acid, heat to boiling, add water to make 250 mL, and filter: a
Preparation as follows. Dissolve an accurately measured 25-mL portion of the filtrate shows no more sulfate than
quantity of the Gel, equivalent to 0.5 g of AI(OH)3' in 20 mL corresponds to 0.20 mL of 0.020 N sulfuric acid (0.6%).
of 7 N sulfuric acid. The limit is 0.001 %, based on the Arsenic, Method I (211 )-Dissolve 1.5 g in 80 mL of 7 N
AI(OH)3 content. sulfuric acid, and dilute with water to 220 mL: 55 mL of the
resulting solution meets the requirements of the test, the
Assay- . addition of 20 mL of 7 N sulfuric acid specified under Procedure
Edetate disodium titrant-Prepare and standardize as being omitted. The limit is 8 ppm.
directed in the Assay under AmmoniumAlum.
Procedure-Transfer an accurately measured quantity of Assay-
Gel, equivalent to about 1.5 g of AI(OH)3' to a beaker, add 15 Edetate disodium titrant-Prepare and standardize as
mL of hydrochloric acid, and heat gently until solution is directed in the Assay under Ammonium Alum.
complete. Cool, transfer to a 500-mL volumetric flask, dilute Procedure-Weigh accurately about 2 g of Gel, and dissolve
with water to volume, and mix. Pipet 20 mL of this solution in 15 mL of hydrochloric acid, with the aid of heat. Cool,
into a 250-mL beaker, and add, in the order named and with transfer to a 500-mL volumetric flask, dilute with water to
continuous stirring, 25.0 mL of Edetate disodium titrant and 20 volume, and mix. Pipet 20 mL of this solution into a 250-mL
mL of acetic acid-ammonium acetate buffer TS, then heat the beaker, and add, in the order named and with continuous
solution near the boiling point for 5 minutes. Cool, and add stirring, 25.0 mL of Edetate disodium titrant and 20 mL of acetic
50 mL of alcohol and 2 mL of dithizone TS.Titrate the solution acid-ammonium acetate buffer TS,then heat the solution near
with 0.05 M zinc sulfate VS until the color changes from the boiling point for 5 minutes. Cool, and add 50 mL of alcohol
green-violet to rose-pink. Perform a blank determination, and 2 mL of dithizone TS.Titrate the solution with 0.05 M zinc
substituting 20 mL of water for the sample, and make any sulfate VS to a bright rose-pink color. Perform a blank
necessary correction. Each mL of 0.05 M Edetate disodium determination, substituting 20 mL of water for the sample
titrant consumed is equivalent to 3.900 mg of AI(OH)3' solution, and make any necessary correction. Each mL of
0.05 M Edetate disodium titrant is equivalent to 3.900 mg of
AI(OH)3'

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 190 Aluminum / Official Monographs USP 43

Identification-
Dried Aluminum Hydroxide Gel Capsules A: Place a quantity of finely ground Tablets, equivalent to
about 500 mg of aluminum hydroxide, in a flask equipped
» Dried Aluminum Hydroxide Gel Capsulescontain with a stopper and glass tubing, the tip of which is immersed
in calcium hydroxide TS in a test tube. Add 5 mL of 3 N
not less than 90.0 percent and not more than hydrochloric acid to the flask, and immediately insert the
110.0 percent of the labeled amount of aluminum stopper: gas evolves in the flask and a precipitate is formed in
hydroxide [AI(OH)3]' the test tube.
B: The solution remaining in the flask responds to the tests
Packaging and storage-Preserve in well-closed for Aluminum (191).
containers. Disintegration (701): 10 minutes, simulated gastric fluid TS
Labeling-The Capsules may be labeled to state the being substituted for water in the test.
aluminum hydroxide content in terms of the equivalent Uniformity of dosage units (905): meet the requirements
amount of dried aluminum hydroxide gel, on the basis that for Weight Variation.
each mg of dried gel is equivalent to 0.765 mg of AI(OH)3' Acid-neutralizing capacity (301)-Not less than 5 mEq of
acid is consumed by the minimum single dose recommended
Identification- in the labeling, and not less than 55.0% of the expected mEq
A: Placea portion of Capsule contents, equivalent to about value, calculated from the labeled quantity of AI(OH)3' is
500 mg of aluminum hydroxide, in a flask equipped with a obtained. Each mg of AI(OHh has an expected
stopper and glass tubing, the tip of which is immersed in acid-neutralizing capacity value of 0.0385 mEq.
calcium hydroxide TS in a test tube. Add 10 mL of 3 N
hydrochloric acid to the flask, and immediately insert the Assay-
stopper: gas evolves in the flask and a precipitate is formed in Edetate disodium titrant-Prepare and standardize as
the test tube. directed in the Assay under Ammonium Alum.
B: The solution remaining in the flask responds to the tests Procedure-Weigh and finely powder not fewer than 20
for Aluminum (191). Tablets. Weigh accurately a portion of the powder, equivalent
Disintegration (701): 10 minutes, simulated gastric fluid TS to about 1.2 g of aluminum hydroxide, add 15 mL of
being substituted for water in the test. hydrochloric acid, and heat until dissolved. Dilute with water
Uniformity of dosage units (905): meet the requirements. to about 100 mL, mix, and filter quantitatively into a 500-mL
Acid-neutralizing capacity (301)-Not lessthan 5 mEq of volumetric flask, washing the filter with water. Proceed as
acid is consumed by the minimum single dose recommended directed in the Assay under DriedAluminumHydroxide Gel,
in the labeling, and not less than 55.0% of the expected mEq beginning with "dilute with water to volume." Each mL of
value, calculated from the labeled quantity of AI(OH)3' is 0.05 M Edetate disodium titrant is equivalent to 3.900 mg of
obtained. Each mg of AI(OH)3 has an expected AI(OH)3'
acid-neutralizing capacity value of 0.0385 mEq.
Assay-
Edetate disodium titrant-Prepare and standardize as
directed in the Assay under Ammonium Alum. Aluminum Phosphate Gel
Procedure-Weigh accurately the contents of not fewer
than 20 Capsules, and mix. Transfer an accurately weighed
portion of the powder, equivalent to about 1,2 g of aluminum Phosphoric acid, aluminum salt (1:1).
hydroxide, to a beaker, add 15 mL of hydrochloric acid, and Aluminum phosphate (1:1) [7784-30-7].
heat until dissolved. Dilute with water to about 100 mL, mix,
and filter quantitatively into a 500-mL volumetric flask, » Aluminum Phosphate Gel is a water suspension
washing the filter with water. Proceed as directed in the Assay containing not less than 4.0 percent and not more
under DriedAluminum Hydroxide Gel beginning with "dilute than 5.0 percent (w/w) of aluminum phosphate
with water to volume." Each mL of 0.05 M Edetate disodium
titrant is equivalent to 3.900 mg of AI(OH)3' (AIP0 4 ) . It may contain sodium benzoate, benzoic
acid, or other suitable agent, in an amount not
exceeding 0.5 percent, as a preservative.
Packaging and storage-Preserve in tight containers.
Dried Aluminum Hydroxide Gel Tablets Identification-
A: A solution of it in hydrochloric acid meets the
» Dried Aluminum Hydroxide Gel Tablets contain requirements of the tests for Aluminum (191).
not less than 90.0 percent and not more than B: A solution of it in 2 N nitric acid meets the requirements
of the tests for Phosphate (191).
110.0 percent of the labeled amount of aluminum pH (791): between 6.0 and 7.2.
hydroxide [AI(OH)3]' Soluble phosphate-Filter 20 g, and wash the residue with
30 mL of water. Add to the filtrate 2 mL of nitric acid, heat to
Packaging and storage-Preserve in well-closed 60°, and add 20 mL of ammonium molybdate TS. Heat at 50°
containers. for 30 minutes, filter, wash the precipitate with dilute nitric
Labeling-Tablets may be labeled to state the aluminum acid (1 in 36), then wash with potassium nitrate solution (1 in
hydroxide content in terms of the equivalent amount of dried 100) until the last portion of the filtrate is not acid to litmus
aluminum hydroxide gel, on the basisthat each mg of dried paper. Dissolve the precipitate in 50.0 mL of 0.5 N sodium
gel is equivalent to 0.765 mg of AI(OH)3' hydroxide VS,' add phenolphthalein TS, and titrate the excess
alkali with 0.5 N hydrochloric acid VS. Each mL of 0.5 N

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 USP 43 Official Monographs / Aluminum 191

sodium hydroxide is equivalent to 2.065 mg of P0 4• The Aluminum Chlorohydrate. Usethe result obtained to calculate
soluble phosphate, calculated asP0 4, does not exceed 0.30%. the Aluminum/chloride atomic ratio.
Sulfate (221)-Add 10 mL of 3 N hydrochloric acid to 109 of Content of chloride-Using Aluminum .
Gel, and heat to boiling. Cool, dilute with water to 250 mL, Sesquichlorohydrate instead of Aluminum Chlorohydrate,
and filter, if necessary. A 1O-mL portion of the solution shows proceed as directed in the test for Content of chloride under
no more sulfate than corresponds to 0.20 mL of 0.020 N Aluminum Chlorohydrate. Use the result obtained to calculate
sulfuric acid: not more than 0.05% is found. the Aluminum/chloride atomic ratio.
Arsenic, Method I (211)-Prepare the Test Preparation by Aluminum/chloride atomic ratio-Divide the
dissolving 5.0 g of Gel in the smallest necessary volume of 3 percentage of aluminum found in the test for Content of
N hydrochloric acid. The limit is 0.6 ppm. aluminum by the percentage of chloride found in the test for
Chloride-Transfer 25 g to a beaker with the aid of about 50 Content of chloride, and multiply by 35.453/26.98, in which
mL of water, add 5 mL of nitric acid, mix, then add, with 35.453 and 26.98 are the atomic weights of chlorine and
stirring, 30.0 mL of 0.1 N silver nitrate VS. Warm on a steam aluminum, respectively: the ratio is between 1.26:1 and
bath for 30 minutes, filter, and wash the precipitate with water 1.90:1.
acidified with nitric acid. To the filtrate add ferric ammonium Assay-Calculate the percentage of anhydrous aluminum
sulfate TS, and titrate the excess silver nitrate with 0.1 N sesquichlorohydrate in the Aluminum Sesquichlorohydrate by
ammonium thiocyanate VS. Each mL of 0.1 N silver nitrate is the formula:
equivalent to 3.545 mg of CI. Not more than 0.16% of
chloride is found. AI({26.98x+ [17.01(3x-l)] + 35.453}/26.98x)
Assay-To about 20 g of Gel, accurately weighed, in a
1OO-mL volumetric flask, add nitric acid to effect solution, in which AI is the percentage of aluminum found in the test
dilute with water to volume, and mix. Transfer 10.0 mL of this for Content of aluminum, x is the aluminum/chloride atomic
solution to a 400-mL beaker, dilute with water to 100 mL, heat ratio found in the test for Aluminum/chloride atomic ratio,
to 60°, add an excess of ammonium molybdate TS, and 26.98 is the atomic weight of aluminum, 17.01 is the
maintain at 50° for 30 minutes. Filter, and wash the precipitate molecular weight of the hydroxide anion (OH), and 35.453 is
with dilute nitric acid (1 in 36), then with potassium nitrate the atomic weight of chlorine (CI).
solution (1 in 100) until the last portion of the filtrate is not
acid to litmus paper. Dissolvethe precipitate in 50.0 mL of 0.5
N sodium hydroxide VS, add phenolphthalein TS, and titrate
the excess sodium hydroxide with 0.5 N sulfuric acid VS. Each
mL of 0.5 N sodium hydroxide is equivalent to 2.651 mg of
Aluminum Sesquichlorohydrate
AIP0 4 • Solution
» Aluminum Sesquichlorohydrate Solution consists
of complex basic aluminum chloride that is
Aluminum Sesquichlorohydrate polymeric and encompasses a range of
aluminum-to-chloride atomic ratios between
AIY(0H)3y_zCl z ' nHzO 1.26: 1 and 1.90: 1. The following solvents may be
Aluminum chlorohydroxide. used: water, propylene glycol, dipropylene glycol,
Aluminum hydroxychloride [11097-68-0]. or alcohol. It contains the equivalent of not less
than 90.0 percent and not more than 110.0
» Aluminum Sesquichlorohydrate consists of,
percent of the labeled concentration of anhydrous
complex basic aluminum chloride that is polymeric
aluminum sesquichlorohydrate.
and loosely hydrated and encompasses a range of
aluminum-to-chloride atomic ratios between Packaging and storage-Preserve in well-closed
1.26:1 and 1.90:1. It contains not less than 90.0 containers.
percent and not more than 110.0 percent of the Labeling-Label Solution to state the solvent used and the
labeled amount of anhydrous aluminum claimed concentration of anhydrous aluminum
sesquichlorohydrate contained therein.
sesquichlorohydrate.
Identification-
Pack'aging and storage-Preserve in well-closed A: A solution containing the equivalent of about 100 mg of
containers. anhydrous aluminum sesquichlorohydrate per mL responds to
Labeling-The label states the content of anhydrous the tests for Aluminum (191) and for Chloride (191).
aluminum sesquichlorohydrate. B: Identification of propylene glycol (where stated on the
Identification-A solution (1 in 10) responds to the tests for label)-Add about 10 mL of isopropyl alcohol to 2 g of
Aluminum (191) and for Chloride (191). Solution, mix, and filter. Evaporate the filtrate to about 1 mL
pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/ on a steam bath: the IR absorption spectrum of a film of this
w)]. solution on a silver chloride disk exhibits maxima only at the
Arsenic, Method I (211): 2 J.1g per g. same wavelengths as that of a similar preparation of a film of
Limit of iron-Using Aluminum Sesquichlorohydrate propylene glycol.
instead of Aluminum Chlorohydrate, proceed as directed in C: Identification of dipropylene glycol (where stated on the
the test for Limit of iron under Aluminum Chlorohydrate. The label)-Add about 10 mL of isopropyl alcohol to 2 g of
limit is 150 I-Ig per g. Solution, mix, and filter. Evaporate the filtrate to about 1 mL
Content of aluminum-Using Aluminum on a steam bath: the IR absorption spectrum of a film of this
Sesquichlorohydrate instead of Aluminum Chlorohydrate, solution on a silver chloride disk exhibits maxima only at the
proceed as directed in the test for Content of aluminum under

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 192 Aluminum / Official Monographs USP 43

same wavelengths asthat of a similar preparation of a film of Packaging and storage-Preserve in well-closed
dipropylene glycol. containers.
D: Identificat~on ~f alcohol(where stated on the label)-Mix Labeling-The label states the content of anhydrous
5 drops of Solution In a small beaker with 1 mL of potassium aluminum sesquichlorohydrate.
pe.rmanganate solution (1 in 100) and 5 drops of 2 N sulfuric
aCl~, and co~er the beaker immediately with filter paper Identification-
n:olsten~d wl~h a freshly prepared solution of 0.1 g of sodium A: A solution (1 in 10) responds to the tests for Aluminum
~Itroferncyanlde and 0.25 g of piperazine in 5 mL of water: an
(191) and for Chloride (191). .
Intense. blue color is produced on the filter paper, the color
becoming paler after a few minutes.
p!" <?91): between 3.0 and 5.0, in a solution prepared by
~: .•.~~~~~.t~g~gc:/pi~~{~~~tit~~~~1~fJIe~ts(19[):,'JfJtfqr~(;J
diluting 3 g of the Solution with water to obtain 10 mL.
Arsenic, Method I (211 )-Prepare the Test Preparation using SfJ~pJr-g~~()PY:}{~7E4(C:N171yli1Y:?()20)-
an accurately weighed quantity of the Solution. The limit is 2 Test specimen-Dissolve 0.5 g in about 40 mL of water, and
I-Ig per g. while mixing adjust with 2.5 N sodium hydroxide to a pH of
Umi~ o~ iron-Using A!uminum Sesquichlorohydrate
9.55 ± 0.05. Filter the suspensionof precipitate thus obtained.
Solution Inst~ad of ~Iumlnum Chlorohydrate Solution, Evaporate about 15 mL of the filtrate to about 1 mL on a hot
proceed as directed In the test for the Limit of iron under plate. Deposit this solution on a silver chloride disk.
Aluminum Chlorohydrate Solution. The limit is 75 I-Ig per g. Standardspecimen: a similar preparation of polyethylene
Content of aluminum-Using Aluminum glycol.
Sesquichlorohydrate Solution instead of Aluminum pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/
Chlorohydrate Solution, proceed asdirected in the test for the w)].
Content of aluminum under Aluminum Chlorohydrate Solution. Arsenic, Method I (211): 2 I-Ig per g.
Use the result to calculate the Aluminum/chlorideatomic ratio. Limit of Iron-Using Aluminum Sesquichlorohydrex
Cont~nt of chloride-Using Aluminum
Polyethylene Glycol instead of Aluminum Chlorohydrate,
Sesquichlorohydrate Solution instead of Aluminum proceed asdirected in the test for Limitof iron under Aluminum
Chlorohydrate Solution, proceed asdirected in the test for the Chlorohydrate The limit is 150 I-Ig per g.
Contentof chloride under AluminumChlorohydrate Solution. Use . Content of aluminum-Using Aluminum
the result to calculate the Aluminum/chlorideatomic ratio Sesquichlorohydrex Polyethylene Glycol instead of Aluminum
Aluminum/chloride atomic ratio-Divide the . Chlorohydrate, proceed as directed in the test for Contentof
percentage of aluminum found in the test for Content of aluminum under Aluminum Chlorohydrate Usethe result
aluminum by the percentage of chloride found in the test for obtained to calculate the Aluminum/chloride atomic ratio.
Content of chloride, and multiply by 35.453/26.98, in which Content of chloride-Using Aluminum Sesquichlorohydrex
35.453 and 26.98 are the atomic weights of chlorine and Polyethylene.Glycol !nstead of Aluminum Chlorohydrate,
aluminum, respectively: the ratio is between 1.26:1 and proceed as directed In the test for Contentof chloride under
1.90:1. Aluminum Chlorohydrate Usethe result obtained to calculate
Assa~-Calculate th~ percentage of anhydrous aluminum
the Aluminum/chloride atomic ratio.
sesqulchlorohydrate In the Solution by the formula: Aluminum/chloride atomic ratio-Divide the
percentage of aluminum found in the test for Contentof
AI({26.98x + [17.01 (3x - 1)] + 35.453}/26.98x) aluminum by the percentage of chloride found in the test for
Contentof chloride, and multiply by 35.453/26.98, in which
in which AI is the percentage of aluminum found in the test 35.453 and 26.98 are the atomic weights of chlorine and
for Content of aluminum, x is the aluminum/chloride atomic aluminum, respectively: the ratio is between 1.26:1 and
ratio found in the test for Aluminum/chlorideatomic ratio 1.90:1.
26.98 is the atomic weight of aluminum, 17.01 is'the ' Assay-Calculate the percentage of anhydrous aluminum
molecular weight of the hydroxide anion (OH) and 35.453 is sesquichlorohydrex in the Aluminum Sesquichlorohydrex
the atomic weight of chlorine (CI). ' Polyethylene Glycol by the formula:

AI({26.98x + [17.01 (3x - 1)] + 35.453}/26.98x)

in which AI is the percentage of aluminum found in the test
Aluminum Sesquichlorohydrex for Content of aluminum, x is the aluminum/chloride atomic
ratio found in the test for Aluminum/chloride atomic ratio
Polyethylene Glycol 26.98 is the atomic weight of aluminum, 17.01 is the '
molecula~ wei~ht of the hydroxide anion (OH), and 35.453 is
Aly(OH)3y_zClz' nH20 . mH(OCH 2CH 2)nOH the atomic weight of chlorine (CI).
Aluminum chlorohydroxide polyethylene glycol complex.
Aluminum hydroxychloride polyethylene glycol complex.

» Aluminu~ Sesquichl~rohydrex Polyethylene Aluminum Sesquichlorohydrex

~Iyco~ consists of aluminum sesquichlorohydrate
In which some of the waters of hydration have
Propylene Glycol
been replaced by polyethylene glycol. It
encompasses a range of aluminum-to-chloride Aly(OH)3Y.zClz· nH 20 . mC3H 802
Aluminum chlorohydroxide propylene glycol complex.
atomic ratios between 1.26:1 and 1.90:1. It Aluminum hydroxychloride propylene glycol complex.
contains not less than 90.0 percent and not more
than 110.0 percent of the labeled amount of
anhydrous aluminum sesquichlorohydrate.

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 USP 43 Official Monographs / Aluminum 193

» Aluminum Sesquichlorohydrex Propylene Glycol Aluminum Subacetate Topical Solution

consistsof aluminum sesquichlorohydrate in which
some of the waters of hydration have been
replaced by propylene glycol. It encompasses a » Aluminum Subacetate Topical Solution yields,
range of aluminum-to-chloride atomic ratios from .each 100 mL, not less than 2.30 9 and not
between 1.26:1 and 1.90:1. It contains not less more than 2.60 9 of aluminum oxide (Alz0 3) , and
than 90.0 percent and not more than 110.0 not less than 5.43 9 and not more than 6.13 9 of
percent of the labeled amount of anhydrous acetic acid (CZH40Z) ' It may be stabilized by the
aluminum sesquichlorohydrate. addition of not more than 0.9 percent of boric acid.
Aluminum Subacetate Topical Solution may be
Packaging and storage-Preserve in well-closed prepared as follows.
containers.
labeling-The label states the content of anhydrous
aluminum sesquich/orohydrate. Aluminum Sulfate . 145 9
Identification- Acetic Acid . 160 mL
A: A solution (1 in 10) responds to the tests for Aluminum
(191) and for Chloride (191). Calcium Carbonate . 70 g
Purified Water, a sufficient quantity,
B. to make . 1000 mL
;~/5ggtn<, , ..
Test specimen-Dissolve 0.5 g in about 40 ml of water, and Dissolve the Aluminum Sulfate in 600 mL of cold
while mixing adjust with 2.5 N sodium hydroxide to a pH of water, filter the solution, and add the Calcium
9.55 ± 0.05. Filterthe suspension of precipitate thus obtained.
Evaporate about 15 ml of the filtrate to about 1 ml on a hot Carbonate gradually, in several portions, with
plate. Deposit this solution on a silver chloride disk. constant stirring. Then slowly add the Acetic Acid,
Standardspecimen: a similar preparation of propylene mix, and set the mixture aside for 24 hours. Filter
glycol. the product with the aid of vacuum if necessary,
pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/ returning the first portion of the filtrate to the
w)].
Arsenic, Method I (211): 2 IJg per g. funnel. Wash the magma on the filter with small
limit of iron-Using Aluminum Sesquich/orohydrex portions of cold water, until the total filtrate
Propylene Glycol instead of Aluminum Chlorohydrate, measures 1000 mL. .
proceed as directed in the test for Limit of iron under Aluminum
Chlorohydrate. The limit is 150 IJg per g. Packaging and storage-Preserve in tight containers.
Content of aluminum-Using Aluminum Identification-It responds to the tests for Aluminum and for
Sesquichlorohydrex Propylene Glycol instead of Aluminum test B under Acetate under Identification Tests-General (191)
Chlorohydrate, proceed as directed in the test for Content of with a deep red color upon the addition of ferric chloride TS.
aluminum under Aluminum Chlorohydrate. Use the result This color is destroyed by the addition of a mineral acid.
obtained to calculate the Aluminum/chlorideatomic ratio. pH (791): between 3.8 and 4.6.
Content of chloride-Using Aluminum Sesquichlorohydrex limit of boric acid-Proceed as directed in the test for Limit
Propylene Glycol instead of Aluminum Ch/orohydrate, of boric acid under AluminumAcetate Topical Solution.
proceed as directed in the test for Content of chloride under
Aluminum Chlorohydrate. Use the result obtained to calculate Assay for aluminum oxide-
the Aluminum/chloride atomic ratio. Edetate disodium titrant-Prepare and standardize as
Aluminum/chloride atomic ratio-Divide the directed in the Assay under Ammonium Alum.
percentage of aluminum found in the test for Content of Procedure-Pipet 20 ml of Topical Solution into a 250-ml
aluminum by the percentage of chloride found in the test for volumetric flask, add 5 ml of hydrochloric acid, dilute with
Content of chloride, and multiply by 35.453/26.98, in which water to volume, and mix. Pipet 25 ml of this dilution into a
35.453 and 26.98 are the atomic weights of chlorine and 250-mL beaker, and proceed as directed for Procedure in the
aluminum, respectively: the ratio is between 1.26:1 and Assay for aluminum oxide under AluminumAcetate Topical
1.90:1. Solution, beginning with "add, in the order named." Each mL
Assay-Calculate the percentage of anhydrous aluminum of 0.05 M Edetate disodium titrant is equivalent to 2.549 mg
sesquich/orohydrate in the Aluminum Sesquichlorohydrex of Al z0 3 •
Propylene Glycol by the formula: Assay for acetic acid-Proceed as directed in the Assay for
aceticacid under Aluminum Acetate Topical Solution.
AI({26.98x + [17.01 (3x - 1)] + 35.453}/26.98x)
in which AI is the percentage of aluminum found in the test
for Content of aluminum, x is the aluminum/chloride atomic
ratio found in the test for Aluminum/chloride atomic ratio, Aluminum Sulfate
26.98 is the atomic weight of aluminum, 17.01 is the
molecular weight of the hydroxide anion (OH), and 35.453 is AIz(S04)3 . xHzO (anhydrous) 342.15
the atomic weight of chlorine (C/).
Sulfuricacid, aluminum salt (3:2), hydrate.

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 194 Aluminum / OfficialMonographs USP 43

Aluminum sulfate (2:3) hydrate [17927-65-0]. It B. IDENTIFICATION TESTs-GENERAL, Sulfate

Anhydrous 342.16 (191 )andCalcium (191)
[10043-01-3]. Sample solution: Suspend 2 g of sample in 50 mL of water
and filter.
» Aluminum Sulfate contains not less than 54.0 Acceptance criteria: Meets the requirements
percent and not more than 59.0 percent of ASSAY
AI2(S04)3' It contains a varying amount of water of • ALUMINUM SULFATE TETRADECAHYDRATIE
crystallization. . Sample solution: Transfer 109 of Aluminum Sulfate and
Calcium Acetate for Topical Solution to a 1000-mL
Packaging and storage-Preserve in well-closed volumetric flask. Add 100 mL of 1.2 N hydrochloric acid
containers. and 250 mL of water. Heat on a steam bath or hot plate
Identification-A solution (1 in 10) responds to the tests for until dissolved. Cool, and dilute with water to volume.
Aluminum and for Sulfate (191). Retain a portion of the Sample solution for the Assay for
pH (791): not less than 2.9, in a solution (1 in 20). Calcium Acetate Monohydrate.
Water Determination, Method I (921): not less than 41.0% Blank: Water
and not more than 46.0%. Titrimetric system
Limit of alkalies and alkaline earths-To a boiling Mode: Residual titration
solution of 1.0 g in 150 mL of water add a few drops of methyl Titrant: 0.02 M zinc sulfate VS
red TS and then add 6 N ammonium hydroxide just until the Endpoint detection: Visual
color of the solution changes to a distinct yellow. Add hot Analysis: Transfer a 5.0-mL aliquot of the Sample solution to
water to restore the volume to 150 mL, and filter while hot. a 250-mL conical flask. Add, in the order named, 40.0 mL
Evaporate 75 mL of the filtrate to dryness, and ignite to of 0.01 M edetate disodium VS and 20 mL of acetic acid-
constant weight: not more than 2 mg of residue remains ammonium acetate buffer TS, and mix by swirling. Add 50
(0.4%). mL of alcohol and 2 mL of dithizone TS, and titrate the
Limit of ammonium salts-Heat 1 g with 10 mL of 1 N excess 0.01 M edetate disodium VS with Titrant until the
sodium hydroxide on a steam bath for 1 minute: the odor of color changes from green-violet to a clear rose-pink.
ammonia is not perceptible. Perform a blank titration, substituting 5.0 mL of water for
Iron-To 20 mL of a solution (1 in 150) add 0.3 mL of the Sample solution.
potassium ferrocyanide TS: no blue color is produced Calculate the percentage of aluminum sulfate
immediately. tetradecahydrate [AI2(S04)3 . 14HzO] in the portion of
Aluminum Sulfate and Calcium Acetate for Topical
Assay- Solution taken:
Edetate disodium titrant-Prepare and standardize as
directed in the Assay under Ammonium Alum. Result = {[O x (VB - Vs) x M x A/w} x 100
Procedure-Transfer about 7.5 g of Aluminum Sulfate,
accurately weighed, to a 250-mL volumetric flask, and dissolve o =dilution factor, 1000/5.0
in water. Dilute with water to volume, mix, and pipet 10 mL VB = blank titration volume (mL)
of the solution into a 250-mL beaker. Proceed as directed in Vs = sample titration volume (mL)
the Assay for aluminum oxide under Aluminum Acetate Topical M = molarity of the Titrant (mmol/mL)
Solution beginning with "add, in the order named." Each mL F =equivalency factor, 297.2 (mg/mmol)
of 0.05 M Edetate disodium titrant is equivalent to 8.554 mg W = weight of sample used (mg)
of Alz(S04)3'
Acceptance criteria: 90.00/0-110.0% of the labeled amount
of aluminum sulfate tetradecahydrate [Alz(S04)3 . 14HzO]
• CALCIUM ACETATE MONOHYDRATE
Sample: Transfer a 5.0-mL aliquot of the Sample solution
Aluminum Sulfate and Calcium Acetate retained from the Assay for Aluminum Sulfate
for Topical Solution Tetradecahydrate to a 250-mL conical flask.
Titrimetric system
DEFINITION Mode: Direct titration
Aluminum Sulfate and Calcium Acetate for Topical Solution Titrant: 0.01 M edetate disodium VS
contains NLT 90.0% and NMT 110.0% of the labeled Endpoint detection: Visual
amounts of aluminum sulfate tetradecahydrate [AIz(S04)3 . Analysis: Add 1-2 mL of 50% triethanolamine to mask the
14HzO] and calcium acetate monohydrate (C4H 6Ca0 4· aluminum, and mix well. With constant stirring, add to the'
H20). Sample, in the order named, 100 mL of water, 15 mL of 1
N sodium hydroxide, and 300 mg of hydroxy naphthol
IDENTIFICATION blue, and titrate with Titrant. The indicator will change from
• A. purple to a clear blue color at the endpoint.
Sample: 0.25 g of Aluminum Sulfate and Calcium Acetate Calculate the percentage of calcium acetate monohydrate
for Topical Solution (C4H 6Ca0 4· H20) in the portion of Aluminum Sulfate and
Analysis: Placethe Sample in a test tube. Add 10 mL of water Calcium Acetate for Topical Solution taken:
and 0.25 g of calcium carbonate. Heat on a steam bath for
10 min, and filter. Add 3-4 drops of ferric chloride TSto the Result = [(0 x Vx M x A/w] x 100
filtrate. [NOTE-After the addition of the ferric chloride TS,
the solution may be heated for 1 min to speedthe reaction.] o = dilution factor, 1000/5.0
Acceptance criteria: A reddish-brown color or precipitate V = sample titration volume (mL)
indicates acetate. M = molarity of the Titrant (mmol/mL)
F = equivalency factor, 176.2 (mg/mmol)
W = weight of sample used (mg)

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 USP 43 Official Monographs / Aluminum 195

Acceptance criteria: 90.00/0-110.0% of the labeled amount • CALCIUM ACIETATE MONOHYDRATE

of calcium acetate monohydrate (C4H6Ca04· H20) Sample: Transfer 20.0 mL of the Sample solution retained
from the Assay for Aluminum Sulfate Tetradecahydrate to a
SPECIFIC TESTS 125-mL conical flask.
• pH (791) Titrimetric system
Sample solution: 1 g of Aluminum Sulfate and Calcium Mode: Direct titration
Acetate for Topical Solution in 200 mL of water Titrant: 0.01 M edetate disodium VS
Acceptance criteria: 4.0-4.8 Endpoint detection: Visual
ADDITIONAL REQUIREMENTS Analysis: With constant stirring, add to the Sample, in the
• PACKAGING AND STORAGE: Preserve in single-unit order named, 0.5 mL of trolamlne, 10 mL of ammonia-
containers, and protect from excessive heat. ammonium chloride buffer TS, and 3 drops of a solution
prepared by dissolving 500 mg of eriochrome black T
trituration in 10 mL of methanol. Titrate with Titrant to a
violet endpoint. Each mL of 0.01 M edetate disodium is
equivalent to 1.762 mg of the labeled amount of calcium
Aluminum Sulfate and Calcium Acetate acetate monohydrate (C4H6Ca04 . H20).
Acceptance criteria: 90.00/0-110.0%
Tablets for Topical Solution
PERFORMANCE TESTS
DEFINITION • DISINTEGRATION (701): 10 min
Aluminum Sulfate and Calcium Acetate Tablets for Topical • UNIFORMITY OF DOSAGE UNITS (905): Meets the
Solution contains NLT 90.0% and NMT 110.0% of the requirements for Weight Variation
labeled amounts of aluminum sulfate tetradecahydrate
[AI2(S04)3 . 14H 20] and calcium acetate monohydrate SPECIFIC TESTS
(C4H6Ca04 . H20). • pH (791)
Sample solution: 2 g of ground Tablet powder in 500 mL
IDENTIFICATION of water
• A. IDENTIFICATION TESTS-GENERAL, Aluminum (191) Acceptance criteria: 4.0-4.8
Sample solution: Suspend 2 g of ground Tablet powder in • Loss ON DRYING (731)
50 mL of water, and filter. Usea portion in the Analysis, and Analysis: Dry ground Tablet powder at 150 0 for 15 min.
retain the remaining filtrate for Identification test B. Acceptance criteria: NMT 18%
Analysis: Mix 2 mL of the Sample solution with 2 mL of water
and 2 drops of 3 N hydrochloric acid. ADDITIONAL REQUIREMENTS
Acceptance criteria: Meets the requirements of test A • PACKAGING AND STORAGE: Preserve in tight containers, and
• B. IDENTIFICATION TESTS-GENERAL, Sulfate (191) and avoid excessive heat.
Calcium (191)
Sample solution: A portion of the filtrate retained from
Identification test A
Acceptance criteria: Meets the requirements
Aluminum Zirconium
ASSAY Octachlorohydrate
• ALUMINUM SULFATE TETRADECAHYDRATE
Sample solution: Finely powder and mix NLT 20 Tablets.
Weigh a portion of the powder, equivalent to 2.8 g of
aluminum sulfate, and transfer to a 1OOO-mL volumetric
AI y Zr(OH) 3y+4-x CI x • nH 2 °
flask. Add 100 mL of 1.2 N hydrochloric acid and 100 mL » Aluminum Zirconium Octachlorohydrate is a
of water, and heat on a steam bath, with occasional polymeric, loosely hydrated complex of basic
swirling, to dissolve the powder. Allow the solution to cool,
and dilute with water to volume. Retain a portion of the aluminum zirconium chloride that encompasses a
Sample solution for the Assay for, Calcium Acetate range of aluminum-to-zirconium atomic ratios
Monohydrate. between 6.0:1 and 10.0:1, and a range of
Blank: Water (aluminum plus zirconium)-to-chloride atomic
Titrimetric system ratios between 1.5:1 and 0.9:1. It contains not less
Mode: Residual titration
Titrant: 0.02 M zinc sulfate VS than 90.0 percent and not more than 110.0
Back-titrant: 0.01 M edetate disodium VS percent of the labeled amount of anhydrous
Endpoint detection: Visual aluminum zirconium octachlorohydrate.
Analysis: Transfer 25.0 mL of the Sample solution to a
250-mL conical flask. Add, in the order named, 40.0 mL of Packaging and storage-Preserve in well-closed
Back-titrant and 20 mL of acetic acid-ammonium acetate containers.
buffer TS, and mix by swirling. Add 50 mL of alcohol and 2 Labeling-The label statesthe content of anhydrous
mL of dithizone TS, and titrate the excess Back-titrantwith aluminum zirconium octachlorohydrate.
Titrant until the color changes from green-Violet to a clear Identification-A solution (1 in 10) responds to the test for
rose-pink. Perform a blank determination. Each mL of 0.01 Chloride (191).
M edetate disodium is equivalent to 2.972 mg of the pH (791): between 3.0 and 5.0, in a solution [15 in 100 (wi
labeled amount of aluminum sulfate tetradecahydrate w)].
[AliS04)3 . 14H 2° ]. Arsenic, Method I (211): 2 I-Ig per g.
Acceptance criteria: 90.0%-110.0%

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 196 Aluminum / OfficialMonographs USP 43

limit of Iron- hafnium content, and 26.98 is the atomic weight of

Standardpreparation-Transfer 2.0 mL of Standard Iron aluminum: the ratio is between 6.0:1 and 10.0:1.
Solution, prepared as directed under Iron (241), to a 50-mL Content of chloride-Transfer about 250 mg of Aluminum
beaker. Zirconium Octachlorohydrate, accurately weighed, to a
Test preparation-Transfer 2.7 g of Aluminum Zirconium 250-mL beaker, add 100 to 120 mL of water and 20 ml of
Octachlorohydrate to a 1OO-mL volumetric flask, dilute with diluted nitric acid, and swirl to dissolve. Titrate with 0.05 N
water to volume, and mix. Transfer 5.0 rnl, of this solution to silver nitrate VS using a calomel electrode and a silver billet
a 50-ml beaker. electrode system, determining the endpoint
Procedure-To each of the beakers containing the Standard potentiometrically. Each mL of 0.05 N silver nitrate is
preparation and the Test preparation add 5 ml of 6 N nitric equivalent to 1.773 mg of chloride (CI). Use the result
acid, cover with a watch glass, and boil on a hot plate for 3 to obtained to calculate the (Aluminum pluszirconium)/chloride
5 minutes. Allow to cool, add 5 ml of Ammonium Thiocyanate atomic ratio.
Solution, prepared as directed under Iron (241), transfer to (Aluminum plus zirconium)/chloride atomic ratio-
separate 50-mL color comparison tubes, dilute with water to Calculate the (aluminum plus zirconium)/chloride atomic ratio
volume, and mix: the color of the solution from the Test by the formula:
preparation is not darker than that of the solution from the
Standard preparation (150 IJg per g). [(AI/26.98) + (Zr/92.97)]/(CI/35.453)
Content of aluminum-Transfer about 0.15 g of Aluminum
Zirconium Octachlorohydrate, accurately weighed, to a in which AI, Zr, and CI are the percentages of aluminum,
150-ml beaker, and add 5 mL of water and 15 mL of zirconium, and chloride as determined in the tests for Content
hydrochloric acid. Heat this solution to boiling, and continue of aluminum, Content of zirconium, and Contentof chloride,
boiling for 5 minutes. Add 40 mL of water and 15.0 ml of 0.1 respectively; 26.98 is the atomic weight of aluminum; 92.97
M edetate disodium VS. Heat the solution to boiling, and is the atomic weight of zirconium corrected for 2% hafnium
continue boiling for 5 minutes. Allowthe solution to cool, add content; and 35.453 is the atomic weight of chlorine: the ratio
10 to 15 ml of acetic acid-ammonium acetate buffer TS, and is between 1.5:1 and 0.9:1.
adjust with ammonium hydroxide to a pH of 4.5 ± 0.1 . Add Assay-Calculate the percentage of anhydrous aluminum
20 mL of alcohol, and adjust with ammonium hydroxide to a zirconium octachlorohydrate in the Aluminum Zirconium
pH of 4.6 ± 0.1 . Add 5 to 10 drops of dithizone TS, and titrate . Octachlorohydrate by the formula:
with 0.1 M zinc sulfate VS until the first permanent purple-pink
color appears. Perform a blank determination, and make any AI({26.98y+ 92.97 + 17.01 [3y+ 4 - (Y+ 1)/z] + 35.453(y+ 1)
necessary correction. Calculate the percentage of aluminum /z} /26. 98 y)
(AI) in the Aluminum Zirconium Octachlorohydrate by the
formula: in which AI is the percentage of aluminum found in the test
for Content of aluminum, y is the aluminum/zirconium atomic
2.698[15.0M e -(zM z + Z e)]/W ratio found in the test for Aluminum/zirconium atomic ratio, Z
is the (aluminum plus zirconium)/chloride atomic ratio found
in which M e is the molarity of the edetate disodium VS; z is in the test for (Aluminum plus zirconium)/chloride atomicratio,
the volume, in ml, of zinc sulfate VS consumed; M z is the 26.98 is the atomic weight of aluminum, 92.97 is the atomic
weight of zirconium corrected for 2% hafnium content, 17.01
molarity of the zinc sulfate VS; W is the quantity, in g, of is the molecular weight of the hydroxide anion (OH), and
Aluminum Zirconium Octachlorohydrate taken; Z e is the
35.453 is the atomic weight of chlorine (CI).
equivalent volume, in mL, of edetate disodium VS consumed
by the zirconium moiety, calculated as follows:
(Zr/M e)(W/92.97)

in which Zr is the percentage of zirconium as determined in

Aluminum Zirconium
the test for Contentof zirconium, 92.97 is the atomic weight Octachlorohydrate Solution
of zirconium corrected for 2% hafnium content, and the other
terms are as defined above. Use the result obtained to
calculate the Aluminum/zirconium atomicratio and the » Aluminum Zirconium Octachlorohydrate
(Aluminum plus zirconium)/chloride atomicratio. Solution consists of complex basic aluminum
Content of zirconium-Transfer about 250 mg of chloride that is polymeric and encompasses a
Aluminum Zirconium.Octachlorohydrate, accurately weighed, range of aluminum-to-zirconium atomic ratios
to a 150-ml beaker, and add 5 ml of water and 15 mL of
hydrochloric acid. Heat this solution to boiling, and continue
between 6.0:1 and 10.0:1, and a range of
boiling for 6 to 8 minutes. Add 30 to 40 ml of water and 5 mL (aluminum plus zirconium)-to-chloride atomic
of hydrochloric acid, and heat to boiling. Add 1 drop of xylenol ratios between 1.5:1 and 0.9:1. The following
orange TS, and, while still hot, titrate with 0.1 M edetate solvents may be used: water, propylene glycol, or
disodium VS until the color of the solution changes from pink dipropylene glycol. It contains the equivalent of
to yellow. Perform a blank determination, and make any
necessary correction. Each mL of 0.1 M edetate disodium is not less than 90.0 percent and not more than
equivalent to 9.297 mg of zirconium (Zr). Use the result 110.0 .percent of the labeled concentration of
obtained to calculate the Aluminum/zirconium atomicratio and anhydrous aluminum zirconium
the (Aluminumplus zirconium)/chloride atomic ratio. octach lorohyd rate.
Aluminum/zirconium atomic ratia-Divide the
percentage of aluminum found in the test for Contentof Packaging and storage-Preserve in well-closed
aluminum by the percentage of zirconium found in the test for containers. .
Contentof zirconium, and multiply by 92.97/26.98, in which
92.97 is the atomic weight of zirconium corrected for 2%

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 USP 43 OfficialMonographs / Aluminum 197

labeling-Label Solution to state the solvent used and the content; and 35.453 isthe atomic weight of chlorine: the ratio
claimed concentration of anhydrous aluminum zirconium is between 1.5:1 and 0.9:1.
octachlorohydrate. Assay-Calculate the percentage of anhydrous aluminum
zirconium octachlorohydrate in the Solution by the formula:
Identification-
A: A solution containing the equivalent of about 100 mg of AI({26.98y+ 92.97 + 17.01 [3y+ 4 - (Y+ l)/z] + 35.453(y+ 1)
anhydrous aluminum zirconium octachlorohydrate per mL /z}/26.98y)
responds to the test for Chloride (191).
8: Identification of propylene glycol (where stated on the in which AI is the percentage of aluminum found in the test
label)-Add about 10 mL of isopropyl alcohol to 2 g of for Content of aluminum, y is the aluminum/zirconium atomic
Solution, mix, and filter. Evaporate the filtrate to about 1 mL ratio found in the test for Aluminum/zirconium atomic ratio, z
on a steam bath: the IR absorption spectrum of a film of this is the (aluminum plus zirconium)/chloride atomic ratio found
solution on a silver chloride disk exhibits maxima only at the in the test for (Aluminum pluszirconium)/chloride atomic ratio,
same wavelengths as that of a similar preparation of a film of 26.98 is the atomic weight of aluminum, 92.97 is the atomic
propylene glycol. weight of zirconium corrected for 2% hafnium content, 17.01
C: Identification of dipropylene glycol (where stated on the is the molecular weight of the hydroxide anion (OH), and
label)-Add about 10 mL of isopropyl alcohol to 2 g of 35.453 is the atomic weight of chlorine (CI).
Solution, mix, and filter. Evaporate the filtrate to about 1 mL
on a steam bath: the IR absorption spectrum of a film of this
solution on a silver chloride disk exhibits maxima only at the
same wavelengths as that of a similar preparation of a film of
dipropylene glycol. Aluminum Zirconium
pH (791): between 3.0 and 5.0, in a solution prepared by
diluting 3 g of the Solution with water to obtain 10 mL. Octachlorohydrex Gly
Arsenic, Method I (211): Preparethe Test Preparation using an
accurately weighed quantity of the Solution. The limit is 2 I-lg » Aluminum Zirconium Octachlorohydrex Gly is a
per g.
Limit of iron-Using Aluminum Zirconium derivative of Aluminum Zirconium
Octachlorohydrate Solution instead of Aluminum Octachlorohydrate in which some of the water
Chlorohydrate Solution, proceed as directed in the test for the molecules have been displaced by glycine, calcium
Limit of iron under AluminumChlorohydrate Solution. The limit glycinate, magnesium glycinate, potassium
is 75 I-lg per g. glycinate, sodium glycinate, or zinc glycinate. It
Content of aluminum-Using about 0.3 g of Aluminum
Zirconium Octachlor6hydrate Solution, accurately weighed, encompasses a range of aluminum-to-zirconium
instead of Aluminum Zirconium Octachlorohydrate, proceed atomic ratios between 6.0:1 and 10.0:1, and a
as directed in the test for the Content of aluminum under range of (aluminum plus zirconium)-to-chloride
Aluminum Zirconium Octachlorohydrate. Usethe result to atomic ratios between 1.5:1 and 0.9:1. It contains
calculate the Aluminum/zirconium atomic ratio andthe not less than 90.0 percent and not more than
(Aluminum plus zirconium)/chloride atomic ratio.
Content of zirconium-Using about 500 mg of Aluminum 110.0 percent of the labeled amount of anhydrous
Zirconium Octachlorohydrate Solution, accurately weighed, aluminum zirconium octachlorohydrate.
instead of Aluminum Zirconium Octachlorohydrate, proceed
as directed in the test for the Content of zirconium under Packaging and storage-Preserve in well-closed
Aluminum Zirconium Octachlorohydrate. Usethe result to containers.
calculate the Aluminum/zirconium atomic ratio and the labeling-The label states the form of glycine used and the
(Aluminum plus zirconium)/chloride atomic ratio. claimed content of anhydrous aluminum zirconium
Aluminum/zirconium atomic ratio-Divide the octachlorohydrate.
percentage of aluminum found in the test for Content of Identification-
aluminum by the percentage of zirconium found in the test for A: A solution (1 in 10) responds to the test for Chloride
Content of zirconium, and multiply by 92.97/26.98, in which (191 ).
92.97 is the atomic weight of zirconium corrected for 2% 8: Place about 0.5 g of it in a 50-mL beaker, add about 20
hafnium content, and 26.98 is the atomic weight of mL of water, and swirl to dissolve. Heat to boiling on a hot
aluminum: the ratio is between 6.0:1 and 10.0:1. plate, and add about 60 mg of ninhydrin: a deep violet color
Content of chloride-Using about 500 mg of Aluminum immediately develops.
Zirconium Octachlorohydrate Solution, accurately weighed, pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/
instead of Aluminum Zirconium Octachlorohydrate, proceed w)].
as directed in the test for the Content of chloride under Arsenic, Method I (211): 2 I-lg per g.
Aluminum Zirconium Octachlorohydrate. Usethe result to Limit of iron-Using Aluminum Zirconium
calculate the (Aluminum plus zirconium)/chloride atomic ratio. Octachlorohydrex Gly instead of Aluminum Zirconium
(Aluminum plus zirconium)/chloride atomic ratio- Octachlorohydrate, proceed as directed in the test for Limit
Calculate the (aluminum plus zirconium)/chloride atomic ratio of iron under AluminumZirconium Octachlorohydrate. The
by the formula: specified result is obtained (150 I-lg per g limit).
Content of aluminum-Using Aluminum Zirconium
[(AI/26.98) + (Zr/92.97)]/(C1/35.453)
Octachlorohydrex Gly instead of Aluminum Zirconium
in which AI, Zt, and CI are the percentages of aluminum, Octachlorohydrate, proceed asdirected in the test for Content
zirconium, and chloride as determined in the testsfor Content of aluminum under AluminumZirconium Octachlorohydrate.
of aluminum, Content of zirconium, and Content of chloride, Use the result obtained to calculate the Aluminum/zirconium
respectively; 26.98 is the atomic weight of aluminum; 92.97 atomic ratio and the (Aluminum pluszirconium)/chloride atomic
is the atomic weight of zirconium corrected for 2% hafnium ratio.

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198 Aluminum / Official Monographs USP 43

Content of zirconium-Using Aluminum Zirconium solvents may be used: water, propylene glycol, or
Octachlorohydrex Gly instead of Aluminum Zirconium dipropylene glycol. It contains the equivalent of
Octachlorohydrate, proceed asdirected in the test for Content
of zirconium under Aluminum Zirconium Octachlorohydrate. Use not less than 90.0 percent and not more than
the result obtained to calculate the Aluminum/zirconium 110.0 percent of the labeled concentration of
atomicratio and the (Aluminum pluszirconium)/chloride atomic anhydrous aluminum zirconium
ratio. octachlorohydrate.
Aluminum/zirconium atomic ratie-Divide the
percentage of aluminum found in the test for Content of Packaging and storage-Preserve in well-closed
aluminum by the percentage of zirconium found in the test for containers.
Content of zirconium, and multiply by 92.97/26.98, in which labeling-Label Solution to state the solvent and form of
92.97 is the atomic weight of zirconium corrected for 2% glycine used and the claimed concentration of anhydrous
hafnium content, and 26.98 is the atomic weight of aluminum zirconium octachlorohydrate.
aluminum: the ratio is between 6.0:1 and 10.0:1.
Content of chloride-Using Aluminum Zirconium Identification-
Octachlorohydrex Gly instead of Aluminum Zirconium A: A solution containing the equivalent of about 100 mg of
Octachlorohydrate, proceed asdirected in the test for Content anhydrous aluminum zirconium octachlorohydrate per ml
of chloride under Aluminum Zirconium Octachlorohydrate. Use responds to the test for Chloride (191).
the result obtained to calculate the (Aluminum pluszirconium) B: Identification of propylene glycol (where stated on the
/ chloride atomic ratio. label)-Add about 10 mL of isopropyl alcohol to 2 g of
(Aluminum plus zirconium)/chloride atomic ratio- Solution, mix, and filter. Evaporatethe filtrate to about 1 mL
Calculate the (aluminum plus zirconium)/chloride atomic ratio on a steam bath: the IRabsorption spectrum of a film of this
by the formula: solution on a silver chloride disk exhibits maxima only at the
same wavelengths as that of a similar preparation of a film of
[(AI/26.98) + (Zr/92.97)]/(CI/35.453) propylene glycol.
C: Identification of dipropylene glycol (where stated on the
in which AI, Zr, and CI are the percentages of aluminum, label)-Add about 10 mL of isopropyl alcohol to 2 g of
zirconium, and chloride asdetermined in the tests for Content . Solution, mix, and filter. Evaporatethe filtrate to about 1 mL
of aluminum, Content of zirconium, and Content of chloride, on a steam bath: the IRabsorption spectrum of a film of this
respectively; 26.98 is the atomic weight of aluminum; 92.97 solution on a silver chloride disk exhibits maxima only at the
is the atomic weight of zirconium corrected for 2% hafnium same wavelengths as that of a similar preparation of a film of
content; and 35.453 isthe atomic weight of chlorine: the ratio dipropylene glycol.
is between 1.5:1 and 0.9:1. D: Identification of glycine-Place about 1 9 of Solution in a
Assay-Calculate the percentage of anhydrous aluminum 50-mL beaker,add about 20 mL of water, and swirl to dissolve.
zirconium octachlorohydrate in the Aluminum Zirconium Heat to bollinq on a hot plate, and add about 60 mg of
Octachlorohydrex Gly by the formula: ninhydrin: a deep violet color immediately develops.
pH (791): between 3.0 and 5.0, in a solution prepared by
AI({26.98y+ 92.97 + 17.01 [3y+ 4 - (Y+ l)/z] + 35.453(y+ 1) diluting 3 g of the Solution with water to obtain 10 mL.
/ z}/26.98y) Arsenic, Method I (211)-Prepare the Test Preparation using
an accurately weighed quantity of the Solution. The limit is 2
in which AI is the percentage of aluminum found in the test I-Ig per g.
for Content of aluminum, y is the aluminum/zirconium atomic limit of iron-Using about 5.4 g of Aluminum Zirconium
ratio found in the test for Aluminum/zirconium atomic ratio, z Octachlorohydrex Gly Solution, accurately weighed, instead
is the (aluminum plus zirconium)/chloride atomic ratio found of Aluminum Zirconium Octachlorohydrate, proceed as
in the test for (Aluminum pluszirconium)/chloride atomic ratio, directed in the test for the Limit of iron under Aluminum
26.98 is the atomic weight of aluminum, 92.97 is the atomic Zirconium Octachlorohydrate. The limit is 75 I-Ig per g.
weight of zirconium corrected for 2% hafnium content, 17.01 Content of aluminum-Using about 0.3 9 of Aluminum
is the molecular weight of the hydroxide anion (OH), and Zirconium Octachlorohydrex Gly Solution instead of
35.453 is the atomic weight of chlorine (CI). Aluminum Zirconium Octachlorohydrate, proceed asdirected
in the test for the Content of aluminum under Aluminum
Zirconium Octachlorohydrate. Use the result to calculate the
Aluminum/zirconium atomic ratio and the (Aluminum plus
zirconium)/chloride atomic ratio.
Aluminum Zirconium Octachlorohydrex Content of zirconium-Using about 500 mg of Aluminum
Gly Solution Zirconium Octachlorohydrex Gly Solution, accurately
weighed, instead of Aluminum Zirconium Octachlorohydrate,
proceed as directed in the test for the Content of zirconium
» Aluminum Zirconium Octachlorohydrex Gly under Aluminum Zirconium Octachlorohydrate. Use the result
Solution is a solution of Aluminum Zirconium to calculate the Aluminum/zirconium atomic ratio and the
Octachlorohydrate in which some of the waters of (Aluminum pluszirconium)/chloride atomic ratio.
Aluminum/zirconium atomic ratie-Divide the
hydration have been displaced by glycine, calcium percentage of aluminum found in the test for Content of
glycinate, magnesium glycinate, potassium aluminum by the percentage of zirconium found in the test for
glycinate, sodium glycinate, or zinc glycinate. It Content of zirconium, and multiply by 92.97/26.98, in which
encompasses a range of aluminum-to-zirconium 92.97 is the atomic weight of zirconium corrected for 2%
hafnium content, and 26.98 is the atomic weight of
ratios between 6.0:1 and 10.0:1, and a range of aluminum: the ratio is between 6.0:1 and 10.0:1.
(aluminum plus zirconium)-to-chloride atomic Content of chloride-Using about 500 mg of Aluminum
ratios between 1.5:1 and 0.9:1. The following Zirconium Octachlorohydrex Gly Solution, accurately

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 USP 43 Official Monographs / Aluminum 199

weighed, instead of Aluminum Zirconium Octachlorohydrate, Content of aluminum-Using Aluminum Zirconium

proceed asdirected in the test for the Contentof chloride under Pentachlorohydrate instead of Aluminum Zirconium
Aluminum Zirconium Octachlorohydrate. Use the result to Octachlorohydrate, proceed asdirected in the test for Content
calculate the (Aluminum plus zirconium)/chloride atomic ratio. of aluminum under Aluminum Zirconium Octachlorohydrate.
(Aluminum plus zirconium)/chloride atomic ratio- Usethe result obtained to calculate the Aluminum/zirconium
Calculate the (aluminum plus zirconium)/chloride atomic ratio atomicratio and the (Aluminum pluszirconium)/chloride atomic
by the formula: ratio.
Content of zirconium-Using Aluminum Zirconium
[(AI/26.98) + (Zr/92.97)]/(C1/35.453) Pentachlorohydrate instead of Aluminum Zirconium
Octachlorohydrate, proceed asdirected in the test for Content
in which AI, Zr, and CI are the percentages of aluminum, of zirconiumunder Aluminum Zirconium Octachlorohydrate. Use
zirconium, and chloride found in the tests for Content of the result obtained to calculate the Aluminum/zirconium
aluminum, Content of zirconium, and Content of chloride, atomicratio and the (Aluminum pluszirconium)/chloride atomic
respectively; 26.98 is the atomic weight of aluminum; 92.97 ratio.
is the atomic weight of zirconium corrected for 2% hafnium Aluminum/zirconium atomic ratio-Divide the
content; and 35.453 isthe atomic weight of chlorine: the ratio percentage of aluminum found in the test for Content of
is between 1.5:1 and 0.9:1. aluminum by the percentage of zirconium found in the test for
Assay-Calculate the percentage of anhydrous aluminum Content of zirconium, and multiply by 92.97/26.98, in which
zirconium octachlorohydrate in the Solution by the formula: 92.97 is the atomic weight of zirconium corrected for 2%
hafnium content, and 26.98 is the atomic weight of
A/({26.98y+ 92.97 + 17.01 [3y+ 4 - (y+ 1)/z] + 35.453(y+ 1) aluminum: the ratio is between 6.0:1 and 10.0:1.
/ z}/26.98y) Content of chloride-Using Aluminum Zirconium
Pentachlorohydrate instead of Aluminum Zirconium
in which AI is the percentage of aluminum found in the test Octachlorohydrate, proceed asdirected in the test for Content
for Content of aluminum, y is the aluminum/zirconium atomic of chloride under AluminumZirconium Octachlorohydrate. Use
ratio found in the test for Aluminum/zirconium atomic ratio, z the result obtained to calculate the (Aluminum plus zirconium)
is the (aluminum plus zirconium)/chloride atomic ratio found /chloride atomic ratio.
in the test for (Aluminum plus zirconium)/chloride atomic ratio, (Aluminum plus zirconium)/chloride atomic ratio-
26.98 is the atomic weight of aluminum, 92.97 is the atomic Calculate the (aluminum plus zirconium)/chloride atomic ratio
weight of zirconium corrected for 2% hafnium content, 17.01 by the formula:
is the molecular weight of the hydroxide anion (OH), and
35.453 is the atomic weight of chlorine (CI). [(AI/26.98) + (Zr/92.97)]/(C1/35.453)
in which AI, Zr, and CI are the percentages of aluminum,
zirconium, and chloride, asdetermined in the testsfor Content
of aluminum, Content of zirconium, and Content of chloride,
Aluminum Zirconium respectively; 26.98 is the atomic weight of aluminum; 92.97
Pentachlorohydrate is the atomic weight of zirconium corrected for 2% hafnium
content; and 35.453 isthe atomic weight of chlorine: the ratio
is between 2.1:1 and 1.51: 1.
AI y Zr(OH) 3y+4.x CI x • nH 2 0 Assay-Calculate the percentage of anhydrous aluminum
zirconium pentachlorohydrate in the Aluminum Zirconium
» Aluminum Zirconium Pentachlorohydrate is a Pentachlorohydrate by the formula:
polymeric, loosely hydrated complex of basic
AI({26.98y+ 92.97 + 17.01 [3y+ 4 - (Y+ 1)/z] + 35.453(y+ 1)
aluminum zirconium chloride that encompasses a / z}/26.98y)
range of aluminum-to-zirconium atomic ratios
between 6.0:1 and 10.0:1, and a range of in which AI is the percentage of aluminum found in the test
(aluminum plus zirconium)-to-chloride atomic for Content of aluminum, y is the aluminum/zirconium atomic
ratio found in the test for Aluminum/zirconium atomic ratio, z
ratios between 2.1 :1 and 1.51 :1. It contains not is the (aluminum plus zirconium)/chloride atomic ratio found
less than 90.0 percent and not more than 110.0 in thee test for (Aluminum plus zirconium)/chloride atomic ratio,
percent of the labeled amount of anhydrous 26.98 is the atomic weight of aluminum, 92.97 is the atomic
aluminum zirconium pentachlorohydrate. weight of zirconium corrected for 2% hafnium content, 17.01
is the molecular weight of the hydroxide anion (OH), and
Packaging and storage-Preserve in well-closed 35.453 is the atomic weight of chlorine (CI).
containers.
Labeling-The label states the content of anhydrous
aluminum zirconium pentachlorohydrate.
Identification-A solution (1 in 10) responds to the test for
Chloride (191). Aluminum Zirconium
pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/ Pentachlorohydrate Solution
w)].
Arsenic, Method I (211): 2 I-Ig per g.
Limit of iron-Using Aluminum Zirconium » Aluminum Zirconium Pentachlorohydrate
Pentachlorohydrate instead of Aluminum Zirconium Solution is a polymeric, loosely hydrated complex
Octachlorohydrate, proceed as directed in the test for Limit of basic aluminum zirconium chloride that
of iron under Aluminum Zirconium Octachlorohydrate. The
specified result is obtained (150 I-Ig per g limit). ' encompasses a range of aluminum-to-zirconium
atomic ratios between 6.0:1 and 10.0:1 and a

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200 Aluminum / OfficialMonographs USP 43

range of (aluminum plus zirconium)-to-chloride Aluminum Zirconium Octachlorohydrate. Usethe result to

atomic ratios between 2.1:1 and 1.51:1. The calculate the (Aluminum pluszirconium)/chloride atomic ratio.
(Aluminum plus zirconium)/chloride atomic ratio-
following solvents may be used: water, propylene Calculate the (aluminum plus zirconium)/chloride atomic ratio
glycol, or dipropylene glycol. It contains the by the formula:
equivalent of not less than 90.0 percent and not
more than 110.0 percent of the labeled [(AI/26.98) + (Zr/92.97)]/(C1135.453)
concentration of anhydrous aluminum zirconium in which AI, Zr, and CI are the percentages of aluminum,
pentachlorohydrate. zirconium, and chloride found in the test for Content of
aluminum, Content of zirconium, and Content of chloride,
Packaging and storage-Preserve in well-closed respectively; 26.98 is the atomic weight of aluminum; 92.97
containers. is the atomic weight of zirconium corrected for 2% hafnium
labeling-Label Solution to state the solvent used and the content; and 35.453 isthe atomic weight of chlorine: the ratio
claimed concentration of anhydrous aluminum zirconium is between 2.1:1 and 1.51:1.
pentachlorohydrate. Assay-Calculate the percentage of anhydrous aluminum
Identification- zirconium pentachlorohydrate in the Solution by the formula:
A: A solution containing the equivalent of about 100 mg of AI{[26.98y+ 92.97 + (17.01)(3y+4 - (Y+ l)/z) + 35.453(y
anhydrous aluminum zirconium pentachlorohydrate per mL + 1)/ z]/26.98y}
responds to the test for Chloride (191).
B: Identification of propylene glycol (where stated on the in which AI is the percentage of aluminum found in the test
label)-Add about 10 mL of isopropyl alcohol to 2 g of for Content of aluminum, y is the aluminum/zirconium atomic
Solution, mix, and filter. Evaporatethe filtrate to about 1 mL ratio found in the test for Aluminum/zirconium atomic ratio, z
on a steam bath: the IRspectrum of a film of this solution on is the (aluminum plus zirconium)/chloride atomic ratio found
a silver chloride disk exhibits maxima only at the same in the test for (Aluminum pluszirconium)/chloride atomic ratio,
wavelengths as that of a similar preparation of a film of 26.98 is the atomic weight of aluminum, 92.97 is the atomic
propylene glycol. weight of zirconium corrected for 2% hafnium content, 17.01
C: Identification of dipropylene glycol (where stated on the is the molecular weight of the hydroxide anion (OH), and
label)-Add about 10 mL of isopropyl alcohol to 2 g of 35.453 is the atomic weight of chlorine (CI).
Solution, mix, and filter. Evaporatethe filtrate to about 1 mL
on a steam bath: the IRspectrum of a film of this solution on
a silver chloride disk exhibits maxima only at the same
wavelengths as that of a similar preparation of a film of
dipropylene glycol. Aluminum Zirconium
pH (791): between 3.0 and 5.0, in a solution prepared by
diluting 3 g of the Solution with water to obtain 10 mL. Pentachlorohydrex Gly
Arsenic, Method I (211)-Prepare the Test Preparation using
an accurately weighed quantity of the Solution. The limit is 2
» Aluminum Zirconium Pentachlorohydrex Gly is a
J.Jg per g.
Limit of iron-Using about 5.4 g of Aluminum Zirconium derivative of Aluminum Zirconium
Pentachlorohydrate Solution, accurately weighed, instead of Pentachlorohydrate in which some of the water
Aluminum Zirconium Octachlorohydrate, proceed asdirected molecules have been displaced by glycine, calcium
in the test for the Limit of iron under Aluminum Zirconium glycinate, magnesium glycinate, potassium
Octachlorohydrate. The limit is 75 J.Jg per g.
Content of aluminum-Using about 0.3 g of Aluminum glycinate, sodium glycinate, or zinc glycinate. It
Zirconium Pentachlorohydrate Solution instead of Aluminum encompasses a range of aluminum-to-zirconium
Zirconium Octachlorohydrate, proceed as directed in the test atomic ratios between 6.0:1 and 10.0:1 and a
for the Content of aluminum under Aluminum Zirconium range of (aluminum plus zirconium)-to-chloride
Octachlorohydrate. Usethe result to calculate the Aluminum/ atomic ratios between 2.1 :1 and 1.51 :1. It contains
zirconium atomic ratio and the (Aluminum pluszirconium)/
chloride atomic ratio. not less than 90.0 percent and not more than
Content of zirconium-Using about 500 mg of Aluminum 110.0 percent of the labeled amount of anhydrous
Zirconium Pentachlorohydrate Solution, accurately weighed, aluminum zirconium pentachlorohydrate.
instead of Aluminum Zirconium Octachlorohydrate, proceed
as directed in the test for the Content of zirconium under Packaging and storage-Preserve in well-closed
AluminumZirconium Octachlorohydrate. Usethe result to containers.
calculate the Aluminum/zirconium atomicratio and the labeling-The label statesthe form of glycine used and the
(Aluminum plus zirconium)/chloride atomicratio. claimed content of anhydrous aluminum zirconium
Aluminum/zirconium atomic ratio-Divide the pentachlorohydrate.
percentage of aluminum found in the test for Content of
aluminum by the percentage of zirconium found in the test for Identification-
Content of zirconium, and multiply by 92.97/26.98, in which A: A solution (1 in 10) responds to the test for Chloride
92.97 is the atomic weight of zirconium corrected for 2% (191).
hafnium content, and 26.98 is the atomic weight of B: Place about 0.5 g of it in a 50-mL beaker, add about 20
aluminum: the ratio is between 6.0:1 and 10.0:1. mL of water, and swirl to dissolve. Heat to boiling on a hot
Content of chloride-Using about 500 mg of Aluminum plate, and add about 60 mg of ninhydrin: a deep violet color
Zirconium Pentachlorohydrate Solution, accurately weighed, immediately develops.
instead of Aluminum Zirconium 'Octachlorohydrate, proceed pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/
as directed in the test for the Content of chloride under w)].

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 USP 43 OfficialMonographs / Aluminum 201

Arsenic, Method I (211): 2 I-Ig per g.

Limit of iron-Using Aluminum Zirconium Aluminum Zirconium
Pentachlorohydrex Gly instead of Aluminum Zirconium Pentachlorohydrex Cly Solution
Octachlorohydrate, proceed as directed in the test for Limit
of iron under AluminumZirconium Octachlorohydrate. The
specified result is obtained (150 I-Ig per g limit). » Aluminum Zirconium Pentachlorohydrex Gly
Content of aluminum-Using Aluminum Zirconium Solution is a solution of Aluminum Zirconium
Pentachlorohydrex Gly instead of Aluminum Zirconium Pentachlorohydrate in which some of the waters of
Octachlorohydrate, proceed as directed in the test for Content
of aluminum under Aluminum Zirconium Octachlorohydrate.
hydration have been displaced by glycine, calcium
Use the result obtained to calculate the Aluminumlzirconium glycinate, magnesium glycinate, potassium
atomic ratio and the (Aluminum pluszirconium)/chloride atomic glycinate, sodium glycinate, or zinc glycinate. It
ratio. encompasses a range of aluminum-to-zirconium
Content of zirconium-Using Aluminum Zirconium ratios between 6.0:1 and 10.0:1 and a range of
Pentachlorohydrex Gly instead of Aluminum Zirconium
Octachlorohydrate, proceed asdirected in the test for Content
(aluminum plus zirconium)-to-chloride atomic
of zirconium under Aluminum Zirconium Octachlorohydrate. Use ratios between 2.1:1 and 1.51:1. The following
the result obtained to calculate the Aluminumlzirconium solvents may be used: water, propylene glycol,· or
atomic ratio and the (Aluminum pluszirconium)/chloride atomic dipropylene glycol. It contains the equivalent of
ratio. not less than 90.0 percent and not more than
Aluminum/zirconium atomic ratio-Divide the
percentage of aluminum found in the test for Contentof 110.0 percent of the labeled concentration of
aluminum by the percentage of zirconium found in the test for anhydrous aluminum zirconium
Contentof zirconium, and multiply by 92.97126.98, in which pentachlorohydrate.
92.97 is the atomic weight of zirconium corrected for 2%
hafnium content, and 26.98 is the atomic weight of Packaging and storage-Preserve in well-closed
aluminum: the ratio is between 6.0:1 and 10.0:1. containers.
Content of chloride-Using Aluminum Zirconium labeling-label Solution to state the solvent and form of
Pentachlorohydrex Gly instead of Aluminum Zirconium glycine used and the claimed concentration of anhydrous
Octachlorohydrate, proceed as directed in the test for Content aluminum zirconium pentachlorohydrate.
of chloride under AluminumZirconium Octachlorohydrate. Use
the result obtained to calculate the (Aluminum plus zirconium) Identification-
1chloride atomic ratio. • A: A solution containing the equivalent of about 100 mg of
(Aluminum plus zirconium)/chloride atomic ratio- anhydrous aluminum zirconium pentachlorohydrate per ml
Calculate the (aluminum pluszirconium)/chloride atomic ratio responds to the test for Chloride (191).
by the formula: B: Identification of propylene glycol (where stated on the
label)-Add about 10 ml of isopropyl alcohol to 2 g of
[(AI126.98) + (ZrI92.97)]/(ClI35.453) Solution, mix, and filter. Evaporatethe filtrate to about 1 ml
on a steam bath: the IR spectrum of a film of this solution on
in which AI, Zr, and CI are the percentages of aluminum, a silver chloride disk exhibits maxima only at the same
zirconium, and chloride asdetermined in the tests for Content wavelengths as that of a similar preparation of a film of
of aluminum, Contentof zirconium, and Content of chloride, propylene glycol.
respectively; 26.98 is the atomic weight of aluminum; 92.97 C: Identification of dipropylene glycol (where stated on the
is the atomic weight of zirconium corrected for 2% hafnium label)-Add about 10 ml of isopropyl alcohol to 2 gof
content; and 35.453 is the atomic weight of chlorine: the ratio Solution, mix, and filter. Evaporate the filtrate to about 1 ml
is between 2.1:1 and 1.51:1. on a steam bath: the IR spectrum of a film of this solution on
Assay-Calculate the percentage of anhydrous aluminum a silver chloride disk exhibits maxima only at the same
zirconium pentachlorohydrate in the Aluminum Zirconium wavelengths as that of a similar preparation of a film of
Pentachlorohydrex Gly by the formula: dipropylene glycol.
D: Identification of glycine-Place about 1 g of Solution in a
AI({26.98y+ 92.97 + 17.01 [3y+ 4- (y+ l)lz] + 35.453(y+ 1) 50-mL beaker,add about 20 mL of water, and swirl to dissolve.
1z}/26.98y) Heat to boiling on a hot plate, and add about 60 mg of
ninhydrin: a deep violet color immediately develops.
in which AI is the percentage of aluminum found in the test pH (791): between 3.0 and 5.0, in a solution prepared by
for Contentof aluminum, y is the aluminum/zirconium atomic diluting 3 g of the Solution with water to obtain 10 ml.
ratio found in the test for Aluminum/zirconium atomic ratio, Z Arsenic, Method I (211)-Prepare the Test Preparation using
is the (aluminum plus zirconium)/chloride atomic ratio found an accurately weighed quantity of the Solution. The limit is 2
in the test for (Aluminumpluszirconium)1chloride atomic ratio, I-Ig per g.
26.98 is the atomic weight of aluminum, 92.97 is the atomic Limit of iron-Using about 5.4 g of Aluminum Zirconium.
weight of zirconium corrected for 2% hafnium content, 17.01 Pentachlorohydrex Gly Solution, accurately weighed, instead
is the molecular weight of the hydroxide anion (OH), and of Aluminum Zirconium Octachlorohydrate, proceed as
35.453 is the atomic weight of chlorine (CI). directed in the test for the Limit of iron under Aluminum
Zirconium Octachlorohydrate. The limit is 75 I-Ig per g.
Content of aluminum-Using about 0.3 g of Aluminum
Zirconium Pentachlorohydrex Gly Solution instead of
Aluminum Zirconium Octachlorohydrate, proceed asdirected
in the test for the Contentof aluminum under Aluminum
Zirconium Octachlorohydrate. Use the result to calculate the
Aluminumlzirconium atomic ratio and the (Aluminum plus
zirconium)/chloride atomic ratio.

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202 Aluminum / OfficialMonographs USP 43

Content of zirconium-Using about 500 mg of Aluminum Packaging and storage-Preserve in well-closed

Zirconium Pentachlorohydrex Gly Solution, accurately containers.
weighed, instead of Aluminum Zirconium Octachlorohydrate, Labeling-The label states the content of anhydrous
proceed as directed in the test for the Content of zirconium aluminum zirconium tetrachlorohydrate.
under Aluminum Zirconium Octachlorohydrate. Usethe result Identification-A solution (1 in 10) responds to the test for
to calculate the Aluminum/zirconium atomicratio and the Chloride (191).
(Aluminumpluszirconium)/chloride atomic ratio. pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/
Aluminum/zirconium atomic ratio-Divide the w)].
percentage of aluminum found in the test for Content of Arsenic,Method I (211): 2 JJg per g.
aluminum by the percentage of zirconium found in the test for Limit of iron-Using Aluminum Zirconium
Contentof zirconium, and multiply by 92.97/26.98, in which Tetrachlorohydrate instead of Aluminum Zirconium
92.97 is the atomic weight of zirconium corrected for 2% Octachlorohydrate, proceed as directed in the test for Limit
hafnium content, and 26.98 is the atomic weight of of iron under Aluminum Zirconium Octachlorohydrate The
aluminum: the ratio is between 6.0:1 and 10.0:1. specified result is obtained (150 JJg per g limit).
Content of chloride-Using about 500 mg of Aluminum Content of aluminum-Using Aluminum Zirconium
Zirconium Pentachlorohydrex Gly Solution, accurately Tetrachlorohydrate instead of Aluminum Zirconium
weighed, instead of Aluminum Zirconium Octachlorohydrate, Octachlorohydrate, proceed asdirected in the test for Content
proceed asdirected in the test for the Content of chloride under of aluminum under Aluminum Zirconium Octachlorohydrate.
Aluminum Zirconium Octachlorohydrate. Use the result to Use the result obtained to calculate the Aluminum/zirconium
calculate the (Aluminum plus zirconium)/chloride atomicratio. atomic ratio and the (Aluminum pluszirconium)/chloride atomic
(Aluminum plus zirconium)/chloride atomic ratio- ratio.
Calculate the (aluminum plus zirconium)/chloride atomic ratio Content of zirconium-Using Aluminum Zirconium
by the formula: Tetrachlorohydrate instead of Aluminum Zirconium
Octachlorohydrate, proceed asdirected in the test for Content
[(AI/26.98) + (Zr/92.97)]/(C1/35.453) of zirconium under Aluminum Zirconium Octachlorohydrate Use
the result obtained to calculate the Aluminum/zirconium
in which AI, Zr, and CI are the percentages of aluminum, atomic ratio and the (Aluminum pluszirconium)/chloride atomic
zirconium, and chloride found in the test for Content of . ratio.
aluminum, Contentof zirconium, and Content of chloride, Aluminum/zirconium atomic ratio-Divide the
respectively; 26.98 is the atomic weight of aluminum; 92.97 percentage of aluminum found in the test for Contentof
is the atomic weight of zirconium corrected for 2% hafnium aluminum by the percentage of zirconium found in the test for
content; and 35.453 isthe atomic weight of chlorine: the ratio Contentof zirconium, and multiply by 92.97/26.98, in which
is between 2.1:1 and 1.51:1. 92.97 is the atomic weight of zirconium corrected for 2%
Assay-Calculafe the percentage of anhydrous aluminum hafnium content, and 26.98 is the atomic weight of
zirconium pentachlorohydrate in the Solution by the formula: aluminum: the ratio is between 2.0:1 and 5.99:1.
Content of chloride-Using Aluminum Zirconium
AI{[26.98y+ 92.97 + (17.01)(3y+ 4 - (Y+ l)/z) + 35.453(y Tetrachlorohydrate instead of Aluminum Zirconium
+ 1)/ z]/26.98 y} Octachlorohydrate, proceed asdirected in the test for Content
of chloride under Aluminum Zirconium Octachlorohydrate Use
in which AI is the percentage of aluminum found in the test the result obtained to calculate the (Aluminum plus zirconium)
for Contentof aluminum, y is the aluminum/zirconium atomic / chloride atomic ratio.
ratio found in the test for Aluminum/zirconium atomicratio, Z (Aluminum plus zirconium)/chloride atomic ratio-
is the (aluminum plus zirconium)/chloride atomic ratio found Calculate the (aluminum plus zirconium)/chloride atomic ratio
in the test for (Aluminum plus zirconium)/chloride atomicratio, by the formula:
26.98 is the atomic weight of aluminum, 92.97 is the atomic
weight of zirconium corrected for 2% hafnium content, 17.01 [(AI/26.98) + (Zr/92.97)]/(C1/35.453)
is the molecular weight of the hydroxide anion (OH), and
35.453 is the atomic weight of chlorine (CI). in which AI, Zr, and CI are the percentages of aluminum,
zirconium, and chloride asdetermined inthe tests for Content
of aluminum, Contentof zirconium, and Contentof chloride,
respectively; 26.98 is the atomic weight of aluminum; 92.97
is the atomic weight of zirconium corrected for 2% hafnium
Aluminum Zirconium content; and 35.453 isthe atomic weight of chlorine: the ratio
Tetrachlorohydrate is between 1.5:1 and 0.9:1.
Assay-Calculate the percentage of anhydrous aluminum
zirconium tetrachlorohydrate in the Aluminum Zirconium
AI y Zr(OH) 3y+4-x CI x • nH 2 0 Tetrachlorohydrate by the formula:

» Aluminum Zirconium Tetrachlorohydrate is a AI({26.98y+ 92.97 + 17.01 [3y+ 4 - (Y+ 1)/z] + 35.453(y+ 1)
polymeric, loosely hydrated complex of basic /z}/26.98 y)
aluminum zirconium chloride that encompasses a in which AI is the percentage of aluminum found in the test
range of aluminum-to-zirconium atomic ratios for Contentof aluminum, y is the aluminum/zirconium atomic
between 2.0:1 and 5.99:1 and a range of ratio found in the test for Aluminum/zirconium atomic ratio, Z
(aluminum plus zirconium)-to-chloride atomic is the (aluminum plus zirconium)/chloride atomic ratio found
in the test for (Aluminum pluszirconium)/chloride atomic ratio,
ratios between 1.5:1 and 0.9:1. It contains not less 26.98 is the atomic weight of aluminum, 92.97 is the atomic
than 90.0 percent and not more than 110.0 weight of zirconium corrected for 2% hafnium content, 17.01
percent of the labeled amount of anhydrous is the molecular weight of the hydroxide anion (OH), and
aluminum zirconium tetrachlorohydrate. 35.453 is the atomic weight of chlorine (CI).

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 USP 43 OfficialMonographs / Aluminum 203

Aluminum/zirconium atomic ratio-Divide the

Aluminum Zirconium percentage of aluminum found in the test for Content of
Tetrachlorohydrate Solution aluminum by the percentage of zirconium found in the test for
Content of zirconium and multiply by 92.97/26.98, in which
92.97 is the atomic weight of zirconium corrected for 2%
» Aluminum Zirconium Tetrachlorohydrate hafnium content, and 26.98 is the atomic weight of
Solution is a polymeric, loosely hydrated complex aluminum: the ratio is between 2.0:1 and 5.99:1.
of basic aluminum zirconium chloride that Content of chloride-Using about 500 mg of Aluminum
Zirconium Tetrachlorohydrate Solution, accurately weighed,
encompasses a range of aluminum-to-zirconium instead of Aluminum Zirconium Octachlorohydrate, proceed
atomic ratios between 2.0:1 and 5.99:1 and a as directed in the test for the Contentof chloride under
range of (aluminum plus zirconium)-to-chloride Aluminum Zirconium Octachlorohydrate. Usethe result to
atomic ratios between 1.5:1 and 0.9:1. The calculate the (Aluminum plus zirconium)/chloride atomic ratio.
following solvents may be used: water, propylene (Aluminum plus zirconium)/chloride atomic ratio-
Calculatethe (aluminum plus zirconium)/chloride atomic ratio
glycol, or dipropylene glycol. It contains the by the formula:
equivalent of not less than 90.0 percent and not
more than 110.0 percent of the labeled [(AI/26.98) + (Zr/92.97)]/(CI/35.453)
concentration of anhydrous aluminum zirconium
in which AI, Zr, and CI are the percentages of aluminum,
tetrachlorohydrate. zirconium, and chloride found in the test for Content of
aluminum, Contentof zirconium, and Content of chloride,
Packaging and storage-Preserve in well-closed respectively; 26.98 is the atomic weight of aluminum; 92.97
containers. is the atomic weight of zirconium corrected for 2% hafnium
Labeling-Label Solution to state the solvent used and the content; and 35.453 is the atomic weight of chlorine: the ratio
claimed concentration of anhydrous aluminum zirconium is between 1.5:1 and 0.9:1.
tetrachlorohydrate. Assay-Calculate the percentage of anhydrous aluminum
Identification- zirconium tetrachlorohydrate in the Solution by the formula:
A: A solution containing the equivalent of about 100 mg of
anhydrous aluminum zirconium tetrachlorohydrate per mL A/({26.98y+ 92.97 + 17.01 [3y+4 - (Y+ l)/z] + 35.453(y+ 1)
responds to the test for Chloride (191). /z}/26.98y)
8: Identification of propylene glycol (where stated on the
label)-Add about 10 mL of isopropyl alcohol to 2 g of in which AI is the percentage of aluminum found in the test
Solution, mix, and filter. Evaporate the filtrate to about 1 mL for Content of aluminum, y is the aluminum/zirconium atomic
on a steam bath: the IR spectrum of a film of this solution on ratio found in the test for Aluminum/zirconium atomic ratio, Z
a silver chloride disk exhibits maxima only at the same is the (aluminum plus zirconium)/chloride atomic ratio found
wavelengths as that of a similar preparation of a film of in the test for (Aluminum plus zirconium)/chloride atomic ratio,
propylene glycol. . 26.98 is the atomic weight of aluminum, 92.97 is the atomic
C: Identification of dipropylene glycol (where stated on the weight of zirconium corrected for 2% hafnium content, 17.01
label)-Add about 10 mL of isopropyl alcohol to 2 g of is the molecular weight of the hydroxide anion (OH), and
Solution, mix, and filter. Evaporatethe filtrate to about 1 mL 35.453 is the atomic weight of chlorine (CI).
on a steam bath: the IR spectrum of a film of this solution on
a silver chloride disk exhibits maxima only at the same
wavelengths asthat of a similar preparation of a film of
dipropylene glycol.
pH (791): between 3.0 and 5.0, in a solution prepared by
Aluminum Zirconium
diluting 3 g of the Solution with water to obtain 10 mL. Tetrachlorohydrex Gly
Arsenic, Method I (211)-Prepare the Test Preparation using
an accurately weighed quantity of the Solution. The limit is 2
I-Ig per g. » Aluminum Zirconium Tetrachlorohydrex Gly is a
Limit of iron-Using about 5.4 g of Aluminum Zirconium derivative of Aluminum Zirconium
Tetrachlorohydrate Solution, accurately weighed, instead of Tetrachlorohydrate in which some of the water
Aluminum Zirconium Octachlorohydrate, proceed as directed molecules have been displaced by glycine, calcium
in the test for the Limit of iron under AluminumZirconium
Octachlorohydrate. The limit is 75 I-Ig per g. glycinate, magnesium glycinate, potassium
Content of aluminum-Using about 0.3 g of Aluminum glycinate, sodium glycinate, or zinc 'qlycinate. It
Zirconium Tetrachlorohydrate Solution instead of Aluminum encompasses a range of aluminum-to-zirconium
Zirconium Octachlorohydrate, proceed as directed in the test .atomic ratios between 2.0:1 and 5.99:1 and a
for the Contentof aluminum under .Alummum Zirconium range of (aluminum plus zirconium)-to-chloride
Octachlorohydrate. Use the result to calculate the Aluminum/
zirconium atomic ratio and the (Aluminum plus zirconium)/ atomic ratios between 1.5:1 and 0.9:1. It contains
chloride atomic ratio. not less than 90.0 percent and not more than
Content of zirconium-Using about 500 mg of Aluminum 110.0 percent of the labeled amount of anhydrous
Zirconium Tetrachlorohydrate Solution, accurately weighed, aluminum zirconium tetrachlorohydrate.
instead of Aluminum Zirconium Octachlorohydrate, proceed
as directed in the test for the Contentof zirconium under Packaging and storage-Preserve in well-closed
AluminumZirconium Octachlorohydrate. Usethe result to containers.
calculate the Aluminum/zirconium atomic ratio and the
(Aluminumplus zirconium)/chloride atomic ratio.

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204 Aluminum / OfficialMonographs USP 43

Labeling-The label states the form of glycine used and the in the test for (Aluminum pluszirconium)/chloride atomicratio,
claimed content of anhydrous aluminum zirconium 26.98 is the atomic weight of aluminum, 92.97 is the atomic
tetrachlorohydrate. weight of zirconium corrected for 2% hafnium content, 17.01
is the molecular weight of the hydroxide anion (OH), and
Identification- 35.453 is the atomic weight of chlorine (CI).
A: A solution (1 in 10) responds to the test for Chloride
(191 ).
B: Place about 0.5 g of it in a 50-mL beaker, add about 20
mL of water, and swirl to dissolve. Heat to boiling on a hot
plate, and add about 60 mg of ninhydrin: a deep violet color Aluminum Zirconium
immediately develops.
pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/ Tetrachlorohydrex Gly Solution
w)].
Anen;c, Method I (211): 2 I-Ig per g. » Aluminum Zirconium Tetrachlorohydrex Gly
Limit of iron-Using Aluminum Zirconium
Tetrachlorohydrex Gly instead of Aluminum Zirconium Solution is a solution of Aluminum Zirconium
Octachlorohydrate, proceed as directed in the test for Limit Tetrachlorohydrate in which some of the waters of
of iron under AluminumZirconium Octachlorohydrate. The hydration have been displaced by glycine, calcium
specified result is obtained (150 I-Ig per g limit). glycinate, magnesium glycinate, potassium
Content of aluminum-Using Aluminum Zirconium
Tetrachlorohydrex Gly instead of Aluminum Zirconium glycinate, sodium glycinate, or zinc glycinate. It
Octachlorohydrate, proceed as directed in the test for Content encompasses a range of aluminum-to-zirconium
of aluminum under Aluminum Zirconium Octachlorohydrate. ratios between 2.0:1 and 5.99:1 and a range of
Use the result obtained to calculate the Aluminum/zirconium (aluminum plus zirconium)-to-chloride atomic
atomicratio and the (Aluminum pluszirconium)/chloride atomic ratios between 1.5:1 and 0.9:1. The following
ratio.
Content of zirconium-Using Aluminum Zirconium solvents may be used: water, propylene glycol, or
Tetrachlorohydrex Gly instead of Aluminum Zirconium dipropylene glycol. It contains the equivalent of
Octachlorohydrate, proceed as directed in the test for Content not less than 90.0 percent and not more than
ofzirconium under AluminumZirconium Octachlorohydrate. Use 110.0 percent of the labeled concentration of
the result obtained to calculate the Aluminum/zirconium anhydrous aluminum zirconium
atomicratio and the (Aluminumpluszirconium)/chloride atomic
ratio. tetrachlorohydrate.
Aluminum/zir~onium atomic ratio-Divide the
percentage of aluminum found in the test for Content of Packaging and storage-Preserve in well-closed
aluminum by the percentage of zirconium found in the test for containers.
Content of zirconium, and multiply by 92.97/26.98, in which Labeling-Label Solution to state the solvent and form of
92.97 is the atomic weight of zirconium corrected for 2% glycine used and the claimed concentration of anhydrous
hafnium content, and 26.98 is the atomic weight of aluminum zirconium tetrachlorohydrate.
aluminum: the ratio is between 2.0:1 and 5.99:1. Identification-
Content of chloride-Using Aluminum Zirconium A: Asolution containing the equivalent of about 100 mg of
Tetrachlorohydrex Gly instead of Aluminum Zirconium anhydrous aluminum zirconium tetrachlorohydrate per mL
Octachlorohydrate, proceed as directed in the test for Content responds to the test for Chloride (191 ).
of chloride under AluminumZirconium Octachlorohydrate. Use B: Identification of propylene glycol (where stated on the
the result obtained to calculate the (Aluminum plus.zirconium) label)-Add about 10 mL of isopropyl alcohol to 2 g of
/chloride atomic ratio. Solution, mix, and filter. Evaporate the filtrate to about 1 mL
(Aluminum plus zirconium)/chloride atomic ratio- on a steam bath: the IR spectrum of a film of this solution on
Calculate the (aluminum plus zirconium)/chloride atomic ratio a silver chloride disk exhibits maxima only at the same
by the formula: wavelengths as that of a similar preparation of a film of
propylene glycol.
[(AI/26.98) + (Zr/92.97)]/(CI/35.453) C: Identification of dipropylene glycol (where stated on the
label)-Add about 10 mL of isopropyl alcohol to 2 g of
in which AI, Zr, and CI are the percentages of aluminum, Solution, mix, and filter. Evaporate the filtrate to about 1 mL
zirconium, and chloride as determined in the tests for Content on a steam bath: the IR spectrum of a film of this solution on
of aluminum, Content of zirconium, and Content of chloride, a silver chloride disk exhibits maxima only at the same
respectively; 26.98 is the atomic weight of aluminum; 92.97 wavelengths as that of a similar preparation of a film of
is the atomic weight of zirconium corrected for 2% hafnium dipropylene glycol.
content; and 35.453 is the atomic weight of chlorine: the ratio D: Identification of glycine-Place about 1 g of Solution in a
is between 1.5:1 and 0.9:1. 50-mL beaker, add about 20 mLof water, and swirl to dissolve.
Assay-Calculate the percentage of anhydrous aluminum Heat to boiling on a hot plate, and add about 60 mg of
zirconium tetrachlorohydrate in the Aluminum Zirconium ninhydrin: a deep violet color immediately develops.
Tetrachlorohydrex Gly by the formula: pH (791): between 3.0 and 5.0, in a solution prepared by
AI({26.98y+ 92.97 + 17.01 [3y+4 - (Y+ l)/z] + 35.453(y+ 1) diluting 3 g of the Solution with water to obtain 10 mL.
Arsenic, Method I (211 )-Prepare the Test Preparation using
/ z}/26.98y)
an accurately weighed quantity of the Solution. The limit is 2
in which AI is the percentage of aluminum found in the test I-Ig per g.
for Contentof aluminum, y is the aluminum/zirconium atomic Limit of iron.......Using about 5.4 g of Aluminum Zirconium
ratio found in the test for Aluminum/zirconium atomicratio, Z Tetrachlorohydrex Gly Solution, accurately weighed, instead
is the (aluminum plus zirconium)/chloride atomic ratio found of Aluminum Zirconium Octachlorohydrate, proceed as

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 USP 43 Official Monographs / Aluminum 205

directed in the test for the Limit of iron under Aluminum range of aluminum-to-zirconium atomic ratios
Zirconium Octachlorohydrate. The limit is 75 IJg per g. between 2.0:1 and 5.99:1 and a range of
Content of aluminum-Using about 0.3 g of Aluminum
Zirconium Tetrachlorohydrex Gly Solution instead of . (aluminum plus zirconium)-to-chloride atomic
Aluminum Zirconium Octachlorohydrate, proceed as directed ratios between 2.1:1 and 1.51 :1. It contains not
in the test for the Contentof aluminum under Aluminum less than 90.0 percent and not more than 110.0
Zirconium Octachlorohydrate. Use the result to calculate the percent of the labeled amount of anhydrous
Aluminum/zirconium atomic ratio and the (Aluminum plus aluminum zirconium trichlorohydrate.
zirconium)/chloride atomic ratio.
Content of zirconium-Using about 500 mg of Aluminum . Packaging and storage-Preserve in well-closed
Zirconium Tetrachlorohydrex Gly Solution, accurately containers.
weighed, instead of Aluminum Zirconium Octachl~rohXdrate, Labeling-The label states the content of anhydrous
proceed as directed in the test for the Contentof zirconium aluminum zirconium trichlorohydrate.
under Aluminum Zirconium Octachlorohydrate. Usethe result Identification-A solution (1 in 10) responds to the test for
to calculate the Aluminum/zirconium atomic ratio and the Chloride (191).
(Aluminumplus zirconium)/chloride atomic ratio. pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/
Aluminum/zirconium atomic ratio-Divide the w)].
percentage of aluminum found in the test for Contentof Arsenic, Method I (211): 2 IJg per g.
aluminum by the percentage of zirconium found in t~e tes~ for Limit of iron-Using Aluminum Zirconium Trichlorohydrate
Content of zirconium, and multiply by 92.97/26.98, In which instead of Aluminum Zirconium Octachlorohydrate, proceed
92.97 is the atomic weight of zirconium corrected for 2% as directed in the test for Limit of iron under Aluminum
hafnium content, and 26.98 is the atomic weight of Zirconium Octachlorohydrate. The specified result is obtained
aluminum: the ratio is between 2.0:1 and 5.99:1.
(150 IJg per g limit). . . . .
Content of chloride-Using about 500 mg of Aluminum Content of aluminum-Using Aluminum Zirconium
Zirconium Tetrachlorohydrex Gly Solution, accurately Trichlorohydrate instead of Aluminum Zirconium
weighed, instead of Aluminum Zirconium OctachlorC?hydrate, Octachlorohydrate, proceed asdirected in the test for Content
proceed asdirected in the test for the Contentof chlorideunder of aluminum under AluminumZirconium Octachlorohydrate.
Aluminum Zirconium Octachlorohydrate. Use the result to Use the result obtained to calculate the Aluminum/zirconium
calculate the (Aluminum plus zirconium)/chloride atomic ratio. atomic ratio and the (Aluminumpluszirconium)/chlorideatomic
(Aluminum plus zirconium)/chloride atomic ra~io-: ratio.
Calculatethe (aluminum plus zirconium)/chloride atomic ratio Content of zirconium-Using Aluminum Zirconium
by the formula: Trichlorohydrate instead of Aluminum Zirconium
Octachlorohydrate, proceed asdirected in the test for Content
[(AI/26.9'8) + (Zr/92.97)]/(CI/35.453) of zirconiumunder AluminumZirconiumOctachlorohydrate. Use
in which AI, Zr, and CI are the percentages of aluminum, the result obtained to calculate the Aluminum/zirconium
zirconium, and chloride found in the tests for Conten.t of atomic ratio and the (Aluminumplus zirconium)/chlorideatomic
aluminum Content of zirconium, and Contentof chloride, ratio.
respectively; 26.98 is the atomic weight of aluminum; 9~.97 Aluminum/zirconium atomic ratio-Divide the
is the atomic weight of zirconium corrected for 2% hafnium percentage of aluminum found in the test for Content of
content; and 35.453 is the atomic weight of chlorine: the ratio aluminum by the percentage of zirconium found in t~e tes~ for
is between 1.5:1 and 0.9:1. . Contentof zirconium, and multiply by 92.97/26.98, In which
Assay-Calculate the percent~ge of anhxdrous aluminum . 92.97 is the atomic weight of zirconium corrected for 2%
zirconium tetrachlorohydrate In the Solution by the formula: hafnium content, and 26.98 is the atomic weight of
aluminum: the ratio is between 2.0:1 and 5.99:1.
AI({26.98y+ 92.97 + 17.01[3y+ 4 - (y+ l)/z] + 35.453(y+ 1) Content of chloride-Using Aluminum Zirconium
/ z}/26.98y) Trichlorohydrate instead of Aluminum Zirconium
Octachlorohydrate, proceed asdirected in the test for Content
in which AI is the percentage of aluminum found i.n the test. of chloride under AluminumZirconium Octachlorohydrate. Use
for Content of aluminum, y is the aluminum/zirconium atomic the result obtained to calculate the (Aluminumplus zirconium)
ratio found in the test for Aluminum/zirconium atomic ratio, z / chloride atomic ratio.
is the (aluminum plus zirconium)/c~loride at0!'1lic rati~ fou~d (Aluminum plus zirconiu~)/ch.loride at~mic ra~io-:
in the test for (Aluminumplus zircon!um)/chlonde. atomicratio, Calculatethe (aluminum pluszlrconlurnj/chlorlde atomic ratio
26.98 is the atomic weight of aluminum, 92.97 IS the atomic by the formula:
weight of zirconium corrected for 2% hafnium content, 17.01
is the molecular weight of the hydroxide anion (OH), and [(AI/26.98) + (Zr/92.97)]/(CI/35.453)
35.453 is the atomic weight of chlorine (CI).
in which AI, Zr, and CI are the percentages of aluminum,
zirconium and chloride asdetermined in the tests for Content
of alumin~m, Contentof zirconium, and Contentof chloride,
respectively; 26.98 is the atomic weight of aluminum; 9~.97
Aluminum Zirconium Trichlorohydrate content; is the atomic weight of zirconiu~ co~rected for 2.% hafnlu~
and 35.453 is the atomic weight of chlorine: the ratio
is between 2.1:1 and 1.51:1.
AI y Zr(OH) 3y+4.x CI x • nH 2 0 Assay-Calculate the perc:ntage of an.hydrou.s alu~inum
zirconium trichlorohydrate In the Aluminum Zirconium
» Aluminum Zirconium Trichlorohydrate is a Trichlorohydrate by the formula:
polymeric, '?osely hydrate9 complex of basic AI({26.98y+ 92.97 + 17.01 [3y+ 4 - (y+ l)/z] + 35.453(y+ 1)
aluminum zircoruum chloride that encompasses a /z}/26.98y)

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206 Aluminum / Official Monographs USP 43

in which AI is the percentage of aluminum found in the test for the Content of aluminum under Aluminum Zirconium
for Contentof aluminum, y is the aluminum/zirconium atomic Octachlorohydrate. Use the result to calculate the Aluminum/
ratio found in the test for Aluminum/zirconium atomic ratio, Z zirconium atomic ratio and the (Aluminum pluszirconium)/
is the (aluminum plus zirconium)/chloride atomic ratio found chloride atomic ratio.
in the test for (Aluminum pluszirconium)/chloride atomic ratio, Content of zirconium-Using about 500 mg of Aluminum
26.98 is the atomic weight of aluminum, 92.97 is the atomic Zirconium Trichlorohydrate Solution, accurately weighed,
weight of zirconium corrected for 2% hafnium content, 17.01 instead of Aluminum Zirconium Octachlorohydrate, proceed
is the molecular weight of the hydroxide anion (OH), and as directed in the test for the Contentof zirconium under
35.453 is the atomic weight of chlorine (CI). AluminumZirconium Octachlorohydrate. Use the result to
calculate the Aluminum/zirconium atomic ratio and the
(Aluminum plus zirconium)/chloride atomic ratio.
Aluminum/zirconium atomic ratio-Divide the
percentage of aluminum found in the test for Contentof
Aluminum Zirconium Trichlorohydrate aluminum by the percentage of zirconium found in the test for
Content of zirconium and multiply by 92.97/26.98, in which
Solution 92.97 is the atomic weight of zirconium corrected for 2%
hafnium content, and 26.98 is the atomic weight of
» Aluminum Zirconium Trichlorohydrate Solution aluminum: the ratio is between 2.0:1 and 5.99:1.
is a polymeric, loosely hydrated complex of basic Content of chloride-Using about 500 mg of Aluminum
Zirconium Trichlorohydrate Solution, accurately weighed,
aluminum zirconium chloride that encompasses a instead of Aluminum Zirconium Octachlorohydrate, proceed
range of aluminum-to-zirconium atomic ratios as directed in the test for the Contentof chloride under
between 2.0:1 and 5.99:1 and a range of Aluminum Zirconium Octachlorohydrate. Usethe result to
(aluminum plus zirconium)-to-chloride atomic calculate the (Aluminum pluszirconium)/chloride atomic ratio.
(Aluminum plus zirconium)/chloride atomic ratio-
ratios between 2.1:1 and 1.51:1. The following Calculate the (aluminum plus zirconium)/chloride atomic ratio
solvents may be used: water, propylene glycol, or . by the formula:
dipropylene glycol. It contains the equivalent of
not less than 90.0 percent and not more than [(AI/26.98) + (Zr/92.97)]/(CI/35.453)
110.0 percent of the labeled concentration of in which AI, Zr, and CI are the percentages of aluminum,
anhydrous aluminum zirconium trichlorohydrate. zirconium, and chloride found in the test for Contentof
aluminum, Contentof zirconium, and Contentof chloride,
Packaging and storage-Preserve in well-closed respectively; 26.98 is the atomic weight of aluminum; 92.97
containers. is the atomic weight of zirconium corrected for 2% hafnium
Labeling-Label Solution to state the solvent used and the content; and 35.453 isthe atomic weight of chlorine: the ratio
claimed concentration of anhydrous aluminum zirconium is between 2.1:1 and 1.51:1.
trichlorohydrate. Assay-Calculate the percentage of anhydrous aluminum
Identification- zirconium trichlorohydrate in the Solution by the formula:
A: A solution containing the equivalent of about 100 mg of
anhydrous aluminum zirconium trichlorohydrate per mL AI({26.98y+ 92.97 + 17.01 [3y+ 4 - (Y+ 1)/z] + 35.453(y+ 1)
responds to the test for Chloride (191). . /z}/26.98y)
8: Identification of propylene glycol (where stated on the in which AI is the percentage of aluminum found in the test
label)-Add about 10 mL of isopropyl alcohol to 2 g of for Contentof aluminum, y is the aluminum/zirconium atomic
Solution, mix, and filter. Evaporatethe filtrate to about 1 mL ratio as determined in the test for Aluminum/zirconium atomic
on a steam bath: the IR spectrum of a film of this solution on ratio, z is the (aluminum plus zirconium)/chloride ratio
a silver chloride disk exhibits maxima only at the same determined in the test for (Aluminum plus zirconium)/chloride
wavelengths as that of a similar preparation of a film of atomic ratio, 26.98 is the atomic weight of aluminum, 92.97
propylene glycol. is the atomic weight of zirconium corrected for 2% hafnium
C: Identification of dipropylene glycol (where stated on the content, 17.01 is the molecular weight of the hydroxide anion
label)-Add about 10 mL of isopropyl alcohol to 2 g of (OH), and 35.453 is the atomic weight of chlorine (CI).
Solution, mix, and filter. Evaporate the filtrate to about 1 mL
on a steam bath: the IRspectrum of a film of this solution on
a silver chloride disk exhibits maxima only at the same
wavelengths as that of a similar preparation of a film of
dipropylene glycol. Aluminum Zirconium Trichlorohydrex
pH (791): between 3.0 and 5.0, in a solution prepared by
diluting 3 g of the Solution with water to obtain 10 mL. Gly
Arsenic, Method 1(211 )-Prepare the Test Preparation using
an accurately weighed quantity of the Solution. The limit is 2
~g per g.
» Aluminum Zirconium Trichlorohydrex Gly is a
Limit of iron-Using about 5.4 g of Aluminum Zirconium derivative of Aluminum Zirconium
Trichlorohydrate Solution, accurately weighed, instead of Trichlorohydrate in which some of the water
Aluminum Zirconium Octachlorohydrate, proceed asdirected molecules have been displaced by glycine, calcium
in the test for the Limit of iron under Aluminum Zirconium glycinate, magnesium glycinate, potassium
Octachlorohydrate. The limit is 75 ~g per g.
Content of aluminum-Using about 0.3 g of Aluminum glycinate, sodium glycinate, or zinc glycinate. It
Zirconium Trichlorohydrate SoJution instead of Aluminum encompasses a range of aluminum-to-zirconium
Zirconium Octachlorohydrate, proceed as directed in the test atomic ratios between 2.0:1 and 5.99:1 and a

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 USP 43 OfficialMonographs / Aluminum 207

range of (aluminum plus zirconium)-to-chloride Assay-Calculate the percentage of anhydrous aluminum

atomic ratios between 2.1:1 and 1.51:l.lt contains zirconium trichlorohydrate in the Aluminum Zirconium
Trichlorohydrex Gly by the formula:
not less than 90.0 percent and not more than
110.0 percent of the labeled amount of anhydrous AI({26.98y+ 92.97 + 17.01 [3y+ 4 - (Y+ l)/z] + 35.453(y+ 1)
aluminum zirconium trichlorohydrate. /z}/26.98y)

Packaging and storage-Preserve in well-closed in which AI is the percentage of aluminum found in the test
containers. for Contentof aluminum, y is the aluminum/zirconium atomic
Labeling-The label statesthe form of glycine used and the ratio found in the test for Aluminum/zirconium atomic ratio, Z
claimed content of anhydrous aluminum zirconium is the (aluminum plus zirconium)/chloride atomic ratio found
trichlorohydrate. in the test for (Aluminum pluszirconium)/chloride atomic ratio,
26.98 is the atomic weight of aluminum, 92.97 is the atomic
Identification- weight of zirconium corrected for 2% hafnium content, 17.01
A: A solution (1 in 10) responds to the test for Chloride is the molecular weight of the hydroxide anion (OH), and
(191). 35.453 is the atomic weight of chlorine (CI).
B: Place about 0.5 g of it in a 50-mL beaker, add about 20
mL of water, and swirl to dissolve. Heat to boiling on a hot
plate, and add about 60 mg of ninhydrin: a deep violet color
immediately develops.
pH (791): between 3.0 and 5.0, in a solution [15 in 100 (w/ Aluminum Zirconium Trichlorohydrex
w)].
Arsenic, Method I (211): 2 IJg per g. Gly Solution
Limit of iron-Using Aluminum Zirconium Trichlorohydrex
Gly instead of Aluminum Zirconium Octachlorohydrate, » Aluminum Zirconium Trichlorohydrex Gly
proceed asdirected in the test for Limitof iron under Aluminum
ZirconiumOctachlorohydrate. The specified result is obtained Solution is a solution of Aluminum Zirconium
(150 IJg per g limit). Trichlorohydrate in which some of the waters of
Content of aluminum-Using Aluminum Zirconium hydration have been displaced by glycine, calcium
Trichlorohydrex Gly instead of Aluminum Zirconium glycinate, magnesium glycinate, potassium
Octachlorohydrate, proceed as directed in the test for Content glycinate, sodium glycinate, or zinc glycinate. It
of aluminum under Aluminum Zirconium Octachlorohydrate.
Usethe result obtained to calculate the Aluminum/zirconium encompasses a range of aluminum-to-zirconium
atomic ratio and the (Aluminum pluszirconium)/chloride atomic ratios between 2.0:1 and 5.99:1 and a range of
ratio. (aluminum plus zirconium)-to-chloride atomic
Content of zirconium-Using Aluminum Zirconium ratios between 2.1:1 and 1.51 :1. The following
Trichlorohydrex Gly instead of Aluminum Zirconium solvents may be used: water, propylene glycol, or
Octachlorohydrate, proceed as directed in the test for Content
of zirconium under Aluminum Zirconium Octachlorohydrate. Use dipropylene glycol. It contains the equivalent of
the result obtained to calculate the Aluminum/zirconium not less than 90.0 percent and not more than
atomic ratio and the (Aluminum pluszirconium)/chloride atomic 110.0 percent of the labeled concentration of
raoo. . anhydrous aluminum zirconium trichlorohydrate.
Aluminum/zirconium atomic ratio-Divide the
percentage of aluminum found in the test for Contentof Packaging and storage-Preserve in well-closed
aluminum by the percentage of zirconium found in the test for containers.
Contentof zirconium, and multiply by 92.97/26.98, in which Labeling-Label Solution to state the solvent and form of
92.97 is the atomic weight of zirconium corrected for 2% glycine used and the claimed concentration of anhydrous
hafnium content, and 26.98 is the atomic weight of aluminum zirconium trichlorohydrate.
aluminum: the ratio is between 2.0:1 and 5.99:1.
Content of chloride-Using Aluminum Zirconium Identification-
Trichlorohydrex Gly instead of Aluminum Zirconium A: A solution containing the equivalent of about 100 mg of
Octachlorohydrate, proceed as directed in the test for Content anhydrous aluminum zirconium trichlorohydrate per mL
of chloride under Aluminum Zirconium Octachlorohydrate. Use responds to the test for Chloride (191).
the result obtained to calculate the (Aluminum plus zirconium) B: Identification of propylene glycol (where stated on the
/ chloride atomic ratio. label)-Add about 10 mL of isopropyl alcohol to 2 g of
(Aluminum plus zirconium)/chloride atomic ratio- Solution, mix, and filter. Evaporate the filtrate to about 1 mL
Calculate the (aluminum pluszirconium)/chloride atomic ratio on a steam bath: the IR spectrum of a film of this solution on
by the formula: a silver chloride disk exhibits maxima only at the same
wavelengths as that of a similar preparation of a film of
[(AI/26.98) + (Zr/92.97)]/(CI/35.453) propylene glycol.
C: Identification of dipropylene glycol (where stated on the
in which AI, Zr, and CI are the percentages of aluminum, label)-Add about 10 mL of isopropyl alcohol to 2 g of
zirconium, and chloride as determined in the tests for Content Solution, mix, and filter. Evaporate the filtrate to about 1 mL
of aluminum, Contentof zirconium, and Contentof chloride, on a steam bath: the IR spectrum of a film of this solution on
respectively; 26.98 is the atomic weight of aluminum; 92.97 a silver chloride disk exhibits maxima only at the same
is the atomic weight of zirconium corrected for 2% hafnium wavelengths as that of a similar preparation of a film of
content; and 35.453 is the atomic weight of chlorine: the ratio dipropylene glycol.
is between 2.1:1 and 1.51:1. D: Identification of glycine-Place about 1 g of Solution in a
50-mL beaker, add about 20 mL of water, and swirl to dissolve.

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208 Aluminum / Official Monographs USP 43

Heat to boiling on a hot plate, and add about 60 mg of

ninhydrin: a deep violet color immediately develops.
Amantadine Hydrochloride
pH (791): between 3.0 and 5.0, in a solution prepared by
diluting 3 g of the Solution with water to obtain 10 mL.
Arsenic, Method 1(211 )-Prepare the Test Preparation using
an accurately weighed quantity of the Solution. The limit is 2
J.lg per g.
Limit of iron-Using about 5.4 g of Aluminum Zirconium C10H 17N . HCI 187.71
Trichlorohydrex Gly Solution, accurately weighed, inst~ad of Tricydo[3.3.1 .13,7]decan-l-amrne, hydrochloride;
Aluminum Zirconium Octachlorohydrate, proceed asdirected l-Adamantanamine hydrochloride [665-66-7].
in the test for the Limit of iron under Aluminum Zirconium
Octachlorohydrate. The limit is 75 J.lg per g. DEFINITION
Content of aluminum-Using about 0.3 g of Aluminum Amantadine Hydrochloride contains NLT 98.0% and NMT
Zirconium Trichlorohydrex Gly Solution instead of Aluminum 102.0% of amantadine hydrochloride (C10H 17N . HCI).
Zirconium Octachlorohydrate, proceed as directed in the test
for the Content of aluminum under AluminumZirconium IDENTIFICATION
Octachlorohydrate. Use the result to calculate the Aluminum/
zirconiumatomic ratio and the (Aluminumplus zirconium)/
chloride atomic ratio.
Content of zirconium-Using about 500 mg of Aluminum • A.~~~~~~~~~~Q~.(;!~~~""I
Zirconium Trichlorohydrex Gly Solution, accurately weighed, Spg<::trp.s<::PPY:i«li9:7,t1.;197.1<,
instead of Aluminum Zirconium Octachlorohydrate, proceed Cell: 1 mm
as directed in the test for the Content of zirconium under Sample solution: 50 mg in 10 mL of 0.1 N hydrochloric
AluminumZirconium Octachlorohydrate. Usethe result to acid and filter. Transfer the filtrate to a suitable separator,
calculate the Aluminum/zirconium atomic ratio and the add'l mL of 5 N sodium hydroxide, and extract with 5 mL
(Aluminumplus zirconium)/chloride atomic ratio. of methylene chloride.
Aluminum/zirconium atomic ratio-Divide the Acceptance criteria: Meets the requirements
percentage of aluminum found in the test for Content of . • B. The retention time of the amantadine peak of the
aluminum by the percentage of zirconium found in the test for Sample solution corresponds to that of the Standard
Content of zirconium and multiply by 92.97/26.98, in which solution, as obtained in the Assay.
92.97 is the atomic weight of zirconium corrected for 2% • C. IDENTIFICATION TESTS-GENERAL (191), Chemical
hafnium content, and 26.98 is the atomic weight of Identification Tests, Chloride
aluminum: the ratio is between 2.0:1 and 5.99:1. ASSAY
Content of chloride-Using about 500 mg of Aluminum • PROCEDURE
Zirconium Trichlorohydrex Gly Solution,' accurately weighed, Internal standard solution: 0.3 mg/mL of adamantane in
instead of Aluminum Zirconium Octachlorohydrate, proceed n-heptane
as directed in the test for the Content of chloride under Standard stock solution: 1 mg/mL of USP Amantadine
AluminumZirconium Octachlorohydrate. Use the result to Hydrochloride RS in water
calculate the (Aluminumplus zirconium)/chloride atomic ratio. Standard solution: Transfer 10 mL of Standardstock
(Aluminum plus zirconium)/chloride atomic ratio- solution and 10 mL of 5 N sodium hydroxide solution to a
Calculate the (aluminum plus zirconium)/chloride atomic ratio separatory funnel. Add 25 mL of Internal standard solution
by the formula: and shakefor 10 min. Collect the upper layer of n-heptane
and swirl with anhydrous sodium sulfate to remove traces
[(AI/26.98) + (Zr/92.97)]/(C1/35.453) of water.
Sample stock solution: 1 mg/mL of Amantadine
in which AI, Zr, and CI are the percentages of aluminum, Hydrochloride in water .
zirconium, and chloride found in the tests for Content of Sample solution: Transfer 10 mL of Sample stocksolution
aluminum, Content of zirconium, and Content of chloride, and 10 mL of 5 N sodium hydroxide solution to a separatory
respectively; 26.98 is the atomic weight of aluminum; 9~.97 funnel. Add 25 mL of Internal standardsolution and shake
is the atomic weight of zirconium corrected for 2% hafnium for 10 min. Collect the upper layer of n-heptane and swirl
content; and 35.453 isthe atomic weight of chlorine: the ratio with anhydrous sodium sulfate to remove traces of water.
is between 2.1:1 and 1.51: l. Chromatographic system
Assay-Calculate the percentage of anhydrous aluminum (See Chromatography (621), System Suitability.)
zirconium trichlorohydrate in the Solution by the formula: Mode: GC
Detector: Flame ionization
AI({26.98y+ 92.97 + 17.01[3y+ 4 - (y+ l)/z] + 35.453(y+ 1)
Column: 0.53-mm x 30-m basedeactivated fused-silica;
/z}/26.98y) coated with 1.0-J.lm film of stationary phase G27
in which AI is the percentage of aluminum found in the test Temperatures
for Content of aluminum, y is the aluminum/zirconium atomic Injection port: 220 0
ratio as determined in the test for Aluminum/zirconium atomic Detector: 300 0
ratio: z is the (aluminum plus zirconium)/chloride atomic ratio Column: See Table 7.
as determined in the test for (Aluminum plus zirconium)/
chloride atomicratio, 26.98 is the atomic weight of aluminum, Table 1
92.97 is the atomic weight of zirconium corrected for 2% Hold Time at
hafnium content, 17.01 is the molecular weight of the Initial Temperature Final Final
Temperature Ramp Temperature Temperature
hydroxide anion (OH), and 35.453 is the atomic weight of
chlorine (CI).
n e/min) e) (min)
120 0 120 3

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USP 43 Official Monographs / Amantadine 209

Table 1 (continued) System suitability

Hold Time at Sample: Standard solution
Initial Temperature Final Final [NOTE-The relative retention times for adamantane
Temperature Ramp Temperature Temperature and amantadine are 0.7 and 1.0, respectively.]
(0) (O/min) e) (min)
Suitability requirements
120 8 250 10 Relative standard deviation: NMT 3.0% for the peak
response ratio of amantadine to adamantane
Carrier gas: Helium Analysis
Flow rate: 4 mL/min Samples: Standard solution and Sample solution
Calculate the percentage of any individual impurity in the
Injection volume: 2 ut, . . . .
Injection type: Split ratio, 5:1 (deactivated split liner with portion of Amantadine Hydrochloride taken:
glass wool)
Result = (Ru/R s) x (Cs/Cu) x 100
System suitability
Sample: Standard solution = peak response ratio of any individualimpurity to
[NOTE-The relative retention times for adamantane adamantane from the Sample solution
and amantadine are about 0.7 and 1.0, respectively. = peak response ratio of amantadine to
] adamantane from the Standard solution
Suitability requirements =concentration of USP Amantadine Hydrochloride
Relative standard deviation: NMT 1.0% for the peak RS in the Standard stock solution (mg/ml)
response ratio of amantadine to adamantane =concentration of Amantadine Hydrochloride in
Analysis the Sample stock solution (mg/mL)
Samples: Standard solution and Sample solution
Calculate the percentage of amantadine hydrochloride Acceptance criteria: See Table 2.
(C10H 17N . HCI) in the portion of Amantadine
Hydrochloride taken: Table 2
Result =(RulR s) x (Cs/Cu) x 100 Relative Acceptance
Retention Criteria,
Name Time NMT (%)
= peak response ratio of amantadine to
adamantane from the Sample solution Amantadine 1.0 -
= peak response ratio of amantadine to AmantadineRelated
adamantane from the Standard solution Compound A 1.2 0.3
=concentration of USP Amantadine Hydrochloride AmantadineRelated
RS in the Standard stock solution (mg/ml) Compound B 1.9 0.3
=concentration of Amantadine Hydrochloride in Anyindividual unspecified
the Sample stock solution (mg/mL) impurity - 0.10
Acceptance criteria: 98.0%-102.0% Total impurities - 1.0

IMPURITIES
• ORGANIC IMPURITIES SPECIFIC TESTS
Internal standard solution: 0.1 mg/mL of adamantane in • pH (791)
n-heptane Sample: 0.2 g/mL in water
Peak identification solution: 0.03 mg/mL each of USP Acceptance criteria: 3.0-5.5
Amantadine Related Compound A RS and USP Amantadine ADDITIONAL REQUIREMENTS
Related Compound B RS in Internal standard solution • PACKAGING AND STORAGE: Preserve in well-closed
prepared as follows. Transfer suitable amounts of USP . containers.
Amantadine RelatedCompound A RS and USP Amantadine • USP REFERENCE STANDARDS (11)
Related Compound B RS to a suitable volumetric flask. Add USP Amantadine Hydrochloride RS
methylene chloride to about 5% of the flask volume to USP Amantadine Related Compound A RS
dissolve, and dilute with Internal standard solution to 1-Chloroadamantane.
volume. C10H 1SCI 170.68
Standard stock solution: 0.03 mg/mL of USP Amantadine USP Amantadine Related Compound B RS
Hydrochloride RS in water N-(Adamantan-l-yl)acetamide.
Standard solution: Transfer 25 mL of Standard stock C12H19NO 193.29
solution and 10 mL of 5 N sodium hydroxide solution to a
separatory funnel. Add 25 mL of Internal standard solution
and shake for 10 min. Collect the upper layer of n-heptane
and swirl with anhydrous sodium sulfate to remove traces
of water. Amantadine Hydrochloride Capsules
Sample stock solution: 10 mg/mL of Amantadine
Hydrochloride in water . DEFINITION
Sample solution: Transfer 25 ml of Sample stock solution Amantadine Hydrochloride Capsules contain NLT 95.0% and
and 10 ml of 5 N sodium hydroxide solution to a separatory NMT 105.0% of the labeled amount of amantadine
funnel. Add 25 mL of Internal standard solution and shak.e hydrochloride (C1oH 17N . HCI).
for 10 min. Collect the upper layer of n-heptane and swirl
with anhydrous sodium sulfate to remove traces of water.
Chromatographic system: Proceedasdirected in the Assay.

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210 Amantadine / OfficialMonographs USP 43

IDENTIFICATION Suitability requirements

Resolution: NLT2.0 between naphthalene and
amantadine
Tailing factor: NMT 2.0 for the amantadine peak
Relative standard deviation: NMT 2.0% for the peak
response ratio of amantadine to naphthalene
mm Analysis
Sample solution: Place the contents of Capsules, equivalent Samples: Standard solution and Sample solution
to 200 mg of amantadine hydrochloride, in a vessel, Calculate the percentage of the labeled amount of
dissolve in 0.1 N hydrochloric acid, and filter. Transfer the amantadine hydrochloride (ClOH 17N . HCI) in the portion
filtrate to a separator, add 1 mL of 5 N sodium hydroxide, of Capsules taken:
and extract with 5 mL of methylene chloride. Filter the
extract through anhydrous sodium sulfate, and rinse the Result = (Ru/Rs) x (Cs/Cu) x 100
anhydrous sodium sulfate with 2 mL of methylene chloride.
• B. The retention time of the amantadine peak of the Ru = peak response ratio of amantadine to
Sample solution corresponds to that of the Standard naphthalene from the Sample solution
solution, as obtained in the Assay. Rs = peak response ratio of amantadine to
naphthalene from the Standard solution
ASSAY Cs = concentration of USP Amantadine Hydrochloride
• PROCEDURE RS in the Standard solution (mg/mL)
Internal standard solution: 0.4 mg/mL of naphthalene in Cu = nominal concentration of amantadine
hexane hydrochloride in the Sample solution (mg/mL)
Standard stock solution: 2 mg/mL of USPAmantadine
Hydrochloride RS in water Acceptance criteria: 95.00/0-105.0%
Standard solution: Transfer 25.0 mL of Standard stock
solution into a 250-mL separator, and add 25 mL of 2 N PERFORMANCE TESTS
sodium hydroxide and 50.0 mL of Internal standard • DISSOLUTION (711)
solution. Shake for about 10 min, and collect the hexane Test 1
layer. Medium: Water; 900 mL
Sample stock solution: Transfer NLT 20 Capsules to a Apparatus 1: 100 rpm
200-mL volumetric flask. Add 40 mL of 0.1 N Time: 45 min
hydrochloric acid and 40 mL of water. Sonicate for 20 min Internal standard solution: 0.054 mg/mL of naphthalene
with intermittent shaking, and dilute with water to volume. in hexane
Centrifuge the solution for 10 min and pass through a Standard stock solution: 0.1 mg/mL of USP Amantadine
suitable filter. Hydrochloride RS in water
Sample solution: Transfer 5.0 mL of the filtrate into a Standard solution: Transfer 15.0 mL of Standard stock
250-mL separator, and add 40.0 mL of 1 N sodium solution into a 50-mL screw-capped test tube, add 5.0 mL
hydroxide and 50.0 mL of Internal standard.solution. Shake of 5 N sodium hydroxide and 10.0 mLof Internal standard
for about 10 min, and collect the hexane layer. solution, and shake for 60 min. Collect the hexane layer.
Chromatographic system Sample solution: Transfer 15.0 mL of the filtered solution
(See Chromatography (621), System Suitability.) under test, and place into a 50-mL screw-capped test
Mode: GC tube. Pipet 5.0 mL of 5 N sodium hydroxide and 10.0 mL
Detector: Flame ionization of the Internal standard solution into the test tube, and
Column: 0.32-mm x 30-m; coated with a 0.25-lJm film of shake for 60 min. Collect the hexane layer.
phase G1 Chromatographic system
Temperatures (See Chromatography (621), System Suitability.)
Injection port: 250 0 Mode: GC
Detector: 300 0 Detector: Flame ionization
Column: See Table 7. Column: 0.32-mm x 30-m; coated with a 0.25-lJm film
of phase Gl
Table 1 Temperatures
Hold Time at
Injection port: 250 0
Initial Temperature Final Final Detector: 300 0
Temperature Ramp Temperature Temperature Column: See Table 2.
e) (O/min) e) (min)
100 a 100 3 Table 2
Hold Time at
100 10 200 2 Initial Temperature Final Final
Temperature Ramp Temperature Temperature
Carrier gas: Helium e) e/min) e) (min)
Flow rate: 1.4 mL/min 100 a 100 3
Injection volume: 2 IJL 100 200 2
10
Injection type: Split
Split flow rate: 20 mL/min
System suitability Carrier gas: Helium
Sample: Standard solution Flow rate: 1.4 mL/min
[NoTE-The relative retention times for naphthalene Injection volume: 2 IJL
and amantadine are 0.90 and 1.0, respectively.] Injection type: Split
Split flow rate: 20 mL/min

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USP 43 OfficialMonographs / Amantadine 211

System suitability Carrier gas: Helium

Sample: Standard solution Flow rate: 1.4 mL/min
[NOTE-The relative retention times for naphthalene Injectiqn volume: 2 IJL
and amantadine are 0.90 and 1.0, respectively.] Injection type: Split
Suitability requirements Split flow rate: 20 mL/min
Resolution: NLT 2.0 between naphthalene and System suitability
amantadine Sample: Standard solution
Tailing factor: NMT 2.0 for the amantadine peak Suitability requirements
Relative standard deviation: NMT 2.0% for the peak Resolution: NLT 2 between naphthalene and
response ratio of amantadine to naphthalene amantadine
Analysis Tailing factor: NMT 2.0 for the amantadine peak
Samples: Standard solution and Sample solution Relative standard deviation: NMT 2.0% for the peak
Calculate the percentage of the labeled amount of response ratio of amantadine to naphthalene
amantadine hydrochloride dissolved: Analysis
Samples: Standard solution and Sample solution
Result = (Ru/ Rs) x (Cs/ L) x V x 100 Calculate the percentage of the labeled amount of
amantadine hydrochloride dissolved:
= peak response ratio of amantadine to
naphthalene from the Sample solution Result = (Ru/R s) x (Cs/L) x Vx 100
= peak response ratio of amantadine to
naphthalene from the Standard solution = peak response ratio of amantadine to
=concentration of USP Amantadine Hydrochloride naphthalene from the Sample solution
RS in the Standard stock solution (mg/mL) = peak response ratio of amantadine to
L =label claim (mg/Capsule) naphthalene from the Standard solution
V = volume of Medium, 900 mL = concentration of USP Amantadine Hydrochloride
RS in the Standard stock solution (mg/mL)
Tolerances: NLT ·75% (Q) of the labeled amount of L = label claim (mg/Capsule)
amantadine hydrochloride (ClOH 17N . HCI) is dissolved. v = volume of Medium, 900 mL
Test 2: If the product complies with this test, the labeling
indicates that it meets USP Dissolution Test 2. . Tolerances: NLT 75% (Q) of the labeled amount of
Medium: Water; 900 mL amantadine hydrochloride (C10H 17N . HCI) is dissolved.
Apparatus 2: 75 rpm, with sinkers. [NOTE-A suitable • UNIFORMITY OF DOSAGE UNITS (90S): Meet the
sinker is available as catalog number CAPWHT-2S from requirements
www.qla-llc.corn or www.tabletdissolution.com or
IMPURITIES
www.labhut.com.]
Time: 45 min • ORGANIC IMPURITIES
Internal standard solution: 0.06 mg/mL of naphthalene Internal standard solution: 0.1 mg/mL of adamantane in
in hexane . n-heptane
Standard stock solution: 0.12 mg/mL of USP Amantadine Peak identification solution: 0.03 mg/mL each of USP
Hydrochloride RS in Medium Amantadine Related Compound A RS and USP Amantadine
Standard solution: Transfer 60.0 mL of the Standard stock Related Compound B RS in Internal standard solution
solution to a 200-mL volumetric flask. Add 20 mL of 5 N prepared as follows. Transfer suitable amounts of USP
sodium hydroxide and 40.0 mL of Internal standard Amantadine RelatedCompound A RS and USP Amantadine
solution. Shake the flask for approximately 10 min, and Related Compound B RS to a suitable volumetric flask. Add
allow the layersto separate. Usethe top layer for injection. methylene chloride to about 5% of the flask volume to
The final concentration is about 0.18 mg/mL. dissolve, and dilute with Internal standard solution to
Sample solution: Transfer 3.0 mL of the solution under volume.
test to a centrifuge tube. Add 1.0 mL of 5 N sodium Standard stock solution: 0.03 mg/mL of USP Amantadine
hydroxide and 2.0 mL of Internal standard solution. Shake Hydrochloride RS in water
the tube for approximately 10 min, and allow the layers Standard solution: Transfer 25 mL of Standard stock
to separate. Use the top layer for injection. solution, 10 mL of 5 N sodium hydroxide solution, and 25
Chromatographic system mL of Internal standard solution to a separatory funnel and
(See Chromatography (621), System Suitability.) shake for 10 min. Collect the upper layer of n-heptane and
Mode: GC swirl with anhydrous sodium sulfate to remove traces of
Detector: Flame ionization water.
Column: 0.32-mm x 30-m; coated with a 0.25-lJm film Sample stock solution (for hard gelatin Capsules):
of phase Gl Nominally 10 mg/mL of amantadine hydrochloride
Temperatures prepared as follows. Combine the contents of NLT 20
Injection port: 250 0 Capsules and transfer a portion equivalent to 500 mg of
Detector: 300 0 amantadine hydrochloride to a 1OO-mL flask. Add 20 mL of
Column: See Table 3. 0.1 N hydrochloric acid using a volumetric pipette. Heat
gently to dissolve and cool. Add 20 mL of water and
Table 3 sonicate for 15 min. Cool and add 10 mL of water.
Centrifuge the solution for 10 min and collect the
Hold Time at supernatant.
Initial Temperature Final Final
Temperature Ramp Temperature Temperature Sample solution
(") ("/min) (") (min) For hard gelatin Capsules: Transfer 25 mL of Sample stock
100 0
solution and 10 mL of 5 N sodium hydroxide solution to
100 3
a separatory funnel. Add 25 mL of Internal standard
100 10 200 2 solution and shake for 10 min. Collect the upper layer of

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n-heptane and swirl with anhydrous sodium sulfate to Table 5 (continued)

remove traces of water. Relative Acceptance
For soft gelatin Capsules: Transfer 2 Capsules to a 200-mL Retention Criteria,
flask and add 10 mL of water. Heat gently and cool. Add Name Time NMT(%)
5.0 mL of 5 N sodium hydroxide, 3.0 mL of concentrated Any individual unspecified
sodium chloride, and 50.0 mL of Internal standard impurity - 0.2
solution. Shake the contents for 20 min and collect the
upper layer of n-heptane. Total impurities - 1.5
Chromatographic system a Process impurity included in the table for identification only. Process impurities
(See Chromatography (621), System SUitability.) are controlledin the drug substance, and are not to be reported or includedin
Mode: GC the total impurities for the drug product.
Detector: Flame ionization
Column: 0.53-mm x 30-m; base deactivated fused-silica ADDITIONAL REQUIREMENTS
coated with a 1.0-~m film of stationary phase G27 • PACKAGING AND STORAGE: Preserve in tight containers.
Temperatures • LABELING: When more than one Dissolution test is given, the
Injection port: 220 0 labeling states the Dissolution test used only if Test 7 is not
Detector: 300 0 used.
Column: See Table 4. • USP REFERENCE STANDARDS (11)
USP Amantadine Hydrochloride RS
Table 4 USPAmantadine Related Compound A RS
Hold Time at 1-Chloroadamantane.
Initial Temperature Final Final C l oH 15CI 170.68
Temperature Ramp Temperature Temperature USP Amantadine Related Compound B RS
e) e/min)' e) (min) N-(Adamantan-l-yl)acetamide.
120 0 120 3 C12H 19NO 193.29
120 8 250 10

Carrier gas: Helium

Flow rate: 4 mL/min Amantadine Hydrochloride Oral
Injection volume: 2 ~L Solution
Injection type: Split ratio, 5:1 (deactivated split liner with
glass wool) DEFINITION
System sultablllty Amantadine Hydrochloride Oral Solution contains NLT 95.0%
Sample: Standard solution and NMT 105.0% of the labeled amount of amantadine
[NoTE-The relative retention times for adamantane hydrochloride (C1oH 17N . Hel).
and amantadine are 0.7 and 1.0, respectively.]
Suitability requirements . IDENTIFICATION
Relative standard deviation: NMT 3.0% for the peak
response ratio of amantadine to adamantane
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of any individual unspecified
impurity in the portion of Capsules taken: mm
Sample solution: Place a volume of Oral Solution,
Result = (Ru/R s) x (CslCu) x 100 equivalent to 200 mg of amantadine hydrochloride, in a
vessel, dissolve in 0.1 N hydrochloric acid, and filter.
Ru = peak response ratio of any individual unspecified Transfer the filtrate to a separator, add 10 mL of 0.5 N
impurity to adamantane from the Sample solution sodium hydroxide, and extract with 5 mL of methylene
Rs = peak response ratio of amantadine to chloride. Filter the extract through anhydrous sodium
adamantane from the Standard solution sulfate, and rinse the anhydrous sodium sulfate with 2 mL
Cs =concentration of USP Amantadine Hydrochloride of methylene chloride.
RS in the Standard stock solution (mg/mL) Acceptance criteria: Meets the requirements
Cu = nominal concentration of amantadine • B. The retention time of the major peak of the Sample
hydrochloride in the Sample stock solution solution corresponds to that of the Standard solution, as
(mg/mL) obtained in the Assay.
Acceptance criteria: See Table 5. ASSAY
• PROCEDURE
Table 5 Internal standard solution: 0.3 mg/mL of adamantane in
Relative Acceptance
n-heptane
Retention Criteria, Standard stock solution: 1 mg/mL of USP Amantadine
Name Time NMT (%) Hydrochloride RS in water
Standard solution: Transfer 10 mL of Standard stock
Amantadine 1.0 - solution to a separatory funnel and add 10 mL of 5 N
Amantadinerelated
- sodium hydroxide solution. Add 25 mL of Internal standard
compound A" 1.2 solution and shake for 10 min. Collect the n-heptane upper
Amantadinerelated layer and swirl with anhydrous sodium sulfate to remove
compound Ba 1.9 - traces of water.

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 USP 43 OfficialMonographs / Amantadine 213

Sample stock solution: Nominally 1 mg/mL of amantadine Related Compound B RS to a suitable volumetric flask. Add
hydrochloride from a portion of Oral Solution in water methylene chloride to about 5% of the flask volume to
Sample solution: Transfer 10 mL of Sample stock solution to dissolve, and dilute with Internal standard solution to
a separatory funnel and add 10 mL of 5 N sodium hydroxide volume.
solution. Add 25 mL of Internal standard solution and shake Standard stock solution: 0.03 mg/mL of USP Amantadine
for 10 min. Collect the n-heptane upper layer and swirl with Hydrochloride RS in water
anhydrous sodium sulfate to remove traces of water. Standard solution: Transfer 25 mL of Standard stock
Chromatographic system solution, 10 mL of 5 N sodium hydroxide solution, and 25
(See Chromatography (621), System Suitability.) mL of Internal standard solution to a separatory funnel and
Mode: GC shake for 10 min. Collect the upper layer of n-heptane and
Detector: Flame ionization swirl with anhydrous sodium sulfate to remove traces of
Column: 0.53-mm x 30-m base deactivated fused-silica; water.
coated with 1.0-~m film of stationary phase G27 Sample stock solution: Nominally 10.0 mg/mL of
Temperatures amantadine hydrochloride from Oral Solution in water
Injection port: 220 0 Sample solution: Transfer 25 mL of Sample stock solution,
Detector: 300 0 equivalent to 250 mg of amantadine hydrochloride, and 10
Column: See Table 7. mL of 5 N sodium hydroxide solution to a separatory
funnel. Add 25 mL of Internal standard solution and shake
Table 1 for 10 min. Collect the upper layer of n-heptane and swirl
HoldTime at with anhydrous sodium sulfate to remove traces of water.
Initial Temperature Final Final Chromatographic system: Proceed as directed in the
Temperature Ramp Temperature Temperature Assay.
(0) e/min) e) (min) System suitability
120 0 120 3 Sample: Standard solution ,
[NoTE-The relative retention times for adamantane
120 8 250 10
and amantadine are 0.7 and 1.0, respectively.]
Suitability requirements
Carrier gas: Helium , Relative standard deviation: NMT 3.0% for the peak
Flow rate: 4 mL/min response ratio of amantadine and adamantane
Injection volume: 2 ~L Analysis
.Injection type: Split ratio, 5:1 (deactivated split liner with Samples: Standard solution and Sample solution
glass wool) Calculate the percentage of any individual unspecified
System suitability impurity in the portion of Oral Solution taken:
Sample: Standard solution
[NOTE-The relative retention times for adamantane Result = (Ru/R s) x (Cs/Cu) x 100
and amantadine are about 0.7 and 1.0, respectively.
] Ru = peak response ratio of any individual unspecified
Suitability requirements impurity to adamantane from the Sample solution
Tailing factor: NMT 2.0 for the amantadine peak Rs = peak response ratio of amantadine to
Relative standard deviation: NMT 2.0% for the peak adamantane from the Standard solution
response ratio of amantadine to adamantane Cs = concentration of USP Amantadine Hydrochloride
Analysis RS in the 'Standard stock solution (mg/mL)
Samples: Standard solution and Sample solution Cu = nominal concentration of amantadine
Calculate the percentage of the labeled amount of , hydrochloride in the Sample stock solution
amantadine hydrochloride (C,oH17N . HCI) in the portion (mg/mL)
of Oral Solution taken:
Acceptance criteria: see Table 2.
Result = (RulR s) x (Cs/Cu) x 100
Table 2
Ru =peak response ratio of amantadine to Relative Acceptance
adamantane from the Sample solution Retention Criteria,
Rs = peak response ratio of amantadine to Name Time NMT(%)
adamantane from the Standard solution Amantadine 1.0 -
Cs = concentration of USP Amantadine Hydrochloride
RS in the Standard stocksolution (mg/mL) Amantadinerelated
compound A" 1.2
-
Cu = nominal concentration of amantadine
hydrochloride in the Sample stock solution Amantadinerelated
-
(mg/mL) compound Ba 1.9
Anyindividual unspecified
Acceptance criteria: 95.00/0-105.0% impurity - 0.2
IMPURITIES Total impurities - 2.0
• ORGANIC IMPURITIES
Internal standard solution: 0.1 mg/mL of adamantane in a Process impurityincludedin the tablefor identification only. Process impurities
are controlledin the drug substance, and are not to be reported or included in
n-heptane the total impurities for the drug product.
Peak identification solution: 0.03 mg/mL each of USP
Amantadine RelatedCompound A RS and USP Amantadine ADDITIONAL REQUIREMENTS
Related Compound B RS in Internal standard solution • PACKAGING AND STORAGE: Preserve in tight containers.
prepared as follows. Transfer suitable amounts of USP Store at controlled room temperature.
Amantadine Related Compound A RS and USP Amantadine

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214 Amantadine / OfficialMonographs USP 43

• liSP REfERENCE STANDARDS (11) .

Sys~~!'l~~it~~ilit~~~!~~i2~,:",I?·5 IJg/ mL of .~J;.l.~ ~.
USP Amantadine Hydrochloride RS aytylp(,lrap~n~~.(p~eLt"~1l9:.?OlQ) and 20 IJg/mL of USP
USPAmantadine Related Compound A RS Al11cinonide RS in Solution B
1-Chloroadamantane. Standard solution: 0.02 mg/mL of USP Amcinonide RS in
C,oH,sCI 170.68 Solution B
USP Amantadine Related Compound B RS Sample solution: 0.02 mg/mL of Amcinonide in Solution B.
N-(Adamantan-1-yl)acetamide. Sonicate for 5 min.
C12H'9NO 193.29 Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing
Amcinonide L1
Flow rate: 2 mL/min
Injection volume: 10 IJL
System suitability
Samples: System suitabilitysolution and Standardsolution
[NoTE-The relative retention times for butylparaben
and amcinonide are 0.78 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 8.0 between butylparaben and
C2sH3SF07 502.57 , amcinonide, System suitability solution
Pregna-1,4-diene-3,20-dione, 21-(acetyloxy)-16,17- Tailing factor: NMT 1.5, Standardsolution
[cyclopentylidenebis(oxy)]-9-fluoro-11-hydroxy-, (11 ~,16a)- Relative standard deviation: NMT 2.0%, Standard
solution
9~Fluoro-11 ~, 16a, 17,21-tetrahydroxypregna-1 ,4-diene-3,20- Analysis
dione cyclic 16,17-acetal with cyclopentanone, 21-acetate Samples: Standard solution and Sample solution
[51022-69-6]. Calculate the percentage of amcinonide (C2sH3SF07) in the
DEFINITION portion of Amcinonide taken:
Amcinonide contains NLT97.0% and NMT 102.0% of
amcinonide (C2sH3SF07), calculated on the dried basis. Result = (rulrs) x (CslC u) x 100
IDENTIFICATIO~ = peak response from the Sample solution
= peak response from the Standardsolution
= concentration of U5P Amcinonide R5 in the
Standard solution (mg/mL)
= concentration of Amcinonide in the Sample
solution (mg/mL)

Acceptance criteria: 97.0%-102.0% on the dried basis

SPECIFIC TESTS
• OPTICAL ROTATION (7815), Procedures, Specific Rotation
Sample solution: 10 mg/mL of Amcinonide in chloroform
Acceptance criteria: +89.4° to +94.0°
ASSAY • Loss ON DRYING (731)
Sample: Amcinonide
Analysis: Dry the Sample at 105° for 4 h.
Acceptance criteria: NMT 1.0%
• PROCEDURE
Solution A: Acetonitrile and water ADDITIONAL REQUIREMENTS
Solution B: Acetonitrile and water~ • PACKAGING AND STORAGE: Preserve in well-closed
Mobile phase: See Table 7. Equilibrate the system containers.
Solution A.

• USP REfERENCE STANDARDS (11 )

J:illl~ SoII.i~i()nA SolUtioijB
(l11/ oj (l}/Cl) (l}/o)
0 100 0
2.5 100 0
10 0 loO
Amcinonide (ream
25 0 100
'26 Jog Q DEFINITION
Amcinonide Cream is Amcinonide in a suitable cream base. It
32 lOO F(O~~/j'2Ati~Ti6j'~5 contains NLT 90.0% and NMT 115.0% of the labeled
amount of amcinonide (C2sH3SF07)'

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USP 43 Official Monographs / Amcinonide 215

IDENTIFICATION Result = (rufr s) x (Cs/Cu) x 100

,Chang¢ = peak response from the Sample solution

=peak response from the Standardsolution
• A.·The ret~ntio =concentration of USP Amcinonide RS in the
Sample solution corre . Standardsolution (mg/mL)
solution, as obtained in t =nominal concentration of amcinonide in the
Sample solution (mg/mL)
ASSAY
Acceptance criteria: 90.00/0-115.0%
Changetor~ad:
PERFORMANCE TESTS
• PROCEDURE • MINIMUM FILL (755): Meets the requirements
Solution A: Acetonitrile and water~(3
Solution B: Acetonitrile and water·(7 . SPECIFIC TESTS
Mobile phase: See Table 1. Equilibrate the system with
Solution A.

:ATable 1
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
SPECIFIED MICROORGANISMS (62): :·The total aerobic
Time SohitionA
(%) ..
Solution 8 mkrobial count isNMT1Q2 du/g and the tota.l·ye~st(,lnd
(mill) (0/0)
mO,ld count isNI'v1T 10 1du/g .• (USPJ:Dec-2019) It meets the
0 100 0 requirements of the tests for absence of Staphylococcus
',100 0
aureus and Pseudomonas aeruginosa.
2.5
• pH (791): 3.5-5.2
10 0 lPO
ADDITIONAL REQUIREMENTS
25 0 ~PQ
26 100 0 change toretid.:
32 109 P~(USP1.Det.~2(119j • PACKAGING AND STORAGE: Preserve in tight
containers.a S n~rolled room
ter:nperature~ 2019)
Syst~msuit!lb,i1itys()luti~~:.1?5 j.Jg/mLof ''ij1J$~
Butylpar-abenRS:A.(usPl~Dec~i()]9) and 20 j.Jg/mLof USP
Amcinonide RS in Solution B
Standard solution: 0.02 mg/mL of USP Amcinonide RS in • USP REFERENCE STANDARDS (11)
Solution B USP Amcinonide RS
Sample stock solution: Nominally 0.2 mg/mL of
"OSP Btitylparaben RS. (USP l-Dec~2019)
amcinonide from Cream prepared as follows. Transfer a
quantity of Cream, equivalent to 10 mg of amcinonide, to
a 50-mL volumetric flask. Add 5 mL of Solution Band 15 mL
of acetonitrile, and heat over a steam bath until dissolved.
Add 20 mL of Solution B while hot, cool to room Amcinonide Ointment
temperature, dilute with Solution B to volume, and
refrigerate for 30 min. Vigorously shake the solution-to DEFINITION
disperse the mixture, and filter while cold. Use the filtrate. Amcinonide Ointment is Amcinonide in a suitable ointment
Sample soluti~n: Nominally 0.02,m~/rnL~famcinonide base. It contains NLT 90.0% and NMT 115.0% of the
from the • Sample stock"soJuJion1.. ~UsPcl.Dec:201~) in Solution B labeled amount of amcinonide (C2sH35F0 7).
Chromatographic system
(See Chromatography (621), System SUitability.) IDENTIFICATION
Mode: LC
Detector: UV 254 nm th
Column: 4.6-mm x 25-cm;j,5..1411'l:A.(UsPl-De~-2~1§j packing
L1.
Flow rate: 2 mL/min
Injection volume: 10 j.JL
System suitability ASSAY
Samples: System suitability solution and Standardsolution
[NOTE-The relative retention times for butylparaben Chan read:
and amcinonide are 0.78 and 1.0, respectively.] .
Suitability requirements • PROCEDURE
Resolution: NLT 8.0 between butylparaben and Solution A: Acetonitrile and water·(35: l-Aug-20:19)
amcinonide, System suitability solution Solution B: Acetonitrile and water·(70:30)... . Pl-Aug-2019)
Tailing factor: NMT 1.5, Standardsolution Mobile phase: See Table 1. Equilibrate the system with
Relative standard deviation: NMT 2.0%, Standard Solution A.
solution
Analysis .
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of
amcinonide (C2sH3SF07) in the portion of Cream taken:

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216 Amcinonide / Official Monographs USP 43

SPECIFIC TESTS
Time Soh.itiol'lA SolutlonB
(niiiJ) (Ok) (%) .. Chtmge to read:
o "too 0 • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
2.5 lQO 0 SPECIFIED MICROORGANISMS (62): • c
microbial count is N 162 du/g an s.and
10 0 foo molds count is NMT cfu/g.I.(USP1-Aug-2019) Meets the
25 0 100 requirements of the tests for absenceof Staphylococcus
aureus and Pseudomonas aeruginosa
26 100 0
ADDITIONAL REQUIREMENTS
32 log g~(uSRi~Au~i~19)
'.,', ,,-.-

Change t
Diluent: Acetonitrile and chloroform (80:20)
System s~itabilitys()l~tio~:12.5 ~g/mL ofj.I.JS~ • PACKAGING AND STORAGE: . Preserve in tight containers.
Butylparaben RS.a: (USP 1.~ug:2019) and 20 ~g/mL of USP ·Store at controlled room temperature.... (USP l-Aug·2019)
Amcinonide RS in Solution 8
Standard solution: 0.02 mg/mL of USP Amcinonide RS in Change to read:
Solution 8
Sample stock solution: Nominally 0.2 rnq/rn], of • USP REFERENCE STANDARDS (11)
amcinonide prepared asfollows. Dissolvea sUita.9le amount USP Amcinonide RS
()f Ointmentin as~itableyol~meof Diluent~·i~~ ·USP Bi.Jtylparaben RS ... (USP i-Aug-2019)
a
NlolL1metricJlasK!t·(uSP i:Alig-2019) by heating in hot water
bath, cooling, and diluting with Diluent to volume. Cool to
room temperature, dilute with acetonitrile to volume, and
filter. Amifostine
Sample solution: Nominally 0.02 mg/mL of amcinonide in
Solution 8 from the Sample stock solution
Chromatographic system
(See Chromatography (621), System SUitability.)
Mode: LC CSH1SN203PS·3H20 268.27
Detector: UV 240 nm Ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrogen
Column: 4.6-hlm x 25-cm; ~$;b1I'1"1";~hJ~"'l~~~~Z~()I~) packing phosphate (ester), trihydrate;
L1 S-[2-(3-Am inopropyl)amino]ethyl]dihydrogen
Flow rate: 2 mL/min phosphorothioate, trihydrate [112901-68-5].
Injection volume: 10 ~L
System suitability DEfiNITION
Samples: System suitability solution and Standard solution Amifostine contains NLT 78.0% and NMT 82.0% of
[NOTE-The relative retention times for butylparaben CSH1SN203PS, calculated on the as-is basis.
and amcinonide are 0.78 and 1.0, respectively.] IDENTifiCATION
Suitability requirements
Resolution: NLT 8.0 between butylparaben and Change torea~:
amcinonide, System sUitability solution ,
Tailing factor: NMT 1.5, Standard solution • A.'&SPECTROSCOPICID
Relative standard deviation: NMT 2.0%, Standard Spectroscopy! l~7K.a:(CN ay.2020)
solution • B. The retention time of the major peak of the Sample
Analysis solution corresponds to that of the Standard solution, as
Samples: Standard solution and Sample solution obtained in the Assay.
Calculate the percentage of the labeled,amount of
amcinonide (C2sH3SF07) in the portion of Ointment taken: ASSAY
• PROCEDURE
Result = (ru/rs) x (Cs/Cu) x 100 Buffer: 0.94 gil of sodium 1-hexanesulfonate. Adjust with
phosphoric acid to a pH of 3.0.
tu = peak response from the Sample solution Mobile phase: Methanol and Buffer (7:18)
rs = peak response from the Standard solution Standard solution: 3 mg/mL of USP Amifostine RS in water.
Cs = concentration of USP Amcinonide RS in the [NOTE-Inject immediately after preparation.]
Sample solution: 3 mg/mL of Amifostine in water.
Standard solution (mg/mL)
Cu = nominal concentration of amcinonide in the [NOTE-Inject immediately after preparation.]
Chromatographic system
Sample solution (mg/mL)
(See Chromatography (621), System Suitability.)
Acceptance criteria: 90.0%-115.0% Mode: LC
Detector: UV 220 nm
PERfORMANCE TESTS Column: 4.6-mm x 25-cm; 5-~m packing L7
• MINIMUM flu (755): Meets the requirements Autosampler temperature: 4°
Flow rate: 1.0 mL/min
Injection size: 10 ~L
System suitability
Sample: Standard solution

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 USP 43 Official Monographs / Amifostine 217

Suitability requirements = concentration of USP Amifostine RS in the

Tailing factor: NMT 2.0 Standard solution (uq/rnl.)
Column efficiency: NLT 1000 theoretical plates = concentration of the Sample solution (lJg/mL)
Relative standard deviation: NMT 2~0%
Analysis Acceptance criteria
Samples: Standard solution and Sample solution Amifostine thiol: NMT 0.3%
Calculate the percentage of CSH1SN203PS in the portion Any individual impurity, excluding amifostine thiol:
of Amifostine taken: NMT 0.1%
Total impurities including amifostine thiol: NMT 0.3%
Result = (ru/rs) x (Cs/Cu) x 100
SPECIFIC TESTS
=peak response from the Sample solution • pH (791): 6.5-7.5, in a solution (5 in 100)
=peak response from the Standard solution • WATER DETERMINATION, Method Ie (921)
= concentration of USP Amifostine RS in the Sample solution: To 100.0 mg of Amifostine, contained in
Standard solution (mg/mL) a stoppered centrifuge tube, add 10.0 mL of the solution
Cu = concentration of Amifostine in the Sample of N-ethylmaleimide in methanol (4 in 100), and sonicate
solution (mg/mL) for 15 min. Shaketo disperse,and sonicate for an additional
15 min. Use 1.0 mL of the supernatant.
Acceptance criteria: 78.0%-82.0% on the as-is basis Acceptance criteria: 19.20/0-21.2%

IMPURITIES ADDITIONAL REQUIREMENTS

ORGANIC IMPURITIES • PACKAGING AND STORAGE: Preserve in tight, light-resistant
• Procedure containers, and store in a refrigerator.
Mobile phase and Chromatographic system: Proceed as • USP REFERENCE STANDARDS (11)
directed in the Assay. USP Amifostine RS
Standard solution: 70 IJg/mL of USP Amifostine Thiol RS and USP Amifostine Thiol RS
16 IJg/mL of USP Amifostine RS in water. [NoTE-Inject Ethanethiol, 2-[(3-aminopropyl)amino]-,
immediately after preparation.] dihydrochloride.
System suitability solution: Use the Standard solution as CSH16N2SCI2 207.17
described in the Assay. [NoTE-Inject immediately after
preparation.]
Sample solution: 15 mg/mL of Amifostine in water.
[NoTE-Inject immediately after preparation.] Amifostine for Injection
System suitability ,
Samples: Standard solution and System suitability solution DEFINITION
Suitability requirements Amifostine for Injection is a sterile, crystalline substance
Column efficiency: NLT 1000 theoretical plates, System suitable for parenteral use. It contains NLT 90.0% and NMT
suitability solution . 110.0% of the labeled amount of amifostine (CsHlsN203PS),
Tailing factor: NMT 2.0, System suitability solution
Relative standard deviation: NMT 15.0%, Standard IDENTIFICATION
solution
Analysis .
Samples: Standard solution and Sample solution
Calculate the percentage of amifostine thiol in the portion
of Amifostine taken: .'
peak of the Sample
Result = (ru/r s) x (Cs/Cu) x (M r1/Mr2) x 100 solution corresponds to that of Standard solution, as
obtained in the Assay.
= peak response of amifostine thiol from the
Sample solution ASSAY
= peak response of amifostine thiol from the • PROCEDURE
Standard solution Buffer: 0.94 gIL of sodium 1-hexanesulfonate. Adjust with
= concentration of USP Amifostine Thiol RS in the phosphoric acid to a pH of 3.0.
Standard solution (mg/mL) Mobile phase: Methanol and Buffer (7:18)
= nominal concentration of amifostine in the Standard solution: 3 mg/mL of USP Amifostine RS in water.
Sample solution (mg/mL) [NOTE-Inject immediately after preparation, or refrigerate
= molecular weight of amifostine thiol, 134.24 until use.] .
= molecular weight of amifostine thiol Sample solution: 3 mg/mL of amifostine from Amifostine
dihydrochloride, 207.17 for Injection, in water. [NOTE-Inject immediately after
preparation, or refrigerate until use.]
Calculate the percentage of any other individual impurity Chromatographic system
in the portion of Amifostine taken: (See Chromatography (621), System SUitability.)
Mode: LC
Result = (ru/rs) x (Cs/Cu) x 100 Detector: UV 220 nm
Column: 4.6-mm x 25-cm; 5-J.lm packing L7
ru =peak response of each individual impurity in the Autosampler temperature: 4°
Sample solution Flow rate: 1.0 mL/min
rs = peak response of amifostine in the Standard Injection size: 10 IJL
solution System suitability
Sample: Standard solution

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218 Amifostine / Official Monographs USP 43

Suitability requirements ru = peak response of sodium thiophosphate or

Column efficiency: NLT 1000 theoretical plates N,N-dimethylformamide from the Sample
Tailing factor: NMT 2.0 solution
Relative standard deviation: NMT 2.0% rs = peak response of sodium thiophosphate or
Analysis N,N-dimethylformamide from Standard solution
Samples: Standardsolution and Sample solution 2
Calculate the percentage of CSH1SN203PS in the portion of Cs = concentration of sodium thiophosphate or
Amifostine for Injection taken: N,N-dimethylformamide in Standard solution2
(mg/mL)
Result =(ru/rs) x (CsICu) x 100 Cu = concentration of amifostine in the Sample
solution (mg/mL)
= peak responses from the Sample solution
= peak responses from the Standardsolution Calculate the percentage of any other individual, unspecified
= concentration of USP Amifostine RS in the impurity in the portion of sample taken:
Standardsolution (mg/mL)
Cu = nominal concentration of amifostine in the Result = (ru/rr) x 100
Sample solution (mg/mL)
ru = peak response of each individual impurity in the
Acceptance criteria: 90.00/0-110.0% Sample solution
rr = total of all peak responses in the Sample solution
PERFORMANCE TESTS
• UNIFORMITY OF DOSAGE UNITS (905): Meets the Acceptance criteria: Nr:'IT 0.1% of sodium thiophosphate;
requirements NMT 0.088% of N,N-dlmethylformamide; NMT 0.1% of any
IMPURITIES other individual unspecified impurity
ORGANIC IMPURITIES • Procedure 2
• Procedure 1 Buffer: 0.4 giL of sodium l-octanesulfonate. Adjust with
Mobile phase and Chromatographic system: Proceed as trifluoroacetic acid to a pH of 2.5 ± 0.1.
directed in the Assay. Mobile phase: Acetonitrile and Buffer (1:3)
Standard solution 1: 70 IJg/mL of USP Amifostine Thiol RS in Standard solution: 46 IJg/mL of USP Amifostine Disulfide RS
water in water
Standard solution 2: 15 IJg/mL of sodium thiophosphate Sample solution: Dilute a quantity of Amifostine for Injection
and 13 IJg/mL of N,N-dimethylformamide in water. in water to prepare a solution equivalent to 10 mg/mL.
[NOTE--:-The retention ~imes of sodium thiophosphate and [NOTE-Inject immediately after preparation.]
N,N-drmethylformamlde are about 2 min and about 3.6 min, Chromatographic system
respectively.] , (See Chromatography (621), System Suitability.)
Sample solution: 2.4 mg/mL of amifostine from Amifostine Mode: LC
for Injection in water. [NOTE-Inject immediately after Detector: UV 247 nm
preparation.] Column: 4.6-mm x 25-cm; 5-lJm packing L1
System suitability Autosampler temperature: 4°
Samples: Standardsolution 1 and Standardsolution2 Flow rate: 1.0 mL/min
Suitability requirements Injection size: 10 IJL
Relati~e standard deviation: NMT 10.0%, Standard
System suitability
solution 1; NMT 4.0%, Standardsolution 2 Sample: Standardsolution
Analysis Suitability requirements
Samples: Standardsolution 1, Standard solution 2 and Tailing factor: NMT 2.5
Sample solution ' Relative standard deviation: NMT 4.0%
Calculate the percentage of amifostine thiol in the portion of Analysis
sample taken: Samples: Standardsolution and Sample solution
Calculate the percentage of amifostine disulfide in the
Result = (ru/rs) x (Cs/Cu) x (M rdM r2) x 100 portion of sample taken:

= peak response of amifostine thiol from the Result =(ru/rs) x (Cs/Cu) x (M r1/Mr2 ) x 100
Sample solution .
= peak response of amifostine thiol from Standard = peak response of amifostine disulfide from the
solution 1 Sample solution
=concentration of USP Amifostine Thiol RS in = peak response of amifostine disulfide from the
Standardsolution 1 (mg/mL) Standardsolution
= concentration of amifostine in the Sample =concentration of USP Amifostine Disulfide RS in
solution (mg/mL) the Standardsolution(mg/mL)
= molecular weight of amifostine thiol, 134.24 = concentration of amifostine in the Sample
solution (mg/mL)
= molecular weight of amifostine thiol
. = molecular weight of amifostine disulfide, 266.47
dihydrochloride, 207.17
= molecular weight of amifostine disulfide
Calcula~e the percenta~e o! sodium thiophosphate or tetrahydrochloride, 412.31
N,N-drmethylformamrde In the portion of sample taken, if
present: Acceptance criteria: NMT 2.0% of total impurities, including
amifostine thiol and amifostine disulfide
Result =(ru/rs) x (Cs/Cu) x 100

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 USP 43 OfficialMonographs / Amikacin 219

SPECIFIC TESTS IDENTIFICATION

II CONSTITUTED SOLUTION: At the time of use, it meets the

requirements for Injections and ImplantedDrug Products (1),

Specific Tests, Completeness and clarity of solutions. When
constituted with 0.9% Sodium Chloride Injection, the • A •. i~ii~,~~I~~~~~~I~!\I.~~~fl·~~~~~~~~~~~~~i(1\Q7),:lnf r(1i~(j
solution must completely dissolve in 45 s. ~pg~Jfo§sgpY:«lQZK<qFliQ7A.l·(<:.NJ~May~2ozo)
II X-RAY DIFFRACTION (941): Its X-ray diffraction pattern II B. The retention time of the peak for amikacin of the

conforms to that of USP Amifostine RS, similarly Sample solution corresponds to that of the Standard
determined. solution, as obtained in the Assay.
II STERILITY TESTS (71): It meets the requirements when
ASSAY
tested as directed for Test for Sterility of the Product to be
Examined, Membrane Filtration. • PROCEDURE
II pH (791): 6.5-7.5, in a solution constituted as directed in
Mobile phase: 134 mM sodium hydroxide, prepared as
follows. Transfer a volume of deionized water to a suitable
the labeling
II WATER DETERMINATION, Method Ie (921)
plastic container, sonicate, degas, and sparge with helium.
Sample solution: To 100.0 mg of Amifostine for Injection, While stirring, slowly add sodium hydroxide solution of a
contained in a stoppered centrifuge tube, add 10.0 mL of suitable concentration.
a solution of N-ethylmaleimide in methanol (4 in 100), and [NOTE-Prepare fresh daily. The Mobile phase readily
absorbs carbon dioxide and produces carbonate that
sonicate for 15 min. Shaketo disperse, and sonicate for an
changes the retention time of amikacin. The use of a
additional 15 min. Use 1.0 mL of the supernatant.
50% (w/w), low-carbonate sodium hydroxide solution
Acceptance criteria: 18.0%-22.0%
is recommended.]
II PARTICULATE MATTER IN INJECTIONS (788): Meets the
System suitability solution: 0.02 mg/mL of USP Arnlkacln
requirements for small-volume injections
RS and 0.008 mg/mL of USP Kanamycin Sulfate RS in water
• BACTERIAL ENDOTOXINS TEST (85): Contains NMT 0.2 USP
Standard solution: 0.02 mg/mL of USP Amikacin RS in
Endotoxin Unit/mg of amifostine
water
• OTHER REQUIREMENTS: Meets the requirements for Labeling
Sample solution: 0.02 mg/mL of Amikacin in water
(7), Labels and Labeling for Injectable Products.
Chromatographic system
ADDITIONAL REQUIREMENTS (See Chromatography (621), System Suitability.)
• PACKAGING AND STORAGE: Preserve as described in Mode: LC
Packaging and Storage Requirements (659), Injection Detector: Electrochemical
Packaging, and store at controlled room temperature. Mode: Integrated amperometric
• USP REFERENCE STANDARDS (11) Electrodes
USP Amifostine RS Working: Gold
USP Amifostine Disulfide RS Reference: Silver-silver chloride
1,3-Propanediamine, N,N-(dithiodi-2,1-ethanediyl)bis, Detector settings: See Table 7.
tetra hydrochloride.
CloH30N4S2CI4 412.32 Table 1
USP Amifostine Thiol RS Time Potential
Ethanethiol,2-[(3-aminopropyl)aminoJ-, Step (s) (V) Integration
dihydrochloride. 1 0.00 +0.04 -
CsH16NzSCIz 207.17
2 0.30 +0.04 Begins
3 0.50 +0.04 Ends
4 0.51 +0.80 -
Amikacin 5 0.70 +0.80 -
6 0.71 -0.80 -
7 0.90 -0.80 -

Column: 4-mm x 25-cm; 7.5-lJm packing L47

[NOTE-A guard column of packing L47 is
recommended.]
Flow rate: 0.5 mL/min
Injection volume: 20 IJL
CZZH43Ns013 585.60 System suitability
o-Streptamine, 0-3-amino-3-deoxy-a.-o-glucopyranosyl- Samples: System suitability solution and Standardsolution
(1 ~6)-O-[ 6-amino-6-deoxy-a.-o-glucopyranosyl-(1 ~4)]- [NOTE-The relative retention times for kanamycin and
Nl-(4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-, (5)-; amikacin are 0.8 and 1.0, respectively.]
0-3-Amino-3-deoxy-a.-o-glucopyranosyl(1 ~4)-O-[6-amino- Suitability requirements
6-deoxy-a.-o-glucopyranosyl(1 ~6) ]_N3_(4-amino-L-2- Resolution: NLT 3 between kanamycin and amikacin,
hydroxybutyryl)-2-deoxy-L-streptamine [37517-28-5]. System suitability solution
DEFINITION Tailing factor: NMT 2, Standardsolution
Amikacin has a potency of NLT 900 IJg/mg of amikacin Relative standard deviation: NMT 3%, Standard
(CZZH43Ns013)' calculated on the anhydrous basis. solution
Analysis
Samples: Standardsolution and Sample solution

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 220 Amikacin / Official Monographs USP 43

Calculate the quantity, in IJg, of amikacin (C22H43Ns013) in C22H43Ns013 . 2H2S04 781.76

each mg of Amikacin taken:
[39831-55-5].
Result =(r vir s) x (C sIC v) x P x F DEFINITION
Amikacin Sulfate having a molar ratio of amikacin to sulfuric
ru =peak response of amikacin from the Sample acid (H2S04) of 1:2 contains the equivalent of NLT 674
solution IJg/mg and NMT 786 IJg/mg of amikacin (C22H43Ns013)'
r5 = peak response of amikacin from the Standard calculated on the dried basis. Amikacin Sulfate having a
solution molar ratio of amikacin to sulfuric acid (H 2S04) of 1:1.8
C5 =concentration of USP Amikacin RS in the Standard contains the equivalent of NLT 691 IJg/mg and NMT 806IJgI
solution(mg/mL)
mg of amikacin (C22H43Ns013), calculated on the dried basis.
Cu =concentration of Amikacin in the Sample solution
(mg/mL) IDENTIFICATION
P =potency of amikacin in USP Amikacin RS
(mg/mg)
F =conversion factor, 1000 IJg/mg
Acceptance criteria: NLT 900 IJg/mg on the anhydrous
• A·~~~~~~~~~~9~i~1,~<
$p~qt!'9~(tqpy;\1i~.~K\Qt;YJ
basis Acceptance criteria: The IR absorption spectrum conforms
IMPURITIES
to that of USP Amikacin Sulfate RS, similarly obtained.
• RESIDUE ON IGNITION (281): NMT 1.0%, the charred residue
• B. The retention time of the peak for amikacin of the
being moistened with 2 mL of nitric acid and 5 drops of Sample solution corresponds to that of the Standard
sulfuric acid solution, as obtained in the Assay.
• C. IDENTIFICATION TESTS-GENERAL (191), Chemical
SPECIFIC TESTS Identification Tests, Sulfate: Meets the requirements
• OPTICAL ROTATION (781 S), Procedures, Specific Rotation
ASSAY
Sample solution: 20 mg/mL in water
.• PROCEDURE
Acceptance criteria: +97 0 to +105 0
• CRYSTALLINITY (695): Meets the requirements
Mobile phase: 134 mM sodium hydroxide, prepared as
follows. Transfer a volume of deionized water to a suitable
• pH (791) plastic container, sonicate, degas, and sparge with helium.
Sample solution: 10 mg/mL in water
Acceptance criteria: 9.5-11.5 While stirring, slowly add sodium hydroxide solution of a
suitable concentration.
• WATER DETER~INATION (921), Method I: NMT 8.5%
[NOTE-Prepare fresh daily. The Mobilephase readily
ADDITIONAL REQUIREMENTS absorbs carbon dioxide and produces carbonate that
• PACKAGING AND STORAGE: Preserve in tight containers. changes the retention time of amikacin. The use of a
• USP REFERENCE STANDARDS (11 ) 50% (w/w), low-carbonate sodium hydroxide solution
USP Amikacin RS is recommended.]
USP Kanamycin Sulfate RS System suitability solution: 0.02 mg/mL of USP Amikacin
RS and 0.008 mg/mL of USP Kanamycin Sulfate RS in water
Standard solution: 0.02 mg/mL of USP Amikacin RS in
water
Sample solution: Equivalent to 0.02 mg/mLof amikacin,
Amikacin Sulfate from Amikacin Sulfate, in water
Chromatographic system
<:0\ H,N O~NH' (See Chromatography (621), System Suitability.)
Mode: LC

""'''"K''''''Q-"" '"
HO bH HO )-\ OH • XH,S0 4
Detector: Electrochemical
Mode: Integrated amperometric
Electrodes
HO--;-.< ...../
Working: Gold
Reference: Silver-silver chloride
H,N OH Detector settings: See Table 1.
C22H43Ns013 . xH2S04 Table 1
D-Streptamine, 0-3-amino-3-deoxy-a-D-glucopyranosyl-
Time Potential
(1-6)-0-[6-amino-6-deoxy-a-D-glucopyranosyl-(1_4)]- Step (5) (V) Integration
N -(4-amino-2-hydroxy-1-oxobutyl)-2-deoxy-, (5)-, sulfate
(1:2 or 1:1.8 salt); 1 0.00 +0.04 -
0-3-Amino-3-deoxy-a-D-glucopyranosyl-(1-4)-0-[6-amino- 2 0.30 +0.04 Begins
6-deoxy-a-D-glucopyranosyl-(1_6)]-N3-(4-amino-L-2-
hydroxybutyryl)-2-deoxy-D-streptamine sulfate (1:2 or 3 0.50 +0.04 Ends
1:1.8 salt); 4 0.51 +0.80 .-
(25)-4-Amino-N-{(1 R,2S,3S,4R,55)-5-amino-2-[(3-amino-3-
deoxy-a-D-glucopyranosyl)oxy]-4-[(6-am ino-6-deoxy-a-D- 5 0.70 +0.80 -
9lucopyranosyl)oxy]-3-hydroxycyclohexyl}-2- 6 0.71 -0.80 -
hydroxybutanamide sulfate (1:2 or 1:1.8 salt).
7 0.90 -0.80 -
C22H43Ns013 . 1.8H 2S04 762.15
[149022-22-0].
Column: 4-mm x 25-cm; 7.5-lJm packing L47

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 USP 43 OfficialMonographs / Amikacin 221

[NOTE-A guard column of packing L47 is Acceptance criteria: NMT 13.0%

recommended.]
Flow rate: 0.5 mL/min ADDITIONAL REQUIREMENTS
Injection volume: 20 IJL • PACKAGING AND STORAGE: Preserve in tight containers.
System sultablllty • LABELING: Label it to indicate whether its molar ratio of
Samples: System suitability solution and Standardsolution amikacin to sulfuric acid (H 2S04) is 1:2 or 1:1.8.
[NOTE-The relative retention times for kanamycin and • USP REFERENCE STANDARDS (11)
amikacin are 0.8 and 1.0, respectively.] USP Amikacin RS
Suitability requirements USP Amikacin Sulfate RS
Resolution: NLT 3 between kanamycin and amikacin, USP Kanamycin Sulfate RS
System suitability solution
Tailing factor: NMT 2, Standardsolution
Relative standard deviation: NMT 3%, Standard
solution
Analysis
Amikacin Sulfate Injection
Samples: Standardsolution and Sample solution DEFINITION
Calculate the quantity, in IJg, of amikacin (C22H43Ns013) in Amikacin Sulfate Injection is a sterile solution of Amikacin
each mg of Amikacin Sulfate taken: Sulfate in Water for Injection, or of Amikacin in Water for
Injection prepared with the aid of Sulfuric Acid. It contains
Result = (r ulr s) x (C siC u) x P x F NLT 90.0% and NMT 120.0% of the labeled amount of
ru = peak response of amikacin from the Sample
amikacin (C22H43Ns013)'
solution IDENTIFICATION
rs = peak response of amikacin from the Standard • A. The retention time of the peak for amikacin of the
solution Sample solution corresponds to that of the Standard
Cs = concentration of USP Amikacin RS in the Standard solution, as obtained in the Assay.
solution (mg/mL)
Cu = concentration of amikacin in the Sample solution ASSAY
(mg/mL)
P = potency of amikacin in USP Amikacin RS
(mg/mg)
F = conversion factor, 1000 IJg/mg • PROCEDURE
Mobile phase:
fol ..
Acceptance critetia: See Table 2.
pi
Table 2
Acceptance .
Ratio of Criteria
Amikacin:Sulfuric Acid (J.lg/mg)a
1:2 674-786
1:1.8 691-806 .

a Calculated on the dried basis.

suitability solution: 0.02 mg/mL of USP Amikacin
and 0.008 mg/mL of USP Kanamycin Sulfate RS in water
IMPURITIES Standard solution: 0.02 mg/mL of USP Amikacin RS in
• RESIDUE ON IGNITION (281): NMT 1.0%, the charred residue
water
being moistened with 2 mL of nitric acid and 5 drops of Sample solution: Nominally 0.02 mg/mL of amikacin, from
sulfuric acid Injection in water
SPECIFIC TESTS Chromatographic system
• OPTICAL ROTATION (781 S), Procedures,Specific Rotation (See Chromatography (621), System SUitability.)
Sample solution: 20 mg/mL inwater Mode: LC
Acceptance criteria: +76 0 to +84 0 Detector: Electrochemical
• CRYSTALLINITY (695): Meets the requirements Detector mode: Integrated amperometric
• pH (791) Electrodes
Sample solution: 10 mg/mL in water Working: Gold
Acceptance criteria: See Table 3. Reference: Silver-silver chloride
Detector settings: See"'til:j]¢';~'.
Table 3
Ratio of Acceptance ~'J{il.1 ~I~,l
Amikacin:Sulfuric Acid Criteria Time Potential
St~p (s) (Ve) lo~~gl'a~i!?ij
1:2 2.0-4.0
1 o.lOO +0;04
1:1.8 6.0-7.3
2 O.~O +0,04 ~~g ilis
• Loss ON DRYING (731) 3 of5(j 'i:°.()4 Ends
Sample: 100 mg
Analysis: Dry the Sample under vacuum at a pressure not 4 0.51 ...0;80
exceeding 5 mm of mercury at 110 0 for 3 h.

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222 Amikacin / Official Monographs USP 43

Amiloride Hydrochloride
Step
CI' NJJ:
5
6
X~ ~
H,N N
..
NH,
NH, . HCI . 2H,O

7 C6H aCIN70 . HCI. 2H20 302.12

Pyrazinecarboxamide, 3,5-diamino-N-(aminoiminomethyl)-
e-chloro-, monohydrochloride dihydrate;
N-Amidino-3,5-diamino-6-chloropyrazinecarboxamide
monohydrochloride dihydrate [17440-83-4].
DEFINITION
rate: 0.5 mL/min Amiloride Hydrochloride contains NLT 98.0% and NMT
Injection volume: 20 IJL 101.0% of C6H aCIN70 . HCI, calculated on the dried basis.
System suitability
Samples: System sUitability solution and Standard solution IDENTIFICATION
[NoTE-The relative retention times for kanamycin and
amikacin are 0.8 and 1.0, respectively.]
Suitability requirements
Resolution: NLT 3 between kanamycin and amikacin,
System suitabilitysolution
Tailing factor: NMT 2, Standard solution
Relative standard deviation: NMT 3%, Standard
solution -
Analysis • B~~~~~$~~9~~9~1.~;I~~~~I~'~~~I~~~'I'~"-;~>J)~>~~)'
Samples: Standard solution CJltr:Q'(i,plgt-Vi$iplg$pectrp$qoPY:>1?:llll/(cNT;MaY;z02()
Calculate the percentage of Sample: 600 IJg/mL of water, diluted quantitatively and
amikacin (C22H43Ns013) in .+.... ;,..,;"~..;,. ,; ...~i;,..,; .... ;,;;" stepwise with 0.1 N hydrochloric acid to a concentration of
Injection taken: about 9.6 IJg/mL
Acceptance criteria: Meets the requirements
Result = (rvlrs) x (CsICv) x P x 100 • C. IDENTIFICATION TESTS-CENERAL, Chloride (191): Meets
the requirements
tu from the ASSAY
• PROCEDURE
rs from the Sample: 450 mg
Analysis: Dissolve the Sample in 100 mL of glacial acetic
Cs =concentration of USP Amikacin RS in the Standard acid, add 10 mL of mercuric acetate TS and 15 mL of
solution(mg/mL) dioxane. Add crystal violet TS. Titrate with 0.1 N perchloric
Cv =nominal concentrati~n of amikacin in the Sample acid VS to a blue endpoint. Perform a blank determination
solution'&(mg/ml).& (ERR 1"Sep~2018) (see Titrimetry(541». Each mL of 0.1 N perchloric acid is
P = potency of amikacin in USP Amikacin RS equivalent to 26.61 mg of C6H aCIN70 . HCI.
(mg/mg) Acceptance criteria: 98.0%-101.0% on the dried basis
Acceptance criteria: 90.0%-120.0% IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1%
SPECIFIC TESTS • ORGANIC IMPURITIES
• pH (791): 3.5-5.5 Standard solutions: Preparea series of solutions, A, B, C, D,
• PARTICULATE MATTER IN INJECTIONS (788): Meets the £, and F, of USP Amiloride Hydrochloride RS in a mixture of
requirements for small-volume injections methanol and chloroform (4:1) havinq concentrations of
• BACTERIAL ENDOTOXINS TEST (85): NMT 0.33 USP 4000, 40, 20, 8, 4, and 2 IJg/mL, respectively.
Endotoxin Units/mg of amikacin Sample solution: 4 mg/mL of Amiloride Hydrochloride in
• OTHER REQUIREMENTS: Meets the requirements under methanol and chloroform (4:1)
Injections and Implanted Drug Products (1) Chromatographic system
ADDITIONAL REQUIREMENTS (See Chromatography (621), Thin-Layer Chromatography.)
• PACKAGING AND STORAGE: Preserve in single-dose or in Mode: TLC
multiple-dose containers, preferably of Type I or Type III Adsorbent: 0.25-mm layer of chromatographic silica gel
glass. previously washed with methanol
Application volume: 5 IJL
Developing solvent system: Tetrahydrofuran and 3 N
ammonium hydroxide (15:2)
• USP REFERENCE STANDARDS (11) Analysis
USP Amikacin RS Samples: Standard solutions A, B, C, 0, E, and Fand Sample
.&:i(CN 1.May;20tS) solution
USP Kanamycin Sulfate RS Proceed as directed in the chapter. Dry the spots with a
stream of nitrogen, and develop the chromatograms in
the solvent system, until the solvent front has moved
about three-fourths of the length of the plate. Remove
the plate from the developing chamber, mark the

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 USP 43 OfficialMonographs / Amiloride 223

solvent front, allow to air-dry, and examine the plate Develop the plate until the solvent is about three-fourths of
under long-wavelength UV light: the R F value of the the length of the plate from the origin. Remove the plate
principal spot of the Sample solution corresponds to that from the developing chamber, air-dry, and examine
of Standard solution A. Estimate the levelsof any under short-wavelength UV light.
additional spots observed in the chromatogram of the Acceptance criteria: The RF value of the principal spot of the
Sample solution by comparison with the principal spots Sample solution corresponds to that of the Standard
in the chromatograms of Standard solutions B, C, 0, E, solution.
and F.
Acceptance criteria: The sum of the intensities of any ASSAY
additional spots observed is NMT that of the principal spot • PROCEDURE
obtained from Standard solution B (equivalent to 1%). Buffer: Dissolve 136 g of monobasic potassium phosphate
in 800 mL of water. Adjust with phosphoric acid to a pH of
SPECIFIC TESTS 3.0. Dilute with water to 1000 mL.
• ACIDITY Mobile phase: Methanol, water, and Buffer(25:71 :4)
Sample: 1.0 g Standard stock solution: 1.0 mg/mL of USP Amiloride
Analysis: Dissolve the Samplein 100 mL of a mixture of Hydrochloride RS in methanol
methanol and water (1:1). Titrate with 0.10 N sodium Standard solution: 0.1 mg/mL of USP Amiloride
hydroxide to a potentiometric endpoint. Hydrochloride RS from Standard stocksolution, prepared as
Acceptance criteria: NMT 0.30 mL is required (0.1% as follows. Transfer 5.0 mL of the Standard stock solution to a
HCI). 50-mL volumetric flask. Add 10.0 mL of methanol, 2.0 mL
• Loss ON DRYING of 0.1 N hydrochloric acid, and dilute with water to volume.
(See ThermalAnalysis (891).) Sample solution: Transferan amount equivalent to 5 mg of
[NoTE-The quantity taken for the determination may be amiloride hydrochloride, from NLT 20 finely powdered
adjusted, if necessary, for instrument sensitivity.] Tablets, to a 50-mL volumetric flask containing 15.0 mL of
Sample: 10 mg methanol and 2.0 mL of 0;1 N hydrochloric acid. Sonicate
Analysis: Determine the percentage of volatile substances for 10 min, dilute with water to volume, sonicate for an
by thermogravimetric analysis on an appropriately additional 10 min, and filter.
calibrated instrument using the Sample. Heat the specimen Chromatographic system
at the rate of 100 Imin between ambient temperature and (See Chromatography(621), System Suitability.)
225 0 in an atmosphere of nitrogen at a flow rate of 40 mLI Mode: LC
min. From the thermogram determine the accumulated Detector: UV 286 nm
loss in weight between ambient temperature and about Column: 3.9-mm x 30-cm; packing L1
200 0 on the plateau. Flow rate: 1 mL/min
Acceptance criteria: 11.0%-13.0% Injection volume: 10 IJL
System suitability
ADDITIONAL REQUIREMENTS Sample: Standard solution
• PACKAGING AND STORAGE: Preserve in well-closed Suitability requirements
containers. Tailing factor: NMT 2.0
• USP REFERENCE STANDARDS (11) Relative standard deviation: NMT 2.0%
USP Amiloride Hydrochloride RS Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
amiloride hydrochloride (C6H aCIN70 . HCI) in the portion
Amiloride Hydrochloride Tablets of Tablets taken:

DEFINITION Result= (rulrs) x (CsICu) x 100

Amiloride Hydrochloride Tablets contain NLT 90.0% and NMT
110.0% of the labeled amount of amiloride hydrochloride tu = peak response of amiloride from the Sample
(C6H aCIN70 . HCI). solution
ts = peak response of amiloride from the Standard
IDENTIFICATION solution
• A. The retention time of the major peak of the Sample Cs =concentration of USP Amiloride Hydrochloride RS
solution corresponds to that of the Standard solution, as in the Standard solution (mg/mL)
obtained in the Assay. Cu = nominal concentration of amiloride
• B. hydrochloride in the Sample solution (mg/mL)
Standard solution: 0.2 mg/mL of USP Amiloride
Hydrochloride RS in methanol Acceptance criteria: 90.0%-110.0%
Sample solution: Prepare from finely ground Tablets, PERFORMANCE TESTS
equivalent to 0.2 mg/mL of amiloride hydrochloride in
• DISSOLUTION (711)
methanol. Filter the solution. Medium: 0.1 N hydrochloric acid; 900 ml,
Chromatographic system
Apparatus 2: 50 rpm
(See Chromatography (621), Thin-Layer Chromatography.)
Time: 30 min
Mode: TLC Instrumental conditions
Adsorbent: 0.25-mm layer of chromatographic silica gel
Mode: UV
mixture
Analytical wavelength: 363 nm
Developing solvent system: Tetrahydrofuran and 3 N
Standard solution: USP Amiloride Hydrochloride RS of
ammonium hydroxide (88:12)
known concentration in Medium. [NOTE-An amount of
Application volume: 10 IJL methanol not to exceed 2% of the total volume of the
Analysis
Samples: Standard solution and Sample solution

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224 Amiloride / Official Monographs USP 43

Standard solution may be used to dissolve the amiloride SUitability requirements

hydrochloride.] Relative standard deviation: NMT 3.0%
Sample solution: Sample per Dissolution (711). Dilute with Analysis
Medium as necessary. Samples: Standard solution and Sample solution
Analysis Calculate the percentage of each individual impurity in the
Samples: Standard solution and Sample solution portion of Tablets taken:
Tolerances: NLT 80% (Q) of the labeled amount of
amiloride hydrochloride (C6H sCIN 70 . HCI) is dissolved. Result =(rulrs) x (CsICu) x 100
• UNIFORMITY OF DOSAGE UNITS (905)
Procedure for content uniformity tu = peak response of each impurity from the Sample
Standard solution: 10 J,lg/mL of USP Amiloride solution
Hydrochloride RS in 0.1 N hydrochloric acid rs = peak response of amiloride from the Standard
Sample solution: 10 J,lg/mL of amiloride hydrochloride solution
prepared as follows. Transfer one finely powdered Tablet Cs = concentration of USP Amiloride Hydrochloride RS
to a 1OO-mL volumetric flask, add 60 mL of 0.1 N in the Standard solution (mg/mL)
hydrochloric acid, and shake by mechanical means for 30 Cu = nominal concentration of amiloride
min. Dilute with 0.1 N hydrochloric acid to volume, and hydrochloride in the Sample solution (mg/mL)
centrifuge a portion of the mixture. Dilute a portion of the
clear supernatant to obtain the required concentration. Acceptance criteria: See Table 7.
Instrumental conditions
Mode: UV Table 1
Analytical wavelength: 363 nm Relative Acceptance
Blank: 0.1 Nhydrochloric acid Retention Criteria,
Name Time NMT (%)
Analysis
Samples: Standard solution and Sample solution Chloropyrazine acida• e 0.15 -
Calculate the percentage of the labeled amount of
Chloropyrazine methyl car-
amiloride hydrochloride (C6H sCIN 70 . HC!) in the Tablet . boxyiate'': e 0.48 -
taken:
Chloropyrazine carboxa-
midec• e 0.56 -
Result = (AulAs) x (Csi Cu) x 100
Ami/oride 1.00 -
= absorbance of amiloride hydrochloride in the
Sample solution Any other unknown impurity - 0.5
= absorbance of amiloride hydrochloride in the TotalIrnpurltles" - 2.0
Standard solution
= concentration of USP Amiloride Hydrochloride RS a 3,5,-Diamin-6-chloropyrazine carboxylic acid.
in the Standard solution (J,lg/mL) . b 3,5,-Diamin-6-chloropyrazine-2-carboxylate.
= nominal concentration of amiloride c N-Ammidine-3-amino-5-hydroxy-6-chloropyrazine-2-carboxamide
hydrochloride in the Sample solution (J,lg/mL) hydrochloride.
d Totalimpurities is the sum of allthe impurities including process-related.
Disregard peakslessthan 0.05%.
Acceptance criteria: Meet the requirements e Process-related impurity, controlledin the drug substance.
IMPURITIES
• ORGANIC IMPURITIES ADDITIONAL REQUIREMENTS
Diluent: Methanol, 1 N hydrochloric acid, and water • PACKAGING AND STORAGE: Preserve in well-closed
(40:4:56) containers.
Buffer: 0.9 giL of sodium l-hexanesulfonate in water. • USP REFERENCE STANDARDS (11)
Initially add water to about 90% of the volume of the flask, USP Amiloride Hydrochloride RS
adjust with diluted phosphoric acid to a pH of 3.0 ± 0.1,
and dilute with water to volume.
Mobile phase: Acetonitrile and Buffer (10:90)
Standard solution: 0.01 mg/mL of USP Amiloride
Hydrochloride RS in Diluent Amiloride Hydrochloride and
Sample solution: Nominally equivalent to 2 mg/mL of Hydrochlorothiazide Tablets
amiloride hydrochloride in Diluent from NLT 20 powdered
Tablets. Initially add methanol to fill about 40% of the DEFINITION
volume of the flask and 1 N hydrochloric acid to about 4% Amiloride Hydrochloride and Hydrochlorothiazide Tablets
of the volume of the flask. Sonicate for 10 min, dilute with contain NLT 90.0% and NMT 110.0% of the labeled
water to volume, and sonicate for another 10 min. Pass amount of amiloride hydrochloride (C6H sCIN 70 . HCI) and
through a suitable filter of 0.45-J,lm pore size. hydrochlorothiazide (C7HBCIN304S2)'
Chromatographic system
(See Chromatography (621), SystemSuitability.) IDENTIFICATION
Mode: LC • A. The retention times of the major peaksof the Sample
Detector: UV 350 nm solution correspond to those of the Standard solution, as
Column: 4.6-mm x 15-cm; 4-J,lm packing L1 obtained in the Assay.
Flow rate: 2 mL/min
Injection volume: 10 J,lL
System suitability
Sample: Standard solution

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USP 43 Official Monographs / Amiloride 225

wavelerigfhsasthat ofthe Stand ·olution

the Assay..·The UVspectrum·of. yd
peakrof the Dili.!ted sample solutionexh ru =peak response of amiloride AA(USP 1~Aog-i(19) from
minima at the same wavelengths as tha the Sample solution ." ,..">
standardsplution, as obtained in the,Asso rs =peak response of amiloride "'A(USP1-Aug~2019) from
the Standard solution .
ASSAY Cs =concentration of USP Amiloride Hydrochloride
RSA~(USP1:AU9-io19) in the Standard solution
,Chqnge. toiead: (mg/mL)
• PROCEDURE Cu =nominal concentration of amiloride
Buffer: Dissolve 136 9 of monobasic potassium phosphate hydrochloride in the Sample solution (mg/mL)
in 800 mL of water. Adjust with phosphoric acid to a pH of
3.0. Dilute with water to 1000 mL. Calculate the percentage of the labeled amount of
Mobile phase: (Methanol, water, and Buffer (25:~1 :~) hydrochlorothiazide (C7HsCIN304Sz) in the portion of
Standard stock solution: 1.0 mg/mL of USP Arniloride Tablets taken:
Hydrochloride RS in methanol
Standard solution: 0.1 mg/mL of USP Amiloride Result = (ru/rs) x (CslCu) x 100
Hydrochloride RS and 1 mg/mL of USP
Hydrochlorothiazide RS, prepared as follows. Transfer 10.0 tu =peak response of hydrochlorothiazide from the
mL of the Standard stock solution to a 1OO-mL volumetric Sample solution
flask containing 100 mg of USP Hydrochlorothiazide RS rs =peak response of hydrochlorothiazide from the
and 20.0 mL of methanol. Add 4.0 mL of 1 N hydrochloric Standard solution
acid, and dilute with water to vol~me .. Cs = concentration of USP Hydrochlorothiazide RS in
the Standard solution (mg/mL)
ADiluted standard solution: 0.005 m
Amiloride rochloride . 5
Cu =nominal concentration of hydrochlorothiazide
in the Sample solution (mg/mL)
Hydrochl a
diluted wit Acceptance criteria: 90.0%-110.0% of the labeled amount
Sample solution: . ~e of amiloride hydrochloride (C6H sCIN70 . HCI) and
hydrochloride and '. /mL of zlde, hydrochlorothiazide (C7HsCIN304Sz)
prepared asfoHow.A(USP.1-Aug-2019) Transfer an equivalent to
5 mg of amiloride hydrochloride Aand 50 rn9,of PERFORMANCE TESTS
hydrochlorothiazideA (USP l-Aug-2019) from powdered Tablets
(NLT 20) to a 50~mL volumetric flask. Add 15.0 mL of
methanol and 2.0 mL of 1 N hydrochloric acid. Sonicate for
10 min, dilute with water to volume, sonicate for an • DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL
additional 10 min, and filter. . . . '.. . ' ..
Apparatus 2: 50 rpm
ADiluted sample solution: Nominally 0.005 mg/mL of
Time: 30 min . . .
amiloride hydrochloride and 0.05 mg/mL of. . . .. '.'_ ADetermi 'ercentage of the labeled amount of
hydrochlorothiazide, from the Sample solution, diluted With
hloride and hydrochlorothiazide
waterA(USP l-Aug-2019) . pectrophotomf!tric procedure or the
Chromatographic system
(See Chromatography (621), System Suitability.)
cedure.
Mode: LC ... Sp .. ho procedure... (USP l-Aug-2019)
Detector: UV 286 nm. AFo'rldentification B . Amiloride standard solution: AO.005 mg/mL of USP
array detector in the range of 200-400 n eH . loride RS, prepared asfollows. Transfer
SUS loride Hydrochloride RS tOA(USP1-Aug-2019)
Column: 3.9-mm x 30-cm; A1 O-l-lrnA (liSP)
L1 a
200-mL volumetric flask. Dissolve in and dilute with
methanol to volume. Transfer 2.0 mL of this solution to a
Flow rate: 1 mL/min
1OO-mL volumetric flask, and dilute with Medium to
Injection volume: 10 I-lL . . .. .,
ARun time: NLT 2 timestheretentiontiQ1e ()f volume.
Hydrochlorothiazide standard solution: 0.01 mg/mL of
amiiorideA(uspl-Aug-2019)
USP Hydrochlorothiazide RS, prepared asfollows. Transfer
System suitability
100 mg of USP Hydrochlorothiazide RS to a 100-mL
Sample: Standard solution
volumetric flask. Dissolve in and dilute with methanol to
[NoTE-The relative retention times for .., .... volume. Transfer 5.0 mL of this solution to a 100-mL
hydrochlorothiazide and arnllorldee A (USi;1~Aug~2019) volumetric flask, and dilute with Medium to volume.
are about 0.7 and 1.0, respectively.] Transfer 10.0 mL of the resulting solution to a 50-mL
Suitability requirements volumetric flask, and dilute with Medium to volume.
Resolution: NLT 2.0.between hydrochlorothiazide and Sample solution A: Pass a portion of the solution under
amiiorideA.A (USP'l~Aug-2019) test through a glass fiber filter of 0.45-~m pore si~e.
Relative standard deviation: NMT 2.0% for Sample solution B: Transfer 5.0 mL of Sample solutionA to
hydrochlorothiazide and amiloride a 25-mL volumetric flask, and dilute with Medium to
Analysis volume.
Samples: Standard solution and Sample solution Blank: Medium
Calculate the percentage of the labeled amo~nt of . Instrumental conditions
amiloride hydrochloride (C6H sCIN70 . HCI) In the portion Mode: UV-Vis
of Tablets taken: Analytical Wavelengths: 363 nm for amiloride
hydrochloride; 270 nm for hydrochlorothiazide

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226 Amiloride / Official Monographs USP43

Analysis Chromatographic system

Samples: Amiloride standard solution, Hydrochlorothiazide (See Chromatography (621), System SUitability.)
standard solution, Sample solution A, and Sample Mode: LC
solution B Detector: UV 286 nm
Calculate the percentage ~of the I~beled Column: 4.6-mm x A1S-cm; 5-J.JmA(USP1-f.~g~2019) packing
amoi.mtJ: (USP 1~Aug-2019) of arnllorlde hydrochloride L1
(C6H aClN 70 . HCI) dissolved: Flow rate: 1 mL/min
Injection volume: 50 J.JL
Result = [(Au x Cs x \I)/(As x L)] x 100 System suitability
Sample: Standard solution
Au =absorbance of Sample solution A Suitability requirements
Cs = concentration oL"USPArTIHoride Hydrochloride Resolution: NLT 2.0 AA(lJSP l-A!J;S919) between
RS in... (USP l-Aug-2019) the Amiloride standard solution hydrochlorothiazideand amiloride
(mg/mL) Relative standard deviation: NMT 2.0%
V =volume of Medium, 900 mL Analysis
As =absorbance of the Amiloride standard solution Samples: Standard solution and Sample solution
L = label claim of amiloride Calculate the Apercentage of tb~labeleCJ'" (Usr.l~Aug,201~j
;·hydrochloride. (USP 1~Aug.2~1'9) (mg/Tablet) amount of amiloride hydrochloride (C6H aCIN70 . HCI)
and hydrochlorothiazide (C7HaCIN304Sz) dissolved:
"'Calculate F, the ratio of eAmiloride
standard solution at 27 3nm: Result = (ru/rs) x (CslL) x Vx 100

Resul~,= Asil~/As~63 tu = peak response of amiloride or

hydrochlorothiazidefrom the Sample solution
AS270 == absorbance of ;t~e Amiloride standard solution at rs = peak response of amiloride or
270 nm - . hydrochlorothiazide from the Standard solution
== absorbance of the Amiloride staridard solution at Cs = concentration of .A . e
. 363nm . RS or USP Hydroc )
in the Standard solution (mg/mL)
Calculate Auo the' absorbance L = label claim of amiloride hydrochloride or
nm, correct.eq f()r the interfere hydrochlorothiazide (mg/Tablet)
V = volume of Medium, 900 mL
.~esult =Av2lo -. [(AU363 x. 17/D]
Tolerances: NLT 80% (Q) of the labeled amount of
AU270 =absorbance of SamplesolLitio CTl amiloride hydrochloride (C6H aCIN 70· HCI) and NLT 75%
AU363 = absorbar:lce of Sample,'soltitkmAaf 3 3 n-m (Q) of the labeled amount of hydrochlorothiazide
F == ratio of the absorban oridestandard (C7HaClN304Sz) are dissolved.
solution at 270 nm t . m
o = diluf ctor of Sampl Change to read:
S... (u 9)
• UNIFORMITY OF DOSAGE UNITS (905), Content Uniformity:
Calculate the percentage ·...ofthe labeled Meet the requirements...... (USP l-At.ig~2019)
amount... (USP 1~Aug-2019) of hydrochlorothiazide IMPURITIES
(C7HaClN304Sz) dissolved: .
Change to read:
Result = [(Aue x Cs x V x D)/(A s x L)] x 100
• AllMIT OF BENZOTHIADIAZIN"E'RELATED COMPOUND
Aue = corrected absorbance of Sample A... (USP 1.Aug·2019) . - .
solution'" 8... (USP1. 019) at 270 nm Buffer, Mobile phase, and Sample solution: Prepare as
=concentration of'" P HydrochlorothiazideRS directed in the Assay.
in... (USP 1-Aug-2019) the Hydrochlorothiazide standard ...System suitability solutio se the' Standard so/utiop
solution (mg/mL) from the Assay.... (USP l-Aug-
V =volume of Medium, 900 mL Standard solution: ... 0.01mg/m( .... (lJSP ~-A~~-1~19) of USP
o = dilution factor of Sample solution B, 5 Benzothiadiazine Related Com ound A RS in Mobile phase
As = absorbance of the Hydrochlorothiazide standard Chromatographic system:
solution Assay, except for the Inject
L = label claim of hydrochlorothiazide (mg/Tablet) Injection volume: 20 IJL.
IJL. ... (USP 1.Aug-2(>19)
AChromatographicprocedure.(US~1:AU9·20jC~)
System suitability
Buffer and Mobile phase: Prepare as directed in the Assay. Sample: ... System suitability solution... (USP1.A~g-2(n9)
Standard stock solution: Usethe Standard solution from
the Assay. [NoTE-The relative retention times for
Standard solution: 0.005 mg/mL of USP Amiloride hydrochlorothiazide and amiloride l'...':(tJsP 1:Aug-~()'9)
Hydrochloride RS and 0.05 mg/mL of USP are about 0.7 and 1.0, respectively.]
Hydrochlorothiazide RS, from the Standard stock solution, Suitability requirements
diluted with Medium Resolution: NLT 2.0 between hydrochlorothiazide and
Sample solution: Pass a portion of the solution under test arnllorldee... (USP l-Aug~2019)
through a filter of 0.45-J.Jm pore size.

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 USP43 OfficialMonographs / Amiloxate 227

Relative standard deviation: NMT 2.0% ASSAY

I"l·Xg~ggfflI9fQltlj~~ig~ii~i!g~~;miJJi~tg:i • PROCEDURE
Analysis Standard solution: 20 mg/mL of USP Amiloxate RS in
Samples: Sample solution and Standard solution tert-butyl methyl ether
Calculate the percentage of benzothiadiazine related Sample solution: 20 mg/mL of Amiloxate in tert-butyl
compound A in the portion of Tablets taken: methyl ether
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
rv = peak response of benzothiadiazine related Detector: Flame ionization
compound A from the Sample solution Column: 0.32-mm x 25-m; coated with a O.t-um film of
ts = peak response of benzothiadiazine related phase Gl
compound A from the Standard solution Temperatures
Cs =concentration of USP Benzothiadiazine Related Injection port: 2400
A solution Detector: 260 0
Column: See Table 7.

the Sample Table 1

Hold Time
Initial Temperature Final at Final
Temperature Ramp Temperature Temperature
e) e/min) e) (min)
Acceptance criteria: NMT 1.0%
60 8 240 10
ADDITIONAL REQUIREMENTS
Carrier gas: Helium
Flow rate: 6 mL/min
• PACKAGING AND STORAGE: . Injection volume: 1 IJL
containers. :6rE~ at:cc.ntl·oll¢drd(~m'.tel1)P~tal~cil:e:Praite~t System suitability
from light. Sample: Standardsolution
• USP REFERENCE (11) Suitability requirements
USP Amiloride Hydrochloride RS Relative standard deviation: NMT 0.73%
USP Benzothiadiazine Related Compound A RS Analysis
4-Amino-6-chloro-l,3-benzenedisulfonamide. Samples: Standard solution and Sample solution
C6HsCIN304S2 285.73 Calculate the percentage of amiloxate (C15H 200 3) in the
USP Hydrochlorothiazide RS portion of Amiloxate taken:
Result = (rv/rs) x (Cs/Cu) x 100
= peak response from the Sample solution
Amiloxate = peak response from the Standard solution
= concentration of USP Amiloxate RS in the
o CH3 Standard solution (mg/mL)

~'
= concentration of Amiloxate in the Samplesolution
(mg/mL) .
H3CO

Acceptance criteria: 98.0%-102.0%

C15H2003 248.32
4-Methoxycinnamic acid, isoamyl ester; IMPURITIES
3-Methylbutyl 3-(4-methoxyphenyl)-(E)-2-propenoate • ORGANIC IMPURITIES
[71617-10-2]. Standard solution, Sample solution, Chromatographic
system, and System suitability: Proceed as directed in the
DEFINITION Assay.
Amiloxate contains NLT 98.0% and NMT 102.0% of amiloxate Analysis
(C15H2003)' Sample: Samplesolution
IDENTIFICATION Calculate the percentage of each impurity in the portion of
Amiloxate taken:
Result = (rufrT) x 100

to = peak response of each impurity from the Sample

solution
rr =sum of all the peak responses, excluding the
solvent peak, from the Sample solution
Acceptance criteria
Sample so utlon: 5.0 IJg/mL in alcohol Individual impurities: NMT 0.5%
• C. The retention time of the major peak of the Sample Total impurities: NMT 2.0%
solution corresponds to that of the Standard solution, as SPECIFIC TESTS
obtained in the Assay. • SPECIFIC GRAVITY (841): 1.037-1.041

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228 Amiloxate / OfficialMonographs USP 43

• REFRACTIVE INDEX (831): 1.556-1 .560 at 20 0 Injection volume: 5 IJl

• ACIDITY System suitability
Sample solution: Transfer 50 ml of alcohol to a suitable Sample: Standard solution
container. Add 1 ml of phenolphthalein TS and sufficient Suitability requirements
0.1 N sodium hydroxide to obtain a persistent pink color. Tailing factor: NMT 2.0
Transfer 50 ml of this solution to a suitable container, and Relative standard deviation: NMT 0.73%
add 5.0 ml of Amiloxate. Analysis
Analysis: Titrate with 0.1 N sodium hydroxide VS. Samples: Standard solution and Sample solution
Acceptance criteria: NMT 0.2 mL of titrant per ml of Calculate the percentage of aminobenzoate potassium
Amiloxate is required for neutralization. (C7H 6KN02) in the portion of Aminobenzoate Potassium
ADDITIONAL REQUIREMENTS taken:
• PACKAGING AND STORAGE: Preserve in tight containers. Result= (rulrs) x (CsICu) x 100
• USP REFERENCE STANDARDS (11)
USPAmiloxate RS to = peak response from the Sample solution
ts = peak response from the Standardsolution
Cs =concentration of USP Aminobenzoate Potassium
RS in the Standard solution (mg/ml)
Aminobenzoate Potassium Cu =concentration of Aminobenzoate Potassium in
the Sample solution (mg/ml)

~ -~ Acceptance criteria: 98.0%-102.0% on the dried basis

- llA NH,
IMPURITIES
C7H6KN02 175.23
Benzoic acid, 4-amino-, potassium salt;
Potassium 4-aminobenzoate [138-84-1].
DEFINITION
Aminobenzoate Potassium contains NlT 98.0% and NMT
102.0% of aminobenzoate potassium (C7H6KN02) ,
calculated on the dried basis. • LIMIT OF ANILINE AND p-TOLUIDINE
Diluent: Methylene chloride
IDENTIFICATION Standard stock solution: 0.1 mg/ml each of USP Aniline RS
and USP p-Toluidine RS in Diluent
Standard solution: 1 IJg/ml each of USP Aniline RS and USP
p-Toluidine RS in Diluent from Standard stock solution
• A•.·.~·.S~~~"'~9~~qr'~I~~~~I~I~~!IPNr"'~$t~(1~• ~);r·'lnf,.a.,.~a Sample solution: 100 mg/ml of Aminobenzoate
Spgc.trg~.c.9PY;3l.~~~.i(CN1.¥Mil.Y~~Q4.0) Potassium in Diluent prepared as follows. Add an
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as appropriate quantity of Aminobenzoate Potassium to a
obtained in the Assay. suitable volumetric flask and dilute with Diluent to volume.
Agitate for 10 min on a shaker and centrifuge at 3000 rpm
• c. for 5 min. Use the supernatant.
Sample solution: 10 mg/ml of Aminobenzoate Potassium
in water - Chromatographic system
Acceptance criteria: The Sample solution imparts a violet (See Chromatography (621), System Suitability.)
color to a non luminous flame. Since the presence of small Mode: GC
quantities of sodium masks the color, screen out the yellow Detectors
Flame ionization: 300 0
color produced by sodium by viewing through a blue filter
that blocks emission at 589 nm (sodium) but is transparent Hydrogen: 40 ml/min
to emission at 404 nm (potassium). [NOTE-Traditionally, Air: 400 ml/min
cobalt glass has been used, but other suitable filters are Column: 30-m x 0.32-mm fused silica capillary; coated
commercially available.] with 0.5-lJm film of phase G27
Temperatures
ASSAY Injection port: 280 0
• PROCEDURE Detector: 300 0
Solution A: 1.5% acetic acid, prepared by mixing 690 ml Column: See Table 1.
of water with 10 ml of glacial acetic acid and passing
through a suitable filter of 0.45-lJm pore size Table 1
Mobile phase: Methanol and Solution A (15:85) Hold Time at
Standard solution: 0.1 mg/ml of USP Aminobenzoate Initial Temperature Final _ Final
Potassium RS in Mobilephase. Sonicate to dissolve. Temperature Ramp Temperature Temperature
(") e/min) (") (min)
Sample solution: 0.1 mg/ml of Aminobenzoate Potassium
in Mobilephase. Sonicate to dissolve. 130 - 130 4
Chromatographic system
130 20 180 5
(See Chromatography (621), System Suitability.)
Mode: lC
Detector: UV 280 nm Carrier gas:- Helium
Column: 3.0-mm x 15-cm; 3.5-lJm packing III Flow rate: 1.0 ml/min
Flow rate: 0.35 ml/min 'Injection volume: 2 IJl

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USP 43 OfficialMonographs / Aminobenzoate 229

Injection type: Split ratio, 1:10 Column: 3.0-mm x 15-cm; 3.5-lJm packing L11
System suitability Flow rate: 0.4 mL/min
Sample: Standard solution Injection volume: 5 IJL
[NOTE-The relative te~~Dti?D ~im~~;.gt~D!I.ige and System suitability
p-toluidine are~()&\~l"lq'J~9(:~(ERgl:$~P+2()18) Samples: System suitabilitysolution and Standard solution
respectively.] SUitability requirements
Suitability requirements Resolution: NLT 1.5 between benzocaine and
Resolution: NLT 7.0 between aniline and p-toluidine 4-nitrobenzoic acid, System suitability solution
Tailing factor: NMT 1.5 for aniline and p-toluidine Relative standard deviation: NMT 3% for the
Relative standard deviation: NMT 6.0% for aniline and aminobenzoate potassium, 4-nitrobenzoic acid, and
p~toluidine benzocaine peaks, Standard solution
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standard solution and Sample solution
Calculate the percentage of p-toluidine or aniline in the Calculate the percentage of 4-nitrobenzoic acid or
portion of Aminobenzoate Potassium taken: benzocaine in the portion of Aminobenzoate Potassium
taken:
Result = (rvirs) x (CsiCv) x 100
Result = (rvi rs) x (CsiCv) x 100
= peak response of p-toluidine or aniline from the
Sample solution = peak response of 4-nitrobenzoic acid or
= peak response of p-toluidine or aniline from the benzocaine from the Sample solution
Standard solution = peak response of 4-nitrobenzoic acid or
=concentration of the corresponding USP benzocaine from the Standard solution
Reference Standard in the Standard solution(mgl = concentration of USP 4-Nitrobenzoic Acid RS or
mL) USP Benzocaine RS in the Standard solution(mgl
= concentration of Aminobenzoate Potassium in mL)
the Sample solution (mg/mL) = concentration of Aminobenzoate Potassium in
the Sample solution(mg/mL)
Acceptance criteria
Aniline: NMT 10 ppm Calculate the percentage of any individual unspecified
p-Toluidine: NMT 20 ppm impurity in the portion of Arnlnobenzoate Potassium
• ORGANIC IMPURITIES taken:
Solution A: 1.5% acetic acid, prepared by mixing 690 mL
of water with 10 rnl, of glacial acetic acid Result = (rvlrs) x (CsICv) x 100
Solution B: Methanol
Mobile phase: See Table 2. = peak response of any unspecified impurity from
the Sample solution
Table 2 = peak response of aminobenzoate from the
Standard solution
Time Solution A Solution B
(min) (%) (%) = concentration of USP Aminobenzoate Potassium
RS in the Standard solution (mg/mL)
0.0 85 15 =concentration of Aminobenzoate Potassium in
4.0 85 15 the Sample solution (mg/mL)

4.1 45 55 Acceptance criteria: See Table 3.

10.0 45 55
Table 3
10.1 85 15
Relative Acceptance
13 85 15 Retention Criteria,
Name Time NMT(%)

Diluent: Methanol and water (85:15) Aminobenzoic acid 1.0 -

System suitability solution: 1 mg/mL of USP Benzocaine 2.0 0.2
Aminobenzoate Potassium RS, 0.01 mg/mL of USP
4·Nitrobenzoic acid 2.1 0.2
4-Nitrobenzoic Acid RS, and 0.01 mg/mL of USP
BenzocaineRS in Diluent prepared asfollows. Transfer 1 mL Any individual
each of 0.1 mg/mL of USP 4-Nitrobenzoic Acid RS in unspecified -
methanol and 0.1 mg/mL of USP Benzocaine RS in Diluent impurity 0.10
to a 1O-mL volumetric flask containing the appropriate
amount of USP Aminobenzoate Potassium RS, and dilute SPECifiC TESTS
with Diluent to volume. • pH (791)
Standard solution: 1 IJg/mL each of USP Aminobenzoate Sample solution: 50 mg/mL of Aminobenzoate Potassium
Potassium RS, USP 4-Nitrobenzoic Acid RS, and USP in water
Benzocaine RS in Diluent Acceptance criteria: 8.0-9.0
Sample solution: 1 mg/mL of Aminobenzoate Potassium in • Loss ON DRYING (731)
Diluent Analysis: Dry at 105° for 2 h.
Chromatographic system Acceptance criteria: NMT 1.0%
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm

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230 Aminobenzoate / Official Monographs USP43

ADDITIONAL REQUIREMENTS Calculate the percentage of the labeled amount of

• PACKAGING AND STORAGE: Preserve in well-closed aminobenzoate potassium (C7H6KN02) in the portion of
containers. Capsules taken:
CD USP REfERENCE STANDARDS (11)

USP Aminobenzoate Potassium RS Result = (rulrs) x (CslCu) x 100

USP Aniline RS
Aniline. = peak response from the Sample solution
C6H 7N 93.13 = peak response from the Standard solution
USP Benzocaine RS = concentration of USPAminobenzoate Potassium
USP 4-Nitrobenzoic Acid RS RS in the Standard solution (mg/mL)
4-Nitrobenzoic acid. Cu = nominal concentration of aminobenzoate
C7H sN04 167.12 potassium in the Sample solution (mg/mL)
USP p-Toluidine RS
4-Methylaniline. Acceptance criteria: 90.0%-110%
C7H 9N 107.15
PERfORMANCE TESTS
• DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 1: 100 rpm
AminobenzoatePotassium Capsules Time: 45 min
Instrumental conditions
DEfINITION Mode: UV
Aminobenzoate Potassium Capsules contain NLT 90.0% and Analytical wavelength: Maximum absorbance at about
NMT 110.0% of the labeled amount of aminobenzoate 270nm
potassium (C 7H 6KN02) . Standard solution: A known concentration of USP
Aminobenzoate Potassium RS in Medium
IDENTIFICATION Sample solution: Filter portions of the solution under test,
CD A. and dilute with Medium, if necessary, in comparison with
Sample: 1 9 of the Capsule contents the Standard solution concentration.
Analysis: Dissolve the Sample in 25 mL of water, add 5 mL Analysis: Calculate the percentage of the labeled amount of
of 3 N hydrochloric acid, and wash the precipitate with two aminobenzoate potassium (C 7H6KN02) dissolved.
5-mL portions of cold water. Recrystallize from alcohol the Tolerances: NLT 75% (Q) of the labeled amount of
precipitate so obtained, and dry at 110° for 1 h. aminobenzoate potassium (C 7H6KN02) is dissolved.
Acceptance criteria: The p-aminobenzoic acid melts • UNIFORMITY OF DOSAGE UNITS (905): Meet the
between 186° and 189°. requirements
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as IMPURITIES
obtained in the Assay. • ORGANIC IMPURITIES
Solution A, Mobile phase, and Chromatographic system:
ASSAY Prepare as directed in the Assay.
• PROCEDURE Standard solution: 1 IJg/mL each of USP Aminobenzoate
Solution A: 1.5% acetic acid prepared as follows. Mix 690 Potassium RS, USP 4-Nitrobenzoic Acid RS, and USP
mL of water with 10 mL of acetic acid and pass through a Benzocaine RS in Mobile phase
filter of 0,45-lJm pore size. Sensitivity solution: 0.1 IJg/mL of USPAminobenzoate
Mobile phase: Methanol and Solutioh A (15:85) . Potassium RS in Mobile phase from the Standard solution
Standard solution: 0.1 mg/mL of USPAminobenzoate Sample solution: Nominally 1 mg/mL of aminobenzoate
Potassium RS in Mobile phase potassium in Mobile phase prepared as follows. Remove as
Sample solution: Nominally 0.1 mg/mL of aminobenzoate completely as possible, and combine, the contents of NLT
potassium prepared as follows. Remove as completely as 10 Capsules. Transfer a portion of the combined contents,
possible, and combine the contents of NLT 10 Capsules. equivalent to 10 mg of aminobenzoate potassium, to a
Transfer a portion of the combined contents, equivalent to 1O-mL volumetric flask. Dissolve in 7 mL of Mobile phase,
10 mg of aminobenzoate potassium to a 100-mL sonicate for 3-4 min, and dilute with Mobile phase to
volumetric flask, dissolve in 70 mLof Mobile phase, sonicate volume.
for 3-4 min, and dilute with Mobile phase to volume. System suitability
Chromatographic system Samples: Standard solution and Sensitivity solution
(See Chromatography (621), System Suitability.) Suitability requirements .
Mode: LC Resolution: NLT 1.5 between benzocaine and
Detector: 280 nm 4-nitrobenzoic acid, Standard solution
Column: 3.0-mm x 15-cm; 3.S-lJm packing L11 Tailing factor: NMT 2.0, Standard solution
Flow rate: 0.35 mL/min Relative standard deviation: NMT 5.0%, Standard
Injection volume: 5 IJL solution
System suitability Signal-to-noise ratio: NLT 10, Sensitivity solution
Sample: Standard solution Analysis
Suitability requirements Samples: Standard solution and Sample solution
Tailing factor: NMT 2.0 Calculate the percentage of any individual 'unspecified
Relative standard deviation: NMT 2.0% degradation product in the portion of Capsules taken:
Analysis
Samples: Standard solution and Sample solution

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 USP 43 OfficialMonographs / Aminobenzoate 231

= peak response of any individual unspecified ASSAY

degradation product from the Sample solution • PROCEDURE
= peak response of aminobenzoate from the Solution A: 1.5% acetic acid, prepared by mixing 690 mL
Standard solution of water with 10 mL of glacial acetic acid, and passing
= concentration of USP Aminobenzoate Potassium through a suitable filter of 0.45-~m pore size
RS in the Standard solution (mg/mL) Mobile phase: Methanol and Solution A (15:85)
= nominal concentration of aminobenzoate Standard solution: 0.1 mg/mL of USP Aminobenzoate
potassium in the Sample solution(mg/mL) Sodium RS in Mobilephase. Sonicate to dissolve.
Sample solution: 0.1 mg/mL of Aminobenzoate Sodium in
Acceptance criteria: See Table 1. Mobilephase. Sonicate to dissolve.
Chromatographic system
Table 1 (See Chromatography (621), System Suitability.)
Relative Acceptance Mode: LC
Retention Criteria, Detector: UV 280 nm
Name Time NMT(%) Column: 3.0-mm x 15-cm; 3.5-~m packing L11
Aminobenzoic acid 1.0 - Flow rate: 0.35 mL/min
Injection volume: 5 ~L
Benzocaine" 2.0 - System suitability
4-Nitrobenzoic acid" 2.1 - Sample: Standard solution
Suitability requirements
Any individual Tailing factor: NMT 2.0
unspecified - Relative standard deviation: NMT 0.73%
degradation product 0.10
Analysis
Total impurities - 1.0 Samples: Standard solution and Sample solution
Calculate the percentage of aminobenzoate sodium
aTheseare processimpurities controlledin the API and are includedin thistable
for identification purposesonly.Theyare not reported in the drug product and (C7 H6NNa02 ) in the portion of Aminobenzoate Sodium
should not be includedin the total impurities. taken:

ADDITIONAL REQUIREMENTS Result = (r ulr s) x (C siC u) x 100

• PACKAGING AND STORAGE: Preserve in well-closed
containers. ru =peak responsefrom the Sample solution
• USP REFERENCE STANDARDS (11) rs = peak response from the Standard solution
USP Aminobenzoate Potassium RS Cs =concentration of USP Aminobenzoate Sodium RS
USP Benzocaine 'RS in the Standard solution (mg/mL)
USP 4-Nitrobenzoic Acid RS Cu =concentration of Aminobenzoate Sodium in the
4-Nitrobenzoic acid. Sample solution(mg/mL)
C7H sN04 167.12
Acceptance criteria: 98.00/0-102.0% on the dried basis
IMPURITIES
• LIMIT OF ANILINE AND p- TOLUIDINE
Aminobenzoate Sodium Diluent: Methylene chloride
Standard stock solution: 0.1 mg/mL each of USP Aniline RS
o and USP p-Toluidine RS in Diluent
.... ~o~ Standard solution: 1.0 ~g/mL each of USP Aniline RS and
USP p-Toluidine RS in Diluent from Standard stock solution
NH. Sample solution: 100 mg/mL of Aminobenzoate Sodium in
Diluent prepared asfollows. Add an appropriate quantity of
C7H6NNa02 159.12 Aminobenzoate Sodium to a suitable volumetric flask and
Benzoic acid, 4-amino-, sodium salt; dilute with Diluent to volume. Agitate for 10 min on a shaker
Sodium 4-aminobenzoate [555-06-6]. and centrifuge at 3000 rpm for 5 min. Use the supernatant.
DEFINITION Chromatographic system
Aminobenzoate Sodium contains NLT 98.0% and NMT (See Chromatography (621), System Suitability.)
102.0% of aminobenzoate sodium (C 7H 6NNa02) , calculated Mode: GC
on the dried basis. Detectors
Flame ionization: 300 0
IDENTIFICATION Hydrogen: 40 mL/min
Air: 400 mL/min
Column: 30-m x 0.32-mm fused silica capillary; coated
with 0.5-~m film of phase G27
• A. Temperatures
$pl!?"" Injection port: 280 0
• B. The retention time of the major peak of the Sample Detector: 300 0
solution corresponds to that of the Standard solution, as Column: See Table 1.
obtained in the Assay.
• C. IDENTIFICATION TESTS-GENERAL (191), Chemical
Identification Tests, Sodium: A solution (1 in 100) meets the
requirements of the flame test.

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232 Aminobenzoate / OfficialMonographs USP 43

Table 1 amount of USP Aminobenzoate Sodium RS, and dilute with

Hold Time at Diluent to volume.
Initial Temperature Final Final Standard solution: 1 IJg/mL each of USP Aminobenzoate
Temperature Ramp Temperature Temperature Sodium RS, USP 4-Nitrobenzoic Acid RS, and USP
(0) e/min) e) (min)
Benzocaine RS in Diluent
130 - 130 4 Sample solution: 1 mg/mL of Arnlnobenzoate Sodium in
130 20 180 5
Diluent
Chromatographic system
(See Chromatography (621), System SUitability.)
Carrier gas: Helium Mode: LC
Flow rate: 1 mL/min Detector: UV 280 nm
Injection volume: 2 IJL Column: 3.0-mm x 15-cm; 3.5-lJm packing L11
Injection type: Split ratio, 1:10 Flow rate: 0.4 mL/min
System suitability Injection volume: 5 IJL
Sample: Standard solution System suitability
[NoTE-The relative retention times of aniline and Samples: System suitability solution and Standard solution
p-toluidine are about 4.1 and 5.1 min, respectively.] Suitability requirements
Suitability requirements Resolution: NLT 1.5 between benzocaine and
Resolution: NLT 7.0 between aniline and p-toluidine 4-nitrobenzoic acid, System suitability solution
Tailing factor: NMT 1.5 for aniline and p-toluidine Relative standard deviation: NMT 3% for the
Relative standard deviation: NMT 6.0% for aniline and aminobenzoate sodium, 4-nitrobenzoic acid, and
p-toluidine benzocaine peaks, Standard solution
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standard solution and Sample solution
Calculate the percentage of p-toluidine or aniline in the Calculate the percentage of 4-nitrobenzoic acid or
portion of Aminobenzoate Sodium taken: benzocaine in the portion of Aminobenzoate Sodium
taken:
Result =(r vir s) x (C siCv) x 100
Result =(r vir s) x (C siCv) x 100
= peak response of p-toluidine or aniline from the
Sample solution = peak response of 4-nitrobenzoic acid or
=peak response of p-toluidine or aniline from the benzocaine from the Sample solution
Standard solution = peak response of 4-nitrobenzoic acid or
= concentration of the corresponding USP benzocaine from the Standard solution
Reference Standard in the Standard solution (mg/ = concentration of USP 4-Nitrobenzoic Acid RS or
mL) . USP BenzocaineRS in the Standard solution (mgl
=concentration of Aminobenzoate Sodium in the mL)
Sample solution (mg/mL) = concentration of Aminobenzoate Sodium in the
Sample solution (mg/mL)
Acceptance criteria
Aniline: NMT 10 ppm Calculate the percentage of any individual unspecified
p-Toluidine: NMT 20 ppm impurity in the portion of Aminobenzoate Sodium taken:
• ORGANIC IMPURITIES
Solution A: 1.5% acetic acid, prepared by mixing 690 mL Result =(r vir s) x (C siCv) x 100
of water with 10 mL of glacial acetic acid
Solution B: Methanol =peak response of any unspecified impurity from
Mobile phase: See Table 2. the Sample solution
= peak response of aminobenzoate from the
Table 2 Standard solution
Time Solution A Solution B =concentration of USP Aminobenzoate Sodium RS
(min) (0/0) (%) in the Standard solution (mg/mL)
0.0 85 15
=concentration of Aminobenzoate Sodium in the
Sample solution (mg/mL)
4.0 85 15
Acceptance criteria: See Table 3.
4.1 45 55
10.0 45 55 Table 3
10.1 85 15 Relative Acceptance
Retention Criteria,
13 85 15 Name Time NMT(%)
Aminobenzoic acid 1.0 -
Diluent: Methanol and water (85:15) Benzocaine 2.0 0.2
System suitability solution: 1 mg/mL of USP
Aminobenzoate Sodium RS, 0.01 mg/mL of USP 4·Nitrobenzoic acid 2.1 0.2
4-Nitrobenzoic Acid RS, and 0.01 mg/mL of USP Any individual unspecified
Benzocaine RS in Diluent prepared asfollows. Transfer 1 mL impurity - 0.10
each of 0.1 mg/mL of USP 4-Nitrobenzoic Acid RS in
methanol and 0.1 mg/mL of USP Benzocaine RS in Diluent
to a 1O-mL volumetric flask containing the appropriate

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USP 43 Official Monographs / Aminobenzoic 233

SPECIIFIC TESTS Relative standard deviation: NMT 1.0%

iii pH (791) Analysis
Sample solution: 50 mg/mL Samples: Standard solution and Sample solution
Acceptance criteria: 8.0-9.0 Calculate the percentage of aminobenzoic acid (C7H 7N02 )
• Loss ON DRYING (731) in the portion of Aminobenzoic Acid taken:
Analysis: Dry at 105° for 2 h.
Acceptance criteria: NMT 1.0% Result = (r ulr s) x (C siC u) x 100
ADDITIONAL REQUIREMENTS ru = peak responsefrom the Sample solution
• PACKAGING AND STORAGE: Preserve in well-closed rs = peak responsefrom the Standardsolution
containers.
Cs = concentration of the Standard solution (mg/mL)
• USP REFERENCE STANDARDS (11)
USP Aminobenzoate Sodium RS Cu = concentration of the Sample solution (mg/mL)
USP Aniline RS
Aniline. Acceptance criteria: 98.00/0-102.0% on the dried basis
C6H 7N 93.13 IMPURITIES
USP Benzocaine RS • RESIDUE ON IGNITION (281): NMT 0.1%
USP 4-Nitrobenzoic Acid RS • ORGANIC IMPURITIES
4-Nitrobenzoic acid. Solution A: Acetonitrile and methanol (70:80)
C7H sN04 167.12 Buffer: 1.5 giL of monobasic potassium phosphate and 2.5
USP p-Toluidine RS giL of octanesulfonic acid sodium salt in water. Adjust with
4-Methylaniline. phosphoric acid to a pH of 2.2.
C7H 9N 107.16 Mobile phase: Solution A and Buffer (20:80)
Standard stock solution: 0.25 mg/mL each of USP
Benzocaine RS and 4-nitrobenzoic acid in methanol
Standard solution: 0.5 IJg/mL each of USP Benzocaine RS
and 4-nitrobenzoic acid in Mobilephase, from the Standard
Aminobenzoic Acid . stock solution
Sample solution: 0.25 mg/mL of Aminobenzoic Acid in
Mobilephase
Chromatographic system
(See Chromatography (621), System SUitability.)
Mode: LC
C7H 7N02 137.14 Detector: UV 270 nm
Benzoic acid, 4-amino; Column: 4.0-mm x 15-cm; 5-lJm packing L7
p-Aminobenzoic acid [150-1 3-0]. Flow rate: 1.0 mL/min
Injection volume: 20 IJL
DEIFINITION Run time: 11 times the retention time of the
Aminobenzoic Acid contains NLT 98.0% and NMT 102.0% of aminobenzoic acid peak
aminobenzoic acid (C 7H7N02) , calculated on the dried basis. Analysis
IDENTIFICATION Samples: Standard solution and Sample solution
Calculate the percentage of 4-nitrobenzoic acid or any
unspecified impurity in the portion of Aminobenzoic Acid
taken:
• A.',
Result =(r ulr s) x (C siC u) x 100
~i?g€tf9~
• B. The retention time 0 t emajor peak of the Sample ru = peak response of 4-nitrobenzoic acid or any
solution corresponds to that of the Standard solution, as unspecified impurity from the Sample solution
obtained in the Assay. rs =peak response of 4-nitrobenzoic acid from the
ASSAY Standard solution
• PROCEDURE Cs = concentration of 4-nitrobenzoic acid in the
Acetic acid solution: Glacial acetic acid and water (1:69) Standardsolution(mg/mL) .
Mobile phase: Methanol and Acetic acid solution(15:85) Cu = concentration of Aminobenzoic Acid in the
Standard solution: 0.1 mg/mL of USP Aminobenzoic Acid Sample solution(mg/mL)
RS in Mobilephase. Sonicate to aid dissolution.
Sample solution: 0.1 mg/mL of Aminobenzoic Acid in Calculate the percentage of benzocaine in the portion of
Mobilephase. Sonicate to aid dissolution. Aminobenzoic Acid taken:
Chromatographic system
(See Chromatography (621), System Suitability.) Result = (r ulr s) x (C siC u) x 100
Mode: LC
Detector: UV 280 nm ru = peak response of benzocaine from the Sample
Column: 3.0-mm x 15-cm; 3.5-lJm packing L11 solution
Flow rate: 0.4 mL/min rs = peak response of benzocaine from the Standard
Injection volume: 5 IJL solution
System suitability Cs = concentration of USP Benzocaine RS in the
Sample: Standard solution Standard solution(mg/mL)
Suitability requirements Cu =concentration of Aminobenzoic Acid in the
Tailing: NMT 1.5 Sample solution(mg/mL)

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234 Aminobenzoic / Official Monographs USP 43

Acceptance criteria: See Table 7. Disregard any impurity Acceptance criteria

peaks lessthan 0.02%. Aniline: NMT 10 ppm
p-Toluidine: NMT 10 ppm
Table 1
SPECIFIC TESTS
Relative Acceptance
Retention Criteria,
• Loss ON DRYING (731)
Name Time NMT(%) Analysis: Dry a sample at 105° for 2 h.
Acceptance criteria: NMT 0.2%
Aminobenzoic acid 1.0 -
ADDITIONAL REQUIREMENTS
4-Nitrobenzoic acid 4.0 0.2 • g:»ACKAGING AND STORAGE: Preserve in tight, light-resistant
Benzocaine 9.0 0.2 containers.
• USP REFERENCE STANDARDS (11)
Any individual unspecified
impurity - 0.1 USP Aminobenzoic Acid RS
USP Benzocaine RS
Total impurities - 0.5 Benzoic acid, 4-amino-, ethyl ester.
C9H 11N02 165.19
• LIMIT OF ANILINE AND p-TOLUIDINE
Diluent: 84 giL of sodium hydroxide in water
Standard solution: 1.0 uq/m], each of aniline and
p-toluidine in methylene chloride
Sample solution: Dissolve1 g of Aminobenzoic Acid in 10.0 Aminobenzoic Acid Gel
mL of Diluent, and extract with two quantities each of 10.0
mL of methylene chloride. Combine, and wash with 5 mL DEFINITION
of water. Filter through anhydrous sodium sulfate, and Aminobenzoic Acid Gel contains NLT 90.0% and NMT
wash the filter with methylene chloride. Evaporate in a 110.0% of the labeled amount of aminobenzoic acid
water bath at 50°-60° to obtain a volume of about 1-5 mL. (C7H 7N02 ) ·
Transfer to a 1O-mL volumetric flask, and dilute with IDENTIFICATION
methylene chloride to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.32;mm x 30-m fused silica capillary, coated
with a 0.5-~m film of phase G27
Temperatures
Injection port: 280°
Detector: 300°
• B~~$~~~~~~S()~I~~~E~~I~.I~~~I().~)~~~~~~,)l~?(~
U.ltrgy,f91~t71,1.isfb.le§pe.gr9s.g9PY:)1~lY~(CN)1;lV1aY42026)
Column: See Table 2. Sample solution: 5 ~g/mL in alcohol
Acceptance criteria: Meets the requirements
Table 2
ASSAY
Hold Time
Initial Temperature Final at Final • PROCEDURE
Temperature Ramp Temperature Temperature Mobile phase: Methanol, glacial acetic acid, and water
e) e/min) (0) (min) (30:1 :69). Allow the mixture to cool, and pass, if necessary,
130 0 130 4 through a suitable microporous membrane filter, and
degas.
130 20 180 5 Internal standard solution: 7 mg/mL of salicylic acid in
methanol; dissolve by sonicating.
Carrier gas: Helium Standard stock solution: 0.42 mg/mL of USP
Flow rate: 1.0 mL/min Aminobenzoic Acid RS in methanol; dissolve by sonicating.
Injection volume: 2 ~L Standard solution: 0.042 mg/mL of USP Aminobenzoic
Split ratio: 1:10 Acid RS in methanol prepared as follows. Transfer 5 mL
Analysis each of Standardstock solution and Internal standard
Samples: Standardsolution and Sample solution solution into a 50-mL volumetric flask, and dilute with
[NoTE-The relative retention times for aniline and methanol to volume. Pass through 0.6.:.~m filter paper.
p-toluidine are about 0.8 and 1.0, respectively.] Throughout the preparation, protect against actinic light.
Calculate, in ppm, the amount of aniline and p-toluidine in Sample solution: Nominally 0.042 mg/mL of
the portion of Aminobenzoic Acid taken: aminobenzoic acid in methanol prepared as follows.
Transfer a quantity of Gel, equivalent to 4.2 mg of
Result = (r vir 5) x (C siC v) x 106 aminobenzoic acid, into a 1OO-mL volumetric flask. Add
10.0 mL of Internal standardsolution and 50 mL of
ru = peak response of each impurity from the Sample methanol. Shake or sonicate, as necessary, and dilute with
solution methanol to volume. Filter, if necessary, through filter
r5 = peak response of the corresponding impurity paper (Whatman No. 41 or equivalent). Pass through
from the Standardsolution 0.6-~m filter paper. Throughout this preparation, protect
C5 = concentration of the corresponding impurity in against actinic light.
the Standardsolution (mg/mL) Chromatographic system
Cu = concentration of Aminobenzoic Acid in the (See Chromatography (621), System Suitability.)
Sample solution (mg/mL) Mode: LC

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 USP 43 OfficialMonographs / Aminocaproic 235

Detector: UV 280 nm IDENTIFICATION

Column: 3.9-mm x 30-cmi packing L11
Flow rate: 1.0 mL/min
Injection volume: 15 ~L
System suitability
Sample: Standard solution
Chromatograph replicate 15-~L injections of the Standard
solution until the response ratio variability is within 1.0% ASSAY
of average. • PROCEDURE
Solution A: 0.55 gIL of sodium l-heptanesulfonate in water
[NOTE-The relative retention times for aminobenzoic
acid and salicylic acid are about 1.0 and 3.0, Mobile phase: Dissolve 109 of monobasic potassium
respectively. ] phosphate in 300 mL of Solution A, and add 250 mL of
Suitability requirements methanol, followed by another 300 mL of Solution A. A~just
the mixture with phosphoric acid to a pH of 2.2, and dilute
Resolution: NLT 3.0 between aminobenzoic acid and
with Solution A to 1 L.
salicylic acid peaks
Analysis Internal standard solution: 1.25 mg/mL of methionine in
water
Samples: Standard solution and Sample solution
Standard stock solution: 12.5 mg/mL of USP Aminocaproic
Calculate the percentage of the labeled amount of
Acid RS in water
aminobenzoic acid (C 7H 7N02) in the portion of Gel
Standard solution: 5.0 mt of Standard stock solution and
taken:
2.0 mL of Internal standard solution in a 1OO-mL volumetric
flask. Dilute with water to volume.
Result = (R vIR s) x (C sIC v) x 100
Sample stock solution: 12.5 mg/mL of Aminocaproic Acid
= peak response ratio of aminobenzoic to salicylic in water
acid from the Sample solution Sample solution: 5.0 mL of Sample stock solution and 2.0
= peak response ratio of aminobenzoic to salicylic mL of Internal standard solution in a 1OO-mL volumetric
acid from the Standard solution flask. Dilute with water to volume.
= concentration of USP Aminobenzoic Acid RS in Chromatographic system
the Standard solution (mg/mL) (See Chromatography (621), System SUitability.)
= nominal concentration of aminobenzoic acid in Mode: LC
the Sample solution (mg/mL) Detector: UV 210 nm
Column: 4.6-mm x 15-cmi packing L1
Acceptance crlterla: 90.0%-110.0% Column temperature: 30°
Flow rate: 0.7 mL/min
OTHER COMPONENTS ' Run time: NLT 2 times the retention time of
• ALCOHOL DETERMINATION, Method /I (611): 42.30/0-54.0% aminocaproic acid
(w/w) of C2H sOH Injection volume: 20 ~L
System suitability
PERFORMANCE TESTS Sample: Standard solution.. . .
• MINIMUM FILL (755): Meets the requirements [NoTE-The relative retention times for arrunocaproic
SPECIFIC TESTS acid and methionine are about 0.76 and 1.0,
• pH (791): 4.0-6.0 respectively. ]
Suitability requirements
ADDITIONAL REQUIREMENTS . Resolution: NLT 2.0 between aminocaproic acid and
• PACKAGING AND STORAGE: Preserve in tight, light-resistant methionine
containers. Relative standard deviation: NMT 2.0%
• USP REFERENCE STANDARDS (11) Analysis
USP Aminobenzoic Acid RS Samples: Standard solution and Sample solution
Calculate the percentage of aminocaproic acid (C 6H nN0 2)
in the portion of Aminocaproic Acid taken:

Aminocaproic Acid Result =(R vIR s) x (C sIC v) x 100

Ru =peak response ratio of aminocaproic acid to the
internal standard from the Sample solution
Rs =peak response ratio of aminocaproic acid .to the
internal standard from the Standard solution
C6H nN0 2 131.17 Cs =concentration of USP Aminocaproic Acid RS in the
Hexanoic acid, 6-amino-i Standard solution (mg/mL)
6-Aminohexanoic acid [60-32-2]. Cu = concentration of Aminocaproic Acid in the
DEFINITION
Sample solution (mg/mL)
Aminocaproic Acid contains NLT 98.5% and NMT 101.5% of Acceptance criteria: 98.50/0-101 .5% on the anhydrous
aminocaproic acid (C 6H nN0 2) , calculated on the anhydrous basis
basis.
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1 %
SPECIFIC TESTS
• WATER DETERMINATION, Method I (921): NMT 0.5%

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236 Aminocaproic / OfficialMonographs USP 43

ADDITIONAL REQUIREMENTS Result = (r vir s) x (C siCv) x 100

- PACKAGING AND STORAGE: Preserve in tight containers.
Store at room temperature. = peak area from the Sample solution
- USP REFERENCE STANDARDS (11) = peak area from the Standard solution
USPAminocaproic Acid RS = concentration of USP Aminocaproic Acid RS in the
Standard solution (mg/ml)
= nominal concentration of aminocaproic acid in
the Sample solution (mg/ml)
Aminocaproic Acid Injection Acceptance criteria: 95.0%-107.5%
DEFINITION SPECIFIC TESTS
Aminocaproic Acid Injection is a sterile solution of • pH (791): 6.0-7.6
Aminocaproic Acid in Water for Injection. It contains NlT • BACTERIAL ENDOTOXINS TEST (85): NMT 0.05 USP
95.0% and NMT 107.5% of the labeled amount of Endotoxin Unit/mg of aminocaproic acid
aminocaproic acid (C6 H13NOz). • OTHER REQUIREMENTS: It meets the requirements in
Injections and Implanted Drug Products (1).
IDENTIFICATION
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in single-dose or
multiple-dose containers, preferably of Type I glass.
-A • USP REFERENCE STANDARDS (11)
,§f£~g~ USP Aminocaproic Acid RS
Sample: Mix 2 ml of Injection,~~added dropwise, with 100
ml of acetone, rapidly stirring the mixture with a glass rod
to induce crystallization. Allow the mixture to stand for 15
min, and pass through a sintered-glass filter of medium
pore size. Wash the crystals with 25 ml of acetone, apply a ~minocaproic Acid Oral Solution
vacuum to remove the solvent, dry at 105° for 30 min, and
cool. Use the residue. DEFINITION
Acceptance criteria: Meets the requirements Aminocaproic Acid Oral Solution contains NlT 95.0% and
NMT 115.0% of the labeled amount of aminocaproic acid
ASSAY (C 6H13 NO z) ·
• PROCEDURE
Mobile phase: Transfer 11 g of sodium l-pentanesulfonate IDENTIFICATION
and 40 g of anhydrous sodium sulfate to a 2-l volumetric
flask, and dissolve in about 500 ml of water. Add 20 ml of
1 N sulfuric acid and 30 ml of acetonitrile, and dilute with
water to volume. . • A.
:5
Standard solution: 2.5 mg/ml of USPAminocaproic Acid
RS in Mobile phase Sample: Mix 1 g of ion-exchange resin (strongly acidic
System suitability solution: Mix 20 ul, of benzyl alcohol styrene-divinylbenzene cation-exchange resin) with 10 ml
with 100 ml of water. Dilute 1.0 ml of this solution with of 1 N hydrochloric acid in a 1OO-ml beaker. Decant and
Standard solution to 10 mL discard the hydrochloric acid, and wash the resin with five
Sample stock solution: Nominally equivalent to ,45 mg/ml 1O-ml portions of water, decanting and discarding the
of aminocaproic acid from a suitable volume of Injection liquid following each washing. Place the washed resin in a
in water 125-ml glass-stoppered, conical flask, and add a volume of
Sample solution: 2.5 mg/ml of Sample stock solution in Oral Solution, nominally equivalent to 250 mg of
Mobile phase aminocaproic acid, and 10 ml of water. Insert the stopper
Chromatographic system in the flask, and shake by mechanical means for 30 min.
(See Chromatography (621), System Suitability.) Transfer the resin slurry to a sintered-glass funnel of
Mode: lC medium pore size. Wash with 100 ml of water, filter by
Detector: UV 210 nm applying suction, and discard the washing. Place a beaker
Column: 4-mm x 30-cm; packing II under the stem ofthe funnel, add 10 ml of 1 N hydrochloric
Flow rate: 2 ml/min acid to the resin, stir for 4-5 min, and filter by applying
Injection volume: 50 ~l suction. Evaporate the filtrate on a steam bath to dryness,
System suitability dry at 105° for 1 h, and cool.
Samples: Standard solution and System suitability solution Acceptance criteria: The residue meets the requirements.
Suitability requirements ASSAY
Resolution: NlT 7.0 between benzyl alcohol and • PROCEDURE
aminocaproic acid, System suitability solution Sample solution: Place an amount nominally equivalent to
[NOTE-The aminocaproic acid peak elutes before the 250 mg of aminocaproic acid from a volume of Oral
benzyl alcohol peak.] Solution in about 80 ml of glacial acetic acid.
Relative standard deviation: NMT 1.0%, Standard Titrimetric system
solution Mode: Direct titration
Analysis Titrant: 0.1 N perchloric acid in dioxaneVS
Samples: Standard solution and Sample solution Endpoint detection: Visual
Calculate the percentage of aminocaproic acid (C6H13NOz) Analysis: To the Sample solution add 10 drops of a l-in-500
in the portion of Injection taken: solution of crystal violet in chlorobenzene. Titrate with
Titrant to a blue endpoint, and perform a blank

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USP 43 Official Monographs / Aminohippurate 237

determination. Each mL of 0.1 N perchloric acid is 500 solution of ~-naphthoquinone-4-sodium sulfonate to

equivalent to 13.12 mg of aminocaproic acid (C6H13N02) . each flask. Swirl to mix, and place the three flasksin a water
Acceptance criteria: 95.0%-115.0% bath maintained at a temperature of 65± 5° for 45 min.
Cool, and dilute the contents of each flask with water to
SPECIFIC TESTS volume. Determine the percentage of the labeled amount
• pH (791): 6.0-6.5 of aminocaproic acid (C6H 13N02) dissolved from
ADDITIONAL REQUIREMENTS absorbances, at the wavelength of maximum absorbance
• PACKAGING AND STORAGE: Preserve in tight containers. at about 460 nm,from the Sample solution in comparison
• USP REFERENCE STANDARDS (11) with those from the Standard solution, using the Blank to
USP Aminocaproic Acid RS set the instrument.
Tolerances: NLT 75% (Q) of the labeled amount of
aminocaproic acid (C6H 13N02) is dissolved.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
Aminocaproic Acid Tablets
ADDITIONAL REQUIREMENTS
DEFINITION • PACKAGING AND STORAGE: Preserve in tight containers.
Aminocaproic Acid Tablets contain NLT 95.0% and NMT • USP REFERENCE STANDARDS (11)
105.0% of the labeled amount of aminocaproic acid USP Aminocaproic Acid RS
(C 6H 13N02 ) ·
IDENTIFICATION
Aminohippurate Sodium Injection
• A.~~~~~~~~-
Sp~strQ$(:,QPX;X:1• ~
Sample: Triturate 2 Tablets with 10 mL of water, and filter
into 100 mL of acetone. Swirl the mixture, and allow to
stand for 15 min to complete crystallization. Pass the C9H9N2Na03 216.17
solution through a sintered-glass filter of medium pore size, Glycine, N-(4-aminobenzoyl)-, monosodium salt;
and wash the crystals with 25 mL of acetone. Apply vacuum Monosodium p-aminohippurate [94-16-6].
to remove the solvent, dryat 105° for 30 min, and cool. Use
the residue. DEFINITION
Acceptance criteria: Meet the requirements Aminohippurate Sodium Injection is a sterile solution of
Aminohippuric Acid in Water for Injection prepared with the
ASSAY aid of Sodium Hydroxide. It contains NLT 95.0% and NMT
• PROCEDURE 105.0% of the labeled amount of aminohippurate sodium
Sample solution: Nominally equivalent to about 500 mg of (C9H9N 2Na03) .
aminocaproic acid from NLT 20 finely powdered Tablets
taken in a beaker in about 100 mL of glacial acetic acid. IDENTIFICATION
Heat gently to effect solution, and cool. • A.
Titrimetric system Sample solution: A volume of Injection equivalent to 100
Mode: Direct titration mg of aminohippuric acid
Titrant: 0.1 N perchloric acid in dioxane VS Analysis: Dilute the Sample solution with water to 50 mL. To
Endpoint detection: Visual 5 mL of this solution add 0.5 mL of 3 N hydrochloric acid,
Analysis: To the Sample solution add 10 drops of a 1-in-500 0.5 mL of sodium nitrite solution (1 in 10), and a solution
solution of crystal violet in chlorobenzene. Titrate with of 0.20 g of 2-naphthol in 10 mL of 6 N ammonium
Titrant to a blue endpoint, and perform a blank hydroxide.
determination. Each mL of 0.1 N perchloric acid is Acceptance criteria: A red color is produced.
equivalent to 13.12 mg of aminocaproic acid (C 6H 13N02) . • B.
Acceptance criteria: 95.00/0-105.0% Sample solution: A volume of Injection equivalent to about
200 mg of aminohippurate sodium
PERFORMANCETESTS Analysis: In the order named, add to the Sample solution2
• DISSOLUTION (711) mL of potassium iodide TS, 10 mL of water, and 5 mL of
Medium: Water; 900 mL sodium hypochlorite TS.
Apparatus 1: 100 rpm Acceptance criteria: A red color is produced.
Time: 45 min • C. A sample imparts an intense yellow color to a
Buffer: Dissolve 6.185 g of boric acid and 7.930 g of non luminous flame.
potassium chloride in about 1000 mL of water, and.add 60
mL of 1.0 N sodium hydroxide. Dilute with water to 2000 ASSAY
mL, and adjust if necessarywith 1.0 N sodium hydroxide • PROCEDURE
to a pH of 9.5 ± 0.1. Sample solution: A volume of Injection equivalent to 1 g of
Standard solution: 0.5 mg/mL of USP Aminocaproic Acid aminohippurate sodium
RS in water Analysis: Transfer the Sample solution to a 200-mL
Sample solution: Filter a portion of the solution under test. volumetric flask, and dilute with water to volume. Transfer
Blank: Water 50.0 mL of the solution to a suitable container, and add 5
Analysis: Into three separate 50-mL volumetric flasks pipet mL of hydrochloric acid. Proceed as directed in Nitrite
1 mL each of Sample solution, Standard solution, and Blank. Titration (451), beginning with "cool to about 15°". Each
Add 20.0 mL of Buffer and 3.0 mt of a freshly prepared 1-in-

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238 Aminohippurate / OfficialMonographs USP43

mL of 0.1 M sodium nitrite is equivalent to 21.62 mg of

aminohippurate sodium (C9H9N2Na03). Aminolevulinic Acid Hydrochloride
Acceptance criteria: 95.0%-105.0% o

SPECIFIC TESTS H'N~OH • Hel

• pH (791): 6.7-7.6 o
• BACTERIAL ENDOTOXINS TEST (85): NMT 0.04 USP
Endotoxin Units/mg of aminohippurate sodium CsH9N0 3 • HCI 167.59
• OTHER REQUIREMENTS: Meets the requirements in Injections 5-Aminolevulinic acid hydrochloride;
and Implanted Drug Products (1) 5-Amino-4-oxopentanoic acid hydrochloride [5451-09-2].

ADDITIONAL REQUIREMENTS DEFINITION

• PACKAGING AND STORAGE: Preserve in single-dose or Aminolevulinic Acid Hydrochloride contains NLT 98.0% and
multiple-dose containers, preferably of Type I glass. NMT 102.0% of aminolevulinic acid hydrochloride
(CsH9N0 3. HC!), calculated on the dried basis.
IDENTIFICATION

Aminohippuric Acid
• A.~;~
~PJs€Q~€()P!
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
C9Hl0N203 194.19 • C. IDENTIFICATION TESTS-GENERAL, Chloride (191): Meets
Glycine, N-(4-aminobenzoyl)-; the requirements
p-Aminohippuric acid [61-78-9].
ASSAY
DEFINITION '. PROCEDURE
Aminohippuric Acid contains NLT 98.0% and NMT 100.5% of Solution A: Water adjusted with 2 M sulfuric acid to a pH of
aminohippuric acid (C9H lON 203), calculated on the dried 2.2
basis. Mobile phase: See Table 1.
IDENTIFICATION Table 1
Time Solution A Methanol
(min) (%) (%)
0 95 5
2 95 5
• 6 60 40
Sample: 10 mg of Aminohippuric Acid .
Analysis: Dissolvethe Sample in 5 mL of water, add 0.5 mL 8 60 40
of 3 N hydrochloric acid, 0.5 mL of sodium nitrite solution
9 95 5
(1 in 10), and a solution of 0.20 g of 2-naphthol in 10 mL
of 6 N ammonium hydroxide. 23 95 5
Acceptance criteria: A red color is produced. .
ASSAY Diluent: Methanol and Solution A (1:3)
• PROCEDURE Standard solution: 4 mg/mL of USP Aminolevulinic Acid
Sample: 150 mg of Aminohippuric Acid Hydrochloride RS in Diluent
Analysis: To the Sample add 5 mL of hydrochloric acid and Sample solution: 4 mg/mL of Aminolevulinic Acid
50 mL of water. Proceed as directed in Nitrite Titration Hydrochloride in Diluent
(451), beginning with "stir until dissolved." Each mL of 0.1 Chromatographic system
M sodium nitrite is equivalent to 19.42 mg of (See Chromatography (621), System Suitability.)
aminohippuric acid (C9Hl0N203)' Mode: LC
Acceptance criteria: 98.0%-100.5% on the dried basis Detector: UV 210 nm
Column: 2.1-mm x 10-cm; 1.7-urn packing L1
IMPURITIES Flow rate: 0.4 mL/min
• RESIDUE ON IGNITION (281): NMT 0.25% Injection volume: 5 fJL
SPECIFIC TESTS System suitability
• Loss ON DRYING (731) Sample: Standard solution
Analysis: Dry at 105° for 2 h. Suitability requirements
Acceptance criteria: NMT 0.25% Tailing factor: NMT 1.6 .
Relative standard deviation: NMT 0.73%
ADDITIONAL REQUIREMENTS Analysis
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Samples: Standardsolution and Sample solution
containers. Calculate the percentage of aminolevulinic acid
• USP REFERENCE STANDARDS (11) hydrochloride (CsH9N0 3. HCI) in the portion of
USP Aminohippuric Acid RS Aminolevulinic Acid Hydrochloride taken:

Result =(rufrs) x (CslCu) x 100

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USP 43 OfficialMonographs / Aminopentamide 239

= peak responsefrom the Sample solution = peak response of aminolevulinic acid related
= peak responsefrom the Standardsolution compound A from the Standardsolution
= concentration of USP Aminolevulinic Acid = concentration of USP Aminolevulinic Acid Related
Hydrochloride RS in the Standardsolution Compound A RS in the Standardsolution
(mg/mL) (mg/mL)
= concentration of Aminolevulinic Acid = concentration of Aminolevulinic Acid
Hydrochloride in the Sample solution (mg/mL) Hydrochloride in the Sample solution (mg/mL)

Acceptance criteria: 98.0%-102.0% on the dried basis Acceptance criteria: See Table 2. Disregard any impurity
peak less than 0.05%.
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.3% Table 2
• ORGANIC IMPURITIES
Relative Acceptance
Mobile phase, Diluent, and Chromatographic system: Retention Criteria,
Proceed as directed in the Assay. Name Time NMT (%)
Standard solution: 0.04 mg/mL each of USP
Aminolevulinic Acid Hydrochloride RS, USP Aminolevulinic
Aminolevulinic acid 1.0 -
Acid RelatedCompound A RS, and USP Aminolevulinic Acid Aminolevulinic acid
Related Compound B RS in Diluent related compound A 7.8 0.15
Sample solution: 40 mg/mL of Aminolevulinic Acid Aminolevulinic acid
Hydrochloride in Diluent related compound B 12.0 0.15
System suitability
Any individual
Sample: Standardsolution unspecified impurity - 0.10
Suitability requirements
Relative standard deviation: NMT 10% Total impurities - 0.5
Analysis
Samples: Standardsolution and Sample solution SPECIFIC TESTS
Calculate the percentage of aminolevulinic acid related • pH (791)
compound A or aminolevulinic acid related compound B Sample solution: 10 mg/mL in carbon dioxide-free water
in the portion of Aminolevulinic Acid Hydrochloride taken: Acceptance criteria: 2.5-2.9
• Loss ON DRYING (731)
Result = (ru/rs) x (CslCu) x 100 Sample: 1 9
Analysis: Dry the Sample over phosphorous pentoxide
rv = peak response of each impurity from the Sample under vacuum at 100°-105° for 5 h.
solution Acceptance criteria: NMT 0.5%
rs = peak response of the corresponding USP
Reference Standard from the Standardsolution ADDITIONAL REQUIREMENTS
Cs = concentration of the corresponding USP • PACKAGING AND STORAGE: Preserve in well-closed
ReferenceStandard in the Standardsolution (mg/ containers, and store at room temperature.
mL) • USP REFERENCE STANDARDS (11)
Cu = concentration of Aminolevulinic Acid USP Aminolevulinic Acid Hydrochloride RS
Hydrochloride in the Sample solution (mg/mL) USP Aminolevulinic Acid Related Compound A RS
3,3' -(Pyrazine-2,5-diyl)dipropionic acid.
Calculate the percentage of any unspecified impurity C,oH12N 204 224.22
eluting before aminolevulinic acid related compound A in USP Aminolevulinic Acid Related Compound B RS
the portion of Aminolevulinic Acid Hydrochloride taken: Methyl 5-(1, 3-dioxoisoindolin-2-yl)-4-oxopentanoate.
C'4H13NOS 275.26
Result =(ru/rs) x (Cs/Cu) x 100

= peak responseof any unspecified impurity eluting

before aminolevulinic acid related compound A
from the Sample solution Aminopentamide Sulfate
= peak response of aminolevulinic acid from the
Standard solution
= concentration of USP Aminolevulinic Acid
Hydrochloride RS in the Standardsolution
(mg/mL)
= concentration of Aminolevulinic Acid
Hydrochloride in the Sample solution (mg/mL)

Calculate the percentage of any unspecified impurity C,9H24N20. H2S04 394.49

eluting after aminolevulinic acid related compound A in a.-[2-(Dimethylamino)propyl]-a.-phenylbenzeneacetamide
the portion of Aminolevulinic Acid Hydrochloride taken: sulfate [60-46-8].

Result = (ru/rs) x (Cs/Cu) x 100 » Aminopentamide Sulfate contains not less than
= peak responseof any unspecified impurity eluting 95.0 percent and not more than 103.0 percent of
after aminolevulinic acid related compound A C19H24 N20 . H2S04 ,
from the Sample solutior:

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240 Aminopentamide / OfficialMonographs USP 43

Packaging and storage-Preserve in tight containers, and Assay-

store at controlled room temperature. Mobile phase-Transfer 14.4 g of sodium lauryl sulfate to a
Labeling-Label it to indicate that it isfor veterinary use only. 500-mL volumetric flask, add 100 mL of glacial acetic acid,
UiP Reference standards (11)- dilute with water to volume, mix, and pass through a filter
USP Aminopentamide Sulfate RS having a 0.5-lJm or finer porosity. Transfer 50 mL of this
Clarity and color of solution-Dissolve 0.5 g in 10 mL of solution to a 1OOO-mL volumetric flask, add 350 mL of
water: the solution is clear and colorless. methanol and 350 mL of acetonitrile, dilute with water to
volume, and mix. Filter and degas before use. Make
Identification- adjustments if necessary (see System Suitability under
Chromatography (621 »
Standardpreparation-Quantitatively dissolve an accurately
weighed quantity of USP Aminopentamide Sulfate RS in water
A: .~·§R~stt~§c()pi~!!g~~.~!(;~~~~~~~;'r:e§t$(.12~Z)~!li)ft1;tfeFj to obtain a solution having a known concentration equivalent
Speftrq~fqpY:}197K.A.(c~J ,MilY..~()~O). to the labeled concentration of aminopentamide sulfate in the
B: It meets the requirements of the tests for Sulfate (191). Injection. .
Melting range (741): between 179° and 186°. Assay preparation-Use the undiluted Injection.
pH (791): between 1.2 and 3.0, in a solution (2.5 in 100). Chromatographic system (see Chromatography (621 ) -The
Loss on drying (731 )-Dry it at 105° for 4 hours: it loses not liquid chromatograph is equipped with a 254~nm detector
more than 4.4% of its weight. and a 3.9-mm x 30-cm column that contains packing L1 and
Residue on ignition (281): not more than 0.5%. is maintained at a constant temperature of about 40°. The flow
Assay-Dissolve about 500 mg of Aminopentamide Sulfate, rate is about 1 mL per minute. Chromatograph the Standard
accurately weighed, in 100 mL of dimethylformamide in a preparation, and record the peak responses as directed for
suitable container. Add 5 drops of thymol blue TS, and titrate Procedure: the tailing factor is not more than 2.5; and the
with 0.1 N lithium methoxide VS in toluene to a deep blue relative standard deviation for replicate injections is not more
endpoint. Perform a blank determination, and make any than 2%.
necessary correction. Each mL of 0.1 N lithium methoxide is Procedure-Separately inject equal volumes (about 20 IJL)
equivalent to 19.72 mg of C19H24N20· H2S04, of the Standardpreparation and the Assay preparation into the
.chromatograph, record the chromatograms, and measure the
areas for the major peaks. Calculate the quantity, in mg, of
aminopentamide sulfate (C 19 H 24 N 20· H 2 SO 4) in each mL
of the Injection taken by the formula:
Aminopentamide Sulfate Injection
C(r ulr s)
» Aminopentamide Sulfate Injection is a sterile
in which C is the concentration, in mg per mL, of USP
solution of Aminopentamide Sulfate in Water for Aminopentamide Sulfate RS in the Standardpreparation,' and
Injection. It contains not less than 90.0 percent and r u and r s are the aminopentamide peak responses obtained
not more than 110.0 percent of the labeled from the Assay preparation and the Standardpreparation,
amount of aminopentamide sulfate (C 19 H 24 N 20 respectively .
. H 2 SO 4)'
Packaging and storage-Preserve in tight, single-dose or
multiple-dose Containers for Injections, as described in Aminopentamide Sulfate Tablets
Packaging and Storage Requirements (659), Injection Packaging.
Store at controlled room temperature.
Labeling-Label Injection to indicate that it is for veterinary » Aminopentamide Sulfate Tablets contain not less
use only. than 95.0 percent and not more than 105.0
USP Reference standards (11)- percent of the labeled amount of aminopentamide
USP Aminopentamide Sulfate RS
Identification-Transfer 10 mL of the Injection to a sulfate (C19H24N20 . H2S04),
separator, add sodium hydroxide TS until alkaline to litmus,
and extract with 25 mL of chloroform. Transfer a few drops of Packaging and storage-Preserve in tight containers, and
the chloroform extract to a KRS-5 plate, and allow to dry. store at controlled room temperature.
Record the IR absorption spectrum by the attenuated total Labeling-Label Tablets to indicate that they are for
reflectance technique (see Mid-Infrared Spectroscopy (854). veterinary use only.
The spectrum thus obtained exhibits maxima only at the same UiP Reference standards (11)-
wavelengths as that of a similar preparation of USP USP Aminopentamide Sulfate RS
Aminopentamide Sulfate RS, concomitantly measured. Identification-Transfer a portion of ground Tablet powder,
Bacterial Endotoxins Test (85) -It contains not more than equivalent to about 2 mg of aminopentamide, to a separator,
25 USP Endotoxin Units per mg of aminopentamide sulfate. add 20 mL of water and 3 mL of 10 N sodium hydroxide, and
Sterility Tests (71) -It meets the requirements when tested mix. Extract with two 20-mL portions of methylene chloride,
as directed for Membrane Filtration under Test for Sterilityof the and evaporate the combined methylene chloride extracts to a
Product to be Examined. volume of about 0.5 mL. Transfer a few drops of the
pH (791): between 2.5 and 4.5. chloroform concentrate to a KRS-5 plate, and allow to dry.
Other requirements-It meets the requirements under Record the IR absorption spectrum by the attenuated total
Injections and Implanted Drug Products (1). reflectance technique (see Mid-InfraredSpectroscopy (854).
The spectrum thus obtained exhibits maxima only at the same
wavelengths as that of a similar preparation of USP
Aminopentamide Sulfate RS, concomitantly measured.

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 USP43 Official Monographs / Aminophylline 241

Disintegration (701): not more than 10 minutes, simulated Anhydrous [317-34-0].

gastric fluid TS being substituted for water in the test. Dihydrate [5897-66-5].
Uniformity of dosage units (90S): meet the requirements.
loss on drying (731 )-Dry about 1 g of powdered Tablets, DEFINITiON
accurately weighed, in vacuum at a pressure of 5 mm of Aminophylline is anhydrous or contains NMT two molecules
mercury or less at 60° for 3 hours: it loses not more than 4.0% of water of hydration. It contains NLT 84.0% and NMT
87.4% of anhydrous theophylline (C7H sN402), calculated on
of its weight.
the anhydrous basis.
Assay-
Mobilephase-Transfer 14.4 g of sodium lauryl sulfate to a IDENTIFICATION
500-mL volumetric flask, add 100 mL of glacial acetic acid
dilute with water to volume, mix, and pass through a filte~
having a 0.5-J.lm or finer porosity. Transfer 50 mL of this
solution to a 1OOO-mL volumetric flask, add 350 mL of
-A.
methanol and 350 mL of acetonitrile, dilute with water to S
volume, and mix. Filter and degas before use. Make Sample: 500 mg
adjustments if necessary (see System Suitability under Analysis: Dissolve the 20 mL of water add with
Chromatography (621». constant stirring, 1 mL of hydrochloric acid, filter (retain
~tandardpreparation-Q~antitativel~dissolve an accurately
the filtrate), wash the precipitate with small portions of cold
weighed quantity of USP Arnlnopentarnlde Sulfate RS in Mobile water, and dry at 105° for 1 h.
phase to obtain a solution having a known concentration of Acceptance criteria: The IR spectrum of theophylline so
about 0.02 mg per mL. obtained corresponds to that of USP Theophylline RS.
Assay preparat~on-Weigh and finely powder not fewer - B. Th.e retention time of the major peak of the Sample
than 10 Tablets. Transfer an accurately weighed portion of the soiution corresponds to that of the Standard solution as
powder, equivalent to about 0.2 mg of aminopentamide, to a obtained in the Assay. '
suitable flask. Add 10.0 mL of Mobilephase, sonicate for 5 - C.
minutes, and stir by mechanical means for about 10 minutes. Sample: The filtrate obtained in Identification A.
Pass this mixture through a filter having a 0.5-J.lm or finer Analysis: To the Sample add 0.5 mL of benzenesulfonyl
porosity, discarding the first 5 mL of the filtrate. Use the clear chloride and 5 mL of 1 N sodium hydroxide to render
filtrate as the Assay preparation. alkaline. Shake by mechanical means for 10 min, add 5
Chromatographic system (see Chromatography (621»-The mL of 3 N hydrochloric acid to acidify, chill, collect the
liquid chromatograph is equipped with a 254-nm detector precipitated disulfonamide of ethylenediamine, wash with
~md ~ 3.~-mm x 30-cm column that contains packing L1, and
water, recrystallize from water, and dry at 105° for 1 h.
IS maintained at a constant temperature of about 40°. The flow Acceptance criteria: The dried precipitate melts at 164°-
rate is about 1 mL per minute. Chromatograph the Standard 171°.
preparation, and record the peak responses as directed for ASSAY
Procedure: the column efficiency is not less than 900 - PROCEDURE
theoretical plates; and the relative standard deviation for Solution A: 10 mM ammonium acetate prepared asfollows.
replicate injections is not more than 2%. Transfer 770.8 mg of ammonium acetate to a 1-L
Procedure-Separately inject equal volumes (about 50 J.lL) volumetric flask, and dissolve in water to 80% of the flask
of the Standardpreparation and the Assay preparation into the v?lume..Adjust with glacial acetic acid to a pH of 5.5, and
chromatograph, record the chromatograms, and measure the dilute with water to volume. Pass through a suitable filter
areas for the major peaks. Calculate the quantity, in mg, of . of 0.2-J.lm pore size.
aminopentamide sulfate (C19H24N20 . H2S04) in the portion of Solution B: Methanol
Tablets taken by the formula: . Mobile phase: See Table 7.
10C(ru/rs) Table 1
Time Solution A Solution B
in which C is the concentration, in mg per mL, of USP (min) (%) (%)
Aminopentamide Sulfate RS in the Standard preparation; and
to and rs are the aminopentamide peak responses obtained 0 98 2
from the Assay preparation and the Standard preparation, 7 50 50
respectively.
7.3 10 90
8.3 10 90
8.31 98 2
Aminophylline 12 98 2

Impurity stock solution: 25 J.lg/mL of USP Theophylline

~NH,
H,N
Related Compound F RS in water
System suitability solution: 0.8 mg/mL of USP
Theophylline RS and 1 J.lg/mL of USP Theophylline Related
Compound FRS in water prepared as follows. Transfer 21
C16H24N1004 420.43 mg of USP Theophylline RS to a 25-mL volumetric flask and
C16H24N1004·2H20 456.46 add 15 mL of water. Sonicate to dissolve, add 1 mL of
1H-Purine-2,6-dione, 3,7-dihydro-1,3-dimethyl-, compd. Impurity stock solution, and dilute with water to volume.
with 1,2-ethanediamine (2: 1); . Standard solution: 0.17 mg/mL of USP Theophylline RS in
Theophylline compound with ethylenediamine (2:1). water. Sonicate to dissolve as needed.

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242 Aminophylline / Official Monographs USP 43

Sample solution: 0.2 mg/mL of Aminophylline in water System suitability

Chromatographic system Samples: System suitability solution and Standard solution
(See Chromatography (621), System Suitability.) [NoTE-See Table 2 for the relative retention tirnes.]
Mode: LC Suitability. requirements
Detector: UV 270 nm Resolution: NLT 2.0 between theophylline and
Column: 2.1-mm x 10-cm; l.7-JJm packing L1 theophylline related compound F, System suitability
Column temperature: 40 0 solution
Flow rate: 0.4 mL/min Relative standard deviation: NMT 3.0% for each peak,
Injection volume: 1 JJL Standard solution
System suitability Analysis
Samples: System suitability solution and Standard solution Samples: Standard solution and Sample solution
Suitability requirements Calculate the percentage of caffeine, theophylline related
Resolution: NLT 2.0 between theophylline and compound B, theophylline related compound C,
theophylline related compound F, System suitability theophylline related compound D, and theophylline
solution related compound F in the portion of Aminophylline
Relative standard deviation: NMT 0.73%, Standard taken:
solution
Analysis Result = (rulrs) x (Cs/Cu) x 100
Samples: Standard solution and Sample solution
Calculate the percentage of theophylline (C7H sN 40 2) in the ru = peak response of caffeine, theophylline related
portion of Aminophylline taken: compound B, theophylline related compound C,
theophylline related compound D, or
Result =.(rulrs) x (CslCu) x 100 theophylline related compound F from the
Sample solution
to = peak response of theophylline from the Sample rs = peak response of the corresponding Reference
solution Standard from the Standard solution
ts = peak response of theophylline from the Standard Cs = concentration of the corresponding Reference
solution Standard in the Standard solution (mg/mL)
Cs = concentration of USP Theophylline RS in the Cu =concentration of Aminophylline in the Sample
Standard solution (mg/mL) solution (mg/mL)
Cu = concentration of Aminophylline in the Sample
solution (mg/mL) Calculate the percentage of dimethyl uric acid,
theobromine, and any other individual unspecified
Acceptance criteria: 84.0%-87.4% of theophylline on the impurity in the portion of Aminophylline taken:
anhydrous basis
Result =(rufrs) x (CslCu) x (l/A x 100
OTHER COMPONENTS
• CONTENT OF ETHYLENEDIAMINE t» = peak response of dimethyl uric acid,
Sample: 500 mg of Aminophylline theobromine, or any other individual unspecified
Diluent: Water impurity from the Sample solution
Titrimetric system rs = peak response of theophylline from the Standard
Mode: Direct titration solution
Titrant: 0.1 N hydrochloric acid VS Cs =concentration of USP Theophylline RS in the
Endpoint detection: Visual Standard solution (mg/mL)
Analysis: Dissolvethe Sample in 30 mL of Diluent, add Cu =concentration of Aminophylline in the Sample
methyl orange TS, and titrate. Each mL of 0.1 N solution (mg/mL)
hydrochloric acid is equivalent to 3.005 mg of F = relative response factor
ethylenediamine (C2H sN 2) .
Acceptance criteria: 157-175 mg of ethylenediamine Acceptance criteria: See Table 2. Disregard peaks less than
(C2H sN 2 ) per gram of theophylline (C7H sN 4 0 2) found in the 0.05%.
Assay
Table 2
IMPURITIES
Relative Relative Acceptance
• RESIDUE ON IGNITION (281): NMT 0.15% Retention Response Criteria,
• ORGANIC IMPURITIES Name Time Factor NMT(%)
Solution A, Solution B, Mobile phase, System suitability
Theophylline related com-
solution, and Chromatographic system: Proceed as pound C 0.36 - 0.10
directed in the Assay.
Standard stock solution: 25.0 JJg/mL each of USP Caffeine Theophylline related com-
pound B 0.63 - 0.10
RS, USP Theophylline RS, USP Theophylline Related
Compound B RS, USP Theophylline Related Compound C Theophylline related com-
pound D 0.69 - 0.10
RS, USP Theophylline Related Compound DRS, and USP
Theophylline Related Compound F RS in water . Dimethyl uricacid" 0.76 0.55 0.10
Standard solution: 1.0 JJg/mL each of USP Caffeine RS, USP
Theophylline RS, USP Theophylline Related Compound B Theobrornlne'' 0.82 1.0 0.10
RS, USP Theophylline Related Compound C RS, USP Theophylline 1.0 - -
Theophylline Related. Compound DRS, and USP
Theophylline Related Compound FRS in water, from Theophylline related com-
pound F 1.09 - 0.10
Standard stock solution
Sample solution: 1.0 mg/mL of Aminophylline in water Caffeine 1.20 - 0.10

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 USP 43 Official Monographs / Aminophylline 243

Table 2 (continued) IDENTIFICATION

Relative Relative Acceptance • A.
Retention Response Criteria, Analysis: Dilute a volume of Injection equivalent to 500 mg
Name Time Factor NMT (%) of aminophylline with water to 20 mL, and add, with
Any other individual unspe- constant stirring, 1 mL of 3 N hydrochloric acid or enough
cified impurity - 1.0 0.10 to completely precipitate the theophylline, and filter. To the
filtrate add 0.5 mL of benzenesulfonyl chloride and 5 mL of
Total impurities - - 0.3
1 N sodium hydroxide to render alkaline. Shake by
a 1,3-Dimethyl-7,9-dihydro-1 H-purine-2,6,8(3H)-trione. mechanical means for 10 min, add 5 mL of 3 N hydrochloric
b 3,7-Dihydro-3,7-dimethylpurine-2,6(1 H)-diane. acid to acidify, chill, collect the precipitated disulfonamide
of ethylenediamine, wash with water, recrystallize from
SPECIFIC TESTS water, and dry at 105 0 for 1 h.
• WATER DETERMINATION (921), MethodI Acceptance criteria: The precipitate melts at 164 0-1 710 •
Sample: 1.5 9 of Aminophylline
Solvent: 50 mL of chloroform and anhydrous methanol
(50:50) in place of anhydrous methanol
Acceptance criteria
Anhydrous: NMT 0.75%
Hydrous: NMT 7.9% A;nalysis: Wash the precipitated ,theophylline from
Identification A with small portions of cold water, and dry
ADDITIONAL REQUIREMENTS at 105 0 for 1 h.
• PACKAGING AND STORAGE: Preserve in tight containers. Acceptance criteria: The IRspectrum of theophylline so
Store at controlled room temperature. obtained corresponds to that of USP Theophylline RS.
• LABELING: Label it to indicate whether it is anhydrous or • C. The retention time of the major peak of the Sample
hydrous, and also to state the content of anhydrous solution corresponds to that of the Standard solution, as
theophylline. obtained in the Assay.
• USP REFERENCE STANDARDS (11)
USP Caffeine RS ASSAY
USP Theophylline RS • PROCEDURE
USP Theophylline Related Compound B RS Solution A: 10 mM ammonium acetate prepared asfollows.
3-Methyl-l H-purine-2,6-dione; Transfer 770.8 mg of ammonium acetate to a l-L
Also known as 3-Methyl-3,7-dihydro-l H-purine-2,6- volumetric flask, and dissolve in water to 80% of the flask
dione. volume. Adjust with glacial acetic acid to a pH of 5.5 and
C6H6N40 2 166:14 dilute with water to volume. Pass through a suitable filter
USP Theophylline Related Compound C RS of 0.2-lJm pore size.
N-( 6-Amino-l,3-dimethyl-2,4-dioxo-l,2,3,4- Solution B: Methanol
tetrahydropyrimidin-5-yl)formamide. Mobile phase: See Table 1.
C7H,oN 40 3 198.18
Table 1
USP Theophylline Related Compound D RS
Theophyllidine; Time Solution A Solution B
(min) (%) (%)
N-Methyl-5-(methylamino)-1 H-imidazole-4-
carboxamide hydrochloride monohydrate.. 0 98 2
C6H,oN40 . HCI . H20 208.65 7 50 50
USP Theophylline Related Compound F RS .
7-(2-Hydroxyethyl)-l,3-dimethyl-3,7-dihydro-l H-purine- 7.3 10 90
2,6-dione. 8.3 10 90
C9H12N 40 3 224.22
8.31 98 2

12 98 2

Aminophylline Injection Impurity stock solution: 25 IJg/mL of USP Theophylline

Related Compound F RS in water
DEFINITION System suitability solution: 0.8 mg/mL of USP
Aminophylline Injection is a sterile solution of Aminophylline Theophylline RS and 1 IJg/mL of USP Theophylline Related
in Water for Injection, or is a sterile solution of Theophylline Compound F RS in water prepared asfollows. Transfer 21
in Water for Injection prepared with the aid of mg of USP Theophylline RS to a 25-mL volumetric flask, and
Ethylenediamine. It contains, in each mL, an amount of add 15 mL of water. Sonicate to dissolve, add 1 mL of
aminophylline (C'6H24N'004) equivalent to NLT 93.0% and Impuritystock solution, and dilute with water to volume.
NMT 107.0% of the labeled amount of anhydrous Standard solution: 0.1 7 mg/mL of USP Theophylline RS in
theophylline (C 7H sN 40 2) . water. Sonicate to dissolve as needed.
Aminophylline Injection may contain an excess of Sample solution: Nominally 0.17 mg/mL of anhydrous
Ethylenediamine, but no other substance may be added for theophylline in water prepared as follows. Transfer 8.5 mg
the purpose of pH adjustment. of anhydrous theophylline from a volume of Injection to a
[NOTE-Do not use the Injection if crystals have separated.l 50-mL volumetric flask. Dissolve and dilute with water to
volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC

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244 Aminophylline / OfficialMonographs USP43

Detector: UV 270 nm Analysis

Column: 2.1-mm x 10-cm; 1.7-um packing L1 Samples: Standard solution and Sample solution
Column temperature: 40° Calculate the percentage of theophylline related
Flow rate: 0.4 mL/min compound D in the portion of Injection taken:
Injection volume: 1 IJL
System suitability Result =(r ulr s) x (C siC u) x 100
Samples: System suitabilitysolution and Standardsolution
Suitability requirements ru = peak responseof theophylline related compound
Resolution: NLT 2.0 between theophylline and D from the Sample solution
theophylline related compound F, System suitability rs =peak responseof theophylline related compound
solution D from the Standard solution
Relative standard deviation: NMT 1.0%, Standard Cs = concentration of USP Theophylline Related
solution Compound D RS in the Standard solution
Analysis (mg/mL)
Samples: Standard solution and Sample solution Cu =nominal concentration of aminophylline in the
Calculate the percentage of the labeled amount of Sample solution (rnq/rnt)
theophylline (C7H sN 40 2) in the portion of Injection taken:
Calculate the percentage of any other individual
Result = (r ulr s) x (C siC u) x 100 unspecified degradation product in the portion of
Injection taken:
= peak response of theophylline from the Sample
solution Result =(r ulr s) x (C siC u) x 100
= peak response of theophylline from the Standard
solution ru = peak responseof any other individual unspecified
= concentration of USP Theophylline RS in the degradation product from the Sample solution
Standard solution (mg/mL) rs =peak response of theophylline from the Standard
Cu = nominal concentration of theophylline in the solution
Sample solution(mg/mL) . Cs =concentration of USP Theophylline RS in the
Standard solution(mg/mL)
Acceptance criteria: 93.0%-107.0% Cu =nominal concentration of aminophylline in the
Sample solution(mg/mL)
OTHER COMPONENTS .
• CONTENT OF ETHYLENEDIAMINE Acceptance criteria: See Table 2. Disregard peaks less than
Sample: A volume of Injection equivalent to 500 mg of 0.05%.
aminophylline
Diluent: Water Table 2
Titrimetric system Relative Acceptance
Mode: Direct titration Retention Criteria,
Titrant: 0.1 N hydrochloric acid VS Name Time NMT(%)
Endpoint detection: Visual Theophylline relatedcompound ca. b 0.36 -
Analysis: If necessary, dilute the Sample with Diluent to 30
mL, add methyl orange TS, and titrate with Titrant. Each Theophylline relatedcompound Ba. c 0.63 -
mL of 0.1 N hydrochloric acid is equivalent to 3.005 mg of Theophylline relatedcompound D 0.69 0.2
ethylenediamine (C2H sN2 ) .
Acceptance criteria: 166-192 mg of ethylenediamine Dimethyl uricacid>. d 0.76 -
(C2H sN 2) per gram of theophylline (C7H sN 4 0 2) found in the Theobrornlnev e 0.82 -
Assay
Theophylline 1.0 -
IMPURITIES
• ORGANIC IMPURITIES
Theophylline relatedcompound F" 1.09 -
Solution A, Solution B, Mobile phase, Impurity stock Caffeinea 1.20 -
solution, System suitability solution, and
Anyother individual unspecified degrada-
Chromatographic system: Proceed as directed in the tion product - 0.2
Assay.
Standard solution: 2.0 IJg/mL each of USP Theophylline RS Totaldegradation products - 0.5
and USP Theophylline Related Compound D RS in water
a Process impurity includedfor identification onlyand not to be includedin the
Sample solution: Nominally 1.0 mg/mL of anhydrous calculation of total degradation products.
aminophylline in water prepared asfollows. Transfer 25 mg b N-(6-Amino-1 ,3-dimethyl-2,4-dioxo-1 ,2,3,4-tetrahydropyrimidin-S-yl)
of anhydrous aminophylline from a volume of Injection to formamide.
a 25-mL volumetric flask. Dissolveand dilute with water to c 3-Methyl-1 H-purine-2,6-dione.
volume. d 1,3-Dimethyl-7,9-dihydro-1 H-purine-2,6,8(3H)-trione.
System suitability e 3,7-Dihydro-3,7-dimethylpurine-2,6(1 H)-dione.
Samples: System sUitability solution and Standardsolution
[NoTE-See Table 2 for the relative retention tirnes.] SPECIFIC TESTS
Suitability requirements • pH (791): 8.6-9.0
Resolution: NLT 2.0 between theophylline and • PARTICULATE MATTER IN INJECTIONS (788): Meets the
theophylline related compound F, System suitability requirements for small-volume injections
solution • OTHER REQUIREMENTS: Meets the requirements in Injections
Relative standard deviation: NMT 3.0% for each peak, and Implanted Drug Products (1)
Standard solution

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USP 43 Official Monographs / Aminophylline 245

It BACTERIAL ENDOTOXINS TEST (85): It contains NMT 1.0 USP and dilute with water to volume. Pass through a suitable
Endotoxin Unit/mg of aminophylline. filter of 0.2-JJm pore size.
Solution B: Methanol
ADDITIONAL REQUIREMENTS Mobile phase: See Table 7.
• PACKAGING AND STORAGE: Preserve in single-dose
containers from which carbon dioxide has been excluded, Table 1
preferably of Type I glass, protected from light. Store at
controlled room temperature. Time Solution A Solution B
(min) (%) (%)
• LABELING: Label the Injection to state the content of
anhydrous theophylline. 0 98 2
• USP REFERENCE STANDARDS (11) 7 50 50
USP Theophylline RS
USP Theophylline Related Compound D RS 7.3 10 90
Theophyllidine; 8.3 10 90
N-Methyl-5-(methylamino)-1 H-imidazole-4-
carboxamide hydrochloride monohydrate. 8.31 98 2
C6H lON 40 . HCI . H20 208.65 12 98 2
USP Theophylline Related Compound F RS
7-(2-Hydroxyethyl)-1 ,3-dimethyl-3,7-dihydro-l H-purine-
2,6-dione. Impurity stock solution: 25 IJg/mL of USP Theophylline
Related Compound F RS in water
C9H12N403 224.22
System suitability solution: 0.8 mg/mL of USP
Theophylline RS and 2 IJg/mL of USP Theophylline Related
Compound F RS in water prepared asfollows. Transfer 1
mg of USP Theophylline RS to a 25-mL volumetric flask and
Aminophylline Oral Solution add 15 mL of water. Sonicate to dissolve, add 2 mL of
Impurity stock solution, and dilute with water to volume.
DEFINITION Standard solution: 0.17 mg/mL of USP Theophylline RS in
Aminophylline Oral Solution is an aqueous solution of water. Sonicate to dissolve, as needed.
Aminophylline, prepared with the aid of Ethylenediamine. It Sample solution: Nominally 0.1 7 mg/mL of anhydrous
contains an amount of aminophylline (C16H24N1004) theophylline in water prepared as follows. Transfer an
equivalent to NLT 90.0% and NMT 110.0% of the labeled appropriate amount of anhydrous theophylline from a
amount of anhydrous theophylline (C7H sN 402). volume of Oral Solution to a suitable volumetric flask.
Aminophylline Oral Solution may contain an excess of Dissolve and dilute with water to volume.
ethylenediamine, but no other substance may be added for Chromatographic system
the purpose of pH adjustment. (See Chromatography (621), System Suitability.)
Mode: LC
IDENTIFICATION Detector: UV 270 nm
Column: 2.1-mm x 10-cm; 1.7-urn packing L1
Column temperature: 40°
Flow rate: 0.4 mL/min
• A. Injection volume: 1 IJL
$j:Jg rbSeb Q~9) System suitability
Analysis: Transfer" a volume of Oral Solution equivalent to Samples: System suitabilitysolution and Standard solution
500 mg of aminophylline to a suitable container and add, Suitability requirements
with constant stirring, 1 mL of 3 N hydrochloric acid or Resolution: NLT 2.0 between theophylline and
enough to completely precipitate the theophylline. Filter theophylline related compound F, System sUitability
(retain the filtrate), wash the precipitate with small portions solution
of cold water until free from chloride, and dry at 105° for 1 Relative standard deviation: NMT 1.0%, Standard
h. solution
Acceptance criteria: The IR spectrum of theophylline so Analysis
obtained matches that of USP Theophylline RS. Samples: Standard solution and Sample solution
• B. Calculate the percentage of the labeled amount of
Analysis: To the filtrate obtained in Identification A add 0.5 theophylline (C7H sN 402) in the portion of Oral Solution
mL of benzenesulfonyl chloride and 5 mL of 1 N sodium taken:
hydroxide to render alkaline. Shake by mechanical means
for 10 min, add 5 mL of 3 N hydrochloric acid to acidify, Result =(rvlrs) x (CsICv) x 100
chill, collect the precipitated disulfonamide of
ethylenediamine, wash with water, recrystallizefrom water, rv = peak responsefrom the Sample solution
and dry at 105° for 1 h. . rs = peak response from the Standard solution
Acceptance criteria: The dried precipitate melts at 164°- Cs =concentration of USP Theophylline RS in the
171°. Standard solution(mg/mL)
ASSAY Cu =nominal concentration of theophylline in the
Sample solution(mg/mL)
• PROCEDURE
Solution A: 10 mM ammonium acetate prepared asfollows.
Acceptance criteria: 90.00/0-110.0%
Transfer an appropriate amount of ammonium acetate to a
volumetric flask and dissolve in water (about 80% of the
flask volume). Adjust with glacial acetic acid to a pH of 5.5

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246 Aminophylline / Official Monographs USP 43

OTHER COMPONENTS Acceptance criteria: See Table 2. Disregard peaks less than
• CONTENT OF ETHYLENEDIAMINE 0.086%.
Sample: A volume of Oral Solution equivalent to 500 mg of
aminophylline Table 2
Diluent: Water Relative Acceptance
Titrimetric system Retention Criteria,
Mode: Direct titration Name Time NMT(%)
Titrant: 0.1 N hydrochloric acid VS Theophylline related compound ca, b 0.36 -
Endpoint detection: Visual
Analysis: If necessary, dilute the Sample with Diluent to 30 Theophylline related compound Ba,c 0.63 -
mL, add methyl orange TS, and titrate. Each mL of 0.1 N Theophylline related compound D 0.69 0.2
hydrochloric acid is equivalent to 3.005 mg of
ethylenediamine (C2H sN 2) . Dimethyl uric acld- d 0.76 -
Acceptance criteria: 176-283 mg of ethylenediamine Theobromine- e 0.82 -
(C2H sN 2 ) per gram of theophylline (C7H sN 4 0 2 ) found in the
Assay Theophylline 1.0 -
IMPURITIES
Theophylline related compound P 1.09 -
• ORGANIC IMPURITIES Caffeine" 1.20 -
Solution A, Solution 8, Mobile phase, Impurity stock Any other individual unspecified degrada-
solution, System suitability solution, and tion product . - 0.2
Chromatographic system: Proceed as directed in the
Assay. Total degradation products - 0.5
Standard solution: 2.0 IJg/mL each of USP Theophylline RS
a Process impurity included for identification only and not to be included in the
and USP Theophylline Related Compound D RS in water calculation of total degradation products.
Sample solution: Nominally 1.0 mg/mL of anhydrous b N-(6-Amino-l ,3-dimethyl-2,4-dioxo-l ,2,3,4-tetrahydropyrimidin-5-yl)
aminophylline in water prepared as follows. Transfer an formamide.
appropriate amount of anhydrous aminophylline from a c 3-Methyl-l H-purine-2,6-dione.
volume of Oral Solution to a suitable volumetric flask. d 1,3-Dimethyl-7,9-dihydro-l H-purine-2,6,8(3H)-trione.
Dissolve and dilute with water to volume. e 3,7-Dihydro-3,7-dimethylpurine-2,6(1 H)-dione.
System suitability
Samples: System suitability solution and Standard solution SPECIFIC TESTS
[NoTE-See Table 2 for relative retention times.] • pH (791): 8.5-9.7
Suitability requirements
ADDITIONAL REQUIREMENTS
Resolution: NLT 2.0 between theophylline and
• PACKAGING AND STORAGE: Preserve in tight containers.
theophylline related compound F, System suitability
• LABELING: Label the Oral Solution to state the content of
solution
anhydrous theophylline.
Relative standard deviation: NMT 3.0% for each peak
• USP REFERENCE STANDARDS (11)
present in the Standard solution
USP Theophylline RS
Analysis
USP Theophylline Related Compound D RS
Samples: Standard solution and Sample solution
Theophyllidine;
Calculate the percentage of theophylline.related
N-Methyl-5-(methylamino)-1 H-imidazole-4-
compound D in the portion of Oral Solution taken:
carboxamide hydrochloride monohydrate.
Result = (rulrs) x (CslCu) x 100 C6H lON4 0 · HCI· H2 0 208.65
USP Theophylline Related Compound F RS
ru =peak response of theophylline related compound 7-(2-Hydroxyethyl)-1, 3-dimethyl-3, 7-dihydro-l H-purine-
D from the Sample solution 2,6-dione.
rs =peak response of theophylline related compound C9H12N403 224.22
D from the Standard solution
Cs =concentration of USP Theophylline Related
Compound D RS in the Standardsolution
(mg/mL)
Cu =nominal concentration of anhydrous theophylline Aminophynine Rectal Solution
in the Sample solution (mg/mL)
DEFINITION
Calculate the percentage of any other individual Aminophylline Rectal Solution is an aqueous solution of
unspecified degradation product in the portion of Oral Aminophylline, prepared with the aid of Ethylenediamine. It
Solution taken: contains an amount of aminophylline equivalent to NLT
90.0% and NMT 110.0% of the labeled amount of
Result =(rulrs) x (CslCu) x 100 anhydrous theophylline (C7H sN 40 2) .
Rectal Solution may contain an excess of ethylenediamine, but
ru =peak response of any other individual unspecified no other substance may be added for the purpose of pH
degradation product from the Sample solution adjustment.
rs = peak response of theophylline from the Standard
solution
Cs = concentration of USP Theophylline RS in the
Standard solution (mg/mL)
Cu = nominal concentratlon of anhydrous theophylline
in the Sample solution (mg/mL)

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 USP 43 Official Monographs / Aminophylline 247

IDENTIFICATION Acceptance criteria: 218-267 mg of ethylenediamine

(C2H sN 2) per g of theophylline (C7H sN 4 0 2 ) found in the
Assay

• A· ., \~~~~~~I~~~
SPECIFIC TESTS
Spectr.().$c()PY:)l~~i.!(. ..' . . . 0) e pH (791): 9.0-9.5
Sample: Dilute an amount of Rectal Solution, equivalent to ADDITIONAL REQUIREMENTS
500 mg of aminophylline, with water to 20 mL. Add, with e PACKAGING AND STORAGE: Preserve in tight, single-dose or
constant stirring, 1 mL of 3 N hydrochloric acid or enough multiple-dose containers at controlled room temperature.
to precipitate the theophylline completely, and filter (save e LABELING: Label the Rectal Solution to state the content of
the filtrate). Wash the precipitate with a small portion of anhydrous theophylline.
cold water until free from chloride, and dry at 105° for 4 h. e USP REFERENCE STANDARDS (11)
Acceptance criteria: The dried precipitate meets the USP Theophylline RS
requirements.
• B.
Analysis: To the filtrate obtained in Identification test A add
0.5 mL of benzenesulfonyl chloride and 5 mL of 1 N sodium
hydroxide to render alkaline, shake by mechanical means Aminophynine Suppositories
for 10 min, and add 5 mL of 3 N hydrochloric acid to acidify.
Chill, collect the precipitated disulfonamide of DEFINITION
ethylenediamine, and wash with water. Recrystallize the Aminophylline Suppositories contain an amount of
washed precipitate from water, and dry at 105° for 1 h. aminophylline equivalent to NLT 90.0% and NMT 110.0%
Acceptance criteria: The dried precipitate melts at 164°- of the labeled amount of anhydrous theophylline
171°. (C7H sN 402) ·
ASSAY IDENTIFICATION
e PROCEDURE eA.
Standard solution: 8 IJg/mL of USP Theophylline RS in Analysis: Evaporate on a steam bath a portion of Sample
dilute hydrochloric acid (1 :100) stocksolution from the Assay, equivalent to 500 mg of
Sample solution: Pipet a volume of Rectal Solution aminophylline, to about one-half its volume. Adjust with 1
equivalent to 500 mg of aminophylline into a 500-mL N sodium hydroxide to a pH of 7.0, chill, and filter the
volumetric flask, and dilute with water to volume. Pipet 5 crystals of theophylline. Retain the filtrate, free from
mL of this solution to a second 500-mL volumetric flask, add washings. Wash the crystals of theophylline with small
50-mL of dilute hydrochloric acid (1:10), and dilute with portions of ice-cold water, and dry at 105° for 1 h.
water to volume. Acceptance criteria: The recrystallized theophylline melts
Instrumental conditions at 270°-274°.
Mode: UV • B.
Analytical wavelength: About 270 nm Analysis: Transfer 10 mg of the dried precipitate from
Cell: 1 em Identification test A to a porcelain dish, and add 1 mL of
Blank: Dilute hydrochloric acid (1:100) hydrochloric acid and 100 mg of potassium chlorate.
Analysis Evaporate on a steam bath to dryness, and invert the dish
Samples: Standardsolution and Sample solution over a vessel containing a few drops of 6 N ammonium
Calculate the percentage of the labeled amount of hydroxide.
anhydrous theophylline (C 7H sN 40 2) in the portion of Acceptance criteria: The residue acquires a purple color,
Rectal Solution taken: . which is destroyed by solutions of fixed alkalies.

Result =(AulAs) x (CsiCu) x 100

• c.
Analysis: To the filtrate obtained in Identification test A add
0.5 mL of benzenesulfonyl chloride and 5 mL of 1 N sodium
=absorbance of the Sample solution hydroxide to render alkaline, shake by mechanical means
=absorbance of the Standardsolution for 10 min, and add 5 mL of 3 N hydrochloric acid to acidify.
=concentration of USP Theophylline RS in the Chill, collect the precipitated disulfonamide of
Standardsolution (lJg/mL) ethylenediamine, and wash with water. Recrystallize the
=nominal concentration of theophylline in the washed precipitate from water, and dry at 105° for 1 h.
Sample solution (lJg/mL) Acceptance criteria: The dried precipitate melts at 164°-
171°.
Acceptance criteria: 90.0%-110.0%
ASSAY
OTHER COMPONENTS • PROCEDURE
e CONTENT OF ETHYLENEDIAMINE Sample stock solution: Transfer NLT 5 Suppositories to a
Sample solution: Measure a volume of Rectal Solution, tared small dish and a glassrod, and heat on a steam bath
equivalent to 500 mg of aminophylline, and dilute with until the suppositories are melted. Mix the melt by stirring
water if necessary to make 30 mL. it with the rod, and cool while stirring. Transfer a portion of
Titrimetric system the cooled melt, equivalent to 1 g of aminophylline, into a
Mode: Direct titration beaker, add 60 mL of hot water and 3 mL of nitric acid, and
Titrant: 0.1 N hydrochloric acid VS heat on a steam bath for 15 min with frequent stirring.
Endpoint detection: Visual Cool, transfer to a separator with the aid of 40 mL of ether,
Analysis: Add methyl orange TSto the Sample solution, and shake well, and allow to separate, using a few mL of alcohol
titrate. Each mL of 0.1 N hydrochloric acid is equivalent to if necessary to bring about separation of any emulsion that
3.005 mg of ethylene diamine (C 2H sN 2) . hasformed. Draw the water layer into a 1OO-mL volumetric
flask; wash the ether with two 15-mL portions of water,

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248 Aminophylline / Official Monographs USP 43

adding the washings to the volumetric flask; and dilute with IDENTIFICATION
water to volume.
Sample solution: Transfer a portion of the Sample stock
solution, equivalent to 250 mg of aminophylline, to a
250-mL conical flask. Add 10 mL of 6 N ammonium
hydroxide and 20 mL of 0.1 N silver nitrate VS, and heat on
a steam bath for 15 min. Cool to between 5° and 10° for Analysis: quantity of Tablets, equivalent to 500
20 min; filter, preferably through a filtering crucible of fine mg of aminophylline, with 25 mL of water, and filter. The
porosity under reduced pressure; and wash the precipitate filtrate is alkaline to litmus. To the filtrate add 1 mL of 3 N
with small portions of water until the last washing gives hydrochloric acid, stir, and if necessary, chill to precipitate
NMT a faint opalescence with hydrochloric acid. Dissolve the theophylline. Filter, and retain the filtrate, free from
the precipitate by pouring over it small volumes of warm 2 washings. Use the filtrate in Identification C. Wash the
N nitric acid, collecting the solution in a conical flask. Wash theophylline crystals so obtained with small quantities of
the filtering crucible a few times with warm water acidified ice-cold water, and dry at 105° for 1 h.
with nitric acid, receiving the washings in the same flask. Acceptance criteria: The IR spectrum of theophylline so
Cool, and add 2 mL of ferric ammonium sulfate TS. obtained corresponds to that of USP Theophylline RS.
Analysis: Titrate with 0.1 N ammonium thiocyanate VS. • B. The retention time of the major peak of the Sample
Each mL of 0.1 N ammonium thiocyanate is equivalent to solution corresponds to that of the Standard solution, as
18.02 mg of theophylline (C 7H aN4 0 2 ) . obtained in the Assay.
Acceptance criteria: 90.0%-11 0.0% • c.
Sample: The filtrate obtained in Identification A
OTHER COMPONENTS Analysis: To the Sample add 0.5 mL of benzenesulfonyl
• CONTENT OF ETHYLENEDIAMINE chloride and 5 mL of 1 N sodium hydroxide to render
Sample solution: Weigh a portion of the stirred, congealed alkaline, shake by mechanical means for 10 min, and add 5
mass of the Suppositories from the Assay, equivalent to 500 mL of 3 N hydrochloric acid to acidify. Chill, collect the
mg of aminophylline, and place in a 500-mL conical flask. precipitated disulfonamide of ethylenediamine, and wash
Add 150 mL of a mixture of equal volumes of alcohol and with water. Recrystallizethe washed precipitate from water,
ether, and warm gently under reflux for 30 min, with and dry at 105° for 1 h.
occasional swirling. Cool to room temperature. Acceptance criteria: The dried precipitate melts at 164°-
Titrimetric system 171°.
Mode: Direct titration
Titrant: 0.1 N hydrochloric acid VS ASSAY
Endpoint detection: Potentiometric • PROCEDURE
Analysis: Titrate the Sample solution using a glass-modified Solution A: 10 mM ammonium acetate prepared asfollows.
calomel electrode system (replace the saturated potassium Transfer 770.8 mg of ammonium acetate to a 1-L
chloride solution of the calomel electrode with methanol volumetric flask, and dissolve in water to 80% of the flask
saturated with lithium chloride). Each mL of 0.1 N volume. Adjust with glacial acetic acid to a pH of 5.5 and
hydrochloric acid is equivalent to 3.005 mg of dilute with water to volume. Pass through a suitable filter
ethylenediamine (C 2H aN2 ) . of 0.2-l.Jm pore size.
Acceptance criteria: 152-190 mg of ethylenediamine Solution B: Methanol
(C2H aN2) per g of theophylline (C7H aN40 2) found in the Mobile phase: See Table 7.
Assay
Table 1
ADDITIONAL REQUIREMENTS Time Solution A Solution B
• PACKAGING AND STORAGE: Preserve in well-closed (min) (%) (%)
containers, in a cold place.
• LABELING: Label the Suppositories to state the content of
0 98 2
anhydrous theophylline. 7 50 50
7.3 10 90
8.3 10 90

Aminophylline Tablets 8.31 98 2

12 98 2
DEFINITION
Aminophylline Tablets contain an amount of aminophylline
equivalent to NLT 93.0% and NMT 107.0% of the labeled Impurity stock solution: 25 I.Jg/mLof USP Theophylline
amount of anhydrous theophylline (C 7H aN40 2 ) . Related Compound F RS in water
System suitability solution: 0.8 mg/mL of USP
[NoTE-The ammoniacal odor present in the vapor space .
Theophylline RS and 1 I.Jg/mLof USP Theophylline Related
above the Tablets is often quite strong, especially when
Compound F RS in water prepared as follows. Transfer 21
bottles having suitably tight closures are newly opened.
mg of USP Theophylline RS to a 25-mL volumetric flask and
This is due to ethylenediamine vapor pressure build-up,
add 15 mL of water. Sonicate to dissolve, add 1 mL of
a natural condition in the case of arnlnophylllne.]
Impurity stock solution, and dilute with water to volume.
Standard solution: 0.17 mg/mL of USP Theophylline RS in
water. Sonicate to dissolve as needed.
Sample solution: Nominally 0.1 7 mg/mL of anhydrous
theophylline from NLT 20 finely powdered Tablets in water
prepared as follows. Transfer 34 mg of anhydrous
theophylline from a portion of the powder to a 200-mL

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USP 43 OfficialMonographs / Aminophylline 249

volumetric flask. Add 20 mL of water and mix for 1 min. Analytical wavelength: UV about 269 nm
Add an additional 140 mL of water and sonicate for 30 min. Analysis
Dilute with water to volume. Pass through a suitable filter Samples: Standard solution and Sample solution
of 0.22-lJm pore size, discarding the first 2-3 mL. Calculate the percentage of the labeledamount of
Chromatographic system theophylline (C7 HsN 4 0 2) dissolved.
(See Chromatography (621), System Suitability.) Tolerances: NLT 75% (Q) of the labeled amount of
Mode: LC theophylline (C7HsN 4 0 2) is dissolved.
Detector: UV 270 nm • UNIFORMITY OF DOSAGE UNITS (905)
Column: 2.1-mm x 10-cm; l.7-lJm packing L1 Procedure for content uniformity
Column temperature: 40° Standard solution: 10 IJg/mL of USP Theophylline RS
Flow rate: 0.4 mL/min Sample solution: Place 1 Tablet in a 250-mL volumetric
Injection volume: 1 IJL flask, add 200 mL of water, and shake by mechanical
System suitability means until disintegration is complete. Add water to
Samples: System suitability solution and Standard solution volume. Filter a portion of the mixture, discarding the first
Suitability requirements 20 mL of the filtrate.
Resolution: NLT 2.0 between theophylline and Instrumental conditions
theophylline related compound F, System suitability Mode: UV
solution Analytical wavelength: About 269 nm
Relative standard deviation: NMT 1.0%, Standard Cell: 1 cm
solution Blank: Water
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of Calculate the percentage of the labeled amount of
theophylline (C7H sN 4 0 2) in the portion of Tablets taken: anhydrous theophylline (C7H sN 4 0 2) in the Tablet taken:
Result =(ru/rs) x (Cs/Cu) x 100 Result = (Au/As) x (CslCu) x 100
= peak response of theophylline from the Sample = absorbance of the Sample solution
solution = absorbance of the Standard solution
= peak response of theophylline from the Standard =concentration of USP Theophylline RS in the
solution Standard solution (uq/rnt)
= concentration of USP Theophylline RS in the =nominal concentration of theophylline in the
Standard solution (mg/mL) Sample solution (lJg/mL)
= nominal 'concentration of theophylline in the
Sample solution (mg/mL) Acceptance criteria: Meet the requirements
Acceptance criteria: 93.0%-1 07.0% IMPURITIES
• ORGANIC IMPURITIES
OTHER COMPONENTS Solution A, Solution B, Mobile phase, Impurity stock
• CONTENT OF ETHYLENEDIAMINE solution, System suitability solution, and
Sample solution: Transfer a portion of the powdered Chromatographic system: Proceed as directed in the
Tablets, equivalent to 350 mg of aminophylline, prepared Assay.
in the Assay, into a 1OO-mL conical flask. Add 20 mL of Standard solution: 2.0 IJg/mL each of USP Theophylline RS
water, and digest at 50°, with frequent shaking, fo~ 30 min. and USP Theophylline Related Compound D RS in water
Cool, filter into a 250-mL conical flask, and wash with water Sample solution: Nominally 1.0 mg/mL of anhydrous
until the last washing is neutral to litmus. Usethe combined aminophylline from NLT 20 finely powdered Tablets in
filtrate and washings. !
water prepared asfollows. Transfer 10 mg of anhydrous
Titrimetric system aminophylline from a portion of the powder to a 10-mL
Mode: Direct titration volumetric flask. Add 5 mL of water and sonicatefor 30 min.
Titrant: 0.1 N hydrochloric acid VS Dilute with water to volume. Pass through a suitable filter
Endpoint detection: Visual of 0.22-lJm pore size, discarding the first 2-3 mL.
Analysis: Add methyl orange TSto the Sample solution, and System suitability
titrate. Each mL of 0.1 N hydrochloric acid is equivalent to Samples: System suitability solution and Standard solution
3.005 mg of ethylenediamine (C2H sN 2) . [NoTE-See Table 2 for relative retention times.]
Acceptance criteria: 140-190 mg of ethylenediamine Suitability requirements ,
(C2H sN 2) per gram of theophylline (C7H sN 4 0 2 ) found in the Resolution: NLT 2.0 between theophylline and
Assay theophylline related compound F, System suitability
PERFORMANCE TESTS
solution
Relative standard deviation: NMT 3.0% for each peak,
• DISSOLUTION (711)
For uncoated or plain coated tablets Standard solution
Medium: Water; 900 mL Analysis
Apparatus 2: 50 rpm Samples: Standard solution and Sample solution
Time: 45 min Calculate the percentage of theophylline related
Standard solution: USP Theophylline RS in Medium compound D in the portion of Tablets taken:
Sample solution: Proceed as directed in ~he chap.ter. fo.r Result = (ru/rs) x (CslCu) x 100
sample. Dilute with water to a concentration that IS similar
to that of the Standard solution. = peak response of theophylline related compound
Instrumental conditions D from the Sample solution
Mode: UV-Vis

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 250 Aminophylline / Official Monographs USP 43

= peak response of theophylline related compound Aminophylline Delayed-Release Tablets

D from the Standard solution
=concentration of USP Theophylline Related DEfiNITION
Compound D RS in the Standard solution Aminophylline Delayed-Release Tablets contain an amount of
(mg/mL) aminophylline equivalent to NLT 93.0% and NMT 107.0%
=nominal concentration of aminophylline in the of the labeled amount of anhydrous theophylline
Sample solution(mg/mL)
(C7H sN 402) ·
Calculate the percentage of any other individual [NoTE-The ammoniacal odor present in the vapor space
unspecified degradation product in the portion of Tablets above Tablets is often quite strong, especially when
taken: bottles having suitably tight closures are newly opened.
This is due to ethylenediamine vapor pressure build-up,
Result = (rulrs) x (CsICu) x 100 a natural condition in the case of aminophylline.]
IDENTIFICATION
=peak response of any other individual unspecified eA.
degradation product from the Sample solution Analysis: Macerate a quantity of Tablets, equivalent to 500
=peak response of theophylline from the Standard mg of aminophylline, with 25 mL of water, and filter: the
solution filtrate is alkaline to litmus. To the filtrate add 1 mL of 3 N
=concentration of USP Theophylline RS in the hydrochloric acid, stir, and if necessary, chill to precipitate
Standard solution(mg/mL) the theophylline. Filter, and re.tain the filtrate, fre~ from.
= nominal concentration of aminophylline in the washings. Wash the theophylline crystals so obtained with
Sample solution(mg/mL) small quantities of ice-cold water, and dry at 105° for 1 h.
Transfer 10 mg of the dried theophylline crystals to a
Acceptance criteria: See Table 2. Disregard peaks less than porcelain dish, add 1 mL of hydrochloric acid and 100 mg
0.05%. of potassium chlorate, evaporate on a steam bath to
dryness, and invert the dish over a vessel containing a few
Table 2 drops of 6 N ammonium hydroxide.
Relative Acceptance Acceptance criteria: The residue acquires a purple color,
Retention Criteria, which is destroyed by solutions of fixed alkalies.
Name Time NMT (0/0)
e B.
Theophylline related compound ca, b 0.36 - Analysis: Recrystallize the dried theophylline crystals from
Identification test A from water, and dry at 105° for 1 h.
Theophylline related compound Ba,c 0.63 - Acceptance criteria: The recrystallized theophylline melts
Theophylline related compound D 0.69 0.2 at 270°-274°.
Dimethyl uric acid a, d 0.76 - e C.
Analysis: To the filtrate obtained in Identification test A add
Theobromine" e 0.82 - 0.5 mL of benzenesulfonyl chloride and 5 mL of 1 N sodium
hydroxide to render alkaline, shake by mechanical means
Theophylline 1.0 - for 10 min, and add 5 mL of 3 N hydrochloric acid to acidify.
Theophylline related compound P 1.09 - Chill, collect the precipitated disulfonamide of
Caffeinea 1.20 - ethylenediamine, and wash with water. Recrystallize the
washed precipitate from water, and dry at 105° for 1 h.
Any other individual unspecified deqrada- Acceptance criteria: The dried precipitate melts at 164°-
tion product - 0.2 171°.
Total degradation products - 0.5
ASSAY
a Process impurity included for identification only and not to be included in the e PROCEDURE
calculation of total degradation products. Sample solution: Transfer an equivalent to 2 g of
b N-(6.Amino.l ,3-dimethyl-2,4.dioxo-l ,2,3,4.tetrahydropyrimidin-5-yl) aminophylline, from powdered Tablets (NLT 20), to a
formamide. 200-mL volumetric flask with the aid of a mixture of 50 mL
c 3-Methyl.l H-purine.2,6.dione. of water and 15 mL of 6 N ammonium hydroxide, and allow
d 1,3-Dimethyl-7,9-dihydro-l H-purine·2,6,8(3H)-trione. to stand for 30 min with frequent shaking, warming to 50°
e 3,7.Dihydro-3,7-dimethylpurine-2,6(1 H)-dione. if necessary to dissolve the aminophylline. Cool the mixture
to room temperature if it has been warmed, and add water
ADDITIONAL REQUIREMENTS to volume. Centrifuge 50 mL of the mixture; pipet a portion
e PACKAGING AND STORAGE: Preserve in tight containers. of the clear supernatant, equivalent to 250 mg of
Store at controlled room temperature. aminophylline, into a 250-mL conical flask; and dilute with
e LABELING: Label the Tablets to state the content of water if necessary to make 40 mL. Add 8 mL of 6 N
anhydrous theophylline. ammonium hydroxide and 20.0 mL of 0.1 N silver nitrate
e USP REFERENCE STANDARDS (11) VS, mix, heat to boiling, and continue boiling for 15 min.
USP Theophylline RS Cool to between 5° and 10° for 20 min, then filter,
USP Theophylline Related Compound D RS preferably through a filtering crucible under reduced
Theophyllidine; . pressure, and wash the precipitate with three 10-mL
N-Methyl-5-(methylamino)-1 H-imidazole-4- portions of water. Acidify the combined filtrate and
carboxamide hydrochloride monohydrate. washings with nitric acid, and add an additional 3 mL of the
C6H lON4 0 . HCI . H2 0 208.65 acid. Cool, and add 2 mL of ferric ammonium sulfate TS.
USP Theophylline Related Compound F RS Titrimetric system
7-(2-Hydroxyethyl)-1 ,3-dimethyl-3,7-dihydro-l H-purine- Mode: Residual titration
2,6-dione. Titrant: 0.1 N ammonium thiocyanate VS
C9H12N4 0 3 224.22 Endpoint detection: Visual

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 USP 43 OfficialMonographs / Aminosalicylic 251

Analysis: Titrate the excess silver nitrate with Titrant. Each

mL of 0.1. N silver nitrate is equivalent to 18.02 mg of
Aminosalicylic Acid
theophylline (C7H sN40Z) ' '
Acceptance criteria: 93.00/0-107.0%
OTHERCOMPONENTS
~OH
H,N)lAoH
• CONTENT OF ETHYLENEDIAMINE
Sampl~ soluti~n: Transfer a portion, equivalent to 350 mg C7H 7N03 153.14
of aminophylline, of the powdered Tablets prepared in the Benzoic acid, 4-amino-2-hydroxy-;
Assay into a 1OO-mL conical flask. Add 20 mL of water and 4-Aminosalicylic acid [65-49-6].
?igest at 50°, with frequent shaking, for 30 min. Cool,'filter
Into a 250-mL conical flask, and wash with water until the DEFINITION
last washing is neutral to litmus. Use the combined filtrate Aminosalicylic Acid contains NLT 98.5% and NMT 100.5% of
and washings. aminosalicylic acid (C7H 7N03) , calculated on the anhydrous
Titrimetric system basis.
Mode: Direct titration [CAUTION-Under no circumstances use a solution prepared
Titrant: 0.1 N hydrochloric acid VS from Aminosalicylic Acid if its color is darker than that of
Endpoint detection: Visual a freshly prepared solution.]
Analysis: Add methyl orange TS to the Sample solution, and IDENTIFICATION
titrate. Each mL of 0.1 N hydrochloric acid is equivalent to
3.005 mg of ethylenediamine (CzHsNz). • A.
Sample stock solution: Dissolve 250 mg in 3 mL of 1 N
Acceptance criteria: 140-1 90 mg of ethylenediamine sodium hydroxide, transfer to a 500-mL volumetric flask
(CzHsN z) per 9 of theophylline (C 7H sN40Z) found in the and dilute with water to volume. '
Assay Sample solution: Transfer a 5-mL aliquot of the Sample stock
PERFORMANCE TESTS solution to a 250-mL volumetric flask containing 12.5 mL of
• DISINTEGRATION (701): 30 min, determined as directed in pH 7 phosphate buffer (see Reagents, Indicators, and
Delayed-Release (Enteric-Coated) Tablets Solutions-Buffer Solutions), and dilute with water to
• UNIFORMITY OF DOSAGE UNITS (905) Volume.
Procedure for content uniformity Analysis: Compare the Sample solution in a suitable
Standard solution: 10 J,Jg/mL of USP Theophylline RS spectrometer against a blank of the same buffer in the same
Sample solution: Place 1 Tablet in a 250-mL volumetric concentration.
flask, add 200 m L of water, and shake by mechanical Acceptance criteria: The Sample solution exhibits
means until disintegration is complete. Add water to absorbance maxima at 265 ± 2 and 299 ± 2 nm, and the
volume. Filter a portion of the mixture, discarding the first ratio Az6s1AZ99 is 1.50-1 .56.
20 mL of the filtrate. • B.
Instrumental conditions Sample: 1 9
Mode: UV Analysis: Place the Sample in a small, round-bottom flask
Analytical wavelength: About 269 nm and add 10 mL of acetic anhydride. Heat the flask on a '
Cell: 1 em steam bath for 30 min, add 40 mL of water, filter, cool, and
Blank: Water allow to stand until the diacetyl derivative has crystallized.
Analysis Collect the precipitate on a filter, wash well with water, and
Samples: Standardsolution and Sample solution dry at 105° for 1 h.
Calculate the percentage of the labeled amount of Acceptance criteria: The diacetyl derivative melts at 191°-
theophylline (C 7H sN40Z) in each Tablet: 197°.
• C.
Result = (AulAs) x (CsICu) x 100 Sample: 0.1 9
Analysis: Shake the Sample with 10 mL of water, and filter.
= absorbance of the Sample solution To 5 mL of the filtrate add 1 drop of ferric chloride TS.
= absorbance of the Standardsolution Acceptance criteria: A violet color is produced.
= concentration of USP Theophylline RS in the ASSAY
Standardsolution (J,Jg/mL) • PROCEDURE
= nominal concentration of theophylline in the Solution A: 12.7 mg/mL of tetrabutylammonium hydroxide
Sample solution (J,Jg/mL) in methanol
Mobile phase: Solution A, 0.05 M dibasic sodium
Acceptance criteria: Meet the requirements phosphate, and 0.05 M monobasic sodium phosphate
ADDITIONAL REQUIREMENTS (150:425:425)
• PACKAGING AND STORAGE: Preserve in tight containers. Internal standard solution: 5 mg/mL of acetaminophen in
• LABELING: Label the Tablets to state the content of Mobile phase
anhydrous theophylline. Standard solution: 0.5 mg/mL of aminosalicylic acid
• USP REFERENCE STANDARDS (11) prepared asfollows. Transfer 12.5 mg of USP Aminosalicylic
USP Theophylline RS Acid RS to a 25-mL low-actinic volumetric flask, add 15 mL
of Mobilephase, and swirl to dissolve. Add 2.5 mL of the
Internal standard solution, and dilute with Mobilephase to
volume.
Sample solution: 0.5 mg/mL of Aminosalicylic Acid
prepared as f9110ws. Transfer 12.5 mg of Aminosalicylic
Acid to a 25-mL low-actinic volumetric flask, add 15 mL of
Mobilephase, and swirl to dissolve. Add 2.5 mL of the

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252 Aminosaficyllc / OfficialMonographs USP 43

Internal standard solution, and dilute with Mobile phaseto Internal standard solution, and dilute with Mobile phase to
volume. volume.
Chromatographic system Chromatographic system
(See Chromatography (621), System Suitability.) (See Chromatography (621), System Suitability.)
Mode: LC Mode: LC
Detector: UV 254 nm Detector: UV 280 nm
Column: 4.6-mm x 25-cm; packing L1 Column: 4.6-mm x 25-cm; 1O-lJm packing L1
Flow rate: 1.5 mL/min Flow rate: 1.5 mL/min
Injection volume: 20 IJL Injection volume: 20 IJL
System suitability System suitability
Sample: Standard solution Sample: Standard solution
[NoTE-The relative retention times for acetaminophen [NoTE-The relative retention times for sulfanilamide
and aminosalicylic acid are 0.83 and 1.0, and m-aminophenol are about 0.66 and 1.0,
respectively.] respectively. ]
Suitability requirements Suitability requirements
Resolution: NLT 1.7 between aminosalicylic acid and Resolution: NLT 2.5 between m-aminophenol and
acetaminophen sulfanilamide
Relative standard deviation: NMT 1.0% for the peak Relative standard deviation: NMT 7%
response ratio of aminosalicylic acid to acetaminophen Analysis
Analysis Samples: Standard solution and Sample solution
Samples: Standard solution and Sample solution After use, wash the column for 30 min with a mixture of
After use, wash the column for 30 min with a mixture of methanol, water, and phosphoric acid (77: 23: 0.6), and
methanol, water, and phosphoric acid (77: 23: 0.6), and then wash for 30 min with a mixture of methanol and
then wash for 30 min with a mixture of methanol and water (50:50).
water (50:50). Calculate the percentage of m-aminophenol in the portion
Calculate the percentage of aminosalicylic acid (C7H 7N0 3) of Aminosalicylic Acid taken:
in the portion of Aminosalicylic Acid taken:
Result = (R viR s) x (C siC v) x 100
Result = (R viR s) x (C siC v) x 100
Ru = peak response ratio of m-aminophenol to
= peak response ratio of aminosalicylic acid to sulfanilamide from the Sample solution
acetaminophen from the Sample solution Rs = peak response ratio of m-aminophenol to
= peak response ratio of aminosalicylic acid to sulfanilamide from the Standard solution
acetaminophen from the Standard solution Cs = concentration of USP m-Aminophenol RS in the
= concentration of USPAminosalicylic Acid RS in Standard solution (mg/mL)
the Standard solution (mg/mL) Cu =concentration of the Sample solution, as
=concentration of Aminosalicylic Acid in the determined in the Assay(mg/mL)
Sample solution (mg/mL) .
Acceptance criteria: NMT 0.25%
Acceptance criteria: 98.5%-100.5% on the anhydrous
SPECIFIC TESTS
basis
• pH (791): 3.0-3.7, in a saturated solution
IMPURITIES • WATER DETERMINATION, Method I (921): NMT 0.5%
• RESIDUE ON IGNITION (281): NMT 0.2% • HYDROGEN SULFIDE, SULFUR DIOXIDE, AND AMYL ALCOHOL
• CHLORIDE AND SULFATE, Chloride (221) Sample: 500 mg
Sample solution: 25 mg/mL in a mixture of nitric acid and Analysis: Dissolve the Sample in 5 mL of 1 N sodium
water (5:15) hydroxide, add 6 mL of 3 N hydrochloric acid, and stir
Acceptance criteria: NMT 0.042%; the solution shows no vigorously.
more chloride than corresponds to 0.30 mL of 0.020 N Acceptance criteria: No odor of hydrogen sulfide or sulfur
hydrochloric acid. dioxide is perceptible, and NMT a faint odorof amyl alcohol
• LIMIT OF m-AMINOPHENOL is perceptible. A piece of moistened lead acetate test paper
Solution A: 12.7 mg/mL of tetrabutylammonium hydroxide held over the mixture does not become discolored.
in methanol • CLARITY AND COLOR OF SOLUTION
Mobile phase: Solution A, 0.05 M dibasic sodium Sample 1: 1 g
phosphate, and 0.05 M monobasic sodium phosphate Analysis 1: Dissolve Sample 1 in 10 mL of sodium
(150:425:425) bicarbonate solution (1 in 15).
Internal standard solution: 5 IJg/mL of sulfanilamide in Acceptance criteria 1: The resulting solution is clear and has
Mobile phase NMT a faint yellow color.
Standard stock solution: 12 IJg/mL of USPm-Aminophenol Sample 2: 1 g
RS in Mobile phase Analysis 2: Dissolve Sample 2 in 50 mL of freshly prepared
Standard solution: 1.2 IJg/mL of USP m-Aminophenol RS 1.6 M nitric acid.
prepared as follows. Transfer 10.0 mL of the Standard stock Acceptance criteria 2: The resulting solution is clear and has
solution and 10.0 mL of the Internal standard solution to a NMT a slight color. .
1OO-mL low-actinic volumetic flask, and dilute with Mobile ADDITIONAL REQUIREMENTS
phase to volume. • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Sample solution: 0.5 mg/mL of Aminosalicylic Acid containers at a temperature not exceeding 30°.
prepared as follows. Transfer 50 mg of Aminosalicylic Acid
• USP REFERENCE STANDARDS (11)
to a 1OO-mL low-actinic volumetic flask, add 50 mL of USP m-Aminophenol RS
Mobile phase, and swirl to dissolve. Add 10.0 mL of the USP Aminosalicylic Acid RS

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 USP 43 OfficialMonographs / Amiodarone 253

= concentration of USP Amiodarone Hydrochloride

Amiodarone Hydrochloride RS in the Standard solution (mg/mL)
Cu = nominal concentration of Amiodarone
Hydrochloride in the Sample solution (mg/mL)

Acceptance criteria: 98.5%-101 .0%, on the dried basis

IMPURITIES
INORGANIC IMPURITIES
• Residue on Ignition (281): NMT 0.1% on a 1-g sample
CzsHz91zN03' HCI _681.77 ORGANIC IMPURITIES
Methanone, (2-butyl-3-benzofuranyl)[4-[2-( diethyl amino) [NOTE-The product meets the requirements for both
ethoxy]-3,5-diiodophenyl]- hydrochloride; Procedure 7 and Procedure 2.]
2-Butyl-3-benzofuranyl 4-[2-( diethylamino)ethoxy]-3,5- • Procedure 1
diiodophenyl ketone hydrochloride [19774-82-4]. Potassium iodobismuthate solution: Dissolve 100 9 of
2-Butyl-3-benzofuranyl 4-[2-(diethylamino)ethoxy]-3,5- tartaric acid in 400 mL of water, and add 8.5 9 of bismuth
diiodophenyl ketone [1951-25-3]. subnitrate. Shake for 1 h, add 200 mL of a 400 giL solution
DEFINITION of potassium iodide, and shakewell. Allow to stand for 24 h,
Amiodarone Hydrochloride contains NLT 98.5% and NMT filter, and protect from light.
101.0% of CzsHz91zN03 . HCI, calculated on the dried basis. Standard solution A: 0.02 mg/mL of USP Amiodarone
Related Compound H RS in methylene chloride
IDENTIFICATION Standard solution B: StandardsolutionA and Sample solution
(1:1).
Sample solution: 100 mg/mL of Amiodarone Hydrochloride
in methylene chloride
Chromatographic system
(See Chromatography (621), Thin-Layer Chromatography.)
• II. iDENTIFICATION TESTS-GENERAL, Chloride (191): Meets Mode: TLC
the requirements Adsorbent: Suitable layer of chromatographic silica gel and
fluorescent indicator with maximum absorbance at 254 nm
ASSAY Application volume
• PROCEDURE Standard solution A: 50 IJL
Buffer: Dissolve 6.80 9 of monobasic potassium phosphate Standard solution B: 100 IJL
in 900 mL of water, and add 1.0 mLoftriethylamine. Adjust Sample solution: 50 IJL .
with phosphoric acid to a pH of 6.00 ± 0.05, and dilute with Developing solvent system: Methylene chloride,
water to 1000 mL. methanol, and anhydrous formic acid (17:2:1)
Diluent: Acetonitrile and water (1:1) Analysis
Mobile phase: Acetonitrile and Buffer(1:1) Samples: StandardsolutionA, Standardsolution B, and
Standard stock solution: 0.5 mg/mL of USP Amiodarone Sample solution
Hydrochloride RS in methanol Develop the plate in the Developing solvent system until the
Standard solution: 0.1 mg/mL USP Amiodarone . solvent front has moved NLT two-thirds the length of the
Hydrochloride RS in Diluent from Standardstock solution plate, and dry in a current of cold air. Spray the plate with
Sample stock solution: 0.5 mg/mL of Amiodarone Potassium iodobismuthate solution and then with 3%
Hydrochloride in methanol hydrogen peroxide solution. Examine immediately in
Sample solution: 0.1 mg/mL of Amiodarone . daylight: the spot from Standards?'ution B d.u~ to
Hydrochloride in Diluent from Sample stocksolution amiodarone related compound H IS clearly VISible.
Chromatographic system Acceptance criteria: Any spot with the same RF as the spot
(See Chromatography (621), System Suitability.) due to amiodarone related compound H from the Sample
Mode: LC solution is not more intense than the spot from Standard
Detector: UV 240 nm solutionA (0.02%).
Column: 3.9-mm x 15-cm; 5-lJm packing L26
• Procedure 2
Flow rate: 1.5 mL/min Buffer: Add 3 mL of glacial acetic acid to 800 mL of water.
Injection size: 10 IJL Adjust with diluted ammonia solution to a pH of 4.9, and
System suitability dilute with water to 1000 mL.
Sample: Standardsolution Mobile phase: Acetonitrile: methanol: Buffer (4:3:3 v/v/v).
Suitability requirements Diluent: Acetonitrile and water (1:1)
Column efficiency: NLT 1000 theoretical plates Standard stock solution: Dissolve equal quantities of USP
Tailing factor: NMT 2.0 Amiodarone Related Compound DRS, USP Amiodarone
Relative standard deviation: NMT 1.0% Related Compound E RS, and USP Amiodarone
Analysis Hydrochloride RS in a known amount of methanol.
Samples: Standardsolution and Sample solution Standard solution: 0.01 mg/mL each of USP Amiodarone
Calculate the percentage of CzsHz91zN03 . HCI in the Related Compound DRS, USP Amiodarone Related
portion of Amiodarone Hydrochloride taken: Compound E RS, and USP Amiodarone Hydrochloride RS, in
Diluent from Standardstock solution .
Result= (ru/rs) x (Cs/Cu) x 100 Sample solution: 5 mg/mL of Amiodarone Hydrochloride in
Diluent
ru =peak response of amiodarone in the Sample Chromatographic system
solution .
(See Chromatography (621), System Suitability.)
rs = peak response of amiodarone in the Standard
solution Mode: LC
Detector: UV 240 nm

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 254 Amiodarone / Official Monographs USP43

Column: 4.6-mm x 15-cm; 5-~m packing L1 Sample solution: To 15.0 mL of Solution A add 1.0 mL of
Column temperature: 300 0.1 M hydrochloric acid and 1.0 mL of 0.05 M potassium
Flow rate: 1 mL/min iodate. Dilute with water to 20.0 mL. Allow to stand
Injection size: 10 ~L protected from light for 4 h.
Run time: 2 times the retention time of amiodarone Analysis: Measure the absorbances of the Standard soluti~n
System suitability and the Sample solution at 420 nm, using a mixture of Ht:O
Sample: Standardsolution mL of Solution A and 1.0 mL of 0.1 M hydrochloric acid
Suitability requirements diluted with water to 20.0 mL to serve as the blank. The
Resolution: NLT 3.5 between amiodarone related absorbance of the Sample solution is NMT half the
compound D and amiodarone related compound E absorbance of the Standard solution.
Analysis Acceptance criteria: NMT 150 ppm
[NOTE-Disregard any peak that is less than 0.05%.] • pH (791): 3.2-3.8. Dissolve 1 g of Amiodarone
Samples: Standardsolution and Sample solution Hydrochloride in water by heating at 80 0 • Cool, and dilute
Calculate the percentage of each impurity in the portion of with water to 20 mL.
Amiodarone Hydrochloride taken: • Loss ON DRYING (731): Use 1 g of sample, and dry under
vacuum (NMT 0.3 kPa) at 50 0 for 4 h: it loses NMT 0.5%
Result = (ru/r s) x (Cs/Cu) x 100 of its weight.

ru =peak response of each impurity in the Sample ADDITIONAL REQUIREMENTS

solution • PACKAGING AND STORAGE: Preserve in light-resistant, tight
rs = peak response of amiodarone in the Standard containers. Store at controlled room temperature.
solution • USP REFERENCE STANDARDS (11)
Cs =concentration of USP Amiodarone Hydrochloride USP Amiodarone Hydrochloride RS
RS in the Standardsolution (mg/mL) USP Amiodarone Related Compound D RS
Cu = nominal concentration of Amiodarone (2-Butylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)
Hydrochloride in the Sample solution (mg/mL) methanone.
C19H161203 546.14
Acceptance criteria USP Amiodarone Related Compound E RS
Individual impurities: See Impurity Table 7. (2-Butylbenzofuran-3-yl)(4-hydroxyphenyl) methanone.
Total impurities: NMT 0.5% C19H1S03 294.34
USP Amiodarone Related Compound H RS
Impurity Table 1 2-Chloro-N,N-diethylethanamine.
I
Relative C6H14CIN 135.64
Reten- Acceptance
tion Criteria,
Name Time NMT (%)

Amiodarone related compound N 0.26 0.2

Amiodarone Hydrochloride Injection
Amiodarone related compound Db 0.29 0.2

Amiodarone related compound E' 0.37 0.2 DEFINITION

Amiodarone Hydrochloride Injection is a sterile solution of
Amiodarone related compound Bd 0.49 0.2 Amiodarone Hydrochloride. It contains NLT 90.0% and
Amiodarone related compound (e 0.55 0.2 NMT 110.0% of the labeled amount of amiodarone
hydrochloride (C2sH2912N03 . HCI). It may contain suitable
Amiodarone related compound Gf 0.62 0.2 preservatives.
Amiodarone related compound F9 0.69 0.2
IDENTIFICATION
Amiodarone hydrochloride 1.00 - • A. The retention time of the major peak of the Sample
solution corresponds to that of the Standardsolution, as
Any other individual impurity - 0.10
obtained in the Assay.
a (2-Butylbenzofuran-3-yl){4-[2-(diethylamine)ethoxy]phenyl}methanone.
b (2-Butylbenzofuran-3 -yl)(4-hydroxy-3,5-diiodophenyl)methanone.
e(2-Butylbenzoturan-3-yl)(4-hydroxyphenyl)methanone.
d (2-Butylbenzofuran-3-yl) {4-[2 -(ethyla mine)eth oxy]-3,5-diiodophenyl}
methanone.
e (2-Butylbenzofuran-3-yl){4-[2-(diethylamine)ethoxy]-3-iodophenyl}
methanone.
f [2-[(1 RS)-1-Methoxybutyl]benzofuran-3-yl][4-[2-(diethylamino)ethoxy]-3,5- ASSAY
diiodophenyl]methanone.
9 (2-Butylbenzofuran-3-yl)(4-hydroxy-3-iodophenyl)methanone.

SPECIFIC TESTS • PROCEDURE

• LIMIT OF IODIDES Buffer: 1.36 giL of potassium phosphate, monobasic in
Solution A: Add 1.50 g of Amiodarone Hydrochloride to 40 water prepared as follows. To 1.36 g of potassium
mL of water at 80 0 , and shake until completely dissolved. phosphate, monobasic in a 1-L volumetric flask add about
Cool, and dilute with water to 50.0 mL. 900 mL of water and 1 mL of triethylamine. Adjust with
Standard solution: To 15.0 mL of Solution A add 1.0 mL of phosphoric acid to a pH of 6.0. Dilute with water to volume.
0.1 M hydrochloric acid, 1.0 mL of an 88.2 mg/L solution Mobile phase: Acetonitrile and Buffer (80:20)
of potassium iodide, and 1.0 mL of 0.05 M potassium Diluent: Acetonitrile and water (60:40)
iodate. Dilute with water to 20.0 mL. Allow to stand Standard solution: 0.025 mg/mL of USP Amiodarone
protected from light for 4 h. Hydrochloride RS in Diluent

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USP 43 Official Monographs / Amiodarone 255

Sampl~ solution: Nominally~O.O.2[5;~(us~~i~i5~g.:iQJ~)mg/mL Detector: 200 0

of arnlodarone hydrochloride in Diluent from a suitable Carrier gas: Nitrogen

volume of Injection Flow rate: 10 mL/min
Chromatographic system Injection volume: 1 IJL
(See Chromatography (6.21), System Suitability.) Injection type: Split; split ratio, 10:1
Mode: LC System suitability
Detector: UV 240 nm. iji Sample: Standard solution
arrayqetectorit'i'tnera Suitability requirements
Column: 4.6-mm x 10-cm; 5-lJm packing L1 Relative standard deviation: NMT 2.0% for the peak
Flow rate: .2 mL/min response ratio of benzylalcohol to phenol
Injection volume: .20 IJL Analysis
Run time: NLT 2 times the retention time of amiodarone Samples: Standard solution and Sample solution
System suitability Calculatethe percentage of the labeled amount of benzyl
Sample: Standard solution alcohol in the portion of Injection taken:
Suitability requirements
Tailing factor: NMT .2.0 Result = (RulR s) x (CslC u) x 100
Relative standard deviation: NMT .2.0%
Analysis = peak response ratio of benzyl alcohol to phenol
Samples: Standard solution and Sample solution from the Sample solution
Calculate the percentage of the labeled amount of = peak response ratio of benzyl alcohol to phenol
amiodarone hydrochloride (CzsHz91zN03 . HC!) in the from the Standard solution
portion of Injectiontaken:
=concentration of USP Benzyl Alcohol RS in the
Standard solution (mg/mL)
Result = (ru/rs) x (Cs/Cu) x 100 = nominal concentration of benzyl alcohol in the
Sample solution (mg/mL)
= peak 1.response. (USP1~~ec~2<>19j of amiodarone
~cceptance criteria: 90.0%-110.0%
from the Sample solution
= peak 1.responseJ.:'(USp 1~oec:1019) of amiodarone IMPURITIES
from the Standard solution
= concentration of USP Amiodarone Hydrochloride
RS in the Standard solution (mg/mL)
= nominal concentration of amiodarone • LIMIT OF IODIDE
hydrochloride in the Sample solution (mg/mL) Usefreshly prepared solutions in amber glasswar~.
Amiodarone stock solution: • Nominally A,. (USP 1:De~-2019) 5
Acceptance criteria: 90.0%-110.0% mg/mL of ~miodarone hydrochloride in water from '
Injection ~~(usPJ~P~t:2019)
OTHER COMPONENTS Potassium iodide solution: 88.2 IJg/mL of potassium
iodide in water
Change to read: Potassium iodate solution: 10.7 gil of potassium iodide in
(if present) water
• CONTENT OF BENZYL ALCOHOL
Internal standard solution: 1 mg/mL of phenol in isopropyl of
Stafldard soll:ltion: 4.41 IJg/mL 1. potassium
alcohol .iodide. (USP 1,Oec-2019) prepared asfollows. Into a suitableflask
Standard stock solution: 1.1.6.of1. (USP j-Oec-2019j mg/mL of pi.p~!15.0 rTlL of Amiodarone stock solution, 1.0 mLof ....0.1
USP Benzyl Alcohol RS in iso ro I alcohol N.~(USP1"Oec-2019)hydrochloric acid, 1.0 mLof Potassium
Standard solution: AO;.2 ' iodide solution, 1.0 mL of Potassium iodate solution, and
of USP B IAlcohol R 2.0 mL of water. Mix, and allowto stand for 4 h. Protect
Internal dard solutio from light.
solution. (USP I-D~c=2019) Sal1lple solution: arone
Sample stock solution: NominaIlY"J;6~' (U'S' hY(:Jr~diloriae " _ Into a
mg/mL of benzyl alcohol in isopropyl alcoho suitable flask p~ipet 15.0 mL ofAmiodarone stock solution,
Inje~tion...: (USP 1~Dec-2019) 1.0 mLof·O.l N. (USP1,:oec-2019)hydrochloric acid, 1.0 mL of
Sample solution: Nominally 0.2 mg/mL of phenol and 0.19 Potassium iodate solution, and 3.0 mLof water. Mix, and
mg/mL of benzyl alcohol in isopropyl alc?h?l~fron-{the allow to stand for 4 h. Protect from light.
internal standard solution and the Sample stock Blank: Intoa suitableflask pipet 15.0 mL of Amiodarone stock
solution1. (USP 1-0ec-2019) , " , solution, 1.0 mL of1.0~1 N1.(USP1-Oec~io19)hydrochloric acid,
Blank: 0.2 mg/mL of phenolirl,isopropyl alcoholl from :1:he and 4.0 mLofwater. Mix, and allowto stand for 4 h. Protect
InternaJ-standardsolutioi). (USrl-0~~2019) , .. ' from light.
Chromatographic system . Instrumental conditions
(See Chromatography (621), System Suitability.) Mode: ~•• (U.SP'1-o~~:2In9jViS
Mode: GC Analytical wavelength: 420 nm
Detector: Flame ionization Cell: 1 ern
Column: 0.32-mm x 30-m Afused'silica Analysis
capillary;1. (USP 1-0ec-Z019) coated with l-lJm Afilrn Samples: Standard solution, Sample solution, and Blank
()f1. (USP 1-0ec-2019) phase G16 ' .. Calculate the amount of iodide, in ppm, in the portion of
Temperature's' Injection taken:
Injection port: 200 0
Result =(Au - As)/[(As - As) - (Au - As)] x (Cs/Cu) x (M rdMr2 )
Column: 150 0

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256 Amiodarone / OfficialMonographs USP 43

= absorbance of the Sample solution Calculate the percentage of amiodarone related compound
= absorbance of the Blank D or amiodarone related compound E in the portion of
= absorbance of the Standardsolution Injection taken:
= concentration of potassium iodide in the Standard
solution (lJg/mL) Result = (ru/rs) x (CslCu) x 100
= An ominaIA(llSP';-Dec-2019) concentration of = peak AresponseA.(tJSPJ-b~-2oi9j of amiodarone
amiodarone hydrochloride in the Sample solution
related compound D or amiodarone related
(g/mL)
= molecular weight of iodide, 126.90 compound Efrom th,e. Sarnple solution
= molecular weight of potassium iodide, 166.00 = peak Ar.esponse A (USP1~De~-,29J!) of amiodarone
related compound D or amiodarone related
Acceptance criteria: NMT 250 ppm compound Efrom the Standard solution
AA (USP 1-Dec~2019)
Change to read: = concentration of USP Amiodarone Related
Compound D RS or USPAmiodarone Related
• ORGANIC IMPURITIES Compound E RS in the Standardsolution
Buffer: AAdd3 mL~faceticacid,glacial to mL of AI.. (U~P 1-Dec-2019) (mg/mL)
water. Adjust with ammonia TS to a pH of Dilute with = nominal concentration of amiodarone
water to 1000 (TlL. A (USP l-Dec-2019) hydrochloride in the Sample solution (mg/mL)
Mobile phase: Acetonitrile, methanol, and Buffer
(40:30:30) Calculate the percentage of any unspecified degradation
Diluent: Acetonitrile, methanol, and water 50:30:20) product in the portion of Injection taken:
ASt stock • P
A ne Hy Result = (rulr s) x (CslCu) x 100
Stan a tock s
_ Related Compoun RS
=peak Aresponsei(USP1.Dec.2()19) of any unspecified
degradation product from the Sample solution
Standard solution: " IJgl f USPAmiodaro
Hy hloride RS i 30 pg/mL of USPA . ' = peak Arespons~A(USP1~[)eC-2019) of amiodar~ne
Co nd DRS, and 2 f.J from the Standard solution.A A'(USP~~~D~C';O;9j
C d ERS inDilu =concentration of USP Amiodarone' ,
S uantityof Hydrochloride RS in the Standardsolution
itable v A;;; (USP 1"Dec-2019) (mg/mL)
f Standa = nominal concentration of amiodarone
. Dilute Diluent u ,,' 19) hydrochloride in the Sample solution (mg/mL)
Sample solution: Nominally 1 mg/mL of amiodarone
hydrochloride in Diluentfrom a suitable volume of Injection Acceptance criteria: See Table 1.
Chromatographic system '
Table 1
(See Chromatography (621), System Suitability.)
Mode: LC Relative Acceptance
Detector: UV 240 nm Retention Criteria,
Name Time NMT (%)
Column: 4.6-mm x 15-cm; 5-lJm packing L1
Temperatures Amiodaronerelated
A:Autosampler: 2°_8° A (USP 1-De~-2019) compound EAA
(USP hOec-2019) 0.39 0.2
Column: 30°
Flow rate: 1 mL/min Arnlodarone related
Injection volume: 10 IJL compound D A.l
Run time: NLT 1.5 times the retention time of (USP 1..fJec-2019) 0.55 3.0
amiodarone for the Standardsolution, and NLT 2 times the Amiodarone 1.00 -
retention time of amiodarone for the Sample solution
System suitability Any unspecified
Sample: Standardsolution"
degradation -
product 0.20
[NoTE-:-See Table 1 for relative retention times.]
Total 'Ade ation
A (USP 1-De(:~.?019)
'product$.l usp lcOec.2019)
- 3.5
Suitability requirements
":Resolution:NLT 3.5 betWeen theamiodarone related
compound E and~miodaronerelated compound D . SPECIFIC TESTS
peaksA (USP1.Dec.2019) ,
Tailing factor: NMT 2.0 Afor th e Change to read:
amiodaronerelated compc>und ,loc1arone
• BACTERIAL ENDOTO~INS 'fEST (85): "Meets.the
related compound,E peaksA(usF! 1~)
requirements A (USP 1-DeC~2019)
Relative standard deviation: NMT 5.0% Afor the
• STERILITY TESTS (71): Meets the requirements
amiodarone,amiodarone related compound D, and
• pH (791): 3.0-5.0
amiodarone related compound E peaks~ (USP 1-Det-2019) • PARTICULATE MAITER IN INJECTIONS (788): Meets the
Analysis requirements for small-volume injections
Samples: Standardsolution '~A (USP1-Dec-2019) and Sample • OTHER REQUIREMENTS: Meets the requirements in Injections
solution and Implanted Drug Products (1)

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 USP 43 Official Monographs / Amiodarone 257

ADDITIONAL REQUIREMENTS Flow rate: 1.5 mL/min

• PACKAGING AND STORAGE: Preserve in single-dose or Injection volume: 10 I.JL
multiple-dose glass containers, protected from light and System suitability
excessive heat. Store at controlled room temperature. Sample: Standard solution
• LABELING: Label it to indicate that it is to be diluted to the [NoTE-The retention time for amiodarone is about 3.6
appropriate strength with a suitable parenteral vehicle prior min.]
to administration. Label it to indicate the type and amount Suitability requirements
of preservative used. Label it to indicate that it is Relative standard deviation: NMT 2.1 % for replicate
preservative free, if no preservative is present. injections
• USP REFERENCE STANDARDS (11) Analysis
USP Amiodarone Hydrochloride RS Samples: Standard solution and Sample solution
USP Amiodarone Related Compound DRS Calculate the percentage of the labeled amount of
(2-Butylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl) amiodarone hydrochloride (CzsHz91zN03 . HCI) in the
methanone. portion of Oral Suspensiontaken:
C19H161z03 546.14
USP Amiodarone Related Compound E RS Result = (rulrs) x (CsICu) x 100
(2-Butylbenzofuran-3-yl)(4-hydroxyphenyl)methanone.
C19H1803 294.34 t» = peak response from the Sample solution
USP Benzyl Alcohol RS rs =peak response from the Standardsolution
Cs = concentration of USP Amiodarone Hydrochloride
RS in the Standard solution (mg/mL)
Cu = nominal concentration of amiodarone
hydrochloride in the Sample solution (mg/mL)
Amiodarone Hydrochloride
Compounded Oral Suspension Acceptance criteria: 90.00/0-110.0%
SPECIFIC TESTS
DEFINITION • pH (791): 5.8-6.8
Amiodarone Hydrochloride Compounded Oral Suspension
contains NLT 90.0% and NMT 110.0% of the labeled ADDITIONAL REQUIREMENTS
amount of amiodarone hydrochloride (CzsHz91zN03 . HCI). • PACKAGING AND STORAGE: Package in tight, light-resistant
Prepare Amiodarone Hydrochloride Compounded Oral containers. Store in a refrigerator or at controlled room
Suspension 5 mg/mL as follows (see Pharmaceutical temperature.
Compounding-Nonsterile Preparations (795». • BEYOND-USE DATE: NMT 90 days after the date on which it
was compounded when stored in a refrigerator; NMT 30
days when stored at controlled room temperature
Amiodarone Hydrochloride tablets" 600 mg of arnlodar-
equivalentto one hydrochloride • LABELING: Label it to state that it is to be well shaken before
use and to state the Beyond-Use Date.
Vehicle: a 1:1 mixture of Ora-Sweet" (regular or sug- • USP REFERENCE STANDARDS (11 )
ar-free) and Ora-Plus, b a sufficient quantity to make 120 mL USP Amiodarone Hydrochloride RS
a Cordarone 200-mg tablets, Wyeth-Ayerst Laboratories, Philadelphia, PA.
b PaddockLaboratories, Minneapolis, MN.

Calculate the required quantity of each ingredient for the total

amount to be prepared. Place the required number of
Amiodarone Hydrochloride tablets in a suitable mortar and
comminute to a fine powder with a pestle. Adjust the pH of
the Vehicle to 6.5 ± 0.5 with a sodium bicarbonate
50-mg/mL solution prepared in Purified Water. Add the
Vehicle in small portions and triturate to make a smooth
paste. Add increasing volumes of the Vehicle to make an
amiodarone hydrochloride liquid that is pourable. Transfer
the contents of the mortar, stepwise and quantitatively, to a
calibrated bottle. Add enough of the Vehicle to bring to final
volume and mix well.
ASSAY
• PROCEDURE
Mobile phase: Methanol, water, and 50 mM monobasic
ammonium phosphate (0.5: 0.5: 99)
Standard solution: 2.5 mg/mL of USP Amiodarone
Hydrochloride RS in Mobilephase
Sample solution: Shake thoroughly by hand each bottle of
the Oral Suspension. Prepare 2.5 mg/mL of amiodarone
hydrochloride from Oral Suspension and Mobilephase.
Chromatographic system
(See Chromatography (621), System SUitability.)
Mode: LC
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 10-l.Jm packing L1

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258 Amiodarone / OfficialMonographs USP 43

a quantity,equivalent to 100mg of ami Cell: ·1 ,em

hydrochloride, from NLT 20 finely po Blank: Medium
1OO-mLvolumetric'flask, Add Mobilep Anal sis
of the final flask volume. Sonicate with Sa dard solution and Sample soltition
to dissolve. Cool the solution and dilu Cal ntage of the labeledamount of ,
to volume, ' ainiodar chloride (C2sH2912N03·· HCI),dissolved:
$ample solution:. Nominally;O~
hydrochloride in Mobilephase Result , ;,- (Au/As) x C~ xp x V x (1 /L)x ,100
Pass a portion of the solutionthro uitab
0.45-~m pore size, discardthefirstfew milliliter Au = absorbance of amiodarone from theSarnple
collectthe filtrate. solution
Chromatographic system ::;-absorbance of amiodarohe from the Standard
(See Chromatography(621), SysiemSuitdDility.) solution "
Mode:, LC = concentration of USP Amiodarone
Detector: UV 240 nm.For Identification 8, usea diode array Hydrochloride RS in the Standard solution
detector in the range of 200~OO nm. (mg/mL) ", ,_. ' _,'
Column:' 4.6:-f)1m x 25-cm;, 5-~rn packing L1 D =dilution fattor for the Sample solution
Column temperature: 30° V =volume of Medium, 1000 mL
Flow,rate: 1 mL/miri L = label claim of amiodarone hydrochloride (mg/
Injection volume: 10 ~L Tablet)
Runtime; NLT 2.5 times theretentiontime of amiodarone
System suitability' .- Tolerances:-NLT 80% (Q) of the la
Sample: Standard solution -amiodarone hydrochlori C2sH
Suitability requirements • MITY OF DOSAGE NITS (9
Tailing factor: NMT 2.0 Irements .
Relative standard deviation: NMT2.0%
Analysis
Samples: Standardsoluti • URITIES
acetic acid,glacial to 800mL of water.
Calculate the percentage mmonia hydroxidesolutionto a pH
amiodarone hydrochlorid r 0 mL'
portion of Tabletstaken: ril , hanoi, and .Buffer
I Result = (ru/r s) x (Cs/("u) x 1 00

= pe~k response of amiodclrone'from the Sample,

solution
= peak response.ofamiodaronefrom the Standard f USP Amiodarone
ar:d solution
solution
= concentration of USP Amiodarone miodarone
Hydrochloride RS in the Standard solution a
(mg/mL) . ride
= nominal concentration of amiodarone
etric
hydrochloride in the Sample solution (mg!mL)
Acceptance criteria: 90.0~11 0.0%
PERFORMANCE TESTS
• DISSOLUTION (711 )
Medium: ,1 % (w/v) sodiumdodecyl sulfate;l,OOO'niL
Apparatus 2: 100 rpm
Time: 60 min
Standard stock solution: 0.2 mg/
Hydrochloride RS pr~pared as
appropriate quantity of USP A
to a suitable volumetric fla
the final f e.Soni
Medium t imes the
S'tandard s' 0.01' m e 0 ution
Hydrochloride RS in Mediu . 0 S iii
Sample solution: Dilute a'portion 0 amp es: andard solution and SensitIvity solution
test with Medium to a c ' ration s Suitability- requirements
Standardsolution. Pass n of the so Relativestandard deviation: NMT 10.0%, Standard
suitablefilter of0.45-~m size, discard the solution
milliliters, and collectth teo , Signal':to-noise ratio: NLT 10, Sensitivity solution
Instrumental condition~ Analysis .. _ .'.
(See,Ultraviolet-Visible' Spectroscopy :(85'7).) Samples: Standard solution and. Sample solution
Mode:UV
Analytical wavelength: 243nm

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USP 43 Official Monographs / Amitraz 259

Methanimidamide,N'-(2,4-dimethylphenyl)-N-[[(2,4-
dimethylphenyl)imino]methyl]-N-methyl-;
N-Methyl-N'-2,4-xylyl-N-(N-2,4-xylylformimidoyl)
formamidine;
N-Methylbis(2,4-xylyliminomethyl)amine [33089-61-1].
DEFINITION
Amitraz contains NlT 95.0% and NMT 101.5% of amitraz
(C'9H23N3), calculated on the anhydrous basis.
IDENTIFICATION

~ ..... Q)
• amitraz peak of t e Sample
solution corresponds to that of the Standardsolution, as
obtained in the Assay.
ASSAY
• PROCEDURE
Internal standard solution: 0.7% v/v solution of squalane
in methyl acetate
Standard solution: 5.0 mg/ml of USP Amitraz RS in Internal
standard solution
Sample solution: 5.0 mg/ml of Amitraz in Internalstandard
.solutton
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 0.53-mm x 15-m fused silica; coated with a
1.5-llm layer of liquid phase G9
Temperatures
Detector: 300 0
Inlet: 230 0
Column: 220 0
Carrier gas: Helium
Flow rate: 12 ml/min
Injection volume: 1 III
System suitability
Sample: Standardsolution
[NOTE-The elution order is amitraz followed by
squalane.]
Suitability requirements
Resolution: NlT 3.0 between amitraz and squalane
Relative standard deviation: NMT 2.0% from the peak
area ratio of amitraz to squalane
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of amitraz (C,9H23N3) in the
portion of Amitraz taken:
Result = (RulR s) x (Cs/Cu) x .1 00
= peak response ratio of amitraz and squalane
from the Sample solution
= peak response ratio of amitraz and squalane
from the Standardsolution
=concentration of USP Amitraz RS in the Standard
solution (mg/ml)
Amitraz = concentration of Amitraz in the Sample solution
(mg/ml)
Acceptance criteria: 95.00/0-101.5% on the anhydrous
basis
IMPURITIES
293.41 • RESIDUE ON IGNITION (281): NMT 0.2%

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260 Amitraz / OfficialMonographs USP 43

• OR.GANIC IMPURITIES Table 2

Standard solution: 0.05 mg/mL of 2,4-dimethylaniline, Relative Acceptance
1.0 mg/mL of USP Amitraz Related Compound A RS, 0.5 Retention Criteria,
mg/mL of USP Amitraz Related Compound B RS, and 1.0 Name Time NMT (%)
mg/mL of USP Amitraz Related Compound C RS in methyl 2,4-0imethylaniline 0.11 0.1
acetate
Sample solution: 50.0 mg/mL of Amitraz in methyl acetate Amitraz related compound A 0.35 2
Chromatographic system Amitraz related compound B 0.40 1
(See Chromatography (621), System Suitability.)
Mode: GC Amitraz related compound C 0.86 2
Detector: Flame ionization Amitraz 1.0 -
Column: 0.53-mm x 1O-mfused silica; coated with a 5-lJm
layer of liquid phase G27 Any other individual impurity - 0.1
Temperatures
Detector: 300 0 SPECIFIC TESTS
Inlet: 230 0 • WATER DETERMINATION, Method1(921): NMT 0.1 %
Column: See Table 7.
ADDITIONAL REQUIREMENTS
Table 1 • PACKAGING AND STORAGE: Preserve in well-closed
containers.
Hold Time at Fi- • LABELING: Label it to indicate that it is for veterinary use
Initial Temperature Final nal
Temperature Ramp Temperature Temperature only.
(0) e/min) (0) (min) • USP REFERENCE STANDARDS (11)
USP Amitraz RS
125 0 125 5
USP Amitraz Related Compound A RS
125 5 270 15 2,4-Dimethylphenyl formamide;
N-(2,4-Dimethylphenyl)formamide.
Carrier gas: Helium C9H ll NO 149.19
Flow rate: 12 mL/min USP Amitraz Related Compound B RS
Injection volume: 1 IJL 2,4-Dimethylphenyl N-methyl-formamidine;
System suitability N'-(2,4-Dimethylphenyl)-N-methylformimidamide.
Sample: Standard solution ClOH 14N 2 162.23
Suitability requirements USP Amitraz Related Compound C RS
Resolution: NLT 3.0 between amitraz related compound Bisformamidine analog;
A and amitraz related compound B N,N'-Bis(2,4-dimethylphenyl)formimidamide.
Analysis C17H 20N2 252.35
Samples: Standard solution and Sample solution
Calculate the percentage of each of amitraz related
compounds A, B, and C in the portion of Amitraz taken:

Result =(rulrs) x (CslCu) x 100 Amitraz Concentrate for Dip

~ peak response of each individual impurity from DEFINITION
the Sample solution . Amitraz Concentrate for Dip contains arnltraz in a suitable
= peak response of the corresponding related vehicle. It may contain a suitable stabilizing agent. It contains
compound from the Standardsolution NLT 90.0% and NMT 120.0% of the labeled amount of
= concentration of the corresponding related amitraz (C19H23N3)'
compound in the Standard solution (mg/mL)
IDENTIFICATION
= concentration of Amitraz in the Sample solution
• A. THIN-LAYER CHROMATOGRAPHY
(mg/mL) Standard solution: 5 mg/mL of USP Amitraz RS in toluene
Sample solution: Nominally 5 mg/mL of amitraz from
Calcula~e t~~ perc.entag~ of
2,4-dime~hylaniline and any
Concentrate for Dip diluted with toluene .
other Indlvldual Impurity In the portion of Amitraz taken:
Chromatographic system
Result = (rufrs) x (CslCu) x 100 (See Chromatography (621), Thin-Layer Chromatography.)
Mode: TLC
= peak response of each individual impurity from Adsorbent: 0.25-mm layer of chromatographic silica gel
the Sample solution mixture
= peak response of 2,4-dimethylaniline from the Application volume: 2 IJL
Standard solution Developing solvent system: Cyclohexane, ethyl acetate
= concentration of 2,4-dimethylaniline in the and triethylamine (5:3:2) ,
Standard solution (mg/mL) Spray reagent: 0.5% solution of N-(l-naphthyl)
= concentration of Amitraz in the Sample solution ethylenediamine dihydrochloride in methanol
(mg/mL) Analysis
Samples: Standard solution and Sample solution .
Acceptance criteria: See Table 2. The reporting level for Stand the plate to a depth of 3.5 cm in a solution prepared
impurities is 0.05%. by dissolving 35 g of acetamide in 100 mL of methanol,
a~ding 100 mL of triethylamine, and diluting to 250 mL
With methanol. Allow the wet plate to stand in a current
of cold air for 30 s. Immediately apply the Samples

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 USP 43 Official Monographs / Amitriptyline 261

separately to the plate, at a level about 1 cm below the for dilution, and the conditions for storage of the
top of the impregnated zone. Promptly develop the plate constituted Concentrate for Dip.
until the solvent front has moved three-fourths of the • USP REFERENCE STANDARDS (11)
length of the plate. Removethe plate from the developing USP Amitraz RS
chamber and allow to air-dry. Examine the plate under
short-wavelength UV light.
Acceptance criteria: The RF value of the principal spot of the
Sample solution corresponds to that of the Standard
solution. Amitriptyline Hydrochloride
• B. The retention time of the amitraz peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.
• Hel
ASSAY
• PROCEDURE
Internal standard solution: 0.7% vlv solution of squalane
in methyl acetate
Standard solution: 5.0 mg/mL of USP Amitraz RS in Internal C2oH23N . HCI 313.86
standardsolution 1-Propanamine, 3-(10, 11-dihydro-5H-dibenzo[a,d]
Sample solution: Nominally equivalent to 5.0 mg/mL of cyclohepten-5-ylidene)-N,N-dimethyl-, hydrochloride;
amitraz from Concentrate for Dip in Internal standard 10,11-Dihydro-N,N-dimethyl-5H-dibenzo[a,d]cycloheptene-
solution dS,r-propylamine hydrochloride [549-18-8].
Chromatographic system
(See Chromatography (621), System Suitability.) DEFINITION
Mode: GC Amitriptyline Hydrochloride contains NLT 98.0% and NMT
Detector: Flame ionization 102.0% of amitriptyline hydrochloride (C2oH23N . HCI),
Column: 0.53-mm x 15-m fused silica; coated with a calculated on the dried basis.
1.5-\..Im layer of liquid phase G9 IDENTIFICATION
Temperatures
Column: 220 0
Inlet: 230 0
Detector: 300 0
• A.
Carrier gas: Helium
Flow rate: 12 mL/min
.
:~e.,
• B. The retention
Injection volume: 1 \..IL solution corresponds to that of
System suitability obtained in the Assay.
Sample: Standardsolution • C. IDENTIFICATION TESTS-GENERAL (191), Chemical
[NoTE-The elution order is amitraz, followed by Identification Tests, Chloride: Meets the requirements
squalane.]
Suitability requirements ASSAY
Resolution: NLT 3.0 between amitraz and squalane • PROCEDURE
Relative standard deviation: NMT 2.0% from the peak Buffer: 1.4 gil of anhydrous dibasic sodium phosphate in
area ratio of amitraz to squalane water, adjusted with 1.5 M phosphoric acid TS to a pH of
Analysis 7.7
Samples: Standardsolution and Sample solution Mobile phase: Methanol and Buffer (70:30)
Calculate the percentage of the labeled amount of amitraz System suitability stock solution A: 1 mg/mL of USP
(C19H23N3) in the portion of Concentrate for Dip taken: Amitriptyline Related Compound A RS in methanol
System suitability stock solution B: 0.4 mg/mL of USP
Result = (Ru/ Rs) x (Cs/Cu) x 100 Amitriptyline Hydrochloride RS, 0.6 mg/mL each of USP
Amitriptyline Related Compound B RS, USP
Ru =peak response ratio of amitraz and squalane Cyclobenzaprine Hydrochloride RS, and USP Nortriptyline
from the Sample solution Hydrochloride RS in Mobilephase
Rs = peak response ratio of amitraz and squalane Standard solution: 0.2 mg/mL of USP Amitriptyline
from the Standard solution Hydrochloride RS in Mobilephase
Cs = concentration of USP Amitraz RS in the Standard System suitability solution: 0.5 \..Ig/mL of USP Amitriptyline
solution (mg/mL) Related Compound A RS, 1 \..Ig/mL of USP Amitriptyline
Cu = nominal concentration of amitraz in the Sample Hydrochloride RS, and 1.5 \..Ig/mL each of USP Amitriptyline
solution (mg/mL) Related Compound B RS, USP Cyclobenzaprine
Hydrochloride RS, and USPNortriptyline Hydrochloride RS
Acceptance criteria: 90.00/0-120.0% from suitable volumes of Standard solution, System
suitability stock solution A, and System suitabilitystock
SPECIFIC TESTS solution B in Mobilephase
• WATER DETERMINATION, Method I (921): NMT 0.15% Sample solution: 0.2 mg/mL of Amitriptyline
ADDITIONAL REQUIREMENTS Hydrochloride in Mobilephase
• PACKAGING AND STORAGE: Preserve in well-closed Chromatographic system
containers. (See Chromatography (621), System SUitability.)
• LABELING: Label it to indicate that it isfor veterinary use only Mode: LC
and that it is to be diluted before use. The label also states Detector: uV 215 nm
the name and quantity of diluent to be used, the directions Column: 4.6-mm x 25-cm; 5-\..Im packing L7
Column temperature: 45 0

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262 Amitriptyline / OfficialMonographs USP 43

Flow rate: 1.5 mL/min = peak responseof amitriptyline related compound

Injection volume: 20 IJL A, amitriptyline related compound B, or
Run time: NLT 1.5 times the retention time of amitriptyline nortriptyline from the Standard solution
System suitability = concentration of USP Amitriptyline Related
Samples: Standard solution and System suitability solution Compound A RS, USP Amitriptyline Related
[NoTE-For relative retention times, see Table 1.] Compound B RS, or USP Nortriptyline
Suitability requirements Hydrochloride RS in the Standard solution
Resolution: NLT 1.5 between amitriptyline related (lJg/m L)
compound B and nortriptyline, System suitability solution = concentration of Amitriptyline Hydrochloride in
Relative standard deviation: NMT 0.73% for the Sample solution (uq/rnt)
amitriptyline, Standard solution
Analysis Calculate the percentage of cyclobenzaprine in the portion
Samples: Standard solution and Sample solution of Amitriptyline Hydrochloride taken:
Calculate the percentage of amitriptyline hydrochloride
(C2oH 23N . HC!) in the portion of Amitriptyline Result =(ru/rs) x (CslCu) x (Mr,/Mrz) x 100
Hydrochloride taken:
to = peak response of cyclobenzaprine from the
Result =(ru/rs) x (Cs/C u) x 100 Sample solution
rs = peak response of cyclobenzaprine from the
ru = peak response from the Sample solution Standard solution
rs = peak response from the Standard solution Cs = concentration of USP Cyclobenzaprine
Cs = concentration of USP Amitriptyline Hydrochloride Hydrochloride RS in the Standard solution
RS in the Standard solution (mg/mL) (lJg/m L) .
Cu = concentration of Amitriptyline Hydrochloride in Cu = concentration of Amitriptyline Hydrochloride in
the Sample solution (mg/mL) the Sample solution (lJg/mL)
M r1 = molecular weight of cyclobenzaprine, 275.39
Acceptance criteria: 98.0%-102.0% on the dried basis Mr2 = molecular weight of cyclobenzaprine
hydrochloride, 311.85
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1% Calculate the percentage of each unspecified impurity in
the portion of Amitriptyline Hydrochloride taken:

Result = (ru/rs) x (CslCu) x 100

• ORGANIC IMPUfUTIES
Buffer, Mobile phase, ru = peak response of any unspecified impurity from
Chromatographic Proceed as the Sample solution
directed in the Assay. rs = peak responseof USP Amitriptyline Hydrochloride
·Sensitivity solution: 0.5JJg/ hi-ipty·line RS from the Standard solution
Hydrochloride RSin Mobile phase. (USP 1 aY·2o;zo~ Cs = concentration of USP Amitriptyline Hydrochloride
Standard solution: Usethe Systemsuitability solution, RS in the Standard solution (lJg/mL)
prepared as directed in the Assay. Cu = concentration of Amitriptyline Hydrochloride in
Sample solution: 1000 IJg/mL of Amitripty.line the Sample solution (lJg/mL)
Hydrochloride in Mobile phase
• System suitability Acceptance criteria: See Table 1. Do not
Sa . 'vitysolut with a relative retention time lessthan 0.22. ~5E1henre[jolitllna
[ . ~11!~$I1Qlcfi$.().().s<>4)f~«Qs~iif~~~~~~~~j
Su
Table 1
Relative Acceptance
Retention Criteria,
Name Time NMT (%)

Amitriptyline related
compound A 0.35 0.05
Amitriptyline related
compound B 0.52 0.15
Nortriptyline 0.60 0.15
Samples: Standard solution and Sample solution
Calculate the percentages of amitriptyline related Cyclobenzaprine 0.76 0.15
compound A, amitriptyline related compound B, and Amitriptyline 1.0 -
nortriptyline hydrochloride in the portion of Amitriptyline
Hydrochloride taken: Any individual
unspecified impurity - 0.10
Result = (ru/rs) x (CslC u) x 100 Total impurities - 1.0

=peak responseof amitriptyline related compound SPECIFIC TESTS

A, amitriptyline related compound B, or
• pH (791)
nortriptyline from the Sample solution Sample: 10 mg/mL in water
Acceptance criteria: 5.0-6.0

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 USP 43 OfficialMonographs / Amitriptyline 263

• Loss ON DRYING (731) Injection volume: 20 ~L

Analysis: Dry at a pressure not exceeding 5 mm of mercury A' '. ·m.e: NLTJ.5 times the retention Jime of
at 60° to constant weight. a . '. tylinej..2s (USP41) , • ' " - '.
Acceptance criteria: NMT 0.5% System suitability
ADDITIONAL REQUIREMENTS Sample: Standard solution
• PACKAGING AND STORAGE: Preserve in well-closed Suitability requirements
containers. ~~2S (USP41)
• USP REFERENCE STANDARDS (11) Tailing factor: NMT 2.0
USP Amitriptyline Hydrochloride RS Relative standard deviation: NMT 2.0%
USP Amitriptyline Related Compound A RS Analysis
10,11-Dihydro-5H-dibenzo[a,d]cyclohepten-5-one; Samples: Standardsolution and Sample solution
Also known as Dibenzosuberone. Calculate the percentage of the labeled amount of
C1sH 120 208.26 amitriptyline hydrochloride (C2oH23N . HC!) in each Tablet
USP Amitriptyline Related Compound B RS taken:
5-[3-(Dimethylamino)propyl]-1 0,11-dihydro-5H-
dibenzo[ a,d]-cyclohepten-5-ol; Result =(rulrs) x (CsICu) x 100
Also known as Amitriptynol.
C2oH 2sNO 295.42
=peak response from the Sample solution
= peak response from the Standard solution
USP Cyclobenzaprine Hydrochloride RS
USP Nortriptyline Hydrochloride RS =concentration of USP Amitriptyline Hydrochloride
RS in the Standard solution (mg/mL)
=nominal concentration of amitriptyline
hydrochloride in the Sample solution (mg/mL)

Amitriptyline Hydrochloride Tablets Acceptance criteria: 90.00/0-110.0%

PERFORMANCE TESTS
DEFINITION
Amitriptyline Hydrochloride Tablets contain NLT 90.0% and
NMT 110.0% of the labeled amount of amitriptyline
hydrochloride (C2oH 23N . HCI). • DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL
IDENTIFICATION
Apparatus 1: 100 rpm
Time: 45 min

• ~.~ ~mm
v-
~91Lfti8t1\
()l:>ti:li.Qe,gi.rt.(tfj~~~~
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as ne )
obtained in the Assay. Instrumental conditions
Analytical wavelength: UV 239 nm
ASSAY Analysis
Sam

• PROCEDURE
Buffer: 11.04 9 of monobasic sodium phosphate in 900
mL of water. Adjust with phosphoric acid to a pH of 2.5
± 0.5, and dilute to make 1000 mL.
Mobile phase: Acetonitrile and Buffer (42:58)
Standard solution: 0.2 mg/mL of USP Amitriptyline
Hydrochloride RS in Mobilephase
Sample solution: Nominally 0.2 mg/mL of amitriptyline
hydrochloride in Mobilephase, prepared asfollows. Transfer
NLT 20 Tablets to a suitable volumetric flask, add 50% of
the flask volume of Mobilephase, and shakethe mixture for
1 h or until the Tablets have disintegrated. Dilute with D
Mobilephase to volume, and filter. Dilute the clear filtrate
with Mobilephase to obtain a solution with a nominal Tolerances: NLT 75% (Q) of the labeled amount of
concentration of 0.2 mg/mL of amitriptyline hydrochloride. amitriptyline hydrochloride (C2oH 23N . HCI) is dissolved.
Chromatographic system • UNIFORMITY OF DOSAGE UNITS (905): Meet the
(See Chromatography (621), System SUitability.) requirements
Mode: LC
Detector: UV nm.

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264 Amitriptyline / OfficialMonographs USP43

IMPURITIES Result = (rul(5) x' (Cs/Cu) ,x 100

= peak responseof any other individual
degradation product from the Sample solution
=.peakresponse of amitriptyline from,the Standard
solution
~concentrqti0n of USP Amitriptyline
Hydrochloride RSin the Standard solution
.(1-I9!ml) :,' ..
= nominal concentration of amitriptyline
hydrochloride in the Sample solution (J.lg!ml)
Acceptance criteria: ,See Table 1.
Table 1
Relative Acceptance
Retention criteria,
Name Thne NMT(%)
Amitriptyline related
compound A 0.32 0.2
Amitript}'IIne related
compound B 0;48 0.2
NortriptYline 0.62 0.2
AmitriptYline 1.0 -
Anyother individual
degradation product - 0.2
5nm Iota) degradation proa-
-
5-cm;5-,..11" packingl7 ucts 1.OJi2s(UsP41)
: 45~
in ADDITIONAL REQUIREMENTS

retentiontimeofamiti-iptyline Change;
S
Sa • PACKAGING AND STORAGE: Preserve in well-closed
[NOTE containers.·Store at controlled room
Suitabil temperature.• 2S(USP4l)
Resolution: eenamitriptyline related
compound ine Change to read: '
Relative stan :NMT5.0%
Analysis • USP REFERENCE STANDARDS (11)
Samples USP Amitriptyline Hydrochloride RS
Calcula A 'triptyline Related Compound ARS
comp dro-5H:·dibenzo[ a,d]-cyclohepten-5-one;
nortriptyline Dibenzosuberone.
taken: 26
Rei ompound BRS
=
Result (tu!fs).X (Cs/Cu) x 100 amino yl]-lO, 11-dihydro-~H-
-cyclohepten-5-ol;
fv = peak res s Amitriptynol.
A, amitri N 295A2
nortriptyli mple ortriptyJine'Hydrochloride RS. 2S(USP.41)
sol .
fs =pe
A, amI
nortr' ndard
solution Amlodipine Compounded Oral
Cs = concehtratlon of US I' Arnitri t I Suspension
CompoundA RS, iJs:~:~~i~
Compound BRS,or .SP N DEFINITION
Hydroc:hloride RS. in the Standard Amlodipine Compounded Oral Suspension contains NLT
(J.l9!ml) 90.0% and NMT 110.0% of the labeled amount of
Cv = nominal concentrqtionofamitriptyline _ amlodipine (CzoHzsClNzOs).
hydrochlorideinthe Sample solution (1-I9!ml) PrepareAmlodipine Compounded Oral Suspension 1 rnq/mt,
as follows (see Pharmaceutical Compounding-Nonsterile
of
CalCulate the percentage any other in'divicual Preparations (795»).
degradation product in the .portion of Tablets taken:

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USP 43 Official Monographs / Amlodipine 265

100 mg of arnlodl- = nominal concentration of amlodipine in the

Amlodipine besylatetablets" equivalentto pine Sample solution (lJg/mL)
Vehicle: a 1:1 mixture of Ora-Sweet" and
Ora-Plus, b a sufficient quantity to make 100 mL Acceptance criteria: 90.00/0-110.0%
SPECIFIC TESTS
a Norvasc 5-mg tablets, Pfizer, Inc., Groton, CT.
b PaddockLaboratories, Minneapolis, MN.
• pH (791): 4.0-5.0
ADDITIONAL REQUIREMENTS
Calculate the required quantity of each ingredient for the total • PACKAGING AND STORAGE: Package in tight, light-resistant
amount to be prepared. Place the required number of tablets containers. Store in a refrigerator or at controlled room
in a suitable mortar and comminute to a fine powder. Add temperature.
the Vehicle in small portions, and triturate to make a smooth • BEYOND-USE DATE: NMT 90 days after the date on which it
paste. Add increasing volumes of the Vehicle to make an was compounded when stored in a refrigerator; NMT 60
amlodipine liquid that is pourable. Transfer the contents of days when stored at controlled room temperature
the mortar, stepwise and quantitatively, to a calibrated • LABELING: Label it to indicate that it is to be well shaken
bottle. Add enough of the Vehicle to bring to final volume, before use, and to state the Beyond-Use Date.
and mix well. [NOTE-To ensure component uniformity, • USP REFERENCE STANDARDS (11)
homogenization is recommended.] USP Amlodipine Besylate RS
ASSAY
• PROCEDURE
Mobile phase: Acetonitrile, methanol, and 40 mM
ammonium acetate (50:15:35). Filter through a nylon 66 Amlodipine and Atorvastatin Tablets
filter of 0.45-lJm pore size, and degas.
Standard stock solution: Dissolve an appropriately
weighed amount of USPAmlodipine Besylate RS in
methanol, equivalent to 1.0 mg/mL of amlodipine
(approximately equal to 1.4 mg/mL of amlodipine DEFINITION
besylate). Amlodipine and Atorvastatin Tablets contain an amount of
Standard solution: Transfer 1.0 mL of the Standardstock amlodipine besylate equivalent to NlT 90.0% and NMT
solution into a 50-mL volumetric flask, and dilute with 110.0% of the labeled amount of amlodipine
Mobilephaseto volume to obtain a solution with a nominal (C2oH2sClN20s) and an amount of atorvastatin calcium
concentration of,about 20 IJg/ml of amlodipine. equivalent to NLT 94.5% 1 the labeled
Centrifuge. amount of atorvastatin It
Sample solution: Shake thoroughly by hand each bottle of may contain suitable ::Int'inv'irl::l,ntc::
Oral Suspension. Pipet 1.0 ml of Oral Suspension into a
50-ml volumetric flask, rinse the pipet three times with IDENTIFICATION
Mobile phase, and dilute with Mobilephase to volume to • A. The UV spectrum of the major peaks of the Sample
obtain a solution with a nominal concentration of about 20 solution exhibits maxima and minima at the same
IJg/ml of amlodipine. Centrifuge. wavelengths as that of the Standard solution, as obtained in
Chromatographic system the Assay.
(See Chromatography (621), System Suitability.) • B. The retention times of the major peaks of the Sample
Mode: LC solution correspond to those of the Standardsolution, as
Detector: UV 240 nm obtained in the Assay. '
Column: 3.0-mm x 15-cm; 5-lJm packing L10 ASSAY
Flow rate: 0.4 mL/min
Injection volume: 10 IJl
System suitability
Sample: Standardsolution • PROCEDURE
[NoTE-The retention time for amlodipine isabout 10.1 Solution A: Dissolve 1.54 g of ammonium acetate in 1000
min.] ml of water and add 2 mL of triethylamine. Adjust with
Suitability requirements acetic acid to a pH of 5.0.
Column efficiency: NLT 4000 theoretical plates Mobile phase: Acetonitrile, methanol, and Solution A
Tailing factor: NMT 2.0 (38:15:47)
Relative standard deviation: NMT 2.0% for replicate Buffer: Transfer 7 mL of triethylamine to a 1000-mL
injections volumetric flask containing 900 ml of water and mix.
Analysis Adjust with dilute phosphoric acid (1 in 100) to a pH of 3.0
Samples: Standardsolution and Sample solution and dilute with water to volume.
Calculate the percentage of the labeled amount of Diluent: Acetonitrile, methanol, and Buffer (3:7:1 0)
amlodipine (C2oH2sCIN20s) in the portion of Oral Standard stock solution 1: 0.35 mg/mL of USP Amlodipine
Suspension taken: Besylate RS in methanol
Standard stock solution 2: 0.44 mg/mL of USPAtorvastatin
Result e (rvirs) x (CsiCu) x 100 Calcium RS in methanol
Standard solution: Prepare solutions of USP Amlodipine
= peak response of amlodipine from the Sample Besylate RS and USP Atorvastatin Calcium RS in Mobile
solution phase at concentrations given in Table 1 from Standard
= peak response of amlodipine from the Standard stocksolution 1 and Standardstock solution 2.
solution
=concentration of amlodiplne in the Standard
solution (lJg/mL)

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266 Amlodipine / OfficialMonographs USP 43

Table 1 Cs = concentration of USP Atorvastatin Calcium RS in

Strength of the Standard solution(mg/mL)
Tablet Concentration of Concentration of Cu = nominal concentration of atorvastatin in the
Amlodipine/Atorvas- Amlodipine Atorvastatin Sample solution(mg/mL)
tatin Besylate Calcium
(mg/mg) (mg/ml) (mg/ml)
M = number of moles of atorvastatin per mole of
atorvastatin calcium, 2
2.5/1 0, 5/20, 10/40 0.028 0.088 M rl = molecular weight of atorvastatin, 558.64
2.5/20, 5/40, 10/80 0.014 0.088 M r2 = molecular weight of atorvastatin calcium,
1155.34
5/10,10/20 0.028 0.044
2.5/40, 5/80 0.014 0.176 Acceptance criteria
Amlodipine: 90.00/0-110.0%
10/10 0.028 0.022 Atorvastatin: 94.5%-105.0%

Sample solution: Transfer NLT10 Tablets to a suitable PERFORMANCE TESTS

volumetric flask. Add about 20% of the final volume of the
volumetric flask size in Diluent and sonicate to disperse the
Tablets. Add about 40% of the final volume of the • DISSOLUTION (711)
volumetric flask size in Diluent, sonicate for 20 min and Solution A, Mobile phase, Standard stock solution 1,
dilute with Diluent to volume. Centrifuge and tran;fer a Standard stock solution 2, Chromatographic system,
suitable quantity of the supernatant to an appropriate and System suitability: Proceed as directed in the Assay.
suitable volumetric flask. Dilute with Mobilephase to Medium: 0.1 % polysorbate 80 in pH 6.8 phosphate buffer;
volume to obtain the nominal concentrations of amlodipine 900 mL
and atorvastatin similar to that of the Standardsolution. Apparatus 2: 75 rpm
Chromatographic system Time: 20 min
(See Chromatography (621), System Suitability.) Standard solution: (L tl900) mg/mL of amlodipine and (L
Mode: LC i900) mg/mL of atorvastatin in Medium from Standard
Detector: UV 237 nm. For Identification A, use a diode
array detector in the range of 200-400 nm. stocksolution 7 and Standard stock solution 2, where L 1 is
Column: 4.6-mm x 15-cm; 5-J,Jm packing L1 the label claim of amlodipine in mg/Tablet, and L 2 is the
Column temperature: 35° label claim of atorvastatin in mg/Tablet
Flow rate: 1 mL/min Sample solution: Centrifuge the solution under test and use
Injection volume: 20 J,JL the supernatant.
Run time: NLT3.5 times the retention time of amlodipine Analysis
System suitability Samples: Standardsolution and Sample solution
Sample: Standardsolution Calculate the percentage of the labeled amount of
Suitability requirements .. amlodipine (C2oH2sCIN20s) dissolved:
Tailing factor: NMT 2.0 for both peaks
Relative standard deviation: NMT 2.0% for both peaks Result = (r vir s) x C s x V x (M ,tiM (2) x (1 I L) x 100
Analysis
Samples: Standardsolution and Sample solution = peak response of amlodipine from the Sample
Calculate the percentage of the labeled amount of solution
amlodipine (C2oH2sCIN20s) in the portion of Tablets = peak response of amlodipine from the Standard
taken: solution
= concentration of USP Amlodipine Besylate RS in
Result = (r vir s) x (C siC v) x (M ,tiM (2) x 100 the Standard solution(mg/mL)
V = volume of Medium, 900mL
= peak response of amlodipine from the Sample M rl = molecular weight of amlodipine, 408.88
solution M r2 = molecular weight of amlodipine besylate, 567.05
= peak response of amlodipine from the Standard L = label claim of amlodipine (mg/Tablet)
solution
= concentration of USPAmlodipine Besylate RS in Calculate the norror,t""..:,

the Standardsolution (mg/mL) atorvastatin

= nominal concentration of amlodipine in the
Sample solution (mg/mL) Result = (r vir s) x C s x V x [M x (M ril M (2)] X (1I L) x 100
M'I = molecular weight of amlodipine, 408.88
M'2 =molecular weight of amlodipine besylate, 567.05 = peak response of atorvastatin from the Sample
solution
Calculate the nor·ror,t"",o = peak response of atorvastatin from the Standard
atorvastatin solution
of Tablets = concentration of USP Atorvastatin Calcium RS in
the Standardsolution(mg/mL)
Result = (r vir s)x (C siC v) x [M x (M ,tiM r2)] X 100 V = volume of Medium, 900 mL
M = number of moles of atorvastatin per mole of
ru = peak response of atorvastatin from the Sample atorvastatin calcium, 2 .
solution M'I = molecular weight of atorvastatin, 558.64
rs =peak response of atorvastatin from the Standard M r2 = molecular weight of atorvastatin calcium,
solution 1155.34
L = label claim of atorvastatin (mg/Tablet)

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USP 43 OfficialMonographs / Amlodipine 267

Tolerances: NLT 80% (Q) of the labeled amount of System suitability .

amlodipine (CzoHzsClNzOs) and atorvastatin Sample: Standard solution 2
~(~ii~~~F('\J2Q~)-i//(~~~'itp~~Z~§j~) are dissolved. Suitability requirements
• U~nFORMITV OF DOSAGE UNITS (905): Meet the Tailing factor: NMT 2.0
requirements Relative standard deviation: NMT 5.0%
Analysis
IMPURITIES Samples: Standard solution 7, Standard solution 2, and
• ORGANIC IMPURITIES RELATED TO AMLODIPINE Sample solution
Buffer 1: Add 7 mL of triethylamine in 1000 mL of water and Calculate the percentage of amlodipine related compound
adjust with phosphoric acid to a pH of 2.5. Add 1.8 g of A in the portion of Tablets taken:
tetrabutylammonium hydrogen sulfate and mix well.
Solution A: Methanol and Buffer 7 (40:60) Result = (r vir s) x (C siC v) x (M rllM r2) X 100
Solution B: Acetonitrile, methanol, and Buffer 7 (40:40:20)
Mobile phase: See Table 2. ru = peak response of amlodipine related compound
A from the Sample solution
Table 2 rs =peak response of amlodipine related compound
Time Solution A Solution B A from Standard solution 7
(min) (%) (%) Cs = concentration of USP Amlodipine Related
Compound A RS in Standard solution 7 (mg/mL)
0 90 10
Cu = nominal concentration of amlodipine in the
2 90 10 Sample solution(mg/mL)
M rl = molecular weight of amlodipine related
7 75 25
compound A free base, 406.86
16 70 30 M r2 = molecular weight of amlodipine related
18 55
compound A fumarate, 522.94
45
24 25 75 Calculate the percentage of atorvastatin-amlodipine
30 90
adduct or any unspecified degradation product in the
10
portion of Tablets taken:
31 0 100
Result = (r vir s) x (C siC v) x (M ,JiM r2) X (1 IF) x 100
35 0 100
36 90 10 ru =peak response of each degradation product
from the Sample solution
40 90 10
rs = peak response of amlodipine from Standard
solution2
Buffer 2: Add 7 mL of triethylamine in 1000 mL of water. Cs = concentration of USP Amlodipine Besylate RS in
Adjust with phosphoric acid to a pH of 3.0. Standard solution 2 (mg/mL)
Diluent 1: Methanol and water (50:50) Cu = nominal concentration of amlodipine in the
Diluent 2: Methanol and Buffer 2 (50:50) Sample solution(mg/mL)
Standard stock solution: 0.7 mg/mL of USP Amlodipine M rl = molecular weight of amlodipine, 408.88
Besylate RS in Diiuen: 2, prepared asfollows. Transfer a M r2 = molecular weight of amlodipine besylate, 567.05
suitable amount of USP Amlodipine Besylate RS to a suitable F = relative responsefactor (see Table 3)
volumetric flask and dissolve in a quantity of methanol,
about 20% of the volume of the flask. Dilute with Diluent 2 Acceptance criteria: See Table 3. Disregard peaks at the
to volume. relative retention times of 2.18, 2.47 (atorvastatin), and
Standard solution 1: 5 ~g/mL of USP Amlodipine Related 2.79 min.
Compound A RS in Diluent 7
Standard solution 2: 3.5 ~g/mL of USP Amlodipine Besylate Table 3
RS from Standardstock solution in Diluent2 Relative Relative Acceptance
Sample solution: Nominally 0.5 mg/mL of amlodipine in Retention Response Criteria,
Dlluent 2, prepared as follows. Finely powder NLT 25 Name Time Factor NMT(%)
Tablets and transfer a portion of the powder, equivalent to
Amlodipine
50 mg of amlodipine to a 1OO-mL volumetric flask. Add related -
about 40 mL of methanol, shake to disperse, and sonicate compound A 0.59 0.50
for 15 min. Add about 40 mL of Buffer 2 and sonicate for
another 10 min. Dilute with Diluent2 to volume, centrifuge, Amlodipine 1.00 - -
and use the supernatant. Pass a portion of the solution Atorvastatin-am-
through a suitable filter of 0.22-~m pore size. Prepare this lodipineadduct- 3.49 0.47 0.50
solution fresh. Any
Chromatographic system unspecified
(See Chromatography (621), System Suitability.) degradation -
Mode: LC product 1.0 0.20
Detector: UV 270 nm for amlodipine related compound
Ai 360 nm for all other impurities
Column: 2.1-mm x 15-cmi 1 .s-um packing L1
Column temperature: 40 0
Flow rate: 0.3 mL/min
Injection volume: 5 ~L

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268 Amlodipine / OfficialMonographs USP 43

Table 3 (continued) Suitability requirements

Relative Relative Acceptance Resolutlon.. NLT 1.0 between atorvastatin pyrrolidone
Retention Response Criteria, analog and atorvastatin related compound A, System
Name Time Factor NMT(%) suitability solution
Total Relative standard deviation: NMT 5.0%, Standard
degradation solution
products for am- - - Analysis .
lodipine 1.0 Samples: Standardsolution and Sample solution
a 3-Ethyl 5-methyl 4-(2-chlorophenyl)-2-(2-{(3R,5R)-7-(2-(4-fluorophenyl)-5-
Calculate the percentage of each atorvastatin specified or
isopropyl-3-phenyl-4-(phenylcarbamoyl)-1 H-pyrrol-1-yl]-3,5- unspecified degradation product in the portion of Tablets
dihydroxyheptanamido}ethoxy)methyl]-6-methyl-l ,4-dihydropyridine-3,5- taken:
dicarboxylate.
Result= (r vir s) x (C siC v) x [M x (M rdM r2)] X (1 IF) x 100
• ORGANIC IMPURITIES RELATED TO ATORVASTATIN
Buffer 1: Dissolve 6.8 g of potassium dihydrogen phosphate ru = peak response of each atorvastatin degradation
in 1000 mLof water and adjust with dilute phosphoric acid product from the Sample solution
(1 in 10) to a pH of 3.4. rs = peak response of atorvastatin from the Standard
Buffer 2: Dissolve 6.8 g of potassium dihydrogen phosphate solution
in 1000 mL of water and adjust with triethylamine to a pH Cs = concentration of USP Atorvastatin Calcium RS in
of 7.0. the Standardsolution (mg/mL)
Solution A: Tetrahydrofuran, acetonitrile, and Buffer 1 Cu = nominal concentration of atorvastatin in the
(5:25:70) Sample solution (mg/mL)
Solution B: Tetrahydrofuran, acetonitrile, and Buffer 2 M = number of moles of atorvastatin per mole of
(5:70:25) atorvastatin calcium, 2
Mobile phase: See Table 4. M rl = molecular weight of atorvastatin, 558.64
M r2 = molecular weight of atorvastatin calcium,
Table 4 1155.34
Time Solution A Solution B F = relative response factor (see Table 5)
(min) (0/0) (0/0)
Acceptance criteria: See Table 5. Disregard any impurity
0 85 15
peaks less than 0.05% and the peaks from amlodipine
30 75 25 related impurities.
70 . 40 60
Table 5
75 25 75 Relative Relative Acceptance
75 Retention Response Criteria,
80 25
Name Time Factor NMT(%)
85 85 15
Atorvastatin
90 85 15 pyrrolidone
analog" 0.86 0.67 0.45

Diluent: Acetonitrile and water (50:50) Atorvastatin related

compound Ab 0.91 - -
System suitability solution: Heat a suitable amount of USP
Atorvastatin Calcium RS at 60° for 1 h for degradation; 0.55 Atorvastatin related
- -
mg/mL of degraded USP Atorvastatin Calcium RS, 3 IJg/mL compound Bb 0.95
each of USP Atorvastatin Related Compound A RS, USP Atorvastatin 1.00 - -
Atorvastatin Related Compound B RS, USP Atorvastatin
Related Compound C RS, and USP Atorvastatin Related Atorvastatin related
compound Cb 1.04 - -
Compound H RS in Diluent. Sonication may be necessary
for complete dissolution. Atorvastatin epoxy
Standard solution: 2.7 IJg/mL of USP Atorvastatin Calcium pyrrolooxazin
RS in Diluent 6-hydroxy analog' 1.35 0.39 0.5
Sample solution: Nominally 0.5 mg/mL of atorvastatin in Atorvastatin epoxy
Diluent, prepared as follows.Transferan amount equivalent pyrrolooxazin
to 50 mg of atorvastatin from a portion of NLT 20 finely 7-hydroxy analoq" 1.40 0.52 0.5
powdered Tablets to a 1OO-mL volumetric flask. Add about Atorvastatin related
10 mLof acetonitrile, shake to disperse, and sonicate for 5 compound H 1.78 1.0 1.0
min. Add about 70 mL of Diluent and sonicate for another Atorvastatin epoxy
20 min. Dilute with Diluent to volume and centrifuge. tetrahydrofuran
Prepare this solution fresh. analog e 1.96 0.63
Chromatographic system 0.59
Atorvastatin oxlrane' 2.23 1.0
(See Chromatography (621), System Suitability.)
Mode: LC Atorvastatin tert-butyl
- -
Detector: UV 246 nm esterb. h 2.55
Column: 4.6-mm x 25-cm; 4-lJm packing L11 Any unspecified
Column temperature: 45° degradation - -
Flow rate: 1.2 mL/min product 0.20
Injection volume: 20 IJL
System suitability
Samples: System suitability solution and Standardsolution

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USP 43 Official Monographs / Amlodipine 269

Table 5 (continued)
Amlodipine and Benazepril
Relative Relative Acceptance
Retention Response Criteria, Hydrochloride Capsules
Name Time Fador NMT (%)
Total degradation products DEfiNITION
for - - Amlodipine and Benazepril Hydrochloride Capsules contain
atorvastatin 2.0 NLT 90.0% and NMT 110.0% of the labeled amounts of each
Total degradation of amlodipine (C2oH2sN20sCI) and benazepril hydrochloride
products' - - 3.0 (C24H2SN20S . HCI).

a (3R,5R)-7-[5-(4-Fluorophenyl)-3-isopropyl-2-oxo-4-phenyl-3- IDENTIFICATION
(phenylcarbamcyh-z.s-dihydro-1H-pyrrol-1-yl]-3,5-dihydroxyheptanoic acid. • A. The retention times of the major peaks of the Sample
b Processimpurityincludedinthe table foridentification only.Process impurities solution correspond to those of the Standard solution, as
are controlled in the drug substance, and are not to be reported or included in obtained in the Assay.
the total impurities for the drug product.
c 4-{6-(4-Fluorophenyl)-7,8-epoxy-6-hydroxy-8a-isopropyl-7-phenyl-8- ASSAY
(phenylcarbamoyl)hexahydro-2H-pyrrolo[2,1 -b][1 ,3]oxazin-2-yl}-3- • PROCEDURE
hydroxybutanoicacid.
d (3 R)-4-(1 b-(4-Fluorophenyl)-7-hydroxy-7-lsopropyl-1a-phenyl-7a- Buffer 1: 0.7% (v/v) of triethylamine in water. Adjust with
(phenylcarbamoyl)hexahydro-1aH-oxireno[2',3':3,4]pyrrolo[2,1-b][1,3]oxazin- phosphoric acid to a pH of 3.0/ and add 1.2 g of tetrabutyl
3-yl)-3-hydroxybutanoic acid. ammonium hydrogen sulfate per 1 L of Buffer. Pass through
e 4-(4-Fluorophenyl)-2,4-dihydroxy-2-isopropyl-N,5-diphenyl-3,6- a suitable filter of 0.45-l.lm pore size.
dioxabicyclo[3.1 .O]hexane-1-carboxamide. Buffer 2: 7.0 mL/L of triethylamine in water. Adjust with
f 3-(4-Fluorobenzoyl)-2-isobutyryl- N, 3-diphenyloxirane-2-carboxamide. phosphoric acid to a pH of 3.0. Pass through a suitable filter
9 Sum of atorvastatinepoxy tetrahydrofuran analog and atorvastatinoxirane. of 0.45-l.lm pore size.
h (3 R,5R)-tert-Butyl 7-(2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4-
(phenylcarbamoyl)-1H-pyrrol~ 1-yl)-3,5-dihydroxyheptanoate. Diluent: Acetonitrile, methanol, and Buffer 2 (20:30:50)
i Sum of the total degradation productsfor amlodipinefrom the test for Organic Mobile phase: Acetonitrile, methanol, and Buffer 1
ImpuritiesRelated to Am/odipine and the total degradation productsfor (10:30:70)
atorvastatinfrom the test for OrganicImpurities Related to Atorvastatin. Standard solution: Prepare the corresponding solutions in
Diluent as directed in Table 1.
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed Table 1
containers. Store at controlled room temperature. Strength of Concentration of Amlodipine Besy-
• USP REFERENCE STANDARDS (11) Capsule late/Benazeprii
USPAmlodipine Besylate RS (Amlodipine (mg)/Benazeprii Hy- Hydrochloride
USPAmlodipine Related Compound A RS drochloride (mg» (mg/mL)
3-Ethyl 5-methyl [2-(2-aminoethoxymethyl)-4-(2- 2.5/10 0.18/0.5
chlorophenyl)-6-methyl-3/5-pyridinedicarboxylate]
fumarate. 5/20 0.18/0.5
C2oH23CIN20S . C4H404 522.94 5/10 0.18/0.25
USPAtorvastatin Calcium RS
10/20 0.36/0.5
USPAtorvastatin Related Compound A RS
Calcium (3 R,5 R)-7-[2-isopropyl-4/5-diphenyl-3- 5/40 and 10/40 0.04/0.16
(phenylcarbamoyl)-1 H-pyrrol-1-yl]- .
3/5-dihydroxyheptanoate (1:2). Pass through a suitable membrane filter of 0.45-l.lm pore
C66H70CaN401O 1119.38 size.
USPAtorvastatin Related Compound B RS Sample solution: Transfer the contents of five Capsules into
(35/5 R)-7.-[3-(Phenylcarbamoyl)-5-(4-fluorophenyl)-2- a volumetric flask as given in Table 2. Add Diluent (about
isopropyl-4-phenyl-1 H-pyrrol-1-yl]-3,5- 70% of the volume of the flask) and keep on a rotary shaker
dihydroxyheptanoic acid calcium salt. for about 45 mini sonicate for 30 min with occasional
C66H6SCaF2N401O 1155.34 shaking, and dilute with Diluent to volume. Centrifuge a
USPAtorvastatin Related Compound C RS portion of the above solution at 3000 rpm for 10 mini and
Calcium (3R,5 R)-7-[2/3-Bis(4-fluorophenyl)-5-isopropyl- pass through a filter of 0.45-l.lm pore size.
4-(phenylcarbamoyl)-1 H-pyrrol-1-yl]-
3/5-dihydroxyheptanoate (1:2). Table 2
C66H66CaF4N40,O 1191.34 Strength of Concentration of Amlodipinel
USPAtorvastatin Related Compound H RS Capsule Benazepril
5-( 4-Fluorophenyl)-1-{2-[(2R,4R)-4-hydroxy-6- (Amlodipine (mg)/Benazeprii Hy- Hydrochloride
oxotetrahydro-2H-pyran-2-yl]ethyl}-2-isopropyl-N,4- drochloride (mq) (mg/mL)
diphenyl-1 H-pyrrole-3-carboxamide. 2.5/10 0.125/0.5
C33H33FN204 540.62
5/20 0.125/0.5
5/10 0.125/0.25
10/20 0.25/0.5
5/40 0.02/0.16
10/40 0.04/0.16

Chromatographic system
(See Chromatography (621)/ System SUitability.)

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270 Amlodipine / Official Monographs USP 43

Mode: LC Table 3
Detector: UV 237 nm Volume of Amlodipine
Column: 4.6-mm x 15-cm; 5-fJm packing L1 Besylate Standard
Flow rate: 1.2 mL/min Strength of the Capsu- Stock Solution/
Injection volume: 10 IJL Ie Volume of Benazepril
(Amlodipine/Benaze- Hydrochloride Stand-
Run time: Two times the retention time of amlodipine pril Hydrochloride) ard Stock Solution Volume of the Flask
System suitability (mg/mg) (ml) (ml)
Sample: Standardsolution
2.5/10 1/5 50
Suitability requirements
Tailing factor: NMT 2.0 for both amlodipine and 5/10 2/5 50
benazepril peaks 5/20 2/10 50
Relative standard deviation: NMT 2.0% for both,
amlodipine and benazepril peaks 10/20 2/5 25
Analysis
Samples: Sample solution and Standardsolution For Tablet strengths 5/40 (Amlodipine/Benazeprii
Calculate the percentage of the labeled amount of Hydrochloride, mg/mg): 0.01 mg/mL of USP Amlodipine
amlodipine (C2oH2sN20sCI) in the portion of Capsules BesylateRS and 0.08 mg/mL of USP Benazepril
taken: Hydrochloride RS in Medium.
For Tablet strengths 10/40 (Amlodipine/Benazepril
Result = (r vIr 5) x (C siC v) x (M rdM r2) X 100 Hydrochloride, mg/mg): 0.02 mg/mL of USP Amlodipine
Besylate RS and 0.08 mg/mL of USP Benazepril
ru =responseof the amlodipine peak from the Sample Hydrochloride RS in Medium.
solution Sample solution: Pass a portion of the solution under test
rs =response of the amlodipine peak from the through a suitable filter of 0.45-lJm pore size.
Standard solution Chromatographic system
Cs = concentration of amlodipine in the Standard (See Chromatography (621), System Suitability.)
solution (mg/mL) Mode: LC
Cu = nominal concentration of amlodipine in the Detector: UV 237 nm
Sample solution (mg/mL) Column: 4.6-mm x 10-cm; 3-lJm packing L1
M rl = molecular weight of amlodipine, 408.88 Flow rate: 1 mL/min
M r2 = molecular weight of amlodipine besylate, 567.05 Injection volume: 50 IJL
System suitability
Calculate the percentage of the labeled amount of Sample: Standard solution
benazepril hydrochloride (C24H28N20s . HCI), in the Suitability requirements
portion of Capsules taken: Tailing factor: NMT 2.0 for both amlodipine and
benazepril peaks
Result = (r vIr 5) x (C sICv) x 100 Relative standard deviation: NMT 2.0% for both
amlodipine and benazepril peaks
=response of the benazepril peak from the Sample Analysis
solution Samples: Standard solution and Sample solution
= response of the benazepril peak from the Calculate the percentage of the labeled amount of
Standard solution amlodipine (C2oH2sN20sCI) dissolved:
= concentration of benazepril hydrochloride in the
Standard solution (mg/mL) Result = (r vIr s) x (C slL) x (M rdM r2) X V x 100
Cv = nominal concentration of benazepril
hydrochloride in the Sample solution (mg/mL) ru = peak response for amlodipine from the Sample
solution
Acceptance criteria: 90.0%-110.0% of the labeled amount rs = peak response for amlodipine from the Standard
of amlodipine free base and 90.0%-110.0% of the labeled solution
amount of benazepril hydrochloride Cs = concentration of USP Amlodipine Besylate RS in
PERFORMANCE TESTS the Standard solution
• DISSOLUTION (711) L = label claim for amlodipine (mg/Capsule)
Medium: 0.01 N hydrochloric acid; 500 mL M rl = molecular weight of amlodipine, 408.88
Apparatus 1: 100 rpm M r2 = molecular weight of amlodipine besylate, 567.05
Time: 30 min V = volume of Medium, 500 mL
Buffer: 2.72 gIL of potassium dihydrogen phosphate in
water. Add 0.2% (v/v) of triethylamine per L. Adjust with Calculate the percentage of the labeled amount of
phosphoric acid to a pH of 3.0. benazepril hydrochloride (C24H28N20s . HCI) dissolved:
Mobile phase: Acetonitrile, methanol, and Buffer
(15:35:50) Result = (r vIr 5) x (C slL) x V x 100
Amlodipine besylate standard stock solution: 0.385
mg/mL of USP Amlodipine Besylate RS in Medium ru = peak response for benazepril from the Sample
Benazepril hydrochloride standard stock solution: 0.225 solution
mg/mL of USP Benazepril Hydrochloride RS in Medium rs = peak response for benazepril from the Standard
Standard solution: Dilute aliquots of the Amlodipine solution
besylate standardstock solution and Benazepril hydrochloride Cs = concentration of benazepril hydrochloride in the
standard stock solution with Medium as per Table 3. Standard solution
L = label claim for benazepril hydrochloride (mgl
Capsule)

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USP 43 OfficialMonographs / Amlodipine 271

v = volume of Medium, 500 mL Cs = concentration of amlodipine related compound

A in the Standard solution (mg/mL)
Tolerances: NLT 80% (Q) of the labeled amounts of Cu = nominal concentration of amlodipine in the
amlodipine (C2oH2sN20sCI) and benazepril hydrochloride Sample solution (mg/mL)
(Cz4Hz8NzOs . HCI) are dissolved. M ,1 = molecular weight of amlodipine related
• UNIFORMITY OF DOSAGE UNITS (905): Meet the compound A, 408.88
requirements M ,2 = molecular weight of amlodipine related
compound A fumarate, 522.93
IMPURITIES
• PROCEDURE Calculate the percentage of benazepril related compound
Buffer 1, Buffer 2, and Diluent: Proceed as directed in the C in the portion of Capsules taken:
Assay.
Solution A: Acetonitrile and Buffer 1 (20:80) Result = (r ulr s) x (C siCu) x 100
Solution B: Methanol and Buffer 1 (80:20)
Mobile phase: See Table 4. ru = response of benazepril related compound C
from the Sample solution
Table 4 rs = response of benazepril related compound C
Time Solution A Solution B from the Standard solution
(min) (%) (%) Cs = concentration of benazepril related compound C
in the Standard solution (mg/mL)
0 85 15
Cu = nominal concentration of benazepril
100 30 70 hydrochloride in the Sample solution (mg/mL)
101 . 85 15
Calculate the percentage of any other impurity in the
110 85 15 portion of Capsules taken:

Standard stock solution: 0.36 mg/mL of amlodipine and Result =(r ulr T) x 100
amlodipine related compound A and 1 mg/mL each of
ru = response of each other impurity from the Sample
benazepril hydrochloride and benazepril related
solution
compound C solution in Diluent
Standard solution: 1 J.lg/mLof amlodipine and amlodipine
r T = sum of responses of all peaksfrom the Sample
solution
related compound A and 3 J.lg/mLeach of benazepril
hydrochloride and benazepril related compound C Acceptance criteria: See Table 5.
solution in Diluent
Sample solution: 0.25 mg/mL of amlodipine from Table 5
powdered Capsules (NLT 20). [NoTE-The benazepril
hydrochloride concentration may vary depending on the Acceptance
Relative Criteria,
ratio of amlodipine to benazepril hydrochloride in the Impurity Name Retention Time NMT (%)
Capsule.] Initially add Diluent, about 70% of the volume of
the flask, sonicate for 30 min with intermittent shaking, and Benazepril related com-
pound C' 0.23 3.0
dilute with Diluent to volume. Pass through a membrane
filter of 0.45-J.lm pore size. . Amlodipine related
Chromatographic system compound Ab 0.44 1.0
(See Chromatography (621), System Suitability.) Amlodipine 1.00 -
Mode: LC
Detector: UV 237 nm Benazepril 1.20 -
Column: 4.6-mm x 25-cm; 5-J.lm packing L1 Any other individual un- -
Column temperature: 40° specified impurity 0.2
Flow rate: 1.2 mL/min
Injection volume: 40 J.lL
Total impurities' - 5.0
System suitability [NOTE-Disregard the peaks at relative retention times of 0.09 and 0.10.]
Sample: Standardsolution
Suitability requirements a {3-[1-Carboxy-3-phenyl-(1 S)-propyl]amino-2,3,4,5-tetrahydro-2-oxo-l H-l-
(3S)-benzazepine}-1-acetic acid.
Tailing factor: NMT 2.0 for both amlodipine and
b 3-Ethyl 5-methyl [2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-
benazepril peaks 3,5-pyridinedicarboxylate].
Resolution: NLT 2.0 between the amlodipine and C Total impurities include the sum of all impurities. The process-related impurities
benazepril peaks are not included.
Analysis
Samples: Standardsolution and Sample solution ADDITIONAL REQUIREMENTS
Calculate the percentage of amlodipine related compound • PACKAGING AND STORAGE: Preserve in well-closed
A in the portion of Capsulestaken: containers, and store at controlled room temperature.
• USP REFERENCE STANDARDS (11)
Result = (r vir s) x (C siC u) x (M ,tiM (2) x 100 USP Amlodipine Besylate RS
USP Amlodipine Related Compound A RS
=response of amlodipine related compound A 3-Ethyl 5-methyl [2-(2-aminoethoxymethyl)-4-(2- .
from the Sample solution chlorophenyl)-6-methyl-3,5-pyridinedicarboxylate]
=response of amlodipine related compound A fumarate.
from the Standard solution CzoHz3CINzOs . C4H404 522.93
USP Benazepril Hydrochloride RS

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272 Amlodipine / OfficialMonographs USP 43

USP Benazepril Related Compound C RS ofolmesartan medoxornil, S0m'

3-(1-Carboxy-3-phenyl-1 (S)-propyl)amino-2,3,4,5- mg ofolme edoxomi' .
tetrahydro-2-oxo-1 H-1-(3S)-benzazepine-1-acetic acid. and 200 m
CZZHZ4NzOs 396.44 volumetric
and sonicate for 5 mi
total volume and soni .
of the total volume and SOniC e
Diluentto volume. Centrifuge apo
1o min.and pass through a filterof 0.4~

Tcible2

. Tablet Strength Nominal

DEFINlrlON Amlodipine/Olmesar- Conceritratioriof
Amlodipine andOlrnes tan Medoxomil Amlodipine
(mg/mg) (mg/mL) (mg/rril,)
amount of Amlo . .
and NMT 110.0 5/20,10/40 0.5
(CzoHz~CIN20s) 5/40 0.25 2
labeled amoun
10/20 0.5

• Sample solution: Nominal centrationsin Diluentfrom

Sample stock solutionareg in Table 3; . .'
rable 3

Tablet Strength Nominal

Nominal
) -' '.
Amlodiplne/Olmesar- Concentration of
ASSAY tan Medoxomil Amladiplne
• EDU8E (mg/mg) (mg/mL)
S ion A: 6.9 giL' iuinphospha . nobasic;
5/20,10/49 0.04 0.16
Adjust withpho cid.to a pH of
Solutio cet rile 5/40 0.02 0.16
Mobile p ase:' See Table 1.
10/20 0;04 0.08
Table 1
Time Sofution"A Solution B
phicsystem
(min) (%) '(0/0) tography (621), System SUi~ability~)
0 68 32 " 254 nm.For Identificatio diode a
12 68 32 array detector in .the range of 200-40 . .
Column: 4.6-mm.x 25~cm; 5~JJm packing p 1
15 31:) 7()
Temperatures
21 30 70 Autosampler: '5 0
Colu 0
23 ,68 32
Flow r a , L/min
25 68 32 Injectionvo ume: 10'~L
System'suitability
Sample: Standardsolution
SuitabiJit . ments
Tailingf T 2.0 for amlodipirle'anq olrnesarta-n
medoxomil p .
Relative standard deviation: . NMT 2.0% for imlodipine
and olmesartan medoxomil peaks' '
Analysis
SampleS: Standardsolution and Sa
Calculatethe percentage of the I
amlodipine (CZOH2SCINzOs)in th
taken: . ,

Result= (ru/rs) x (Cs/Cu) 'x (M,dM,z) x100

=peak response of amlodipine'fromihe Sample

solution .
= peak resppnse of amJodipine from the Standard
solution . - .'..... ,
= 'concentration of USPAinlodipineBesylate R5 in
the Standard solution (mg/mL)

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USP 43 Official Monographs / Amlodipine 273

::;. nominalco'ncentrat n;ofcfml6djplpe In)he ,Result=(ruhs) xCsx' V x'. (Mrtl Mr2) >; (l/L)'x 10'0
. Sample solution (m . . . '" . . ..
:;::molecula~ weigb.t of a. . dil?ine,.4()8..8~ 'fJ sponse of amlodlpine fromthe Sample.
== molecularweightofamlocflphie De~y(~te,\?67:Q5,
rs . onse of amlodipinefrom,-the:Stcmdara
Car late.thepercentag e'labeledamouiifpt ..
01 n medoxomil !;130N606)Jn the. J:>0~io!1 of q
Ta aken:
'\1
Re~ult= (ru/rs)x.(Cic.J) ·x,1.00. Mr1 ipirr~;4()8.88
Mr2 ~lTlol.ecur~rweigh!. of amlqdipi.neh~sYlate, $67.05
= peakresponseofq.lmesartan rTt~d.oxorrii(troITi,;!Iie, ,~. :=='I,abeldaim of arnlocfiplne '(mgtralJlet)
Sample solution ... " '. ....
s
r =peak "'7' ...•.•••

response ofolmesartan .medqxomilfron,lhe

•••

Standard solution
= concentration of USP Ofrl1esartan]\i1edoxoh,ifRS
intheStandard solutio g/ l" . .
Cu = nominal concentration. . 1m an medo><o.mil Result,= (ruN's) xCs
in the Sample solution (mg/ml
(u seaf olniesartan medoxomIl fromthe
Acceptance criteria lion at the 30~ or 45-min time point
Amlodipine: 90.0%-11 °0 rs ofolmesartanmedoxomil from t~e
Olmesartan medoxomil .oo/~i j 0.00/0
C~ USP-dlmesartan MedoxomjJ'RS
P NCETESTS
solution (nig/nil)
• .. ON (711)'. "
Me.: 6.8 gil of potassium phosphate, monobasic
Adjust with 0.2 N sodiumhydroxide solution toapH of 6J3;
900mL
Apparatus 2: .50 rpm
TO s

L of olmesartan medoxomil(mg/
Tablet)

nt, Stand.ard

lodipine
edoxornil

me .' _ .. . .. _ e Besylate
Qelativestandard deviation: NMT 2.0%:for amlodipine Olind A
alld almesartan megoxomilpe~kS . - . . . i1:RS in .
Analysis . , .. ." . ._ __.. .gndard stock
Samples: •Sta,ndard solution and $ample so{ution _.
Calculate the percentage of the labeled amount of oliition; 0.28"~·g/rI1t.of O$p Arnlod-ipine
amlodipine(C2oH2SCIN20S) dissc>lved:: t~ RS; _0~5 IJg/mLof USPAmlodipine Related

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274 Amlodipine / Official Monographs USP 43

Compound A ~S, andO.8IJg!mL ofUSPOlrnes~lft~n Cu :; nominal

Medoxomil RSin Diluentfrom Sta . . inthe S
Sample solution: .U.$e'th~ Sample st repared.as
directed in the Assay. Aq:eptance crite'i-fa: 5e'e table:4:
System suitability ,
Samples: Standard solution a Table 4
[NoTE-See' h
Suitability require
Tailing fact Name
compoun BenzenesulfoniC aCid" ().13
peaks{ St
Relative $t Olmesartan~
related co A Ipine relat~d
medoxomll undN .0:5
SigniJl-to-noise. ra
Amlodipine OA7
compound A amI
peaks, Sensitivity solution bime~artan medoxomjlT.O
Analysis Olrnesartan medoxomil
Samples: Standard solution and ,tion relatedcompound
Calculate the percentage of ami. ed compound Ad,e 1.13
Afree base in the portion of Tci Olmesartan olefinic
impuritY', ~ T.sq
Result = (rvlr;):~ (Cs!Cv) x(!y1ril¥f2,:x-l00
Olmesarti:ui N-alkyl
= peak response of impurity9,e
A from th Any unspecified
= peak res amlodipine or
A fro olmesartan
= concen ra 10 lated me mil
relapurityh
Compound A RS ution
(mg/mL) Totalimpuritiesl
= nom' ,
. Sam to the couriterionand isnot to kle~eporieet or\n~ucl~ lr1the
=ffiole
compou
= molecular eo
compound A
Calculate the percentage .of~m lodipine
related impurity in the portion n:
Result = (rv/rs) x (CsICv) x (MrdMr2) x 100
rv = peak respo
related'
rs = pea ' dard
solution
Cs = concentra in€! Besylate ~Sin
the Stand solutio )
Cv = nominal con dlplne,inthe
Sample soluti ADDITiON
M r1 = molecular weight mlodipine,408.88 • PACKAGIN
Mr2 =fTlolecular weight of amlaalpine besylate, 567.05 store
·USPR
Calculate the percentage of b USPAm
olmesartan medoxomil relat USPAm
Tablets taken: ~ . ' . 3-Ethyl ethyl [ -
chlorophenyl),.6~methy - ,
Result.= (ivlrs)x'(CsICv) x lob fumarate.
= peak respons CzoH23C1N;Ps' t~H40
olmesa USP Olmesartan Medo
Sampl
= peak r olmesartan m~d6x6mil from1tle
Standards n
= concentrat USP blmesartan Medoxomll RS
in the StandardsoJutiof] (mg/mL) . ,

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USP 43 Official Monographs / Amlodipine 275

Amlodipine and Valsartan Tablets Suitability requirements

Tailing factor: NMT 1.5 for both amlodipine and
DEFINITION valsartan
Amlodipine and Valsartan Tablets contain NLT 90.0% and Relative standard deviation: NMT 2.0% for amlodipine
NMT 110.0% of the labeled amount of amlodipine and valsartan
(C2oH2sCIN20s) and valsartan (C24H29Ns03)' Analysis
Samples: Standardsolution, Sample solutionA, and Sample
IDENTIFICATION solution B
• A. The UV absorption spectra of the major peaks of Sample Calculatethe percentage of the labeled amount of
solution A and Sample solution B and those of the Standard amlodipine (C2oH2SCIN20S) in the portion of Tablets
solution exhibit maxima and minima at the same taken:
wavelengths, as obtained in the Assay.
• B. The retention times of the major peaksof Sample solution Result =(r vir s) x (C siC v) x (M ,JiM (2) x 100
A and Sample solutionB correspond to those of the Standard
solution, as obtained in the Assay. = peak response of amlodipine from Sample
solutionA
ASSAY = peak response of amlodipine from the Standard
• PROCEDURE solution
Solution A: Water and triethylamine (1000:10). Adjustwith = concentration of USP Amlodipine Besylate RS in
phosphoric acid to a pH of 2.8. the Standardsolution (mg/mL)
Solution B: Methanol and acetonitrile (700:300) = nominal concentration of amlodipine in Sample
Mobile phase: See Table 7. solutionA (mg/mL)
M'1 = molecular weight of amlodipine, 408.88
>Table 1 M,2 = molecularweight of amlodipine besylate, 567.05
Time Solution A Solution B
(min) (%) (%) Calculatethe percentage of the labeledamount ofvalsartan
0 50 50 (C24H29Ns03) in the portion of Tabletstaken:
3 50 50 Result = (r vir s) x (C siC v) x 100
15 30 70
= peak response of valsartanfrom Sample solutionB
20 30 70 = peak response of valsartan from the Standard
20.1 I
50 50 solution
= concentration of USP Valsartan RS in the Standard
25 50 50
solution (mg/mL)
Cv = nominal concentration of valsartan in Sample
Diluent: SolutionA and Solution B(50:50) solution B (mg/ml)
Standard solution: 0.14 mg/mL of USP Amlodipine
Besylate RS and 0.16 mg/mL of USP Valsartan RS. Add Acceptance criteria: 90.0%-110.0%
methanol to 5% of the final volume to dissolve, and dilute
with Diluent to volume. PERFORMANCE TESTS
Sample stock solution: Transfer NLT 10 Tablets into a • DISSOLUTION (711)
suitable volumetric flask. Initially add water to 10% of the Test 1
final volume, and sonicate to disperse as needed. Add Buffer: Dissolve 6.805 9 of monobasic potassium
Diluent, using about 70% of the final volume, and> shake for phosphate and 0.896 9 of sodium hydroxideinwater, and
up to 45 min to disperse. Following dispersion, sonicate for dilute with water to 1000 ml. Adjustwith 0.2 N sodium
15 min, and shakefor 30 min. Dilutewith Diluent to volume hydroxide or 1 M phosphoric acid to a pH of 6.8.
to obtain a solution containing known nominal Medium: Buffer; 1000 ml
concentrations of 0.1-0.2 mg/mL of amlodipine and 1.6- Apparatus 2: 75 rpm
6.4 mg/mL of valsartan. Centrifuge the solution for about Time: 30 min
10 min at 3000 rpm. Mobile phase: Acetonitrile, water, and trifluoroacetic acid
Sample solution A: Nominally equivalent to 0.1 mg/mL of (500:500:2)
amlodipine in Diluent from the Sample stock solution Diluent: 1 mg/ml of polysorbate 80 in Buffer
Sample solution B: Nominallyequivalentto 0.16 mg/mL of System suitability solution: 0.4 mg/mL each of USP
valsartan in Diluent from the Sample stock solution AmlodipineBesylate RS and USP Valsartan RS, prepared as
Chromatographic system follows. Initially dissolve in methanol to 40% of the total
(See Chromatography(621), System Suitability.) volume, and dilute with Bufferto volume.
Mode: LC Standard stock solution A: 0.072 mg/mL of USP
Detector: UV 237 nm. For Identification A, use a diode Amlodipine Besylate RS, prepared as follows. Initially
array detector in the range of 200-400 nm. dissolve in methanol to 4% of the final volume, and dilute
Column: 3.9-mm x 15-cm; 5-lJm packing L1 with Diluent to volume.
Temperatures Standard stock solution B: 2.2 mg/mL of USP Valsartan
Autosampler: 10° RS in> methanol
Column: 30° Standard solution: (L Jil000) mg/ml of amlodipine and
Flow rate: 1.0 mL/min (L 211 000) mg/mL of valsartan in Diluent from Standard
Injection volume: 10 IJL stocksolutionA and Standardstock solution B, where L 1 is
System suitability the label claim of amlodipine in mg/Tablet, and L 2 is the
Sample: Standard solution label claim of valsartan in mg/Tablet

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276 Amlodipine / OfficialMonographs USP 43

Sample solution: Pass a portion of the solution under test Table 2

through a suitable filterof 0.45-l.Im pore size. Discard the Time Solution A Solution B
first 10 mLof the filtrate. (min) (%) (%)
Chromatographic system 80 20
0
(See Chromatography (621), System Suitability.)
Mode: LC 7 30 70
Detector: UV 230 nm 8 80 20
Column: 4.6-mm x 15-cm; 4-l.Im packing L11
Column temperature: 40° 10 80 20
Flow rate: 1.2 mL/min
Injection volume: 10 I.IL Standard stock solution A: 0.14 mg/mL of USP
Run time: NLT 2 times the retention time of amlodipine Amlodipine Besylate RS, prepared as follows. Initially
System suitability . dissolve in 10% of the final volume of methanol, and
Samples: System suitability solution and Standardsolution dilute with Medium to volume.
Suitability requirements Standard stock solution B: 1.6 mg/mL of USP Valsartan
Resolution: NLT 2.0 between amlodipine and RS in methanol
valsartan, System suitability solution Standard solution: (L ,/1000) mg/mL of amlodipine and
Tailing factor: NMT 2.0 for amlodipine and valsartan, (L il 000) mg/mL of valsartan in Medium from Standard
Standardsolution stock solutionA and Standardstock solution 8, where L 1 is
Relative standard deviation: NMT 2.0% for the label claim of amlodipine in mg/Tablet, and L 2 is the
amlodipine and valsartan, Standard solution
Analysis labelclaim of valsartan in mg/Tablet
Samples: Standardsolution and Sample solution Sample solution: Pass a portion of the solution under test
Calculate the percentage of the labeled amount of through a suitable filter of t-urn pore size.
amlodipine (C2oHzsCIN20S) dissolved: Chromatographic system
(See Chromatography (621), System Suitability.)
Result =(r vir s) xC s x Vx (M ,,/M (2) x (l1L 1) x 100 Mode: LC
Detector: UV 237 nm
= peak response of amlodipine from the Sample Column: 4.6-mm x 15-cm; 5-l.Im packing L1
solution Temperatures
= peak response of amlodipine from the Standard Autosampler: 10°
solution Column: 50°
= concentration of USP Amlodipine Besylate RS in Flow rate: 1.5 mL/min
the Standardsolution (mg/mL) Injection volume: 20 I.IL
V = volume of Medium, 1000 mL System suitability
M rl = molecular weight of amlodipine, 408.88 Sample: Standardsolution
=molecular weight of amlodipine besylate, 567.05 Suitability requirements
M r2 Tailing factor: NMT 2.0 for amlodipine and valsartan
L1 = label claim of amlodipine (mg/Tablet) Relative standard deviation: NMT 2.0% for
amlodipine and valsartan
Calculate the percentage of the labeled amount of Analysis
valsartan (C24H29Ns03) dissolved: Samples: Standardsolution and Sample solution
Calculatethe percentage of the labeled amount of
Result = (r vir s) xC s x Vx (l1L 2) x 100 amlodipine (C2oH2sClN20s) dissolved:
= peak response of valsartanfrom the Sample Result = (r vir s) xC s x VX (M r,/M r2) X (l1L 1) x 100
solution
= peak response of valsartan from the Standard = peak response of amlodipine from the Sample
solution solution
= concentration of USP Valsartan RS in the Standard
= peak response of amlodipine from the Standard
solution (mg/mL) solution
=volume of Medium, 1000 mL = concentration of USP Amlodipine Besylate RS in
= label claim of valsartan (mg/Tablet) the Standardsolution (mg/ml)
V = volume of Medium, 1000 mL
Tolerances: NLT 80% (Q) of the labeled amount of = molecular weight of amlodipine, 408.88
M rl
amlodipine (C2oHzsCIN20S) and valsartan (C24H29Ns03) is = molecularweight of amlodipine besylate, 567.05
dissolved. M'2
L1 = label claim of amlodipine (mg/Tablet)
Test 2: Ifthe product complies with this test, the labeling
indicates that the product meets USP Dissolution Test 2.
Medium and Time: Proceed as directed in Dissolution Test Calculate the percentage of the labeled amount of
1; 1000 mL. valsartan(C24H29Ns03) dissolved:
Apparatus 2: 50 rpm
Buffer: Mix 7.0 mLof triethylamine with 1000 mLof Result =(r vir s) x C s x V x (1IL 2) x 100
water. Adjust with phosphoric acid to a pH of 3.0. =peak response of valsartan from the Sample
Solution A: Acetonitrile and Buffer(1 0:90) solution
Solution B: Acetonitrile and Buffer (90:1 0) = peak response of valsartan from the Standard
Mobile phase: See Table 2. solution
= concentration of USP Valsartan RS in the Standard
solution (mg/mL)
v = volume of Medium, 1000 mL

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USP 43 Official Monographs / Amlodipine 277

L2 = label claim of valsartan (mg/Tablet) C5 = concentration of USP Amlodipine Besylate RS in

the Standard solution (mg/mL)
Tolerances: NLT 75% (Q) of the labeled amount of V '= volume of Medium, 1000 mL
amlodipine (C2oH2sCIN20s) is dissolved and NLT 80% (Q) M rl = molecular weight of amlodipine, 408.88
of the labeled amount of valsartan (C24H29Ns03) is M r2 =molecular weight of amlodipine besylate, 567.05
dissolved. LI = label claim of amlodipine (mg/Tablet)
Test 3: Ifthe product complies with this test, the labeling
indicates that the product meets USP Dissolution Test 3. Calculate the percentage of the labeled amount of
Medium, Apparatus 2, and Time: Proceed as directed in valsartan (C24H29Ns03) dissolved:
Dissolution Test 1.
Solution A: Acetonitrile, trifluoroacetic acid, and water ResuIt = (r ulr s) x C 5 X V x (1 I L 2) x 100
(10: 0.1: 90)
Solution B: Acetonitrile, trifluoroacetic acid, and water ru = peak response of valsartanfrom the Sample
(90: 0.1: 10) solution
Mobile phase: See Table 3. rs = peak response of valsartanfrom the Standard
solution
Table 3 C5 =concentration of USP Valsartan RS in the Standard
Time Solution A Solution B solution (mg/mL)
(min) (%) (%) V =volume of Medium, 1000 mL
0.01 90 10
L2 = label claim of valsartan (mg/Tablet)
2.5 10 90 Tolerances: NLT 75% (Q) of the labeled amount of
3.0 90 10
amlodipine (CzoH2sCIN20s) isdissolved and NLT 80% (Q)
of the labeled amount of valsartan (C24Hz9Ns03) is
5.0 90 10 dissolved.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
Diluent: Acetonitrile and water (50:50) requirements
Standard stock solution A: 0.14 mg/mL of USP IMPURITIES
Amlodipine Besylate RS, prepared as follows. Initially • ORGANIC IMPURITIES
dissolve in Diluent about 4% ofthe final volume, and Mobile phase, Diluent, Sample solution A, Sample
dilute with Medium to volume. solution B, and Chromatographic system: Proceed as
Standard stock solution B: 1.6 mg/mL of USP Valsartan directed in the Assay.
RS, prepared as follows. Initially dissolve in about 20% of Standard stock solution A: Prepare as directed for the
the final volume of Diluent, and dilute with Medium to Standard solution in the Assay.
volume. System suitability solution: Dissolve a suitable quantity of
Standard solution: (L Iil000) mg/mL of amlodipine and USP Valsartan Related Compound BRS in Standard stock
(L il 000) mg/mL of valsartan in Medium from Standard solution A to obtain a solution containing 0.08 mg/mL of
stock solution A and Standard stock solution B, where L I is USP Valsartan RelatedCompound BRS, 0.14 mg/mL of USP
the label claim of amlodipine in mg/Tablet, and L 2 is the Amlodipine Besylate RS, and 0.16 mg/mL of USP Valsartan
label claim of valsartan in mg/Tablet RS.
Sample solution: Pass a portion of the solution under test Sensitivity solution: 0.14 IJg/mL of USP Amlodipine
through a suitable filter of 0.45-lJm pore size and discard Besylate RS and 0.16 IJg/mL of USP Valsartan RS in Diluent
the firstfew milliliters of the filtrate. from Standard stocksolution A
Chromatographic system Standard stock solution B: 0.1 mg/mL of USP Amlodipine
(See Chromatography (621), System Suitability.) Related Compound A RS as free base, prepared as follows.
Mode: LC Add methanol to 5% of the final volume to dissolve, and
Detector: UV 237 nm for amlodipine and UV 270 nm for dilute with Diluent to volume.
valsartan Standard solution: 0.0005 mg/mL of USP Amlodipine
Column: 4.6-mm x 10-cm; 5-lJm packing L1 Related Compound A RS as free base, and 0.0003 mg/mL
Flow rate: 1.5 mL/min each of USP Amlodipine Besylate RS and USP Valsartan RS
Injection volume: 10 IJL in Diluent from Standard stock solution A and Standard stock
System suitability solution B, respectively
Sample: Standard solution System suitability
Suitability requirements Samples: System suitability solution, Sensitivity solution, and
Tailing factor: NMT 2.0 for amlodipine and valsartan Standard solution
Relative standard deviation: NMT 2.0% for Suitability requirements
amlodipine and valsartan Resolution: More than 4.0 between amlodipine and
Analysis valsartan related compound Band more than 4.0
Samples: Standard solution and Sample solution between valsartan related compound Band valsartan,
Calculate the percentage of the labeled amount of System suitability solution
amlodipine (C2oH2SCIN20S) dissolved: Relative standard deviation: NMT 5.0% for amlodipine
related compound A, amlodipine, and valsartan,
Result =(r ulr 5) xC 5 X Vx (M rliM r2) X (l1L 1) x 100 Standard solution
Signal-to-noise ratio: NLT 10 for amlodipine and
ru = peak response of amlodipine from the Sample valsartan, Sensitivity solution
solution Analysis '
rs = peak response of amlodipine from the Standard Samples: Sample solution A, Sample solution B, and
solution Standard solution

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278 Amlodipine / Official Monographs USP 43

Calculate the percentage of amlodipine related compound Table 4 (continued)

A free base in the portion of Tablets taken: Relative Acceptance
Retention Criteria,
Result = (r vir s) x (C siC v) x (M r7IM (2) x 100 Name Time NMT(%)
Valsartan related degradation
ru = peak response of amlodipine related compound product 3C 1.44 0.2
A from Sample solutionA
rs = peak response of amlodipine related compound Valsartan 1.74 -
Afrom the Standardsolution Valsartan relateddegradation
Cs =concentration of USP Amlodipine Related product 4C 2.06 0.2
Compound A RS in the Standardsolution
Valsartan ethylester" 2.32 0.2
(mg/mL)
Cu = nominal concentration of amlodipine in Sample Any other unspecified
-
solutionA (mg/mL) degradation product 0.2
M ,1 = molecular weight of amlodipine related 1.2; 2.0, if valsar-
compound A free base, 406.86 tan related com-
M ,2 = molecular weight of am/odipine related - pound A is a po-
tential degrada-
compound A fumarate, 522.93 Totaldegradation products' tion product
Calculate the percentage of valsartan related degradation a N-{[2'-(1 H-Tetrazole-5-yl)biphenyl-4-yl]methyl}-L-valine.
products other than va/sartan related compound A in the b 3-Ethyl 5-methyl[2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-
portion of Tablets taken: 3,5-pyridinedicarboxylate].
C Theseare specified unidentified degradation products. No informationis
Result =(r vir s) x (C siC v) x 100 available about chemical structuresor chemical names for these impurities.
d N-Butyryl-N-{[2'-(1 H-tetrazole-5-yl)biphenyl-4-yl]methyl}-L-valine.
ru =peak response of va/sartan related degradation e N-Valeryl-N-{[2'-(1 H-tetrazole-5-yl)biphenyl-4-yl]methyl}-L-valine ethyl ester.
f Ifvalsartanrelated compound Aisa potentialdegradation product, the total
product from Sample solution B
degradation products limitdoes not include valsartanrelatedcompound Aand
rs = peak response of valsartan from the Standard amlodipinerelated compound A.
solution
Cs =concentration of USP Valsartan RS in the Standard • LIMIT OF VALSARTAN RELATED COMPOUND A
solution (mg/mL) [NoTE-Valsartan related compound A is a process
Cu = nominal concentration of valsartan in Sample impurity and a formulation-specific degradation
soluti?n B (mg/mL) product.]
Mobile phase: n-Hexane, 2-propanol, and trif/uoroacetic
Calculate the percentage of each unspecified degradation acid (850: 150:1)
product in the portion of Tablets taken: System suitability solution: 0.04 mg/mL each of USP
Valsartan Related Compound A and USP Valsartan RS in
Result =(r vir s) x (C siC v) x (M ,dM ;2) x 100 Mobilephase
ru = peak response of each unspecified degradation Standard solution: 0.001 mg/mL of USP Va/sartan Related
product from Sample solutionA Compound A RS in Mobilephase
rs = peak response of amlodipine from the Standard Sample solution: Nominally 0.5 mg/mL of va/sartan in
solution Mobile phase from a suitable amount of finely crushed
Cs =concentration of USPAmlodipine Besylate RS in powder from NLT 20 Tablets. Sonication may be necessary
the Standardsolution (mg/mL) . for complete dissolution. Pass through a suitable filter of
Cu = nominal concentration of amlodipine in Sample 0,45-lJm pore size.
solutionA (mg/mL) Chromatographic system
M ,1 = molecular weight of amlodipine, 408.88 (See Chromatography (621), System Suitability.)
Mode: LC
M ,2 = molecular weight of am/odipine besylate, 567.05
Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-lJm packing L40
Acceptance criteria: See Table 4. Disregard va/sartan Temperatures
related compound B, the benzenesulfonic acid peak at Autosampler: 10°
relative retention time 0.19, and any peaks below 0.1 %. Column: 30°
Flow rate: 0.8 mL/min
Table 4
Injection volume: 20 IJL .
Relative Acceptance Run time: NLT 3.5 times the retention time of valsartan
Retention Criteria, related compound A
Name Time NMT(%)
System suitability
Devaleryl valsertan- 0.24 0.2 Samples: System suitability solution and Standardsolution
Amlodipine related [NOTE-The relative retention times for va/sartan
compound Ab 0.50 0.5 related compound A and valsartan are about 0.7 and
1.0, respectively.]
Valsartan related degradation Suitability requirements
product 1C 0.54 0.2
Resolution: NLT 2.0 between valsartan and valsartan
Valsartan related degradation related compound A, System suitability solution
product 2C 0.81 0.2 Relative standard deviation: NMT 5.0% for va/sartan
Amlodipine 1.00 - related compound A, Standard solution
Analysis
Valsartan related compound Bd 1.34 - Samples: Standardsolution and Sample solution

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 USP 43 OfficialMonographs / Amlodipine 279

Calculatethe percentage of valsartan related compound A Solution B: Acetonitrile, water, and phosphoric acid
in the portion of Tablets taken: (950:50:1 )
Mobile phase: See Table 1.
Result = (r vir s)x (C siC v) x 100
Table 1
ru = peak response of valsartan related compound A Time Solution A Solution B
from the Sample solution . (min) (%) (%)
rs = peak response of valsartan related compound A
from the Standard solution 0 95 5
Cs = concentration of USP Valsartan Related 3 50 50
Compound A RS in the Standard solution
(mg/mL) 6 40 60
Cu =nominal concentration of valsartan in the Sample 10 5 95
solution (mg/mL)
10.1 95 5
Acceptance criteria: NMT 1.0% 15 95 5
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Store at controlled room Diluent: Acetonitrile and water (500:500)
temperature, in tight containers, and in a dry place. 0.1% Phosphoric acid: Water and phosphoric acid
• LABELING: When more than one Dissolution test isgiven, the (1000:1)
labeling states the Dissolution test used only if Test 1 is not Standard solution: 0.14 mg/mL of USP Amlodipine
used. Besylate RS, 0.064 mg/mL of USP Valsartan RS, and 0.025
• USP REFERENCE STANDARDS (11) mg/mL of USP Hydrochlorothiazide RS in Diluent
USP Amlodipine Besylate RS Sample stock solution: Transfer NLT 10 Tablets into a
USP Amlodipine Related Compound A RS suitable volumetricflask. Add 0.1% Phosphoric acid to 4%
3-Ethyl 5-methyl [2-(2-aminoethoxymethyl)-4-(2- of the total volume to disperse the Tablets. Sonicate for 10
chlorophenyl)-6-methyl-3,5-pyridinedicarboxylate] min. Add 4% of the total volume of acetonitrile, swirl to
fumarate. mix, and add 60% of the total volume of Diluent. Sonicate
CzoHz3CINzOs . C4H404 522.93 for 20 min. Dilute with Diluentto volumeto obtain solutions
USP Valsartan RS of nominal concentrations stated in Table 2. Centrifuge,
USP Valsartan Related Compound A RS and use the clear supernatant.
N-Valeryl-N-{[2'-(1 H-tetrazole-5-yl)biphenyl-4-yl]
methyl}-o-valine. Table 2
CZ4H29Ns03 435.52 Tablet Strength
USP Valsartan Related Compound B RS Amlodipine/ Nominal
Valsartan/ Nominal Nominal Concentration
N-Butyryl-N-{[2'-(1 H-fetrazole-5-yl)biphenyl-4-yl] Hydrochloro- Concentration Concentration of Hydrochloro-
methyl}-L-valine. thiazide of Amlodlpine of Valsartan thiazide
CZ3Hz7Ns03 421.49 (mg/mg/mg) (mg/mL) (mg/mL) (mg/mL)
5/160/12.5 0.1 3.2 0.25
10/160/12.5 0.2 3.2 0.25
5/160/25 0.1 3.2 0.5
Amlodipine, Valsartan, and
10/160/25 0.2 3.2 0.5
Hydrochlorothiazide Tablets
10/320/25 0.1 3.2 0.25
DEFINITION
Amlodipine, Valsartan, and Hydrochlorothiazide Tablets Sample solution A: Nominally equivalent to 0.1 mg/mL of
contain NLT 92.5% and NMT 107.5% each of the labeled amlodipine in Diluent from Sample stock solution
amounts of amlodipine (CzoHzsCINzOs), valsartan Sample solution B: Nominally equivalent to 0.064 mg/mL
(Cz4H29Ns03), and hydrochlorothiazide (C7HaCIN304S2)' of valsartan in Diluent from Sample stock solution
Sample solution C: Nominally equivalent to 0.025 mg/mL
IDENTIFICATION of hydrochlorothiazide in Diluentfrom Sample stock solution
• A. The UV absorption spectra of the amlodipine, valsartan, Chromatographic system
and hydrochlorothiazide peaks of Sample solutionA, Sample (See Chromatography (621), System Suitability.)
solution B, and Sample solution C, and those of the Standard Mode: LC
solution exhibit maxima and minima at the same Detector: UV 225 nm. For Identification A, use a diode
wavelengths, as obtained in the Assay. array detector in the range of 200-400 nm.
• B. The retention times of the amlodipine, valsartan, and Column: 4.6-mm x 15-cm; 3-lJm packing L1
hydrochlorothiazide peaks of Sample solutionA, Sample Column temperature: 40°
solution B, and Sample solution Ccorrespond to those of the Flow rate: 1.5 mL/min
Standard solution, as obtained in the Assay.
Injection volume: 10 IJL
ASSAY System suitability
• PROCEDURE Sample: Standard solution
Use amber glasswarefor all solutions containing drug Suitability requirements
substances. Tailing factor: NMT 2.0 for amlodipine, valsartan, and
Solution A: Acetonitrile, water, and phosphoric acid hydrochlorothiazide
(50:950:1) Relative standard deviation: NMT 2.0% for amlodipine,
valsartan, and hydrochlorothiazide

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280 Amlodipine / OfficialMonographs USP 43

Analysis Solution B: Acetonitrile, water, and phosphoric acid

Samples: Standardsolution, Sample solutionA, Sample (950:50:1)
solution B, and Sample solution C Mobile phase: See Table 3.
Calculate the percentage of the labeled amount of
amlodipine (C2oH2sCIN20s) in the portion of Tablets Table 3
taken: Time Solution A Solution B
(min) (%) (%)
Result =(r vir s) x (C siC v) x (M rtiM r2) X 100
0.00 67 33
rv =peak response of amlodipine from Sample 2.50 23 77
solutionA
67
rs = peak response of amlodipine from the Standard 2.51 33
solution 4.00 67 33
Cs =concentration of USP Amlodipine Besylate RS in
the Standardsolution (mg/mL) Diluent: 1 mg/mL of polysorbate 80 in Buffer
Cu = nominal concentration of amlodipine in Sample Standard stock solution A: 0.07 mg/mL of USP
solution A (mg/mL) Amlodipine Besylate and 0.124 mg/mL of USP
M rl =molecular weight of amlodipine, 408.88 Hydrochlorothiazide RS. Initially dissolve with 4% of the
M r2 = molecular weight of amlodipine besylate, 567.05 total volume of methanol, and dilute with Diluent to
volume.
Calculate the percentage of the labeled amount of valsartan Standard stock solution B: 3.2 mg/mL of USP Valsartan
(C24H29Ns03) in the portion of Tablets taken: RS in methanol
Standard solution: 0.014 mg/mL of USPAmlodipine
Result = (i vir s) x (C siC v) x 100 Besylate RS, 0.16 mg/mL of USP Valsartan RS, and 0.0248
mg/mL of USP Hydrochlorothiazide RS in Diluent from
ru = peak response of valsartan from Sample solutionB Standardstock solution A and Standard stock solution B,
rs = peak response of valsartan from the Standard respectively
solution Sample solution: Pass a portion of the solution under test
Cs = concentration of USP Valsartan RS in the Standard through a suitable filter of 0,45-lJm pore size. Discard at
solution (mg/mL) least the first 10 mL of the filtrate.
Cu = nominal concentration of valsartan in Sample Chromatographic system
solution B (mg/mL) (See Chromatography (621), System SUitability.)
Mode: LC
Calculate the percentage of the labeled amount of Detector: UV 250 nm
hydrochlorothiazide (C7HsCIN304S2) in the portion of Column: 4.6-mm x 5-cm; 3-lJm packing L1
Tablets taken: Column temperature: 30°
Flow rate: 1.5 mL/min
Result = (r vir s) x (C siC v) x 100 Injection volume
For 10/320/25 (mg/mg/mg) of Tablet strengths
ru = peak response of hydrochlorothiazide from (amlodipine/valsartan/hydrochlorothiazide): 5 IJL
Sample solution C For 5/160/12.5, 10/160/12.5,5/160/25, and
rs = peak response of hydrochlorothiazide from the 10/160/25 (mg/mg/mg) of Tablet strengths
Standardsolution (amlodipine/valsartan/hydrochlorothiazide):
Cs = concentration of USP Hydrochlorothiazide RS in 10 IJL
the Standardsolution (mg/mL) System suitability
Cu = nominal concentration of hydrochlorothiazide in Sample: Standardsolution
Sample solution C (mg/mL) Suitability requirements
Resolution: NLT 3.0 between amlodipine and valsartan
Acceptance criteria: 92.50/0-107.5% Tailing factor: NMT 2.0 for amlodipine, valsartan, and
PERFORMANCE TESTS hydrochloroth iazide
• DISSOLUTION (711) Relative standard deviation: NMT 2.0% for
Test 1 amlodipine, valsartan, and hydrochlorothiazide
Buffer: Dissolve 6.805 9 of monobasic potassium Analysis
phosphate and 0.896 9 of sodium hydroxide in 1000 mL Samples: Standardsolution and Sample solution
of water. Adjust with 0.2 N sodium hydroxide or 1 M Calculate the percentage of the labeled amount of
phosphoric acid to a pH of 6.8. amlodipine (C2oH2sCIN20s) dissolved:
Medium: Buffer; 900 mL
Apparatus 2 Result =(r vir s) x C s x V x (M rtfM r2) X (1 ILl) x 100
For 5/160/12.5, 10/160/12.5, 5/160/25, and
10/160/25 (mg/mg/mg) of Tablet strengths = peak response of amlodipine from the Sample
(amlodipine/valsartan/hydrochlorothiazide): solution
50 rpm = peak response of amlodipine from the Standard
For 10/320/25 (mg/mg/mg) of Tablet strengths solution
(amlodipine/valsartan/hydrochlorothiazide): =concentration of USP Amlodipine Besylate RS in
55 rpm the Standardsolution (mg/mL)
Time: 30 min V = volume of Medium, 900 mL
Solution A: Acetonitrile, water, and phosphoric acid M rl = molecular weight of amlodipine, 408.88
(50:950:1) M r2 =molecular weight of amlodipine besylate, 567.05
L1 = label claim of amlodipine (mg/Tablet)

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USP 43 Official Monographs / Amlodipine 281

Calculate the percentage of the labeled amount of Standard stock solution B: 1.6 mg/ml of USP Valsartan
valsartan (C24H29Ns03) dissolved: RS in methanol
Standard stock solution C: 0.7 mg/ml of USP
Result = (r vir s) x C s x V x (1I L 2) x 100 Hydrochlorothiazide RS, prepared as follows. Initially.
dissolve in 25% of the final volume of methanol and dilute
ru = peak response of valsartan from the Sample with Medium to volume.
solution Standard solution: (L dl000) mg/ml of amlodipine, (L
rs = peak response of valsartan from the Standard 2/1 000) mg/ml of valsartan, and (L 3/1000) mg/mL of
solution hydrochlorothiazide in Medium from Standardstock
Cs = concentration of USP Valsartan RS in the Standard solutionA, Standard stock solution 8, and Standardstock
solution (mg/ml) solution C, where L 1 is the label claim of amlodipine in
V = volume of Medium, 900 ml mg/Tablet, L 2 isthe label claimof valsartan in mg/Tablet,
L2 = label claim of valsartan (mg/Tablet)
and L 3is the label claim of hydrochlorothiazide in mgl
Calculate the percentage of the labeled amount of Tablet
hydrochlorothiazide (C7HsCIN304S2) dissolved: Sample solution: Pass a portion of the solution under test
through a suitable filter of t-urn pore size.
Result = (r vir s) x C s x V x (1I L 3) x 100 Chromatographic system
(See Chromatography (621), System Suitability.)
= peak response of hydrochlorothiazide from the Mode: lC
Sample solution Detector: UV 237 nm
= peak response of hydrochlorothiazide from the Column: 4.6-mm x 15-cm; 5-lJm packing II
Standardsolution Temperatures
= concentration of USP Hydrochlorothiazide RS in Autosampler: 10°
the Standardsolution (mg/ml) Column: 50°
= volume of Medium, 900 ml Flow rate: 1.5 mL/min
= label claim of hydrochlorothiazide (mg/Tablet) Injection volume: 20 IJl
System suitability
Tolerances: NlT 75% (Q) of the labeled amount of Sample: Standard solution
amlodipine (C2oH2sClN20s) is dissolved, Nl T 80% (Q) of Suitability requirements
the labeled amount of valsartan (C24H29Ns03) isdissolved, Tailing factor: NMT 2.0 for each peak
Relative standard deviation: NMT 2.0% for each peak
and NlT 80% (Q) of the labeled amount of Analysis
hydrochloroth,iazide (C7HsCIN304S2) is dissolved. Samples: Standardsolution and Sample solution
Test 2: Ifthe product complies with this test, the labeling Calculate the percentage of the labeled amount of
indicates that the product meets USP Dissolution Test 2. amlodipine (C2oH2sCIN20s) dissolved:
Medium: Proceed as directed under Dissolution Test 7;
900 ml. Result = (r vir s) x C s x Vx (M rdM r2) X(l1L 1) x 100
Apparatus 2
For Tablets labeled to contain amlodipine/valsartan/ =peak response of amlodipine from the Sample
hydrochlorothiazide, 5/160/12.5, 10/160/12.5, solution
5/160/25, 10/160/25, and 5/80/12.5 (mg/mg/mg): = peak response of amlodipine from the Standard
50 rpm . solution
For Tablets labeled to contain amlodipine/valsartan/ =concentration of USP Amlodipine Besylate RS in
hydrochlorothiazide, 10/320/25 (mg/mg/mg): 55 the Standard solution(mg/ml)
rpm V = volume of Medium, 900 mL
Times M rl = molecular weight of amlodipine, 408.88
For valsartan and hydrochlorothiazide: 30 min M r2 = molecularweight of amlodipine besylate, 567.05
For amlodipine: 45 min L1 = label claim of amlodipine (mg/Tablet)
Buffer: Mix 7.0 ml of triethylamine with 1000 ml of
water. Adjust with phosphoric acid to a pH of 3.0. Calculate the percentage of the labeled amount of
Solution A: Acetonitrile and Buffer (10:90) valsartan (C24H29Ns03) dissolved:
Solution B: Acetonitrile and Buffer (90:1 0)
Mobile phase: See Table 4. Result = (r vir s) x Cs x Vx (l1L 2) x 100
Table 4 = peak response of valsartan from the Sample
Time Solution A Solution B solution
(min) (%) (%) = peak response of valsartan from the Standard
0 90 10 solution .
= concentration of USP Valsartan RS in the Standard
7 30 70 solution (mg/mL)
8 90 10 =volume of Medium, 900 mL
15 90 10
=label claim of valsartan (mg/Tablet)
Calculate the percentage of the labeled amount of
Standard stock solution A: 0.35 mg/ml of USP hydrochlorothiazide (C7HsCIN304S2) dissolved:
Amlodipine Besylate RS, prepared as follows. Initially
dissolve in 10% of the finalvolume of methanol and dilute Result = (r vir s) x Cs x V x (1 I L 3) x 100
with Medium to volume.

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282 Amlodipine / Official Monographs USP43

= peak response of hydrochlorothiazide from the and L 3 is the label claim of hydrochlorothiazide in mgl
Sample solution Tablet
= peak response of hydrochlorothiazide from the Sample solution: Pass a portion of the solution under test
Standardsolution through a suitable filter of 0.45-~m pore size. Discard at
= concentration of USP Hydrochlorothiazide RS in least the first few milliliters of the filtrate.
the Standardsolution (mg/mL) Chromatographic system
=volume of Medium, 900 mL (See Chromatography (621), System Suitability.)
= label claim of hydrochlorothiazide (mg/Tablet) Mode: LC
Detectors
Tolerances: NLT 75% (0) of the labeled amount of For amlodipine: UV 237 nm
amlodipine (C2oH2sCIN20s) is dissolved, NLT 80% (0) of For valsartan and hydrochlorothiazide: UV 270 nm
the labeled amount of valsartan (C24H29Ns03) is dissolved, Column: 4.6-mm x 1O-cm; 5-~m packing L1
and NLT80% (0) of the labeled amount of Flow rate: 1.5 mL/min
hydrochlorothiazide (C7HaCIN304S2) is dissolved. Injection volume: 10 ~L
Test 3: If the product complies with this test, the labeling System suitability
indicates that the product meets USP Dissolution Test 3. Sample: Standardsolution
Medium: Dissolve 6.80 9 of monobasic potassium Suitability requirements
phosphate in 1000 mL of water. Adjust with 10% sodium Tailing factor: NMT 2.0 for each peak
hydroxide solution to a pH of 6.8; 1000 mLfor valsartan Relative standard deviation: NMT 2.0% for each peak
and hydrochlorothiazide; 900 mL for amlodipine. Analysis
Apparatus 2 Samples: Standardsolution and Sample solution
For valsartan and hydrochlorothiazide: 50 rpm Calculate the percentage of the labeled amount of
For amlodipine in Tablets labeled to contain amlodipine (C2oH2sCIN20s) dissolved:
amlodipine/valsartan/hydrochlorothiazide,
10/320/25 (mg/mg/mg): 55 rpm Result =(r vir s) x C s x Vx (M rdM r2) X (l1L 1) x 100
For amlodipine in Tablets labeled to contain
amlodipine/valsartan/hydrochlorothiazide, = peak response of amlodipine from the Sample
5/160/12.5, 10/160/12.5, 5/160/25, 10/160/25, and solution
5/80/12.5 (mg/mg/mg): 50 rpm = peak response of amlodipine from the Standard
Times solution
For valsartan and hydrochlorothiazide: 30 min =concentration of USP Amlodipine Besylate RS in
For amlodipine: 45 min the Standardsolution (mg/mL)
Solution A: Acetonitrile, trifluoroacetic acid and water (10: V = volume of Medium, 900 mL
0.1: 90) M rl = molecular weight of amlodipine, 408.88
Solution B: Acetonitrile, trifluoroacetic acid and water (90: Mr2 =molecular weight of amlodipine besylate, 567.05
0.1:10) L1 = label claim of amlodipine (mg/Tablet)
Mobile phase: See Table 5.
Calculate the percentage of the labeled amount of
Table 5 valsartan (C24H29Ns03) dissolved:
Time Solution A Solution B
(min) (%) (%) Result = (r vir s) xes x Vx (l/L 2) x 100
0.01 90 10 = peak response of valsartanfrom the Sample
2.5 10 90 solution
3.0 90 10
= peak response of valsartan from the Standard
solution
5.0 90 10 =concentration of USP Valsartan RS in the Standard
solution (rnq/rnl.)
Diluent: Acetonitrile and water (50:50) =volume of Medium, 1000 mL
Standard stock solution A: 0.15 mg/mL of USP
= label claim of valsartan (mg/Tablet)
Amlodipine Besylate RS in Medium, prepared as follows.
Initiallydissolve and sonicate in 5% of the final volume of Calculate the percentage of the labeled amount of
Diluent, and dilute with Medium to volume. hydrochlorothiazide (C7HaCIN304S2) dissolved:
Standard stock solution B: 1.6 mg/mL of USP Valsartan
RS in Medium, prepared as follows. Initiallydissolve and Result =(r vir s) xes x V x (11L 3) x 100
sonicate in 20% of the final volume of Diluent, and
dilute with Medium to volume.
ru = peak response of hydrochlorothiazide from the
Sample solution
Standard stock solution C: 0.25 mg/mL of USP rs = peak response of hydrochlorothiazide fromthe
Hydrochlorothiazide RS in Medium, prepared as follows. Standardsolution
Initially dissolve and sonicate in 10% of the final volume Cs =concentration of USP Hydrochlorothiazide RS in
of Diluent, and dilute with Medium to volume. the Standardsolution (mg/mL)
Standard solution: (L dl000) mg/mL of amlodipine, (L V = volume of Medium, 1000 mL
211000) mg/mL of valsartan, and (L 3/1000) mg/mL of
hydrochlorothiazide in Diluent from Standardstock
L3 =label claim of hydrochlorothiazide (mg/Tablet)
solutionA, Standardstocksolution 8, and Standardstock Tolerances
solution C, where L 1 is the label claim of amlodipine in For Tablets labeled to contain amlodipine/valsartan/
mg/Tablet, L 2 is the label claim of valsartan in mg/Tablet, hydrochlorothiazide, 5/160/12.5, 10/160/12.5,
5/160/25, and 10/160/25 (mg/mg/mg): NLT 75%

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USP 43 OfficialMonographs / Amlodipine 283

(Q) of the labeled amount of amlodipine Cu = nominal concentration of amlodipine in Sample

(CzoHzsCINzOs) isdissolved, NLT 80% (Q) of the labeled solution A (mg/mL)
amount of valsartan(Cz4Hz9Ns03) is dissolved, and NLT M rl = molecular weight of amlodipine related
80% (Q) of the labeled amount of hydrochlorothiazide compound Afree base, 406.86
(C7HsCIN304Sz) is dissolved. M r2 = molecular weight of amlodipine related
For Tablets labeled to contain amlodipine/valsartan/ compound A fumarate, 522.93
hydrochlorothiazide, 5/160/25, and 10/320/25 (mg/
mg/mg): NLT 70% (Q) of the labeled amount of Calculate the percentage of any valsartan related
amlodipine (CzoHzsCINzOs) isdissolved, NLT 80% (Q) of degradation product in the portion of Tablets taken:
the labeled amount of valsartan (Cz4Hz9Ns03) is Result = (r vir s) x (C siCv) x 100
dissolved, and NLT 80% (Q) of the labeled amount of
hydrochlorothiazide (C7HgCIN304Sz) is dissolved. ru = peak response of any valsartan related
• UNIFORMITY OF DOSAGE UNITS (905): Meet the degradation product from Sample solution B
requirements rs = peak response of valsartan from the Standard
IMPURITIES solution
• ORGANIC IMPURITIES Cs = concentration of USP Valsartan RS in the Standard
Use amber glasswarefor all solutions containing drug solution (mg/mL)
substances. Cu = nominal concentration of valsartan in Sample
Mobile phase, Diluent, Sample solution A, Sample solution B (mg/mL)
solution B, Sample solution C, and Chromatographic
system: Proceed as directed in the Assay. Calculate the percentage of benzothiadiazine related
System suitability solution: 0.02 mg/mL each of USP compound Ain the portion of Tablets taken:
Benzothiadiazine Related Compound A RS and USP Result =(r vir s) x (C siCv) x 100
Valsartan Related Compound B RS, 0.005 mg/mL of USP
Amlodipine Related Compound A RS, 0.14 mg/mL of USP rv = peak response of benzothiadiazine related
Amlodipine Besylate RS, 0.064 mg/mL of USP Valsartan compound A from Sample solution C
RS,. and 0.025 mg/mL of USP Hydrochlorothiazide RS in rs = peak response of benzothiadiazine related
Diluent compound A from the Standard solution .
Sensitivity solution: 0.14 IJg/mL of USP Amlodipine Cs =concentration of USP Benzothiadiazine Related
Besylate RS, 0.064 IJg/mL of USP Valsartan RS, and 0.025 Compound A RS in the Standard solution
IJg/mL of USP Hydrochlorothiazide RS in Diluent (mg/mL)
Standard solution: 0.0005 mg/ml of USP Amlodipine Cu = nominal concentration of hydrochlorothiazide in
Related Compound A RS, 0.0001 mg/mL of USP Sample solution C (mg/mL)
Benzothiadiazine Related Compound A RS, 0.0003 mg/mL
of USP Amlodipine Besylate RS, 0.00015 mg/mL of USP Calculate the percentage of chlorothiazide and
Valsartan RS, and 0.00005 mg/mL of USP hydrochlorothiazidedimer inthe portion ofTablets taken:
Hydrochlorothiazide RS in Diluent
System suitability . Result = (r vir s) x (C siCv) x 100
Samples: System suitability solution, Sensitivity solution, and
Standard solution ru = peak response of chlorothiazide or
Suitability requirements hydrochlorothiazidedimer from Sample solution
Resolution: NLT 2.0 between any adjacent peaks of C
benzothiadiazine related compound A, . rs = peak response of hydrochlorothiazide from the
hydrochlorothiazide, amlodipine related compound A, Standard solution
amlodipine, valsartan related compound B, and Cs =concentration of USP Hydrochlorothiazide RS in
valsartan, System suitability solution the Standard solution (mg/mL)
Relative standard deviation: NMT 5.0% for amlodipine Cu = nominal concentration of hydrochlorothiazide in
related compound A, benzothiadiazine related Sample solution C (mg/mL)
compound A, amlodipine, valsartan, and
hydrochlorothiazide, Standard solution Calculate the percentage of each unspecified degradation
Signal-to-noise ratio: NLT 10 for amlodipine, valsartan, product in the portion of Tablets taken:
and hydrochlorothiazide, Sensitivity solution
Analysis Result = (r vir s) x (C siCv) x (M rtiM r2) X 100
Samples: Sample solution A, Sample solution B, Sample
solution C, and Standard solution ru = peak response of each unspecified degradation
Calculate the percentage of amlodipine related compound product from Sample solution A
A in the portion of Tablets taken: rs = peak response of amlodipine from the Standard
solution
Result = (r vir s) x (C siCv) x (M rtiM r2) X 100 Cs =concentration of USP Amlodipine Besylate RS in
the Standard solution (mg/mL)
= peak response of amlodipine related compound Cu = nominal concentration of amlodipine in Sample
Afrom Sample solution A solution A (mg/mL)
= peak response of amlodipine related compound M rl = molecular weight of amlodipine, 408.88
Afrom the Standard solution M r2 = molecularweight of amlodipine besylate, 567.05
= concentration of USP Amlodipine Related
Compound A RS in the Standard solution Acceptance criteria: See Table 6. Disregard the amlodipine
(mg/mL) ethyl analog peak, the valsartan related compound Bpeak,
and any peaks below 0.1 %.

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 284 Amlodipine / Official Monographs USP 43

Table 6 for complete dissolution. Pass through a suitable filter of

Relative Acceptance 0,45-J.lm pore size.
Retention Criteria, Chromatographic system
Name . Time NMT (%) (See Chromatography (621), System Suitability.)
Benzothiadiazine related Mode: LC
compound As 0.60 1.0 Detector: UV 230 nm
Column: 4.6-mm x 25-cm; 5-J.lm packing L40
Chlorothlazlde'' 0.62 0.50
Temperatures
Hydrochlorothiazide 0.64 - Autosampler: 10°
Devaleryl valsartan- 0.71 0.2
Column: 30°
Flow rate: 0.8 mL/min
Hydrochlorothiazide dirner" 0.89 0.50 Injection volume: 20 J.lL
Amlodipine related
Run time: NLT 3.5 times the retention time of valsartan
compound Ae 0.96 0.5 related compound A
System suitability
Amlodipine 1.00 - Samples: System suitability solution and Standardsolution
Valsartan related [NoTE-The relative retention times of valsartan related
degradation compound Aand valsartan are about 0.65 and 1.0,
product l' 1.04 0.2 respectively.]
Amlodipine ethyl Suitability requirements
analog9 1.08 - Resolution: NLT 2.0 between valsartan and valsartan
related compound A, System suitability solution
Valsartan related Relative standard deviation: NMT 5.0% for valsartan
compound Bh 1.22 -
related compound A, Standard solution
Valsartan related Analysis
degradation Samples: Standardsolution and Sample solution
product 2' 1.27 0.2
Calculate the percentage of the valsartan related
Valsartan 1.36 - compound A in the portion of Tablets taken:
Valsartan related
degradation Result = (r ulr s) x (C sIC u) x 100
product 3' 1.51 0.2
= peak response of valsartan related compound A
Valsartan related from the Sample solution
degradation ,
product 4' 1.62 0.2 = peak response of valsartan related compound A
from the Standard solution
Anyother unspecified
degradation product! - 0.2
=concentration of USP Valsartan Related
Compound A RS in the Standardsolution
Total degradation (mg/mL)
-
products 2.0 Cu =nominal concentration of valsartan in the Sample
solution (mg/mL)
a 4-Amino-6-chloro-1,3-benzenedisulfonamide.
b 6-Chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide l,l-dioxide. Acceptance criteria: NMT 1.0 %
c N-([2'-(1 H-Tetrazole-5-yl)biphenyl-4-yl]methyl}-L-valine.
d 6-Chloro-N-[( 6-chloro-7-sulfamoyl-2,3-dihydro-4H-1,2,4-benzothiadiazine- ADDITIONAL REQUIREMENTS
4-yll,l-dioxide)methyl]3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide • PACKAGING AND STORAGE: Store at controlled room
l,l-dioxide. temperature in tight containers in a dry place.
e 3-Ethyl 5-methyl [2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-
3,5-pyridinedicarboxylate]. • LABELING: When more than one Dissolution test isgiven, the
fThese are specified unidentifieddegradation products. No informationis labeling states the Dissolution test used only if Test 7 is not
availableabout chemicalstructures or chemicalnames for these impurities. used.
9 Diethyl 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-l,4- • USP REFERENCE STANDARDS (11)
dihydropyridine-3,5-dicarboxylate. Processrelated impuritygivenfor USP Amlodipine Besylate RS
informationonly.
h N-Butyryl-N-([2'-(1 H-tetrazole-5-yl)biphenyl-4-yl]-methyl}-L-valine. Process
USP Amlodipine Related Compound A RS
related impuritygiven for information only. 3-Ethyl 5-methyl [2-(2-aminoethoxymethyl)-4-(2-
i Benzenesulfonic acid isthe counter ion to the amlodipine,and peaksat RRT of chlorophenyl)-6-methyl-3,5-pyridinedicarboxylate]
0.33 and 0.42 are not considered as degradation products. fumarate.
CZOHZ3CINzOs . C4H404 522.93
• LIMIT OF VALSARTAN RELATED COMPOUND A USP Benzothiadiazine Related Compound A RS
[Nors-Valsartan related compound A is a process 4-Amino-6-chloro-l,3-benzenedisulfonamide.
impurity and a formulation specific degradation C6HsCIN304SZ 285.73
product.] USP Hydrochlorothiazide RS
Mobile phase: n-Hexane, 2-propanol, and trifluoroacetic USP Valsartan RS
acid (850:150:1) USP Valsartan Related Compound A RS
System suitability solution: 0.04 mg/mL each of USP N-Valeryl-N-{[2'-(1 H-tetrazole-5-yl)biphenyl-4-yl]
Valsartan Related Compound A and USP Valsartan RS in methyl}-o-valine.
Mobile phase CZ4Hz9Ns03 435.52
Standard solution: 0.001 mg/mL of USP Valsartan Related USP Valsartan Related Compound B RS
Compound A RS in Mobilephase
Sample solution: Nominally 0.5 mg/mL of valsartan in
Mobilephase from a suitable amount of finely crushed
powder from NLT 20 Tablets. Sonication may be necessary

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 USP 43 OfficialMonographs / Amlodipine 285

Application volume: 10 IJL.

Amlodipine Besylate Developing solvent system-Use the upper layer of a mixture of
methyl isobutyl ketone, water, and glacial acetic acid
(50:25:25).
Procedure-Proceed as directed for Thin-Layer
Chromatography under Chromatography (621). Dry the plate
for 15 minutes at 80 0 • Examinethe plate under UV light at 254
nm and 365 nm. The chromatogram from the System
suitability solution shows two clearly separated minor spots
with RFvalues of about 0.18 and 0.22. Compare the intensities
CzoHzsCINzOs' C6 H60 3S 567.05 of any secondary spots observed in the chromatogram of the
3,5-Pyridinedicarboxylic acid, 2-[(2-aminoethoxy)methyl]-4- Test solution with those of the principal spots in the
(2-chlorophenyl)-1,4-dihydro-6-methyl-, 3-ethyl 5-methyl chromatograms of the Standardsolutions. Any spot obtained
ester, (±)-, monobenzenesulfonate. from the Test solution, except for the principal spot, is not
3-Ethyl 5-methyl (±)-2-[(2-aminoethoxy)methyl]-4-(0- greater in size than the spot obtained from Standardsolution
chlorophenyl)-1,4-dihydro-6-methyl-3,5- 7 (0.3%), and at most two spots are more intense than the
pyridinedicarboxylate, monobenzenesulfonate [111470- spot obtained from Standardsolution 2 (0.1%).
99-6]. TEST 2-
Monohydrate 585.07 pH 3.0 Buffer and Mobilephase-Prepare as directed in the
Assay.
» Amlodipine Besylate is anhydrous or hydrated System SUitability solution-Dissolve about 5 mg of Amlodipine
and contains not less than 97.0 percent and not Besylatein 5 mL of hydrogen peroxide, and heat at 70 0 for 45
minutes.
more than 102.0 percent of C2oH2SCIN20S . Standardsolution-Dissolve an accurately weighed quantity of
C6 H6 0 3 S, calculated on the anhydrous basis. USP Amlodipine Besylate RS in Mobilephase to obtain a
solution having a known concentration of about 0.003 mg per
Packaging and storage-Preserve in tight containers, mL.
protected from light. Store at room temperature. Test solution-Transfer about 50 mg of Amlodipine Besylate,
USP Reference standards (11)- accurately weighed, to a 50-mL volumetric flask, dissolve in
USP Amlodipine Besylate RS and dilute with Mobilephase to volume, and mix.
Labeling-Where it is the hydrated form, the label so Chromatographic system (see Chromatography (621 »-
indicates. Prepare as directed in the Assay. Chromatograph the System
suitability solution, and record the peak responses as directed
Identification- I
for Procedure: the resolution, R, between amlodipine impurity
A and amlodipine is not less than 4.5.
[NOTE-For the purpose of identification, the relative
retention times are about 0.2 for benzene sulfonate, 0.5
for amlodipine impurity A, and 1.0 for amlodipine.
Amlodipine impurity A is 3-ethyl 5-methyl
of major peak in the 2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-
chromatogram of the Assay preparation corresponds to that in 6-methylpyridine-3,5-dicarboxylate.]
the chromatogram of the Standardpreparation, as obtained Chromatograph the Standardsolution, and record the peak
in the Assay. responses asdirected for Procedure: the standard deviation for
Optical rotation (781A): between -0.10 0 and +0.10 0 , replicate lnjections is not more than 10.0%.
measured at 20 0 • •
Procedure-Separately inject equal volumes (about 10 IJL) of
Test solution: 10 mg per mL, in methanol. the Standardsolution and the Test solution into the
Water Determination, Method I (921): not more than 0.5% chromatograph, record the chromatograms for a period of
for the anhydrous form. If labeled as the hydrated form, the time that is about 3 times the retention time of amlodipine,
limit is between 3.1% and 5.0%. and measure the peak responses. Calculate the percentage of
Residue on ignition (281): not more than 0.2%. each impurity in the portion of Amlodipine Besylatetaken by
Related compounds- the formula:
TEST 1-
Adsorbent: 0.25-mm layer of chromatographic silica gel 100(11f)(C sIC T)(r;lr s)
mixture.
Test solution-Transfer 140 mg of Amlodipine Besylateto a in which Fis the relative responsefactor, which is equal to 0.5
2-mL volumetric flask, dissolve in and dilute with methanol to for amlodipine impurity A and to 1.0 for other impurities; C s
volume, and mix. and C T are the concentrations, in mg per mL, of amlodipine
System suitability solution-Transfer about 14 mg of USP besylate in the Standardsolution and the Test solution,
Amlodipine Besylate RS to a suitable container, dissolve in 0.2 respectively; r i is the peak responsefor each impurity obtained -
mL of methanol, and mix. from the' Test solution: and r s is the peak response for
Standardstocksolution-Dissolve an accurately weighed amlodipine besylate obtained from the Standardsolution: not
quantity of USP Amlodipine BesylateRS in methanol to obtain more than 0.3% of amlodipine impurity A is found, and not
a solution containing 7.0 mg per mL. . more than 0.3% of total other impurities is found. Disregard
Standardsolution 1-Transfer 3.0 mL of the Standardstock any peak less than 0.03%, and disregard any peak due to
solution to a 1OO-mL volumetric flask, dilute with methanol to benzene sulfonate. .
volume, and mix.
Standardsolution 2-Transfer 1.0 mL of the Standardstock
solution to another 1OO-mL volumetric flask, dilute with
methanol to volume, and mix.

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286 Amlodipine / Official Monographs USP43

Assay- Standard solution: 0.0275 mg/mL of USP Amlodipine

pH 3.0 Buffer-Dissolve 7.0 mL of triethylamine in 800 mL Besylate RS and 0.0025 mg/mL of USP Amlodipine Related
of water. Adjust with phosphoric acid to a pH of 3.0 ± 0.1, and Compound A RS in Mobilephase
dilute with water to 1 L. Sample solution: Nominally 0.02 mg/mL of amlodipine in
Mobilephase-Prepare a filtered and degassed mixture of Mobilephase prepared as follows. Place NLT 5 Tablets in a
pH 3.0 Buffer, methanol, and acetonitrile (50:35:15). Make suitable volumetric flask, and add sufficient quantity of
adjustments if necessary(see System Suitability under Mobilephaseto disintegrate the Tablets. Shake for 30 min,
Chromatography (621 »). and dilute with Mobilephase to volume. Pass the sample
Standardpreparation-Dissolve an accurately weighed through a syringe tip filter of 0.45-~m pore size. Discard the
quantity of USP Amlodipine Besylate RS in Mobilephase to first few mL of the filtrate.
obtain a solution having a known concentration of about 0.05 Chromatographic system
mg per mL. (See Chromatography (621), System Suitability.)
Assay preparation-Transfer about50 mg of Amlodipine Mode: LC
Besylate, accurately weighed, to a 50-mL volumetric flask, Detector: UV 237 nm
dissolve in and dilute with Mobilephase to volume, and mix. Column: 3.9-mm x 15-cm; 5-~m packing L1
Transfer 5.0 mL of this solution to a 1OO-mL volumetric flask, Flow rate: 1 mL/min
dilute with Mobilephase to volume, andrnlx. Injection volume: 50 ~L
Chromatographic system (see Chromatography (621»)-The Run time: NLT 3 times the retention of the amlodipine
liquid chromatograph is equipped with a 237-nm detector peak
and a 3.9-mm x 15-cm column that contains packing L1. The System suitability
flow rate is about 1.0 mL per minute. Chromatograph the Sample: Standard solution
Standard preparation, and record the peak responses as [NOTE-The relative retention times for amlodipine and
directed for Procedure: the standard deviation for replicate amlodipine related compound A are about 1.0 and
injections is not more than 2.0%. 0.5 respectively.]
Procedure-Separately-inject equal volumes (about 10 ~L) Suitability requirements
of the Standard preparation and the Assay preparation into the Resolution: NLT 8.5 between amlodipine and
chromatograph, record the chromatograms, and measure the amlodipine related compound A
responses for the major peaks. Calculate the percentage of Tailing factor: NMT 2.0 for both amlodipine and
CzoHzsCINzOs . C6H6 0 3S in the portion of Amlodipine Besylate amlodipine related compound A
taken by the formula: Relative standard deviation: NMT 5.0% for amlodipine
related compound A
1OO(C siC v)(r vir s) Analysis
Samples: Standardsolution and Sample solution
in which C sand 'C u are the concentrations, in mg per mL, of Calculate the percentage of the labeled amount of
amlodipine besylate in the Standard preparation and the Assay amlodipine (CzoHzsCINzOs) in the portion of Tablets
preparation, respectively; and r u and r sare the peak responses taken:
obtained from the Assay preparation and the Standard
preparation, respectively. Result = (r vir s) x (C siC v) x (M ,tiM (2) x 100

ru = peak response from the Sample solution

rs = peak response from the Standard solution
Cs = concentration of USP Amlodipine Besylate RS in
Amlodipine Besylate Tablets the Standard solution(mg/mL)
Cu = nominal concentration of amlodipine in the
DEfiNITION Sample solution(mg/mL)
Amlodipine Besylate Tablets contain NLT 90% and NMT M ,1 = molecular weight of amlodipine, 408.88
110% of the labeled amount of amlodipine (CzoHzsCINzOs)' _M ,2 = molecular weight of amlodipine besylate, 567.05
IDENTIFICATION
Acceptance criteria: 900/0-110% of the labeled amount of
amlodipine (CzoHzsCINzOs) .
PERFORMANCE TESTS
• A~~I~i~~!
$pg<;:Ar;o§b9P'/i; (»
• DISSOLUTION (711)
[NOTE-Do not expose any of the solutions to stainless
Standard solution and Sample solution: Prepare as steel because of the degradation of amlodipine.]
directed in the test for Dissolution. Medium: 0.01 N hydrochloric acid; 500 mL
Acceptance criteria: Meet the requirements Apparatus 2: 75 rpm. [NoTE-Use paddles covered with
• B. The retention time of the major peak of the Sample Teflon or made of any inert material except stainless
solution corresponds to that of the Standard solution, as steel.]
obtained in the Assay. Time: 30 min
ASSAY Standard solution: Make appropriate dilutions of USP
• PROCEDURE Amlodipine Besylate RS in Medium to obtain the following
Buffer: Add 7.0 mL of triethylamine into a 1OOO-mL flask concentrations: 0.00695 mg/mL for Tablets labeled to
containing 900 mL of water. Adjust the solution with contain 2.5 mg, 0.0139 mg/mL for Tablets labeled to
phosphoric acid to a pH of 3.0 ± 0.1. Dilute with water to contain 5 mg, and 0.0278 mg/mL for Tablets labeled to
volume, and mix well. contain 10 mg. These solutions are stable for 1 day.
Mobile phase: Methanol, acetonitrile, and Buffer Sample solution: Pass a portion of the solution under test
(35:15:50) through a suitable filter of 0.45-~m pore size.

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USP 43 Official Monographs / Ammonia 287

Analysis ru = peak response of the amlodipine glucosel

Samples: Standard solution and Sample solution galactose adduct, amlodipine lactose adduct, or
Determine the amount of amlodipine (CzoHzsCINzOs) . any unspecified degradation product from the
dissolved by using UV absorption at the wavelength of Sample solution
maximum absorbance at about 239 nm on portions of the rs = peak response of amlodipine from the Standard
Sample solution in comparison with the Standard solution, solution
using a l-cm quartz cell and the Medium as the blank. Cs = concentration of USP Amlodipine Besylate RS
Calculate the percentage of the labeled amount of from the Standard solution (mg/mL)
amlodipine (CzoHzsCINzOs) dissolved: Cv = nominal concentration of amlodiplne in the
Sample solution (mg/mL)
Result =(A viA s) x C s x 0 x (M ,tiM '2) x VX (1IL) x 100 Mr = molecular weight of amlodipine, 408.9
M ,2 = molecular weight of amlodipine besylate, 567.05
Au = absorbance of the Sample solution
As =absorbance of the Standard solution Acceptance criteria: See Table 1.
Cs = concentration of the Standard solution (mg/mL)
o =dilution factor of the Sample solution Table 1
M ,1 = molecular weight of amlodipine, 408.88 Relative Acceptance
M ,2 = molecular weight of amlodipine besylate, 567.05 Name
Retention
Time
Criteria,
NMT(%)
V = volume of Medium, 500 mL
L = label claim (mg/Tablet) Amlodipine related com-
pound A" 0.50 1.0
Tolerances: NLT 75% (Q) of the labeled amount of Amlodipine lactose adduct" 0.80 0.5
amlodipine (CzoHzsCINzOs) is dissolved.
• UNIFORMITY OF DOSAGE UNITS (90S): Meet the Amlodipine glucose/galac-
tose
requirements adduct" 0.90 0.5
IMPURITIES Arnlodlplne besylate 1.0 -
• ORGANIC IMPURITIES
Buffer, Mobile phase, Standard solution, Any unspecified degradation
product - 0.2
Chromatographic system, and System suitability:
Proceed as directed in the Assay. a 3-Ethyl 5-methyl [2-(Z-aminoethoxymethyl)-4-(Z-chlorophenyl)-6-methyl-
Sample solution: Place a suitable number of Tablets into a 3,5-pyridinedicarboxylate] fumarate. .
25-mL volumetric flask to obtain a solution with a final b Formulation-specific impurities.
nominal concentration of 0.4 mg/mL of amlodipine. Add
about 10 mL of Mobile phase to the flask. Swirl to ADDITIONAL REQUIREMENTS
disintegrate the Tablets, then sonicate for 5 min to • PACKAGING AND STORAGE: Preserve in tight, light-resistant
completely dissolve, and cool the sample to room containers. Store at controlled room temperature.
temperature. Dilute with Mobile phase to volume. Stir for • USP REFERENCE STANDARDS (11)
an additional 15 min using a magnetic stir bar, and passthe USPAmlodipine Besylate RS
sample through a syringe tip filter of 0.45-J.lm pore size, USPAmlodipine Related Compound A RS
discarding the first 5 mL. 3-Ethyl 5-methyl [2-(2-aminoethoxymethyl)-4-(2-
Analysis chlorophenyl)-6-methyl-3,5-pyridinedicarboxylate]
Samples: Standard solution and Sample solution fumarate.
Calculate the percentage of amlodipine related compound CZOHZ3CINzOs' C4H4 0 4 522.93
A in the portion of Tablets taken:

Result = (r vir s) x (C siCv) x (M ,tiM '2) x 100

ru =peak response of amlodipine related compound Aromatic Ammonia. Spirit
A from the Sample solution
rs =peak response of amlodipine related compound DEFINITION
A from the Standard solution - Aromatic Ammonia Spirit is a hydroalcoholic solution that
Cs =concentration of USP Amlodipine Related contains, in each 100 mL, NLT 1.7 g and NMT 2.1 g of total
Compound A RS in the Standard solution ammonia (NH 3) and Ammonium Carbonate corresponding
(mg/mL) to NLT 3.5 g and NMT 4.5 g of ammonium carbonate
Cu =nominal concentration of amlodipine in the [(NH 4)zC0 3] ·
Sample solution (mg/mL)
M r = molecular weight of amlodipine related ASSAY
compound A, 406.86 • TOTAL AMMONIA (NH 3)
M ,2 = molecular weight of amlodipine related Sample: 10.0 mL
compound A fumarate, 522.93 Titrimetric system
(See Titrimetry (541), Residual Titrations.)
Calculate the percentage of amlodipine glucoselgalactose Mode: Residual titration
adduct or amlodipine lactose adduct, if present, and any Titrant: 0.5 N sodium hydroxide VS
unspecified degradation product in the portion of Tablets Analysis: Transfer the Sample to a 250-mL conical flask
taken: containing 50 mL of water. Add 30.0 mL of 0.5 N sulfuric
acid VS,and boil until the solution becomes clear. Cool, add
Result = (r vir 5) x (C siCu) x (M ,dM '2) x 100 methyl red TS, and titrate the excess acid with Titrant.

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 288 Ammonia / Official Monographs USP 43

Perform a blank determination. Each mL of 0.5 N sulfuric

acid is equivalentto 8.515 mg of ammonia (NH 3). Ammonium Chloride Injection
• AMMONIUM CARBONATE
Sample: 10.0 mL »Ammonium Chloride Injection is a sterile solution
Titrimetric system
(See Titrimetry (541), Residual Titrations.)
of Ammonium Chloride in Water for Injection. It
Mode: Residual titration contains not less than 95.0 percent and not more
Titrant: 0.5 N sulfuric acid VS than 105.0 percent of the labeled amount of
Analysis: Transfer the Sample to a 300-mL flask. Add 30 mL NH 4CI. Hydrochloric acid may be added to adjust
of 0.5 N sodium hydroxide, and boil the mixture, replacing
the water lost by evaporation, until the vapors no longer
the pH.
turn moistened red litmus paper blue. Cool, dilute with 100 Packaging and storage-Preserve in single-dose or
mL of cold, carbon dioxide-free water, add 6 drops of
multiple-dose containers, preferably of Type I or Type" glass.
phenolphthalein TS, then add just enough 0.5 N sulfuric labeling-The label states the content of ammonium
acid VS to discharge the color of the phenolphthalein. Add chloride in terms of weight and· of milliequivalents in a given
methyl orange TS, and titrate with Titrant. Perform a blank volume. The label states also the total osmolar concentration
determination. Each mL of Titrant consumed in the titration in mOsmol per L or per mL. The label states that the Injection
with methyl orange TS is equivalent to 48.04 mg of is not for direct injection but is to be diluted with Sodium
ammonium carbonate [(NH4)2C03]' Chloride Injection to the appropriate strength before use.
Acceptance criteria: In each 100 mL, NLT 1.7 g and NMT Identification-It responds to the tests for Ammonium (191)
2.1 g of total ammonia (NH 3) and Ammonium Carbonate and for Chloride (191).
corresponding to NLT 3.5 g and NMT 4.5 g of ammonium Bacterial Endotoxins Test (85) -It contains not more than
carbonate [(NH4)2C03] 1.72 USP Endotoxin Units per mEq of chloride.
OTHER COMPONENTS
pH (791): between 4.0 and 6.0, in a concentration of not
more than 100 mg of ammonium chloride per mL.
• ALCOHOL DETERMINATION, Method 1(611): 62.0%-68.0%
Particulate Matter in Injedions (788): meets the
ADDITIONAL REQUIREMENTS .requirements for small-volume injections.
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Chloride content-Transfer an accurately measured volume
containers at a temperature not exceeding 30°. of Injection, evaporated, if necessary, equivalent to about 2 g
of ammonium chloride, to a 200-mL volumetric flask, dilute
with water to volume, and mix. Transfer 10.0 mL of this
solution to a conical flask, add 10 mL of glacial acetic acid, 75
mL of methanol, and 0.5 mL of eosin Y TS. Titrate, with
Ammonium Chloride shaking, with 0.1 N silver nitrate VS to a pink endpoint.· Each
mL of 0.1 N silver nitrate is equivalent to 3.545 mg of CI. The
content of CI is between 63.0% and 70.3% of the labeled
NH 4C1 53.49
amount of ammonium chloride.
Ammonium chloride. Other requirements-It meets the requirements under
Ammonium chloride [12125-02-9]. Injections and Implanted Drug Products (1 ).
Assay-Transfer an accurately measured volume of Injection,
»Ammonium Chloride contains not less than 99.5 equivalent to about 200 mg of ammonium chloride, to a
percent and not more than 100.5 percent of 500-mL Kjeldahl flask, dilute with water to 200 mL, mix, and
NH 4CI, calculated on the dried basis. add 50 mL of sodium hydroxide solution (2 in 5). Immediately
connect the flask by means of a distillation trap to a
Packaging and storage-Preserve in tight containers. well-cooled condenser, the delivery tube of which dips into 40
Identification-A solution (1 in 10) responds to the tests for mL of boric acid solution (1 in 25) contained in a suitable
Ammonium (191) and for Chloride (191). receiver. Heat to boiling, and distill about 200 mL. Cool the
pH (791): between 4.6 and 6.0, in a solution (1 in 20). liquid in the receiver, if necessary, then add methyl red TS, and
Loss on drying (731 )-Dry it over silica gel for 4 hours: it loses titrate with 0.1 N sulfuric acid VS. Perform a blank
not more than 0.5% of its weight. determination, and make any necessary correction. Each mL
Residue on ignition (281 )-Add 1 mL of sulfuric acid to about of 0.1 N sulfuric acid is equivalent to 5.349 mg of NH 4CI.
2 g, accurately weighed, and heat the mixture gently until
volatilization is complete: the residue is white, and when
ignited, not more than 0.1 % of nonvolatile substance remains.
limit of thiocyanate-Acidify 10 mL of a solution (1 in 10)
with hydrochloric acid, and add a few drops of ferric chloride Ammonium Chloride Delayed-Release
TS: no orange-red color is produced. Tablets
Assay-Transfer about 100 mg of Ammonium Chloride,
accurately weighed, to a conical flask, add 10 mL of water, and
swirl to dissolve. Add 10 mL of glacial acetic acid, 75 mL of » Ammonium Chloride Delayed-Release Tablets
methanol, and 0.5 mL of eosin YTS. Titrate, with shaking, with contain not less than 94.0 percent and not more
0.1 N silver nitrate VS to a pink endpoint. Each mL of 0.1 N than 106.0 percent of the labeled amount of
silver nitrate is equivalent to 5.349 mg of NH 4CI.
NH 4CI. Ammonium Chloride Delayed-Release
Tablets are enteric-coated.
Packaging and storage-Preserve in tight containers.
Identification-A filtered solution of finely powdered
Tablets, equivalent to ammonium chloride solution (1 in 10),

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 USP43 OfficialMonographs / Ammonium 289

responds to the tests for Ammonium (191) and for Chloride Standardsolutions-Transfer 0.25, 0.50, 1.0, and 3.5 mL of
(191 ). StandardMercurySolution to four separate glass-stoppered
Disintegration (701): 2 hours, determined as directed for bottles, such as biological oxygen-demand bottles, of about
Enteric-Coated Tablets. 300-mL capacity. Dilute the contents of each bottle with water
limit of thiocyanate-Powder and dissolve in water a to 100 mL, and mix. Thesesolutions contain the equivalent of
sufficient number of Tablets to make about 25 mL of 2.5, 5.0, 10.0, and 35.0 ng of mercury per mL, respectively.
ammonium chloride solution (1 in 10), and filter. Acidify 10 Test solution-Transfer about 1.000 g of Ferric Ammonium
mL of the solution with hydrochloric acid, and add a few drops Citrate, accurately weighed, to a 200-mL centrifuge bottle
of ferric chloride TS: no reddish orange color is produced. with a polytef-Iined screw cap, and add 5 mL of nitric acid and
Assay-Weigh and finely powder not fewer than 20 Tablets. 5 mL of hydrochloric acid. Close the bottle tightly, digest on
Transfer an accurately weighed portion of the powder, a steam bath for 1 hour, and cool. Quantitatively transfer the
equivalent to about 200 mg of ammonium chloride, to a solution to a suitable glass-stoppered bottle, dilute with water
500-mL Kjeldahl flask, and add 200 mL of water and 50 mL of to 100 mL, and bubble air through the solution for 2 minutes.
sodium hydroxide solution (2 in 5). Immediately connect the Prepare a reagent blank in the same manner.
flask by means of a distillation trap to a well-cooled condenser, Procedure-Add 5 mL of Stannous Chloride Solution to each
the delivery tube of which dips into 40 mL of boric acid solution, and immediately insert the bubbler of the Aeration
solution (1 in 25) contained in a suitable receiver. Heat to Apparatus. Obtain the absorbances as directed by the
boiling, and distill about 200 mL. Cool the liquid in the instrument manufacturer's operating instructions. Perform a
receiver, if necessary, then add methyl red TS, and titrate with blank determination, and make any necessary correction. Plot
0.1 N sulfuric acid VS. Perform a blank determination, and the absorbances of the Standard solutions versus
make any necessary correction. Each mL of 0.1 N sulfuric acid concentrations, in IJg per mL, of mercury, and draw the
is equivalent to 5.349 mg of NH 4CI. straight line best fitting the plotted points. From the graph so
obtained, determine the concentration, in IJg per g, of
mercury in the Test solution: not more than 10 IJg per g is
found.
limit of lead-
Ferric Ammonium Citrate Standardstock solution-Dissolve about 159.8 mg of lead
nitrate, accurately weighed, in 100 mL of water containing 1
» Ferric Ammonium Citrate contains not less than mL of nitric acid. Dilute with water to 1000.0 mL, and mix.
16.5 percent and not more than 18.5 percent of Standardsolution- [NOTE-Preparethis solution on the day
of use.] Transfer 10.0 mL of Standard stock solution to a 500,.mL
iron (Fe). volumetric flask, dilute with water to volume, and mix. Each
mL contains the equivalent of 2 IJg of lead (Pb).
Packaging and storage-Preserve in tight, light-resistant Test solution-Transfer about 15 g of Ferric Ammonium
containers, in a cool place. Citrate, accurately weighed, to a 1OO-mL volumetric flask
Identification- (previously rinsed with nitric acid and water), dissolve in a
A: Ignite about 0.5 g: it chars, and leavesa residue of iron mixture of 50 mL of water and 1 mL of nitric acid, dilute with
oxide. water to volume, and mix.
8: To 10 mL of a solution of Ferric Ammonium Citrate (1 in Procedure-Using a suitable atomic absorption
100) add 6 N ammonium hydroxide dropwise: the solution spectrophotometer (see AtomicAbsorption Spectroscopy (852»
darkens, but no precipitate forms. . equipped with a deuterium arc background corrector, a digital
C: To 5 mL of a solution of Ferric Ammonium Citrate (1 in readout device, and a burner head capable of handling 15%
100) add 0.3 mL of potassium permanganate TS and 4 mL of solids content, perform a blank determination with water,
mercuric sulfate TS, and heat the mixture to boiling: a white following the manufacturer's operating instructions.
precipitate forms. Separately aspirate portions of the Standard solution and the
Ferric citrate-To a solution of Ferric Ammonium Citrate (1 Test solution, and record the absorbances. Calculate the lead
in 100) add potassium ferrocyanide TS: no blue precipitate is content, in IJg per g, in the portion of Ferric Ammonium
formed. Citrate taken by the formula:
Sulfate (221)-Dissolve 100 mg in 1 mL of 2.7 N hydrochloric
acid, and dilute with water to 30 mL. Add 3 mL of barium 1OO( C/\tV)(A u/ As)
chloride TS, dilute with water to 50 mL, and mix: any turbidity
formed after 10 minutes is not greater than that produced in in which C is the concentration, in IJg per mL, of lead in the
a similarly treated control solution containing 0.31 mL of Standardsolution; W is the weight, in g, of Ferric Ammonium
0.020 N sulfuric acid (0.3%). Citrate taken; and Au and As are the absorbances of the Test
Oxalate-Transfer 1 g to a 125-mL separator, dissolve in 10 solution and the Standardsolution, respectively: not more than
I!1Lof water, add 2 mL of hydrochloric acid, and extract 10 IJg per g is found.
successively with one 50-mL portion and one 20-mL portion Assay-Transfer about 1 g of Ferric Ammonium Citrate,
of ether. Transfer the combined ether extracts to a 150-mL accurately weighed, to a 250-mL conical flask, and dissolve in
beaker, add 10 mL of water, and remove the ether by 25 mL of water and 5 mL of hydrochloric acid. Add 4 g of
evaporation on a steam bath. Add 1 drop of glacial acetic acid potassium iodide, insert the stopper, and allow to stand
and 1 mL of calcium acetate solution (1 in 20): no turbidity is protected from light for 15 minutes. Add 100 mL of water, and
produced within 5 minutes. titrate the liberated iodine with 0.1 N sodium thiosulfate VS,
using starch TS as the indicator. Perform a blank
Mercury- determination, and make any necessary correction. Each mL
MercuryStock Solution and Standard MercurySolution- of 0.1 N sodium thiosulfate is equivalent to 5.585 mg of iron
Proceed as directed for Method I under Mercury(261). (Fe).
MercuryDetection Instrument, AerationApparatus,and
Stannous Chloride Solution-Proceed as directed for Method
lIa and Method lib under Mercury (261).

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 290 Ammonium / Official Monographs USP 43

4.0. Add saturated hexamethylenetetramine solution to

Ferric Ammonium Citrate for Oral achieve a pH of 5-6.
Solution Analysis: Heat the Sample solution to 60°, and add 0.2 mL
of 0.1% 4-[2-pyridylazo]resorcinol solution in alcohol.
Titrate with 0.1 M lead nitrate VSfrom the yellow color to
» Ferric Ammonium Citrate for Oral Solution the first permanent pink endpoint: Carry out ~ blank
contains Ferric Ammonium Citrate and an titration. Each mL of 0.1 M lead nitrate IS equivalent to
effervescent mixture of a suitable organic acid and 17.66 mg of (NH4)6Mo70z4 . 4HzO.
an alkali metal bicarbonate. It contains not less Acceptance criteria: 99.3%-101.8%
than 90.0 percent and not more than 110.0 IMPURITIES
percent of the labeled amount of Fe. It may contain INORGANIC IMPURITIES
one or more suitable flavors, colors, or stabilizing • Arsenate, Phosphate, and Silicate
Sample solution: Dissolve 2.5 g of the analyte in 70 mL of
agents. water in a container other than glass.
Control solution: Dissolve 0.5 g of the analyte in 70 mL of
Packaging and storage-Preserve in tight, light-resistant water in a container other than glass, and add an amount of
containers, and store in a cool place. sodium silicate solution equivalent to 0.02 mg of silica (SiOz).
Identification-A 6-g portion dissolves in 600 mL of water Analysis
with effervescence. The collected gas meets the requirements Samples: Sample solution and Controlsolution
of the test for Bicarbonate (191), and the resulting solution Adjust with 1.2 N hydrochloric acid to a pH of between 3
meets the requirements of the tests for Iron (191) and for and 4, transfer to a glass container, add 2 mL of bromine
Citrate (191). TS, and adjust with 1.2 N hydrochloric acid to a pH of 1.8
Uniformity of dosage units (905)- ± 0.1. Heat almost to boiling, and cool to room
FOR POWDER PACKAGED IN SINGLE-UNIT CONTAINERS: meets the temperature. Dilute with water to 90 mL, add 10 mL of
requirements. hydrochloric acid, and transfer to a separator. Add 1 mL
of butyl alcohol and 30 mL of 4-methyl-2-pentanone,
Deliverable volume (698)- 'shake vigorously, and allow the phases to separate.
FOR POWDER PACKAGED IN MULTIPLE-UNIT CONTAINERS: meets the Discard the aqueous phase, and wash the ketone phase
requirements. with three successive 1O-mL portions of 1.2 N
Assay-Transfer about 6 g of Ferric Ammonium Citrate for hydrochloric acid, discarding the washings. To the
Oral Solution, accurately weighed, to a 250-mL conical flask, washed ketone phase add 10 mL of 1.2 N hydrochloric
and dissolve in 100 mL of water. Allow the gas to escape, add acid to which has just been added 0.2 mL of a freshly
5 mL of hydrochl6ric acid and 4 g of potassium iodide, insert prepared solution (1 in 50) of stannous chloride in
the stopper, and allow to stand protected from light for 15 hydrochloric acid.
minutes. Add 25 mL of water, and titrate the liberated iodine Acceptance criteria: Any blue color in the Sample solution
with 0.1 N sodium thiosulfate VS, using starch TS as the does not exceed that in the Control solution (NMT 10 ppm).
indicator. Perform a blank determination, and make any • Chloride and Sulfate, Chloride (221 ): A0.5-g portion shows
necessary correction. Each mL of 0.1 N sodium thiosulfate is no more chloride than 0.30 mLof 0.001 N hydrochloric acid
equivalent to 5.585 mg of Fe. (NMT 20 ppm).
• Chloride and Sulfate, Sulfate (221): A0.25-g portion shows
no more sulfate than corresponds to 1.0 mL of 0.001 N
sulfuric acid (NMT 200 ppm).
• INSOLUBLE SUBSTANCES
Ammonium Molybdate Sample: 20 g
Analysis: Dissolve the Sample in 200 mLof water in a beaker,
(NH4)6M070Z4 ·4H zO 1236.00
heat to boiling, cover, and heat on a steam bath for 1 h.
Molybdate (M0 70 Z46-), hexaammonium, tetrahydrate; Pass the hot solution through a tared filtering crucible, wash
Hexaammonium molybdate tetrahydrate [12054-85-2]. the insoluble residue with hot water, and dry at 105° for
DEFINITION 2 h.
Ammonium Molybdate contains NLT99.3% and NMT - Acceptance criteria: The weight of the residue is NMT 1 mg
101.8% of (NH4)6M070z4 ·4H zO. (0.005%).
• NITRATE
,IDENTIFICATION Sample: 1 g
• PROCEDURE Analysis: Dissolve the Sample in 10 mL of water containing
Sample: 0.6 g 5 mg of sodium chloride, and add 0.10 mL of a solution (1
Analysis: Dissolve the Sample in 1.4 mLof water and 1.45 in 1000) of indigo carmine in 3.6 N sulfuric acid.
mL of ammonium hydroxide. Cool this mixture, and add Acceptance criteria: The blue color is not completely
slowly, with mixing, 7.2 mL of a well-cooled mixture of 3.2 discharged in 5 min.
mL of nitric acid and 4 mL of water. Allow to stand for 24- • MAGNESIUM AND ALKALI SALTS
48 h, and pass through a sintered-glass filter. To 5 mL of Sample: 5.0 g
the filtrate add 2 mL of dibasic sodium phosphate TS. Analysis: Dissolve the Sample in 50 mL of water, and filter.
Acceptance criteria: A yellow precipitate is formed, and it To the filtrate add 0.5 g of sodium carbonate and 25 mL of
is soluble in an excess of 6 N ammonium hydroxide. 2.5 N sodium hydroxide. Boil the solution gently for 5 min,
cool, and pass through an ignited and tared filter. Wash the
ASSAY
filter with 1 N ammonium hydroxide. Ignite the filter at 800
• PROCEDURE ± 25° for 30 min.
Sample solution: Dissolve 0.7 g of Ammonium Molybdate Acceptance criteria: The weight of the residue does not
in 100 mL water. Adjust with dilute nitric acid to a pH of exceed 1 mg (NMT 0.02%).

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 USP 43 OfficialMonographs / Amobarbital 291

• PHOSPHATE Sodium sulfate solution-Dissolve 1 g of sodium sulfate in

Standard solution: Dissolve 143.3 mg of dried monobasic water to make 100 mL.
potassium phosphate in water to make 1000 mL, and then Molybdenum stock solution-Transfer about 1.84 g of
dilute 1.0 mL of this solution with 3 N ammonium previously assayed Ammonium Molybdate, accurately
hydroxide to 100 mL. weighed, to a 1OOO-mL volumetric flask, dilute with
Sample solution: Dissolve 20 g of the analyte in 100 mL of Ammonium hydroxide diluent to volume, and mix. This solution
3 N ammonium hydroxide. contains the equivalent of 1000 ~g of molybdenum per mL.
Analysis Standard preparations-Transfer 0, 1.0, 2.0, 3.0, and 4.0
Samples: Standard solution and Sample solution mL, respectively, of Molybdenum stock solution to separate
Add 3.5 mL of ferric nitrate solution (1 in 10), and allow 1OO-mL volumetric flasks, and to the respective flasks add 5.0,
to stand for 15 min. Warm gently to coagulate the 4.0, 3.0, 2.0, and 1.0 mL of Ammonium hydroxide diluent. To
precipitate, and filter. Wash the filter several times with each flask add 10 mL of Sodium sulfate solution, dilute with
1.5 N ammonium hydroxide, then wash the filter with water to volume, and mix. These Standard preparations
60 mL of warm 4 N nitric acid to dissolve the residue on contain, respectively, 0, 10, 20, 30, and 40 ~g of molybdenum
the filter, collecting the filtrate in a glass-stoppered, per mL.
250-mL conical flask. Add 13 mL of ammonium Assay preparation-Transfer an accurately measured
hydroxide, warm to 40°, add 50 mL of ammonium volume of Injection, equivalent to about 500 ~g of
molybdate TS, shakefor 5 min, and allow to stand at 40° molybdenum, to a 25-mL volumetric flask, add 1.25 mL of
for 2 h. Ammonium hydroxide diluent and 2.5 mL of Sodium sulfate
Acceptance criteria: Any yellow precipitate formed from solution, dilute with water to volume, and mix.
the Sample solution does not exceed that from the Standard Procedure-Concomitantly determine the absorbances of
solution (5 ppm). the Standard preparations and the Assay preparation at the
molybdenum emission line of 313.3 nm with a suitable atomic
ADDITIONAL REQUIREMENTS absorption spectrophotometer (see AtomicAbsorption
• PACKAGING AND STORAGE: Preserve in tight containers. Spectroscopy (852») equipped with a molybdenum
hollow-cathode lamp and a nitrous oxide-acetylene reducing
flame, using water as the blank. Plot the absorbances of the
Standard preparations versus concentration, in ~g per mL, of
molybdenum, and draw the straight line best fitting the five
Ammonium Molybdate Injection plotted points. From the graph so obtained, determine the
concentration, in ~g per mL, of molybdenum in the Assay
» Ammonium Molybdate Injection is a sterile preparation. Calculate the quantity, in ~g, of molybdenum
(Mo) in each mL of the Injection taken by the formula:
solution of Ammonium Molybdate in Water for
Injection. It contains not less than 85.0 percent and 25C/V
not more than 115.0 percent of the labeled
amount of molybdenum (Mo). in which C is the concentration, in ~g per mL, of molybdenum
in the Assay preparation; and V is the volume, in mL, of
Packaging and storage-Preserve in single-dose or Injection taken.
multiple-dose containers, preferably of Type I or Type II glass.
Labeling-Label the Injection to indicate that it is to be
diluted to the appropriate strength with Sterile Water for
Injection or other suitable fluid prior to administration.
Amobarbital Sodium
Identification-
A: The Assay preparation, prepared as directed in the
Assay, exhibits an absorption maximum at about 313 nm CllH17N2Na03 248.25
when tested as directed for Procedure in the Assay. 2,4,6(1 H,3H,5H)-Pyrimidi netrione, 5-ethyl-5-(3-methyl
B: Add 0.3 mL of alkaline mercuric-potassium iodide TS to butyl)-, monosodium salt.
5 mL of Injection: a reddish-brown color develops. Sodium 5-ethyl-5-isopentylbarbiturate [64-43-7].
C: Evaporate 50 mL of Injection on a steam bath to a
volume of about 0.3 mL, and add 0.3 mL of ammonium » Amobarbital Sodium contains not less than 98.5
hydroxide. Cool, and add slowly, with mixing, a well-cooled percent and not more than 100.5 percent of
mixture of 1 mL of nitric acid and 1.2 mL of water. Allow to (11 H17N2Na03/ calculated on the dried basis.
stand for 24 to 48 hours, and pass through a sintered-glass
filter. To the filtrate add 0.5 mL of dibasic sodium phosphate Packaging and storage-Preserve in tight containers.
TS: a yellow precipitate is formed, and it dissolves in an excess Labeling-Where it is intended for usein preparing injectable
of 6 N ammonium hydroxide. dosage forms, the label states that it is sterile or must be
Pyrogen (151 )-It meets the requirements, the test dose subjected to further processing during the preparation of
being 10 mL of Injection per kg. injectable dosage forms.
pH (791): between 3.0 and 6.0. VSP Reference standards (11)-
Particulate Matter in Injections (788): meets the USP Amobarbital RS
requirements for small-volume injections. Completeness of solution (641)-Mix 1.0 g with 10 mL of
Other requirements-It meets the requirements under carbon dioxide-free water: after 1 minute, the solution is clear
Injections and ImplantedDrug Products (1). and free from undissolved solid.
Assay-
Ammonium hydroxide diluent-Dilute 40 mL of ammonium
hydroxide with water to 1000 mL. Store in a plastic bottle.

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 292 Amobarbital/Official Monographs USP 43

Identification- glass filter funnel into a tared beaker. Wash the separator
and the filter with several small portions of chloroform. Use
the combined chloroform extractions and the washings.
Test for completeness of extraction: Extract the
.4:.. :~$" remaining solution in the separator with an additional
$pg¢tr;9~ 1O-mL portion of chloroform, and evaporate the solvent;
Assay. ' NMT 0.5 mg of residue remains.
B: Ignite about 200 mg: the residue effervesces with acid Analysis: Evaporate the Sample solution on a steam bath
and responds to the tests for Sodium (191). with the aid of a current of air. Dry the residue at 105 0 for
pH (791): not more than 11.0, in the solution prepared for 30 min, cool, and weigh.
the test for Completeness of solution. Calculate the percentage of the labeled amount of
Loss on drying (731 )-Dry about 1 g, accurately weighed, at amobarbital sodium (CllH 17N 2Na0 3) in the portion of
105 0 for 4 hours: it loses not more than 2.0% of its weight. Amobarbital Sodium for Injection taken:
Other requirements-Where the label states that
Amobarbital Sodium is sterile, it meets the requirements for Result = (W iW s) x (M ,,1M (2) x 100
Sterility and Bacterial endotoxins under AmobarbitalSodium for
Injection. Where the label states that Amobarbital Sodium WR = weight of the residue from the Analysis (mg)
must be subjected to further processing during the Ws = nominal weight of amobarbital sodium in the
preparation of injectable dosage forms, it meets the Sample solution (mg)
requirements for Bacterial endotoxins under Amobarbital M ,1 = molecular weight of amobarbital sodium, 248.25
Sodium for Injection. M ,2 = molecular weight of amobarbital, 226.27
Assay-Dissolve about 500 mg of Amobarbital Sodium,
accurately weighed, in about 15 mL of water in a separator. Acceptance criteria: 98.5%-100.5% on the dried basis
To the solution add 2 mL of hydrochloric acid, shake, and
completely extract the liberated amobarbital with 25-mL PERFORMANCE TESTS
portions of chloroform. Test for completeness of extraction by • UNIFORMITY OF DOSAGE UNITS (905): Meets the
extracting with an additional 1O-mLportion of chloroform and requirements
evaporating the solvent: not more than 0.5 mg of residue SPECIFIC TESTS
remains. Filter the combined extract through a glass filter • INJECTIONS AND IMPLANTED DRUG PRODUCTS (1), Specific
funnel into a tared beaker, and wash the separator and the Tests, Completeness and clarity of solutions: At the time of
filter with several small portions of chloroform. Evaporate the use, it meets the requirements.
combined filtrate and washings On a steam bath with the aid • COMPLETENESS OF SOLUTION (641)
of a current of air, dry the residue at 105 0 for 30 minutes, cool, Sample solution: 1 g of amobarbital sodium from
and weigh. The weight of the residue, multiplied by 1.097, Amobarbital Sodium for Injection in 10 mL of carbon
represents the weight of CllH17N2Na03' dioxide-free water
Acceptance criteria: After 1 min, the solution is clear and
free from undissolved solid.
• Loss ON DRYING (731)
Sample: 1 g
Amobarbital Sodium for Injection Analysis: Dry the Sample at 105 0 for 4 h.
. DEFINITION . Acceptance criteria: NMT 1.0%
Amobarbital Sodium for Injection is Amobarbital Sodium • pH (791)
suitable for parenteral use. It contains NLT 98.5% and NMT Sample solution: Nominally 100 mg/mL of amobarbital
100.5% of the labeled amount of amobarbital sodium sodium from Amobarbital Sodium for Injection in carbon
(CllH17N2Na03), calculated on the dried basis. dioxide-free water
Acceptance criteria: NMT 11.0
IDENTIFICATION • STERILITY TESTS (71): Meets the requirements
• BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.4 USP
Endotoxin Units/mg of amobarbital sodium.
• OTHER REQUIREMENTS: It meets the requirements in
•A.>.~'~~
~P¢~~[(;J> r. :J •.'. )
Labeling (7), Labels and Labeling for Injectable Products.
ADDITIONAL REQUIREMENTS
Sample: Residue obtained in the Assay
Acceptance criteria: Meets the requirements • PACKAGING AND STORAGE: Preserve as described in
Packaging and Storage Requirements (659), Injection
• B. IDENTIFICATION TESTS-GENERAL, Sodium (191)
Sample: Nominally 200 mg of amobarbital sodium from
Packaging, Packaging for constitution.
• USP REFERENCE STANDARDS (11)
Amobarbital Sodium for Injection
USP Amobarbital RS
Analysis: Ignite the Sample.
Acceptance criteria: The residue effervesces with acid and
meets the requirements of the tests.
ASSAV
• PROCEDURE Amodiaquine
Sample solution: Transfer nominally 500 mg of
amobarbital sodium from Amobarbital Sodium for Injection
to a suitable separator, and dissolve in 15 mL of water. Add
2 mL of hydrochloric acid, and shake. Completely extract
the amobarbital with 25-mL portions of chloroform.
Combine the chloroform extractions, and pass through a 355.86

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 USP 43 Official Monographs / Amodiaquine 293

Phenol, 4-[(7-chloro-4-quinolinyl)amino]-2-[(diethylamino)- Standard solution B: StandardsolutionA and chloroform

methyl]-; (saturated with ammonium hydroxide) (1 in 200)
4-[ (7-Chloro-4-quinolyl)amino ]-a-(diethylamino)-o-cresol Sample solution: 15 mg/mL of Amodiaquine in chloroform
[86-42-0]. (saturated with ammonium hydroxide)
Chromatographic system
DEfiNITION (See Chromatography (621), Thin-Layer Chromatography.)
Amodiaquine contains NLT 97.0% and NMT 103.0% of Mode: TLC
amodiaquine (C2oH22CIN30), calculated on the anhydrous Adsorbent: 0.25-mm layer of chromatographic silica gel
basis. mixture
IDENTifiCATION Application volume: 10 IJL
Developing solvent system: Chloroform (saturated with
ammonium hydroxide) and dehydrated alcohol (9:1)
Analysis
• A. Samples: Standardsolution A, Standardsolution B, and
c~p ,) Sample solution
Standard: Dissolve 20 mg of USP Amodiaquine Develop the chromatogram until the solvent front has
Hydrochloride RS in 10 mL of water in a separator, add 1 moved about three-fourths of the length of the plate.
mL of ammonium hydroxide, and extract by shaking with Remove the plate from the developing chamber, mark
25 mL of chloroform. Draw off and evaporate the the solvent front, allow the solvent to evaporate, and
chloroform extract, and dry the residue at 105° for 2 h. examine the plate under short-wavelength UV light.
Acceptance criteria: Meets the requirements Acceptance criteria: The chromatograms show principal
spots at about the same RF value; and no secondary spot, if
present in the chromatogram from the Sample solution, is
more intense than the principal spot obtained from
• B. Standardsolution B.
QlL, . ' c , ; y...•..• 'c;.. )
SPECIFIC TESTS
Sample solution: 1 IJg/mL in 0.1 N Y roc loric acid.
• .WATER DETERMINATION, Method I (921): NMT 0.5%
Acceptance criteria: Meets the requirements
ADDITIONAL REQUIREMENTS
ASSAY • PACKAGING AND STORAGE: Preserve in tight containers.
• PROCEDURE • USP REFERENCE STANDARDS (11)
Standard solution: 15 IJg/mL of USP Amodiaquine
USP Amodiaquine Hydrochloride RS
Hydrochloride RS in 0.1 N hydrochloric acid
Sample solution:' 15 IJg/mL of Amodiaquine in 0.1 N
hydrochloric acid
Instrumental conditions
(See Ultraviolet- Visible Spectroscopy (857).) Amodiaquine Hydrochloride
Mode: UV
Analytical wavelength: 342 nm C2oH22C1N30 . 2HCI ·2M 20 464.81
Cell: 1 cm
Blank: 0.1 N hydrochloric acid C2oH22CIN30 . 2HCI 428.79
Analysis Phenol, 4-[(7 -chloro-4-quinolinyl)amino]-2-[(diethylamino)-
Samples: Standardsolution and Sample solution methyl]-, dihydrochloride, dihydrate;
Calculate the percentage of amodiaquine (C2oH22CIN30) in 4-[(7-Chloro-4-quinolyl)amino ]-a-(diethylamino)-o-cresol
the portion of Amodiaquine taken: dihydrochloride dihydrate [6398-98-7].
Anhydrous [69-44-3].
Result = (Au/As) x (Cs/Cu) x (MrdMr2 ) x 100 DEFINITION
Amodiaquine Hydrochloride contains NLT 97.0% and NMT
Au = absorbance of the Sample solution 103.0% of amodiaquine hydrochloride (C2oH22CIN30 .
As =absorbance of the Standardsolution 2HCI), calculated on the anhydrous basis.
Cs =concentration of USP Amodiaquine
Hydrochloride RS in the Standardsolution IDENTIFICATION
(lJg/ mL)
Cu =concentration of Amodiaquine in the Sample
solution (lJg/mL)
MrI = molecular weight of amodiaquine, 355.86
Mrl = molecular weight of anhydrous aminodiaquine p ., (.?Q)
hydrochloride, 428.79 Sample: mg of Amodiaquine Hydrochloride in
10 mL of water in a separator. Add 1 mL of ammonium
Acceptance criteria: 97.0%-103.0% on the anhydrous hydroxide, and extract by shaking with 25 mL of
basis chloroform. Draw off and evaporate the chloroform extract,
and dry the residue at 105° for 2 h.
IMPURITIES Acceptance criteria: Meets the requirements
• RESIDUE ON IGNITION (281): NMT 0.2%
• ORGANIC IMPURITIES
Standard solution A: To 20 mg of USP Amodiaquine
Hydrochloride RS in a glass-stoppered test tube add 1.0 mL
of chloroform (saturated with ammonium hydroxide), and
shake vigorously for 2 min. Allow the solids to settle, and
decant the liquid into a second test tube.

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294 Amodiaquine / Official Monographs USP 43

Sample solution: 10 jJg/mL in dilute hydrochloric acid (1 Calculate the percentage of each impurity in the portion of
in 100) Amodiaquine Hydrochloride taken:
Acceptance criteria: Meets the requirements
• C. IDENTIFICATION TESTS-GENERAL, Chloride (191): Meets Result = (rulI".r) x 100
the requirements
• D. The retention time of the major peak of the Sample to =response of each impurity from the Sample
solution corresponds to that of the Standardsolution, as solution
obtained in the Assay. rr =sum of the responses from the Sample solution
ASSAY Acceptance criteria
• PROCEDURE Individual impurity: NMT 0.5%
Buffer: 6.8 giL of monobasic potassium phosphate in water.
Add 1.0 mL of perchloric acid to each 1 L of solution, adjust SPECIFIC TESTS
with phosphoric acid to a pH of 2.5 ± 0.5, and passthrough • WATER DETERMINATION, Method J (921): 7.00/0-9.0%
a filter of 0,45-jJm pore size. • COMPLETENESS Of SOLUTION (641): A solution of 200 mg in
Mobile phase: Methanol and Buffer(22:78) 10 mL of water is clear.
System suitability solution: 0.15 mg/mL of USP ADDITIONAL REQUIREMENTS
Amodiaquine Hydrochloride RS and 0.'15 mg/mL of USP • PACKAGING AND STORAGE: Preserve in tight containers.
Chloroquine Phosphate RS in water
• USP REFERENCE STANDARDS (11)
Standard solution: 0.15 mg/mL of USP Amodiaquine USP Amodiaquine Hydrochloride RS
Hydrochloride RS in water USP Chloroquine Phosphate RS
Sample solution: 0.15 mg/mL in water
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 224 nm Amodiaquine Hydrochloride Tablets
Column: 4.6-mm x 10-cm; 5-lJm packing L1
Column temperature: 25° ± 5° .DEFINITION
Flow rate: 1.2 mL/min Arnodlaquine Hydrochloride Tablets contain an amount of
Injection volume: 10 IJL amodiaquine hydrochloride (C2oH22CIN30 . 2HCI . 2H 20)
System suitability equivalent to NLT 93.0% and NMT 107.0% of the labeled
Sample: System suitability solution amount of amodiaquine (C2oH22CIN30).
[NoTE-The relative retention times for chloroquine
phosphate and amodiaquine hydrochloride are 0.8 IDENTIFICATION
and 1.0, respectively.]
Suitability requirements
Resolution: NLT 1.5 between amodiaquine
hydrochloride and chloroquine phosphate
Tailing factor: NMT 1.5 for amodiaquine hydrochloride
and chloroquine phosphate or more and transfer a portion
Relative standard deviation: NMT 2.0% for of powder, equivalent to 50 mg of amodiaquine, to a
amodiaquine hydrochloride and chloroquine phosphate 125-mL separator. Add 20 mL of water, and shakefor 1 min.
Analysis . . Add 25 mL of chloroform and 1 mL of ammonium
Samples: Standardsolution and Sample solution hydroxide, shake for 2 min, and when settled, filter the
Calculate the percentage of amodiaquine hydrochloride chloroform extract through cotton that previously has been
(C2oH22CIN30 . 2HCI) in the portion of Amodiaquine rinsed with chloroform, collecting the extract in a vessel
Hydrochloride taken: suitable for evaporation. Evaporatethe chloroform, and dry
the residue at 105° for 1 h.
Result = (rulrs) x (CsICu) x 100 Acceptance criteria: Meet the requirements
• B. The retention time of the amodiaquine hydrochloride
t» = response from the Sample solution peak of the Sample solution corresponds to that of the
rs = response from the Standardsolution Standardsolution, as obtained in the Assay.
Cs = concentration of USP Amodiaquine ASSAY
Hydrochloride RS in the Standardsolution • PROCEDURE
(mg/mL) Buffer: 6.8 giL of monobasic potassium phosphate in water.
Cu = concentration of Amodiaquine Hydrochloride in Add 1.0 mL of perchloric acid to each 1 L of solution, adjust
the Sample solution (mg/mL) with phosphoric acid to a pH of 2.5, and passthrough a
filter of 0,45-lJm pore size.
Acceptance criteria: 97.0%-103.0% on the anhydrous Diluent: 1% (v/v) hydrochloric acid in water
basis Mobile phase: Methanol and Buffer(22:78)
IMPURITIES Standard solution: 0.15 mg/mL of USP Amodiaquine
• RESIDUE ON IGNITION (281): NMT 0.2% Hydrochloride RS and 0.15 mg/mL of USP Chloroquine
• ORGANIC IMPURITIES Phosphate RS in water
Buffer, Mobile phase, System suitability solution, Sample solution: Transfer a quantity equivalent to 7.5 mg
Standard solution, Sample solution, Chromatographic of amodiaquine hydrochloride from finely powdered
system, and System suitability: Proceedasdirected in the Tablets (NLT 20) to a 50-mL volumetric flask, and dissolve
Assay. in and dilute with Diluent to volume. Sonicate for 25 min at
Analysis 29°. Pass 10 mL through a nylon filter of 0.2-lJm pore size,
Sample: Sample solution discarding the first 4 mL. Use 2 mL for the analysis.

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 USP 43 OfficialMonographs / Amoxapine 295

Chromatographic system
(See Chromatography (621), System Suitability.)
Amoxapine
Mode: LC
Detector: UV 224 nm
Column: 4.6-mm x 1O-cm; 5-~m packing L1
Flow rate: 1.2 mL/min
Injection volume: 10 ~L
System suitability
Sample: Standardsolution
[NoTE-The relative retention times for the chloroquine
C17H16CIN 30 313.78
and amodiaquine peaks are 0.8 and 1.0, Dibenz[b,fj[l ,4]oxazepine, 2-chloro-l1-(1-piperazinyl)-;
respectively.] 2-Chloro-11-(1-piperazinyl)dibenz[b,fj[1,4]oxazepine
Suitability requirements [14028-44-5].
Resolution: NLT 1.5 between amodiaquine DEFINITION
hydrochloride and chloroquine phosphate Amoxapine contains NLT 98.0% and NMT 102.0% of
Tailing factor: NMT 1.5 for amodiaquine hydrochloride amoxapine (C 17H16C/N 30), calculated on the dried basis.
and chloroquine phosphate
Relative standard deviation: NMT 2.0% for IDENTIFICATION
amodiaquine hydrochloride and chloroquine phosphate
AM~ili .
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of
amodiaquine (C2oH22C1N30) in the portion of Tablets
taken: of the peak of the Sample
solution corresponds to that of Standardsolution, as
Result = (rulr s) x (CsICu) x 100 obtained in the Assay.
ASSAY
tu =peak response from the Sample solution • PROCEDURE
rs = peak response from the Standardsolution Buffer: 3.9 gIL of ammonium acetate in water adjusted with
Cs =concentration of amodiaquine in USP acetic acid or diluted ammonia solution to a pH of 7.3
Amodiaquine Hydrochloride RS in the Standard Mobile phase: Acetonitrile and Buffer(30:70)
solution (mg/mL) Diluent: Acetonitrile and Buffer (70:30)
Cu = nominal concentration of amodiaquine in the System suitability solution: 0.1 mg/mL each of USP
Sample solution (mg/mL) Amoxapine RS and USP Amoxapine Related Compound G
RS in Diluent
Acceptance criteria: 93.0%-107.0% Standard solution: 0.1 mg/mL of USP Amoxapine RS in
PERFORMANCE TESTS Diluent
• DISSOLUTION (711) Sample solution: 0.1 mg/mL of Amoxapine in Diluent
Medium: Water; 900 mL Chromatographic system
Apparatus 2: 50 rpm (See Chromatography (621), System Suitability.)
Time: 30 min Mode: LC
Detector: UV 342 nm Detector: UV 254 nm
Standard solution: USP Amodiaquine Hydrochloride RS in Column: 4.6-mm x 7.5-cm; 2.5-~m or 2.7-~m packing L1
Medium Column temperature: 35°
Sample solution: Filter portions of the solution under test, Flow rate: 1.2 mL/min
. SUitably diluted with water, if necessary, in comparison Injection volume: 10 ~L
with a Standardsolution having a known concentration of System suitability
USP Amodiaquine Hydrochloride RS. Samples: System suitability solution and Standardsolution
Analysis [NoTE-The relative retention times for amoxapine and
Samples: Standardsolution and Sample solution amoxapine related compound G are 1.0 and 1.3,
Determine the amount of amodiaquine hydrochloride respectively.]
(CzoHz2CIN30 . 2HCI . 2H 20) dissolved from UV Suitability requirements
absorbances. Resolution: NLT 1.5 between amoxapine and
Tolerances: An amount of amodiaquine hydrochloride amoxapine related compound G, System suitability
(C2oH22CIN30· 2HCI· 2H 20 ) equivalent to NLT 75% (Q) of solution
Tailing factor: 0.8-1.8, Standard solution
the labeled amount of amodiaquine (CzoH22CIN30) is
Relative standard deviation: NMT 0.73%, Standard
dissolved.
solution
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
Analysis
requirements
Samples: Standardsolution and Sample solution
ADDITIONAL REQUIREMENTS Calculate the percentage of amoxapine (C 17H16CIN 30) in
• PACKAGING AND STORAGE: Preserve in tight containers. the portion of Amoxapine taken:
• USP REFERENCE STANDARDS (11)
USP Amodiaquine Hydrochloride RS Result = (rulrs) x (CsICu) x 100
USP Chloroquine Phosphate RS
tu = peak response of amoxapine from the Sample
solution
rs = peak response of amoxapine from the Standard
solution

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296 Amoxapine / Official Monographs USP 43

Cs = concentration of USP Amoxapine RS in the Cu = concentration of Amoxapine in the Sample

Standard solution (mg/mL) solution (lJg/mL)
Cu =concentration of Amoxapine in the Sample
solution (mg/mL) Calculatethe percentage of any other impurity in the
portion of Amoxapine taken:
Acceptance criteria: 98.00/0-102.0% on the dried basis
Result = (rulrs) x (CsICu) x 100
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1% = peak response of any other impurityfrom the
• ORGANIC IMPURITIES Sample solution
Solution A: 3.9 giL of ammonium acetate in water adjusted = peak response of amoxapine from the Standard
with acetic acid or diluted ammonia solution to a pHof 7.3 solution
Solution B: Acetonitrile = concentration of USP Amoxapine RS in the
Mobile phase: See Table 7. Standard solution (pq/rnl)
=concentration of Amoxapine in the Sample
Table 1 solution (lJg/mL)
Time Solution A Solution B
(min) (%) (%) Acceptance criteria: See Table 2. Disregard peaks that are
0 70 30 less than 0.02% of the amoxapine peak.
5 70 30 Table 2
7.5 60 40 Relative Acceptance
Retention Criteria,
15 60 40 Name Time NMT (0/0)
20 20 80 Chlorophenoxyanillne
urea
25 20 80 analog" 0.57 0.10
30 70 30 Amoxapine 1.0 -
35 70 30 Amoxapine related
compound G 1.4 0.15
Diluent: Solution A and Solution B(30:70) Amoxapine related
System suitability solution: 1 mg/mL of USP AmoxapineRS compound D 1.7 0.15
and 1.5 IJg/mL of USP AmoxapineRelated Compound G RS Chlorophenoxyantllne'' 2.9 0.10
in Diluent
Standard solution: 1 IJg/mL of USP Amoxapine RS, and 1.5 Chlorophenoxyaniline
IJg/mL each of USP Amoxapine Related Compound G RS carbamate- 3.8 0.10
and USP Amoxapine Related Compound D'RS in Diluent N·Carbamoyl
Sample solution: 1000 IJg/mL of Amoxapine in Diluent amoxaplne" 4.3 0.10
Chromatographic system: Proceed as directed in the Amoxapine dimer' 5.0 0.10
Assay.
System suitability Any individual
Samples: System suitability solution and Standard solution unspecified -
impurity 0.10
[NoTE-See Table 2 for the relative retention tlrnes.]
Suitability requirements Total impurities - 0.50
Peak-to-valley ratio: NLT 3 between amoxapine and
amoxapine related compound G, System suitability a N-[2.(4-Chlorophenoxy)phenyl]piperazine-1-carboxamide.
solution b 2-(4.Chlorophenoxy)aniline.
Relative standard deviation: NMT 5.0% each for c Ethyl[2-(4-Chlorophenoxy)phenyl]carbamate.
d 4-(2.Chlorodibenzo[b,fj[1 ,4]oxazepin·11.yl)-N.[2-(4·chlorophenoxy)phenyl]
amoxapine, amoxapine related compound G, and piperazine-1-carboxamide.
amoxapine related compound D, Standard solution e l,4-Bis(2-chlorodibenzo[b,fj[1 ,4]oxazepine·11-yl)piperazine.
Analysis
Samples: Standard solution and Sample solution SPECIFIC TESTS
Calculatethe percentage of amoxapine related compound • Loss ON DRYING (731)
G and amoxapine related compound D in the portion of Analysis: Dryat 105° for 4 h.
Amoxapinetaken: Acceptance criteria: NMT 0.5%
Result = (rulrs) x (CsICu) x 100 ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers.
to = peak response of amoxapine related compound • USP REFERENCE STANDARDS (11)
G or amoxapine related compound D from the USP Amoxapine RS
Sample solution USP Amoxapine Related Compound D RS
ts = peak response of amoxapine related compound 2-Chlorodibenzo[b,f]-[l,4]-oxazepin-ll-one.
G or amoxapine related compound D from the C13HsCIN0 2 245.66
Standard solution USP Amoxapine Related Compound G RS .
Cs = concentration of USP Amoxapine Related 3-Chloro-ll-(piperazin-l-yl)dibenzo[b,f][1,4]oxazepine.
Compound G RS or USP Amoxapine Related C17H 16CIN 30 313.78
Compound D RS in the Standard solution
(lJg/mL)

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USP 43 Official Monographs / Amoxicillin 297

Amoxaplae Tablets Cu = nominal concentration of amoxapine in the

Sample solution(mg/mL)
DEFINITION
Acceptance criteria: 90.0%-110.0%
Amoxapine Tablets contain NLT 90.0% and NMT 110.0% of
the labeled amount of amoxapine (C17H 16C1N 30). PERFORMANCE TESTS
• DISSOLUTION (711)
IDENTIFICATION Medium: Simulated gastric fluid (without enzyme); 900 mL
Apparatus 2: 50 rpm
Time: 30 min
• A. i~· Sample solution: Sample per Dissolution (711 ).
Standard solution: USP Amoxapine RS having a
'§pg .f.Q~€gfi
concentration similar to the expected Sample solution in
Sample: Triturate a quantity 0 finely ground Tablets,
Medium
equivalent to 50 mg of amoxapine, with 10 mL of
Instrumental conditions
chloroform, and filter. Evaporate the filtrate on a steam bath
Analytical wavelength: 294 nm
to dryness (about 30 min).
Analysis
• B. The retention time of the major peak of the Sample
Samples: Sample solution and Standard solution
solution corresponds to that of the Standard solution, as
Determine the percentage of the labeled amount of
obtained in the Assay.
amoxapine (C17H 16CIN30) dissolved from UVabsorbances
ASSAY of filtered portions of the Sample solution, suitably
• PROCEDURE diluted with Medium, if necessary.
Solution A: 1.38 giL of monobasic sodium phosphate in Calculate the percentage of the labeled amount of
water amoxapine (C17H16C1N30) dissolved:
Solution B: 11 3 giL of tetramethylammonium chloride in
water Result = (AulAs) x Cs x V x (1 I L) x 100
Mobile phase: Transfer 20.0 mL of Solution 8, 4.0 mL of
dilute phosphoric acid (1 in 5), and 720 mL of acetonitrile Au = absorbance of the Sample solution
to a 2000-mL volumetric flask. Dilute with Solution A to As =absorbance of the Standard solution
volume. Cs = concentration of the Standard solution (mg/mL)
Standard stock solution: 1 mg/mL of USP Arnoxaplne RS in V =volume of the Medium, 900 mL
acetonitrile. Shake by mechanical means to dissolve, and L =label claim (mg/Tablet)
then dilute with acetonitrile to volume.
Standard solution: 0.1 mg/mL from the Standard stock Tolerances: NLT 80% (Q) of the labeled amount of
solution diluted with Mobilephase - amoxapine (C 17H 16CIN30) is dissolved.
Sample stock solution: Nominally 1 mg/mL of amoxapine • UNIFORMITY OF DOSAGE UNITS (905): Meet the
from NLT 20 finely powdered Tablets prepared as follows. requirements
Transfer a suitable quantity of the powder to a volumetric
flask. Add 80% of the flask volume of Mobilephase, and ADDITIONAL REQUIREMENTS
shake vigorously by mechanical means for 20 min. Dilute • PACKAGING AND STORAGE: Preserve in well-closed
with Mobilephase to volume, and filter. containers.
Sample solution: 0.1 mg/mL from the Sample stock solution • USP REFERENCE STANDARDS (11)
diluted with Mobilephase USPAmoxapine RS
Chromatographic system
(See Chromatography (621 ),System Suitability.)
Mode: LC
Detector: . UV 254 nm
Column: 4.6-mmx 25-cm; packing L1 AmoxicUlin
Flow rate: 1.5 mL/min
Injection volume: 10 fJL
System suitability
Sample: Standardsolution
Suitability requirements
Column efficiency: NLT 1200 theoretical plates
Tailing factor: NMT 1.8 C16H19N30SS·3H20 419.45
Relative standard deviation: NMT 2.0% 4-Thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid,6-
Analysis [[ amino(4-hydroxyphenyl)acetyl]amino]-3, 3-dimethyl-7-
Samples: Standard solution and Sample solution oxo-, trihydrate [2S-[2a,5a,6~(S*)]]-;
Calculate the percentage of the labeled amountof (2S,5R,6R)-6-[(R)-(-)-2-Amino-2-(p-hydroxyphenyl)
amoxapine (C17H 16CIN30) in the portion of Tablets acetamido]-3, 3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]
taken: heptane-2-carboxylic acid trihydrate [61336-70-7].
Anhydrous 365.41
Result = (rulrs) x (CsICu) x 100 [26787-78-0].
tu = peak response of amoxapine from the Sample DEFINITION
solution Amoxicillin contains NLT 900 fJg/mg and NMT 1050 fJg/mg
rs = peak response of amoxapine from the Standard of amoxicillin (C16H19N30SS), calculated on the anhydrous
solution basis.
Cs =concentration of USP Amoxapine RS in the
Standardsolution (mg/mL)

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298 Amoxicillin / Official Monographs USP43

IDENTIFICATION System suitability solution: 12.5 IJg/mL each of US~ ..

Amoxicillin Related Compound A RS and USP Amoxlclllln
Related Compound D RS in Solution A
Standard solution: 12.5 IJg/mL of USP Amoxicillin RS in
• A·.. ~~~~~l~~~ Solution A
Spg&(fQ~&QPY;Y)Y.< ... ~~()j . Sample solution: 1.25 mg/mL of Amoxicillin in Solution A.
• B. The retention time of the major peak of the Sample [NOTE-Store this solution at 4 0 and use within 4 h.]
solution corresponds to that of the Standard solution, as Chromatographic system
obtained in the Assay. (See Chromatography (621); System Suitability.)
Mode: LC
ASSAY
Detector: UV 210 nm
• PROCEDURE Column: 4.6-mm x 10-cm; 5-lJm packing L7
Diluent: 6.8 giL of monobasic potassium phosphate in
Temperatures
water. Adjust with a 45% (w/w) solution of potassium
Autosampler: 4 0
hydroxide to a pH of 5.0 ± 0.1.
Column: 40°
Mobile phase: Acetonitrile and Diluent (1:24)
Flow rate: 1.5 mL/min
Standard solution: 1.2 mg/mL of USP Amoxicillin RS in
Injection volume: 10 IJL
Diluent. [NOTE-Use this solution within 6 h.]
System suitability
Sample solution: 1.2 mg/mL of Amoxicillin in Diluent. Samples: System suitabilitysolution and Standard solution
[NOTE-Use this solution within 6 h.]
[NOTE-Identify peaksby the relative retention times in
Chromatographic system
Table 2.]
(See Chromatography (621), System Suitability.) Suitability requirements
Mode: LC
Resolution: NLT 1.5 between amoxicillin related
Detector: UV 230 nm compound A and the second peak for amoxicillin related
Column: 4-mm x 25-cm; packing L7 compound D, System suitability solution
Flow rate: 1.5 mL/min Relative standard deviation: NMT 10%, Standard
Injection volume: 10 IJL solution
System suitability
Analysis
Sample: Standard solution Samples: Standard solution and Sample solution
Suitability requirements Calculate the percentage of each impurity in the portion of
Tailing factor: NMT 2.5 Amoxicillin taken:
Relative standard deviation: NMT 2.0%
Analysis Result = (r vir s) x (C siC v) x F x 100
Samples: Standard solution and Sample solution
Calculate the quantity, in IJg/mg, of amoxicillin = peak responseof each impurity from the Sample
(C16H19N30SS) in the portion of Amoxicillin taken: solution
= peak response of amoxicillin from the Standard
Result = (r vir s) x (C siC v) x. p solution
= concentration of USP Amoxicillin RS in the
ru = peak responsefrom the Sample solution Standard solution (lJg/mL)
rs = peak response from. the Standard solution = concentration of Amoxicillin in the Sample
Cs =concentration of USP Arnoxlclllln RS in the solution (mg/mL)
Standard solution (mg/mL) F = unit conversion factor, 0.001 mg/lJg
Cu = concentration of Amoxicillin in the Sample
solution (mg/mL) . Acceptance criteria: See Table 2. [NOTE-The reporting limit
P = potency of amoxicillin in USP Amoxicillin RS is 0.03 times the amoxicillin peak from the Standard
(lJg/mg) solution.]
Acceptance criteria: 900-1050 IJg/mg of amoxicillin Table 2
(C16H19N30SS) on the anhydrous basis Relative Acceptance
Retention Criteria,
IMPURITIES Name Time NMT (%)
• ORGANIC IMPURITIES
Solution A: 2.72 giL of monobasic potassium phosphate. Amoxicillin related compound la
(o-hydroxyphenylglycine) 0.32 1.0
Adjust with 7 N potassium hydroxide or 20% phosphoric
add to a pH of 5.0 ± 0.1. 0.53 1.0
Solution B: Methanol Amoxicillin related compound Db, c (arnoxl-
cillin open ring) 0.68 1.0
Mobile phase: See Table 7.
Amoxicillin related compound Ad
Table 1 (6-aminopenicillanic acid) 0.78 0.5

Time Solution A Solution B Amoxicillin related compound Be, f

(min) (%) (%) (l-amoxicillin) 0.87
-
0 97 3 Amoxicillin 1.0 -
10 97 3 Amoxicillin related compound G9
(o-hydroxyphenyl glycylamoxicillin) 2.9 1.0
22 75 25
Amoxicillin related compound Eh,l (amoxicil-
26 97 3 lin penilloic dei-ivative) 4.5 1.0

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 USP 43 OfficialMonographs / Amoxicillin 299

Table 2 (continued) dosage forms, it contains NMT 0.25 USP Endotoxin

Relative Acceptance Units/mg of amoxicillin.
Retention Criteria,
Name Time NMT(%} ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers, and
Amoxicillin related compound Ml store at controlled room temperature.
[N-(penicillan-6-yl} open ring amoxicillina-
mide] 6.0 1.0 • LABELING: Where it is intended for use in preparing
injectable dosage forms, the label states that it is intended
Amoxicillin related compound Fe, k (phenyl- for veterinary use only and that it is sterile or must be
pyrazinediol) 6.3 -
subjected to further processing during the preparation of
Amoxicillin related compound CI (amoxicillin injectable dosage forms. Label all other Amoxicillin to
rearrangement product) 6.4 1.0 indicate that it is to be used in the manufacture of
Amoxicillin related compound Eh, i (amoxicil- nonparenteral drugs only.
linpenilloic derivative) 6.7 1.0 • USP REFERENCE STANDARDS (11)
USP Amoxicillin RS
Amoxicillin related compound l'" (amoxicillin USP Amoxicillin Related Compound A RS
open ringdimer) 8.8 1.0
(2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-
Amoxicillin related compound L" azabicyclo[3.2.0]heptane-2-carboxylic acid;
[N-(penicillan-6-yl)amoxicillinamide] 9.0 1.0 6-Aminopenicillanic acid.
Any unspecified individual impurity - 1.0 CSH12N 20 3S 216.26
USP Amoxicillin Related Compound D RS
Total impurities - 5.0
(4S)-2-{[(R)-2-Amino-2-(4-hydroxyphenyl)acetamido]
a (R)-2-Amino-2-(4-hydroxyphenyl)acetic acid. (carboxy)methyl}-5,5-dimethylthiazolidi ne-4-carboxylic
bThe chromatographicsystem resolves two penicilloic acidsfrom each other. acid; -
c (45)-2-{[( R)-2-Amino-2-(4-hydroxyphenyl)acetamido](carboxy)methyl}-S,5- Amoxicillin open ring. -
dimethylthiazolidine-4-carboxylic acid. C16H21N306S 383.42
d (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-
2-carboxylic acid.
eThese compounds are listed for informationonly and are not to be reported.
f (2S,5R,6R)-6-[(5)-2-Amino-2-(4-hydroxyphenyl)acetamido]-3,3-dimethyl-7-
oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid.
9 (2S,5R,6R)-6-{( R)-2-[(R)-2-Amino-2-(4-hydroxyphenyl)acetamido]-2-(4-
Amoxicillin Boluses
hydroxyphenyl)acetamido}-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]
heptane-2-carboxylic acid.
h The chromatographicsystem resolves two penilloic acidsfrom each other. » Amoxicillin Boluses contain not less than 90.0
i (45)-2-{[( R)-2 -Amino-2-(4-hydroxyphenyl)acetamido]methyl}-S ,5- percent and not more than 110.0 percent of the
dimethylthiazolidine-4-carboxylic acid.
j (2S,5R,6R)-6-(2-[(R)-2-Amino-2-(4-hydroxyphenyl}acetamido]-2-«45)-4-
labeled amount of amoxicillin (C16H19N30SS).
carboxy-S,S-dimethylthiazolidin-2-yl}acetamido)-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylic acid. Packaging and storage-Preserve in tight containers, and
k3-(4-Hydroxyphenyl)pyrazin-2-ol. store at controlled room temperature.
I(45)-2-[5-(4-Hydroxyphenyl}-3, 6-dioxopiperazin-2-yl]-S,5- Labeling-Label Boluses to indicate that they are for
dimethylthiazolidine-4-carboxylic acid. veterinary use only:
m (2S,5R,6R)-6-((2R)-2-{2-[(R)-2-Amino-2-(4-hydroxyphenyl)acetamido]-2-
[(45)-4-carboxy-5,5-dimethylthiazolidin-2-yl]acetamido}-2-(4-hydroxyphenyl) VSP Reference standards (11)-
acetamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2- USP Amoxicillin RS
carboxylic acid.
n (2S,5R,6R)-6-{(2S,5R,6R)-6-[(R)-2-Amino-2-(4-hydroxyph enyl)acetamido]- Identification-
3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxamido}-3,3- Test solution-To a portion of powdered Boluses add 0.1 N
dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid. hydrochloric acid to obtain a Test solution containing about 4
mg of amoxicillin per mL. Use within 10 minutes after
SPECIFICTESTS preparation.
• CRYSTALLINITY (695): Meets the requirements Application volume, Developing solvent system, Procedure-
• DIMETHYLANILINE (223): Meets the requirements Proceed as directed for the Identification test under
• pH (791) Amoxicillin Tablets.
Sample solution: 2 mg/mL Disintegration (701): 30 minutes, simulated gastric fluid
Acceptance criteria: 3.5-6.0 being used instead of water.
• WATER DETERMINATION (921), Method I: 11.50/0-14.5% Water Determination, Method I (921): not more than 7.5%.
• STERILITY TESTS (71), Test for Sterility of the Product to Be
Examined, DirectInoculation of the Culture Medium: Where Assay-
the label states that Amoxicillin is sterile, it meets the Diluent, Mobilephase, Standard preparation, and
requirements, except to use Fluid Thioglycollate Medium Chromatographic system-Prepare as directed in the Assay
containing polysorbate 80 solution (5 mg/mL) and an under Amoxicillin.
amount of sterile penicillinase sufficient to inactivate the Assay preparation-Weigh and finely powder not fewer
amoxicillin in each tube, to use Soybean-Casein Digest than 5 Boluses. Transfer an accurately weighed portion of the
Medium containing polysorbate 80 solution (5 mg/mL) powder, equivalent to about 250 mg of amoxicillin, to a
and an amount of sterile penicillinase sufficient to inactivate 250-mL volumetric flask, add Diluent to volume, and mix.
the amoxicillin in each tube, and to shake the tubes once Sonicate if necessaryto ensure complete dissolution of the
daily. amoxicillin. Pass a portion of this solution through a filter of
• BACTERIAL ENDOTOXINS TEST (85): Where the label states 1-J.Im 'or finer porosity, and use the filtrate as the Assay
that Amoxicillin is sterile or Amoxicillin must be subjected preparation. [NoTE-Use this solution within 6 hours.]
to-further processing during the preparation of injectable Procedure-Proceed as directed for Procedure in the Assay
under Amoxicillin. Calculate the quantity, hi mg, of

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300 Amoxicillin / Official Monographs USP 43

amoxicillin (C16H19N30SS) in the portion of Boluses taken by PERFORMANCE TESTS

the formula: • DISSOLUTION (711)
Test 1
0.25CP(ru/rs) Medium: Water; 900 mL
Apparatus 1: 100 rpm, for Capsules containing 250 mg
in which the terms are as defined therein. Apparatus 2: 75 rpm, for Capsules containing 500 mg
Time: 60 min
Analytical wavelength: UV 272 nm
Standard solution: USP Arnoxklllln RS in Medium
Sample solution: Pass a portion of the solution under test
AmoxicUUn Capsules through a suitable filter. Dilute with Medium, if necessary,
to a concentration that is similar to that of the Standard
DEFINITION solution.
Amoxicillin Capsulescontain the equivalent of NLT 90.0% and Tolerances: NLT 80% (Q) of the labeled amount of
NMT 120.0% of the labeled amount of amoxicillin amoxicillin (C16H19N30SS) is dissolved.
(C16H19N30SS), Test 2: If the product complies with this test, the labeling
IDENTIFICATION indicates that it meets USP Dissolution Test 2.
• A. The retention time of the major peak of the Sample Medium: Water; 900 mL
solution corresponds to that of the Standardsolution, as Apparatus 1: 100 rpm
obtained in the Assay. Time: 90 min
Analytical wavelength: UV 272 nm
ASSAY Standard solution: USP Amoxicillin RS in Medium
• PROCEDURE Sample solution: Pass a portion of the solution under test
Buffer: Dissolve 6.8 giL of monobasic potassium phosphate through a suitable filter. Dilute with Medium, if necessary,
in water. Adjust with 45% potassium hydroxide TS to a pH to a concentration that is similar to that of the Standard
of 5.0 ± O.l. solution.
Mobile phase: Acetonitrile and Buffer (1 :24) Tolerances: NLT 80% (Q) of the labeled amount of
Standard solution: 1.2 mg/mL of USP Amoxicillin RS in amoxicillin (C16H19N30SS) is dissolved.
Buffer. [NoTE-Use this solution within 6 h.] • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Sample solution: Remove, as completely as possible, the requirements
contents of NLT 20 Capsules. Mix the combined contents,
and transfer a quantity, equivalent to 200 mg of anhydrous IMPURITIES
amoxicillin, to a 200-mL volumetric flask. Add Buffer to
volume. Sonicate if necessary to ensure complete
dissolution. [NoTE-Use this solution within 6 h.]
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 230 nm
Column: 4-mm x 25-cm; 10-lJm packing L1
Flow rate: 1.5 mL/min
Injection volume: 10 IJL
System suitability tI"~bl~:-"i
Sample: Standardsolution ]M!j~ S9!ijti(jgtA
Suitability requirements (mill) ~ (9(0) (0/0)
Tailing factor: NMT 2.5
0 100 0
Relative standard deviation: NMT 2.0%
Analysis 5 100 0
Samples: Standardsolution and Sample solution
25 94 6
Calculate the percentage of the labeled amount of
amoxicillin (C16H19N30SS) in the portion of Capsules 40 84 t6
taken: 50 ,84 16
Result =(rufrs) x (Cs/Cu) x P x F x 100 §;l 9
ru =peak response from the Sample solution .60 100 0
rs = peak response from the Standardsolution
Cs = concentration of USP Amoxicillin RS in the
Standardsolution (mg/mL)
Cu = nominal concentration of amoxicillinin the
Sample solution (mg/mL)
P = potency of amoxicillin in USP Amoxicillin RS
(lJg/mg)
F = conversion factor, 0.001 mg/lJg
Acceptance criteria: 90.00/0-120.0%

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USP 43 OfficialMonographs / Amoxicillin 301

Table 2 (continued)
Rel~i:ive ,it~ Acceptanc-e
Retention R ,Criteria;
nmEi NMT{%)

0.8~

~.Q

4.03

-4.39,4.15

Analysis l?.18,6.40;
Sam 6.56
Calcu duct if)
the
7.02 O·M 2.0
7.96
rlj

rs 1.Q
.dllinRS in
." "
tne
u.

~Oxkmininttie

p PAmo><lcilliri RS

Accepteiri~e, criteria:-See.TabJi2: [)lsrega'ra:~D)ip-ea.k:Jess

than 0.05%. '

(J.B

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 302 Amoxicillin / OfficialMonographs USP 43

Table Z (continued) amount of amoxicillin (C16H19N30SS). It contains a

Relative Relative Accept.mce suitable dispersing agent and preservative.
Retention Response Criteria;
Nanie Ihne F.u:to,; NMIT(%)
Packaging and storage-Preserve in well-closed disposable
Total impurities ~ ~
f~Q syringes.
Labeling-Label it to indicate that it is intended for veterinary
'trolled Inthe drugsubstance..,Ihey use only.
t to be reported.
USPReference standards (11)-
Cid;
)~cetamldo]( carboxy)rnethyl}-S,S;
USP Amoxicillin RS
Identification-Transfer a quantity of Intramammary
Ive the peaks fre>m'isomerS:'and the Infusion, equivalent to about 60 mg of amoxicillin, to a 50-mL
centrifuge tube, add 25 mL of toluene, mix, and centrifuge.
Decant and discard the toluene. Wash the residue with four
25-mL portions of toluene, sonicating for about 30 seconds
after each addition of toluene. Dry the residue in vacuum over
silica gel. Add 15 mL of 0.1 N hydrochloric acid to the residue,
and mix. The solution obtained responds to the
/DENT/FICA T/ON test under Amoxicillin Capsules.
Water Determination, Method I (921): not more than 1.0%,
20 mL of a mixture of toluene and methanol (7:3) being used
in place of methanol in the titration vessel.
Assay-Proceed asdirected for amoxicillin under Antibiotics-
Microbial Assays (81). Expel the contents of 1 syringe of
Intramammary Infusion into a high-speed glass blender jar
containing 499.0 mL of Buffer B.3 and 1.0 mL of polysorbate
80, and blend for 3 to 5 minutes. Allow to stand for about 10
minutes, and dilute an accurately measured volume of the
aqueous phase quantitatively and stepwise with BufferB.3 to
obtain a Test Dilution having a concentration assumed to be
equal to the median dose level of the Standard.

SPECIFIC TESTS
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
SPECIFIED MICROORGANISMS (62): The total aerobic
microbial count does not exceed 10 3 du/g, and the total Amoxicillin for Injectable Suspension
combined molds and yeastscount does not exceed 102 du/
g. .
» Amoxicillin for Injectable Suspension is a sterile
ADDITIONAL REQUIREMENTS mixture of Amoxicillin and one or more suitable
• PACKAGING AND STORAGE: Preserve in tight containers, and buffers, preservatives, stabilizers, and suspending
store at controlled room temperature. .
agents. It contains not less than 90.0 percent and
• LABELING: When more than one Dissolution test is given, the
labeling states the Dissolution test used only if Test 1 is not not more than 120.0 percent of the labeled
used. amount of amoxicillin (C16H19N30SS).
Change to read: Packaging and storage-Preserve as described in
Packaging and Storage Requirements (659), Injection Packaging,
• USP REFERENCE STANDARDS (11) Packaging for constitution.
USP Amoxicillin RS Labeling-Label it to indicate that it isfor veterinary use only.
·USP Amoxieillin Related Compoundt.RS USPReference standards (11)-
(45)-2-[5-(4-Hydroxyphenyl)~3,6-dioxopiperazin-2 :yl]- USP Amoxicillin RS
5,5-dimethylthiazolidine-4-carboxylic acid. Identification-Prepare a test solution containing the
C16H19N30SS -:365.40 equivalent of.4 mg of amoxicillin per mL by adding 0.1 N
USP Amoxicillin Related Compound tI RS hydrochloric acid to Amoxicillin for Injectable Suspension.
(R)-2-(4-HydroxyphenyJ)-2-pivalamidoacetic acid; Allow the solution to stand for 5 minutes before use: the
C13H 17N04 251.28 ... (Postponed on l-May-20H~) solution responds to the /DENTIFICA T/ON test under
Amoxicillin Capsules.
Bacterial fndotoxins Test (85) -It contains not more than
0.25 Endotoxin Unit per mg of amoxicillin.
Sterility Tests (71) -It meets the requirements when tested
Amoxicillin Intramammary Infusion as directed in the section DirectInoculation of the Culture
Medium under Test for Sterilityof the Product to be Examined,
except to use Fluid Thioglycollate Medium containing
» Amoxicillin lntramarnrnary Infusion is a polysorbate 80 solution (1 in 200) and an amount of sterile
suspension of Amoxicillin in a suitable vegetable oil penicillinase sufficient to inactivate the amoxicillin in each
vehicle. It contains not less than 90.0 percent and tube, to use Soybean-Casein Digest Medium containing
polysorbate 80 solution (1 in 200) and an amount of sterile
not more than 120.0 percent of the labeled penicillinase sufficient to inactivate the amoxicillin in each
tube, and to shake the tubes once daily.

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 USP 43 OfficialMonographs / Amoxicillin 303

pH (791): between 5.0 and 7.0, in the suspension constituted Assay-

as directed in the labeling. Standardpreparation-Prepare as directed for Standard
Water Determination, Method 1(921): between 11.0% and Preparation under lodometric Assay-Antibiotics (425), using
14.0%. USP Amoxicillin RS.
Assay preparation-Using the dosing pump, deliver a
Assay-
number of dosesof Oral Suspension, equivalent to about 250
Diluent, Mobile phase, Standardpreparation, and mg of amoxicillin, to a separator containing 100 mL of
Chromatographic system-Prepare as directed in the Assay hexanes, and shake vigorously. Add 140 mL of water, and
under Amoxicillin.
shakefor 5 minutes. Allow the layersto separate, and drain the
Assay preparation 7 (where it is represented as being in a lower, aqueous layer into a 250-mL volumetric flask. Repeat
single-dose container)-Constitute Amoxicillin for Injectable the extraction with two 50-mL portions of water. Combine the
Suspension as directed in the labeling. Withdraw all of the aqueous extracts in the volumetric flask, dilute with water to
withdrawable contents, using a hypodermic needle and volume, and mix.
syringe, and quantitatively dilute with Diluent to obtain a Procedure-Proceed with Oral Suspension as directed for
solution containing about 1 mg of anhydrous amoxicillin per Procedure under lodometricAssay-Antibiotics (425), using USP
mL. Pass a portion of this solution through a suitable filter of Amoxicillin RS. Calculate the quantity, in mg, of amoxicillin
t-urn or finer porosity, and usethe filtrate as Assay preparation
(C16H19N30SS) in each dose of Oral Suspension taken by the
7. Use this solution within 6 hours.
formula:
Assay preparation 2 (where the label states the quantity of
amoxicillin in a given volume of constituted suspension)- (2501N)(FI2000)(B - f)
Constitute Amoxicillin for Injectable Suspension as directed in
the labeling. Quantitatively dilute an accurately measured in which N is the number of doses taken, and the other terms
volume of the constituted suspension with Diluent to obtain a are as defined therein.
solution containing about 1 mg of anhydrous amoxicillin per
mL. Pass a portion of this solution through a suitable filter of
I-urn or finer porosity, and usethe filtrate as Assay preparation
2. Use this solution within 6 hours.
Procedure-Proceed as directed for Procedure in the Assay AmoxicUlin for Oral Suspension
under Amoxicillin. Calculate the quantity, in mg, of
amoxicillin (C16H19N30SS) in the container, or in the portion of DEfiNITION
constituted Suspension taken by the formula: Amoxicillin for Oral Suspensioncontains the equivalent of NLT
90.0% and NMT 120.0% of the labeled amount of
(L/D)(CP/1000)(r vir s) amoxicillin (C16H19N30SS), It contains one or more suitable
buffers, colors, flavors, preservatives, stabilizers, sweeteners,
in which L is the labeled quantity, in mg, of anhydrous and suspending agents.
amoxicillin in the container, or in the volume of constituted
suspension taken; D is the concentration, in mg of anhydrous IDENTifiCATION
amoxicillin per mL, of Assaypreparation 7 or of Assay • The retention time of the major peak of the Sample solution
preparation 2 on the basis of the labeled quantity in the corresponds to that of the Standardsolution, as obtained in
container or in the portion of constituted suspension taken, the Assay.
respectively, and the extent of dilution; and the other terms ASSAY
are as defined therein. • PROCEDURE
Buffer: Dissolve6.8 giL of monobasic potassium phosphate
in water. Adjust with a 45% (w/w) solution of potassium
hydroxide to a pH of 5.0 ± 0.1.
Mobile phase: Acetonitrile and Buffer (1:24)
Amoxicillin Oral Suspension Standard solution: 1.2 mg/mL of USP Amoxicillin RS in
Buffer. [NoTE-Use this solution within 6 h.]
» Amoxicillin Oral Suspension is a suspension of Sample solution: Dilute a measured volume of Amoxicillin
for Oral Suspension, constituted asdirected in the labeling,
Amoxicillin in Soybean Oil. It contains not less than freshly mixed and free from air bubbles, quantitatively and
90.0 percent and not more than 120.0 percent of stepwise in Buffer to obtain a solution containing nominally
the labeled amount of amoxicillin (C16H19N30SS). 1 mg/mL of anhydrous amoxicillin. Pass a portion of this
solution through a suitable filter. [NoTE-Use this solution
Packaging and storage-Preserve in multiple-dose within 6 h.]
containers equipped with a suitable dosing pump. Chromatographic system
Labeling-Label it to indicate that it isfor veterinary useonly. (See Chromatography (621), System Suitability.)
USPReference standards (11) - Mode: LC
USP Amoxicillin RS Detector: UV 230 nm
Identification-Shake a portion of Oral Suspension with a Column: 4-mm x 25-cm;1 O-l.Jm packing L1
mixture of acetone and 0.1 N hydrochloric acid (4:1) to obtain Flow rate: 1.5 mL/min
a solution containing about 1 mg of amoxicillin per mL. The Injection size: 10 I.lL
solution responds to the Identification test under Amoxici/lin System suitability
Capsules. Sample: Standardsolution
Water Determination, Method I (921): not more than 2.0%, Suitability requirements
20 mL of a mixture of toluene and methanol (7:3) being used Tailing factor: NMT 2.5
in place of methanol in the titration vessel. Relative standard deviation: NMT 2.0%
Analysis
Samples: Standardsolution and Sample solution

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 304 Amoxicillin / Official Monographs USP 43

Calculate the percentage of C16H19N30SS in the Amoxicillin to volume, add a magnetic stirring bar, and stir for 30 min.
for Oral Suspension taken: Centrifuge a portion of this solution.] ,
Pass a portion of the clear supernatant through a suitable
Result = (ru/rs) x (CsICu) x P x F x 100 filter. [NOTE-Use this solution within 6 h.]
Chromatographic system
= peak response from the Sample solution (See Chromatography (621), System Suitability.)
= peak response from the Standardsolution Mode: lC
= concentration of USP Amoxicillin RS in the Detector: UV 230 nm
Standardsolution (mg/ml) Column: 4-mm x 25-cm; 10-J.lm packing II
= nominal concentration of anhydrous amoxicillin Flow rate: 1.5 ml/min
in the Sample solution (mg/ml) Injection volume: 10 ul,
P = potency of amoxicillin in USP Amoxicillin RS System suitability
(J.lg/mg) Sample: Standardsolution
F = conversion factor, 0.001 mglJ.lg Suitability requirements
Tailing factor: NMT 2.5
Acceptance criteria: 90.0%-120.0% Relative standard deviation: NMT 2.0%
Analysis
PERFORMANCE TESTS Samples: Standard solution and Sample solution
• UNIFORMITY OF DOSAGE UNITS (905) Calculate the percentage of the labeled amount of
For solids packaged in single-unit containers: Meets the amoxicillin (C16H19N30SS) in the portion of Tablets taken:
requirements
• DELIVERABLE VOLUME (698): Meets the requirements Result = (rvlrs) x (CsICv) x P x F.x 100
SPECIFIC TESTS
• pH (791): 5.0-7.5, in the suspension constituted as directed rv = peak response of amoxicillin from the Sample
in the labeling solution
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR ts = peak response of amoxicillin from the Standard
SPECIFIED MICROORGANISMS (62): The total aerobic solution
microbial count does not exceed 10 3 cfu/g, and the total Cs =concentration of USP Amoxicillin RS in the
combined molds and yeastscount does not exceed Standardsolution (mg/ml)
102 cfu/g. Cv = nominal concentration of amoxicillin in the
Sample solution (mg/mL)
ADDITIONAL REQUIREMENTS P = potency of amoxicillin in USP Amoxicillin RS
• PACKAGING AN,D STORAGE: Preserve in tight containers, and (J.lg/mg)
store at controlled room temperature. F = conversion factor, 0.001 mglJ.lg
• USP REFERENCE STANDARDS (11)
USP Amoxicillin RS Acceptance criteria: 90.00/0-120.0%
PERFORMANCE TESTS
• DISSOLUTION (711)
Medium: Water; 900 ml
Amoxicillin Tablets Apparatus 2: 75 rpm
Time: 30 min
DEfiNITION Determine the amount of amoxicillin (C16H19N30SS)
Amoxicillin Tablets contain NlT 90.0% and NMT 120.0% of dissolved by using the following method.
the labeled amount of amoxicillin (C16H19N30SS).' Buffer: 27.2 g of monobasic potassium phosphate in 3 lof
water. Adjust with 45% potassium hydroxide TS to a pH of
IDENTIFICATION . . 5.0 ± 0.1. Dilute with water to obtain 4 l of solution.
• A. The retention time of the major peak of the Sample Mobile phase: Acetonitrile and Buffer(1:39)
solution corresponds to that of the Standard solution, as Standard solution: 0.05 mg/ml of USP Amoxicillin RS in
obtained in the Assay. Buffer. [NOTE-Use this solution within 6 h.] .
ASSAY Sample solution: Pass a portion of the sample through a
• PROCEDURE suitable filter of 0.5-J.lm pore size. Quantitatively dilute a
Buffer: 6.8 gIL of monobasic potassium phosphate in volume of the filtrate with water to obtain an estimated
water. Adjust with 45% potassium hydroxide TS to a pH of concentration of 0.045 mg/ml of amoxicillin. Use this
5.0 ± 0.1. solution within 6 h.
Mobile phase: Acetonitrile and Buffer (1 :24) Chromatographic system
Standard solution: 1.2 mg/ml of USP Amoxicillin RS in (See Chromatography (621), System Suitability.)
Buffer. [NOTE-Use this solution within 6 h.] Mode: lC
Sample solution: Place NlT 5 Tablets in a high-speed glass Detector: UV 230 nm
blender jar containing Buffer sufficient to yield a Columns
concentration of 1 mg/ml of anhydrous amoxicillin. Blend Guard: 2-mm x 2-cm; packing l2
for 4 ± 1 min, allow to stand for 5 min, and centrifuge a Analytical: 3.9-mm x 30-cm; packing II
portion of the mixture. [NOTE-Where the volume of Buffer Column temperature: 40 ± 10
required would exceed 500 ml, place 5 Tablets in a Flow rate: 0.7 mL/min
volumetric flask of such capacity that when finally diluted Injection volume: 10 J.lL
to volume, a concentration of 1 mg of anhydrous System suitability
amoxicillin per milliliter would be obtained. Add a volume Sample: Standardsolution
of Buffer equivalent to three-fourths of the capacity of the Suitability requirements
volumetric flask, and sonicate for 5 min. Dilute with Buffer Tailing factor: NMT 2.5
, Relative standard deviation: NMT 1.5%

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USP 43 OfficialMonographs / Amoxicillin 305

Analysis fla~kvolume. SOlliCate, to dissolve and

ailutE(with Solution
Samples: Standard solution and Sample solution Atovolume. ,.,',' .: ..
Calculate the percentage of the labeled amount of System suita'biUtY solution:
amoxicillin (C16H19N30SS) dissolved: RS.a'nd 0.015mg/mC each
CompOlmdCRS and US
Result = (rvlrs) x (CslL) x Vx 0 x P x Fx 100 HRS' S ' 'repa
a oxic'
rv =peak response of amoxicillin from the Sample f d , " . A~to
solution priate volume of mp
ts =peak response of amoxicillin from the Standard volurn " lask. Sonitateto dis'
solution Solut' volume. ' • " .'
Cs =concentration of USP Amoxicillin RS in the ~tand 0 ution:0.011 mg/rnL'of US
Standard solution (mg/mL) Solut . SoniCate ifne todissol
L = label claim (mg/Tablet) imrnecUateliafterpr' . '
V =volume of the dissolution medium, 900 mL Sample solution: No
D = dilution factor for the Sample solution Solution A from the p,
P = potency of amoxicillin in USP Amoxicillin RS Transferpowdered Ta
(~g/mg)
F =conversion factor, 0.001 mg/~g amoxicillin into a 50;.
fill 60% ofthe finalflas
Tolerances: NLT 75% (Q) of the labeled amount of dilute with Solution A to
amoxicillin (C16H19N30SS) is dissolved. filte . - .m pore size
For products labeled as chewable Tablets: Proceed as aft .
directed above. Chr system
For chewable Tablets labeled to contain 200 or 400 mg (See .chro graphy{621), 'system Su'itablJit'y')
Time: 20 min Mode:LC
Tolerances: NLT 70% (Q) of the labeled amount of Detector: ,UV 230 no'
amoxicillin (C16H19N30SS) is dissolved. Column: 4.6-mm x 15-c::m; 5-~m packing L7
For chewable Tablets labeled to contain 125 or 250 mg eolu ature: 40 0

Time: 90 min Flo min

Tolerances: NLT 70% (Q) of the labeled amount of 's I . 20pL
: ..... ,
amoxicillin (C16H19N30SS) is dissolved.
For veterinary products: Proceed as directed above, except S
use Apparatus 2 at 100 rpm. [ E
Suitabili
IMPURITIES ResQlu
~ompou d amoxicillin r~lated co
.System ~uitabjJity solution
Relative'standard deviation; NMT5.0%~:Staf1C1iird
solution
Analysis
Samples: ,Standard solution and Sample ion
Calculate the percentage of each degrad product in
the portion of Tabletstaken:

:nme Sofution)\ S()f/.ltfO~;1J = peak respoh gradation product

(min) (%) (0/0)
fromth n
0 100 0 ;'peakres cillln'from'the-Stiiiiikiid
solution
5 100 Q
~ concentration' P Arnoxicillin RS:'rl:1 the
25 94 ~ Standard soluti gtmL)" . , .. .
~ nominal tonce I') ofamoxicillin'in the
40 84 l6 Sample solution (mg/mL) ,
,5,0 84 ,16, p ,;.; potency of amoxicil/inin .USP Amoxicillin RS
51 100 0 (~g/mg)
= conversionfactor, 0.001 mg/JJ9
60 10Q 0 = relativeresponse factor (see Table.2)

I k solution: Acceptance criteria: See Table 2~ [)i~regard any pe~k less

, , .clelated Com than :0.05%.
RelatedCompound H'R
TransfE:!r a weighed a '
Compound C RSan
H'RS to a s,uitable vol'
J 0% of the flaskvolunie Cil) .

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306 Amoxicillin / Official Monographs USP 43

Table 2 (continued)
Relative Relative Relative Acceptance
Iteteniiori Retention Respol1~e Criteria;
Name tiro~ ' Name 'Time' . factor Nl\1T(%)

iOta.lirtlpurities - ;.....;. 10,1)

0.19

o:~ 2..0

:0:66

1'.0 1.0

,1.'0

nyl)acetamido]-
yl]amino]-3,3-
acid.
~.63;3'.84 1:~ 1,0
oXicillin••
(Postponed on l-May:2018)
4.03 SPECIFIC TESTS
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
4.12 1",1 1.0 SPECIFIED MICROORGANISMS (62): The total aerobic
microbial count does not exceed 10 3 du/g, and the total
combined molds and yeastscount does not exceed 102 du/g.

4,39,,4.75 0.64 1.0

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers, and
store at controlled room temperature.
• LABELING: Label chewable Tablets to indicate that they are
0.46 1;0 to be chewed before swallowing. Tablets intended solely
for veterinary use are so labeled.

Change to read:
7.0~ 0.64 2.5
• USP REFERENCE STANDARDS (11)
7.96 ~ - USPAmoxicillin RS
·US
--: (4
1.0 5,
C 5
US oxicilli Related Compound,IjRS
(R}..2-(4-Hydroxyphenyl)-2:-pivalamidoacetic acid.
Cl~H17N04 25128... (Postponedon 17May~2018)

Amoxicillin Tablets for Oral Suspension

DEFINITION
Amoxicillin Tablets for Oral Suspension contain NLT 90.0%
and NMT 110.0% of the labeled amount of arnoxiclllln
(C16H19N30SS).

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USP 43 Official Monographs / Amoxicillin 307

IDENTIFICATION P = potency of amoxicillin in USP Amoxicillin RS

• A. THIN-LAVER CHROMATOGRAPHIC IDENTIFICATION TEST (J.lg/mg)
(201) F = conversion factor, 0.001 mg/J.lg
Standard solution: 4 mg/mL of USP Amoxicillin RS in 0.1 N
hydrochloric acid. Use within 10 min of preparation. Acceptance criteria: 90.0%-110.0%
Sample solution: An aqueous dispersion of Tablets for Oral
Suspensionin 0.1 N hydrochloric acid containing 4 mg/mL PERFORMANCE TESTS
of amoxicillin. Use within 10 min of preparation. • DISINTEGRATION (701)
Chromatographic system . Medium: Water at 20 ± 5°
Ad~orbent: 0.25-mm layer of chromatographic silica gel
Time: 3 min
mixture Acceptance criteria: Meet the requirements
Application volume: 5 J.lL • DISSOLUTION (711)
Developing solvent system: Methanol, chloroform, Medium: Water; 900 mL
pyridine, and water (90:80:1 :30) Apparatus 2: 75 rpm
Spray reagent: 3 mg/mL of ninhydrin in alcohol Time: 30 min
Analysis Buffer: 27.2 9 of monobasic potassium phosphate in 3 L of
Samples: Standard solution and Sample solution water. Adjust with a 45% (w/w) solution of potassium
Proceed as directed in the chapter. Dry the plate with the hydroxide to a pH of 5.0 ± 0.1, and dilute with water to
aid of a current of warm air for 10 min. Spraylightly with obtain 4 L of solution.
Spray reagent, and dry at 110° for 15 min. Mobile phase: Acetonitrile and Buffer (10:390). Pass
Acceptance criteria: The RF value of the principal spot of the through a filter of 0.5-J.lm or finer pore size.
Standard solution: 0.05 mg/mL of USP Amoxicillin RS in
Sample solution corresponds to that of the Standard
solution. . Buffer. Use this solution within 6 h.
Sample solution: Pass a portion of the sample through a
ASSAY filter of 0.5-J.lm or finer pore size. Dilute a suitable aliquot
• PROCEDURE of the filtrate with water to obtain a concentration of 0.045
Diluent: 6.8 giL of monobasic potassium phosphate in mg/mL of amoxicillin. Use this solution within 6 h.
water. Adjust with a 45% (w/w) solution of potassium Chromatographic system
hydroxide to a pH of 5.0 ± 0.1. (See Chromatography (621), System SUitability.)
Mobile phase: Acetonitrile and Diluent (1:24). Decrease the Mode: LC
acetonitrile concentration to increasethe retention time of Detector: UV 230 nm
amoxicillin. Columns
Standard solution: 1.2 mg/mL of USP Amoxicillin RS in Guard: 2-mm x 2-cm; packing L2
Diluent. Use this solution within 6 h. Analytical: 3.9-mm x 30-cm; packing L1
Sample solution: Prepare a dispersion of 20 Tablets for Oral Column temperature: 40 ± 1°
Suspension using a suitable aliquot of water. Dilute a Flow rate: 0.7 mL/min
portion of the dispersion with Diluent to obtain a solution Injection volume: 10 J.lL
containing 1.2 mg/mL of amoxicillin. Pass a portion of the System suitability
solution through a filter of 'l-urn or finer pore size. Usethis Sample: Standard solution
solution within 6 h. Suitability requirements
Chromatographic system Capacity factor: 1.1-2.8
(See Chromatography (621), System Suitability.) Column efficiency: NLT 1700 theoretical plates
Mode: LC Tailing factor: NMT 2.5
Detector: UV 230 nm Relative standard deviation: NMT 1.5%
Column: 4-mm x 25-cm; packing L1 Analysis
Flow rate: 1.5 mL/min Samples: Standard solution and Sample solution
Injection volume: 10 J.lL Calculate the percentage of the labeled amount of
System suitability amoxicillin (C16H19N30SS) dissolved:
Sample: Standardsolution
Suitability requirements Result = (rulrs) x Cs x Vx D x P x Fx (lIL) x 100
Capacity factor: 1.1-2.8
Column efficiency: NLT 1700 theoretical plates to =peak responsefrom the Sample solution
Tailing factor: NMT 2.5 ts = peak responsefrom the Standard solution
Relative standard deviation: NMT 2.0% Cs = concentration of USP Amoxicillin RS in the
Analysis Standard solution (mg/mL)
Samples: Standardsolution and Sample solution V =volume of medium, 900 mL
Calculate the percentage of the labeled amount of D = dilution factor
amoxicillin (C16H19N30SS) in the portion of Tabletsfor Oral P =potency of amoxicillin in USP Amoxicillin RS
Suspension taken: (J.lg/mg)
F = conversion factor, 0.001 mg/J.lg
Result = (rufrs) x (CsIC u) x P x F x 100 L = label claim (mg/Tablet)
= peak response from the Sample solution Tolerances: NLT 80% (Q) of the labeled amount of
= peak response from the Standard solution amoxicillin (C16H19N30SS) is dissolved.
= concentration of USP Amoxicillin RS in the • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Standardsolution (mg/mL) requirements
= nominal concentration of the Sample solution
SPECIFIC TESTS
(mg/mL) • DISPERSION FINENESS: Place 2 Tabletsfor Oral Suspension in
100 mL of water, and stir until completely dispersed. A

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308 Amoxicillin / OfficialMonographs USP 43

smooth dispersion that passes through a No. 25 sieve is = peak response of amoxicillin from the Standard
obtained. solution
= concentration of USP Amoxicillin RS in the
ADDITIONAL REQUIREMENTS
Standardsolution(mg/mL)
• PACKAGING AND STORAGE: Preserve in tight containers.
• USP REFERENCE STANDARDS (11)
= nominal concentration of amoxicillin in the
Sample solution (mg/mL)
USP Amoxicillin RS
P =potency of amoxicillin in USP Amoxicillin RS
(j.lg/mg)
F =conversion factor, 0.001 mg/j.lg
Amoxicillin and Clavulanate Potassium Calculate the percentage of the labeled amount of.
c1avulanic acid (CSH 9NOs) in the Amoxicillin and
for Oral Suspension Clavulanate Potassium for Oral Suspension taken:
DEFINITION Result = (rulrs) x (CsICu) x P x 100
Amoxicillin and Clavulanate Potassium for Oral Suspension
contains the equivalent of NLT 90.0% and· NMT 120.0% of ru = peak response of c1avulanic acid from the
the labeled amount of amoxicillin (C16H19N30SS) and the Sample solution
equivalent of NLT 90.0% and NMT 125.0% of the labeled ts = peak response of c1avulanic acid from the
amount of c1avulanic acid (CSH 9NOs). It contains one or more Standardsolution
suitable buffers, colors, flavors, preservatives, stabilizers, Cs =concentration of USP Clavulanate Lithium RS in
sweeteners, and suspending agents. the Standardsolution(mg/mL)
Cu = nominal concentration of c1avulanic acid in the
IDENTIFICATION Sample solution (mg/mL)
• A. The retention times of the major peaks of the Sample P = potency of c1avulanic acid in USP Clavulanate
solution correspond to those of the Standardsolution, as Lithium RS (mg/mg)
obtained in the Assay.
ASSAY Acceptance criteria: 90.0%-120.0% of the labeled amount
• PROCEDURE of amoxicillin (C16H19N30SS) and 90.0%-125.0% of the
Buffer: 7.8 9 of monobasic sodium phosphate in 900 mL of labeled amount of c1avulanic acid (CSH 9NOs)
water. Adjust with phosphoric acid or 10 N sodium PERFORMANCE TESTS
hydroxide to a pH of 4.4 ± 0.1, and dilute with water to • DELIVERABLE VOLUME (698)
1000 mL. For powder packaged in multiple-unit containers: Meets
Mobile phase: Methanol and Buffer(1 :19). Pass through a the requirements
suitable filter.
• UNIFORMITY OF DOSAGE UNITS (905)
Standard solution: 0.5 mg/mL of USPAmoxicillin RS and For powder packaged in single-unit containers: Meets the
0.2 mg/mL of USP Clavulanate Lithium RS in water requirements
Sample solution: Nominally 0.5 mg/mL of amoxicillin in
water, prepared asfollows. Constitute Amoxicillin and SPECIFIC TESTS
ClavuJanatePotassium for Oral Suspension with water using • pH (791)
the volume specified in the labeling. Stir by mechanical Sample solution: Constitute as directed in the labeling, and
means for 10 min, and filter. Use within 1 h. perform the test immediately after constitution. Acceptance
Chromatographic system criteria: 3.8-6.6
(See Chromatography (621), System Suitability.) . • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Mode: LC SPECIFIED MICROORGANISMS (62): The total aerobic
Detector: UV 220 nm microbial count does not exceed 103 cfu/g, and the total
Column: 4-mm x 30-cm; 3- to 10-j.lm packing L1 combined molds and yeasts count does not exceed 10 2 cfu/g.
Flow rate: 2 mL/min
Injection volume: 20 pt, ADDITIONAL REQUIREMENTS
System suitability • PACKAGING AND STORAGE: Preserve in tight containers, at
Sample: Standardsolution controlled room temperature.
[NOTE-The relative retention times for c1avulanic acid • USP REFERENCE STANDARDS (11)
and amoxicillin are about 0.5 and 1.0, respectively.] USP Amoxicillin RS
Suitability requirements USP Clavulanate Lithium RS
Resolution: NLT 3.5 between the amoxicillin and
c1avulanic acid peaks
Tailing factor: NMT 1.5 for each analyte peak
Relative standard deviation: NMT 2.0% for each
analyte peak Amoxiciliin and Clavulanate Potassium
Analysis .
Samples: Standardsolution and Sample solution Tablets
Calculate the percentage of the labeled amount of
amoxicillin (C16H19N30SS) in the Amoxicillin and DEFINITION
Clavulanate Potassium for Oral Suspension taken: Amoxicillin and Clavulanate Potassium Tablets contain the
equivalent of NLT 90.0% and NMT 120.0% of the labeled
Result = (rufr s) x (CsICu) x P x F x 100 amounts of amoxicillin (C16H19N30SS) and c1avulanic acid
(CSH 9NOs)'
t» = peak response of amoxicillin from the Sample
solution

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USP 43 Official Monographs / Amoxicillin 309

IDENTIFICAnON Acceptance criteria: 90.0%-120.0%

• The retention times of the major peaks of the Sample
solution correspond to those of the Standard solution, as PERFORMANCE TESTS
obtained in the Assay. • DISINTEGRATION (701): Tablets labeled for veterinary use
only; 30 min, simulated gastric fluid TS being substituted
ASSAY for water in the test
• PROCEDURE • DISSOLUTION (711)
Buffer: 7.8 g of monobasic sodium phosphate in 900 mL of [NOTE-Tablets labeled for veterinary use only are
water. Adjust with phosphoric acid or 10 N sodium exempt from this requirernent.]
hydroxide to a pH of 4.4 ± 0.1, and dilute with water to Test 1
1000 mL. Medium: Water; 900 mL
Mobile phase: Methanol and Buffer (1:19). Pass through a Apparatus 2: 75 rpm
suitable filter. Time: 30 min; or 45 min where the Tablets are labeled as
Standard solution: 0.5 mg/mL of USP Amoxicillin RS and chewable
0.2 mg/mL of USP Clavulanate Lithium RS in water Analysis: Determine the amount of C16H19N30SS and
Sample stock solution: Dissolve NLT 10 Tablets in water CSH 9NOs dissolved, using the Analysis set forth in the
with the aid of mechanical stirring. Transfer to a suitable Assay, making any necessary volumetric adjustments.
volumetric flask, and dilute with water to volume. Tolerances: NLT 85% (Q) of the labeled amount of
Sample solution: Dilute a suitable volume of the Sample C16H19N30SS and NLT 80% (Q) of the labeled amount of
stock solution with water to obtain a solution containing 0.5 CSH 9NOs are dissolved.
mg/mL of amoxicillin. [NoTE-Use the Sample solution For Tablets labeled as chewable: NLT 80% (Q) of the
within 1 h.] labeled amounts of C16H19N30SS and CSH9NO s is
Chromatographic system
dissolved in 45 min.
(See Chromatography (621), System Suitability.)
Test 2: If the product complies with this test, the labeling
Mode: LC
indicates that it meets USP Dissolution Test 2.
Detector: UV 220 nm
Medium, Apparatus 2, and Analysis: Proceed as
Column: 4-mm x 30-cm; 3- to 10-l.Jm packing L1
directed for Test 7.
Flow rate: 2 mL/min
Times: 45 min for amoxicillin, and 30 min for c1avulanic
Injection size: 20 I.JL acid
System suitability Tolerances: NLT 85% (Q) of the labeled amount of
Sample: Standardsolution
C16H19N30SS and NLT 80% (Q) of the labeled amount of
[NOTE-The relative retention times for c1avulanic acid
and amoxicillin are 0.5 and 1.0, respectively.] CSH 9NOs are dissolved.
Suitability requirements • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Resolution: NLT 3.5 between the amoxicillin and requirements
c1avulanic acid peaks SPECifiC TESTS
Tailing factor: NMT 1.5 for each analyte peak • WATER DETERMINATION, Method I (921):
Relative standard deviation: NMT 2.0%
Analysis
Acceptance
Samples: Standardsolution and Sample solution Tablet label Claim Criteria,
Calculate the percentage of C16H19N30SS in each Tablet Amoxicillin (mg/Tablet) NMT(%)
taken: .
5250 7.5
Result = (ru/r s) x (Cs/Cu) x P x F x 100 >250 and 5500 10.0

= peak response of amoxicillin from the Sample >500 11.0

solution
= peak response of amoxicillin from the Standard For products labeled as chewable Tablets:
solution
=concentration of USP Amoxicillin RS in the Acceptance
Standardsolution (mg/mL) Tablet label Claim Criteria,
= nominal concentration of amoxicillin in the Amoxicillin (mg/Tablet) NMT (%)
Sample solution (mg/mL) 5125 6.0
P = potency of USP Amoxicillin RS (l.Jg/mg)
F = conversion factor, 0.001 mg/l-lg >125 8.0

Calculate the percentage of CSH 9NOs in each Tablet taken: For Tablets labeled for veterinary use only: NMT 10.0%
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Result = (ru/rs) x (Cs/Cu) x P x 100 SPECIFIED MICROORGANISMS (62): The total aerobic
microbial count does not exceed 103 cfu/g, and the total
= peak response of c1avulanic acid from the combined molds and yeastscount does not exceed 102 cfu/
Sample solution g.
= peak response of c1avulanic acid from the
Standardsolution ADDITIONAL REQUIREMENTS
= concentration of USP Clavulanate Lithium RS in • PACKAGING AND STORAGE: Preserve in tight containers.
the Standardsolution (mg/mL) • LABELING: Label chewable Tablets to include the word
Cu = nominal concentration of c1avulanic acid in the "chewable" in juxtaposition to the official name. The
Sample solution (mg/mL) labeling indicates that chewable Tablets may be chewed
P = potency of c1avulanicacid in USP Clavulanate before being swallowed or may be swallowed whole.
Lithium RS (mg/mg) Tablets intended for veterinary use only are so labeled.

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310 Amoxicillin / Official Monographs USP 43

When more than one Dissolution test is given, the labeling Calculate the percentage of the labeled amount of
states the Dissolution test used only if Test 1 is not used. c1avulanic acid (C SH 9NOs) in the portion of Tablets taken:
• USP REFERENCE STANDARDS (11)
USP Amoxicillin RS Result =(rufr s) x (Cs/Cu) x P x 100
USP Clavulanate Lithium RS
tu =response of c1avulanic acid from the Sample
solution
rs = response of c1avulanic acid from the Standard
solution
AmoxiciUin and Clavulanic Acid Cs = concentration of USP Clavulanate Lithium RS in
Extended-Release Tablets the Standard solution (lJg/mL)
Cu = nominal concentration of c1avulanic acid in the
DEFINITION Sample solution (lJg/mL)
Amoxicillin and Clavulanic Acid Extended-Release Tablets P =potency of c1avulanic acid in USP Clavulanate
contain NLT 90.0% and NMT 110.0% of the labeled Lithium RS (mg/mg)
amounts of amoxicillin (C16H19N30SS) and c1avulanic acid
Acceptance criteria: 90.0%-110.0%
(CSH9NO s) ·
PERFORMANCE TESTS
IDENTIFICATION
• DISSOLUTION (711)
• A. The retention times of the major peaks of the Sample
Test 1
solution correspond to those of the Standard solution, as
Medium: Water; 900 mL
obtained in the Assay.
Apparatus 2: 75 rpm
ASSAY Times
• PROCEDURE Amoxicillin: 1, 3, and 5 h
Buffer: 6.9 gil of monobasic sodium phosphate adjusted Clavulanic acid: 1·h
with phosphoric acid to a pH of 4.2 Mobile phase, Chromatographic system, and System
Mobile phase: Methanol and Buffer(5:95) suitability: Proceed as directed in the Assay.
Standard solution: 1 mg/mL of USP Amoxicillin RS and Standard solution: USP Amoxicillin RS and USP
62.5 IJg/mL of USP Clavulanate Lithium RS in water. Store Clavulanate Lithium RS in Medium at known
the solution at 4°, and inject within 10 h. concentrations similar to those expected in the Sample
Sample solution: Equivalent to 1 mg/mL of amoxicillin and solution
62.5 IJg/mL of c1avulanic acid from finely powdered Tablets Sample solution: Pass a portion of the solution under test
(NLT 6) in wafer. Stir for about 60 min. Store the solution through a suitable filter, and dilute with Medium, if
at 4°, and inject within 12 h. necessary.
Chromatographic system Analysis
(See Chromatography (621), System Suitability.) Samples: Standard solution and Sample solution
Mode: LC . Calculate the amounts of amoxicillin (C16H19N30SS) and
Detector: UV 229 nm c1avulanic acid (CSH 9NOs) dissolved.
Column: 8-mm x 10-cm; 5-lJm packing L1 Tolerances
Flow rate: 2 mL/min Amoxicillin: The percentage of the labeled amount of
Injection volume: 20 IJL amoxicillin (C16H19N30SS) dissolved at the times
Autosampler temperature: 4° specified conforms to Table 7.
System suitability
Sample: Standard solution Table 1
Suitability requirements Time Amount Dissolved
Resolution: NLT 2.0 between the amoxicillin and (h) (%)
c1avulanic acid peaks
Tailing factor: NMT 1.8 for the amoxicillin and c1avulanic 1 50-65
acid peaks 3 65-85
Relative standard deviation: NMT 1.0% for the
5 NLT 85
amoxicillin and c1avulanic acid peaks
Analysis
Samples: Standard solution and Sample solution Clavulanic acid: NLT 80% (Q) of the labeled amount of
Calculate the percentage of the labeled amount of c1avulanic acid (CSH9NOs) is dissolved in 1 h.
amoxicillin (C16H19N30SS) in the portion of Tablets taken: Test 2
Medium: Water; 900 mL
Result = (ru/rs) x (Cs/Cu) x P x F x 100 Apparatus 2: 75 rpm
Times
ru = response of amoxicillin from the Sample solution Amoxicillin: 1, 3, and 5 h
rs = response of amoxicillin from the Standardsolution Clavulanic acid: 45 min
Cs = concentration of USP Amoxicillin RS in the Mobile phase, Chromatographic system, and System
Standard solution (mg/mL) suitability: Proceed as directed in the Assay.
Cu = nominal concentration of amoxicillin in the Standard solution: USP Amoxicillin RS and USP
Samplesolution (mg/mL) Clavulanate Lithium RS in Medium at known
P = potency of amoxicillin in USP Amoxicillin RS concentrations similar to those expected in the Sample
(lJg/ mg) solution .
F = conversion factor, 0~001 mg/lJg Sample solution: Pass a portion of the solution under test
through a suitable filter, and dilute with Medium, if
necessary.

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USP 43 OfficialMonographs / Amoxicillin 311

Analysis System suitability

Samples: Standard solution and Sample solution Samples: System suitabilitysolution and Standardsolution
Calculate the amounts of amoxicillin (C'6H,gN30SS) and Suitability requirements
c1avulanic acid (CsHgNOs) dissolved. Resolution: NLT 1.25 between the amoxicillin and
Tolerances amoxicillin related compound D peaks at a relative
Amoxicillin: The percentage of the labeled amount of retention time of 0.83, System suitability solution
amoxicillin (C'6H,gN30SS) dissolved at the times Tailing factor: NMT1.8 for the amoxicillin peak,
specified conforms to Table 2. Standard solution
Relative standard deviation: NMT 1.0% for the
Table 2 amoxicillin peak, Standard solution
Analysis
Time Amount Dissolved
(h) (%) Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of
1 50-70 Tablets taken:
3 65-90
Result = (ru/rs) x (Cs/Cu) x P x F7 x (1/F2 ) x 100
5 NLT85
ru =response of each impurity from the Sample
Clavulanic acid: NLT 85% (Q) of the labeled amount of solution
c1avulanic acid (CsHgNOs) is dissolved in 45 min. ts = responseof amoxicillin from the Standardsolution
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Cs = concentration of USP Amoxicillin RS in the
requirements Standard solution (mg/mL)
Cu = nominal concentration of amoxicillin in the
IMPURITIES Sample solution (mg/mL)
• ORGANIC IMPURITIES P = potency of amoxicillin in USP Amoxicillin RS
Buffer: 13.8 gil of monobasic sodium phosphate in water (lJg/ mg)
adjusted with phosphoric acid to a pH of 4.2 F7 = conversion factor, 0.001 mg/lJg
Solution A: Methanol and Buffer (1 :199) F2 = relative response factor (see Table 4)
Solution B: Methanol and Buffer (10:90)
Mobile phase: See Table 3. Acceptance criteria: See Table 4. The reporting limit is
0.003%.
Table 3
Time I Solution A Solution B Table 4
(min) (%) (0/0)
Relative Relative Acceptance
0 100 0 Retention Response Criteria,
Name Time Factor NMT(%)
8 70 30
Amoxicillin related com-
13 70 30 pound I
(D-hydroxyp.henyl- - -
13.01 40 60 glycine)a.b 0.15
18 40 60 Amoxicillin related com-
pound A
18.01 100 0 (6-aminopenicillanic - -
acid)a. c 0.30
25 100 0

30 100 0
Clavulanic acid 0.39 - -
Amoxicillin related com-
pound 0
[NoTE-These gradient elution times are established on an (amoxicillin open -
HPLC system with a dwell volume of approximately 5 mL. ring)a,d,e 0.63 0.74
The gradient elution times in Table 3 can be adjusted as
Amoxicillin related com-
necessaryto achieve the separation described.] pound B - -
System suitability solution: 0.4 mg/mL of USP Amoxicillin (t-amoxkllllnjv f 0.78
RS and 30 IJg/mL of USP Amoxicillin Related Compound
Amoxicillin related com-
DRS in water. Store the solution at 4°. pound 0
Standard solution: 0.4 mg/mL of USP Amoxicillin RS in (amoxicillin open ring)d,e 0.83 0.74 2.5
water. Store the solution at 4°, and inject within 24h.
Sample solution: 1 mg/mL of amoxicillin and 62.5 IJg/mL Amoxicillin 1.0 - -
of c1avulanic acid from finely powdered Tablets (NLT 2) in Amoxicillin related com-
water. Stir for about 60 min. Store the solution at 4°, and pound G
- -
use within 24 h. (D-hydroxyphenyl-
glycylamoxicillin)a, 9 2.57
Chromatographic system
(See Chromatography (621), System Suitability.) Amoxicillin related com- 2.63
Mode: LC pound E
(amoxicillin penilloic de- - -
Detector: UV 230 nm rivatives)" h. i 3.00
Column: 4.6-mm x s-cm; 3-lJm packing L1
Flow rate: 1.5 mL/min Amoxicillin related com-
pound C
Injection volume: 20 IJL (amoxicillin rearrange-
Autosampler temperature: 4° ment product)' 3.22 1.1 2.5

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 312 Amoxicillin / OfficialMonoqraphs USP 43

Table 4 (continued) DEFINITION

Relative Relative Acceptance Amphetamine Sulfate contains NLT 98.0% and NMT 102.0%
Retention Response Criteria, of (C9 H13N)2 . H2 S0 4 on the dried basis.
Name Time Factor NMT (%)
IDENTIFICATION
Amoxicillin open ring
methylester' k 3.38 - -
Amoxicillin relatedcom-
pound J
(amoxicillin open ring
dirner)' 4.07 1.0 4.5
peak of the Sample
Anyindividual unspecified
impurity - - 0.5
solution corresponds to that of Standardsolution, as
obtained in the Assay.
a Theseare syntheticprocessimpurities, which are controlledin the drug • C. IDENTIFICATION TESTS-GENERAL, Sulfate (191): Meets
substance. Theyare listed here for referenceonlyand are not to be reported. the requirements
b (R)-2-Amino-2-(4-hydroxyphenyl)acetic acid. Sample solution: 100 mg/mL
c (25,5R,6R)-6-Amino-3,3-dimethyl-7 -oxo-4-thia-l-azabicyclo[3.2.0]heptane-
2-carboxylic acid. ASSAY
d The chromatographicsystem resolves two isomers of amoxicillin open ring. • PROCEDURE
e (4S)-2-{[(R)-2-Amino-2-(4-hydroxyphenyl)acetamido](carboxy)methyl}-5,5- Solution A: Add 5.0 mL of trifluoroacetic acid to 900 mL of
dimethylthiazolidine-4-carboxylic acid. water, adjust with ammonium hydroxide to a pH of 2.2
f (25,5R, 6R)-6-[(5)-2-Amino-2-(4-hydroxyphenyl)acetamido]-3,3-dimethyl-7- ± 0.1, and add 100 mL of acetonitrile.
oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid. Solution B: Use degassedacetonitrile.
9 (25,5R,6R)-6-{(R)-2-[(R)-2-Amino-2-(4-hydroxyphenyl)ac etamido]-2-(4-
hydroxyphenyl)acetamido)-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0] Mobile phase: See the gradient table below.
heptane-2-carboxylic acid.
h The chromatographicsystem resolves two amoxicillin penilloic derivatives. Time Solution A Solution B
i (4S)-2-{[(R)-2-Amino-2-(4-hydroxyphenyl)acetamido]methyl}-5,5- (min) (%) (%)
dimethylthiazolidine-4-carboxylic acid.
j (45)-2-[5-(4-Hydroxyphenyl)-3,6-dioxopiperazin-2-yl]-5,5- 0 100 0
dimethylthiazolidine-4-carboxylic acid.
k (4S)-2-{[(R)-2-Amino-2-(4-hydroxyphenyl)acetamido]
15 65 35
methoxycarbonylmethyl}-5,5-dimethylthiazolidine-4-carboxylic acid. 20 0 100
I(25,5R,6R)-6-«2R)-2-{2-[(R)-2-Amino-2-( 4-hydroxyphenyl)acetamido]-2-
[(45)-4-carboxy-5,5-dimethylthiazolidin-2-yl]acetamldo)-2-(4- 22 0 100
hydroxyphenyl)acetamido)-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]
heptane-2-carboxylic acid. 23 100 0
30 100 0
SPECIFIC TESTS
• MICROBIAL ENUMERATION TESTS (61) and TE~TS FOR
SPECIFIED MICROORGANISMS (62): The total aerobic Standard solution: 2.0 mg/mL of USP Dextroamphetamine
microbial count does not exceed 103 cfu/g, and the total Sulfate RS in Solution A
combined molds and yeastscount does not exceed System suitability solution: Transfer 40 mL of the Standard
102cfu/g. solution to a 50-mL volumetric flask. Using a microliter
syringe, add 1 ~L each of USP Oextroamphetamine Related
ADDITIONAL REQUIREMENTS Compound A RS and USP Dextroamphetamine Related
• PACKAGING AND STORAGE: Preserve in tight containers and Compound B RS. Dilute with Standardsolution to volume,
store at controlled room temperature. . and mix.
• LABELING: When more than one Dissolution test is given, the Sample solution: 2.0 mg/mL of Amphetamine Sulfate in
labeling states the test used only if Test 1 is not used. Solution A
• USP REFERENCE STANDARDS (11) Chromatographic system
USP Amoxicillin RS (See Chromatography (621), System Suitability.)
USP Amoxicillin Related Compound D RS Mode: LC
(4S)-2.,{[(R)-2-Amino-2-( 4-hydroxyphenyl)acetamido] Detector: UV 257 nm
(carboxy)methyl}-5,5-dimethylthiazolidine-4-carboxylic Column: 4.6-mm x 15-cm; 5-~m packing L1
acid. Column temperature: 40°
C16H21N 30 6S 383.42 Flow rate: 1.5 mL/min
USP Clavulanate Lithium RS Injection size: 20 ~L
System suitability
Samples: Standardsolution and System suitability solution
[NoTE-Identify the peaks by the relative retention
times in Impurity Table 1 under OrganicImpurities.
Amphetamine Sulfate Amphetamine and dextroamphetamine have exactly
the same retention time.]
Suitability requirements
Resolution: NLT 3.0 between dextroamphetamine
related compound A and dextroamphetamine related
(C9H13N)2' H2S04 368.49 compound B, System suitability solution. .
Tailing factor: NMT 3.0, Standard solution
Benzeneethanamine, c-rnethyl-, sulfate (2:1), (±)-;
Relative standard deviation: NMT 2.0%, Standard
(±)-a-Methylphenethylamine sulfate (2:1) [60-13-9].
solution
Analysis
Samples: Standardsolution and Sample solution

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 USP 43 Official Monographs / Amphetamine 31 3

Calculate the percentage of (C9H 13N)2 . H2S04 in the ADDITIONAL REQUIREMENTS

portion of Amphetamine Sulfate taken: • PACKAGING AND STORAGE: Preserve in well-closed
containers.
Result = (ru/rs) x (Cs/Cu) x 100 • USP REFERENCE STANDARDS (11)
USP Dextroamphetamine Sulfate RS
ru = peak response for amphetamine sulfate from the USP Dextroamphetamine Related Compound A RS
Sample solution l-Phenyl-2-propanol.
rs = peak response for dextroamphetamine sulfate C9H120 136.20
from the Standard solution [CAS-14898-87-4].
C, =concentration of USP Dextroamphetamine USP Dextroamphetamine Related Compound B RS
Sulfate RS in the Standard solution(mg/mL) Phenyl acetone.
Cu =concentration of Amphetamine Sulfate in the C9H 100 134.18
Sample solution (mg/mL) [CAS-l03-79-7].
Acceptance criteria: 98.00/0-102.0% on the dried basis
IMPURITIES
INORGANIC IMPURITIES
• Residue on Ignition (281): NMT 0.2% Amphetamine Sulfate Tablets
ORGANIC IMPURITIES
DEFINITION
• Procedure Amphetamine Sulfate Tablets contain NLT 93.0% and NMT
Solution A, Solution B, Mobile phase, System suitability
107.0% of the labeled amount of amphetamine sulfate
solution, Standard solution, Sample solution,
[(C9H13N)2' H2S04 ] ,
Chromatographic system, and System suitability:
Proceed as directed in the Assay. IDENTIFICATION
Analysis • A. MELTING RANGE ORTEMPERATURE, Class 1(741)
Samples: Standard solution and Sample solution Sample: Macerate a quantity of powdered Tablets,
[NoTE-Identify the impurities by the relative retention . equivalent to 50 mg of amphetamine sulfate, with 10 mL
times in Impurity Table 1.] of water for 39 min, and filter into a small flask. To the
Calculate the percentage of each impurity in the portion of filtrate add 3 mL of 1 N sodium hydroxide. Cool to 10°-15°,
Amphetamine Sulfate taken: add 1 mL of a mixture of absolute ether and benzoyl
chloride (2:1), insert the stopper, and shake well for 3 min.
Result = (ru/rs) x (Cs/Cu) x (1 IF) x 100 Filter the precipitate, wash with 15 mL of cold water, and
,
recrystallize twice from diluted alcohol. Dry the residue at
=peak response for each impurity from the 80° for 2 h.
Sample solution Acceptance criteria: The crystals of the benzoyl derivative
= peak response for dextroamphetamine from the of amphetamine melt between 131° and 135°.
Standardsolution • B. The retention time of the major peak of the Sample
= concentration of USP Dextroamphetamine solution corresponds to that of the Standardsolution, as
Sulfate RS in the Standard solution(mg/mL) obtained in the Assay.
=concentration of Amphetamine Sulfate in the
Sample solution (mg/mL) . ASSAY
F = relative response factor (see Impurity Table 1) • PROCEDURE
Diluted acetic acid: 14 mL of glacial acetic acid in 100 mL
Acceptance criteria of water
Individual impurities: See Impurity Table 1. Mobile phase: Dissolve 1.1 9 of sodium l-heptanesulfonate
Total impurities: NMT 1.0% in 525 mL of water. Add 25 mL of diluted acetic acid and
450 mL of methanol. Adjust with glacial acetic add to a pH
Impurity Table 1 of 3.3 ± 0.1. Pass through a 0.5-f.Jm membrane filter.
Relative Relative Acceptance Standard solution: 0.3 mg/mL of USP
Retention Response Criteria, Dextroamphetamine Sulfate RS in 0.12 N phosphoric acid
Name Time Factor NMT (%) Sample solution: Nominally 0.3 mg/mL of amphetamine
Cathinone 0.81 55.6 0.25 sulfate from NLT 20 finely powdered Tablets prepared as
follows. Transfer a suitable amount of the powdered tablets
Amphetamine 1.0 1.0 - to a suitable volumetric flask. Add 80% of the flask volume
Benzaldehyde 1.73 105.3 0.25 of 0.12 N phosphoric acid, and sonicate for 15 min. Dilute
with 0.12 N phosphoric acid to volume. Pass through a
Dextroamphetamine 0.5-f.Jm membrane filter, discarding the first 20 mL of the
related compound A 1.88 1.5 0.25
filtrate.
Dextroamphetamine Chromatographic system
related compound B 2.05 1.8 0.25 (See Chromatography (621), System Suitability.)
Individual unspecified Mode: LC
impurity - 1.0 0.1 Detector: UV 254 nm
Column: 3.9-mm x 30-cm; 10-f.Jm packing L1
SPECIFIC TESTS Flow rate: 2 mL/min. [NOTE-A 4.6-mm x 25-cm column;
• Loss ON DRYING (731): Dry a sample at 105° for 2 h: it loses 5-f.Jm packing L1 may be used with a flow rate of 1 mLI
NMT 1.0% of its weight. min.] .
• DEXTROAMPHETAMINE: A solution (20 mg/mL) is optically Injection size: 50 f.JL
inactive. . System suitability
Sample: Standard solution

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 314 Amphetamine / OfficialMonographs USP 43

Suitability requirements
Tailing factor: NMT 3 Amphotericin B
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of HO
H3C"".
'.
o~'~"
OI~O ,.~
0 OH OH OH
",OH
.,' OH
amphetamine sulfate [(C9H nN)2 . H2S04] in the portion of IflCH3 !
Tablets taken: H3C"'" ~~~~~~~ : 0

Result = (rulr s) x (CslCu) x 100

=peak response from the Sample solution

= peak response from the Standardsolution
=concentration of USP Dextroamphetamine C47H 73N017 924.08
Sulfate RS in the Standardsolution (mg/mL)
= nominal concentration of amphetamine sulfate Amphotericin B.
in the Sample solution (mg/mL) Amphotericin B.
[1 R-(1 R*,3S*,5R*,6R*,9R*, 11 R*, 15S*,16R*,17 R*, 18S*,19£,
Acceptance criteria: 93.0%-107.0% 21 £,23£,25£,27£,29£,31 £,33R*,35S*,36R*,37S*)]-33- [(3-
Amino- 3,6-dideoxy-p-o-mannopyranosyl)oxy]-
PERFORMANCE TESTS 1,3,5,6,9,11,17,37-octahydroxy-15,16,18-trimethyl-13-
• DISSOLUTION, Procedure for a Pooled Sample (711) oxo-14,-39-dioxabicyclo[33.3.1 ]nonatriaconta-
Medium: Water; 500 mL 19,21,23,25,27,29,31-heptaene-36-carboxylic acid
Apparatus 1: 100 rpm [1397-89-3].
Time: 45 min
Standard solution: USP Dextroamphetamine Sulfate RS in » Amphotericin Bhas a potency of not less than
Medium having a known concentration of USP 750 JJg of C47H73N017 per mg, calculated on the
Dextroamphetamine Sulfate RS similar to the concentration
expected in the sample. dried basis.
Sample solution: Pass a portion of the solution under test
through a suitable filter. Packaging and storage-Preserve in tight, light-resistant
Diluted acetic acid: 14 mL of glacial acetic acid in1 00 mL containers, and store in a cold place.
of water Labeling-Label it to state whether it is intended for use in
Mobile phase: 1.1 g of sodium 1-heptanesulfonate in 575 preparing dermatological and oral dosage forms or parenteral
mL of water. Add 25 mL of Diluted aceticacid and 400 mL dosage forms.
of methanol. Adjust with glacial acetic acid to a pH of 3.3 USPReference standards (11)-
± 0.1. USP Amphotericin B RS
Chromatographic system . USP Nystatin RS
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 3.9-mm x 30-cm; packing L1 Ide~!i!i~~tiol1,.~~~~~t~~~~~~l~';!
Flow rate: 1 mL/min l.IItrqviqlet"\lis(f>lg!$pg9PI"Q$9QPy:YY1~
Injection size: 500 IJL Spectral range 1: 240 to 320 nm.
System suitability Solution 1: prepared as directed for Test preparation in the
Sample: Standardsolution Limit of amphotericin A, and compare its absorbance to that of
Suitability requirements the Amphotericin B standardpreparation. An extra peak may
Relative standard deviation: NMT 2.0% occur at 304 nm in the spectrum of this solution.
Analysis Spectral range 2: 320 to 400 nm.
Calculate the percentage of the labeled amount of Solution 2: prepared as directed for Test preparation in the
amphetamine sulfate [(C9H nN)2 . H2S04] dissolved: Limit of amphotericin A and then diluted with 9 volumes of
methanol. Compare its absorbance to that of a similar dilution
Result =(rulr s) x (Csf L) x V x 100 of the Amphotericin Bstandardpreparation.
Loss on drying (731)-Dry about 1OOmg, accurately
to =peak response from the Sample solution weighed, in a capillary-stoppered bottle in vacuum at a
ts = peak response from the Standardsolution pressure not exceeding 5 mm of mercury at 60° for 3 hours:
Cs = concentration of the Standardsolution (mg/mL) it loses not more than 5.0% of its weight.
Residue on ignition (281): not more than 0.5%, the charred
L =label claim (mg/Tablet) residue being moistened with 2 mL of nitric acid and 5 drops
V = volume of Medium, 500 mL
of sulfuric acid. [NoTE-Amphotericin B intended for use in
Tolerances: NLT 75% (Q) of amphetamine sulfate preparing dermatological creams, lotions, and ointments and
[(C9H13N)2' H2S04] is dissolved. oral suspensions and capsules, yields not more than 3.0%.]
• UNifORMITY Of DOSAGE UNITS (905): Meet the Limit of amphotericin A-
requirements Test preparation-Dissolve about 50 mg of Amphotericin B,
accurately weighed, in 10.0 mL of dimethyl sulfoxide in a
ADDITIONAL REQUIREMENTS
50-mL volumetric flask, dilute with methanol to volume and
• PACKAGING AND STORAGE: Preserve in well-closed
mix. Transfer 4.0 mL of this solution to a 50-mL volumetric
containers.
flask, dilute with methanol to volume, and mix.
• USP REfERENCE STANDARDS (11)
USP Dextroamphetamine Sulfate RS

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 USP 43 OfficialMonographs / Amphotericin 315

Nystatin standardpreparation-Dissolve about 20 mg of

USP Nystatin RS, accurately weighed, in 40.0 mL of dimethyl Amphotericin B for Injection
sulfoxide in a 200-mL volumetric flask, dilute with methanol
to volume, and mix. Transfer 4.0 mL of this solution to a 50-mL
» Amphotericin B for Injection is a sterile complex
volumetric flask, dilute with methanol to volume, and mix.
Amphotericin Bstandardpreparation-Dissolve about 50 mg of Amphotericin B and deoxycholate sodium and
of USP Amphotericin B RS, accurately weighed, in 10.0 mL of one or more suitable buffers. It contains not less
dimethyl sulfoxide in a 50-mL volumetric flask, dilute with than 90.0 percent and not more than 120.0
methanol to volume, and mix. Transfer 4.0 mL of this solution percent of the labeled amount of C47H73N017'
to a 50-mL volumetric flask, dilute with methanol to volume,
and mix. Prepare this solution fresh daily. Packaging and storage-Preserve as described in
Procedure-Concomitantly determine the absorbances of Packaging and Storage Requirements (659), InjectionPackaging,
the Nystatin and Amphotericin Bstandardpreparations and the Packaging for constitution, in a refrigerator and protected from
Test preparation in 1-cm cells at 304 nm and at 282 nm, with light.
a suitable spectrophotometer, using a 1 in 62.5 solution of labeling-Label it to indicate that it is intended for use by
dimethyl sulfoxide in methanol as the blank. Calculate the intravenous infusion to hospitalized patients only, and that the
percentage of amphotericin A taken by the formula: solution should be protected from light during administration.
USPReference standards (11)-
USP Amphotericin B RS
25WN[(ABm X A U304) - (A B304 X Aum)l!
Bacterial Endotoxins Test (85) -It contains not more than
[(AB282 X A N304) - (A B304 X A Nm)] Wu 5.0 USP Endotoxin Units per mg of amphotericin B. For
products used or labeled for intrathecal injection, it contains
not more than 0.9 USP Endotoxin Unit per mg of amphotericin
in which WN is the weight, in mg, of USP Nystatin RS taken, B.
A8282 and A8304 are the absorbances of the Amphotericin B Sterility Tests (71) -It meets the requirements when tested
standard preparation at 282 nm and 304 nm, respectively, as directed in the section Membrane Filtration under Test for
AN2 82 and AN 304 are the absorbances of the Nystatin standard Sterility of the Product to be Examined, 50 mg from each
preparation at 282 nm and 304 nm, respectively, AU282 and container being tested.
AU304 are the absorbances of the Test preparation at 282 nm pH (791): between 7.2 and 8.0, in an aqueous solution
and 304 nm, respectively, and Wu is the weight, in mg, of the containing 10 mg of amphotericin B per mL.
Amphotericin B taken: not more than 5%, calculated on the Loss on drying (731 )-Dry about 100 mg in a
dried basis, is found. [NoTE-Amphotericin B intended for use capillary-stoppered bottle in vacuum at a pressure not
in preparing dermatological creams, lotions, and ointments, exceeding 5 mm of mercury at 60° for 3 hours: it loses not
and oral suspensions and capsules, contains not more than more than 8.0% of its weight.
15% of amphotericin A, calculated on the dried basis.] Other requirements-It meets the requirements for
Assay-Proceed with amphotericin B as directed under Uniformity of Dosage Units(905) and for Labeling (7), Labels and
Antibiotics-Microbial Assays (81). Labeling for Injectable Products.
Assay-
Assay preparation 7 (where it is packaged as a single-dose
container)-Constitute Amphotericin B for Injection as
directed in the labeling. Withdraw all of the withdrawable
Amphotericin B Cream contents, using a suitable hypodermic needle and syringe, and
dilute quantitatively and stepwise with dimethyl sulfoxide to
obtain a solution containing about 20 I-Ig of amphotericin B
» Amphotericin B Cream contains not less than per mL.
90.0 percent and not more than 125.0 percent of Assay preparation 2 (where the labeling states the quantity
the labeled amount of Amphotericin B. of amphotericin B in a given volume of constituted solution)
-Constitute Amphotericin B for Injection as directed in the
Packaging and storage-Preserve in collapsible tubes, or labeling. Withdraw an accurately measured volume of the
other well-closed containers. resultant solution, using a suitable hypodermic needle and
USPReference standards (11)- syringe, and dilute quantitatively and stepwise with dimethyl
USP Amphotericin B RS sulfoxide to obtain a solution containing about 20 I-Ig of
Minimum fill (755): meets the requirements. amphotericin B per mL.
Assay-Proceed as directed for amphotericin B under Procedure-Proceed as directed for amphotericin B under
Antibiotics-Microbial Assays (81), blending a suitable Antibiotics-Microbial Assays (81), using an accurately
accurately weighed portion of Cream in a high-speed blender measured volume of Assay preparation diluted quantitatively
with a sufficient accurately measured volume of dimethyl and stepwise with Buffer B.1 0 to obtain a Test Dilution having
sulfoxide to give a convenient concentration. Quantitatively a concentration assumed to be equal to the median dose level
dilute an accurately measured volume of this solution with of the Standard.
dimethyl sulfoxide to obtain a stock solution having a
concentration of about 20 I-Ig of amphotericin B per mL.
Quantitatively dilute an accurately measured volume of this
stock solution with Buffer B. 70 to obtain a Test Dilution having
a concentration assumed to be equal to the median dose level
of the Standard.

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 316 Amphotericin / Official Monographs USP 43

Amphotericin B Lotion Ampicillin

o

00x~~
»Amphotericin BLotion contains not less than 90.0
percent and not more than 125.0 percent of the
labeled amount of amphotericin B.
NH2

Packaging and storage-Preserve in well-closed C16H19N304S (anhydrous) 349.41

containers.
4-Thia-1-azabicyclo[3.2.0]heptane-2 carboxylic acid, [6-
USP Reference standards (11) -
(aminophenylacetyl)amino]-3,3-dimethyl-7-oxo-, [2S-[2u,
USP Amphotericin B RS
5u,6~(S*)]]-;
Minimum fill (755): meets the requirements. (2S,5R,6R)-6-[(R)-2-Amino-2-phenylacetamido]-3,3-
pH (791): between 5.0 and 7.0. dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-
Assay-Proceed as directed for amphotericin B under
carboxylic acid [69-53-4].
Antibiotics-Microbial Assays (81), quantitatively dissolving a Trihydrate 403.46
suitable accurately measured volume of Lotion in sufficient
[7177-48-2].
dimethyl sulfoxide to give a convenient concentration.
Quantitatively dilute an accurately measured volume of this DEFINITION
solution with dimethyl sulfoxide to obtain a stock solution Ampicillin is anhydrous or contains three molecules of water
having a concentration of about 20 ~g of amphotericin B per of hydration. It contains NLT 900 ~g/mg and NMT 1050 ~gl
mL. Quantitatively dilute an accurately measured volume of mg of ampicillin (C16H,9N304S), calculated on the anhydrous
this stock solution with Buffer 8.10 to obtain a Test Dilution basis.
having a concentration assumed to be equal to the median
dose level of the Standard. IDENTIFICATION

(Jf~~)~I~lt?l(j,",gg
Amphotericin B Ointment ~/Jl~ p Except that where the
specimen under test is the trihydrate, both it and the USP
Ampicillin Trihydrate RS are undried.
» Amphotericin B Ointment is Amphotericin B in a
ASSAY
suitable ointment base. It contains not less than
• PROCEDURE
90.0 percent and not more than 125.0 percent of Solution A: 6.54 giL of monobasic potassium phosphate
the labeled amount of amphotericin B. and 0.34 giL of dibasic potassium phosphate, adjusted with
1 N sodium hydroxide or 1 N phosphoric acid to a pH of
Packaging and storage-Preserve in collapsible tubes, or 5.5 before final dilution
other well-closed containers. Solution B: Acetonitrile and Solution A (2:23)
USP Reference standards (11) - Solution C: Acetonitrile and Solution A (3:7)
USP Amphotericin B RS Mobile phase: See Table 1.
Minimum fill (755): meets the requirements.
Water Determination, Method I (921): not more than 1.0%, Table 1
20 mL of a mixture of toluene and methanol (7:3) being used Time Solution B Solution C
in place of methanol in the titration vessel. (min) (0/0) (0/0)
Assay-Proceed as directed for amphotericin B under
Antibiotics-Microbial Assays (81), using an accurately weighed 0 100 0
portion of Ointment, equivalent to about 30 mg of 6 100 0
amphotericin B, mixed with 10.0 mL of ether in a suitable 0,
15 100
glass-stoppered conical flask and allowed to stand, with
intermittent shaking, for 1 hour. Add 20.0 mL of dimethyl 16 0 100
sulfoxide and shake by mechanical means for 10 minutes.
18 100 0
Dilute quantitatively and stepwise with dimethyl sulfoxide to
a concentration of approximately 20 ~g per mL. Quantitatively 20 100 0
dilute an accurately measured volume of this stock solution
with BufferB.1 0 to obtain a Test Dilution havlnq a Solution D: 46.3 giL of monobasic potassium 'phosphate
concentration assumed to be equal to the median dose level and 27.8 giL of dibasic potassium phosphate, adjusted with
of the Standard. 1 N sodium hydroxide or 1 N phosphoric acid to a pH of
6.5 before final dilution
System suitability solution: 0.5 mg/mL of USP Ampicillin
RS and 0.1 mg/mL of USP Amoxicillin RS in acetonitrile,
water, and Solution 0 (4:91 :5) prepared asfollows. Dissolve
first in a mixture of acetonitrile, water, andSolution 0
(4:30:5), sonicating if necessary, and dilute with water to
volume. .
Standard solution: 0.5 mg/mL of USP Ampicillin RS in
acetonitrile, water, and Solution 0 (4:91 :5) prepared as
follows. Dissolvefirst in a mixture of acetonitrile, water, and

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USP 43 OfficialMonographs / Ampicillin 317

Solution 0 (4:30:5), sonicating if necessary, and dilute with Tailing factor: NMT 1.4 for ampicillin, System suitability
water to volume. Analyze immediately after preparation. solution
Sample solution: 0.5 mg/mL of Ampicillin in acetonitrile, Relative 'standard deviation: NMT 10.0%, Standard
water, and Solution 0 (4:91 :5) prepared as follows. Dissolve solution
first in a mixture of acetonitrile, water, and Solution 0 Analysis
(4:30:5), sonicating if necessary, and dilute with water to Samples: Standardsolution and Sample solution
volume. Analyze immediately after preparation. Calculate the percentage of each impurity in the portion of
Chromatographic system Ampicillin taken:
(See Chromatography (621), System Suitability.)
Mode: LC Result =(r vir s) x (C siC v) x P x F x 100
Detector: UV 230 nm
Column: 4.6-mm x 15-cm; 5-lJm packing L1 ru = peak response of each impurity from the Sample
Flow rate: 1.5 mL/min solution
Injection volume: 20 IJL rs = peak response of ampicillin from the Standard
System suitability solution
Samples: System suitability solution and Standard solution Cs =concentration of USPAmpicillin RS in the
Suitability requirements Standardsolution (mg/mL)
Resolution: NLT 10 between ampicillin and amoxicillin, Cu =concentration of Ampicillin in the Sample solution
System suitability solution (mg/mL)
Tailing factor: NMT 1.4 for ampicillin, System suitability P =potency of USPAmpicillin RS (lJg/mg)
solution F = conversion factor, 0.001 mg/lJg
Relative standard deviation: NMT 2.0%, Standard
solution Acceptance criteria: See Table 2.
Analysis
Samples: Standardsolution and Sample solution Table 2
Calculate the quantity, in IJg, of ampicillin (C16H19N304S) in Relative Acceptance
each mg of Ampicillin taken: Retention Criteria,
Name Time NMT(%)
Result =(r vir s) x (C siC v) x P o-Phenyiqlyclne" 0.27 0.5

rv = peak response from the Sample solution Amoxicillin related

compound A
rs = peak response from the Standardsolution (6-aminopenicillanic acid)" 0.31 0.5
Cs =concentration of USP Ampicillin RS in the Ampicilloic acid' 0.45 1.0
Standardsolution (mg/mL)
Cu = concentration of Ampicillin in the Sample solution Ampicillin thiazepine analoq" 0.65 0.3
(mg/mL)
P = potency of USPAmpicillin RS (lJg/mg)
Ampicillin 1.0 -
Ampicillin rearrangement
Acceptance criteria: 900-1050 IJg/mg on the anhydrous product (isomer l)e 1.8 0.4
basis Ampicillin rearrangement
product (isomer 2)e 2.0 0.3
IMPURITIES
• ORGANIC IMPURITIES, PROCEDURE 1 Ampicillin oligomer 21 2.2 0.6
OrganicImpurities, Procedure 1 is recommended when the
D-Phenylglycylampicillin9 2.5 0.8
impurity profile includes ampicillin thiazepine.
Solution A, Solution 8, Solution C, Mobile phase, Solution Ampicillin oligomer 1 (dlrner)" 2.6 1.0
0, System suitability solution, Sample solution, and
Ampicillin oligomer 1 (trimer)' 2.9 0.4
Chromatographic system: Prepare as directed in the
Assay. Anyindividual unspecified im-
-
Standard stock solution: Prepare as directed for the purity 0.25
Standardsolution in the Assay. . Total impurities - 3.0
Standard solution: 0.005 mg/mL of ampicillin in Solution 0
and water (1:19) from Standardstock solution. Transfer an a (R)-2-Amino-2-phenylacetic acid.
aliquot of the Standardstock solution to a suitable b (2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1-azabicyc1o[3.2.0]
volumetric flask, add Solution 0, using about 5% of the final heptane-2-carboxylicacid.
volume, and dilute with water to volume. Analyze c(4S)-2-{[(R)-2-Amino-2-phenylacetamido](carboxy)methyl}-5,5-
dimethylthiazolidine-4-carboxylic acid.
immediately after preparation. d (S)-6-[(R)-2-Amino-2-phenylacetamido]-2,2-dimethyl-7-oxo-2,3,4,7-
SensitiVity solution: 0.5 IJg/mL of ampicillin in Solution 0 tetrahydro-1,4-thiazepine-3-carboxylic acid.
and water (1 :19) from the Standardsolution. Transfer an e (4S)-2-(3,6-Dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-
aliquot of the Standardsolution to a suitable volumetric carboxylic acid.
flask, add Solution 0, using about 5% of the final volume, f (4S)-2-{1-[(R)-2-Amino-2-phenylacetamido]-2-[(1 R)-2-{carboxy[(45)-4-
and dilute with water to volume. carboxy-5,5-dimethylthiazolidin-2-yl]methylamino}-2-oxo-1-
phenylethylamino]-2-oxoethyl}-5,5-dimethylthiazolidine-4-carboxylic acid.
System suitability 9 (25,5R,6R)-6-{(R)-2-[(R)-2-Amino-2-phenylacetamido]-2- phenylacetamido}-
Samples: Sensitivity solution, System suitabilitysolution, and 3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid.
Standardsolution h (25,5R,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(45)-4-
Suitability requirements carboxy-5,5-dimethylthiazolidin-2-yl]acetamido}-2-phenylacetamido]-3,3-
Signal-to-noise ratio: NLT 3, Sensitivity solution dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid. .
Resolution: NLT 10 between ampicillin and amoxicillin, i (45,4'5)-2,2'-{(1 R,7R,13R)-1-Amino-14-[(2S,5R,6R)-2-carboxy-3,3-dimethyl-
7-oxo-4-thia-1-azabicyclo[3.2.0]heptan-6-ylamino]-2,5,8,11,14-pentaoxo-
System suitability solution l,7,13-triphenyl-3,6,9,12-tetraazatetradecane-4,l 0-diyl}bis(5,5-
dimethylthiazolidine-4-carboxylic acid).

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318 Ampicillin / OfficialMonographs USP 43

• ORGANIC IMPURITIES, PROCIEDURE_ 2, DIMETHYLANILINE =concentration of Ampicillin in the Sample solution

(223): Meets the requirements
Organic Impurities, Procedure 2 is recommended when P . '11'In In
(mg/mL)0 f ampict
= potency • USP A rnprci
. '11'In RS
dimethylaniline is used during the production of (~g/mg)
Ampicillin. F =conversion factor, 0.001 mg/~g
• ORGANIC IMPURITIES, PROCEDURE 3
OrganicImpurities, Procedure 3 is recommended when the Acceptance criteria: See Table 4 and Table 5. The limits in
impurity profile includes phenylpyrazinol, pivaloyl Table 5 are to be used only where Ampicillin is intended for
phenylglycine, pivaloyl aminopenicillanic acid, use in preparing veterinary products.
diphenyldiketopiperazine, and open ring dimer.
Solution A: 4 giL of monobasic sodium phosphate Table 4
L dihydrate adjusted with 1 N sodium hydroxide to a pH of Relative Acceptance
5.0 Retention Criteria,
Solution B: Acetonitrile Name Time NMT (%)
Mobile phase: See Table 3. o-Phenylqlycine- 0.15 1.0

Table 3 Amoxicillin related

compound A
Time Solution A Solution B (6-aminopenicillanic add)" 0.21 1.0
(min) (%) (%)
0.40
0 98 2
Ampicilloic acidc,d 0.58 1.0
20 90 10
t-Amplclllln" 0.65 1.0
40 85 15
Ampicillin 1.0 -
50 80 20
1.16
55 75 '25
, Ampilloic acid" 9 1.40 1.0
60 75 25
1.25
62 98 2 Ampicillin rearrangement prod-
ucth,l 1.48 1.0
70 98 2
Phenylpyrazinol l 1.75 1.0
Diluent: Acetonitrile and SolutionA (2:98) Pivaloyl phenylqlydne" 1.87 1.0
System suitability solution: 1.5 mg/mL of USP Ampicillin
Dlphenyldiketoplperazlne' 1.94 1.0
System Suitability Mixture RS in Diluent
Standard solution: 15 ~g/mL of USP Ampicillin RS in Diluent Ampicillin oligomer 2m 2.08 1.0
Sample solution: 1.5 mg/mL of Ampicillin in Diluent. Store
o-Phenylglycylampicillin" 2.25 1.0
the sample in the refrigerator, and discard after 60 min,
Chromatographic system Pivaloyl aminopenicillanic acld" 2.54 1.0
(See Chromatography(621), System Suitability.) 2.87
Mode: LC
Detector: UV 220 nm 2.97
Column: 4.6-mm x 15-cm; 5-~m packing L7 Open ring dimerP' q 3.03 1.0
Column temperature: 40°
Flow rate: 1.5 mL/min Ampicillin oligomer 1 (dimer)' 3.15 1.0
Injection volume: 20 ~L Ampicillinyl-o-phenylglycine' 3.86 1.0
Autosampler temperature: 4°
System suitability Ampicillin oligomer 1 (trimer)' 4.19 1.0
Samples: System suitability solution and Standardsolution Any individual unspecified im-
Suitability requirements purity - 0.10
Resolution: NLT 1.5 between the pivaloyl phenylglycine
and diphenyldiketopiperazine peaks, System sUitability
solution
Tailing factor: NMT 2.0, Standardsolution
Relative standard deviation: NMT 5.0%, Standard
solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of
Ampicillin taken:

Result = (r vir s) x (C siC v) x P x Fx 100

ru = peak response of each impurity from the Sample

solution
rs = peak response of ampicillin from the Standard
solution
Cs = concentration of USP Ampicillin RS in the
Standard solution (mg/mL)

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 USP 43 OfficialMonographs / Ampicillin 319

Table 4 (continued) Table 5 (continued)

Relative Acceptance Relative Acceptance
Retention Criteria, Retention Criteria,
Name Time NMT(%) Name Time NMT (%)
Total impurities - 5.0 Pivaloyl phenylglycine' 1.87 2.0

a (R)-2-Amino-2-phenylacetic acid. Dlphenyldlketoplperazine" 1.94 2.0

b (25,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-l-azabicycl0[3.2.0] Ampicillin oligomer2" 2.08 2.0
heptane-2-carboxylic acid.
c (4S}-2-{[(R)-2-Amino-2-phenylacetamido](carboxy)methyl}-5,5- o-Phenylqlycylarnpldllln'' 2.2S 2.0
dimethylthiazolidine-4-carboxylic acid.
d The system resolves the two isomers of ampicilloic acid. The sum of the two Pivaloyl aminopenicillanic
isomersis reported. acidP 2.S4 2.0
e (25,5R,6R)-6-[(S}-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-l- 2.87 O.SO
azabicyclo[3.2.0]heptane-2-carboxylic acid.
f (4S}-2-{[(R)-2-Amino-2-phenylacetamido]methyl}-5,5-dimethylthiazolidine-4- 2.97 O.SO
carboxylic acid.
9 The system resolves the two isomers of ampilloic acid. The sum of the two Open ring dimer'!' r 3.03 O.SO
isomersis reported.
Ampicillin oligomer1 (dimer)' 3.1S 4.S
h(4S}-2-(3,6-Dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-
carboxylic acid. Ampicillinyl-o-phenylglycinet 3.86 2.0
i The system resolves the two isomers of ampicillin rearrangement product. The
sum of the two isomersis reported. Ampicillin oligomer1 (trimer)" 4.19 2.0
j 3-Phenylpyrazin-2-01.
k(R)-2-Phenyl-2-pivalamidoacetic acid. Anyindividual unspecified lrn- -
purity O.S
13,6-Diphenylpiperazine-2,5-dione.
m (4S}-2-{1-[(R)-2-Amino-2-phenylacetamido]-2-[(1 R)-2-{carboxy[(4S}-4- Total impurities - S.O
carboxy-5,S-dimethylthiazolidin-2-yl]~ethylan:ino}:2~oxo-1- ..
phenylethylamino]-2-oxoethyl}-S,S-dlmethylthlazolldtne-4-carboxyllc add. a (R)-2-Amino-2-phenylacetic acid.
n (25,SR,6R)-6-{(R)-2-[(R)-2-Amino-2-phenylacetamido]-2-phenyl~cet~m ido) ­ b(25,SR,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]
3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxyhc add. heptane-2-carboxylic acid.
o (25,SR,6R)-3,3-Dimethyl-7 -oxo-6-pivalamido-4-thia-l-azabicyclo[3.2.0] c(4S}-2-{[( R)-2-Amino-2 -phenylacetamido](carboxy)methyl}-3-formyl-S ,S-
heptane-2-carboxylic acid. dimethylthiazolidine-4-carboxylic acid.
p (4S}-2-{1-[(R)-2-amino-2-phenylacetamido]-2-[(1 R)-2-{[(4S}-~-carboxy-S,S­ d (4S}-2-{[(R)-2-Amino-2-phenylacetamido](carboxy)methyl}-S ,S-
dimethylthiazolidin-2-yl]methylamino}-2-oxo-l-phenylethylamlno]-2- dimethylthiazolidine-4-carboxylic acid.
oxoethyl}-S,S-dimethylthiazolidine-4-carboxylic acid. e The system resolves the two isomers of ampicilloic acid. The sum of the two
q The system may resolve the three isomers of open ring dimer. The sum of the isomersis reported.
three isomers is reported. f (25,5R,6R)-6-[(S}-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-l-
r (25,SR,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetami~0]-2-[(4S}.-4-ca rboxy­ azabicyclo[3.2.0]heptane-2-carboxylic acid.
S,S-dimethylthiazolidin-2-yl]acetamido}-2-phenyl~cet~mldo]- 3,3-dlmethyl-7- 9 (4S}-2-{[( R)-2-Amino-2-phenylacetamido]methyl}-S,S-dimethylthiazolidine-
oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxyhc acid. , 4-carboxylic acid.
s (R)-2-((25,SR,6R)-6-((R)-2-Amino-2-phenylacetamido)-3,3-dir:nethy'-7-oxo-4- hThe system resolves the two isomers of ampilloic acid.The sum of the two
thia-l-azabicyclo[3.2.0]heptane-2-carboxamido)-2-phenylacetle acid, isomers is reported.
t (45,4'S}-2,2'-{(1 R,7R, 13R)-1-Amino-14-[(25,SR,6R)-2-carboxy-3,3-dimethyl-
i (4S}-2-(3,6-Dioxo-S-phenylpiperazin-2-yl)-5,S-dimethylthiazolidine-4-
7-oxo-4-thia-l-azabicyclo[3.2.0]heptan-6-ylamino]-2,S,8,11,14-pentaoxo- carboxylic acid.
1,7,13-triphenyl-3,6,9,12-tetraazatetradecane-4,10-diyl}bis(S,5-
dimethylthiazolidine-4-carboxylic acid). . j The system resolves the two isomers of ampicillin rearrangement product. The
sum of the two isomers is reported.
k3-Phenylpyrazin-2-01.
Where it is intended for use in preparing veterinary i (R)-2-Phenyl-2-pivalamidoacetic acid.
products: m 3,6-Diphenylpiperazine-2,S-dione.
n(4S}-2-{1-[(R)-2-Amino-2-phenylacetamido]-2-[(1 R)-2-{carboxy[(4S}-4-
Table 5 carboxy-S S-dimethylthiazolidin-2-yl]methylamino}-2-oxo-l-
Relative Acceptance phenylethylamino]-2-oxoethyl}-S,S-dimethylthiazolidine-4-carboxylic acid.
Retention Criteria, o (25,SR, 6R)-6-{(R)-2-l(R)-2-Amino-2-phenylacetamido]-2-phenyl~cet~mido)­
Name Time NMT(%) 3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxyhc acid.
p (2S,SR,6R)-3,3-Dimethyl-7-oxo-6-pivalamido-4-thia-l-azabicyclo[3.2.0]
o-Phenylqlyclne- O.lS 2.0 heptane-2-carboxylic acid. ,
q (4S}-2-{1-[(R)-2-amino-2-phenylacetamido]-2-[(1 R)-2-{[(4S}-~-carboxy-S,S­
Amoxicillin related compound dimethylthiazolidin-2-yl]methylamino}-2-oxo-1-phenylethylamlno]-2-
A oxoethyl}-S,S-dimethylthiazolidine-4-carboxylic acid.
(6-aminopenicillanic add)" 0.21 2.0
rThe system may resolve the three isomers of open ring dimer.
N-Formyl ampicilloic acid' 0.26 1.0 s (25,SR,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(4S}-4-carboxy-
S,S-dimethylthiazolidin-2-yl]acetamido}-2-phenyl~cet~mido]- 3,3-dimethyl-7-
0040 oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxyhc add,
t (R)-2-((25,SR, 6 R)-6-((R)-2-Amino-2-phenyl~cetamido)- 3,3-dir~1eth'yl- 7-oxo-4-
Ampicilloic acidd, e 0.S8 2.0 thia-l-azabicyclo[3.2.0]heptane-2-carboxamldo)-2-phenylacetlc acid.
u (45,4'S}-2,2'-{(1 R,7R, 13R)-1-Amino-14-[(25,S.R,6R)-2-carboxy-3,3-dimethyl-
t-Arnplclllln' 0.6S 2.0
7-oxo-4-thia-l-azabicyclo[3.2.0]heptan-6-ylamlno]-2,S,8,11,14-pentaoxo-
Ampicillin 1.0 - 1,7,13-triphenyl-3,6,9,12-tetraazatetradecane-4,1O-diyl}bis(S,S-
dimethylthiazolidine-4-carboxylic acid).
1.16
Ampilloic acid9' h lAO 2.0 • ORGANIC IMPURITIES, PROCEDURE 4
OrganicImpurities, Procedure 4 is recommended when the
1.2S impurity profile includes ampilloyl aminopenicillanic acid
Ampicillin rearrangement
product-I 1048 2.0 and penldllanyl ampicillinamide.

Phenylpyrazlnol" 1.7S 2.0

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320 Ampicillin / OfficialMonographs USP43

Solution A: 3.4 giL of dibasic sodium phosphate Table 7

dodecahydrate and 1.4 giL of monobasic potassium Relative Acceptance
phosphate adjusted with phosphoric acid to a pH of 5.5 Retention Criteria,
Solution B: Acetonitrile Name Time NMT (%)
Mobile phase: See Table 6. o-Phenylqlyclne- 0.21 0.5

Table 6 Amoxicillin related compound A

(6-aminopenicillanic acld)" 0.32 0.5
Time Solution A Solution B
(min) (%) (%) 0.46 1.0
0 99 1 Ampicilloic acld- 0.57 1.0
1.5 95 5 Ampicillin thiazepine analogd, e 0.72 -
6.5 90 10 t-Arnplclllln' 0.84 0.5
7.5 89 11 Ampilloyl aminopenicillanic acid9 0.87 0.5
13.5 84 16 Ampicillin 1.00 -
16.5 75 25 1.15 1.0
18 60 40 Ampilloic add" 1.34 1.0
25 99 1 Ampicillin rearrangement product! 1.24 1.0
Pivaloyl phenylqlyclnev j 1.47 -
Standard solution: 30 IJg/mL of USP Amoxicillin Related
Compound A RS, 30 IJg/mL of o-phenylglycine, and 25 IJgI Phenylpyrazlnolv k 1.84 -
mL of USP Ampicillin RS in Solution A Dlphenyldlketoptperazlnev I 1.94 -
Sample solution: 2.5 mg/mL of Ampicillin in Solution A.
Storethe Sample solution in the refrigerator, and usewithin Pivaloyl aminopenicillanic
acide,m 1.95 -
9 h.
Chromatographic system o-Phenylglycylampicillin n 2.08 1.0
(See Chromatography (621), System Suitability.)
Ampicillin oligomer 1 (dlmer)" 2.16 1.0
Mode: LC
Detector: UV 220 nm Penicillanyl ampicillinamideP 2.27 1.0
Column: 4.0-mm x 15-cm; 3-lJm packing L1
Arnpldlllnyl-o-phenylqlyclnes 2.64 1.0
Column temperature: 40°
Flow rate: 1.3 mL/min Anyindividual unspecified impurity - 1.0
Injection volume: 5 IJL Total impurities - 5.0
Autosampler temperature: 4°
System suitability a (R)-2-Amino-2-phenylacetic acid.
Sample: Standardsolution b (25,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]
Suitability requirements heptane-2-carboxylic acid.
Resolution: NLT 1.5 between o-phenylglycine and c(45)-2-{[(R)-2-Amino-2-phenylacetamido](carboxy)methyl}-5,5-
amoxicillin related compound A dimethylthiazolidine-4-carboxylic acid.
Analysis d (5)-6-[(R)-2-Amino-2-phenylacetamido]-2,2-dimethyl-7-oxo-2,3,4,7-
tetrahydro-l ,4-t~iazepine-3-carboxylic acid.
Samples: Standardsolution and Sample solution eThese impurities are listedfor information only.They are not to be reported.
Calculate the percentage of each impurity in the portion of They are not to be included in total impurities.
Ampicillin taken: f (25,5R,6R)-6-[(5)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-l-
azabicyclo[3.2.0]heptane-2-carboxylic acid.
Result =(r vir s) x (C siC v) x P x F x 100 9 (25,SR,6R)-6-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(45)-4-carboxy-5,5-
dimethylthiazolidin-2-yl]acetamidoH,3-dimethyl-7-oxo-4-thia-l-
ru = peak response of each impurity from the Sample azabicyclo[3.2.0]heptane-2-carboxylic acid.
h (45)-2-{[(R)-2-Amino-2-phenylacetamido]methyl}-5,5-dimethylthiazolidine-
solution 4-carboxylic acid.
rs = peak response of ampicillin from the Standard i (45)-2-(3,6-Dioxo-5-phenylpiperazin-2-yl)-5,5-dimethylthiazolidine-4-
solution carboxylic acid.
Cs =concentration of USP Ampicillin RS in the j (R)-2-Phenyl-2-pivalamidoacetic acid.
Standardsolution (mg/mL) k3-Phenylpyrazin-2-ol.
Cu =concentration of Ampicillin in the Sample solution I3,6-Diphenylpiperazine-2,5-dione.
(mg/mL) m (25,SR,6R)-3,3-Dimethyl-7-oxo-6-pivalamido-4-thia-l-azabicyclo[3.2.0]
P =potency of ampicillin in USP Ampicillin RS heptane-2-carboxylic acid.
n (25,5R,6R)-6-{(R)-2-[(R)-2-Amino-2-phenylacetamido]-2- phenylacetamido}-
(lJg/mg) 3,3-dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid.
F = conversion factor, 0.001 mg/lJg o (2S,5R,6R)-6-[(2R)-2-{2-[(R)-2-Amino-2-phenylacetamido]-2-[(45)-4-
carboxy-5,5-dimethylthiazolidin-2-yl]acetamido}-2-phenylacetamido]-3,3-
Acceptance criteria: See Table 7. Disregard any peak with dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid.
an area less than 0.03 times the area of the ampicillin peak p (25,5R,6R)-6-{(2S,5R,6R)-6-[(R)-2-Amino-2-phenylacetam ido]-3,3-dimethyl-
in the System suitability solution. 7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-carboxamido}-3,3-dimethyl-7-oxo-
4-thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid.
q (R)-2-«2S,5R,6R)-6-«R)-2-Amino-2-phenylacetamido)-3,3-dimethyl-7-oxo-4-
thia-l-azabicyclo[3.2.0]heptane-2-carboxamido)-2-phenylacetic acid,

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 USP 43 Official Monographs / Ampicillin 321

SPECIFIC TESTS USP/Reference standards (11)-

• STERILITY TIESTS (71) USP Ampicillin RS
Sample solution: Dissolve 6 gin 800 mL of FluidD Identification-Powder 1 or more Boluses, and prepare a
containing sufficient sterile penicillinase to inactivate the solution containing the equivalent of 10 mg of ampicillin per
ampicillin, and swirl the vessel until dissolution is complete mL in a mixture of acetone and 0.1 N hydrochloric acid (4:1):
before filtering. the resulting solution responds to the Identification test under
Acceptance criteria: Where the label states that Ampicillin Ampicillin Capsules.
is sterile, it meets the requirements when tested as Uniformity of dosage units (905): meet the requirements.
directed for Test for Sterility of the Product to Be Examined, Loss on drying (731): not more than 5.0%.
Membrane Filtration.
• CRYSTALLINITY (695): Meets the requirements
Assay-
• pH (791)
Standardpreparation-Prepare as directed for Standard
Sample solution: 10 mg/mL Preparation under todometrk: Assay-Antibiotics (425), using
Acceptance criteria: 3.5-6.0 USP Ampicillin RS.
• WATIER DETERMINATION, Method I (921): NMT 2.0% where
Assay preparation-Place not fewer than 5 Boluses in a
it is labeled asAmpicillin (anhydrous); between 12.0% and high-speed glass blender jar containing an accurately
15.0% where it is labeled asAmpicillin (trihydrate) measured volume of water, and blend for 4 ± 1 minutes. Dilute
• BACTERIAL ENDOTOXINS TEST (85): Where the label states
an accurately measured volume of this stock solution
that Ampicillin is sterile or must be subjected to further quantitatively and stepwise with water to obtain an Assay
processing during the preparation of injectable dosage preparation containing about 1.25 mg of ampicillin per mL.
forms, it contains NMT 0.15 USP Endotoxin Unit/mg of Procedure-Proceed as directed for Procedure under
ampicillin. lodometricAssay-Antibiotics (425). Calculate the quantity, in
mg, of ampicillin (C16H19N304S) in each Bolus taken by the
ADDITIONAL REQUIREMENTS formula:
• PACKAGING AND STORAGE: Preserve in tight containers.
• LABELING: Label to indicate whether it is anhydrous or is the (T/D)(F/2000)(B - f)
trihydrate. Where the quantity of ampicillin is indicated in
the labeling of any preparation containing Ampicillin, this hi which T is the labeled quantity, in mg, of ampicillin in each
shall be understood to be in terms of anhydrous ampicillin Bolus; and D is the concentration, in mg per mL, of ampicillin
(C16H19N304S), Where it is intended for use in preparing in the Assay preparation on the basisof the labeled quantity in
injectable dosage forms, the label states that it is the each Bolus and the extent of dilution.
trihydrate and that it is sterile or must be subjected to
further processlnq during the preparation of injectable
dosage forms.
If a test for OrganicImpurities other than Procedures 7 and 2
is used, then the labeling states with which Organic
Ampicillin Capsules
Impurities test the article complies. Where it.is intended DEFINITION
for use in preparing veterinary products, the label so Ampicillin Capsules contain an amount of ampicillin
states. (anhydrous or as the trihydrate) equivalent to NLT 90.0%
• USP REFERENCE STANDARDS (11)
and NMT 120.0% of the labeled amount of ampicillin
USP AmoxicillinRS
(C16H19N304S),
USP Amoxicillin Related Compound A RS
(2S,5R,6R)-6-Amino-3,3-dimethyl-7-oxo-4-thia-1- IDENTIFICATION
azabicyclo[3.2.0]heptane-2-carboxylic acid. • A. THIN-LAYER CHROMATOGRAPHY
C16H14NzOz 266.29 Diluent: Acetone and 0.1 N hydrochloric acid (4:1)
USP Ampicillin RS Standard solution: 5 mg/mL of USP Ampicillin RS in Diluent
USP Ampicillin System Suitability Mixture RS Sample solution: 5mg/mL of ampicillin in Diluent from the
This is a mixture which contains ampicillin, pivaloyl contents of Capsules
phenylglycine [(R)-2-phenyl-2-pivalamidoacetic acid; Chromatographic system
C13H17N03; 235.28], diphenyldiketopiperazine (See Chromatography (621), Thin-Layer Chromatography.)
(3,6-diphenylpiperazine-2,5-dione; C16H14NzOz; Adsorbent: 0.25-mm layer of chromatographic silica gel
266.29), and other related compounds. mixture
USP Ampicillin Trihydrate RS Application volume: 2 ~L
Developing solvent system: Acetone, toluene, glacial
acetic acid, and water (650:100:25:100)
Spray reagent: 3 mg/mL of ninhydrin in alcohol
Analysis
Ampicillin Boluses Samples: Standard solution and Sample solution
Apply the Standard solution and the Sample solution to the
plate, and develop the chromatogram using the
» Ampicillin Boluses contain an amount of Developing solvent system. When the solvent front has
ampicillin (as the trihydrate) equivalent to not less moved about three-fourths of the length of the plate,
than 90.0 percent and not more than 120.0 remove the plate from the chamber, mark the solvent
percent of the labeled amount of ampicillin front, and allow to air-dry. Locate the spots on the plate
by spraying lightly with Spray reagent, and dry at 90° for
(C16H19N304S). 15 min.
Acceptance criteria: The RF value of the principal spot of the
Packaging and storage-Preserve in tight containers. Sample solution corresponds to that of the Standard
labeling-Label the Boluses to indicate that they are for solution.
veterinary use only.

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322 Ampicillin / Official Monographs USP 43

ASSAY mUminute
• PROCEDURE Thenumbers represent
Standard solution: Prepare as directed for Standard the reagents as follows: 1) 0.42
Preparation in lodometricAssay-Antibiotics (425), using Air 0.60
(1) Hydroxylamine (2) 0.32
USP Ampicillin RS. . ... hydrochloride solution;
Sample solution: Nomrnally 1.25 mg/mL of arnplclllln (2) Acetate buffer; 0.32
prepared as follows. Place NLT 5 Capsules in a high-speed (3) 3.3 N sulfuric acid;
glass blender jar containing a suitable volume of water, and (4) Ferric nitrate solution. (3) 0.32
blend for 4 ± 1 min. Dilute a suitable aliquot with water.
Analysis: Proceed as directed for Procedure in lodometric (4) 0.60
Assay-Antibiotics (425).
Calculatethe percentage of the labeled amount of
ampicillin (C16H19N304S) in the portion of Capsules taken:
Result = (B - I) x (Ftl2) x (l/C u) x F2 x 100 Sampler
(1) 0.42 Rate: 40
B = volume of 0.01 N sodium thiosulfate consumed Air 0.60 perhour
in the Blank Determination (mL) (3) 0.32
= volume of 0.01 N sodium thiosulfate consumed
in the Inactivation and Titration of the Sample 0.32
solution (mL)
(2) 0.32
F, = factor as calculated in lodometricAssay-
Antibiotics(425) (4) 0.60
Cu = nominal concentration of ampicillin in the Sample Blank
solution (mg/mL) 480 nm
= conversion factor, 0.001 mg/J.lg
Spectrophotometer
Acceptance criteria: 90.0%-120.0%
Figure 1
PERFORMANCE TESTS
• DISSOLUTION, Procedure for a Pooled Sample (711 ) Analysis: With the sample line pumping water, the other
Medium: Water; 900 mL lines pumping their respective reagents, and the
Apparatus 1: 1 00 rpm spectrophotometer set at 480 nm, standardize the system
Time: 45 min ' until a steady absorbance baseline has.been established.
Standard solution: L/900 mg/mL of USP Ampicillin RS in Transfer portions of the Standard s~/utlon and the Sample
water, where L is the labeled amount of ampicillin in mg/ solution to sampler cups, and place Inthe sampler. Start the
Capsule sampler and conduct determinations of the Standard
Sample solution: Usea filtered portion of the solution under solution'and the Sample solution typically at the ra~e of 40/
test. h using a ratio of about 2:1 for sample and wash time.
Solution A: 1 in 1000 solution of polyoxyethylene (23) Calculatethe percentage of the labeled amount of
lauryl ether in water . . ampicillin (C16H19N304S) dissolved:
Solution B: Dissolve 20 9 of hydroxylarnlne hydrochloride
in 5 mLof SolutionA, and add water to make 1000 mL. Result =(Au/As) x Cs x V x P x F x (l/L) x 100
Buffer: 26 mg/mL of sodium hydroxide and 3.1 rnq/rnl, of
sodium acetate in water Au =absorbance of the Sample solution
Ferric nitrate solution: Suspend 233 9 of ferric nitrate in As =absorbance of the Standardsolution
about 600 mLof water, add 2.8 mL of sulfuric acid, stir until
the ferric nitrate is dissolved, add 1 mLof polyoxyethylene Cs = concentration of USP Ampicillin RS in the
(23) lauryl ether, dilute with water to 1000 mL, and mix. Standardsolution (mg/mL)
Apparatus: Automatic analyzer consistingof (1) a liquid V = volume of Medium, 900 mL
sampler, (2) a proportioning pump, (3) suitable P =potency of ampicillin in USP Ampicillin RS
spectrophotometers equipped with matched flow c~lIs and (J.lg/mg)
analysis capability at 480 nm, (4) a means of recording F = conversionfactor, 0.001 mg/J.lg
spectrophotometric readings, and/or c~mputer f?r .data L = label claim (mg/Capsule)
retrieval and calculation, and (5) a manifold consisting of Tolerances: NLT 75% (Q) of the labeled amount of
the components illustrated in Figure 7.
ampicillin (C16H,9N304S) is dissolved.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
SPECIFIC TESTS
• WATER DETERMINATION, Method I (921): NMT 4.0% where
the Capsules contain anhydrous ampicillin, or between
10.0% and 15.0% where the Capsules contain ampicillin
trihydrate
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers, and
store at controlled room temperature.

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USP 43 OfficialMonographs / Ampicillin 323

• LABELING: Label the Capsulesto indicate whether the Result = (r vIr s) x (C sIC u) x P x (1 IF) x 100
ampicillin therein is in the anhydrous form or is the
trihydrate. ru = peak response from the Sample solution
• USP REFERENCE STANDARDS (11) rs = peak response from the Standard solution
USP Ampicillin RS Cs =concentration of USP Ampicillin RS in the
Standard solution (mg/mL)
Cu =concentration of Sample solution 7 or Sample
solution 2 (mg/mL)
Ampicillin for Injection P =potency of ampicillin in USP Ampicillin RS
(j.Jg/mg)
F = conversion factor, 0.001 mg/j.Jg
DEFINITION
Ampicillin for Injection contains an amount of Ampicillin Where the test for Uniformity of Dosage Units has been
Sodium equivalent to NLT 90.0% and NMT 115.0% of the performed using the Procedure for content uniformity, use
labeled amount of ampicillin (C16H19N304S), the average of these determinations as the Assay value.
ASSAY Acceptance criteria: 90.00/0-115.0%
• PROCEDURE PERFORMANCE TESTS
Mobile phase: Acetonitrile, water, 1 M monobasic • UNIFORMITY OF DOSAGE UNITS (905)
potassium phosphate, and 1 N acetic acid (80:909:10:1) Procedure for content uniformity
Diluent: Water, 1 M monobasic potassium phosphate, and Analysis: Perform the Assay on individual containers using
1 N acetic acid (989:10:1) Sample solution 7 or Sample solution 2, or both, as
Standard solution: 1 mg/mL of USP Ampicillin RS in appropriate.
Diluent. Shake and sonicate, if necessary, to dissolve. Use Acceptance criteria: Meets the requirements
this solution promptly after preparation.
System suitability solution: 0.12 mg/mL of caffeine in the SPECIFICTESTS
Standardsolution • CRYSTALLINITY (695): Meets the requirements. Freeze-dried
Sample solution 1 (where it is represented as being in a products are exempt from this requirement.
single-dose container): 1 mg/mL of ampicillin in Diluent. • pH (791)
Constitute Ampicillin for Injection in a volume of Diluent, Sample solution: 10.0 mg/mL of ampicillin
corresponding to the volume of solvent specified in the Acceptance criteria: 8.0-10.0
labeling. Withdraw all of the withdrawable contents, using • WATER DETERMINATION, Method I (921): NMT 2.0%
a suitable hypodermic needle and syringe, and dilute with • PARTICULATE MATTER IN INJECTIONS (788): Meets the
Diluent. Use this solution promptly after preparation. requirements for small-volume injections
Sample solution 2 (where the label states the quantity of • STERILITY TESTS (71): Meets the requirements
ampicillin in a given volume of constituted solution): 1 mgl • BACTERIAL ENDOTOXINS TEST (85): NMT 0.15 USP
mL of ampicillin in Diluent. Constitute 1 container of Endotoxin Units/mg of ampicillin
Ampicillin for Injection in a volume of Diluent, . • CONSTITUTED SOLUTION: At the time of use, it meets the
corresponding to the volume of solvent specified in the requirements for Injections and ImplantedDrug Products (1),
labeling. Dilute a suitable aliquot of the constituted Specific Tests, Completeness and clarity of solutions.
solution with Diluent. Use this solution promptly after • OTHER REQUIREMENTS: It meets the requirements of the
preparation. tests for Identification in Ampicillin Sodium. It also meets the
Chromatographic system requirements in Labeling (7), Labels and Labeling for
(See Chromatography (621), System Suitability.) Injectable Products.
Mode: LC ADDITIONAL REQUIREMENTS
Detector: UV 254 nm • PACKAGING AND STORAGE: Preserve as described in
Columns Packaging and Storage Requirements (659), Injection
Precolumn: 4-mm x 5-cm; 5- to 10-j.Jm packing L1 Packaging, Packaging for constitution. Protect the
Analytical: 4-mm x 30-cm; 5- to 10-j.Jm packing L1 constituted solution from freezing.
Flow rate: 2 mL/min • USP REFERENCE STANDARDS (11)
Injection volume: 20 j.JL USP Ampicillin RS
System suitability USP Ampicillin Sodium RS
Samples: Standardsolution and System suitability solution
[NoTE-The relative retention times for ampicillin and
caffeine are 0.5 and 1.0, respectively, System suitabilty
solution.]
Suitability requirements Ampicillin Soluble Powder
Resolution: NLT 2.0 between caffeine and ampicillin,
System suitability solution
Tailing factor: NMT 1.4, Standard solution » Ampicillin Soluble Powder is a dry mixture of
Capacity factor: NMT 2.5, Standard solution Ampicillin (as the trihydrate) and one or more
Relative standard deviation: NMT 2.0%, Standard suitable diluents and stabilizing agents. It contains
solution
Analysis not less than 90.0 percent and not more than
Samples: Standardsolution and Sample solution 7 or 120.0 percent of the labeled amount of ampicillin
Sample solution2 (C16H19N304S),
Calculate the percentage of the labeled amount of
ampicillin (C16H19N304S) in the container or in the volume Packaging and storage-Preserve in tight containers.
of constituted solution taken: Labeling-Label it to indicate that it isfor veterinary use only.

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 324 Ampicillin / OfficialMonographs USP 43

USPReference standards (11)- Other requirements-It meets the requirements for

USP Ampicillin RS Uniformity of Dosage Units (90S), and for Labeling (7), Labels
Identification-Dissolve a quantity of it in a mixture of and Labeling for Injectable Products.
acetone and 0.1 N hydrochloric acid (4:1) to obtain a solution
containing 10 mg of ampicillin per mL: the resulting solution Assay-
responds to the Identification test under Ampicillin Capsules. Phosphate buffer solution-Accurately weigh 68 g of
pH (791): between 3.5 and 6.0, in an aqueous solution monobasic potassium phosphate, and transfer to a 500-mL
containing the equivalent of 20 mg of ampicillin per mL. volumetric flask. Dissolve in and dilute with water to volume.
Water Determination, Method 1(921): not more than 5.0%. Mobilephase-Prepare a suitable mixture of water,
acetonitrile, Phosphate buffersolution, and glacial acetic acid
Assay- (3600:360:40:4). Pass through a 0.45-lJm nylon filter, and
Standardpreparation-Prepare "as directed for Standard degas.
Preparation under lodometricAssay-Antibiotics (425), using Standardpreparation-Dissolve, with sonication, an
USP Ampicillin RS. accurately weighed quantity of USP Ampicillin RS in water to
Assay preparation-Transfer an accurately weighed prepare a solution having 0.5 mg per mL. Pass through a
quantity of Soluble Powder, equivalent to about 125 mg of 0.45-lJm PTFE filter, discarding the first 3 mL of the' filtrate.
ampicillin, to a 1OO-mL volumetric flask, dissolve in and dilute Caffeine solution-Transfer about 30 mg of caffeine,
with water to volume, and mix. accurately weighed, to a 50-mL volumetric flask. Add 25 mL
Procedure-Proceed as directed for Procedure under of water, sonicate to dissolve,and dilute with water to volume.
lodometricAssay-Antibiotics (425). Calculate the quantity, in Pass through a 0.45-lJm PTFE filter, discarding the first 3 mL
mg, of ampicillin (C'6H'9N304S) in the portion of Soluble of the filtrate.
Powder taken by the formula: System suitability solution-Prepare a solution of 1.0 mL of
Caffeine solution and 9.0 mL of Standardpreparation, and mix.
(F/20)(B - I). Assay preparation-Quantitatively dilute an accurately
measured volume of Ampicillin for Injectable Suspension,
constituted as directed in the labeling, with water to obtain a
solution containing about 0.5 mg per mL. Pass through a
'0.45-lJm PTFE filter, discarding the first 3 mL of the filtrate.
Chromatographic system (see Chromatography (621)-The
Ampicillin for Injectable Suspension liquid chromatograph is equipped with a 254-nm detector
and a 3.9-mm x 30-cm analytical column that contains 10-lJm
packing L1. The flow rate is about 2.0 mL per minute. The
» Ampicillin for Injectable Suspension is a dry
column temperature ismaintained at 40°. Chromatograph the
mixture of ampicillin trihydrate and one or more System SUitability solution and the Standardpreparation, and
suitable buffers, preservatives, stabilizers, and record the peak responses as directed for Procedure: the order
suspending agents. It contains the equivalent of of elution is ampicillin followed by caffeine; the resolution, R,
not less than 90.0 percent and not more than between ampicillin and caffeine is greater than 2; the column
efficiency is not lessthan 2000 theoretical plates for the
120.0 percent of the labeled amount of ampicillin ampicillin peak; the tailing factor is not greater than 1.4; and
(C16H19N304S), the relative standard deviation for replicate injections of the
Standardpreparation is not more than 2.0%.
Packaging and storage-Preserve as described in Procedure-Separately inject equal volumes (about 20 IJL)
Packaging and Storage Requirements (659), Injection Packaging, of the Standardpreparation and the Assay preparation into the
Packaging for constitution. chromatograph, record the chromatograms, and measure the
USPReference standards (11)- areas of the major peaks. Calculate the quantity, in mg, of
USP Ampicillin RS ampicillin (C'6H19N304S) in each mL of the constituted
Identification-Dissolve a quantity in a mixture of acetone solution of Ampicillin for Injectable Suspension taken by the
and 0.1 N hydrochloric acid (4:1) to obtain a solution formula:
containing 5 mg of ampicillin per mL: the resulting solution
responds to the Identification test under Ampicillin Capsules.
Bacterial fndotoxins Test (85) -It contains not more than
0.15 Endotoxin Unit per mg of ampicillin. in which C is the concentration, in mg per mL, of USP
pH (791): between 5.0 and 7.0, in the suspension constituted Ampicillin RS in the Standardpreparation; D is the dilution
as directed in the labeling. factor used in preparing the Assay preparation; and r u and r s
Water Determination, Method I (921): between 11.4% and are the average peak responses of the ampicillin peaks
14.0%. obtained from the Assay preparation and the Standard
Sterility Tests (71) -It meets the requirements when tested preparation, respectively.
as directed for Antibiotic Solids, Bulks; and Blends in the section
Membrane Filtration under Test for Sterility of the Product to be
Examined, except to use Fluid 0, to which has been added
sufficient sterile penicillinase to inactivate the ampicillin and to
swirl the vessel until solution is complete before filtering. If it Ampicillin for Oral Suspension
does not dissolve completely, proceed asdirected for Solids in
the section DirectInoculation of the Culture Medium under Test DIEFINITION
for Sterility of the Product to be Examined, except to use Fluid Ampicillin for Oral Suspension contains an amount of
Thioglycollate Medium and Soybean-Casein Digest Medium Ampicillin (anhydrous or asthe trihydrate) equivalent to NLT
containing sufficient penicillinase to inactivate the ampicillin 90.0% and NMT 120.0% of the labeled amount of ampicillin
in each vessel. (C'6H'9N304S), when constituted as directed. It contains one
or more suitable buffers, colors, flavors, preservatives, and
sweetening ingredients.

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 USP 43 OfficialMonographs / Ampicillin 325

IDENTIFICATION ADDITIONAL REQUIREMENTS

It A. THIN-LAYER CHROMATOGRAPHY • PACKAGING AND STORAGE: Preserve in tight containers.
Diluent: Acetone and 0.1 N hydrochloric acid (4:1) • LABELING: Label to indicate whether the ampicillin therein
Standard solution: 5 mg/mL of USP Ampicillin RS in Diluent is in the anhydrous form or is the trihydrate.
Sample solution: Nominally 5 mg/mL of ampicillin in • USP REFERENCE STANDARDS (11)
Diluent USP Ampicillin RS
Chromatographic system
(See Chromatography (621), Thin-Layer Chromatography.)
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica gel
mixture Ampicillin Tablets
Application volume: 2 IJL
Developing solvent system: Acetone; toluene, glacial DEFINITION
acetic acid, and water (650:100:25:100) Ampicillin Tablets contain an amount of Ampicillin (anhydrous
Spray reagent: 3 mg/mL of ninhydrin in alcohol form or trihydrate form) equivalent to NLT 90.0% and NMT
Analysis 120.0% of the labeled amount of ampicillin (C16H19N304S),
Samples: Standardsolution and Sample solution IDENTIFICATION
Apply the Standardsolution and the Sample solution to the • A. THIN-LAYER CHROMATOGRAPHY
plate, and develop the chromatogram in the Developing Diluent: Acetone and 0.1 N hydrochloric acid (4:1)
solvent system. When the solvent front has moved about Standard solution: 5 mg/mL of USP Ampicillin RS in Diluent
three-fourths of the length of the plate, remove the plate Sample solution: 5 mg/mL of ampicillin from powdered
from the chamber, mark the solvent front, and allow to Tablets in Diluent
air-dry. Locate the spots on the plate by spraying Chromatographic system
lightly with Sprayreagent, and dry at 90° for 15 min. (See Chromatography (621), Thin-Layer Chromatography.)
Acceptance criteria: The RF value of the principal spot of the Adsorbent: 0.25-mmlayer of chromatographic silica gel
Sample solution corresponds to that of the Standard mixture
solution. . Application volume: 2 IJL
ASSAY Developing solvent system: Acetone, toluene, glacial
• PROCEDURE acetic acid, and water (650:100:25:100)
Standard solution: Prepareas directed for Standard Spray reagent: 3 mg/mL of ninhydrin in alcohol
preparation in lodometricAssay-Antibiotics (425), using Analysis
USP Ampicillin R~. Samples: Standardsolution and Sample solution
Sample solution: Nominally 1.25 mg/mL of ampicillin Apply the Standardsolution and the Sample solution to the
prepared as follows. Dilute a suitable aliquot of Ampicillin plate, and develop the chromatogram using the
for Oral Suspension, constituted as directed in the labeling, Developing solventsystem. When the solvent front has
freshly mixed and free from air bubbles, with water. moved about three-fourths of the length of the plate,
Analysis: Proceed as directed for lodometricAssay- remove the plate from the chamber, mark the solvent
Antibiotics (425), Procedure. front, and allow to air-dry. Locate the spots on the plate
Calculate the percentage of the labeled amount of by spraying lightly with the Spray reagent, and dry at 90°
ampicillin (C16H19N304S) in the portion of Ampidllin for for 15 min.
Oral Suspension taken: Acceptance criteria: The RF value of the principal spot of the
Sample solution corresponds to that of the Standard
Result =(B - I) x F x (l/Cu) x 100 solution.
ASSAY
B =volume of 0.01 N sodium thiosulfate consumed • PROCEDURE
in the BlankDetermination (mL) Standard solution: Prepare as directed for Standard
= volume of 0.01 N sodium thiosulfate consumed Preparation in lodometricAssay-Antibiotics (425), using
in the Inactivation and Titration (mL) USP Ampicillin RS.
F = factor as calculated in lodometricAssay- Sample solution: Place NLT 5 Tablets in a high-speed glass
Antibiotics(425) blender jar containing an accurately measured volume of
Cu =nominal concentration of ampicillin in the Sample water, and blend for 4 ± 1 min. Dilute a suitable aliquot with
solution (mg/ml) water to obtain a concentration of 1.25 mg/mL of
ampicillin.
Acceptance criteria: 90.0%-120.0%
Analysis
PERFORMANCE TESTS Samples: Standardsolution and Sample solution
• DELIVERABLE VOLUME (698): Meets the requirements Proceed as directed for Procedure in lodometricAssay-
It UNIFORMITY OF DOSAGE UNITS (905) Antibiotics(425).
For single-unit containers Calculate the percentage of the labeled amount of
Acceptance criteria: Meets the requirements ampicillin (C16H19N304S) in the portion of Tablets taken:
SPECIFIC TESTS Result = (B - I) x (F,/2) x (l/Cu) x F2 x 100
• pH (791)
Sample solution: Constitute as directed in the labeling. B =volume of 0.01 N sodium thiosulfate consumed
Acceptance criteria: 5.0-7.5 in the Blank Determination (mL)
• WATER DETERMINATION, Method1(921): NMT 2.5% where =volume of 0.01 N sodium thiosulfate consumed
the solid for Oral Suspensioncontains anhydrous ampicillin in the Inactivation and Titration of the Sample
or NMT 5.0% if it contains ampicillin trihydrate and the solution (mL)
equivalent of 100 mg/mL of ampicillin when constituted as
directed in the labeling

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326 Ampicillin / Official Monographs USP 43

F] =factor as calculated in lodometricAssay- spectrophotometer set at 480 nm, standardize the

Antibiotics (425) system until a steady absorbance baseline has been
Cu = nominal concentration of ampicillin in the Sample established. Transfer portions of the Standard solution
solution (mg/mL) and the Sample solution to sampler cups, and place in
F2 =conversion factor, 0.001 mg/j.Jg the sampler. Start the sampler, and conduct
determinations of the Standard solution and the Sample
Acceptance criteria: 90.0%-120.0% solution typically at the rate of 40/h using a ratio of about
2:1 for sample and wash time.
PERFORMANCE TESTS
Calculate the percentage of the labeled amount of
• DISSOLUTION, Procedure for a Pooled Sample (711)
ampicillin (C16H19N304S) dissolved:
Medium: Water; 900 mL
Apparatus 1: 100 rpm Result =(Au/As) x Cs x V x P x F x (1/ L) x 100
Time: 45 min
Standard solution: L/900 mg/mL of USP Ampicillin RS in = response of the Sample solution
water, where L is the labeled amount of ampicillin in mg/ = response of the Standard solution
Tablet
= concentration of USP Ampicillin RS in the
Sample solution: Usea filtered portion of the solution under
test.
Standardsolution (mg/mL)
V = volume of medium, 900 mL
Solution A: 1 in 1000 solution of polyoxyethylene (23)
lauryl ether in water
P = potency of ampicillin in USP Ampicillin RS
(j.Jg/mg)
Solution B: Dissolve 20 9 of hydroxylamine hydrochloride
in 5 mL of Solution A, and add water to make 1000 mL.
F =conversion factor, 0.001 mg/j.Jg
Buffer: 26 mg/mL of sodium hydroxide and 3.1 mg/mL of
L = label claim (mg/Tablet)
sodium acetate in water Tolerances: NLT 75% (Q) of the labeled amount of
Ferric nitrate solution: Suspend 233 9 of ferric nitrate in ampicillin (C16H19N304S) is dissolved.
about 600 mL of water, add 2.8 mL of sulfuric acid, stir until
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
the ferric nitrate is dissolved, add 1 mL ofpolyoxyethylene
requirements
(23) lauryl ether, dilute with water to 1000 mL, and mix.
Apparatus: Automatic analyzer consisting of (1) a liquid SPECIFIC TESTS
sampler, (2) a proportioning pump, (3) suitable • WATER DETERMINATION, Method I (921)
spectrophotometers equipped with matched flow cells and
analysis capability at 480 nm, (4) a means of recording
spectrophotometric readings, and/or computer for data
Type of Tablets Form of Ampicillin limit (0/0)
retrieval and calculation, and (5) a manifold consisting of Nonchewable Anhydrous NMT4.0
the components illustrated in Figure 7. Nonchewable Trihydrate 9.5-12.0
Chewable Anhydrous NMT3.0
The numbers represent Chewable Trihydrate NMT 5.0
the reagents as follows:
(1) Hydroxylamine Tablets labeled for veterinary
use only Trihydrate NMT 13.0
hydrochloride solution;
(2) Acetate buffer; 0.32
(3) 3.3 N sulfuric acid; ADDITIONAL REQUIREMENTS
(4) Ferric nitrate solution. (3) 0.32 • PACKAGING AND STORAGE: Preserve in tight containers, and
store at controlled room temperature.
(4) 0.60 • LABELING: Label the Tablets to indicate whether the
ampicillin therein is in the anhydrous form or is the
trihydrate. Label chewable Tablets to indicate that they are
to be chewed before swallowing. Tablets intended for
veterinary use only are so labeled.
• USP REFERENCE STANDARDS (11)
Sampler USP Ampicillin RS
1) 0.42 Rate: 40
Air 0.60 per hour
(3) 0.32
0.32
(2) 0.32
Ampicillin and Probenecid for Oral
Suspension
(4) 0.60
» Ampicillin and Probenecid for Oral Suspension
Spectrophotometer contains an amount of Ampicillin (as the
trihydrate) equivalent to not less than 90~0 percent
Figure 1 and not more than 120.0 percent of the labeled .
amount of ampicillin (C16H19N304S) and not less
Analysis than 90.0 percent and not more than 110.0
Samples: Standardsolution and Sample solution percent of the labeled amount of probenecid
With the sample line pumping water, the other lines
pumping their respective reagents, and the

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 USP 43 OfficialMonographs / Ampicillin 327

(C13H19N04S). It contains one or more suitable Ampicillin and Probenecid for Oral Suspension taken by the
formula:
colors, flavors, and suspending agents.
C(L/D)(Au/As)
Packaging and storage-Preserve in tight, unit-dose
containers. in which C is the concentration, in mg per mL, of USP
USP Reference standards (11)- Probenecid RS in the Standard preparation; L is the labeled
USP Ampicillin RS quantity, in mg, of probenecid in the Ampicillin and
USP Probenecid RS Probenecid for Oral Suspension; D is the concentration, in mg
Uniformity of dosage units (905)- per mL, of probenecid in the Assay preparation on the basis of
FOR SOLID PACKAGED IN SINGLE-UNIT CONTAINERS: meets the the labeled quantity in the Ampicillin and Probenecid for Oral
requirements for Content Uniformity with respect to ampicillin Suspension and the extent of dilution; and Au and As are the
and probenecid. absorbances of the solutions from the Assay preparation and
Deliverable volume (698): meets the requirements. the Standard preparation, respectively.
pH (791): between 5.0 and 7.5, in the suspension constituted
as directed in the labeling.
Water Determination, Method I (921): not more than 5.0%.
Assay for ampicillin- Ampicillin Sodium
Standard preparation-Prepare as directed for Standard
Preparation under lodometric Assay-Antibiotics (425), using C16H1SN3Na04S 371.39
USP Ampicillin RS. 4-Thia-l-azabicyclo[3.2.0]heptane-2-carboxylic acid, [6-
Assay preparation-Constitute Ampicillin and Probenecid (aminophenylacetyl)amino]-3, 3-dimethyl-7 -oxo-,
for Oral Suspension as directed in the labeling, and mix. monosodium salt, [2S-[2a,5a,6p(S*)]]-;
Transfer the resulting suspension to a high-speed glassblender Monosodium D-(- )-6-(2-amino-2-phenylacetamido)-3,3-
jar containing sufficient water to make 500.0 mL, and blend dimethyl-7-oxo-4-thia-l-azabicyclo[3.2.0]heptane-2-
for about 10 minutes. Quantitatively dilute an accurately carboxylate [69-52-3].
measured volume of this stock solution with water to obtain
an Assay preparation containing about 1.25 mg of ampicillin DEFINITION
per mL. . Ampicillin Sodium has a potency equivalent to NLT 845 ~g
Procedure-Proceed as directed for Procedure under and NMT 988 ~g of ampicillin (C16H19N304S) per mg,
lodometric Assay-:-Antibiotics (425). Calculate the quantity, in calculated on the anhydrous basis.
mg, of ampicillin (C,6H19N304S) in the Ampicillin and IDENTIFICATION
Probenecid for Oral Suspension taken by the formula:

(L/D)(F/2000)(B - /)
.N;!"'~i"'~jK1~t()i;1nfra,.;kl
in which L is the labeled quantity, in mg, of ampicillin in the ~p'ggitL~~@"Il iYt..zQ.?Q). ..
Ampicillin and Probenecid for Oral Suspension; and D is the • B. IDENTIFICATION TESTS-GENERAL, Sodium (191)
concentration, in mg per mL, of ampicillin in the Assay
preparation on the basis of the labeled quantity in the ASSAY
Ampicillin and Probenecid for Oral Suspension and the extent • PROCEDURE
of dilution. Diluent: Water, 1 M monobasic potassium phosphate, and
1 N acetic acid (989:10:1)
Assay for probenecid- Mobile phase: Acetonitrile, water, 1 M monobasic
Standard preparation-Dissolve an accurately weighed potassium phosphate, and 1 N acetic acid (80:909:10:1)
portion of USP Probenecid RS in sodium carbonate solution (1 Standard solution: 1 mg/mL of USP Ampicillin RS in Diluent
in 100) to obtain a solution having a known concentration of using shaking and sonication, if necessary, to dissolve. Use
about 1 mg per mL. this solution promptly after preparation.
Assay preparation-Constitute Ampicillin and Probenecid System suitability solution: 0.12 mg/mL of caffeine in
for Oral Suspension as directed in the labeling, and mix. Standard solution
Quantitatively dilute the resulting suspension with sodium Sample solution: [NOTE-Ampicillin Sodium is hygroscopic.
carbonate solution (1 in 100) to obtain a solution containing Minimize exposure to the atmosphere, and weigh
about 1 mg of probenecid per mL, mix, and filter. '
promptly.] Equivalent to 1 mg/mL of anhydrous
Procedure-Transfer 2.0 mL of the clear Assay preparation ampicillin in Diluent. [NOTE-Usethis solution promptly after
to a 125-mL separator, and add 8.0 mL of 1.0 N hydrochloric
preparation.]
acid. Extract this solution with four 20-mL portions of
Chromatographic system
chloroform, filtering each extract through a glasswool pledget
(See Chromatography (621), System Suitability.)
and 6 g of chloroform-washed anhydrous sodium sulfate into
Mode: LC
a 1OO-mL volumetric flask. Wash the pledget arid the sodium Detector: UV 254 nm
sulfate with chloroform, collecting the washings in the 100-mL Column
volumetric flask, dilute with chloroform to volume, and mix.
Pre-column: 4-mm x 5-cm; 5- to 1O-~m packing L1
Treat 2.0 mL of the Standard preparation in the same manner.
Analytical column: 4-mm x 30-cm; 5- to 1o-urn packing
Concomitantly determine the absorbances of the solutions
Ll
from the Assay preparation and the Standard preparation at the
Flow rate: 2 mL/min
wavelength of maximum absorbance at about 257 nm, with
Injection size: 20 ~L
a suitable spectrophotometer, using chloroform washed with
System suitability
sodium carbonate solution (1 in 100) as the blank. Calculate
Samples: Standard solution and System suitability solution
the quantity, in mg, of probenecid (C13H 19N0 4S) in the

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328 Ampicillin / Official Monographs USP 43

[NOTE-The relative retention times for ampicillin and = concentration of methylene chloridein the
caffeine are 0.5 and 1.0, respectively, System Standard solution (mg/mL)
suitability solution.] Cu =nominal concentration of Ampicillin Sodium in
Suitability requirements the Sample solution (mg/mL)
Resolution: NLT 2.0 between the caffeine and the
ampicillin peaks, System suitability solution Acceptance criteria: NMT 0.2%.
Tailing factor: NMT 1.4, Standardsolution • Procedure 2: Dimethylaniline (223): Meets the
Capacity factor: NMT 2.5, Standardsolution requirement
Relative standard deviation: NMT 2.0%, Standard
solution SPECIFIC TESTS
Analysis • CRYSTALLINITY (695): Meets the requirements..
[NoTE-Ampicillin Sodium in the freeze-dried form is
Samples: Standardsolution and Sample solution
exempt from this requirement.]
Calculate the quantity, in IJg, of C16H19N304S in each mg
• pH (791): 8.0-10.0
of Ampicillin Sodium taken:
Sample solution: 10.0 mg/mL of ampicillin
• WATlER DlETEMINATION, Method I (921): NMT 2.0%
Result =(ru/rs) x (Cs/Cu) x p • STERILITY TESTS (71): Where the label statesthat Ampicillin
= peak response from the Sample solution Sodium is sterile, it meets the requirements.
• BACTERIAL ENDOTOXINS TEST (85): Where the label states
= peak response from the Standardsolution that Ampicillin Sodium is sterile or the label states that
= concentration of USP Ampicillin RS in the Ampicillin Sodium must be subjected to further processing
Standardsolution (mg/mL) during the processing of injectable dosage forms, it
Cu = nominal concentration of Ampicillin Sodium in contains NMT 0.15 USP Endotoxin Unit/mg of ampicillin.
the Sample solution (mg/mL)
P = potency of USP Ampicillin RS (lJg/mg) ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers.
Acceptance criteria: 845-988 J,Jg/mg on the anhydrous • LABELING: Where it is intended for use in preparing
basis . injectable dosage forms, the label states that it is sterile or
must be subjected to further processing during the
IMPURITIES
preparation of injectable dosage forms.
ORGANIC IMPURITIES
• USP REFERENCE STANDARDS (11)
• Procedure 1: Limit of Methylene Chloride USP Ampicillin RS
Internal standard solution: 2.1 mg/mL of dioxane in
USP Ampicillin Sodium RS
dimethyl sulfoxide
Standard solution: 0.33 mg/mL of methylene chloride in
Internal standard solution
Sample solution: 166.7 mg/mL of Ampicillin Sodium in
Internal standard solution Ampicillin and Sulbactam for Injection
Chromatographic system .
(See Chromatography (621), System Suitability.)
Mode: GC » Ampicillin and Sulbactam for Injection is a sterile,
Detector: Flame ionization dry mixture of Ampicillin Sodium and Sulbactam
Column: 1.8-m x 4-mm glass column packed with a 10%
phase G390n unsilanized support Sl A
Sodium. It contains the equivalent of not less than
Temperature 90.0 percent and not more than 115.0 percent of
Column: 65° the labeled amounts of ampicillin (C16H19N304S)
Injector: 100° and sulbactam (CSH 11NOsS), the labeled amounts
Detector block: 260°
Carrier gas: Nitrogen representing proportions of ampicillin to
Flow rate: 60 mL/min sulbactam of 2:1. It contains not less than 563 I-Ig
Injection size: 1 IJL of ampicillin and 280 I-Ig of sulbactam per mg,
System suitability calculated on the anhydrous basis.
Sample: Standardsolution
[NOTE-The relative retention times for methylene Packaging and storage-Preserve as described in
chloride and dioxane are 0.5 and 1.0, respectively.] Packaging and Storage Requirements (659), Injection Packaging,
Suitability requirements Packaging for constitution.
Resolution: NLT 4 between methylene chloride and USPReference standards (11)-
dioxane USP Ampicillin RS
Relative standard deviation: NMT 5% USP Sulbactam RS
Analysis . Constituted solution-At the time of use, it meets the
Samples: Standardsolution and Sample solution requirements for Injections and ImplantedDrug Products (1),
Calculate the percentage of methylene chloride in the Specific Tests, Completeness. and clarity of solutions.
portion of Ampicillin Sodium taken: Identification-The retention times of the major peaks in
the chromatogram of the Assay preparation correspond to
Result = (Ru/Rs) x (Cs/Cu) x 100 those in the chromatogram of the Standard preparation, as
obtained in the Assay.
= peak response ratio of methylene chloride to Bacterial Endotoxins Test (85) -It contains not more than
dioxane from the Sample solution 0.17 USP Endotoxin Unit in a portion equivalent to 1 mgof a
= peak response ratio of methylene chloride to mixture of ampicillin and sulbactam (0.67 and 0.33 mg,
dioxane from the Standardsolution respectively).

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 USP 43 Official Monographs / Amprolium 329

Sterility Tests (71) -It meets the requirements when tested degradation product is not less than 4.0. Chromatograph the
asdirected for Membrane Filtration under Test for Sterility of the Standard preparation, and record the responses asdirected for
Product to be Examined. Procedure: the relative retention times are about 0.35 for
pH (791): between 8.0 and 10.0, in a solution containing 10 ampicillin and 1.0 for sulbactam; the column efficiency
mg of ampicillin and 5 mg of sulbactam per mL. determined from the sulbactam peak is not lessthan 3500
Water Determination, MethodI (921): not more than 2.0%. theoretical plates; the tailing factor is not more than 1.5; and
Particulate Matter in Injections (788): meets the the relative standard deviation for replicate injections is not
requirements for small-volume injections. more than 2.0%.
Other requirements-It meets the requirements for Procedure-Separately inject equal volumes (about 10 IJL)
Uniformityof Dosage Units(905) and for Labeling (7), Labels and of the Standard preparation and the appropriate Assay
Labeling for Injectable Products. preparation into the chromatograph, record the
chromatograms, and measure the areas for the major peaks.
Assay- Calculate the quantities, in IJg, of ampicillin (C'6H'9N304S) and
0.005 M Tetrabutylammonium hydroxide-Dilute 6.6 mL of
of sulbactam (CSH 1,NOsS) in the portion of Ampicillin and
a 40% solution of tetrabutylammonium hydroxide with water
to obtain 1800 mL of solution. Adjust with 1 M phosphoric Sulbactam for Injection taken by the same formula:
acid to a pH of 5.0 ± 0.1, dilute with water to 2000 mL, and
mix. (C s PIC v)(r vir s)
Mobilephase-Prepare a filtered and degassed mixture of in which C s is the concentration, in mg per mL, of the
0.005 M Tetrabutylammonium hydroxide and acetonitrile
(1650:350). Make adjustments if necessary (see System appropriate USP Reference Standard in the Standard
Suitability under Chromatography (621». preparation; P is the assigned content, in I-Ig per mg, of the
Standard preparation-Quantitatively dissolve accurately appropriate USP Reference Standard; C u is the concentration,
weighed quantities of USP Ampicillin RS and USP Sulbactam in mg per mL, of Ampicillin and Sulbactam for Injection in
RS in Mobilephase to obtain a solution having known Assay preparation 1, based on the weight, in mg, of powder
concentrations of about 0.6 mg of-ampicillin per mL and 0.3 removed from the container and the extent of dilution; and r u
mg of sulbactam per mL. [NoTE-Inject this solution promptly.] and r s are the peak areasfor the appropriate analyte obtained
Resolution solution-Prepare a solution of USP Sulbactam RS from Assay preparation 1 and the Standard preparation,
in 0.01 N sodium hydroxide containing 0.3 mg per mL, and respectively. Calculate the quantities of ampicillin
allow to stand for 30 minutes. Adjust with phosphoric acid to (C'6H'9N304S) and of sulbactam (CSH ll NOsS)withdrawn from
a pH of 5.0 ± 0.1. Transfer 5 mL of the solution to a 25-mL the container, or in the volume of constituted solution taken
volumetric flask, add 4.25 mL of acetonitrile, dilute with 0.005 by the same formula:
M Tetrabutylammonium hydroxide to volume, and mix.
Transfer 1 mL of this solution to a second 25-mL volumetric (LIO)(C s P)(r vir s)
flask, add 15 mg of USP Ampicillin RS, dilute with Mobile
phaseto volume, and mix. [NoTE-Inject this solution in which L is the labeled quantity, in mg, of ampicillin or
promptly.] sulbactam, as appropriate, in the container or in the volume
Assay preparation 1-Mix the contents of a container of of constituted solution taken; D is the concentration, in mg
Ampicillin and Sulbactam for Injection. Quantitatively dissolve per mL, of ampicillin or sulbactam in Assay preparation 2 or
an accurately weighed portion of the powder in Mobilephase Assay preparation 3, on the basis of the labeled quantity, in
to obtain a solution having a concentration of about 1 mg of mg, of ampicillin or sulbactam, as appropriate, in the
the powder per mL. [NoTE-Inject this solution promptly.] container and the extent of dilution; r u and r s are the peak
Assay preparation 2 (where it is represented as being in a areas for the appropriate analyte obtained from Assay
single-dose container)- Constitute a container of Arnplclllln preparation 2 or Assay preparation 3 and the Standard
and Sulbactam for Injection with a volume of water, accurately preparation, respectively; and the other terms are as defined
measured, corresponding to the volume of solvent specified above.
in the labeling. Withdraw the total withdrawable contents
from the container, using a suitable hypodermic needle and
syringe, and dilute quantitatively, and stepwise if necessary,
with Mobilephase to obtain a solution containing about 0.6
mg of ampicillin per mL and 0.3 mg of sulbactam per mL. Amprolium
[NoTE-Inject this solution promptly.] .
Assay preparation 3 (where the label statesthe quantities of
ampicillin and sulbactam in a given volume of constituted CI • HCI

solution)- Constitute a container of Ampicillin and

Sulbactam for Injection with a volume of water, accurately
measured, corresponding to the volume of solvent specified C,4H,9CIN4'HCI 315.24
in the labeling. Dilute an accurately measured volume of the 1-[(4-Amino-2-propyl-5-pyrimidinyl)methyl]-2-
constituted solution quantitatively, and stepwise if necessary, methylpyridinium chloride monohydrochloride;
with Mobilephase to obtain a solution containing about 0.6 1-[(4-Amino-2-propyl-5-pyrimidinyl)methyl]-2-picolinium
mg of ampicillin per mL and 0.3 mg of sulbactam per mL. chloride monohydrochloride [137-88-2].
[NoTE-Inject this solution promptly.]
Chromatographic system (see Chromatography (621»-The DEFINITION
liquid chromatograph is equipped with a 230-nm detector Amprolium contains NLT 97.0% and NMT 101.0% of
and a 4-mm x 30-cm column containing packing L1. The flow amprolium (C,4H,9CIN4 . HCI), calculated on the dried basis.
rate is about 2 mL per minute. Chromatograph the Resolution
solution, and record the responses as directed for Procedure:
the relative retention times are about 0.7 for ampicillin and 1.0
for sulbactam alkaline deqradatlon product; and the
resolution, R, between ampicillin and sulbactam alkaline

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 330 Amprolium / OfficialMonographs USP 43

IDENTIFICATHON ADDITIONAL REQUIREMENTS

o PACKAGING AND STORAGE: Preserve in well-closed
containers.
• LABELING: label it to indicate that it is for veterinary use
o A.ASPECTROSCOPICIDENTiFICATIONTESTS(197),"/nfrared only.
Spectroscopy: 197K.. (CNT-MaY.2020) . . . " o USP REFERENCE STANDARDS (11)
Sample: Previously dried USP Amprolium RS
Acceptance criteria: Meets the requirements

o B~ t~~i~~~~P.S~P"'~i,~~~~~~~g!l' Amprolium Soluble Powder

£.!ltrqViQle.~'7~isJpJec$p~~trQ§g9P.Y:••l~~"';(
Analytical wavelength: 246 nm DEFINITION
Sample solution: 10 IJg/ml in 0.1 N hydrochloric acid Amprolium Soluble Powder contains NlT 95.0% and NMT
Acceptance criteria: Absorptivities, calculated on the dried 105.0% of the labeled amount of amprolium (C14H19CIN4 .
basis, do not differ by more than 3.0%. HCI).
ASSAY IDENTIFICATION
o PROCEDURE
Diluent: Methanol, acetonitrile, and water (45:5:50)
Mobile phase: 6 g of sodium 1-heptanesulfonate in 500 ml
of water. Add 12 ml of glacial acetic acid, 2.0 ml of o A.
triethylamine, 450 rnl, of methanol, and 50 ml of l!1l~rg;<.< . .....i~lp[g
acetonitrile. Pass through a suitable filter of 0.5-lJm or finer Sample solution: 1() IJg/ml (filtered) in
pore size. acid
System suitability solution: 0.5 mg/n;l ~f U~P . Acceptance criteria: Meets the requirements
Amprolium RS and 0.2 mg/ml of 2-plcohne I~ D"uen~ "ASSAY
Standard solution: 0.5 mg/ml of USP Amprohum RS m o PROCEDURE
Diluent Diluent: Methanol, acetonitrile, and water (45:5:50)
Sample solution: 0.5 mg/ml of Amprolium in Diluent Mobile phase: 6 9 of sodium 1-heptanesulfonate in 500 ml
Chromatographic system of water. Add 12 ml of glacial acetic acid, 2.0 ml of
(See Chromatography (621), System Suitability.) triethylamine, 450 ml of met~anol, ~nd 50 ml of .
Mode: lC I
acetonitrile. Pass through a suitable filter of 0.5-lJm or finer
Detector: UV 254 nm pore size.
Column: 4.6-mm x 25-cm; packing l13 System suitability solution: 0.5 mg/n;l ~f U~P .
Flow rate: 0.6 ml/min Amprolium RS and 0.2 mg/ml of 2-plcolme I~ Dlluen~
Injection volume: 10 IJl Standard solution: 0.5 mg/ml of USP Amprohum RS m
System suitability Diluent
Sample: System suitability solution Sample solution: Nominally 0.5 mg/ml of ~mprolium in
Suitability requirements . . . Diluent, prepared as follows. Transfer a I?0rtlon of Soluble
Resolution: NlT 7 between amprollum.and 2-plcollne Powder, equivalent to 50 mg of ~mprohum, to.a 100-ml
Column efficiency: NlT 6500 theoretical plates from the volumetric flask add 75 ml of Diluent, and sonkate for 10
amprolium peak . . min. Allow to c~ol to room temperature, and dilute with
Tailing factor: NMT 2.3 for the amprollum peak Diluent to volume. Pass through a suitable filter of 0.5-lJm
Relative standard deviation: NMT 1.0% for amprolium or finer pore size, and use the clear filtrate.
Analysis Chromatographic system
Samples: Standardsolution and Sample solution (See Chromatography (621), System SUitability.)
Calculate the percentage of amprolium (C14H19CIN4 . HCI) Mode: lC
in the portion of Amprolium taken: Detector: UV 254 nm
Column: 4.6-mm x 25-cm; packing l13
Result= (rv/rs) x (Cs/Cv) x 100 Flow rate: 0.6 ml/min
Injection volume: 10 IJl
=peak response from the Sample solution System suitability
= peak response from the Standard solution Sample: System suitability solution
= concentration of USP Amprolium RS in the Suitability requirements
Standardsolution (mg/ml) Resolution: NlT 7 between amprolium and 2-picoline
Cv = concentration of Amprolium in the Sample Column efficiency: NlT 6500 theoretical plates from the
solution (mg/ml) amprolium peak
Tailing factor: NMT 2.3 for the amprolium peak
Acceptance criteria: 97.0%-1 01.0%on the dried basis Relative standard deviation: NMT 1.0% for amprolium
SPECIFIC TESTS Analysis
o Loss ON DRYING (731) Samples: Standardsolution and Sample solution
Analysis: Dry a sample at a pressure not exceeding 5 mm of Calculate the percentage of the labeled amount of
mercury at 100° for 3 h. amprolium (C14H 19CIN4 . HCI) in the portion of Soluble
Acceptance criteria: NMT 1.0% Powder taken:

Result= (rufrs) x (Cs/Cv) x100

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 USP 43 OfficialMonographs / Amyl 331

= peak response from the Sample solution Procedure-Separately inject equal volumes (about 10 IJL)
= peak responsefrom the Standardsolution of the Standardpreparation and the Assay preparation into the
= concentration of USP Amprolium RS in the chromatograph, record the chromatograms, and measure the
Standard solution (mg/mL) peak ar~as for amprolium. Calculate the quantity, in mg, of
Cu = nominal concentration of amprolium in the arnproliurn (C14H19C1N4 . HCI) in each mL of the Oral Solution
Sample solution (mg/mL) taken by the formula:

Acceptance criteria: 95.0%-105.0%

ADDITIONAL REQUIREMENTS in which C is the concentration, in mg per mL, of USP
• PACKAGING AND STORAGE: Preserve in tight containers. Amprolium RS in the Standard preparation; V is the volume, in
• LABELING: Label it to indicate that it is for veterinary use mL, of Oral Solution taken t? prepare the Assay preparation;
only. and tu and rs are the amprohum peak areas obtained from the
• USP REFERENCE STANDARDS (11 > Assay preparation and the Standard preparation, respectively.
USP Amprolium RS

Amprolium Oral Solution Amyl Nitrite

CSHllN0 2 117.15
» Amprolium Oral Solution contains not less than Mixture of nitrous acid, 2-methylbutyl ester, and nitrous acid,
93.0 percent and not more than 107.0 percent of 3-methylbutyl ester [8017-89-8; 110-46-3].
the labeled amount of amprolium (C14H19CIN4 .
HCI). » Amyl Nitrite is a mixture of the nitrite esters of
3-methyl-l-butanol and 2-methyl-l-butanol. It
Packaging and storage-Preserve in tight containers, contains not less than 85.0 percent and not more
protected from light. Store at a temperature between 5° and than 103.0 percent of CsH llN02 •
30°, in a dry place.
Labeling-Label it to indicate that it is for veterinary use only. [CAUTION-Amyl Nitrite is very flammable. Do not
USP Reference standards (11)- . use where it may be ignited.]
USP Amprolium RS I

Packaging and storage-Preserve in tight containers, and

store in a cool place, protected from light.
USP Reference standards (11) -
USP Benzyl Benzoate RS
Ide~~i!i_~~tion, . ~~~~~l~~~~.;c;c·
tJlt[qyiQlgt..\Iisib1gSpggJ[()§~PPY;(Yi ···..A Identification-
Solution: 10 IJg per mL, filtered. A: The NMR spectrum recorded as directed in the Assay
Medium: 0.1 N hydrochloric acid. exhibits, among other peaks, a doublet with a band centered
pH (791): between 2.5 and 3.0. at about 1 ppm and a multiplet with a band centered at about
Assay- 4.8 ppm representing methyl protons and methylene protons
Mobile phase-To 4.5 g of sodium 1-hexanesulfonate add alpha to the nitrite group, respectively, both relative to the
1500 mL of water, 400 mL of methanol, and 100 mL of tetramethylsilane singlet at 0 ppm.
acetonitrile, mix, and allow to cool to room temperature. B: To a few drops of it add a mixture of 1 mL of ferrous
Adjust with phosphoric acid to a pH of 5.1, and pass through sulfate TS and 5 mL of 3 N hydrochloric acid: a greenish brown
a filter having a 0.5-lJm or finer porosity. Make adjustments if color is produced.
necessary(see System Suitability under Chromatography Specific gravity (841): between 0.870 and 0.876.
(621 ».
Standard preparation--Quantitatively dissolve an accurately
Acidity-To 0.30 mL in a glass-stoppered cylinder add a
mixture of 0.60 mL of 0.1 N sodium hydroxide, 10 mL of
weighed quantity of USP Amprolium RS in water to obtain a water, and 1 drop of phenolphthalein TS, and invert the
solution having a known concentration of about 0.5 mg per cylinder three times: the red tint of the water layer is still
mL. perceptible.
Assay preparation-Transfer an accurately measured limit of nonvolatile residue-Allow 10 mL to evaporate
volume of Oral Solution, equivalent to about 960 mg of at room temperature in a tared evaporating dish, in a
amprolium, to a 1OO-mL volumetric flask, dilute with water to well-ventilated hood, and dry the residue at 105° for 1 hour:
volume, and mix. Transfer 5.0 mL of this stock solution to a the weight of the residue does not exceed 2 mg (0.02%).
second 1OO-mL volumetric flask, dilute with water to volume, Con!ent of total nitriteS-Inject a portion of Amyl Nitrite
and mix. of suitable volume, but not more than 2 IJL, into a suitable gas
Chromatographic system (see Chromatography (621»-The chromatograph (see Chromatography (621» equipped with a
~hermal conductiyity detector. Under typical conditions, the
liquid chromatograph is equipped with a 268-nm detector
and a 3.9-mm x 30-cm column that contains packing L11. The Instrument contains a 3-mm x 2-m column packed with a
column is maintained at a constant temperature of about 45°. methyl polysiloxane oil, 25% by weight on suitable calcined
The flow rate is about 1 mL per minute. Chromatograph the diatomite, the column is maintained at about 80°, the
Standardpreparation, and record the peak responses as injection port and detector block are maintained about 10°
directed for Procedure: the tailing factor is not more than 1.5; above the temperature of the column, and helium is used as
and the relative standard deviation for replicate injections is a carrier gas at a flow rate of about 60 mL per minute. From
not more than 2.0%. the ar~a ~nder the curve, calculate the percentage (a/a) of
total nitrites, represented by the area under the main peak of

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 332 Amyl/Official Monographs USP 43

the chromatogram, in the Amyl Nitritetaken: not lessthan Procedure-Remove the gauze or other covering from 1 or
97.0% is found. more Inhalant ampuls containing a total of 300 t,o 400 ut, of
amyl nitrite. Weigh accuratelythe clean and dry Intact glass
Assay- ampul(s), and place the weighed spe~imen in ~ freezer for not
Solvent: carbon tetrachloride.
lessthan 15 minutes. Transferthe chilled specimen to a
Internal standard-tJSP Benzyl Benzoate RS.
glass-stoppered 25-ml conical flask containing a solution of
Procedure-Transfer 4 to 5 mEq of Internal standard, 4 to 5 mEq of I~ternal standard, accurately weigh~d, in 1 to 2
accurately weighed, to a semimicrosampling tube, add 2 to ml of carbon tetrachloride. Break the ampul(s) with a glass
3 ml of carbon tetrachloride, apply a sampling valve and rod and rinseany sample or glassfragments adhering to the
septum,· thereby sealing the tube, and determine the weight gla;s rod with 1 ml of carbon tetrachloride into the main assay
of the sealed assembly. Open the valve, introduce about 500 solution. Insertthe stopper in the flask immediat:ly, .mix, and
IJl of Amyl Nitritewith a syringe, close the valve, and proceed as directed for Absolute Method of QuantltatlOn under
determine the weight of the sealed assemblywhen it has Nuclear Magnetic Resonance (761,{, b~ginning. wi~h "Whe~
attained constant weight. Shakethe sampling tube and valve dissolution has been completed. With no spinning, or with
assembly, and transfer about 500 IJl of the solution to a the spinning adjusted so that the spinning side bands of
precision NMR tube as directed for Absolute Method of . neither the substance under assay nor the Internal standard
Quantitation under Nuclear Magnetic Resonance (761). ':"'It~ no interfere with the regions to be integrated, record as As the
spinning, or with the spinning adjusted so that the spmnmq average area of the Internal standard singlet appearing at
side bands of neither the substance under assay nor the about 5.3 ppm, representing the methylene protons of b~nzyl
Internal standard interfere with the regions to be integrated,
benzoate and record as Au the average area of the multiplet
record as As the average area of the Internal standard singlet
appearing at about 5.3 ppm, representing the methylene with a band center at-about 4.8 ppm, representing the alpha
protons of benzylbenzoate, and record as Au the average area methylene protons of amyl nitrite, with reference to thE7
tetramethylsilane singlet at 0 ppm. Calculate the quantity of
of the multiplet with a band center at about 4.8 pp.m! . CSHllN0 2 in the Inhalant taken, using 58.57 as the equivalent
representing the alpha methylene protons of amyl nitnte, with weight of amyl nitrite (EWu) and 106.12 as that of benzyl
reference to the tetramethylsilanesinglet at 0 ppm. Calculate
the quantity of CSH11N02 in the Amyl Nitrite taken, using benzoate (EWs). Rinse the flask containing the assay
58.57 as the equivalent weight of amyl nitrite (EWu) and ,preparation with three 5-ml portions of ether, decanting each
106.12 as that of benzyl benzoate (EWs) . rinsingcarefully to avoidlossofglassfragments, and ev~porate
any remaining ether with the aid of a current of dry air.
Transferthe dry glassfragments to a tared watch glass,weigh,
and subtract the weight of the glass fragments from that of
the intact ampul(s) to obtain the weight of the Inhalant taken.
Amyl Nitrite Inhalant
» Amyl Nitrite Inhalant contains a mixture of the
Anagrelide Hydrochloride
nitrite esters of 3-methyl-l-butanol and 2-methyl-
l-butanol. It contains not less than 80.0 percent
and not more than 105.0 percent of CsH" N02 • It
contains a suitable stabilizer.
[CAUTIoN-Amyl Nitrite Inhalant is very flammable.
ClOH 7CI2N30 . HCI ' H20 310.56
Do not use where it may be ignited.] Anhydrous 292.55
Packaging and storage-Preserve in tight, unit-dose glass [58579-51-4].
Im~dazo[2,l-b]quinazolin-2qH)-one, 6,7-dichloro-1,5-
containers, wrapped loosely in gauze or other sUita~le
material, and store in a cool place, protected from light. dlhydro-, monohydrochlonde, monohydrate;
USPReference standards (11)- 6,7-Dichloro-1,5-dihydroimidazo[2,1-b]-quinazolin-2(3H)-
USP Benzyl Benzoate RS one monohydrochloride, monohydrate [823178-43-4].
Specific gravity (841): between 0.870 and 0.880. DEFINITION
Content of total nitrites-Remove the gauze or other Anagrelide Hydrochloride contains NlT 98.0% and NMT
covering place the glasscontainer of Inhalant upright in a dry 102.0% of anagrelide hydrochloride(CloH7C12N30 . HCI),
ice-acet~ne slurry, and cool for 10 minutes. Dry the container calculated on the anhydrous basis.
of Inhalant, place it in a pointed glass tube, and break the
container with a glass rod. Proceed as directed for Total IDENTIFICATION
nitrites under Amyl Nitrite: not less than 95.0% is found.
Other requirements-It responds to the Identification tests
and meets the requirements of the test for Acidity- under
Amyl Nitrite. • A.
S
Assay- • B.The retention time of the lTIajor peak of the Sample
Solvent: carbon tetrachloride. solution corresponds to that of the Standard solution, as
Internal standard-tJSP Benzyl Benzoate RS. obtained in the Assay.
• C. IDENTIFICATION TESTS-GENERAL, Chloride (191): Meets
the requirements
• Suitable sampling tubes, sampling valves, and septums are available,
respectively, as catalog Nos. K-749000, K-7491 00, and K-749102 (50
septums) or K-749101 (100 septums), from Kontes Glass Company,
Vineland, NJ 08360.

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 USP 43 OfficialMonographs / Anagrelide 333

ASSAY volumetric flask, add acetonitrile equivalent to fill about

• PROCEDURE 50% of the final volume and a small amount of 2 N
Use freshly prepared standard and sample solutions and hydrochloric acid (3 drops per 200 mL of the final volume).
inject within 2 h. Sonicate to dissolve, heat in the hot water bath if necessary,
Solution A: 6.8 giL of monobasic potassium phosphate in and dilute with acetonitrile to volume.
water. Adjust with phosphoric acid to a pH of 2.5. Standard stock solution C: 0.1 mg/mL of anagrelide
Mobile phase: Acetonitrile and Solution A (1:3) hydrochloride in acetonitrile. Transfer USP Anagrelide
Diluent: Acetonitrile and water (1:1) Hydrochloride RS into a suitable volumetric flask, add
Standard stock solution: 0.5 mg/mL of anagrelide acetonitrile equivalent to fill about 80% of the final volume
hydrochloride in acetonitrile prepared as follows. Transfer and a small amount of 0.12 N hydrochloric acid (1 mL per
USP Anagrelide Hydrochloride RS into a suitable volumetric 100 mL of the final volume). Sonicateto dissolve, and dilute
flask, add a small amount of 2 N hydrochloric acid (3 drops with acetonitrile to volume.
per every 50 mL of the final volume) and acetonitrile System suitability solution: 0.25 IJg/mL of each of
equivalent to fill about 80% of the final volume. Sonicate anagrelide related .cornpound A and anagrelide related
to dissolve, and dilute with acetonitrile to volume. compound B in Mobile phasefrom Standardstock solution
Standard solution: 0.05 mg/mL of anagrelide A and Standardstock solution B
hydrochloride in Diluent from Standardstocksolution Standard solution: 0.05 IJg/mL of anagrelide
Sample stock solution: Weigh Anagrelide Hydrochloride, hydrochloride in Mobilephasefrom Standardstock solution
equivalent to 25 mg of anhydrous salt, into a 50-mL C
volumetric flask, add 3 drops of 2 N hydrochloric acid and Sample stock solution: Weigh Anagrelide Hydrochloride,
40 mL of acetonitrile. Sonicateto dissolve, and dilute with equivalent to 25 mg of anhydrous salt, into a 50-mL
acetonitrile to volume. volumetric flask. Add 45 mL of acetonitrile, sonicate, and
Sample solution: TransferS mL of Sample stock solution to swirl the flask until the preparation turns into a cloudy
a 50-mL volumetric flask, and dilute with Diluent to volume. liquid. Add 1 drop of 0.12 N hydrochloric acid, swirl the
Chromatographic system flask until the liquid turns to clear, and dilute with
(See Chromatography (621), System Suitability.) acetonitrile to volume.
Mode: LC Sample solution: Transfer 5 mL of Sample stock solution into
Detector: UV 254 nm a 50-mL volumetric flask, and dilute with Diluent B to
Column: 4.6-mm x 15-cm; 4-lJm packing L11 volume.
Flow rate: 1.2 mL/min Chromatographic system
Injection volume: 20 IJL (See Chromatography (621), System Suitability.)
System suitability Mode: LC
Sample: Standarpsolution Detector: UV 254 nm
Suitability requirements Column: 4.6-mm x 15-cm; 4-l-'m packing L11
Column efficiency: NLT 3000 theoretical plates Autosampler temperature: 5°
Tailing factor: NMT 2.0 Flow rate: 1.2 mL/min
Relative standard deviation: NMT 2.0% Injection volume: 50 IJL
Analysis System suitability
Samples: Standardsolution and Sample solution Samples: System suitability solution and Standardsolution
Calculate the percentage of anagrelide hydrochloride Suitability requirements
(C,oH7CI2N 30 · HCI) in the portion of Anagrelide Resolution: NLT 2.0 between anagrelide related
Hydrochloride taken: . compound Band anagrelide related compound A,
System suitability solution
Result = (r vir 5) x (C siC u) x 100 Column efficiency: NLT 3000 theoretical plates,
Standardsolution
ru = peak response of anagrelide from the Sample Tailing factor: NMT 2.0, Standard solution
solution Relative standard deviation: NMT 10.00/0, Standard
r5 = peak response of anagrelide from the Standard solution
solution Analysis
C5 = concentration of USP Anagrelide Hydrochloride Samples: Standardsolution and Sample solution
RS in the Standardsolution (mg/mL) Calculate the percentage of each impurity in the portion of
Cu =concentration of Anagrelide Hydrochloride in the Anagrelide Hydrochloride, on the anhydrous basis, taken:
Sample solution (mg/mL)
Result = (r vir 5) x (C siC u) x (1 IF) x 100
Acceptance criteria: 98.0%-102.0% on the anhydrous
basis ru = peak response of each impurity from the Sample
solution
IMPURITIES r5 = peak response of anagrelide from the Standard
• RESIDUE ON IGNITION (281): NMT 0.1% solution
• ORGANIC IMPURITIES C5 = concentration of USP Anagrelide Hydrochloride
Use freshly prepared standard and sample solutions and RS in the Standardsolution (mg/mL)
inject within 2 h.
Mobile phase: Proceed as directed in the Assay.
Cu =concentration of Anagrelide Hydrochloride
(anhydrous) in the Sample solution (mg/mL)
Diluent A: Usethe Diluent as described in the Assay. F =relative response factor for each individual
Diluent B: Acetonitrile and water (1 :3) impurity (see Table 7)
Standard stock solution A: 0.05 mg/mL of USP Anagrelide
Related Compound A RS in Diluent A Acceptance criteria See Table 1. Disregard any impurity
Standard stock solution B: 0.05 mg/mL of anagrelide peak less than 0.05%.
related compound B in acetonitrile. Transfer USP
Anagrelide Related Compound B RS into a suitable

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334 Anagrelide / Official Monographs USP 43

Table 1 the final volume). Sonicate to dissolve, and dilute with

Relative Relative Acceptance acetonitrile to volume.
Retention Response Criteria, Standard solution: 0.01 mg/ml of anagrelide free base in
Name Time Factor NMT (%) Diluent from Standard stock solution
Anagrelide related Sample solution: Nominally 0.01 mg/ml of anagrelide free
compound Ba 0.40 0.43 0.3 base prepared from the contents of NlT 20 Capsules. Add
Diluent (80% of the volume of the flask), sonicate for 10
Anagrelide related
cornpound A" 0.55 0.37 0.15 min, and stir for 15 min. Further dilute with Diluent to
volume, centrifuge for 15 min at 4000 rpm, and use the
Anagrelide open ring meth- supernatant for analysis.
yl ester (if present)" 0.80 0.51 0.25 Chromatographic system
Anagrelide 1.00 1.0 - (See Chromatography (621), System Suitability.)
Mode: lC
Anagrelide related Detector: UV 254 nm. For Identification A, use a diode
compound Cd 1.41 0.32 0.15
array detector in the range of 200-400 nm.
Anagrelide trichloro Column: 4.6-mm x 15-cm; 4-lJm packing L11
derivatives 2.44 1.0 0.15 Column temperature: 60°
Any unspecified Flow rate: 1.0 ml/min
impurity - 1.0 0.1 Injection volume: 20 IJL
System suitability
Total impurities - - 1.0
Sample: Standardsolution
a(2-Amino-5,6-dichloroquinazolin-3(4H)-yl)acetic acid. Suitability requirements
b Ethyl 2-(6-amino-2,3-dichlorobenzylamino)acetate. Tailing factor: NMT 2.0
c Methyl2-(5,6 dichloro-2-imino-l ,2-dihydroquinazo/ln-3(4H)-yl)acetate. Relative standard deviation: NMT 2.0%
d Ethyl 2-(5~6-dichloro-2·imino.l ,2-dihydroquinazolin-3(4H)-yl)acetate Analysis
hydrobrornlde. Samples: Standardsolution and Sample solution
e 6,7,8-Trichloro-3,5-dihydroimidazo[2,1·b]quinazolin.2(1 H)-one. Calculate the percentage of the labeled amount of
anagrelide (ClOH 7CI2N 30) in the portion of Capsules
SPECIFIC TESTS taken:
• WATER DETERMINATION, Method I (921): 4.50/0-7.5%
ADDITIONAL REQUIREMENTS Result = (rulrs) x (CslCu) x 100
• PACKAGING AND STORAGE: Preserve in tight, light-resistant = peak response of anagrelide from the Sample
containers. Store in a cold place.
solution
• USP REFERENCE STANDARDS (11) = peak response of anagrelide from the Standard
USP Anagrelide Hydrochloride RS
solution
USPAnagrelide Related Compound A RS
Ethyl 2-(6-amino-2, 3-dichlorobenzylamino)acetate.
=concentration of anagrelide in the Standard
solution (mg/mL)
c.,H14CI2N202 277.15 Cu = nominal concentration of anagrelide in the
USPAnagrelide Related Compound B RS Sample solution (mg/ml)
(2-Amino-5,6-dichloroquinazolin-3(4H)-yl)acetic acid.
CloHgCI2N302 274.10 Acceptance criteria: 90.00/0-110.0%
PERFORMANCE TESTS
• DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 ml
Anagrelide Capsules Apparatus 1: 100 rpm
Time: 15 min
DEFINITION Mobile phase: Prepare as directed in the Assay.
Anagrelide Capsules contain NlT 90.0% and NMT 110.0% of Standard solution: Transfer about 30.32 mg of USP
the labeled amount of anagrelide (ClOH 7C1 2N30). Anagrelide Hydrochloride RS, equivalent to 25.00 mg of
anagrelide, to a 1OO-ml volumetric flask. Add about 80
IDENTIFICATION ml of acetonitrile and 3 drops of 2 N hydrochloric acid.
• A. The UV absorption spectrum of the major peak of the Sonicate until dissolved. Dilute with acetonitrile to volume.
Sample solution exhibits maxima and minima at the same Dilute this solution with Medium to obtain a final
wavelengths as those of the corresponding peak of the concentration of about (Lll 000) mgfrnl, where L is the
Standardsolution, as obtained in the Assay. label claim in mg/Capsule. .
• B. The retention time of the major peak of the Sample Sample solution: Pass a portion of the solution under test
solution corresponds to that of the Standardsolution as through a suitable filter of 0,45-lJm pore size.
obtained in the Assay. ' Chromatographic system
ASSAY (See Chromatography (621), System Suitability.)
• PROCEDURE Mode: lC
Solution A: 1.0 gil of sodium hexanesulfonate. Add 1.0 Detector: UV 274 nm
mL of phosphoric acid and filter. Column: 4.6-mm x 15-cm; 5-lJm packing l7
Mobile phase: Acetonitrile and Solution A (7:13) Sample cooler temperature: 5°
Diluent: Acetonitrile and water (1:1) Flow rate: 1.0 ml/min
Standard stock solution: 0.25 mg/mL of USP Anagrelide Injection volume: 20 IJL
Hydrochloride RS in acetonitrile. Initially add acetonitrile System suitability
(about 80% of the volume of the flask) and a small quantity Sample: Standardsolution
of 2 N hydrochloric acid (about 0.2 ml for every 100 ml of

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USP 43 OfficialMonographs / Anagrelide 335

Suitability requirements Suitability requirements

Tailing factor: NMT 2.0 Resolution: NLT 2.0 between anagrelide hydrochloride
Relative standard deviation: NMT 2.0% and anagrelide related compound C, and between
Analysis anagrelide hydrochloride and anagrelide related
Samples: Standardsolution and Sample solution compound A, System suitability solution
Calculate the percentage of the labeled amount of Tailing factor: NMT 2.0, Standardsolution
anagrelide dissolved: Relative standard deviation: NMT 2.0%, Standard
solution
Result = (rulrs) x (CsICu) x (M,tlM'2) x (VIL) x 100 Analysis
Samples: Standardsolution and Sample solution
to = peak response of anagrelide from the Sample Calculate the percentage of each impurity in the portion of
solution Capsules taken:
rs = peak response of anagrelide from the Standard
solution Result = (ru/r s) x (CsICu) x (1 IF) x 100
Cs = concentration of USP Anagrelide Hydrochloride
RS in the Standardsolution (mg/mL) ru = peak response of each individual impurity from
Cu = nominal concentration of anagrelide in the the Sample solution
Sample solution (mg/mL) rs = peak response of anagrelide from the Standard
M'I = molecular weight of anagrelide, 256.09 solution
M'2 = molecular weight of anagrelide hydrochloride, Cs = concentration of anagrelide in the Standard
292.55 solution (mg/mL)
V = volume of Medium, 900 mL Cu = nominal concentration of anagrelide in the
L = label claim (mg/Capsule) Sample solution (mg/mL)
F = relative response factor (see Table 1)
Tolerances: NLT 80% (Q) of the labeled amount of
anagrelide is dissolved. Acceptance criteria: See Table 1.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements Table 1
IMPURITIES Relative Relative Acceptance
Retention Response Criteria,
• O~GANIC IMPURITIES Name Time Factor NMT(%)
Buffer: 6.8 giL of monobasic potassium phosphate. Adjust
with diluted phosphoric acid to a pH of 3.50 ± 0.05. Mix Anagrelide related corn-
pound Aa 0.86 - -
well and filter. '
Mobile phase: Acetonitrile and Buffer(27:73) Anagrelide
Diluent: Acetonitrile and water (7:13) hydrochloride 1.0 - -
Related compound A stock solution: 10 IJg/mL of USP Anagrelide related corn-
Anagrelide Related Compound A RS in Diluent' pound Bb 0.3 0.34 1.0
Related compound C stock solution: 10 IJg/mL of USP Anagrelide related corn-
Anagrelide Related Compound C RS in Diluent pound C· 1.15 - -
System suitability solution: 0.2 IJg/mL each of USP
Anagrelide Related Compound A RS and USP Anagrelide Anagrelide trichloro derlva-
tive' 1.8-2.3 1.0 0.15
Related Compound C RS and 0.02 mg/mL of USP
Anagrelide Hydrochloride RS prepared asfollows. lnitially Any other individual impurity - - 0.2
dissolve USP Anagrelide Hydrochloride RS in Diluent (about
80% of the volume of the flask), sonicate for 10 min, and
Total impurities - - 1.5
stir for 15 min. Add appropriate quantities of Related a This is a process-related impurity and controlled in the drug substance.
compoundA stock solution and Related compound Cstock b [2-Amino.5,6.dichloroquinazoline.3(4H)-yl]acetic acid.
solution, and dilute with Diluent to volume. c 6,7,8-Trichloro.3,5-dihydroimidazo[2,1.b]quinazolin.2(1 H)-one.
Standard stock solution: Prepare as directed in the Assay.
Standard solution: 0.10 IJg/mL of anagrelide free base in ADDITIONAL REQUIREMENTS
Diluent from Standardstock solution • PACKAGING AND STORAGE: Preserve in tight containers,
Sample solution: Nominally 0.02 mg/mL of anagrelide free protected from light. Storeat controlled room temperature.
base from NLT 20 Capsules. Initially add Diluent to about • USP REFERENCE STANDARDS (11)
80% of the volume of the flask, sonicate for 10 min, stir for USP Anagrelide Hydrochloride RS
about 15 min, and dilute with Diluent to volume. Centrifuge USP Anagrelide Related Compound A RS
the solution at about 4000 rpm for 15 min, and use the Ethyl 2-(6-amino-2, 3-dichlorobenzylamino)acetate.
supernatant for analysis. CllH14CI2N202 277.15
Chromatographic system USP Anagrelide Related Compound C RS
(See Chromatography (621), System Suitability.) Ethyl 2-(5,6-dichloro-2-imino-1,2-dihydroquinazolin-
Mode: LC 3(4H)-yl)acetate hydrobromide.
Detector: UV 254 nm C12H13CI2N302' HBr 383.07
Column: 4.6-mm x 15-cm; 4-lJm packing L11
Column temperature: 45°
Flow rate: 1 mL/min
Injection volume: 30 IJL
System suitability
Samples: System suitability solution and Standard solution

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336 Anastrozole / OfficialMonographs USP 43

Anastrozole System suitability

Sample: Standard solution
Suitability requirements
H3~C
CH
3
H
3C
CH
3

Tailing factor: Between 0.9 and 1.4

NC I "" CN
Relative standard deviation: NMT 0.73%
h
Analysis
Samples: Standard solution and Sample solution
(~
N
Calculate the percentage of anastrozole (C17H19N s) in the
portion of Anastrozole taken:
C17H19N s 293.37
l,3.-Benzenediacetonitrile, u,u,u',u'-tetramethyl-5-(l H;.l,2,4- Result = (r vir s) x (C siCv) x 100
tnazol-l-ylmethyl)-;
u,u,u',u'-Tetramethyl-5-(l H-l,2,4-triazol-l-ylmethyl)-m- =peak area of anastrozole from the Sample solution
benzenediacetonitrile [120511-73-1]. = peak area of anastrozole from the Standard
solution
DEFINITION = concentration of USP Anastrozole RS in the
Anastrozole contains NLT 98.0% and NMT 102.0% of Standard solution (mg/mL)
anastrozole (C17H 19N s), calculated on the anhydrous and = concentration of Anastrozole in the Sample
solvent-free basis. . solution (mg/mL)
IDENTIFICATION
Acceptance criteria: 98.0%-102.0% on the anhydrous and
solvent-free basis
IMPURITIES
• A.~~~~~~~~~ • RESIDUE ON IGNITION (281): NMT 0.1 %
Spectrosc:oPy:'l?7I<.i. . +M~}'t~Q:ZQ) • ORGANIC IMPURITIES
• B. The retention time of the major peak of the Sample Solution A, Solution B, and Chromatographic system:
solution corresponds to that of the Standard solution, as Proceed as directed in the Assay.
obtained in the Assay. Standard stock solution: 0.2 mg/mL of USPAnastrozole RS
ASSAY prepared as follows. Dissolve in acetonitrile, using 40% of
the final volume, and then dilute with Solution A to volume.
• PROCEDURE
Solution A: Acetonitrile, methanol, trifluoroacetic acid, and Standard solution: 0.02 mg/mL of USP Anastrozole RS in
water (100: 3PO: 0.5: 600) Solution A from the Standard stock solution
Solution B: Acetonitrile, methanol, trifluoroacetic acid, and Sample solution: 2 mg/mL of Anastrozole prepared as
water (150: 450: 0.5: 400) follows. Transfer 50 mg of Anastrozole to a 25-mL
Mobile phase: See Table 1. volumetric flask. Add 10 mL of acetonitrile. Dissolve in and
dilute with Solution A to volume.
Table 1 Blank solution: Transfer 10 mL of acetonitrile into a 25-mL
volumetric flask, and dilute with Solution A to volume.
Time Solution A Solution B System suitability
(min) (%) (%)
Sample: Standard solution
0 100 0 Suitability requirements
10 100 0
Tailing factor: Between 0.9 and 1.4
Relative standard deviation: NMT 5%
40 0 ioo Analysis
41 100 0 Samples: Standard solution, Sample solution, and Blank
solution. [NOTE-Adjustthe peak areas for any interference
56 100 0 from the Blank solution.]
Calculate the percentage of each individual impurity in the
[NOTE-These gradient elution times are established on portion of Anastrozole taken:
an HPLC system with a dwell time of approximately 0
min. The gradient elution times in the table can be Result = (r vir s) x (C siCv) x 100
adjusted by subtracting the dwell time to achieve the
separation described.] = peak area of each individual impurity from the
Standard solution: 0.5 mg/mL of USPAnastrozole RS Sample solution
prepared as follows. Transfer USP Anastrozole RS into a = peak area of anastrozole from the Standard
suitable volumetric flask. Dissolve in acetonitrile, using 40% solution
of the final volume, and then dilute with Solution A to = concentration of USP Anastrozole RS in the
volume. Standard solution (mg/mL)
Sample solution: 0.5 mg/mL of Anastrozole prepared as =concentration of Anastrozole in the Sample
follows. Transfer 25 mg of Anastrozole to a 50-mL solution (mg/mL)
volumetric flask, add 20 mL of acetonitrile to dissolve.
Dilute with Solution A to volume. Acceptance criteria: See Table 2. Disregard any impurity of
Chromatographic system less than 0.05%.
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 21 5 nm
Column: 3.2-mm x 1O-cm; 5-~m packing L42
Flow rate: 0.75 mL/min .
Injection volume: 10 ~L

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 USP 43 OfficialMonographs / Anastrozole 337

Table 2 Tablets to a suitable volumetric flask. Add 40% of the flask

Relative Acceptance volume of water, and shake on a rotary shakerfor 10 min
Retention Criteria, to disintegrate the Tablets. Add 40% of the flask volume of
Name Time NMT (%) acetonitrile, and sonicate for 15 min with intermittent
Oesmethyl anastrozole- 0.6 0.2 shaking, maintaining the sonicator temperature at 25°.
Dilute with Diluent to volume. Centrifuge a portion of the
Anastrozole 1.0 - solution at 3500 rpm for 10 min, and usethe clear solution
Anastrozole dimer" 2.0 0.2 for analysis.
Chromatographic system
S-Bromomethyl anastrozole- 4.3 0.1 (See Chromatography (621), System Suitability.)
S-Oibromomethyl anastrozole" 5.4 0.1 Mode: LC
Detector: UV 215 nm
Individual unspecified impurity - 0.1 Column: 4.6-mm x 15-cm; 5-lJm packing L1
Total impurities - 0.5 Flow rate: 1 mL/min
Injection volume: 20 IJL
a 2-(3-(1-Cyanoethyl)-S-(1 H-l ,2,4-triazol-l-ylmethyl)phenyl)-2- System suitability
methylpropionitrile. Sample: Standardsolution
b 2,3-Bis(3-(1-cyano-l-methylethyl)-S-(1 H-' ,2,4-triazol-'-ylmethyl)phenyl)-2- Suitability requirements
methylpropionitrile.
c 2,2'-(S-(Bromomethyl)-', 3-phenylene)bis(2-methylpropionitrile).
Tailing factor: NMT 2.0
d 2,2'-(S-(Oibromomethyl)-' ,3-phenylene)bis(2-methylpropionitrile).
Relative standard deviation: NMT 2.0%
Analysis
SPECIFIC TESTS Samples: Standardsolution and Sample solution
• WATER DETERMINATION, Method Ie (921): NMT 0.3% Calculate the percentage of the labeled amount of
anastrozole (C17H,9NS) in the portion of Tablets taken:
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed Result =(rulrs) x (CsICu) x 100
containers. Store at room temperature.
• USP REFERENCE STANDARDS (11) = peak area from the Sample solution
USP Anastrozole RS = peak area from the Standard solution
=concentration of USP Anastrozole RS in the
Standardsolution (mg/mL)
Cu = nominal concentration of anastrozole in the
Sample solution (mg/mL)
Anastrozole tablets
Acceptance criteria: 90%-110%
DEFINITION
Anastrozole Tablets contain NLT 90% and NMT 110% of the PERFORMANCE TESTS
labeled amount of anastrozole (C17H19Ns)' • DISSOLUTION (711)
Test 1
IDENTIFICATION Medium: Water; 900 mL, deaerated
Apparatus 2: 50 rpm
Time: 15 min
Mobile phase: Acetonitrile and water (40:60)
• A•..t~.~~~!~~~~~~'~;~.~~~~~~I~jI!9NtI"~$'I7$!(Oif!;~);;ilnti:i:Ji:~C:I Diluent: Acetonitrile and water (50:50)
Sp~~trp~eQPY:·J~?~~(CN1 ..f\i1aY@205 . Standard stock solution: 0.2 mg/mL of USP Anastrozole
Sample: Transfer the finely ground Tablet powder RS in Diluent
containing 8 mg of anastrozole into a suitable container. Standard solution: Dilute the Standardstocksolution with
Add 10 mL of diethyl ether and sonicate for 5 min. Aspirate Medium to obtain a final concentration of (L/1000)
the supernatant and pass through a nylon filter of 0.45-lJm mg/mL, where L is the label claim in mg/Tablet.
pore size into another suitable container containing 400 Sample solution: Pass a portion of the solution under test
mg of potassium bromide. Evaporate the mixture to through a suitable filter of 0.45-lJm pore size. Discard the
dryness under nitrogen. Further dry it under vacuum at 50° first few mL of the filtrate.
for 1 h. Add an additional 400 mg of potassium bromide Chromatographic system
for preparation of pellet and analysis. (See Chromatography (621), System SUitability.)
Acceptance criteria: The spectrum obtained from the Mode: LC
Sample shows bands at approximately 2235, 1606, 1500, Detector: UV 215 nm
1359, 1205, 1137, 1013, and 875 crrr', similar to the Column: 4.6-mm x 15-cm; 5-lJm packing L1
spectrum from the Reference Standard similarly obtained. Flow rate: 1 mL/min
• B. The retention time of the major peak of the Sample Injection volume: 50 IJL
solution corresponds to that of the Standardsolution, as System suitability
obtained in the Assay. Sample: Standardsolution
ASSAY Suitability requirements
• PROCEDURE Tailing factor: NMT 2.0
Mobile phase: Acetonitrile and water (40:60) Relative standard deviation: NMT 2.0%
Diluent: Acetonitrile and water (50:50) Analysis
Standard solution: 40 IJg/mL of USP Anastrozole RS in Samples: Standardsolution and Sample solution
Diluent. Sonication may be used to aid dissolution. Calculate the percentage of the labeled amount of
Sample solution: Nominally equivalent to 40 IJg/mL of anastrozole (C17H19N s) dissolved:
anastrozole in Diluent, prepared asfollows. Transfer NLT 10

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 338 Anastrozole / Official Monographs USP 43

Result =(rulr s) x (CslL) x Vx 100 Table 1

Time Solution A Solution B
tu = peak response from the Sample solution (min) (%) (%)
rs = peak response from the Standard solution 0 100 0
Cs = concentration of the Standard solution(mg/mL)
L = label claim (mg/Tablet) 25 100 0
V = volume of Medium, 900 mL 25.1 0 100
Tolerances: NLT 80% (Q) of the labeled amount of 30 0 100
anastrozole (C17H19N s) is dissolved. 31 100 0
Test 2: Ifthe product complies with this test, the labeling
indicates that it meets USP Dissolution Test 2. 40 100 0
Medium: Water; 1000 mL, deaerated
Apparatus 2: 50 rpm Diluent: Acetonitrile, trifluoroacetic acid, and water (200:
Time: 15 min 0.8: 800)
Mobile phase: Acetonitrile, trifluoroacetic acid, and water System suitability stock solution: 0.5 mg/mL of USP
(300:1 :700) Anastrozole RS and 0.3 mg/mL of ethyl
Standard stock solution: 0.2 mg/mL of USP Anastrozole 4-hydroxybenzoate in Diluent prepared as follows. Transfer
RS prepared as follows. Transfer USP Anastrozole RS into USP Anastrozole RS and ethyl 4-hydroxybenzoate into a
a suitable volumetric flask and add acetonitrile equivalent suitable volumetric flask and add Diluent equivalent to 50%
to 8% of the final volume. Sonicate to dissolve and of the final volume. Sonicate to dissolve and dilute with
dilute with water to volume. Diluent to volume.
Standard solution: Dilute the Standard stock solution with System suitability solution: 10 uq/rnl, of USP Anastrozole
Medium to obtain a final concentration of (L/l 000) RS and 6 uq/rn], of ethyl4-hydroxybenzoate in Diluentfrom
mg/mL, where L is the label claim in mg/Tablet. the System suitabilitystock solution
Sample solution: Pass a portion of the solution under test Standard stock solution: 0.5 mg/mL of USP Anastrozole RS
through a suitable filter of 0.45-~m pore size. Discard the in Diluent prepared as follows. Transfer USP Anastrozole RS
first few mL of the filtrate. into a suitable volumetric flask and add Diluent equivalent
Chromatographic system to 50% of the final volume. Sonicate to dissolve and
(See Chromatography (621), System Suitability.) dilute with Diluent to volume.
Mode: LC Standard solution: 10 ~g/mL of USP Anastrozole RS in
Detector: UV 215 nm Diluent from the Standard stock solution
Column: 3.2-mm x 1O-cm; 5-~m packing L42 Sample solution: Nominally equivalent to 1.0 mg/mL of
Flow rate: 0.75 mL/min anastrozole from NLT 25 finely powdered Tablets, prepared
Injection volume: 100 ~L as follows. Transfer a weighed quantity of powdered
System suitability Tablets, equivalent to 10 mg of anastrozole, to a suitable
Sample: Standardsolution container and add 10.0 mL of Diluent. Sonicate for 30 min
Suitability requirements and allow to cool to room temperature. Pass through a
Tailing factor: 0.9-1.4 suitable filter of 0.45-~m pore size, and discard the first few
Relative standard deviation: NMT 1.5% mL of the filtrate. Ifthe filtrate is not clear, pass again
Analysis . through a suitable filter of 0.2-~m pore size, and discard the
Samples: Standardsolution and Sample solution first few mL of the filtrate.
Calculate the percentage of the labeled amount of Chromatographic system
anastrozole (C17H19N s) dissolved: . (See Chromatography (621), System Suitability.)
Mode: LC
Result = (rulrs) x (CslL) x Vx 100 Detector: UV 215 nm
Column: 3.2-mm x 1O-cm; 5-~m packing L42
to = peak response from the Sample solution Flow rate: 1.0 mL/min
ts = peak response from the Standard solution Injection volume: 10 ~L
Cs =concentration of the Standard solution(mg/mL) Analysis time: 25 min
L = label claim (mg/Tablet) System suitability
V = volume of Medium, 1000 mL Sample: System suitabilitysolution
[NoTE-The relative retention times for ethyl
Tolerances: NLT 80% (Q) of the labeled amount of 4-hydroxybenzoate and anastrozole are 0.7 and 1.0,
anastrozole (C17H19N s) is dissolved. respectively.]
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Suitability requirements
requirements Resolution: Greater than 4 between the ethyl
4-hydroxybenzoate and anastrozole peaks
IMPURITIES Tailing factor: 0.9-1.3 for the anastrozole peak
• ORGANIC IMPURITIES Relative standard deviation: NMT 5% for the
Solution A: Methanol, acetonitrile, trifluoroacetic acid, and anastrozole peak
water (200: 100: 0.7: 700) Analysis
Solution B: Methanol, acetonitrile, trifluoroacetic acid, and Samples: Standard solution and Sample solution
water (500: 250: 0.7: 250) Calculate the percentage of each impurity in the portion of
Mobile phase: See Table 1. Tablets taken: .

Result =(rulrs) x (CsICu) x 100

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 USP 43 Official Monographs / Anileridine 339

= peak response of each individual impurity from DEFINITION

the Sample solution Anileridine contains NLT 98.5% and NMT 101.0% of
= peak response of anastrozole from the Standard anileridine (CzzHzsNzOz), calculated on the anhydrous basis.
solution
=concentration of USP Anastrozole RS in the IDENTIFICATION
Standard solution (mg/mL) • A.
= nominal concentration of anastrozole in the Buffer solution: Dissolve 5.68 g of anhydrous dibasic
Sample solution (mg/mL) sodium phosphate and 3.63 g of monobasic potassium
phosphate in water to make 1000 mL: the pH is 7.0 ± 0.05.
Acceptance criteria: See Table 2. Disregard any impurity Sample stock solution: Dissolve40 mg of Anileridine in 2.3
peak lessthan 0.1 %. mL of 0.1 N hydrochloric acid in a 1OO-mL volumetric flask
and dilute with water to volume. '
Table 2 Sample solution A: Sample stock solution, Buffer solution,
Relative Acceptance
and water (4:25:71)
Retention Criteria, Sample solution B: Sample stock solution, Buffer solution,
Name Time NMT (%) and water (20:25:55)
Acceptance criteria: The UV absorption spectrum of Sample
Anastrozole dlarnlde- 0.11 0.5
solution A exhibits a maximum at 234 ± 1 nm; and the UV
Anastrozole monoacid rnonoarnlde" 0.26 0.5 absorption spectrum of Sample solution B exhibits a
Anastrozole monoamide mononi-
maximum at 285 ± 2 nm. The ratio 5Az34/AzS5 is 8.8.
trlle- 0.30 0.5 • lB.
Sample solution: 0.2 mg/mL of Anileridine in 0.1 N
Desmethyl anastrozole" 0.51 - hydrochloric acid
Anastrozole diacld" 0.71 0.5 Analysis: To 5 mL of the Sample solution add 2 mL of a
solution of p-dimethylaminobenzaldehyde in alcohol (1 in
Anastrozole monoacid 100).
rnononltrlle' 0.87 0.5
Acceptance criteria: A yellow color develops immediately.
Anastrozole 1.00 - ASSAY
Anyindividual unspecified • PROCEDURE
impurity - 0.5 Sample: 350 mg of Anileridine
Total impurities - 1.0 Titrimetric system
Mode: Direct titration
a 2,2'-(5-[(1 H-1,2,4-Triazol~ 1-yl)methyl]-l,3-phenylene}bis(2- Titrant: 0.1 N perchloric acid VS
methylpropanamide). Endpoint detection: Visual
b 2-(3-[(1 H-1,2,4-Triazol-1-yl)methyl]-S-(1-amino-2-methyl-1-oxopropan-2-yl)
phenyl}-2-methylpropanoic acid.
Analysis: Dissolve the Sample in 50 mL of glacial acetic acid,
c 2-(3-[(1 H-1,2,4-Triazol-1-yl)methyl]-S-(2-cyanopropan-2-yl)phenyl}-2- add 1 drop of crystal violet TS, and titrate with Titrant to a
methylpropanamide. blue-green endpoint. Perform a blank determination, and
d 2-(3-(1-Cyanoethyl)-S-(1 H-1,2,4-triazol-1-ylmethyl)phenyl)-2- make any necessary correction. Each mL of Titrant is
methylpropanenitrile. This processimpurity is controlled in the drug substance equivalent to 17.62 mg of anileridine·(CzzHzsNzOz).
monograph. It is included in the table for identification only,and it is not to be Acceptance criteria: 98.5%-101.0% on the anhydrous
reported in the total impurities.
e ~,2'-(S-[(1 H-1,2,4-Triazol-1-yl)methyl]-l,3-phenylene}bis(2-niethylpropanoic basis
add),
f 2-{3-[(1 H-1,2,4-Triazol-1-yl)methyl]-S-(2-cyanopropan-2-yl)phenyl}-,z-
IMPURITIES
methylpropanoic acid. • RESIDUE ON IGNITION (281): NMT 0.1%
• CHLORIDE AND SULFATE, Chloride (221)
ADDITIONAL REQUIREMENTS Sample: 180 mg
• PACKAGING AND STORAGE: Preserve in tight containers, and Analysis: Dissolve the Sample in a mixture of 1 mL of nitric
store at controlled room temperature. acid and 40 mL of water.
• LABELING: When more than one Dissolution test is given, the Acceptance criteria: NMT 400 PPm.; the solution shows no
labeling states the test used only if Test 7 is not used. more chloride than corresponds to 0.10 mL of 0.020 N
• USP REFERENCE STANDARDS (11) hydrochloric acid.
USP Anastrozole RS SPECIFIC TESTS
• WATER DETERMINATION, Method I (921): NMT 1.0%
ADDITIONAL REQUIREMENTS
• PACKAGING A~D STORAGE: Preserve in tight, light-resistant
Anileridine containers. Store at room temperature.

Anileridine Injection
DEFINITION
Anileridine Injection is a sterile solution of Anileridine in Water
CZ2H2SNzOz 352.47
for Injection, prepared with the aid of Phosphoric Acid. It
4-Piperidinecarboxylic acid, 1-[2-(4-aminophenyl)ethyl]-4- contains NLT 90.0% and NMT 115.0% of the labeled
phenyl-, ethyl ester;
amount of anileridine (CzzHzsNzOz), as the phosphate.
Ethyl 1-(p-aminophenethyl)-4-phenylisonipecotate [144-
14-9].

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 340 Anileridine / Official Monographs USP 43

IDENTIFICATION
CD A. Anileridine Hydrochloride
Sample solution: 0.25 mg/mL of anileridine from Injection
diluted with water C22H2sN202 .2HCI 425.39
Analysis: To 5 mL of the Sample solution add 2 mL of a 4-Piperidinecarboxylic acid, 1-[2-(4-aminophenyl)ethyl]-4-
1O-mg/mL solution of p-dimethylaminobenzaldehyde in phenyl-, ethyl ester, dihydrochloride;
alcohol. Ethyl l-(p-am inophenethyl)-4-phenylisonipecotate
Acceptance criteria: A yellow color develops immediately. dihydrochloride [126-12-5].
o B. A volume of Injection, diluted with water to a DEFINITION .
concentration of 0.025 mg/mL of anileridine, exhibits Anileridine Hydrochloride contains NLT 96.0% and NMT
absorbance maxima at 234 ± 1 and 285 ± 2 nm. 102.0% of anileridine hydrochloride (C22H2sN202 . 2HCI),
ASSAY calculated on the dried basis.
o PROCEDURE IDENTIFICATION
Standard solution: Prepare 250 IJg/mL of USP Anileridine
Hydrochloride RS in 0.1 N hydrochloric acid on the day of
the assay. Each mg of anileridine hydrochloride is
equivalent to 0.8286 mg of anileridine.
Sample solution: Nominally equivalent to 200 IJg/mL of
anileridine, from a volume of Injection diluted with 0.1 N
hydrochloric acid Buffer solution: 5.68 giL of anhydrous dibasic sodium
Blank: 0.1 N hydrochloric acid phosphate and 3.63 giL of monobasic potassium
Instrumental conditions phosphate in water: the pH, determined
Analytical wavelength: 560 nm potentiometrically, is 7.0 ± 0.05.
Cell: 1 cm Sample stock solution: 0.5 mg/mL of anileridine
Analysis hydrochloride in water
Samples: Standard solution, Sample solution, and Blank Sample solution A: Sample stock solution, pH 7.0 buffer
Transfer 5.0 mL each of the Standard solution, Sample solution, and water (4:25:71)
solution, and Blank to separate 200-mL volumetric flasks. Sample solution B: Sample stock solution, pH 7.0 buffer
To eachflask add 25 mL of water,S mL of 1 N hydrochloric solution, and water (20:25:55)
acid, and 5 mL of 1-mg/mL sodium nitrite solution. Allow Acceptance criteria: The UV absorption spectrum of Sample
to stand for 2 min, then add to each flask 5 mL of solutionA exhibits a maximum at 234 ± 1 nm, and the UV
5-mg/mL ammonium sulfamate solution. Allow to stand absorption spectrum of Sample solution B exhibits a
for 3 min, then add 5 mL of 1-mg/mL N-(l-naphthyl) maximum at 285 ± 2 nm.
ethylenediamine dihydrochloride solution. Allow to stand o C.
for 1 h, and dilute with water to volume. Use the reagent Sample solution: 0.2 mg/mL of Anileridine Hydrochloride
blank to set the instrument. Analysis: To 5 mL of the Sample solution add 2 mL of a
Calculate the percentage of anileridine (C2zH2sN202) in the solution of 10 mg/mL of p-dimethylaminobenzaldehyde
volume of Injection taken: in alcohol.
Acceptance criteria: A yellow color develops immediately.
Result = (A viA s) x (C siC v) x (M rdM r2) X 100 o D. IDENTIFICATION TESTS-GENERAL, Chloride (191)
Sample: 1O-mg/mL solution of Anileridine Hydrochloride
Au =absorbance of the Sample solution Acceptance criteria: Meets the requirements
As =absorbance of the Standard solution
Cs = concentration of USP Anileridine Hydrochloride ASSAY
o PROCEDURE
RS in the Standard solution (lJg/mL)
Cu = nominal concentration of anileridine in the Sample: 200 mg of Anileridine Hydrochloride
Sample solution (lJg/mL) Titrimetric system
M r7 =molecular weight of anileridine, 352.48 Mode: Direct titration
M r2 = molecular weight of anileridine hydrochloride, Titrant: 0.1 N perchloric acid VS
425.40 Endpoint detection: Visual
Analysis: Dissolve the Sample in 10 mL of glacial acetic acid
Acceptance criteria: 90.00/0-115.0% by heating it on a steam bath. Cool immediately in a cold
water bath, add 5 mL of mercuric acetate TS, 20 mL of
SPECIFIC TESTS acetone, and 0.5 mL of indicator solution (70 mg of
o pH (791): 4.5-5.0 a-naphtholbenzein, 10 mg of crystal violet, and 40 mg of
o BACTERIAL ENDOTOXINS TEST (85): It contains NMT 7.2 USP quinaldine red in 100 mL of glacial acetic acid). Titrate with
Endotoxin Units/mg of anileridine. Titrant to a gray-green endpoint. Perform a blank
o OTHER REQUIREMENTS: It meets the requirements in determination, and make any necessary correction. Each
Injections and ImplantedDrug Products (1). mL of Titrant is equivalent to 21.27 mg of anileridine
hydrochloride (C22H2sN202 . 2HCI).
ADDITIONAL REQUIREMENTS
Acceptance criteria: 96.00/0-1 02.0% on the dried basis
o PACKAGING AND STORAGE: Preserve in single-dose or
multiple-dose containers, preferably of Type I glass, OTHER COMPONENTS
protected from light. o CONTENT OF CHLORIDE
o USP REFERENCE STANDARDS (11) Sample: 200 mg
USP Anileridine Hydrochloride RS Analysis: Dissolve the Sample in 50 mL of water in a
glass-stoppered flask. Add 25.0 mL of 0.1 N silver nitrate
VS, then add 5 mL of 2 N nitric acid and 5 mL of
nitrobenzene, shake vigorously, and add 2 mL of ferric

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 USP 43 Official Monographs / Anileridine 341

ammonium sulfate TS. Titrate the excess silver nitrate with acid and 100 mL of water, and heat on a water bath. Cool,
0.1 N ammonium thiocyanate VS. Each mL of 0.1 N silver and dilute with water to volume. Filter the solution,
nitrate is equivalent to 3.545 mg of CI. discarding the first 25 mL of the filtrate.
Acceptance criteria: 16.00/0-17.2% Blank: 0.1 N hydrochloric acid
Instrumental conditions
IMPURITIES AnalytiCal wavelength: 560 nm
CD RESIDUE ON IGNITION (281): NMT 0.1%
Cell: 1 cm
SPECIFIC TESTS Analysis
CD pH (791) Samples: Standard solution, Sample solution, and Blank
Sample solution: 50 mg/mL Transfer 5.0 mL each of the Standardsolution, Sample
Acceptance criteria: 2.5-3.0 solution, and Blank to separate 200-mL volumetric flasks.
CD Loss ON DRYING (731) _, To each flask add 25 mL of water, 5 mL of 1 N
Analysis: Dry a sample at a pressurebelow 5 mm of mercury hydrochloric acid, and 5 mL of 1-mg/mL sodium nitrite
at 100 0 for 2 h. solution. Allow to stand for 2 min, then add to each flask
Acceptance criteria: NMT 1.0% 5 mL of 5-mg/mL ammonium sulfamate solution: Allow
to stand for 3 min, then add 5 mL of 1-mg/mL
ADDITIONAL REQUIREMENTS N-(1 -naphthyl)ethylenediamine dihydrochloride
CD PACKAGING AND STORAGE: Preserve in tight, light-resistant solution. Allow to stand for 1 h, and dilute with water to
containers. volume. Usethe reagent blank to set the instrument.
CD USP REFERENCE STANDARDS (11) Calculate the percentage of anileridine (C22H2sN202) in the
USP Anileridine Hydrochloride RS portion of Tablets taken:

Result = (Au/As) x (CsICu) x (MrdMr2 ) x 100

Anileridine Hydrochloride Tablets =absorbance of the solution from the Sample

solution
DEFINITION
=absorbance of the solution from the Standard
solution
Anileridine Hydrochloride Tablets contain an amount of = concentration of USP Anileridine Hydrochloride
anileridine hydrochloride (C22H2sN202 . 2HCI) equivalent to RS in the Standardsolution (J,Jg/mL)
NLT 95.0% and NMT 105.0% of the labeled amount of = nominal concentration of anileridine in the
anileridine (C22H2SN202)' Sample solution (J,Jg/mL)
IDENTIFICATION • = molecular weight of anileridine, 352.48
• A.
=molecular weight of anileridine hydrochloride,
.Sample solution: Transfer an equivalent to 50 mg of 425.40
anileridine, from finely powdered Tablets, to a 250-mL
volumetric flask. Add 100 mL of water, and heaton a steam Acceptance criteria: 95.0%-105.0%
bath. Cool, dilute to volume, and filter. PERFORMANCE TESTS
Analysis: To 5 mL of the Sample solution add 2 mL of a • DISSOLUTION (711)
1O-mg/mL solution of p-dimethylaminobenzaldehyde in Medium: 0.01 N hydrochloric acid; 900 mL
alcohol. . Apparatus 1: 100 rpm
Acceptance criteria: A yellow color develops immediately. Time: 45 min
• B. Standard solution: USP Anileridine Hydrochloride RS of a
Buffer solution: 5.68 giL of anhydrous dibasic sodium known concentration, in Medium
phosphate and 3.63 giL of monobasic potassium Sample solution: Filtered portion of the solution under test
phosphate in water: the pH, determined Analysis
potentiometrically, is 7.0 ± 0.05. Samples: Standard solution and Sample solution
Sample stock solution: Transfer an equivalent to 50 mg of Calculate the percentage of anileridine (C22H2sN202)
anileridine, from finely powdered Tablets, to a 100-mL dissolved, using the Analysis set forth in the Assay and in
volumetric flask. Add 30 mL of water, and heat on a steam comparison to the Standardsolution.
bath. Cool, dilute with water to volume, and filter. Tolerances: NLT 65% (Q) of the labeled amount of
Sample solution A: Sample stock solution, Buffer solution, anileridine (C22H2sN202) is dissolved.
and water (4:25:71) CD UNIFORMITY OF DOSAGE UNITS (905): Meet the
Sample solution B: Sample stock solution, Buffer solution, requirements
and water (20:25:55)
Acceptance criteria: The UV absorption spectrum of Sample ADDITIONAL REQUIREMENTS
solution A exhibits a maximum at 234 ± 1 nm, and the UV CD PACKAGING AND STORAGE: Preserve in tight, light-resistant

absorption spectrum of Sample solution B exhibits a containers.

maximum at 285 ± 2 nm. • USP REFERENCE STANDARDS (11)
USP Anileridine Hydrochloride RS
ASSAY
CD PROCEDURE

Standard solution: 250 J,Jg/mL of USP Anileridine

Hydrochloride RS in 0.1 N hydrochloric acid. (Each mg of
anileridine hydrochloride is equivalent to 0.8286 mg of
anileridine.) Prepare on the day of the assay.
Sample solution: Transfer an equivalent to 50 mg of
anileridine, from finely powdered Tablets (NLT 20), to a
250-mL volumetric flask. Add 25 mL of 1 N hydrochloric

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342 Antazoline / OfficialMonographs USP 43

Antazoline Phosphate rs = peak response from the Standard solution

Cs = concentration of USP Antazoline Phosphate RS in
the Standard solution (mg/mL)
Cv = concentration of AntazoJine Phosphate in the
Sample solution (mg/mL)
Acceptance criteria: 98.0%-102.0% on the dried basis
C17H19N 3· H3P04 363.35 IMPURITIES
1H-lmidazole-2-methanamine, 4,5-dihydro-N-phenyl- • ORGANIC IMPURITIES
N-(phenylmethyl)-, phosphate (1:1); Solution A: Dilute 1.0 mL of formic acid with water to 1000
2-[(N-Benzylanilino)methyl]-2-imidazoline phosphate (1:1) mL.
[154-68,.7]. Mobile phase: Acetonitrile and Solution A (17:83)
Diluent: Acetonitrile and water (17:83)
DEFINITION Standard solution A: 5 J.Ig/mL of USP Antazoline Related
AntazoJine Phosphate contains NLT 98.0% and NMT 102.0% Compound A RS and 1.0 mg/mL of USP Antazoline
of antazoJine phosphate (C17H19N 3 . H3P04), calculated on Phosphate RS in Diluent
the dried basis. Standard solution B: 1 J.Ig/mL of USP Antazoline Phosphate
IDENTIFICAnON RS in Diluent
Sample solution: 1.0 mg/mL of Antazoline Phosphate in
Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
peak of the Sample Detector: UV 240 nm
solution corresponds to that of Standard solution, as Column: 2.1-mm x 10-cm; 1.7-urn packing L11
obtained in the Assay. Temperatures
Column: 35 0
ASSAY Autosampler: 4 0
• PROCEDURE Flow rate: 0.5 mL/min
Solution A: Dilute 1.0 mL of formic acid with water to 1000 Injection volume: 5 J.lL
mL. Run time: NLT 4 times the retention time of antazoline
Mobile phase: Acetonitrile and Solution A (17:83) System suitability
Diluent: Acetonitrile and water (17:83) Samples: Standard solution A and Standard solution B
Standard solution: 0.2 mg/mL of USP Antazoline [NoTE-The relative retention times of antazoline and
Phosphate RS in Diluent . antazoline related compound A are 1.0 and 1.1,
System suitability solution: 1 J.Ig/mL of USP Antazoline respectively.]
Related Compound A RS in the Standard solution Suitability requirements
Sample solution: 0.2 mg/mL of Antazoline Phosphate in Resolution: NLT 2.0 between antazoline and antazoline
Diluent related compound A peaks, Standard solution A
Chromatographic system Relative standard deviation: NMT 5.0% for antazoline,
(See Chromatography (621), System Suitability.) Standard solution B
Mode: LC Analysis
Detector: UV 240 nm Samples: Standard solution A, Standard solution B, and
Column: 2.1-mm x 10-cm; 1.7-J.Im packing L11 Sample solution
Temperatures Calculate the percentage of antazoline related compound
Column: 35 0 A in the portion of Antazoline Phosphate taken:
Autosampler: 4 0

Flow rate: 0.5 mL/min Result =(rulrs) x (Cs!Cu) x 100

Injection volume: 5 J.IL
System suitability to =peak response of antazoline related compound A
Samples: Standard solution and System suitability solution from the Sample solution
[NoTE-The relative retention times of antazoline and rs = peak response of antazoline related compound
antazoline related compound A are 1.0 and 1.1, Afrom Standard solution A
respectively. ] Cs = concentration of USP Antazoline Related
Suitability requirements Compound A RS in Standard solution A (mg/mL)
Resolution: NLT2.0 between antazoline and antazoline Cv =concentration of Antazoline Phosphate in the
related compound A, System sUitability solution Sample solution (mg/mL)
Tailing factor: NMT 1.5 for antazoline, Standard solution
Relative standard deviation: NMT 0.73% for Calculate the percentage of any individual unspecified
antazoline, Standard solution impurity in the portion of Antazoline Phosphate taken:
Analysis
Samples: Standard solution and Sample solution Result = (rulrs) x (Cs!Cu) x 100
Calculate the percentage of antazoline phosphate
(C17H19N 3· H3P04) in the portion of Antazoline Phosphate rv = peak response of any impurity from the Sample
taken: solution
rs = peak response ofantazoline phosphate from
Result = (ru/rs) x (CsICu) x 100 Standard solution B
C, =concentration of USP Antazoline Phosphate RS in
t» = peak response from the Sample solution Standard solution B(mg/mL)

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 USP 43 Official Monographs / Anthralin 343

Cu = concentration of Antazoline Phosphate in the Internal standard solution: 0.5 mg/mL of o-nitroaniline in
Sample solution (mg/mL) n-hexane prepared asfollows. First dissolve o-nitroaniline
in a small quantity of dichloromethane, and then dilute
Acceptance criteria: See Table 7. Disregard any impurity with n-hexane.
peaks less than 0.05%. System suitability stock solution: 0.1 mg/mL of USP
Anthralin RS and 0.2 mg/mL of danthron in
Table 1 dichloromethane
Relative Acceptance System suitability solution: Transfer 5 mL of the System
Retention Criteria, suitability stock solution into a 25-mL volumetric flask, add
Name Time NMT(%) 5 mL of n-hexane, and dilute with Mobile phase to volume.
Antazoline phosphate 1.0 - Solvent blank solution: Mobile phase, n-hexane, and
dichloromethane (3:1:1)
Antazoline relatedcompound A" 1.1 0.5 Standard stock solution: 0.25 mg/mL of USP Anthralin RS
Anyindividual unspecified impurity - 0.5 in dichloromethane
Standard solution: Transfer 5 mL each of Standard stock
Total impurities - 1.0 solution and Internal standard solution into a 25-mL
a N-(2-Aminoethyl)-2-[benzyl(phenyl)amino]acetamide.
volumetric flask, and dilute with Mobile phase to volume.
Sample stock solution: 0.25 mg/ml of Anthralin in
SPECIFIC TESTS dichloromethane
Sample solution: Transfer 5 mL each of Sample stock
• pH (791) solution and Internal standard solution into a 25-mL
Sample: 20 mg/mL
volumetric flask, dilute with Mobile phase to volume.
Acceptance criteria: 4.0-5.0
Chromatographic system
• Loss ON DRVING (731)
(See Chromatography (621), System Suitability.)
Analysis: Dry at 105° for 4 h.
Mode: LC
Acceptance criteria: NMT 0.5%
Detector: UV 354 nm
ADDITIONAL REQUIREMENTS Column: 4.6-mm x 25-cm; packing L3
• PACKAGING AND STORAGE: Preserve in tight containers. Flow rate: 2 ml/min
• USP REFERENCE STANDARDS (11) Injection volume: 10 I-/L
USP Antazoline Phosphate RS System suitability
USP Antazoline Related Compound A RS Samples: System suitability solution, Solvent blank solution,
N-(2-Aminoethyl)-2-[benzyl(phenyl)amino]acetamide. and Standard solution
C17H21N30 28~.38 [NOTE-The relative retention times for anthralin,
danthron, dianthrone, and o-nitroaniline are 1.0, 1.2,
1.7, and 2.3, respectively.]
Suitability requirements
Resolution: NLT 1.3 between anthralin and danthron,
Anthralin System suitability solution
Tailing factor: NMT 1.5, System suitability solution
Relative standard deviation: NMT 2.0% of the ratio of
the peak responses, Standard solution
Interference: No discernible signal is observed at the
retention time of anthralin, Solvent blank solution
C14H lO0 3 226.23 Analysis
9(10H)-Anthracenone, 1,8-dihydroxy-; Samples: Standard solution and Sample solution
1,8-Dihydroxy-9-anthrone [1143-38-0]. Calculate the percentage of anthralin (C14H lO0 3) in the
portion of Anthralin taken:
DEFINITION
Anthralin contains NLT 97.0% and NMT 102.0% of anthralin Result = (Ru/R s) x (Cs/Cu) x 100
(C14H lO0 3) , calculated on the dried basis.
IDENTIFICATION = peak response ratio of anthralin to o-nitroaniline
from the Sample solution
= peak response ratio of anthralin to o-nitroaniline
from the Standard solution
= concentration of USP Anthralin RS in the Standard
solution (l-/g/mL)
=concentration of Anthralin in the Sample solution
(l-/g/mL)

• B. i~S Acceptance criteria: 97.0%-102.0% on the dried basis

J.J]tfJ, IMPURITIES
Sample solution: 10 I-/g/mL in chloroform • RESIDUE ON IGNITION (281): NMT 0.1%
Acceptance criteria: Meets the requirements • CHLORIDE AND SULFATE, Chloride (221)
ASSAY Sample: 1 g
• PROCEDURE
Analysis: To 15 ml of water add the Sample, mix, and filter.
[NOTE-Use low-actinic glassware.] Acidify 5 ml of.the filtrate with nitric acid, and add a few
Mobile phase: n-Hexane, dichloromethane, and glacial drops of silver nitrate TS.
acetic acid (82:12:6)

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344 Anthralin / Official Monographs USP 43

Acceptance criteria: No more opalescence is produced Sample solution: Transfer a volume of Sample stocksolution
immediately than is present in a 5-mL portion of the filtrate equivalent to 0.5 mg of anthralin and 2 mL of Internal
to which nothing has been added. standard solution into a 25-mL volumetric flask, and dilute
o CHLORIDE AND SULFATE, Sulfate (221) with Mobile phase to volume.
Sample: 5 mL of the untreated filtrate obtained in the test Chromatographic system
for Chloride (See Chromatography (621), System Suitability.)
Analysis: To the Sample add 3 drops of 3 N hydrochloric Mode: LC
acid and 5 drops of barium chloride TS. Detector: UV 354 nm
Acceptance criteria: Column: 4.6-mm x 25-cm; packing L3
No more turbidity is produced than is present in a 5-mL Flow rate: 2 mL/min
portion of the filtrate to which nothing has been added. Injection volume: 10 IJL
System suitability
SPECIFIC TESTS Samples: System suitability solution, Solvent blank solution,
o MELTING RANGE OR TEMPERATURE, Class 1(741): 178
0-181 0
and Standard solution
o Loss ON DRYING (731)
[NOTE-The relative retention times for anthralin,
Analysis: Dry a sample over silica gel for 4 h. danthron, dianthrone, and o-nitroaniline are 1.0, 1.2,
Acceptance criteria: NMT 0.5% 1.7, and 2.3, respectively.]
o ACIDITY OR ALKALINITY
Suitability requirements
Analysis: Suspend a sample in water, and filter. Resolution: NLT 1.3 between anthralin and danthron,
Acceptance crlterla. The filtrate is neutral to litmus. System suitability solution
ADDITIONAL REQUIREMENTS Tailing factor: NMT 1.5, System suitability solution
o PACKAGING AND STORAGE: Preserve in tight containers in a Relative standard deviation: NMT 2.0% of the ratio of
cool place. Protect from light. the peak responses, Standard solution
o USP REFERENCE STANDARDS (11) Interference: No discernible signal is observed at the
USP Anthralin RS retention time of anthralin, Solvent blank solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of anthralin (C14H1003) in the
portion of Cream taken:
Anthralin Cream
Result = (Ru/R s) x (Cs/Cu) x 100
DEFINITION
Anthralin Cream,is Anthralin in an aqueous (oil-in-water) or Bo =peak response ratio of anthralin to o-nitroaniline
. oily (water-in-oil) cream vehicle. Cream labeled to contain from the Sample solution
more than 0.1% of anthralin contains NLT 90.0% and NMT Rs =peak response ratio of anthralin to o-nitroaniline
115.0% of the labeled amount of anthralin (C14H1003), and peak from the Standard solution
Cream labeled to contain 0.1% or less of anthralin contains Cs = concentration of USP Anthralin RS in the Standard
NLT 90.0% and NMT 130.0% of the labeled amount of solution (lJg/mL)
anthralin (C14HlO0 3). Cu = nominal concentration of anthralin in the Sample
solution (lJg/mL)
ASSAY
o PROCEDURE Acceptance criteria: 90.00/0-115.0% for Cream labeled to
[NOTE-Use low-actinic glassware.] contain more than 0.1% of anthralin; 90.00/0-1 30.0% for
Mobile phase: n-Hexane, dichloromethane, and glacial Cream labeled to contain 0.1% or less of anthralin.
acetic acid (82:12:6)
Internal standard solution: 0.5 mg/mL of o-nitroaniline in ADDITIONAL REQUIREMENTS
n-hexane prepared as follows. First dissolve o-nitroaniline o PACKAGING AND STORAGE: Preserve in tight containers, in
in a small quantity of dichloromethane, and then dilute a cool place. Protect from light.
with n-hexane. o LABELING: Label it to indicate whether the cream vehicle is
System suitability stock solution: 0.1 mg/mL of USP aqueous or oily.
Anthralin RS and 0.2 mg/mL of danthron in o USP REFERENCE STANDARDS (11)
dichloromethane USP Anthralin RS
System suitability solution: Transfer 5 mL of the System
suitability stock solution into a 25-mL volumetric flask, add
5 mL of n-hexane, and dilute with Mobile phase to volume.
Solvent blank solution: Mobile phase, n-hexane, and
dichloromethane (3:1:1) Anthralin Ointment
Standard stock solution: 0.25 mg/mL of USP Anthralin RS
DEFINITION
in dichloromethane Anthralin Ointment is Anthralin in a petrolatum or other
Standard solution: Transfer 2 mL each of Standard stock oleaginous vehicle. Ointment labeled to contain more than
solution and Internal standard solution into a 25-mL 0.1 % of anthralin contains NLT 90.0% and NMT 115.0% of
volumetric flask, and dilute with Mobile phase to volume. the labeled amount of anthralin (C14HlO0 3), and Ointment
Sample stock solution: Weigh 5 g of Cream into a 100-mL
beaker. Add 20 mL of dichloromethane and 10 mL of glacial labeled to contain 0.1% or less of anthralin contains NLT
acetic acid, and stir to disperse the Cream. Transfer the 90.0% and NMT 130.0% of the labeled amount of anthralin
contents of the beaker to a filter paper (Whatman No.4, or (C14Hl003)'
equivalent) with the aid ofdichloromethane, and filter into ASSAY
a 1OO-mL volumetric flask. Thoroughly wash the precipitate o PROCEDURE
with dichloromethane, and allow the washings to drain into [NOTE-Use low-actinic glassware.]
the flask. Dilute with dichloromethane to volume.

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USP 43 OfficialMonographs / Anticoagulant 345

Mobile phase: n-Hexane, dichloromethane, and glacial Acceptance criteria: 90.0%-115.0% for Ointment labeled
acetic acid (82:12:6) to contain more than 0.1% of anthralin; 90.00/0-130.0% for
Internal standard solution: 0.5 mg/mL of o-nitroaniline in Ointment labeled to contain 0.1% or less of anthralin
n-hexane prepared asfollows. First dissolve o-nitroaniline
in a small quantity of dichloromethane, and then dilute ADDITIONAL REQUIREMENTS
with n-hexane. • PACKAGING AND STORAGE: Preserve in tight containers, in
System suitability stock solution: 0.1 mg/mL of USP a cool place. Protect from light.
Anthralin RS and 0.2 mg/mL of danthron in • USP REFERENCE STANDARDS (11)
dichloromethane USP Anthralin RS
System suitability solution: Transfer 5 mL of the System
suitability stock solution into a 25-mL volumetric flask, add
5 mL of n-hexane, and dilute with Mobile phase to volume.
Solvent blank solution: Mobile phase, n-hexane, and Anticoagulant Citrate Dextrose
dichloromethane (3:1:1)
Standard stock solution: 0.25 mg/mL of USP Anthralin RS Solution
in dichloromethane
Standard solution: Transfer 2 mL each of Standard stock DEFINITION
solution and Internal standard solution into a 25-mL Anticoagulant Citrate Dextrose Solution is a sterile solution of
volumetric flask, and dilute with Mobile phase to volume. Citric Acid, Sodium Citrate, and Dextrose in Water for
Sample stock solution: Weigh 5 g of Ointment into a Injection. It contains no antimicrobial agents, and in each
1OO-mL beaker. Add 20 mL of dichloromethane and 10 mL 1000 mL it contains:
of glacial acetic acid, and stir to disperse the Ointment.
Transfer the contents of the beaker to a filter paper Solution A Solution B
(Whatman No.4, or equivalent) with the aid of
dichloromethane, and filter into a 1OO-mL volumetric flask. NLT 20.599 NLT12. 379
Total Citrate, expressedas
Thoroughly wash the precipitate with dichloromethane, anhydrous citric acid (C6H s0 7) NMT 22.759 NMT 13.67 9
and allow the washings to drain into the flask. Dilute with
NLT 23.28 9 NLT 13.96 9
dichloromethane to volume.
Sample solution: Transfer a volume of Sample stock solution Dextrose (C6H120 6 • H2O) NMT 25.739 NMT 15.44 9
equivalent to 0.5 mg of anthralin and 2 mL of Internal
NLT 4.909 NLT 2.949
standard solution into a 25-mL volumetric flask, and dilute
with Mobile phase to volume. Sodium (Na) NMT5.429 NMT 3.259
Chromatographic system
(See Chromatography (621), System Suitability.) Prepare Anticoagulant Citrate Dextrose Solution as follows.
Mode: LC
Detector: UV 354 nm
Column: 4.6-mm x 25-cm; packing L3 Solution A Solution B
Flow rate: 2 mL/min Citric Acid (anhydrous) 7.39 4.4 9
Injection volume: 10 IJL
System suitability Sodium Citrate (dihydrate) 22.09 13.29
Samples: System suitability solution, Solvent blank solution, Dextrose (monohydrate) 24.59 14.7 9
and Standard solution .
Water for Injection, a
[NOTE-The relative retention times for anthralin, sufficient quantity to make 1000 mL 1000 mL
danthron, dianthrone, and o-nitroaniline are 1.0, 1.2,
1.7, and 2.3, respectively.]
Suitability requirements Dissolvethe ingredients, and mix. Filterthe solution until clear,
Resolution: NLT 1.3 between anthralin and danthron, place immediately in suitable containers, and sterilize.
System suitability solution If desired, 8 g and 4.8 g of monohydrated citric acid may be
Tailing factor: NMT 1.5, System suitability solution used instead of the indicated, respective amounts of
Relative standard deviation: NMT 2.0% of the ratio of anhydrous citric acid; 19.3 g and 11.6 g of anhydrous sodium
the peak responses, Standard solution citrate may be used instead of the indicated, respective
Interference: No discernible signal is observed at the amounts of dihydrated sodium citrate; and 22.3 g and 13.4
retention time of anthralin, Solvent blank solution g of anhydrous dextrose may be used instead of the
Analysis indicated, respective amounts of monohydrated dextrose.
Samples: Standard solution and Sample solution IDENTIFICATION
Calculate the percentage of anthralin (C14H100 3) in the • A. DEXTROSE
portion of Ointment taken: Analysis: Add a few drops of solution (1 in 20) to 5 mL of
hot alkaline cupric tartrate TS.
Result= (Ru/R s) x (Cs/Cu) x 100 Acceptance criteria: A copious red precipitate of cuprous
oxide is formed.
Ru =peak response ratio of anthralin to o-nitroaniline • B. IDENTIFICATION TESTS-GENERAL, Citrate (191): Meets
from the Sample solution . the requirements when concentrated to one-half its volume
Rs = peak response ratio of anthralin to o-nitroaniline • C. IDENTIFICATION TESTS-GENERAL, Sodium (191): Meets
from the Standard solution the requirements when concentrated to one-half its volume
Cs = concentration of USP Anthralin RS in the Standard
solution (lJg/mL)
Cu = nominal concentration of anthralin in the Sample
solution (lJg/mL)

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346 Anticoagulant / Official Monographs USP 43

ASSAY Acceptance criteria: Each 1000 mL should contain 4.90-

• TOTAL CITRATE 5.42 g in Solution A and 2.94-3.25 g in Solution B of
Mobile phase, Standard preparation 1, and sodium.
Chromatographic system: Proceed as directed in Assay • DEXTROSE
for Citric Acid/Citrate and Phosphate (345). Analysis: Determine tHe angular rotation of Solution in a
Sample solution: Pipet 5 mL of Solution into a suitable suitable polarimeter tube (see Optical Rotation (781 ».
volumetric flask, and proceed as directed in Assay for Citric Where Solution is labeled to contain anhydrous dextrose,
Acid/Citrate and Phosphate (345), Sample solution (for the calculate the percentage, in gil 00 mL, of anhydrous
assay of citric acid/citrate). dextrose (C6H120 6) in the portion of Solution taken:
Analysis
Samples: Standardpreparation 1 and Sample solution Result =(1 001F) x A x R
Proceed as directed in Assay for Citric Acid/Citrate and
Phosphate (345), Procedure. F =midpoint of the specific rotation range for
Calculate the quantity, in g, of anhydrous citric acid anhydrous dextrose, 52.9°
(C6 Hs0 7) in the volume of Solution taken: A = 100 mm divided by the length of the polarimeter
tube (mm)
Result =(r ulr s) xC s x (M ,tiM (2) x Fx D R =observed rotation e)
ru = peak area of citrate from the Samplesolution Where Solution is labeled to contain dextrose
rs =peak area of citrate from Standardpreparation 1 monohydrate, calculate the percentage, in gl100 mL, of
dextrose (C6 H12 0 6 • H20) in the portion of Solution taken:
Cs =concentration of citrate in Standardpreparation 1
(~g/mL)
M ,1 =molecular weight of anhydrous citric acid, 192.12 Result = (1 001F) x (M ,tiM (2) x A x R
M r2 = molecular weight of citrate (C6H s0 7) , 189.10 F = midpoint of the specific rotation range for
F = conversion factor, 0.000001 g/~g anhydrous dextrose, 52.9°
D = dilution factor M r7 =molecular weight for dextrose monohydrate,
198.17
Acceptance criteria: Each 1000 mL should contain 20.59- M ,2 =molecular weight for anhydrous dextrose, 180.16
22.75 g in Solution A and 12.37-13.67 g in Solution B of
total citrate expressed as anhydrous citric acid.
A =100 mm divided by the length of the polarimeter
tube (mm)
• SODIUM R = observed rotation (0)
Solution A: Transfer 1.04 g of lithium nitrate to a 1000-mL
volumetric flask, add a suitable non ionic surfactant, add Acceptance criteria: Each 1000 mL should contain 23.28-
water to volume, and mix. This solution contains 15 mEql 25.73 g in Solution A and 13.96-15.44 g in Solution B of
1000 mL of lithium.
dextrose monohydrate.
Standard solution: Transfer 8.18 g of sodium chloride,
previously dried at 105° for 2 h to a 1OOO-mL volumetric IMPURITIES
flask, dilute with water to volume, and mix. This solution • CHLORIDE AND SULFATE, Chloride (221): A 1O-mL portion
contains 140 mEqll 000 mL of sodium. Transfer 50 ~L of shows no more chloride than corresponds to 0.50 mL of
this solution to a 1O-mLvolumetric flask, dilute with Solution 0.020 N hydrochloric acid (0.0035%).
A to volume, and mix.
Sample solution: Transfer 25 mL of Solution to a 50-mL SPECIFIC TESTS
volumetric flask, and dilute with water to volume. Transfer • pH (791): 4.5-5.5
50 ~L of this solution to a 1O-mL volumetric flask, dilute • OTHER REQUIREMENTS: Meets the requirements in Injections
with SolutionA to volume, and mix. and Implanted Drug Products (1)
Analysis • BACTERIAL ENDOTOXINS TEST (8,): It contains NMT 5.56
Samples: Standard solution and Sample solution USP Endotoxin Units/mL.
Using a suitable flame photometer, adjusted to read zero ADDITIONAL REQUIREMENTS
with SolutionA, concomitantly determine the sodium
flame emission readings for the Standard solution and the
Sample solution at the wavelength of maximum emission
at 589 nm. • PACKAGING AND STORAGE: Preserve in single-dose
Calculate the quantity, in g, of sodium (Na) in 1000 mL of containers of colorless, transparent e I or II
Solution taken:
o~. . ?f ~ .~~i!~R!~ ..el.~~!iS .•~~~~~i~I,~s~~
fJlil~t~riq]"(~fJCl<?ct.Q!Slilii;gJlf!J'lJ.yt9g~JJi;'I£~§. )
Result = (r ult s) x (A ,/ M ,) x W x F • LABELING: Label to indicate the number of mL of Solution
required per 100 mL of whole blood or the number of mL
ru = sodium emission readlnqs from the Sample of Solution required per volume of whole blood to be
solution collected.
rs = sodium emission readings from the Standard
• USP REFERENCE STANDARDS (11)
solution USP Citric Acid RS
Ar = atomic weight of sodium, 22.99
M r = molecular weight of sodium chloride, 58.44
W = weight of sodium chloride taken to make the
Standard solution, 8.1 8 g
F = conversion factor, 2

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 USP 43 Official Monographs / Anticoagulant 347

Anticoagulant Citrate Phosphate Result = (r vIrs) x C s x (M ,,1Mr2) X Fx 0

Dextrose Solution ru = peak area of citrate from the Sample solution for
total citrate
DEFINITION rs = peak area of citrate from Standard preparation 2
Anticoagulant Citrate Phosphate Dextrose Solution is a sterile Cs =concentration of citrate in Standard preparation2
solution of Citric Acid, Sodium Citrate, Monobasic Sodium (f.Jg/ m L)
Phosphate, and Dextrose in Water for Injection. It contains, M rl = molecular weight of anhydrous citric acid
in each 1000 mL, NLT 2.11 g and NMT 2.33 9 of monobasic (C6H a0 7) , 192.12
sodium phosphate (NaH 2P04 • H20); NLT 24.22 9 and NMT M r2 = molecular weight of citrate (C6H s0 7) , 189.10
26.78 9 of dextrose (C6H1206 • H20); NLT 19.16 g and NMT F = conversion factor, 0.000001 g/f.Jg
21.18 9 of total citrate, expressed as anhydrous citric acid o = dilution factor
(C6H a0 7) ; and NLT 6.21 g and NMT 6.86 g of Sodium (Na).
It contains no antimicrobial agents. Acceptance criteria: Each 1000 mL of Solution should
Prepare Anticoagulant Citrate Phosphate Dextrose Solution as contain 19.16 g-21.18 g of total citrate expressed as
follows. anhydrous citric acid.
Analysis: Proceed as directed in Assay for CitricAcid/Citrate
Citric Acid (anhydrous) 2.99 9 and Phosphate (345), Procedure.
Calculate the quantity of phosphate, in g, expressed as
Sodium Citrate (dihydrate) 26.3 9 monobasic sodium phosphate (NaH2P04 • H20), in the
Monobasic Sodium Phosphate (monohy- volume of Solution taken:
drate; NaH2P04 • H2O) 2.22 9
Result = (r vIrs) x C s x (M rtlM r2) X Fx 0
Dextrose (C6H1206 • H2O) 25.5 9
Water for Injection, a sufficient quantity to = peak area of phosphate from the Sample solution
make 1000 mL for total phosphate
r,S = peak area of phosphate from Standard
Dissolvethe ingredients, and mix. Filter the solution until clear, preparation2
place immediately in suitable containers, and sterilize. =concentration of phosphate in Standard
If desired, 3.27 9 of monohydrated citric acid may be used preparation 2 (f.Jg/mL)
instead of the indicated amount of anhydrous citric acid; M rl = molecular weight of monobasic sodium
23.06 9 of anhydrous sodium citrate may be used instead of phosphate monohydrate (NaH2P04 • H20),
the indicated amount of dihydrated sodium citrate; 1.93 g 137.99
of anhydrous monobasic sodium phosphate may be used M r2 = molecular weight of phosphate (P0 4) , 94.97
instead of the indicated amount of monohydrated F =conversion factor, 0.000001 g/f.Jg
monobasic sodium phosphate; and 23.2 g of anhydrous o = dilution factor
dextrose may be used instead of the indicated amount of
monohydrated dextrose. Acceptance criteria: Each 1000 mL of Solution should
contain 2.11-2.33 g of monobasic sodium phosphate
IDENTIFICATION (NaH 2P04 • H20).
• A. DEXTROSE • DEXTROSE
Analysis: Add a few drops of solution (1 in 20) to 5 mL of Sample: 5.0 mL of Solution
hot alkaline cupric tartrate TS. Analysis: Tare a clean, medium-porosity filtering crucible
Acceptance criteria: A copious red precipitate of cuprous containing several carborundum boiling chips or glass
oxide is formed. beads. Pipet 50 mL of freshly mixed alkaline cupric tartrate
• B. IDENTIFICATION TESTS-GENERAL, Phosphate (191): TS into a 400-mL beaker. Add the boiling chips or glass
Meets the requirements beads from the tared crucible, 45 mL of water, and 5.0 mL
• C. IDENTIFICATION TESTS-GENERAL, Citrate (191): Meets of Solution to the beaker. Heat the beaker and contents
the requirements when concentrated to one-half its volume over a burner that has been adjusted to causeboiling of the
• D. IDENTIFICATION TESTS-GENERAL, Sodium (191): Meets solution to start in 3.5-4 min. Boil the solution for 2 min,
the requirements when concentrated to one-half its volume accurately timed, and filter immediately through the tared
ASSAY crucible, taking care to transfer all of the boiling chips or
• TOTAL CITRATE AND TOTAL PHOSPHATE glass beads to the crucible. Wash the precipitate with hot
Mobile phase, Standard preparation 2, and water and 10 mL of alcohol. Dry the crucible and contents
Chromatographic system: Proceed as directed in Assay at 110° to constant weight. Perform a blank determination,
for CitricAcid/Citrate and Phosphate (345). and correct the weight of the precipitate from the sample
Sample solution for total citrate: Pipet 10 mL of Solution for any precipitate obtained in the blank.
into a suitable volumetric flask, and proceed as directed in Each mg of cuprous oxide precipitate of the substance under
Assay for CitricAcid/Citrate and Phosphate (345), Sample assay is equivalent to 0.496 mg of dextrose (C6H1206 • H20).
solution (for the assay of citric acid/citrate). Acceptance criteria: Each 1000 mL of Solution should
Sample solution for total phosphate: Pipet 5 mL of contain 24.22-26.78 9 of dextrose (C6H1206 • H20).
Solution into a suitable volumetric flask, and proceed as • SODIUM
directed in Assay for CitricAcid/Citrateand Phosphate (345), Solution A: Transfer 1.04 g of lithium nitrate to a 1000-mL
Sample solution (for the assay of phosphate). volumetric flask, add a suitable nonionic surfactant, add
Analysis: Proceed as directed in Assay for CitricAcid/Citrate water to volume, and mix. This solution contains 15 mEq/
and Phosphate (345), Procedure. 1000 mL of lithium.
Calculate the quantity, in g, of anhydrous citric acid Standard solution: Transfer 8.18 9 of sodium chloride,
(C6H a0 7) in the volume of Solution taken: previously dried at 105° for 2 h to a 1OOO-mL volumetric
flask, dilute with water to volume, and mix. This solution

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 348 Anticoagulant / OfficialMonographs USP 43

contains 140 mEql1000 mL of sodium. Transfer 50 IJL of 2.33 9 of monobasic sodium phosphate (NaH 2P04 • H20);
this solution to a 1O-mLvolumetric flask, dilute with Solution NLT 30.30 9 and NMT 33.50 9 of dextrose (C6H1206 • H20);
A to volume, and mix. NLT 19.16 9 and NMT 21.18 9 of total citrate, expressed as
Sample solution: Transfer 25 mL of Solution to a 50-mL citric acid, anhydrous (C 6H s0 7) ; NLT 6.21 9 and NMT 6.86
volumetric flask, and dilute with water to volume. Transfer 9 of sodium (Na); and NLT 0.247 9 and NMT 0.303 9 of
50 IJL of this solution to a 1O-mL volumetric flask, dilute adenine (CsHsN s). It contains no antimicrobial agents.
with Solution A to volume, and mix.
Prepare Anticoagulant Citrate Phosphate Dextrose Adenine
Analysis
Solution as follows.
Samples: Standardsolution and Sample solution
Using a suitable flame photometer, adjusted to read zero
with Solution A, concomitantly determine the sodium Citric Acid (anhydrous) 2.99 9
flame emission readings for the Standardsolution and the Sodium Citrate (dihydrate) 26.3 9
Sample solution at the wavelength of maximum emission
at 589 nm. Monobasic Sodium Phosphate (monohydrate; NaH2 P0 4
. H2O) 2.22 9
Calculate the quantity, in g, of sodium (Na) in 1000 mL of
Solution taken: Dextrose (monohydrate) 31.9 9

Adenine (CsHsNs) 0.275 9

Result = (r vir s) x (A,IM ,) x W x F
Water for Injection, a sufficient quantity to make 1000 mL
rv = sodium emission readings from the Sample
solution
rs = sodium emission readings from the Standard Dissolvethe ingredients, and mix. Filter the solution until clear,
solution place immediately in suitable containers, and sterilize.
Ar = atomic weight of sodium, 22.99 If desired, 3.27 9 of monohydrated citric acid may be used
instead of the indicated amount of anhydrous citric acid;
Mr =molecular weight of sodium chloride, 58.44 23.06 9 of anhydrous sodium citrate may be used instead of
W =weight of sodium chloride taken to make the the indicated amount of dihydrated sodium citrate; 1.93 9
Standardsolution, 8.18 9 of anhydrous monobasic sodium phosphate may be used
F = conversion factor, 2 instead of the indicated amount of monohydrated
monobasic sodium phosphate; and 29.0 9 of anhydrous
Acceptance criteria: Each 1000 mL of Solution should dextrose may be used instead of the indicated amount of
contain 6.21-6.86 9 of sodium. monohydrated dextrose.
IMPURITIES
ASSAY
• CHLORIDE AND. SULFATE, Chloride (221): A 1O-mL portion
• TOTAL CITRATE AND TOTAL PHOSPHATE
shows no more chloride than corresponds to 0.50 mL of Mobile phase, Standard preparation 2, and
0.020 N hydrochloric acid (0.0035%). Chromatographic system: Proceed as directed in Assay
SPECIFIC TESTS for CitricAcid/Citrateand Phosphate (345).
• pH (791): 5.0-6.0 Sample solution for total citrate: Pipet 10 mL of Solution
• BACTERIAL ENDOTOXINS TEST (85): It contains NMT 5.56 _ into a suitable volumetric flask, and proceed as directed in
USP Endotoxin Units/mL. Assay for CitricAcid/Citrateand Phosphate (345), Sample
• OTHER REQUIREMENTS: It meets the requirements in solution (for the assay of citric acid/citrate).
Injections and Implanted Drug Products (1); . Sample solution for total phosphate: Pipet 5 mL of
Solution into a suitable volumetric flask, and proceed as
ADDITIONAL REQUIREMENTS directed in Assay for CitricAcid/Citrate and Phosphate (345),
Sample solution (for the assay of citric acid/citrate).
Analysis
Samples: Standardpreparation 2 and Sample solutionfor
• PACKAGING AND STORAGE: Preserve in single-dose total citrate
containers of colorless, transparent Tel or T .e" Proceed as directed in Assay for CitricAcid/Citrate and
()~)~!)~. .•.~~i~~RI~el.~.~!i;.;,:,m&~~a~t~ei9rilgja~'i~£s~e!et~!
~c:lg~[iqlgJJgQtQxinanq
Phosphate (345), Procedure.
Calculate the quantity, in g, of anhydrous citric acid
• LABELING: Label it to indicate the number of mL of Solution (C6H s0 7) in the volume of Solution taken:
required per 100 mL of whole blood or the number of mL
of Solution required per volume of whole blood to be Result =(r vir s) x C s x (M ,tiM (2) x Fx D
collected.
• USP REFERENCE STANDARDS (11) ru = peak area of citrate from the Sample solution for
USP Citric Acid RS total citrate
rs = peak area of citrate from Standardpreparation2
Cs = concentration of citrate in Standardpreparation 2
(lJg/mL)
Anticoagulant Citrate Phosphate M'I = molecular weight of anhydrous citric acid, 192.12
M ,2 = molecular weight of citrate (C6H s0 7) , 189.10
Dextrose Adenine Solution F = conversion factor, 0.000001 g/lJg
D = dilution factor
DEFINITION
Anticoagulant Citrate Phosphate Dextrose Adenine Solution is Acceptance criteria: Each 1000 mL of Solution should
a sterile solution of Citric Acid, Sodium Citrate, Monobasic contain NLT 19.16g and NMT 21.18 9 of total citrate
Sodium Phosphate, Dextrose, and Adenine in Water for expressed as anhydrous citric acid (C6H s0 7) .
Injection. It contains, in each 1000 mL, NLT 2.11 9 and NMT

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USP 43 Official Monographs / Anticoagulant 349

Analysis beads from the tared crucible, 45 mL of water, and 5.0 mL

Samples: Standard preparation 2 and Sample solution for of Solution to the beaker. Heat the beaker and contents
total phosphate over a burner that has been adjusted to causeboiling of the
Calculate the quantity of phosphate, in g, expressed as solution to start in 3.5-4 min. Boil the solution for 2 min,
monobasic sodium phosphate (NaH 2P04 • H20), in the accurately timed, and filter immediately through the tared
volume of Solution taken: crucible, taking care to transfer all of the boiling chips or
glass beads to the crucible. Wash the precipitate with hot
Result = (r vir s) x C s x (M rdM r2) X Fx D water and 10 mL of alcohol. Dry the crucible and contents
at 110° to constant weight. Perform a blank determination,
ru = peak area of phosphate from the Sample solution and make any necessary correction.
for total phosphate Each mg of cuprous oxide precipitate obtained is equivalent
rs = peak area of phosphate from Standard to 0.496 mg of dextrose (C6H1206 • H20).
preparation 2 Acceptance criteria: Each 1000 mL of Solution should
Cs = concentration of phosphate in Standard contain 30.30-33.50 g of dextrose (C6H1206 • H20).
preparation 2 (~g/mL) • ADENINE
M rl = molecular weight of monobasic sodium Mobile phase: Dissolve 3.45 g of ammonium dihydrogen
phosphate monohydrate, 137.99 phosphate in 950 mL of water, add 10 mL of glacial acetic
M r2 = molecular weight of phosphate (P04) , 94.97 acid, dilute with water to 1000 mL, and mix. Pass the
F = conversion factor, 0.000001 g/~g solution through a membrane filter with a t-urn or finer
D = dilution factor pore size, and degas.
System suitability solution: 0.275 mg/mL of each USP
Acceptance criteria: Each 1000 mL of Solution should Adenine RS and purine in dilute hydrochloric acid (1 in
contain 2.11-2.33 g of monobasic sodium phosphate 120)
(NaH 2P04 • H20). Standard solutions: Place quantities of USP Adenine RS in
• SODIUM dilute hydrochloric acid (1 in 120) in three separate
Solution A: Transfer 1.04 g of lithium nitrate to a 1000-mL volumetric flasks, and dilute with the dilute hydrochloric
volumetric flask, add a suitable non ionic surfactant, add acid solution to volume to obtain Standard solutions having
water to volume, and mix. This solution contains 15 mEql known concentrations of 0.25, 0.275, and 0.30 mg of
1000 mL of lithium. adenine per mL, respectively. Protect from light.
Standard solution: Transfer 8.18 g of sodium chloride, Chromatographic system
previously dried at 105° for 2 h to a 1OOO-mL volumetric (See Chromatography (621), System Suitability.)
flask, dilute with water to volume, and mix. This solution Mode: LC
contains 140 mE~/l 000 mL of sodium. Transfer 50 ~L of Detector: UV 254 nm
this solution to a 1,0-mLvolumetric flask, dilute with Solution Column: 4-mm x 30-cm stainless steel; packing L9
A to volume, and mix. Flow rate: 2.0 mL/min
Sample solution: Transfer 25 mL of Solution to a 50-mL Injection volume: 20 ~L
volumetric flask, dilute with water to volume, and mix. System suitability
Transfer 50 ~L of this solution to a 1O-mL volumetric flask, , Sample: System sUitability solution (NLT four injections)
dilute with Solution A to volume, and mix. SUitability requirements
Analysis Resolution: NLT 3.0 between adenine and purine
Samples: Standard solution and Sample solution Relative standard deviation: NMT 2.5% for adenine
Using a suitable flame photometer, adjusted to read zero peak and NMT 2.0% for the retention time of adenine
with Solution A, concomitantly determine the sodium peak
flame emission readings for the Standard solution and the Analysis
Sample solution at the wavelength of maximum emission Samples: Standard solutions and Solution
at 589 nm. Plot the responses against the concentrations, in mg, of
Calculate the quantity, in g, of sodium (Na) in 1000 mL of USP Adenine RS per mL of the Standard solutions.
Solution taken: Calculate the quantity, in mg, of adenine (CsHsN s) in each
mL of the Solution taken as the value read directly from
Result =(r vir s) x (A j M r) x W x F the Standard curve corresponding to the response
. obtained from the portion of the Solution
rv = sodium emission readings from the Sample chromatographed.
solution Acceptance criteria: Each 1000 mL of Solution should
rs = sodium emission readings from the Standard contain 0.247-0.303 g of adenine (CsHsN s).
solution
Ar = atomic weight of sodium, 22.99 IMPURITIES
M r = molecular weight of sodium chloride, 58.44 • CHLORIDE AND SULFATE, Chloride (221): A 1O-mL portion
W = weight of sodium chloride taken to make the shows no more chloride than corresponds to 0.50 mL of
Standard solution, 8.18 g 0.020 N hydrochloric acid (0.0035%).
F = conversion factor, 2
SPECIFIC TESTS
Acceptance criteria: Each 1000 mL of Solution should • pH (791): 5.0-6.0
contain 6.21 g-6.86 g of sodium. • BACTERIAL ENDOTOXINS TEST (85): It contains NMT 5.56
USP Endotoxin Units/mL.
• DEXTROSE
Sample: 5 mL of Solution • OTHER REQUIREMENTS: It meets the requirements in
Analysis: Tare a clean, medium-porosity filtering crucible Injections and ImplantedDrug Products (1).
containing several carborundum boiling chips or glass
beads. Pipet 50 mL of freshly rnlxed alkaline cupric tartrate
TS into a 400-mL beaker. Add the boiling chips or glass

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350 Anticoagulant / OfficialMonographs USP 43

ADDITIONAL REQUIREMENTS M r2 = molecularweight of citrate (C6H s0 7), 189.10

D = dilution factor
F =conversion factor, 0.000001 g/l-lg
• PACKAGING AND STORAGE: Preserve in single-dose Acceptance criteria: In each 100 mL, 3.80-4.20 9 of
containers, of colorless, transparent, Type I or Typ_e !I glass, sodium citrate dihydrate (C6HsNa307. 2H20)
or of a suitable plastic material(see fAMecli vke
BacterialEnaotoxinand Pyrogen Tests(16l. . .1-M9) SPECIFIC TESTS
• LABELING: Label to indicate the number of mLof solution • ptf (791): 6.4-7.5
required per 100 mLof whole blood or the number of mL • BACTERIAL ENDOTOXINS TEST (85): Contains NMT 5.56 USP
of solution required per volume of whole blood to be Endotoxin Units/mL
collected. • OTHER REQUIREMENTS: Meets the requirements in Injections
• USP REFERENCE STANDARDS (11) and Implanted Drug Products (1)
USP Adenine RS ADDITIONAL REQUIREMENTS
USP CitricAcid RS • PACKAGING AND STORAGE: Preserve in single-dose
containers, preferablyof Type I or Type II glass.
• USP REFERENCE STANDARDS (11)
USP CitricAcid RS
Anticoagulant Sodium Citrate Solution
DEFINITION
Anticoagulant Sodium Citrate Solution is a sterile solution of Antimony Potassium Tartrate
Sodium Citrate in Waterfor Injection. Itcontains, in each 100
mL, NLT 3.80 g and NMT 4.20 g of sodium citrate dihydrate
(C6HsNa307· 2H20). It contains no antimicrobial agents.
Prepare Anticoagulant Sodium Citrate Solution as follows.

Sodium Citrate (dihydrate) 40 9

Waterfor Injection,a sufficient quantity to make 1000 ml
CgH4K2012Sb2·3H20 667.87
[NOTE-Anhydrous sodium citrate (35.1 g) may be used CgH4K2012Sb2 613.82
instead of the 'dihydrate.] Antimonate(2-), bis[I-I-[2,3-dihydr6xybutanedioato(4-)-
Dissolve the Sodium Citrate in sufficient Waterfor Injection to Ol,02:03,Q4]]-di-, dipotassium, trihydrate, stereoisomer;
make 1000 mL, and filter until clear. Place the solution in Dipotassium bis[I-I-[l-(+)-tartrato(4-)]]diantimonate(2-)
suitable containers, and sterilize. trihydrate [28300-74-5].
IDENTIFICATION Anhydrous [11071-15-1].
• A. IDENTIFICATION TESTS-GENERAL, Citrate (191): Meets DEFINITION
the requirements when evaporated to a concentration of 1 Antimony PotassiumTartrate contains NLT 99.0% and NMT
in 20 103.0% of antimony potassium tartrate (CgH4K2012Sb2'
• B. IDENTIFICATION TESTS-GENERAL, Sodium (191): Meets 3H20 ).
the requirements when evaporated to a concentration of 1
in 20 IDENTIFICATION
ASSAY • A.
• PROCEDURE
Sample: An appropriate quantity
Mobile phase, Standard preparation 1, and Analysis: Heat the Sample to redness.
Chromatographic system: Proceed as directed in Assay Acceptance criteria: It chars, emits an odor resembling that
for CitricAcid/Citrateand Phosphate (345). of burning sugar, and leaves a blackened residue. This
Sample solution: Pipet 10 mLof Solution into a suitable residue has an alkaline reaction, and when a smallfragment
volumetric flask, and proceed as directed in Assay for Citric of it is held in a nonluminous flame, the flame is tinted
Acid/Citrateand Phosphate (345), Sample solution (for the violet.
assay of citric acid/citrate). • B.
Analysis Sample solution: Antimony PotassiumTartrate (1 in 20) in
Samples: Standardpreparation 1 and Sample solution water acidified with hydrochloric acid
Proceed as directed in Assay for CitricAcid/Citrateand Analysis: Add hydrogen sulfide TSto the Sample solution.
Phosphate (345), Procedure. Acceptance criteria: It yields an orange-red precipitate,
Calculate the quantity, in g, of sodium citrate dihydrate which issoluble in ammonium sulfideTSand in 1 N sodium
(C6HsNa307 . 2H20) in the volume of Solution taken: hydroxide.
• C. IDENTIFICATION TESTS-GENERAL, Tartrate (191): Meets
Result =(r'ulr s) xes x (M rllM r2) X 0 X F
the requirements
ASSAY
= peak area of citrate from the Sample solution • PROCEDURE
= peak area of citrate from Standard preparation 1 Sample: 500 mg
= concentration of citrate in Standardpreparation 1 Analysis: Dissolve the Sample in 50 mLof water, add 5 9 of
(l-Ig/mL) potassium sodium tartrate, 2 g of sodium borate, and 3 mL
M rl = molecular weight of sodium citrate dihydrate, of starch TS, and immediately titrate with 0.1 N iodine VS
294.10 to the production of a persistent blue color. Each mLof 0.1

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 USP 43 OfficialMonographs / Antipyrine 351

N iodine is equivalent to 16.70 mg of antimony potassium of starch TS, and immediately titrate with 0.1 N iodine VS
tartrate (CsH4K2012Sb2 . 3H20). to the production of a persistent blue color. Each mL of 0.1
Acceptance criteria: 99.00/0-103.0% N iodine is equivalent to 14.54 mg of antimony sodium
tartrate (CSH4Na2012Sb2)'
IMPURITIES Acceptance criteria: 98.00/0-101.0% on the dried basis
• ARSENIC
Sample solution: Dissolve 100 mg in 5 mL of hydrochloric IMPURITIES
acid. Add 10 mL of a recently prepared solution of 20 9 of • ARSENIC, Method /I (211): NMT 8 ppm
stannous chloride in 30 mL of hydrochloric acid. • LEAD (251): NMT 20 ppm
Blank: Add 5 mL of hydrochloric acid to 10 mLof a recently
prepared solution of 20 9 of stannous chloride in 30 mL of SPECIFIC TESTS
hydrochloric acid. • Loss ON DRYING (731)
Analysis: Transfer the Sample solution to a color-comparison Analysis: Dry at 105 0 to constant weight.
tube, and allow to stand for 30 min. Acceptance criteria: NMT 6.0%
Acceptance criteria: NMT 0.015%; viewed downward over • ACIDITY OR ALKALINITY
a white surface, the color of the solution appears no deeper Sample solution: Dissolve 1.0 9 in 50 mL of carbon
than that of the Blank to which has been added 15 IJg of dioxide-free water.
arsenic. . Analysis: Titrate the Sample solution with 0.010 N
• LEAD (251): NMT 20 ppm hydrochloric acid or 0.010 N sodium hydroxide to a pH of
4.5.
SPECIFIC TESTS Acceptance criteria: NMT 2.0 mL
• COMPLETENESS OF SOLUTION (641)
Sample: 750 mg ADDITIONAL REQUIREMENTS
Solvent: Water • PACKAGING AND STORAGE: Preserve in well-closed
Acceptance criteria: Meets the requirements containers.
• Loss ON DRYING (731)
Analysis: Dry at 105 0 to constant weight.
Acceptance criteria: NMT 2.7%
• ACIDITY OR ALKALINITY
Sample solution: Dissolve 1.0 9 in 50 mL of carbon
Antipyrine
dioxide-free water. o
Analysis: Titrate the Sample solution with 0.010 N
hydrochloric acid or 0.010 N sodium hydroxide to a pH of
4.5.
jjN-Q
H,C \
CH,
Acceptance criteria: NMT 2.0 mL
ADDITIONAL REQUIREMENTS CllH,2N20 188.23
• PACKAGING AND STORAGE: Preserve in well-closed 1,2-Dihydro-1 ,5-dimethyl-2-phenyl-3H-pyrazol-3-one;
containers. 2,3-Dimethyl-l-phenyl-3-pyrazolin-5-one [60-80-0].
DEFINITION
Antipyrine contains NLT 98.0% and NMT 102.0% of
antipyrine (CllH 12N20), calculated on the dried basis.
Antimony Sodium Tartrate IDENTIFICATION

• A.
~
• B. The retention time of the peak of the Sample
solution corresponds to that of Standardsolution, as
obtained in the Assay.
CsH4Na20,2Sb2 581.61
Antimonate(2-), bis[IJ-[2, 3-dihydroxybutanedioato(4-)- ASSAY
0',02: 0 3, Q4]]di-, disodium, stereoisomer; • PROCEDURE
Disodium bis[IJ-[L-(+)-tartrato(4-)]]diantimonate(2-) Solution A: 0.77 giL of ammonium acetate in water. Adjust
[34521-09-0]. with diluted ammonium hydroxide to a pH of 7.0 and pass
through a filter of 0.2-lJm pore size.
DEFINITION Solution B: Acetonitrile
Antimony Sodium Tartrate contains NLT 98.0% and NMT Mobile phase: See Table 7.
101.0% of antimony sodium tartrate (CsH4Na2012Sb2),
calculated on the dried basis. Table 1
IDENTIFICATION Time Solution A Solution B
• A. IDENTIFICATION TESTS-GENERAL, Antimony (191), (min) (%) (%)
Sodium (191), and Tartrate (191) 0 75 25
ASSAY 1.0 75 25
• PROCEDURE
5.0 20 80
Sample: 500 mg
Analysis: Dissolve the Sample in 50 mL of water, add 5 9 of 5.01 75 25
potassium sodium tartrate, 2 9 of sodium borate, and 3 mL

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352 Antipyrine / Official Monographs USP 43

Table 1 (continued) Suitability requirements

Time Solution A Solution B Resolution: NLT 3.0 between antipyrine related
(min) (%) (%) compound A and antipyrine
8.0 75 25
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of antipyrine related compound
System suitability solution: 0.12 mg/mL of USP A in the portion of Antipyrine taken:
Antipyrine RS and 0.12 jJg/mL of USP Antipyrine Related
Compound A RS in water Result =(r vir s) x (C siC v) x 100
Standard solution: 0.12 mg/mL of USP Antipyrine RS in
water ru =peak response of antipyrine related compound A
Sample solution: 0.12 mg/mL of Antipyrine in water from the Sample solution
Chromatographic system rs =peak response of antipyrine related compound A
(See Chromatography (621), System Suitability.) from the Standardsolution
Mode: LC Cs = concentration of USP Antipyrine Related
Detector: UV 240 nm Compound A RS in the Standard solution
Column: 2.1-mm x 10-cm; 1.8-jJm packing L1 (jJg/mL)
Column temperature: 35° Cu =concentration of the Sample solution (uq/rnt)
Flow rate: 0.4 mL/min
Injection volume: 1 jJL Calculate the percentage of any individual unspecified
System suitability impurity in the portion of Antipyrine taken:
Samples: System suitability solution and Standardsolution
Suitability requirements Result = (r vir s) x (C siC v) x 100
Resolution: NLT 2.0·between antipyrine and antipyrine
related compound A, System suitability solution ru = peak response of any individual unspecified
Tailing factor: NMT 2.0, Standardsolution impurity from the Sample solution
Relative standard deviation: NMT 0.73%, Standard r5 =peak response of antipyrine from the Standard
solution solution
Analysis C5 =concentration of USP Antipyrine RS in the
Samples: Standardsolution and Sample solution Standard solution (jJg/mL)
Calculate the percentage of antipyrine (C11H12N zO) in the Cu = concentration of the Sample solution (uq/rnl)
portion of Antipyrine taken:
Acceptance criteria: See Table 2. Disregard any impurity
Result =(r vir s) x (C siC v) x 100 peak less than 0.03%.

= peak response from the Sample solution Table 2

= peak response from the Standardsolution Relative Acceptance
=concentration of USP Antipyrine RS in the Name
Retention
Time
Criteria,
NMT (%)
Standardsolution (mg/mL)
= concentration of Antipyrine in the Sample solution Antipyrine related compound A 0.8 0.05
(mg/mL)
Antipyrine 1.0 -
Acceptance criteria: 98.0%-102.0% on the dried basis Individual unspecified impurity - 0.05
IMPURITIES Total impurities - 0.1
• RESIDUE ON IGNITION (281): NMT 0.15%
• ORGANIC IMPURITIES SPECIFIC TESTS
Buffer: Dissolve6.8 9 of monobasic potassium phosphate in • Loss ON DRYING (731)
1 L of water, add 2 mL of triethylamine, and adjust with 5 Analysis: Dry at 60° for 2 h.
N sodium hydroxide solution to a pH of 7.0. Acceptance criteria: NMT 1.0%
Mobile phase: Methanol and Buffer(43:1 00)
System suitability solution: 5 jJg/mL each of USP ADDITIONAL REQUIREMENTS
Antipyrine RS and USP Antipyrine RelatedCompound A RS • PACKAGING AND STORAGE: Preserve in tight containers.
in Mobile phase • USP REFERENCE STANDARDS (11)
Standard solution: 0.5 jJg/mL of USP Antipyrine RS and USP Antipyrine RS
0.25 jJg/mL of USP Antipyrine Related Compound A RS in USP Antipyrine Related Compound A RS
Mobilephase 3-Methyl-1-phenyl-1 H-pyrazol-5(4H)-one.
Sample solution: 500 jJg/mL of Antipyrine in Mobilephase ClOH lON 20 174.20
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 6.0-mm x 15-cm; 5-jJm packing L1 Antipyrine and Benzocaine Otic
Flow rate: 1 mL/min Solution
Injection volume: 10 jJL
Run time: 3 times the retention time of antipyrine DEFINITION
System suitability Antipyrine and Benzocaine Otic Solution is a solution of
Sample: System suitability solution Antipyrine and Benzocaine in Glycerin. It contains NLT

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USP 43 OfficialMonographs / Antipyrine 353

90.0% and NMT 110.0% of the labeled amount of antipyrine rs = peak response of antipyrine or benzocaine from
(C11 H12NzO) and benzocaine (C9H 11 NO z). the Standard solution
[NoTE-In the preparation of this Otic Solution, useGlycerin Cs = concentration of USP Antipyrine RS or USP
that has a low water content, in order that the Otic Benzocaine RS in the Standard solution (mg/mL)
Solution may comply with the limit for Water Cu = nominal concentration of antipyrine or
Determination. This may be ensured by using Glycerin benzocaine in the Sample solution (mg/mL)
having a specific gravity of NLT 1.2607, corresponding to
a concentration of 99.5%.] Acceptance criteria: 90.0%-110.0%
IDENTIFICATION IMPURITIES
• A. The UV spectrum of the antipyrine and benzocaine peaks • ORGANIC IMPURITIES
of the Sample solution exhibit maxima and minima at the Solution A, Solution B, Mobile phase, and
same wavelengths as those of the Standard solution, as Chromatographic system: Proceed as directed in the
obtained in the Assay. Assay.
• B. The retention times of the major peaks of the Sample System suitability solution: 0.1 mg/mL of USP Antipyrine
solution correspond to those of the Standard solution, as RS and 0.01 mg/mL of USP Antipyrine Related Compound
obtained in the Assay. A RS in Diluent
Standard solution: 0.001 mg/mL each of USP Benzocaine
ASSAY RS and USP Ethyl4-Nitrobenzoate RS, and 0.004 mg/mL of
• PROCEDURE USP Antipyrine RS in Diluent
Solution A: 50 mM monobasic potassium phosphate and Sample solution: Nominally 3.2 mg/mL of antipyrine and
0.2% triethylamine. Adjust with 10 N sodium hydroxide 0.8 mg/mL of benzocaine prepared as follows. Transfer a
to a pH of 7.0. suitable quantity of Otic Solution to a suitable volumetric
Solution B: Acetonitrile flask. Dilute with Diluent to volume.
Mobile phase: See Table 7. System suitability
Samples: System suitability solution and Standard solution
Table 1 [NoTE-See Table 2 for the relative retention times.]
Time Solution A Solution B Suitability requirements
(min) (%) (%) Resolution: NLT 2.0 between antipyrine related
0 85 15 compound A and antipyrine, System suitability solution
Relative standard deviation: NMT 5%, Standard
20 50 50 solution
23 50 50 Analysis
Samples: Standardsolution and Sample solution
23.1 85 15 Calculate the percentage of ethyl 4-nitrobenzoate in the
25 85 15 portion of Otic Solution taken:

Result = (rulrs) x (CsICu) x 100

Diluent: Acetonitrile and water (1:1)
Standard solution: 0.014 mg/mL of USP BenzocaineRS and ru =peak response of ethyl4-nitrobenzoate from the
0.054 mg/mL of USP Antipyrine RS in Diluent Sample solution
Sample solution: Nominally 0.014 mg/mL of benzocaine ts = peak response of ethyl 4-nitrobenzoate from the
and 0.054 mg/mL of antipyrine prepared as·follows. Standard solution
Transfer a suitable quantity of Otic Solution to a suitable Cs =concentration of USP Ethyl 4-Nitrobenzoate RS in
volumetric flask. Dilute with Diluent to volume. the Standard solution(mg/mL)
Chromatographic system Cu = nominal concentration of benzocaine in the
(See Chromatography (621), System Suitability.) Sample solution (mg/mL)
Mode: LC
Detector: UV 270 nm. For Identification A, use a diode Calculate the percentage of any individual, unspecified
array detector in the range of 200-400 nm. degradation product in the portion of Otic Solution taken:
Column: 4.6-mm x 15-cm; 5-lJm packing L7
Flow rate: 1 mL/min Result = (rulrs) x (CsICu) x 100
Injection volume: 20 IJL
System suitability t» = peak response of each individual, unspecified
Sample: Standard solution degradation product from the Sample solution
[NoTE-See Table 2 for the relative retention times for rs =peak response of benzocaine from the Standard
antipyrine and benzocaine.] solution
Suitability requirements Cs = concentration of USP Benzocaine RS in the
Tailing factor: NMT 2.0 Standard solution (mg/mL)
Relative standard deviation: NMT 2.0% Cu = nominal concentration of benzocaine in the
Analysis Sample solution(mg/mL)
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of Acceptance criteria: See Table 2. Disregard any impurity
antipyrine (C llH 12N zO) and benzocaine (C9H l1NO z) in the peaks less than 0.1%.
portion of Otic Solution taken:
Table 2
Result = (r ulis) x (CsfCu) x 100 Relative Acceptance
Retention Criteria,
ru = peak response of antipyrine or benzocaine from Name Time NMT (%)
the Sample solution .
Antipyrine related compound N 0.46 -

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354 Antipyrine / OfficialMonographs USP43

Table 2 (continued) Acceptance criteria: NMT 0.2%

Relative Acceptance
Retention Criteria, SPECIFIC TESTS
Name Time NMT (%) • WATER DETERMINATION (921), Method I: NMT 1.0%
Antipyrine 0.53 - ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
Benzocaine 1.0 - containers. Store at controlled room temperature.
Ethyl 4-nitrobenzoateb 1.6 0.2 • USP REFERENCE STANDARDS (11)
Any individual, unspecified
USP Aminobenzoic Acid RS
degradation product - 0.2 p-Aminobenzoic acid.
C7H 7N02 137.14
Totaldegradation products' - 2.0 USP Antipyrine RS
USP Antipyrine Related Compound A RS
a Process impurities are controlled in the drug substance and are not to be
reported here. They are not includedin the calculation of total impurities. 3-Methyl-l-phenyl-1 H-pyrazol-5(4 H)-one.
b Specified impurityrelated to benzocaine. C,oH,oN20 174.20
USP Benzocaine RS
• LIMIT OF AMINOBENZOIC ACID USP Ethyl 4-Nitrobenzoate RS
Solution A: Glacial acetic acid and water (1 :69) Ethyl p-nitrobenzoate.
Solution B: Methanol C9H9N04 195.1 7
Mobile phase: See Table 3.

Table 3
Time - Solution A Solution B
(min) (%) (%) Antipyrine, Benzocaine, and
0 90 10 Phenylephrine Hydrochloride Otic
10 90 10 .Solution
10.1 15 85
13 15 85 » Antipyrine, Benzocaine, and Phenylephrine
Hydrochloride Otic Solution is a solution of
13.1 90 10
Antipyrine, Benzocaine, and Phenylephrine
16 . 90 10 Hydrochloride in a suitable nonaqueous solvent. It
contains not less than 90.0 percent and not more
Diluent: Solution A and Solution B (9: 1) than 110.0 percent of the labeled amounts of
Standard solution: 0.001 mg/mL of USP Aminobenzoic
Acid RS in Diluent . antipyrine (Cn H12 N2 0 ), benzocaine (C9H n N02) ,
Sample solution: Nominally 3.2 mg/mL of antipyrine and and phenylephrine hydrochloride (C9H 13 N0 2 •
0.8 mg/mL of benzocaine prepared as follows. Transfer a HC!).
suitable quantity of Otic Solution to a suitable volumetric
flask. Dilute with Diluent to volume. . Packaging and storage-Preserve in tight, light-resistant
Chromatographic system containers.
(See Chromatography (621 ),. System Suitability.) VSP Reference standards (11)-
Mode: LC USP Antipyrine RS
Detector: UV 280 nm USP Benzocaine RS
Column: 3.0-mm x 15-cm; 3.5-lJm packing L11 USP Phenylephrine Hydrochloride RS
Flow rate: 0.4 mL/min Identification-The retention times of the major peaks in
Injection volume: 5 IJL the chromatograms of the Assay preparations correspond to
System suitability those in the chromatogram of the Standardpreparation, as
Sample: Standard solution obtained in the Assay.
Suitability requirements
Tailing factor: NMT 2.0 Assay-
Relative standard deviation: NMT 5.0% Mobile phase-Mix 480 mL of acetonitrile, 3520 mL of a
Analysis 0.005 M solution of sodium l-heptanesulfonate in water, and
Samples: Standard solution and Sample solution 4 mL of phosphoric acid.
Calculate the percentage of aminobenzoic acid in the Standard preparation-Accurately weigh about 25 mg of
portion of Otic Solution taken: USP Antipyrine RS, about 25 mg of USP Benzocaine RS, and
about 25 mg of USP Phenylephrine Hydrochloride RS into a
Result = (rvlrs) x (Cs/Cv) x 100 250-mL volumetric flask. Add 5 mL of a 0.5 mg per mL
solution of p-aminobenzoic acid in Mobile phase. Add 150 mL
t» = peak response of aminobenzoic acid from the of Mobile phase, and mix to effect solution, sonicating if
Samplesolution necessary. Dilute with Mobile phase to volume, and mix.
rs = peak response of aminobenzoic acid from the Assaypreparation A-Transfer an accurately measured
Standard solution volume of Otic Solution, equivalent to about 100 mg of
C, =concentration of USP Aminobenzoic Acid RS in antipyrine, to a 50-mL volumetric flask, dilute with Mobile
the Standard solution (mg/mL) phase to volume, and mix. Pipet 5 mL of this solution into a
Cv = nominal concentration of benzocaine in the 1OO-mL volumetric flask, dilute with Mobile phase to volume,
Samplesolution (mg/mL) and mix.

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 USP 43 OfficialMonographs / Antithrombin 355

Assay preparation B-Transfer an accurately measured by transfusion of blood or blood derivatives. The
volume of Otic Solution, equivalentto about 100 mg of manufacturing steps are shown to removeor inactivate
benzocaine, to a 50-mL volumetric flask, dilute with Mobile known agents of infection. If substances are used for
phase to volume, and mix. Pipet 5 mLof this solution into a inactivation of viruses during production, the subsequent
1OO-mL volumetric flask, dilute with Mobilephaseto volume, purification procedure must be validated to demonstrate
and mix. that the concentration of these substances is reduced to an
Assay preparation P-Transfer an accuratelymeasured acceptable level and that any residues are such as not to
volume of Otic Solution, equivalentto about 5 mg of compromisethe safetyof the preparation for patients. When
phenylephrine hydrochloride, to a 50-mL volumetric flask, reconstituted in the recommended volumeof diluent, the
dilute with Mobilephaseto volume, and mix. potency is NLT 25 International Units (IU)/mL. It contains
Chromatographic system (see Chromatography (621 »)- 80%-120% of the potency stated on the label.
The liquid chromatograph is equipped with a 272-nm
detector and a 4.6-mm x 30-cmcolumn that contains packing IDENTIFICATION
L11. The flow rate is about 1.5 mL per minute. • A. Meets the requirements of the Assay
Chromatograph the Standard preparation, and record the ASSAY
peak responsesas directed for Procedure: the relative retention • PROCEDURE
times are about 0.19 for p-aminobenzoic acid, 0.26 for Solution A: Dissolve tris(hydroxymethyl)aminomethane,
phenylephrine, 0.64 for antipyrine, and 1.0 for benzocaine; edetic acid, and sodium chloride in water to obtain a
the resolution, R, between phenylephrine and aminobenzoic solution having concentrations of 0.050, 0.0075, and
acid is not less than 1.5, and the relative standard deviation 0.175 M, respectively. Adjust with hydrochloric acid or
for replicate injections is not more than 3.0%. sodium hydroxide solution to a pH of 8.4.
Procedure-5eparately injectequal volumes(about 20 or 25 Solution B: 0.05% (w/v) of albumin human in Solution A
IJL) of the Standard preparation and each of the Assay Solution C: 10 mg/mL of polybrene in Solution B
preparations into the chromatograph, record the Solution D: Reconstitute thrombin bovine(factor lIa), and
chromatograms, and measure the responsesfor the major dilute with Solution B to obtain a solution having a
peaks. Calculate the quantity, in mg, of antipyrine concentration of 4 Thrombin IU/mL.
(Cll H12N 20) in each mL of the Otic Solution taken by the Solution E: Preparea solution of chromogenic substrate for
formula: amidolytic test (see Reagents, Indicators, and Solutions-
Reagent Specifications) for thrombin (factor lJa) in Solution
Cto obtain a solutionhavinga concentrationof about 40.0
mM.
inwhich Cis the concentration, in IJg per mL of U5P Antipyrine Solution F: Resuspend USP Heparin Sodium for Assays RS
R5 in the Standard preparation; V isthe volume, in mL, of Otic accordingto the U5P Certificate and diluteto 3 U5P Heparin
Solution taken; and 'ru and rs are the antipyrine peak responses Units/mL in Solution A.
obtained from Assay preparation A and the Standard Standard solutions: Preparesevendilutions from U5P
preparation, respectively. Calculate the quantity, in mg, of Antithrombin III Human R5 within the linearrange of the
benzocaine (C9H ll N0 2) in each mL of the Otic Solution taken assay in Solution F (for example, 1.7, 1.5, 1.2, 1.0, 0.8, 0.6,
by the formula: and 0.4 IU/mL).
Sample solutions: Preparethree or more dilutions in
Solution Fwithin the linear range of the assay.
Blank: Solution A
in which C is the concentration, in IJg per mL, of USP Analysis: [NOTE-The procedure also can be performed
Benzocaine R5 in the Standard preparation; V isthe volume, in using alternative platforms.]
mL, of Otic Solution taken; and rv and ts are the benzocaine Foreach dilution of the Standard solution and Sample
peak responses obtained from Assay preparation B and the solution, at least duplicatesshould be tested. Label a
Standardpreparation, respectively. Calculatethe quantity, in suitable number of tubes depending on the number of
IJg of phenylephrine hydrochloride (C9H 13N02 • HCI) in each replicates that will be tested. For example, iffive blanks
mL of the Otic Solution taken by the formula: will be used: Bl, B2, B3, B4, and B5 for the blanks; Tl, T2,
and T3 each at least in duplicatefor the dilutions of the
50( CI V)(rvi rs) Sample solutions; and 51, 52, 53, 54, 55, 56, and 57 each
at least in duplicate for the dilutions of the Standard
in which C is the concentration, in IJg per mL, of U5P solutions. Distribute the blanks overthe series in such a
Phenylephrine Hydrochloride R5 in the Standard preparation; way that they accuratelyrepresentthe behaviorof the
V is the volume, in mL, of Otic Solution taken; and tu and rs reagents during the experiments. [NOTE-Treat the tubes
are the phenylephrine peak responsesobtained from Assay in the order Bl, Sl, 52, 53, 54, S5, 56, 57, B2, Tl, T2, T3,
preparation P and the Standard preparation, respectively. B3, Tl, T2, T3, B4, S1, S2, S3, 54, S5, 56, 57, B5.]
Prewarm Solution D and Solution Eat 37°. Pipet50 IJL each
of the Standard solutions, Sample solutions, and Blank into
suitable tubes placed in a water bath set at 37°. Add 350
IJL of prewarmed Solution D to each tube, mix, and
Antithrombin III Human incubate for 1 min. Add 100 IJL of prewarmed Solution E
to each tube in the same order and mix. Follow the
[9000-94-6]. change in absorbance for each solution over 1 min at 405
DEFINITION nm using a suitable spectrophotometer (see
Antithrombin III Human is a glycoprotein, which is the major Ultraviolet-Visible Spectroscopy (857»). Calculate the
inhibitor of thrombin and other activated clotting factors, change in absorbance/min. Plotstandard concentrations
includingfactors IX, X, XI, and XII. It isobtained from human against resulting absorbance values and determine
plasma of healthy donors who have been tested and shown potency by interpolatingfrom the standard curve using
to be free from detectable agents of infection transmissible mean sample absorbances.

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356 Antithrombin / OfficialMonographs USP 43

System suitability Acceptance criteria: NMT 0.1 USP Heparin Unit!

Samples: Standardsolutions and Sample solutions Antithrombin III IU
The R2 value of the standard curve is NLT0.99. The initial
and final blanks differ by NMT 10%. The absorbances of SPECIFIC TESTS
the three dilutions of the Sample solution must fall within • STERILITY TESTS (71), Test for Sterility of the Product to Be
the range of absorbances of the standard curve. The Examined, Direct Inoculation of the Culture Medium: Meets
three dilutions of the Sample solution give potency the requirements
estimates that differ by NMT 10%. • WATER DETERMINATION (921), Method I: NMT 3.0%
Acceptance criteria: 80%-120% of the potency stated on • PYROGEN TEST (151): Inject 50 U5P Antithrombin III Units
the label. per kg of the rabbit's weight, calculated from the activity
stated on the label: meets the requirements.
IMPURITIES • GENERAL SAFETY: Meets the requirements for biologics in
• HEPARIN CONTENT Biological Reactivity Tests, In Vivo (88), SafetyTests-
Solution A: 9 giL of sodium chloride Biologicals
Sheep plasma substrate: Use sheep plasma suitable for the • OSMOLALITY AND OSMOLARITY (785), Osmolality:
test procedure. Iffrozen, thaw at 37°. Reconstitute with the diluent according to the
APTT reagent: Use a suitable activated partial manufacturer's instruction: NLT 240 mOsmol/kg for the
thromboplastin time (APTI) reagent containing solution.
phospholipid and a contact activator at a dilution giving a • pH (791): Reconstitute with the diluent according to the
suitable blank recalcification time not exceeding 60 s. manufacturer's instruction: 6.0-7.5.
Calcium chloride solution: 3.7 giL of calcium chloride • MOLECULAR WEIGHT DISTRIBUTION
System suitability solution: 5 IU/mLof heparin sodium for Mobile phase: 0.05 M sodium phosphate (dibasic), 0.05 M
assays in USP Antithrombin III Human RS sodium phosphate (monobasic), 0.4 M arginine
Standard solutions: Make three or more dilutions of U5P monohydrochloride, and 0.05% sodium azide. Adjust with
Heparin Sodium for Assays R5 to known concentrations in 1 N sodium hydroxide to a pH of 6. Degas and filter.
U5P Heparin Units/mL that are in the expected range of the Solution A: 4-5' mg/mL of thyroglobulin in Mobile phase
sample (for example, 0.5-1.5 U5P Heparin Units/mL). Sample solution: 8-10 mg/mL of Antithrombin III Human
Sample solutions: Make three or more dilutions of . Chromatographic system
Antithrombin III Human in the range of the Standard (5ee Chromatography (621), System SUitability.)
solution dilutions. Mode: LC
Blank: Solution A Detector: UV 280 nm
Analysis: [NoTE-The procedure also can be performed Columns
using alternative platforms.] Guard: 7.5 mm x 7.5 cm guard column containing
For each System suitability solution, Standardsolution, and packing L59
Sample solution, at least duplicates should be tested. Label Analytical: 7.5 mm x 30 cm analytical column
a suitable number of tubes depending on the number of containing packing L59
replicates that will be tested. For example, if five blanks Temperatures
will be used: 81, 82, 83, 84, and 85 for the blanks; Tl, T2, Autosampler: r
and T3 each at least in duplicate for the dilutions of the Column: Ambient
Sample solutions; and 51, 52, and 53 each at least in Flow rate: 0.5 mL/min maintained constant to ±1%
duplicate for the dilutions of the Standardsolutions and 5S Injection volume: 20 J.lL
for the System suitability solution. Distribute the blanks System suitability
over the series in such a way that they accurately represent Sample: Sample solution
the behavior of the reagents during the experiments. Suitability requirements
[NOTE-Treat the tubes in the order 81, Sl, 52, 53, 82, 55, Column efficiency: NLT 2000 theoretical plates
Tl, T2, T3, 83, Tl, T2, T3, S5, 84, Sl, 52, 53, 85.] In the Tailing factor: 0.9-1.3
following order add 1.0 mL of thawed Sheep plasma Analysis
substrate to 1.0 mL of the Standardsolution dilutions or Samples: Solution A and Sample solution
the Sample solution dilutions or the System suitability Acceptance criteria: Note the retention times of the major
solution. After each addition, mix but do not allow bubbles peak in the Solution A chromatogram. The relative peak area
to form. Transfer each tube to a water bath at 37°, allow of the high-molecular weight peak eluting at about the
to equilibrate at 3r for about 15 min, and add to each same retention time as the major peak in the Solution A
tube 1 mL of APTT reagent previously heated to 3r. After chromatogram, or earlier, is NMT 13%.
an appropriate time for the APTT reagent used, usually 2- • TOTAL PROTEIN CONTENT
5 min, add 1 mL of Calcium chloride solution previously Solution A: 1000 mg/mL of trichloroacetic acid in water
heated to 3r and determine the clotting time. Plot Sample solution: 7.5 mg/mL of Antithrombin III Human in
standard concentrations against resulting clotting times 0.15 M sodium chloride solution
and determine heparin content by interpolating from the Blank: 0.15 M solution of sodium chloride
standard curve using mean sample clotting times. For Analysis: To each of 2.0 mL of the Sample solution and the
samples with clotting times longer than the lowest Blank in suitable centrifuge tubes, add 1.5 mL of Solution
standard dilution, report the result as NMT the lowest A. Mix, allow to stand for at least 10 min, centrifuge for 5
Standardsolution concentration. min, and decant the supernatant. Resuspend the
System suitability precipitates in 1.5 mL of Solution A, centrifuge for 5 min,
Samples: Standardsolutions and Sample solutions decant the supernatant, and hold the tubes inverted on a
The R2 value of the standard curve is NLT 0.99. The three filter paper to drain. Quantitatively transfer the residues
dilutions of the Sample solution give heparin content with a minimum quantity of water to a micro-Kjeldahl flask,
estimates that differ by NMT 10%. The heparin content and determine the nitrogen content (see Nitrogen
in the System SUitability solution is in the range of 4.0-7.5 Determination (461), Method II). Multiply the result,
IU/mL. corrected for the Blank, by 6.25 to calculate the quantity of
protein.

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 USP 43 OfficialMonographs / Apomorphine 357

ADDITIONAL REQ.UIREMENTS Table 1

• PACKAGING AND STORAGE: Usea Type I glasscontainer with Time Solution A Solution B
an appropriate stopper and seal. Store protected from light (min) (%) (0/0)
between 2° and 8°, excursions permitted up to 25°.
0 85 15
• LABELING: The labeling should state the content of
antithrombin III in USP Antithrombin III Units. The diluent 2 85 15
and the volume to be used to reconstitute the preparation 32 68 32
are indicated.
• USP REFERENCE STANDARDS (11) 37 68 32
USP Antithrombin III Human RS
USP Heparin Sodium for Assays RS Return to original conditions and re-equilibrate the system.
System suitability solution: 0.25 mg/mL each of USP
Apomorphine Hydrochloride RS and boldine in Diluent.
[NOTE-Boldine is 2,9-dihydroxy-
l,10-dimethoxyaporphine.]
Apomorphine Hydrochloride Standard solution: 2.5 ~g/mL of USP Apomorphine
Hydrochloride RS in Diluent

•~
:
Sensitivity solution: 0.14 ~g/mL of USP Apomorphine
'CH3
• HCI • Y.H20
Hydrochloride RS in Diluent from the Standardsolution.
H
N
HO [NOTE-The peak response of this solution is equivalent to
\": that of a solution containing 1.25 ~g/mL of morphine
HO fi
hydrochloride, taking into account the relative response
C17H17N02 • HCI . Y2H 20 312.79 factor of this impurity (see Table 2).]
Sample solution: 2.5 mg/mL of Apomorphine
C17H17N02 • HCI 303.79 Hydrochloride in Diluent
4H-Dibenzo[de,g]quinoline-l O,ll-diol, 5,6,6a,7-tetrahydro- Chromatographic system
6-methyl-, hydrochloride, hemihydrate, (R)-; (See Chromatography (621), System Suitability.)
6a~-Aporphine-l 0,ll-diol hydrochloride hemihydrate Mode: LC
[41372-20-7]. Detector: UV 280 nm
Anhydrous [314-19-2]. Column: 4.6-mm x 15-cm; 5-~m end-capped packing L1
Column temperature: 35°
DEFINITION
Flow rate: 1.5 mL/min
Apomorphine Hydrochloride contains NLT 98.5% and NMT Injection volume: 10 ~L
101.5% of apom6rphine hydrochloride (C17H17N02 • HCI), System suitability
calculated on the dried basis. Samples: System suitability solution and Sensitivity solution
IDENTIFICATION [NoTE-The typical relative retention times for boldine
and apomorphine are about 0.9 and 1.0,
respectively.]
Suitability requirements
• A. Resolution: NLT 2.5 between boldine and apomorphine,
S System suitability solution
• B. IDENTIFICATION TESTS--GENERAL, Chloride (191) Signal-to-noise ratio: NLT 10, SensitiVity solution
Sample solution: 10 mg/mL of Apomorphine Analysis
Hydrochloride in carbon dioxide-free water Samples: Standardsolution and Sample solution
Analysis: To 2 mL ofthe Sample solution add 0.1 mLof nitric Calculate the percentage of any individual impurity in the
acid. Mix, filter, and use the filtrate. portion of Apomorphine Hydrochloride taken:
Acceptance criteria: Meets the requirements
Result =(r vir s) x (C d C u) x (1IF) x 100
ASSAY
• PROCEDURE ru = peak response of each impurity from the Sample
Sample solution: Dissolve 250 mg of Apomorphine solution _
Hydrochloride in a mixture of 5.0 mL of 0.01 N hydrochloric rs =peak responseof apomorphine from the Standard
acid and 50 mL of alcohol. solution
Analysis: Titrate the Sample solution with 0.1 N sodium Cs =concentration of USP Apomorphine
hydroxide VS. Read the volume added between the first two Hydrochloride RS in the Standardsolution
points of inflexion. Each mL of 0.1 N sodium hydroxide is (mg/mL)
equivalent to 30.38 mg of apomorphine hydrochloride Cu =concentration of Apomorphine Hydrochloride in
(C17H17N02 • HCI). the Sample solution (mg/mL)
Acceptance criteria: 98.5%-101 .5% on the dried basis F =relative response factor (see Table 2)
IMPURITIES Acceptance criteria: See Table 2. Disregard any peak below
• RESIDUE ON IGNITION (281): NMT 0.1 % 0.05%.
• ORGANIC IMPURITIES
Diluent: Glacial acetic acid and water (1:99) Table 2
Solution A: 1 .1-g/L solution of sodium octanesulfonate,
Relative Relative Acceptance
adjusted with diluted phosphoric acid (1:1) to a pH of 2.2 Retention Response Criteria,
Solution B: Acetonitrile Name Time Factor NMT (%)
Mobile phase: See Table 1.
Morphine 0.4 0.11 0.15

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358 Apomorphine / Official Monographs USP 43

Table 2 (continued) Place a quantity of powdered Tabl~t

Relative Relative Acceptance mgofaponiorphine .hydrochloride i
Retention Response Criteria, sma.llsize,add 10.0 mL of cold
Name Time Factor NMT (%) stopper in the test tube, -
Apomorphine 1.0 - - dissolves; if necessary,fil
Anyother individual lrn-
pledget'of cotton. The
purity - 1.0 0.10 promptly, after preparati . ' ..... .
the.color stai1dard~UsecJoselymatched t
Total impurities - - 0.5 comparison;
Identification-ToS mL of
SPECIFIC TESTS containing about 10 mgof
• OPTICAL ROTATION, Specific Rotation (781 S) a slight excess of sodium.bic
Sample solution: 10 mg/mL in Diluent white or greenish-white prec'
Diluent: 2.06-g/L solution of hydrochloric acid in water iodine TS,and shake vig
Acceptance criteria: -48° to -52°, determined at 20° produced. Add 5 mL of e
• Loss ON DRVING (731) allow the layers to separate: e.e
Analysis: Dry a sample at 105° for 2 h. while the water layer retains its
Acceptance criteria: 2.0%-4.2% Disintegration ( 701) :
• COLOR OF SOLUTION Uniformity of dosage units (9
Sample solution: Place 100 mg of Apomorphine Procedurefor contentuniformity
Hydrochloride in a suitable test tube, add 10 mL of cold, volumetricflask containing 10
oxygen-free water, and agitate gently until dissolved. and shake for 15 minutes.
Standard solution: Dissolve5 mg of Apomorphine to volume, mix, and filter, dl
Hydrochloride in 100.0 mLof water. Transfer 1.0 mLof this Dilute a portion of tile subs
solution to a test tube of the same size as that used for the stepwise; if necessary, w·
Sample solution. Dilute with 6 mL of water, add 1 mL of a a solution containing ap
50-mg/mL sodium bicarbonate solution, and then add 0.50 .hydrochloride per mL. Con
mL of iodine TS. Allowto stand for 30 s, add 0.60 ml, of a absorbances of this solution
25-mg/mL sodium thiosulfate solution, and dilute with Apomorphine. HydrochlorideR
water to 10 mL. a known concentration of abou
Acceptance criteria: The color of the Sample solution, apomorphine hydrochloride
observed promptly after the Apomorphine Hydrochloride wavelength of maximum,ab
has dissolved, Is not more intense than that of a color of the .asuitable spectrophotomete
Standardsolution. as the blank. Calculate the qu
ADDITIONAL REQUIREMENTS . lhHzO in the Tablet taken by . e
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers. (312.80/303.79)(TC/ D)(A~/As)
• USP REFERENCE STANDARDS (11)
USP Apomorphine Hydrochloride RS in which 312.80 and 303.79 .
apomorphine hydrochl .
apomorphine hydrochl
quantity, in mg, ·of apomor
C isthe concentration, i
apomorphine hydrochl
concentration, in J../g per .
in the solution from the T
quantity per Tablet and th
are the absorbancesof the luti
Standard solution, respective .
Assay-Weigh and finely po
Dissolve. an accurately wei
equivalent to out 5
25 mL in as
bicarbo d co
portionso.e er. Co
and w'ash them w'
combined water. " ings
ether to the combined ethe
solutions with 20.0 mL of 0.02
with three 5-mL:portions of water
and washings in a beaker, and war
any residual ether. Cool, addm
excess acid with 0.02 N sodium
Titrations under Tit/'imetry.(541)
acid is equivalent to 6.256 mg Q
,V2H;O. A (USP 1~Ma~-2020)

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 USP 43 OfficialMonographs / Apraclonidine 359

100r;lr t
Apraclonidine Hydrochloride
inwhich r, isthe responseof each peakother than the principal
peak, and r t is the sum of the responses of all of the peaks,
excluding that of the solvent peak: not more than 1.0% for
any individual impurity and not more than 2.0% total
impurities are found.
Assay-Dissolve about 125 mg of Apraclonidine
C9H1oClzN4' ao 281.57 Hydrochloride, accurately weighed, in 40 mL of glacial acetic
1,4-Benzenediamine, 2,6-dichloro-N 1-2-imidazolidinylidene- acid. Add 10 mL of mercuricacetate TS, and titrate with 0.1
, monohydrochloride. N perchloric acid VS, determining the endpoint
2-[(4-Amino-2,6-dichlorophenyl)imino]imidazolidine potentiometrically from the second inflection point, using a
monohydrochloride [73218-79-8]. calomel-glass electrode system (see Titrimetry (541 »). Perform
a blank determination, and make any necessary correction.
» Apraclonidine Hydrochloride contains not less Each mLof 0.1 N perchloric acid is equivalentto 14.08 mg of
than 98.0 percent and not more than 102.0 C9H1oClzN4 . HCI.
percent of C9H lOCI 2N4 . HCI, calculated on the dried
basis.
Packaging and storage-Preserve in tight, light-resistant
containers. Apraclonidine Ophthalmic Solution
VSPReference standards (11 )-
USP Apraclonidine Hydrochloride RS » Apraclonidine Ophthalmic Solution is a sterile,
Identification- aqueous solution of Apraclonidine Hydrochloride.
It contains an amount of apraclonidine
hydrochloride (C9H lOCI 2N4 . HC/) equivalent to not
less than 90.0 percent and not more than 115.0
percent of the labeled amount of apraclonidine
(191 ). (C9HloCI2N4)'
pH (791): 5.0 and 6.6 in a solution (1 in 100).
Loss on drying (73~ )-Dry it in vacuum at 105° for 3 hours: Packaging and storage-Preserve in tight, light-resistant
it loses not more than 1.0% of its weight. containers.
Residue on ignition (281): not more than 0.1%. VSP Reference standards (11 )-
Chromatographic purity- USP Apraclonidine Hydrochloride RS
Phosphate buffer-Transfer 6.8 mL of phosphoric acid to a Identification-
2000-mLvolumetric flask, add about 1900 mL of water, and A: The retention time of the major peak in the
mix. Adjust with sodium hydroxide solution (1 in 2) to a pH of chromatogram of the Assay preparationcorrespondsto that of
3.0, dilute with water to volume, and mix. the major peak in the chromatogram of the Standard
Mobilephase-Prepare a filtered and degassed mixture of preparation, as obtained in the Assay.
acetonitrile, Phosphate buffer, and methanol (56:40:4). Make B: Apply 2 IJL of Apraclonidine Ophthalmic Solution and 2
adjustments if necessary (see System Suitability under. IJL of a Standard solution of USP Apraclonidine Hydrochloride
Chromatography (621 », RS in methanol containing about 11.5 mg per mL to a suitable
System suitability solution-Prepare a solution in Mobile high performance thin-layer chromatographic plate (see
phase containing about 0.8 mg of USP Apraclonidine Chromatography (621») coated with a 0.2-mm layer of
Hydrochloride RS per mL. chromatographic silica gel mixture, or equivalent. Allow the
Test solution-Transfer about 20 mg of Apraclonidine applicationsto dry, and develop the chromatogram in a
Hydrochloride, accuratelyweighed, to a 25-mLvolumetric solvent system consisting of a mixture of chloroform,
flask, dissolve in and dilute with Mobilephaseto volume, and methanol, and ammonium hydroxide (74:22:4) until the
mix. solventfront has moved about three-fourthsof the length of
Chromatographic system (see Chromatography (621 »)- the plate. Remove the plate from the developing chamber,
The liquid chromatograph is equipped with a 220-nm mark the solventfront, and allow the solventto evaporate.
detector and an 8-mm x 100-mni column that contains locate the spots on the plate by viewing under
packing L7. The flow rate is about 3 mL per minute. short-wavelength UV light. [NOTE-The apraclonidine spot
Chromatograph the System suitabilitysolution, and record the should appear as a blue spot.] Spray the plate with
peak responses as directed for Procedure: the tailingfactor for fluorescamine solution, prepared by dissolving about 25 mg
the apraclonidine peak is not more than 2.2, and the relative offluorescamine in25 mL of acetone. [NOTE-Avoid prolonged
standard deviation for replicateinjections is not more than or repeated breathing of the aerosol from the fluorescamine
2.0%. spray. Also avoid prolonged or repeated contact with skin.
Procedure-/nject about 20 IJL of the Test solution into the Huorescarnlne solution should be sprayed only in a hood.]
chromatograph, record the chromatograrn, and measure the Examine the plate under normal light and long-wavelength
areas for the major peaks. [NOTE-Allow about five times the UV light. [NOTE-The apraclonidine spot should appear as a
elution time of apraclonidine before making the next yellowspot under normal light and as a white spot under
injection.] Calculate the percentage of each peak, other than long-wavelength UV light.]The RF valueand appearance ofthe
the solvent peak and the apraclonidine peak, in the specimen principal spot obtained from the test solution corresponds to
of Apraclonidine Hydrochloride taken by the same formula: that obtained from the Standard solution.

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 360 Apraclonidine / OfficialMonographs USP43

Sterility Tests (71) -It m~ets ~he requirements wh~~ tested

as directed for Membrane hltraticn under Test for Stenltty of the Aprepitant
Product to be Examined.
pH (791): between 4.4 and 7.8.
Assay-
Phosphate buffer-Prepare as directed!n.the test for .
Chromatographic purity- un?er Apraclomdme Hydro~hlonde.
Mobilephase-Prepare a filtered and degassed mixture of
Phosphate buffer, acetonitrile, and meth.ano.I.(68:30:2). Make
adjustments if necessary(see System SUItabIlity under C23 H21 F7N4o 3 534.43
Chromatography (621 ». . 3H-1,2,4-Triazol-3-one, 5-[[(2R,35)-2-[(1 R)-1-[3,5-
Standardpreparation-Dissolve an accurately weighed
quantity of USP Apraclonidine Hydrochloride RS i~ water, and bis(trifluoromethyl)phenyl]ethoxy]-3-(4-fluorophenyl)-4-
dilute quantitatively, and stepwise !f necessary, with water.to morphoJinyl]methyl]-1,2-dihydro-;
obtain a Stock standardsolution having a known concentration 3-[[(2R 35)-3-(p-Fluorophenyl)-2-[[(aR)-a-methyl-3,5-
of about 0.23 mg per mL. Transfer2.5 ml of this solution to bis(trifl uoromethyl)benzyl]oxy]morphoJino] methytj-A 2_
a 50-ml volumetric flask, dilute with Mobilephase to volume, 1,2,4-triazoJin-5-one;
and mix to obtain a Standardpreparation having a known 3-[[(2R, 35)-2-[(R)-1-[3,5-Bis(trifluoromethyl) phenyl]~thoxy]­
concentration of about 11.5 J.Jg of USP Apraclonidine 3-(4-fluorophenyl)morpholino]methyl]-l H-1 ,2,4-trrazol-
Hydrochloride RS per ml (equivalent to about 10 J.Jg of 5(4H)-one; ,
5-[[(2R 35)-2-[(R)-1-[3,5-Bis(trifluoromethyl)phenyl]ethoxy]-
apracJonidine per ml). . 3-(4-fluorophenyl)-4-morpholinyl]methyl]-1 ,2-dihydro-3H-
Resolution solution-Transfer about 1 mLof propiophenone
to a 1OO-mL volumetricflask, dilute with methanol to volume, 1,2,4-triazol-3-one [170729-80-3].
and mix.Transfer 3.0 ml of this solution to a 50-ml volumetric DEFINITION
flask dilute with methanol to volume, and mix. Transfer 1.0 Aprepitant contains NlT 98.0% and NMT 102.0% of
ml ~f this solution and 5.0 ml of the Stock standardsolution aprepitant (C23H21F7N403), calculated on the anhydrous and
to a 1OO-ml volumetricflask, dilute with Mobile phase to solvent-free basis.
volume, and mix.
Assay preparation-Transfer an accurately measured IDENTIFICATION
volume of Ophthalmic Solution,equiyalent to ~bout ?Omg of
apraclonidine, to a 1OO-ml volurnetrlc fl~sk, dll~te With water
to volume,and mix.Transfer2.5 ml of this solution to a 50-"!1l
volumetric flask, dilute with Mobilephase to volume, and mix.
Chromatographic system (see Chromatography (621 »- •
!?()2()
time of the major peak of the Sample
The liquid chromatograph is equipped with a 254-nn:t
detector and an 8-mm x 1OO-mm column that contains solution corresponds to that of the Standardsolution, as
packing l7. The flow rate is about 3 ml per minute. obtained in the Assay.
Chromatograph the Resolution solution, an~ record t~e p~ak ASSAY
responsesas directed for Procedure: the relative re~entlon times • PROCEDURE
are about 0.6 for apraclonidine and 1.0 for proplophen~ne; Dilute phosphoric acid: Dilute 1 ml of phosphoric acid
the column efficiency determined from the analyte peak IS not with water to 1 l.
less than 1000 theoretical plates; the tailing factor for the Mobile phase: Acetonitrile and Dilutephosphoric acid
analyte peak is not more than 2.2; the resolution, R, between
the analyte and propiop~en.one peaks i.s not .Ie~s t~an ~.O; and (48:52) . . ..
Diluent: Acetonitrile and DIlutephosphonc acid (50:50)
the relative standard deviation for replicate injections IS not Standard solution: 0.2 mg/mL of USP Aprepitant RS in
more than 2.0%. Diluent; sonicate as needed ...
Procedure-Separately inject equal volumes (ab?ut .20 J.JL) Sample solution: 0.2 mg/ml of Aprepitant In DIluent;
of the Standardpreparation and the Assay preparation Intothe sonicate as needed
chromatograph, record the chromatograms, a~d ~easure the Chromatographic system . ..
areas for the major peaks. Calculatethe quantity, In mg~ of (See Chromatography (621), System SUItabIlity.)
apraclonidine (CgH 1OCI 2N 4) in each ml of the Ophthalmic Mode: lC
Solution taken by the formula: Detector: UV 210 nm
Column: 4.6-mm x 25-cm; 5-J.Jm packing L1
(245.11/281.57)(2CIV)(rulrs) Column temperature: 35°
Flow rate: 1.5 ml/min
in which 245.11 and 281.57 are the molecular weights of Injection volume: 20 J.Jl
apraclonidine and apraclonidine hydrochloride, respe~ti.vely; System suitability
C is the concentration, in J.Jg per mL, of USP Apradonidlne Sample: Standardsolution
Hydrochloride RS in the Standardpreparation; Vis the volume, Suitability requirements
in mL, of Ophthalmic Solution taken; and tu and rs are the Tailing factor: NMT 2.0
apracJonidine peak responses obtaine~ from the ~ssay Relative standard deviation: NMT 0.73%
preparation and the Standardpreparation, respectively. Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of aprepitant (C23H21F7N403) in
the portion of Aprepitant taken:
Result = (rulr s) x (CsICu) x 100

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USP 43 Official Monographs / Aprepitant 361

= peak response from the Sample solution Acceptance criteria: See Table 2. Disregard any peak below
= peak response from the Standard solution 0.05%. I

= concentration of the Standard solution (mg/mL)

=concentration of the Sample solution (mg/mL) Table 2
Relative Acceptance
Acceptance criteria: 98.0%-102.0%, on the anhydrous Retention Criteria,
Name Time NMT(%)
and solvent-free basis
Desfluoro aprepitant 0.85 0.15
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1% Aprepitant 1.0 -
• ORGANIC IMPURITIES Any individual
Dilute phosphoric acid and Diluent: Proceed as directed unspecified impurity - 0.10
in the Assay.
Solution A: Dilute phosphoric acid Total impurities - 0.30
Solution B: Acetonitrile
Mobile phase: See Table 7 • LIMIT OF S,R,S-ENANTIOMER (if present)
[NoTE-Perform this test ifthis impurity is possible from
Table 1 the manufacturing process.]
Time Solution A Solution B Mobile phase: Hexane and dehydrated alcohol (90:10)
(min) (%) (%) System suitability solution: 0.08 mg/mL of USP
Aprepitant RS and 0.08 mg/mL of USP Aprepitant Related
0 58 42
Compound B RS in Mobilephase
25 58 42 Sample solution: 0.5 mg/mL of Aprepitant in Mobilephase
Chromatographic system
45 30 70
(See Chromatography (621), System Suitability.)
50 30 70 Mode: LC
Detector: UV 210 nm
Return to original conditions and re-equilibrate the system. Column: 4.6-mm x 25-cm; packing L51
System suitability solution: 2.0 mg/mL of USP Aprepitant Column temperature: 30°
RS and 0.003 mg/mL of USP Desfluoro Aprepitant RS in Flow rate: 0.5 mL/min
Diluent, using sonication as necessary to dissolve Injection volume: 20 IJL
Sensitivity solution: 0.001 mg/mL of USP Aprepitant RS in System suitability
Diluent I
Sample: System suitabilitysolution
Standard solution: 0.003 mg/mL of USPAprepitant RS in Suitability requirements
Diluent, using sonication as necessary to dissolve Resolution: Greater than 2.0 between the enantiomer
Sample solution: 2.0 mg/mL of Aprepitant in Diluent, using peaks. [NoTE-The elution order is the S,R,5-enantiomer
sonication as necessary to dissolve . followed by the aprepitant peak, which is the
Chromatographic system R,S, R-enantiomer.]
(See Chromatography (621), System Suitability.) Analysis
Mode: LC Sample: Sample solution ,
Detector: UV 210 nm Calculate the percentage of the S,R,5-enantiomer in the
Column: 4.6-mm x 25-cm; 5-~m packing L1 portion of Aprepitant taken:
Column temperature: 35°
Flow rate: 1.0 mL/min Result = (rv/rr) x 100
Injection volume: 10 ~L t» = peak response of the S,R,5-enantiomer
System suitability
Samples: System suitabilitysolution, Sensitivity solution, and rr =sum of the peak responses of aprepitant and the
Standardsolution S, R,5-enantiomer
Suitability requirements Acceptance criteria: NMT 0.10% of the S,R,5-enantiomer
Resolution: NLT 3.0 between the desfluoro aprepitant
and aprepitant peaks, System suitability solution SPECIFIC TESTS
Signal-to-noise ratio: NLT 10, Sensitivity solution • WATER DETERMINATION, Method la or Ie (921): NMT 0.5%
Relative standard deviation: NMT 5.0%, Standard • OPTICAL ROTATION, Specific Rotation (781 S)
solution Sample solution: 10 mg/mL in methanol
Analysis Acceptance criteria: +66.0° to +71.0°
Samples: Standard solution and Sample solution
Calculate the percentage of each individual impurity in the ADDITIONAL REQUIREMENTS
portion of Aprepitant taken: • PACKAGING AND STORAGE: Preserve in well-closed
containers, protected from light. Store at room
Result =(rvlrs) x (CsICv) x 100 temperature.
• USP REFERENCE STANDARDS (11)
= peak response for each impurity from the USP Aprepitant RS
Sample solution USP Aprepitant Related Compound B RS
= peak response for aprepitant from the Standard S, R, 5-Enantiomer: 3-[[(25,3R)-2-[(5)-1-[3,5-
solution Bis(trifluoromethyl)phenyl]ethoxy]-3-(4-fluorophenyl)
= concentration of USP Aprepitant RS in the morpholino]methyl]-l H-1 ,2,4-triazol-5(4H)-one.
Standard solution (mg/mL) CZ3HzlF7N403 534.43
=concentration of Aprepitant in the Sample
solution (mg/mL)

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362 Aprepitant / OfficialMonographs USP 43

USP Desfluoro Aprepitant RS Calculatethe percentage of the labeled amount of

5-[[(2R,3S)-2-[(R)-1-[3,5-Bis(trifluoromethyl)phenyl] aprepitant (C23H21F7N403) in the portion of Capsules
ethoxy]-3-phenylmorpholino]methyl]-2H-l,2,4-triazol- taken:
3(4H)-one.
C23H22F6N403 516.44 Result =(rulrs) x (CslCu) x 100
= peak response from the Sample solution
= peak response from the Standard solution
= concentration of USP Aprepitant RS in the
Aprepitant Capsules Standard solution(mg/mL)
= nominal concentration of aprepitant in the
DEFINITION Sample solution(mg/mL)
Aprepitant Capsulescontain NLT 95.0% and NMT 105.0% of
the labeled amount of aprepitant (C23H 21F7N403). Acceptance criteria: 95.0%-105.0%
IDENTIFICATION PERFORMANCE TESTS

• A~~~~~~~'~~~~~I~; • DISSOLUTION (711)

f..!!trqY{()1~t2fll~l§1~~p ey: Test 1
Wavelength range: 200-400 nm Medium: 2.2% sodium dodecyl sulfate in water; 900 mL
Standard solution: 0.1 mg/mL of USP Aprepitant RS in Apparatus 2: 100 rpm, with sinkers. [NOTE-A suitable
methanol. Use sonication to dissolve. sinker is available from VanKel, www.chem.agilent.com.
Sample solution: Transferthe contents of Capsules, catalog number 12-3050. Proper placement of the
equivalentto 100 mg of aprepitant, to a 1OO-mL volumetric Capsules is in the sinkers with the cap facing the fixed
flask, add about 75 mLof methanol, and sonicate for about prong end.]
5 min with intermittent shaking.Cool,dilutewith methanol Time: 20 min
to volume,further dilute with methanol to obtain a solution Dilute phosphoric acid: Dilute 1 mL of phosphoric acid
containing 0.1 mg/mL of aprepitant, and pass through a with water to 1 L.
nylon filterof 0.45-l..Im pore size. Mobile phase: Acetonitrile and Dilutephosphoric acid
Acceptance criteria: Meet the requirements (50:50)
• B. The retention time of the major peak of the Sample Standard solution: (Lj900) mg/mL of USP Aprepitant RS
solution corresponds to that of the Standard solution, as in Medium, where L is the labelclaim in mg/Capsule.
obtained in the Assay. Dissolve first in a minimal amount of methanol (using
NMT 2% of the final volume) prior to diluting with
ASSAY Medium.
• PROCEDURE Sample solution: Pass a portion of the solution under test
Dilute phosphoric acid: Dilute 1 mLof phosphoric acid through a suitable filter.
with water to 1 L. ~
Chromatographic system
Mobile phase: Acetonitrile and Dilutephosphoric acid (See Chromatography (621), System Suitgbility.)
(45:55) Mode: LC
Standard solution: 0.05 mg/mL of USP Aprepitant RS in Detector: UV 220 nm
Mobilephase. Use sonication as necessaryto dissolve. Column: 4.6-mm x 15-cm; 5-l..Im packing L7
Sample solution: Nominally 0.05 mg/mL of aprepitant in Flow rate: 1.5 mL/min
Mobilephase, prepared as follows. Mix the contents of NLT Injection volume: 50 I..IL for Capsules containing 40 mgl
20 Capsules, and transfer a portion of the contents, Capsule; 10 I..IL for all other strengths
equivalentto 100 mg of aprepitant, to a 1OO-mL volumetric System suitability
flask. Add about 75 mLof Mobilephase and sonicate for Sample: Standard solution
about 10 min with intermittent shaking. Cool, dilute to Suitability requirements I
volume with Mobilephase, further dilute with Mobilephase Relative standard deviation: NMT 2.0%
to obtain a solution containing 0.05 mg/mL of aprepitant, Analysis
and pass through a nylon filter of 0.45-l..Im pore size. Samples: Standard solution and Sample solution
Chromatographic system Calculate the percentage of the labeled amount of
(See Chromatography (621), System Suitability.) aprepitant (C23H21F7N40 3) dissolved:
Mode: LC
Detector: UV 210 nm Result = (rulrs) x Cs x (VIL) x 100
Column: 4.6-mm x 15-cm; 5-l..Im packing L1
Column temperature: 40° tu = peak response from the Sample solution
Flow rate: 1.5 mL/min rs = peak response from the Standard solution
Injection volume: 10 I..IL Cs = concentration of USP Aprepitant RS in the
System suitability Standard solution(mg/mL)
Sample: Standard solution V =volume of Medium, 900 mL
Suitability requirements L = label claim (mg/Capsule)
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% Tolerances: NLT 80% (Q) of the labeled amount of
Analysis
Samples: Standard solution and Sample solution aprepitant (C23H.21F.?~4~?)is . ~i~.s?I~~cl.
Test 2:.. . tlt:·.·~Jj~iRrHcli~~~i~9('n.pl.i.~~Y¥i;tm.;.jt~i~<1~$~)/"l~·,.~·~~li:~9,
indicatesthat .it meetsUSP.pis$oluJi9.r).7fg$t4·~ (RB] "May~2()lg)

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USP 43 OfficialMonographs / Aprepitant 363

Medium: 2.2% sodium dodecyl sulfate in water; 900 mL CakulatEdhe percentageofthe 'labeled amount of
Apparatus 2: 100 rpm, with wire helix sinkers or other ap,.epit_~nt(Cz3Hzl F7N 403) di~solved:
suitable sinkers
Time: 30 min R.esult ::(ru/rs5 x CsxV Xc 0 x OIL) x'l 00
Dilute phosphoric acid and Mobile phase: Proceed as
directed in the Assay. =~Reak re~ponse from the Sample solution
Standard solution: (L/900) mg/mL of USP Aprepitant RS ~'p~a~ r~sp9nSe-froJTi the St~ngardso/~tion
in Medium, where L is the label claim in mg/Capsule. , SP Aprepitant RS inthe
Dissolve first in a minimal amount of methanol (using )
NMT 2% of the final volume) prior to diluting with
Medium.
v ~
[j e solution
Sample solution: Pass a portion of the solution under test {
through a suitable filter of 0.45-lJm pore size.
Chromatographic system: Proceed as directed in the Tolera.. NLT75% (Q).ofthe lab~ledamountof
Assay, except use an autosampler temperature of 15°.
aprepita C23Hzl7N403) is dissolved.~ (RB l-May.2018)
System suitability
• UNIFORMITV OF DOSAGE'UNITS (905): Meet the
Sample: Standardsolution
Suitability requirements requirements
Relative standard deviation: NMT 2.0% IMPURITIES
Analysis • ORGANIC IMPURITIES
Samples: Standardsolution and Sample solution Dilute phosphoric acid: Dilute 1 mL of phosphoric acid
Calculate the percentage of the labeled amount of with water to 1 L.
aprepitant (CZ3H Z1 F7N40 3) dissolved: Solution A: Acetonitrile and Dilute phosphoric acid (5:95)
Solution B: Acetonitrile and Dilute phosphoric acid (95:5)
Result = (ru/rs) x Cs x (V/L) x 100 Diluent: Acetonitrile and Dilute phosphoric acid (50:50)
Mobile phase: See Table 1.
to = peak response from the Sample solution
rs = peak response from the Standardsolution Table 1
Cs =concentration of USP Aprepitant RS in the Time Solution A Solution B
Standardsolution (mg/mL) (min) (%) (%)
V = volume of Medium, 900 mL
60
0 40
L = label claim (mg/Capsule)
20 58 42
Tolerances: NLT 80% (Q) of the labeled amount of
25 35 65
aprepitant (Cz3H z1F7N40 3) is dissolved.
"'Test 3:lfthepr tc 33 35 65
indicates that it
Medium:' 2.2% sodium Return to original conditions and re-equilibrate the system
Apparatus 2: "1 00 rpm, with _ for 10 min.
vessels. System suitability solution: 0.6 mg/mL of USP Aprepitant
Time:.30 min RS and 0.0012 mg/mL each of USP Desfluoro Aprepitant RS
Dilute phosphoriC acid: Prepare and USP Aprepitant Related Compound A RS in Diluent
Mobile phase: Dilute phosphoric Standard solution: 0.0012 mg/mL of USP Aprepitant RS in
(52:48) Diluent
Standard stock solutio . RS Sample solution: Nominally 0.6 mg/mL of aprepitant,
in Mobile phase. Sonka prepared as follows. Transfer the contents of Capsules,
dissolution. equivalent to 120 mg of aprepitant, to a 200-mL volumetric
Standard solution: 44 J.l. flask, add about 150 mL of Diluent, and sonicate for about
Standardsto . 10 min with intermittent shaking. Cool, dilute with Diluent
Sample sol t to volume, and pass through a nylon filter of 0.45-lJm pore
through.as size.
necessary,w Chromatographic system
of the (See Chromatography (621), System Suitability.)
Chroma aphic syste'm Mode: LC
Mode: LC Detector: UV 210 nm
Detector' Column: 4.6-mm x 15-cm; 5-lJm packing L1
Column temperature: 35°
Col
Flow rate: 1.0 mL/min
Col Injection volume: 10 IJL
Fl System suitability
In Samples: System suitability solution and Standardsolution
Sys Suitability requirements
Sa Resolution: NLT 3.0 between the desfluoro aprepitant
Suitabil and aprepitant peaks, System suitability solution
Tailin _ Relative standard deviation: NMT 5.0%, Standard
.__ Relati\l~ s!ancJ ,•. ti<?rtN.MT· 7.0~- solution
An~lysis. _ _ . _ .' -, _', __ " .' Analysis
Samples: ,Standardsolutioij:and S,dmplesolution Samples: Standardsolution and Sample solution

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 364 Aprepitant / Official Monographs USP 43

Calculate the percentage of any individual impurity in the l-Arginyl-l-prolyl-l-aspartyl-l-phenylalanyl-l-cysteinyl-l-

portion of Capsules taken: leucyl-L-glutamyl-l-prolyl-l-prolyl-L-tyrosyl-l-threonylglycyl-
l-prolyl-l-cysteinyl-l-Iysyl-L-alanyl-L-arginyl-l-isoleucyl-L-
Result = (ru/rs) x (CslCu) x 100 isoleucyl-L-arginyl-l-tyrosyl-l-phenylalanyl-l-tyrosyl-l-
asparaginyl-l-alanyl-l-Iysyl-l-alanylglycyl-l-Ieucyl-l-
tu = peak response of each impurity from the Sample cysteinyl-l-glutaminyl-L-threonyl-l-phenylalanyl-l-valyl-l-
solution tyrosylglycylglycyl-l-cysteinyl-L-arginyl-L-alanyl-l-Iysyl-L-
ts = peak response of aprepitant from the Standard arginyl-l-asparaginyl-l-asparaginyl-l-phenylalanyl-L-Iysyl-l-
solution seryl-l-alanyl-l-glutamyl-L-aspartyl-l-cysteinyl-L-methionyl-
Cs = concentration of USP Aprepitant RS in the l-arginyl-l-threonyl-L-cysteinylglycylglycyl-l-alanine cyclic
Standard solution (mg/mL) (5~55), (14~38), (30~51) tris(disulfide) [9087-70-1].
Cv = nominal concentration of aprepitant in the
Sample solution (mg/mL) DEFINITION
Aprotinin is a polypeptide consisting of a chain of 58 amino
Acceptance criteria: See Table 2. acid residues, which inhibits stoichiometrically the activity of
several proteolytic enzymes such as chymotrypsin, kallikrein,
Table 2 plasmin, and trypsin. Aprotinin is obtained from bovine
Acceptance
tissues and purified by a suitable' process, and is stored as a
Relative
Retention Criteria, bulk solution or lyophilized powder. Its potency, calculated
Name Time NMT (%) on the dried basis, is NLT 3 USP Aprotinin Units/mg. In
.:» addition, the method of manufacture is validated to result in
Desfluoro aprepitant 0.85
NMT 0.2 IJg of histamine per 3 USP Aprotinin Units using
Aprepitant 1.0 - validated methods. The origin and sourcing of bovine
material must be specified in compliance with FDA
Aprepitantdiastereomers .:»
(R,R,R and R,S,S)b 1.3 requirements. The manufacturing process is validated to
demonstrate the clearance of potential infectious agents (i.e.,
Anyother individual viruses, TSE agents). One USP Aprotinin Unit is equivalent to
impurity - 0.2
1800 Kallikrein Inhibition Units (K.I.U.).
Total impurities - 0.2
IDENTIFICATION
a Processimpurityincludedin the table foridentification only. Process impurities • A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
are controlled in the drug substance, and are not to be reported or included in (201)
the total impurities for the drug product. Cupric chloride solution: 10 mg/mL of cupric chloride
b The diastereomers are not separated bythis' procedure and shouldbe identified Sample solution: A solution of Aprotinin in water
based on the retention time of aprepitant related compound A (R,R,R-
diastereomer), which is a component of the System suitability solution. containing about 15 USP Aprotinin Units/mL
Chromatographic system
ADDITIONAL REQUIREMENTS Developing solvent system: To a mixture of glacial acetic
• PACKAGING AND STORAGE: Preserve in tight containers. acid and water (5:4) add 100 gil of sodium acetate.
Store at controlled room temperature. Spray reagent: Dissolve 0.1 9 of ninhydrin in a mixture
• LABELING: When more than one Dissolution test is given, the containing 6 mL of Cupric chloride solution, 21 mL of
labeling states the Dissolution test used only if Test 1 is not glacial acetic acid, and 70 mL of alcohol.
used. Analysis: Proceed, as directed in the chapter, except spray
• USP REFERENCE STANDARDS (11) the plate with the Spray reagent, and heat at 60° to visualize
USP Aprepitant RS the spots.
USPAprepitant Related Compound A RS • B. The retention time of the major peak of the Sample
R,R,R-Diastereomer: 3-[[(2R,3R)-2-[(R)-1-[3,5- solution corresponds to that of the System suitability
Bis(trifluoromethyl)phenyl]ethoxy]-3-(4-fl uorophenyl) solution, as obtained in Limit of N-Pyroglutamyl-Aprotinin
morpholino]methyl]-l H-l ,2,4-triazol-5(4H)-one. and Related Compounds.
C23H21F7N403 534.43 ASSAY
USP Desfluoro Aprepitant RS • PROCEDURE
5-[[(2R,3S)-2-[(R)-1-[3,5-Bis(trifluoromethyl)phenyl] Buffer: Transfer about 0.93 9 of boric acid into a 1000-mL
ethoxy]-3-phenylmorpholino]methyl]-2H-l ,2,4-triazol- volumetric flask, dissolve in 900 mL of water, adjust with 5
3(4H)-one. N sodium hydroxide to a pH of 8.0, and dilute with water
C23H22F6N403 516.44 to volume. Transfer 100 mL of this solution into a 1000-mL
volumetric flask, and dilute with water to volume.
Sample solution: A solution of Aprotinin in Buffer
containing about 1.67 USP Aprotinin Units/mL (about 0.6
mg/mL)
Aprotinin Trypsin solution: 4300 USP Trypsin Units/mL of USP Trypsin
Crystallized RS in 0.001 N hydrochloric acid. Use a freshly
RPDFCLEPPY TGP~KARIIR YFYNAKAGLC KRNNFKSAED prepared solution, and store in ice water.
'?'IRTtGGA I Trypsin and aprotinin solution: To 4.0 mL of the Trypsin
solution add 1.0 mL of the Sample solution. Dilute
C284H432N84079S7 6511.44 immediately with Buffer to 40.0 mL. Allow to stand at room
Trypsin inhibitor, pancreatic basic; temperature for 10 min, then keep in ice water. Use within
6 h of preparation.
Dilute trypsin solution: Dilute 0.5 mL of the Trypsin
solution with Buffer to 10.0 mL. Allow to stand at room
temperature for 10 min, then store in ice water.

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USP 43 Official Monographs / Aprotinin 365

Substrate solution: 6.9 mg/mL of N-benzoyl-L-arginine Capillary rinse procedure: Rinsethe capillary for NLT 1 min
ethyl ester hydrochloride in water. Use within 2 h. with NLT 10 total capillary volumes of 0.1 N sodium
Analysis: Mix 9.0 mL of Buffer and 1.0 mL of Substrate hydroxide, followed by at least 10 total capillary volumes
solution in a jacketed glass vessel with a capacity of about of water and by at least 20 capillary volumes of Buffer
30 mL and containing a stirring device. The lid of the between injections.
reaction vessel should contain five holes to accommodate Electrophoretic system
the electrodes, the tip of a buret, a tube for the admission Mode: CE
of nitrogen, and the introduction of reactants. An Detector: UV 214 nm
automated or manual titration apparatus may be used. Capillary: 75-lJm x 45- to 60-cm uncoated fused silica. Use
Adjust with 0.1 N sodium hydroxide VS to a pH of 8.0. Bufferas the electrolyte in both buffer reservoirs.
Maintain an atmosphere of nitrogen within the vessel, and Capillary temperature: 25°
stir continuously. When the temperature has reached Injection procedure: Transfer a volume of the Sample
equilibrium at 25 ± 0.1°, add 1.0 mLof Trypsin andaprotinin solution, approximately 15 nL, into the anodic end of the
solution, and start a timer. Maintain at a pH of 8.0 by the capillary. Apply differential pressure of 3.5 kPa for 3 s
addition of 0.1 N sodium hydroxide VS, and record the either by vacuum or pressure.
volume added every 30 s. Continue the reaction for 6 min. Applied voltage: 0.2 kV/cm
Carry out a similar titration using 1.0 mL of the Dilutetrypsin Run time: 30 min
solution. System sultablllty: The baseline is stable and shows little
For the lyophilized powder, calculate the aprotinin activity drift.
in USP Aprotinin Units/mg: Sample: System suitabilitysolution
Suitability requirements
Result = F, x [(F 2 X n 2 - n ,)/m] Migration time: 19-25 min for aprotinin
Relative migration times: About 0.98 for
F1 = conversion factor, 4000 des-Ala-des-Gly-aprotinin, 0.99 for des-Ala-aprotinin,
F2 = difference in the amount of trypsin used in Trypsin and 1 for aprotinin
and aprotinin solution and Dilutetrypsin solution, Resolution: NLT 0.8 between the
2 des-Ala-des-Gly-aprotinin and des-Ala-aprotinin peaks,
n2 = volume of 0.1 N sodium hydroxide added per and NLT 0.5 between the des-Ala-aprotinin and
second, after adding Dilute trypsin solution aprotinin peaks
(mL/s) Tailing factor: NMT 3 for aprotinin (see Chromatography
n1 = volume of 0.1 N sodium hydroxide added per (621) for calculation)
second, after adding Trypsin and aprotinin Analysis
solution (mL/s) Sample: Sample solution
m = quantity of Aprotinin used to prepare 1 mLof the Calculate the percentage of des-Ala-des-Gly-aprotinin and
Sample solution (mg) des-Ala-aprotinin:
For the concentrated solution, calculate the USP Aprotinin Result = (r vir r) x 100
Units/mL: .
ru = peak response of des-Ala-des-Gly-aprotinin or
Result = FIx (F 2 X n 2 - n 1) x D des-Ala-aprotinin
rr = sum of the peak responses of
= conversion factor, 4000 des-Ala-des-Gly-aprotinin, des-Ala-aprotinin,
= difference in the amount of trypsin used in Trypsin and aprotinin
and aprotinin solution and Dilute trypsin solution,
2 Acceptance criteria: See Table 1.
n2 = volume of 0.1 N sodium hydroxide added per
second, after adding Dilute trypsin solution Table 1
(mL/s) Relative Acceptance
n1 = volume of 0.1 N sodium hydroxide added per Migration Criteria,
second, after adding Trypsin and aprotinin Name Time NMT(%)
solution (mL/s) des-Ala-des-Gly-aproti-
D = dilution factor of the concentrated solution used nin 0.98 8.0
to prepare the Sample solution
des-Ala-aprotinin 0.99 7.5
Acceptance criteria: NLT 3 USPAprotinin Units/mg on the Aprotinin 1 -
dried basis
IMPURITIES • LIMIT OF N-PVROGLUTAMVL-ApROTININ AND RELATED
• LIMIT OF DES-ALA-ApROTININ AND COMPOUNDS
DES-ALA-DES-GLV-APROTININ Solution A: 3.52 giL of monobasic potassium phosphate
Buffer: Dissolve 8.21 g of monobasic potassium phosphate and 7.26 giL of dibasic sodium phosphate. Filter, and
in 400 mL of water. Adjust with phosphoric acid to a pH of degas.
3.0, and dilute with water to 500 mL. Solution B: 3.52 giL of monobasic potassium phosphate,
System suitability solution: Dilute USP Aprotinin RS with 7.26 giL of dibasic sodium phosphate, and 66.07 giL of
water to obtain a solution containing about 4-7 USP ammonium sulfate. Filter, and degas.
Aprotinin Units/mL. Mobile phase: See Table 2.
Sample solution: Dilute a concentrated solution of
Aprotinin with water, or weigh out Aprotinin and dissolve
in water to obtain about 4-7 USP Aprotinin Units/mL of
Aprotinin.

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366 Aprotinin / OfficialMonographs USP 43

Table .2 Mode: LC
Time Solution A Solution B Detector: UV 280 nm
(min) (%) (%) Columns: Series of three 7.8-mm x 30-cm columns;
0 92 8 packing L33
Flow rate: 1.0 mL/min
21 64 36 Injection volume: 100 J.1L
30 0 100 System suitability
Sample: System suitability solution
31 92 8 Suitability requirements
40 92 8 Retention time: 24.5-25.5 min for aprotinin
Relative retention times: 0.9 and 1.0 for the dimer and
aprotinin, respectively
System suitability solution: USP Aprotinin System Resolution: NLT 1.3 between the dimer and aprotinin
SUitability RS in Solution A, with 5 USP Aprotinin Units/mL peaks
Sample solution: Aprotinin in Solution A, with 5 USP Tailing factor: NMT 2.5 for aprotinin
Aprotinin Units/mL Analysis
Chromatographic system Sample: Sample solution
(See Chromatography (621), System Suitability.) Calculate the percentage of each oligomer peak:
Mode: LC
Detector: UV 210 nm Result = (r vir r) x 100
Column: 7.5-mm x 7.5-cm; packing L52
Column temperature: 40° ru = response of each peak with a retention time less
Flow rate: 1 mt/rnln than that of the aprotinin monomer
Injection volume: 40 ~L rr =sum of all the peak responses
System suitability
Sample: System sUitabilitysolution Acceptance criteria: Sum of all the oligomer peakresponses
Suitability requirements is NMT 1.0%.
Retention time: 17-20 min for aprotinin
Relative retention times: 0.9 and 1.0 for SPECIFIC TESTS
N-pyroglutamyl-aprotinin and aprotinin, respectively • ABSORBANCE
Resolution: NLT 1.0 between N-pyroglutamyl-aprotinin (See Ultraviolet-Visible Spectroscopy (857).)
and aprotinin Sample solution: 3.0 USP Aprotinin Units/mL
Tailing factor: NMT 2.0 for aprotinin Acceptance criteria: The Sample solution exhibits an
Analysis I
absorption maximum at 277 nm. The absorbance at the
Sample: Sample solution maximum is NMT 0.80.
Calculate the percentage of each impurity peak: • BIOLOGICAL REACTIVITY TESTS, IN VIVO, Safety Tests-
Biologicals (88)
Result = (i vir r) x 100 Sample solution: Prepare a solution of Aprotinin that
contains 4 USP Aprotinin Units/mL using a sufficient
ru = peak response of each impurity quantity of Water for Injection.
rr = sum of all the peak responses from the Sample Acceptance criteria: Meets the requirements
solution • SPECIFIC ACTIVITY OF THE DRY RESIDUE
This test should only be performed when product is a
Acceptance criteria: See Table 3. concentrated solution.
Sample solution: 25.0 mL of Aprotinin concentrated
Table 3 solution
Relative Acceptance Analysis: Evaporatethe Sample solutionto drynessin a water
Retention Criteria, bath, dry the residue at 110° for 15 h, and weigh. From the
Name Time NMT (%) weight of the residue and the activity determined in the
N-Pyroglutamyl-aproti-
Assay, calculate the number of USP Aprotinin Units/mg of
nin 0.9 1.0 dry residue.
Acceptance criteria: NLT 3.0 USP Aprotinin Units/mg of
Aprotinin 1.0 - dried residue
Any other impurity - 0.5 • Loss ON DRYING (731)
This test should only be performed on the lyophilized
Sum of all unknown im-
purities - 1.0
powder.
Sample: 100 mg
Analysis: Dry the Sample in a capillary-stoppered bottle
• LIMIT OF HIGH MOLECULAR WEIGHT PROTEINS under vacuum at a pressure of NMT 5 mm of mercury at
Mobile phase: Acetonitrile, glacial acetic acid, and water 60° for 3 h.
(1:1 :3). Filter and degas. Acceptance criteria: NMT 6.0%
System suitability solution: Aprotinin solution that • BACTERIAL ENDOTOXINS TEST (85)
contains about 5 USP Aprotinin Units/mL with about 2% Sample solution: 6 USP Aprotinin Units/mL
aprotinin oligomers. [Nora-This solution can be obtained Acceptance criteria: NMT 0.14 USP Endotoxin Unit per USP
by heating lyophilized aprotinin at 112° for about 2 hand Aprotinin Unit
dissolving the solid at the specified concentration in water.]
Sample solution: Aprotinin in water, with about 5 USP ADDITIONAL REQUIREMENTS
Aprotinin Units/mL • PACKAGING AND STORAGE: For lyophilized powder,
Chromatographic system preserve in tight containers, and store in a cold place.
(See Chromatography (621), System Suitability.) Protect from light. For bulk solution, preserve in tight

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USP 43 OfficialMonographs / Aprotinin 367

containers at a temperature not exceeding 25°. Avoid = conversion factor, 4000

freezing . = difference in the amount of trypsin used in Trypsin
.. LABELING: The labeling statesthe source of the material and and aprotinin solution and Dilute trypsin solution,
the number of Kallikrein Inhibition Units/mg or the number 2
of Kallikrein Inhibition Units/mL. nz = volume of 0.1 N sodium hydroxide added per
.. USP REFERENCE STANDARDS (11) second, after adding Dilute trypsin solution
USP Aprotinin RS (mL/s)
USP Aprotinin System Suitability RS n1 =volume of 0.1 N sodium hydroxide added per
USP Trypsin Crystallized RS second, after adding Trypsin and aprotinin
solution(mL/s)
o = dilution factor usedto prepare the Sample solution

Acceptance criteria: 90.0%-110.0% of the potency stated

Aprotinin Injection on the label, expressed in K.I.U./mL
DEFINITION OTHER COMPONENTS
Aprotinin Injection is a sterile solution of Aprotinin in Water for .. CONTENT OF SODIUM CHLORIDE
Injection that also contains sodium chloride. One USP Sample: 5.0 mL of Injection
Aprotinin Unit is equivalent to 1800 Kallikrein Inhibition Units Analysis: Pipet the Sample into a beaker containing 50 mL
(K.I.U.). It contains NLT 90.0% and NMT 110.0% of the of water. Add 10 mL of 25% nitric acid. Titrate with 0.1 N
potency stated on the label, expressed in K.I.U./mL. silver nitrate VSto a potentiometric endpoint, using a silver
combination electrode. Perform a blank determination (see
IDENTIFICATION »,
Titrimetry (541 and make any necessary correction. Each
• A. The retention time of the major peak of the Sample mL of 0.1 N silver nitrate is equivalent to 5.844 mg of
solution corresponds to that of the System suitability sodium chloride.
solution, as obtained in the test for Limit of Acceptance criteria: 42.5-47.5 mg
N-Pyroglutamyl-Aprotinin and Related Compounds.
• B. It meets the requirements in the Assay. IMPURITIES
• LIMIT OF N-PVROGLUTAMVL-ApROTININ AND RELATED
ASSAY
COMPOUNDS
• PROCEDURE Solution A: 3.52 gIL of monobasic potassium phosphate
Buffer: Transfer about 0.93 g of boric acid into a 1000-mL and 7.26 gIL of dibasic sodium phosphate. Filter, and
volumetric flask, dissolve in 900 mL of water, adjust with 5 degas.
N sodium hydroxide to a pH of 8.0, and dilute with water Solution B: 3.52 gIL of monobasic potassium phosphate,
to volume. Transfer 100 mL of this solution into a 1000-mL 7.26 gIL of dibasic sodium phosphate, and 66.07 gIL of
volumetric flask, and dilute with water to volume. ammonium sulfate. Filter, and degas.
Sample solution: A solution of aprotinin in Buffercontaining Mobile phase: See Table 1.
about 1.67 USP Aprotinin Units/mL (about 0.6 mg/mL)
Trypsin solution: 4300 USP Trypsin Units/mL of USP Trypsin Table 1
Crystallized RS in 0.001 N hydrochloric acid.Usea freshly
prepared solution, and store in ice water. Time Solution A Solution B
(min) (%) (%)
Trypsin and aprotinin solution: To 4.0 mL of the Trypsin
solution, add 1.0 mL of the Sample solution. Dilute 0 92 8
immediately with Bufferto 40.0 mL. Allow to stand at room 21 64 36
temperature for 10 min, then keep in ice water. Use within
6 h of preparation. 30 0 100
Dilute trypsin solution: Dilute 0.5 mL of the Trypsin 31 92 8
solution with Bufferto 10.0 mL. Allow to stand at room
temperature for 10 min, then store in ice water. 40 92 8
Substrate solution: 6.9 mg/mL of N-benzoyl-L-arginine
ethyl ester hydrochloride. Use within 2 h. System suitability solution: USP Aprotinin System
Analysis: Mix 9.0 mL of Buffer and 1.0 mL of Substrate Suitability RS in Solution A with 5 USP Aprotinin Units/mL
solution in a jacketed-glass vessel with a capacity of about Sample solution: Aprotinin in Solution A, with 5 USP
30 mL and containing a stirring device. The lid of the Aprotinin Units/mL
reaction vessel should contain five holes to accommodate Chromatographic system
the electrodes, the tip of a buret, a tube for the admission (See Chromatography (621), System Suitability.)
of nitrogen, and the introduction of reactants. An Mode: LC
automated or manual titration apparatus may be used. Detector: UV 210 nm
Adjust with 0.1 N sodium hydroxide VS to a pH of 8.0. Column: 7.5-mm x 7.5-cm; packing L52
Maintain an atmosphere of nitrogen within the vessel, and Column temperature: 40°
stir continuously. When the temperature has reached Flow rate: 1 mL/min
equilibrium at 25 ± 0.1°, add 1.0 mL of Trypsin andaprotinin Injection volume: 40 lJL
solution, and start a timer. Maintain at a pH of 8.0 by the System suitability
addition of 0.1 N sodium hydroxide VS, and record the Sample: System suitabilitysolution
volume added every 30 s. Continue the reaction for 6 min. Suitability requirements
Carry out a similar titration using 1.0 mL of the Dilute trypsin Retention time: 17-20 min for aprotinin
solution. Relative retention times: 0.9 and 1.0 for
Calculate the potency in USP Aprotinin Unlts/rnl: N-pyroglutamyl-aprotinin and aprotinln, respectively
Resolution: NLT 1.0 between N-pyroglutamyl-aprotinin
Result = F I x (F 2 X n 2 - n 1) x 0 and aprotinin

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 368 Aprotinin / OfficialMonographs USP 43

Tailing factor: NMT 2.0 for the aprotinin peak • PARTICULATE MAlTER IN INJECTIONS (788): Meets the
Analysis requirements
Sample: Sample solution • pH (791): 4.5-6.5
Calculate the percentage of each impurity peak: • INJECTIONS AND IMPLANTED DRUG PRODUCTS (1): Meets the
requirements
Result =(r ulr r) x 100 • BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.14
USP Endotoxin Units per USP Aprotinin Unit.
ru = peak response of each impurity
rr = sum of all the peak responses from the Sample ADDITIONAL REQUIREMENTS
solution • PACKAGING AND STORAGE: Preserve in single-dose
containers. Store at up to 25°, and avoid freezing.
Acceptance criteria: See Table 2. • USP REFERENCE STANDARDS (11)
USPAprotinin RS
Table 2 USP Aprotinin System Suitability RS
USPTrypsin Crystallized RS
Relative Acceptance
Retention Criteria,
Name Time NMT (%)
N~Pyroglutamyl-aproti-
n1n 0.9 1.0
Aprotinin 1.0 -
Anyother impurity - 0.5
Argatroban
Sum of all unknownim-
purities - 1.0

• LIMIT OF HIGH MOLECULAR WEIGHT PROTEINS

Mobile phase: Acetonitrile, glacial acetic acid, and water
(1:1 :3). Filter, and degas.
System suitability solution: Aprotinin solution that
contains about 5 USPAprotinin Units/mL with about 2%
aprotinin oligomers. [NOTE-This solution can be obtained C23H36N60sS . H20 526.65
by heating lyophilized aprotinin at 112° for about 2 hand 2-Piperidinecarboxylic acid, 1-[(S)-5-[(aminoiminomethyl)
dissolvlnq the solid at the specified concentration in water.] amino]-1-oxo-2-{[(1,2,3,4-tetrahydro-3-methyl-8-
Sample solution: Aprotinin in water, with 5 USP Aprotinin quinolinyl)sulfonyl]amino}pentyl]-4-methyl-, (2R,4R)-
Units/mL monohydrate;
Chromatographic system (2R,4R)-4-Methyl-l-{N2-[(1,2,3,4-tetrahydro-3-methyl-8-
(See Chromatography (621), System Suitability.) quinolyl)sulfonyl]-L-arginyl}pipecolicacid, monohydrate
Mode: LC [141396-28-3].
Detector: UV 280 nm DEFINITION
Column: Series of three 7.8-mm x 30-cm columns; Argatroban contains NLT98.0% and NMT 102.0% of
packing L33 argatroban (C23H36N60sS), calculated on the anhydrous and
Flow rate: 1.0 mL/min . solvent-free basis.
Injection volume: 100 ~L
System suitability IDENTIFICATION
Sample: System suitability soluiior:
Suitability requirements .
Retention time: 24.5-25.5 min for aprotinin
Relative retention times: 0.9 and 1.0 for the dimer and
aprotinin, respectively
Resolution: NLT 1.3 between the dimer and aprotinin • B. times e peaks of the Sample
peaks solution correspond to those of Standardsolution, as
Tailing factor: NMT 2.5 for the aprotinin peak obtained in the Assay.
Analysis ASSAY
Sample: Sample solution • PROCEDURE
Calculate the percentage of each oligomer peak: [NOTE-It is recommended to keep all solutions
containing argatroban at about 4°.]
Result = (rulr r) x 100 Solution A: 10 mM ammonium acetate and 5 mM sodium .
ru = response of each peak with a retention time less 1-heptanesulfonate
than that of the aprotinin monomer Solution B: Acetonitrile and methanol (500:300)
rt = sum of all the peak responses Mobile phase: See Table 1.

Table 1
Acceptance criteria: Sum of allthe oligomer peak responses
is NMT 1.5%. Time Solution A Solution B
(min) (%) (%)
SPECIFIC TESTS
0 60 40
• STERILITY TESTS (71): It meets the requirements when
tested as directed in Test for Sterilityof the Product to Be 20 60 40
Examined, Membrane Filtration. 35 50 50

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 USP 43 Official Monographs / Argatroban 369

Table 1 (continued) Analysis

Time Solution A Solution B Samples: Standardsolution and Sample solution
(min) (%) (%) Calculate the percentage of argatroban related compound
50 20 80
A and argatroban related compound B in the portion of
Argatroban taken:
60 20 80
60.1 60 40
Result =(r vir s) x (C siC v) x100
72.1 60 40 = peak response of argatroban related compound
A or argatroban related compound Bfrom the
Standard solution: 4 mg/mL of USP Argatroban RS in Sample solution
methanol ' = peak response of argatroban related compound
Sample solution: 4 mg/mL of Argatroban in methanol A or argatroban related compound Bfrom the
Chromatographic system Standardsolution
(See Chromatography (621 >, System Suitability.) = concentration of USP Argatroban Related
Mode: LC Compound A RS or USP Argatroban Related
Detector: UV 259 nm Compound B RS in the Standardsolution
Column: 4.6-mm x 25-cm; 3-l..Im packing L1 (mg/mL)
Temperatures = concentration of Argatroban in the Sample
Column: 50° solution (mg/mL)
Autosampler: 4°
Flow rate: 0.6 mL/min Calculate the percentage of any unspecified impurity in the
Injection volume: 10 I..IL portion of Argatroban taken:
System suitability
Result = (r vir s) x (C siC v) x 100
Sample: Standardsolution
Suitability requirements = peak response of any unspecified impurity from
Tailing factor: NMT 1.5 for both peaks the Sample solution
Relative standard deviation: NMT 1.0% for the sum of =sum of the peak responses of (R)-argatroban and
the peak responses of (R)-argatroban and (S)-argatroban (S)-argatroban from the Standard solution
Analysis =concentration of USP Argatroban RS in the
Samples: Standardsolution and Sample solution Standardsolution (mg/mL)
Calculate the percentage of argatroban (C23H36N60SS) in =concentration of Argatroban in the Sample
the portion of Argatroban taken: solution (mg/mL)
Result = (r vir s) x (C siC v) x 100 Acceptance criteria: See Table 2. Disregard any peak below
0.05%.
= sum of the peak responses of (R)-argatroban and
(S)-argatroban from the Sample solution Table 2
= sum of the peak responses of (R)-argatroban and
(S)-argatroban from the Standardsolution Relative Acceptance
Retention Criteria,
= concentration of USP Argatroban RS in the Name Time NMT (%)
Standardsolution (mg/mL) .
Cv = concentration of Argatroban in the Sample Argatroban related
compound A" 0.23 0.15
solution (mg/mL) .
Argatroban related
Acceptance criteria: 98.0%-102.0% on the anhydrous and compound Bb 0.39 0.15
solvent-free basis (R)-Argatroban C 1.00 -
IMPURITIES (5)-Argatroban d 1.03 -
- RESIDUE ON IGNITION (281 >: NMT 0.1 %
- ORGANIC IMPURITIES Any unspecified impurity - 0.10
[NOTE-It is recommended to keep all solutions Total impurities" - 0.5
containing argatroban at about 4°.]
Mobile phase, Sample solution, and Chromatographic a (2R,4R)-1-[N 8-Nitro-N 2-(3-methylquinoline-8-sulfonyl)-l-arginyl]-4-
system: Proceed as directed in the Assay. methylpiperidine-2-carboxylic acid.
Sensitivity solution: 2 I..Ig/mL of USP Argatroban RS in b Ethyl (4R)-1-[N 8-nitro-l-arginyl]-4-methylpiperidine-2-carboxylate.
methanol c (2R,4R)-4-Methyl-l-{N 2-[«R)·1,2,3,4-tetrahydro-3-methyl-8-quinolyl)
sulfonyl]-l-arginyl}pipecolic acid.
Standard solution: 4 I..Ig/mL each of USP Argatroban RS, d (2R,4R)-4-Methyl-l-{N 2-[«5)-1,2,3,4-tetrahydro-3-methyl-8-quinolyl)
USP Argatroban Related Compound A RS, and USP sulfonyl]-l-arginyl}pipecolic acid.
Argatroban Related Compound B RS in methanol erotal impurities include specified and unspecified impurities and argatroban
System suitability related compound C from the test for Contentof ArgatrobanRelated Compound
Samples: Sensitivity solution and Standardsolution C.
Suitability requirements
Resolution: NLT 1.2 between (R)-argatroban and -CONTENT OF ARGATROBAN RELATED COMPOUND C
(S)-argatroban, Standardsolution [NOTE-It is recommended to keep all solutions
Signal-to-noise ratio: NLT 10 for (R)-argatroban, containing argatroban at about 4°.]
Sensitivity solution Buffer: 10 mM ammonium acetate and 5 mM sodium
Relative standard deviation: NMT 5% for all peaks. For 1-heptanesulfonate
argatroban, use the sum of the peak responses of Solution A: Acetonitrile, dehydrated alcohol, and Buffer
(R)-argatroban and (S)-argatroban, Standard solution. (80:240:680)

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 370 Argatroban / OfficialMonographs USP 43

Mobile phase: See Table 3. Column: 4.6-mm x 25-cm; 3-lJm packing L1

Column temperature: 50°
Table 3 Flow rate: 0.6 mL/min
Time Solution A Acetonitrile Injection volume: 10 IJL
(min) (%) (%) Run time: NLT 1.4 times the retention time of
0 100 0
(R)-argatroban
System suitability
70 100 0 Sample: Standard solution
71 30 70
Suitability requirements .
Resolution: NLT 1.2 between (R)-argatroban and
91 30 70 (S)-argatroban
92 100 0 Relative standard deviation: NMT 2.0% for
(R)-argatroban and (S)-argatroban
102 100 0 Analysis
Sample: Sample solution
Sensitivity solution: 4 IJg/mL of USP Argatroban Related Calculate the percentage of (R)-argatroban and
Compound C RS in methanol (S)-argatroban in the portion of Argatroban taken:
System suitability solution: 10 mg/mL of USP Argatroban
RS and 0.1 mg/mL of USP Argatroban Related Compound Result = [(r u or r s)/(ru + r 5)] x 100
C RS in methanol
Sample solution: 10 mg/mL of Argatroban in methanol ru = peak response of (R)-argatroban from the
Chromatographic system: Proceed as directed in the Sample solution
Assay. r5 = peak response of (S)-argatroban from the
System suitability Sample solution
Samples: Sensitivity solution and System suitability solution
Suitability requirements Acceptance criteria: See Table 5.
Resolution: NLT 1.4 between argatroban related Table 5
compound C and (R)-argatroban, System suitability
solution Relative Acceptance
Retention Criteria,
Signal-to-noise ratio: NLT 10, Sensitivity solution Name Time (%)
Relative standard deviation: NMT 2.5% for argatroban
related compound C, System suitability solution (R)-Argatroban a 1.00 63-67
Analysis , (S)-Argatrobanb 1.06 33-37
Sample: Sample solution
Calculate the percentage of argatroban related compound a (2R,4R)-4-Methyl-l-{N 2-[«R).1 ,2,3,4-tetrahydro-3-methyl-8-quinolyl)
C in the portion of Argatroban taken: sulfonyl]-L-arginyl)pipecolic acid.
b (2R,4R)-4-Methyl-1-{N 2-[«S)-1,2,3,4-tetrahydro-3-methyl-8-quinolyl)
Result = (rvIr r) x 100 sulfonyl]-L-arginyl)pipecolic acid.

ru = peak response of argatroban related compound SPECIFIC TESTS

C from the Sample solution • WATER DETERMINATION (921), Method la: 3.0%-6.0%
rr =total of all peak responses from the Sample • BACTERIAL ENDOTOXINS TEST (85): NMT 2.0 USP Endotoxin
solution Units/mg of argatroban
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Acceptance criteria: See Table 4. SPECIFIED MICROORGANISMS (62): The total aerobic
microbial count is NMT 10 z du/g, and the total combined
Table 4 molds and yeasts count is NMT 10 z cfu/g.
Relative Acceptance ADDITIONAL REQUIREMENTS
Retention Criteria,
Name Time NMT (%) • PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers, and store at controlled room temperature.
Arqatroban related • USP REFERENCE STANDARDS (11)
compound c- 0.94 0.15
USP Argatroban RS
(R)-Argatroban 1.00 - USP Argatroban Related Compound A RS
(2R,4R)-1-[NS-Nitro-NZ-(3-methylquinoline-8-sulfonyl)-L-
(S)-Argatroban 1.07 - arginyl]-4-methylpiperidine-2-carboxylic acid.
a (2R,4R)-1-[N 8-Amino-N 2-(3-methyl-1,2,3,4-tetrahydroquinoline-8-sulfonyl)- Cz3H31N707S 549.60
L-arginyl]-4-methylpiperidine-2-carboxylic acid. USP Argatroban Related Compound BRS
Ethyl (4R)-1-[NS-nitro-L-arginyl]-4-methylpiperidine-2-
• CONTENT OF STEREOISOMERS carboxylate dihydrochloride.
[NOTE-It is recommended to keep all solutions ClsHz8N60S·2HCI 445.34
containing argatroban at about 4°.] USP Argatroban Related Compound C RS
Mobile phase: Methanol and water (520:480) (2R,4R)-1-[NS-Amino-NZ-(3-methyl-1,2,3,4-
Standard solution: 0.16 mg/mL of USP Argatroban RS in tetrahydroquinoline-8-sulfonyl)-L-arginyl]-4-
methanol methylpiperidine-2-carboxylic acid.
Sample solution: 0.16 mg/mL of Argatroban in methanol CZ3H37N70SS 523.65
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC .
Detector: UV 259 nm

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 USP 43 OfficialMonographs / Arginine 371

Developing solvent system: Isopropyl alcohol and

Arginine ammonium hydroxide (7:3)
J:~~OH 0
Analysis '
Samples: Standard solution, Sample solution, and System
H,N
suitability solution
NH,
Proceed as directed under Chromatography (621),
C6H,4N402 174.20 Thin-Layer Chromatography. Dry the plate between 100°
L-Arginine [74-79-3]. and 105° until the ammonia disappears completely.
Spray with Spray reagent, and heat between 100° and
DEfiNITION 105° for about 15 min. Examine the plate under white
Arginine contains NLT 98.5% and NMT 101.5% of light. The chromatogram obtained from the System
C6H,4N402, as L-arginine, calculated on the dried basis. suitability solution exhibits two clearly separated spots.
Acceptance criteria
IDENTIfiCATION Individual impurities: Any secondary spot from the
Sample solution is not larger or more intense than the
principal spot from the Standard solution, NMT 0.5%
Total impurities: NMT 2.0%
SPECifiC TESTS
• OPTICAL ROTATION, Specific Rotation (781 S): +26.3° to
ASSAY +27.7°
• PROCEDURE Sample solution: 80 mg/mL in 6 N hydrochloric acid
Sample: 80 mg of Arginine • loss ON DRYING (731): Dry a sample at 105° for 3 h: it loses
Titrimetric system NMT 0.5% of its weight.
(See Titrimetry(541 ).)
Mode: Direct titration ADDITIONAL REQUIREMENTS
Titrant: 0.1 N perchloric acid VS • PACKAGING AND STORAGE: Preserve in well-closed
Endpoint detection: Potentiometric containers.
Blank: 3 mL of formic acid and 50 mL of glacial acetic acid • 'USP REFERENCE STANDARDS (11)
Analysis: Dissolve the Sample in a mixture of 3 mL of formic USP L-Arginine RS
acid and 50 mL of glacial acetic acid, and titrate with USP L-Lysine Hydrochloride RS
Titrant. Calculate the percentage of C6H,4N402 in the
portion taken:
I

Result = [(V - B) x N x F x 100]/W

Arginine Hydrochloride
V = Sample titrant volume (mL)
B
N
= Blank titrant volume (mL)
= titrant normality (mEq/mL)
J:~~OH
H,N
0
• Hel

F = equivalency factor: 87.10 mg/mEq NH,

W = weight of Sample (mg)
C6H,4N402' HCI 210.66
Acceptance criteria: 98.5%-101.5% on the-dried basis L-Arginine monohydrochloride;
L-(+)-Arginine monohydrochloride [1119-34-2].
IMPURITIES
INORGANIC IMPURITIES DEfiNITION
• Residue on Ignition (281): NMT 0.3% Arginine Hydrochloride contains NLT 98.5% and NMT
• Chloride and Sulfate, Chloride (221 ): A 1.0-g portion shows 101.5% of arginine hydrochloride (C6H,4N402 . HCI),
no more chloride than corresponds to 0.70 mL of 0.020 N calculated on the dried basis.
hydrochloric acid (0.05%).
• Chloride and Sulfate, Sulfate (221): A 1.O-g portion shows
IDENTifiCATION
no more sulfate than corresponds to 0.30 mL of 0.020 N
sulfuric acid (0.03%).
• Iron (241): NMT 30 ppm
ORGANIC IMPURITIES
• Procedure
Adsorbent: 0.25-mm layer of chromatographic silica gel ASSAY
mixture • PROCEDURE
Standard solution: 0.05 mg/mL of USP L-Arginine RS in 0.1 Sample: 100 mg of Arginine Hydrochloride
N hydrochloric acid. [NOTE-This solution has a Titrimetric system
concentration equivalent to 0.5% of that of the Sample (See Titrimetry (541 ).)
solution.] Mode: Direct titration
Sample solution: 10 mg/mL of Arginine in 2 N hydrochloric Titrant: 0.1 N perchloric acid VS
acid Endpoint detection: Potentiometric
System suitability solution: 0.4 mg/mL each of USP Blank: 50 mL of glacial acetic acid and 3 mL of 98% formic
L-Arginine RS and USP L-Lysine Hydrochloride RS in 0.1 N acid. Add 6 mL of mercuric acetate TS.
hydrochloric acid Analysis: Dissolve the Sample in 3 mL of 98% formic acid
Spray reagent: 2 mg/mL of ninhydrin in a mixture of butyl and 50 mL of glacial acetic acid. Add 6 mL of mercuric
alcohol and 2 N acetic acid (95:5) acetate TS and titrate with the Titrant.
Application volume: 5 IJL Calculate the percentage of arginine hydrochloride
(C6H,4N402' HCI) in the Sample taken:

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 372 Arginine / OfficialMonographs USP43

Result = [(V- B) x Nx Fx 100]/W Result = (Vx N x Fx 100)/W

V = Sample titrant volume (mL) V = Sample titrant volume (mL)
B = Blank titrant volume (mL) N = titrant normality (mEq/mL)
N = titrant normality (mEq/mL) F = equivalency factor, 35.45 mg/mEq
F = equivalency factor, 105.3 mg/mEq W = weight of Sample (mg)
W = weight of Sample (mg)
Acceptance criteria: 16.5%-17.1 %
Acceptance criteria: 98.5%-101 .5% on the dried basis
ADDITIONAL REQUIREMENTS
IMPURITIES • PACKAGING AND STORAGE: Preserve in well-closed
• RESIDUE ON IGNITION (281): NMTO.l% containers.
• CHLORIDE AND SULfATE, Sulfate (221): A 1.6-g portion • USP REFERENCE STANDARDS (11)
shows no more sulfate than corresponds to 0.50 mL of USP Arginine Hydrochloride RS
0.020 N sulfuric acid (0.03%). USP L-Lysine Hydrochloride RS
• CHROMATOGRAPHIC PURITY
System suitability solution: 0.4 mg/mL each of USP
Arginine Hydrochloride RS and USP L-Lysine Hydrochloride
RS in water
Standard solution: 0.05 mg/mL of USP Arginine Add thefoJlowing:
Hydrochloride RS in water. [NoTE-This solution has a
concentration equivalent to about 0.5% of that of the ~ArginineHydrochloride Compoun
Sample solution.] Oral Solution
Sample solution: 10 mg/mL of Arginine Hydrochloride in
water ION
Chromatographic system Argl ,Hydrochloride Compounded. I Solution ~ontains
(See Chromatography (621), Thin-Layer Chromatography.) NLT 90.0% and NMT 110.0 0 t eledamount of
Mode: TLC arginine hydrochloride (C6H 1 ).' ~ .
Adsorbent: 0.25-mm layer of chromatographic silica gel
mixture Prepare Arginine Hydrochloride ompo ion
Application volume: 5 IJL 100 mg/mL asfollows (see Pharmaceutic. ,ing":";'
Developing solvent system: Isopropyl alcohol and Nonsterile Preparations (795»).
ammonium hydroxide (70:30)
Spray reaqente 2 mg/mL of ninhydrin in a mixture of butyl ArginineI-fydrochloride powder 109
alcohol and 2 N acetic acid (95:5) Methylparaben O~oSg
Analysis
Samples: System suitabilitysolution, Standard solution, and Propylparaben 0.O~5g
Sample solution ' Purified Water,a sufficientquantity
Proceedasdirected in the chapter. Dry the plate between to make 100 (TlL
100° and 105° until the ammonia disappears
completely. Spray with Spray reagent, and heat between
100° and 105° for about 15 min. Examine the plate In an appropriately sized container dd the Methylpa 'abeh
under white light. The System suitabilitysolution exhibits and Propylparaben to about 80 ofPurified Wat
two clearly separated spots. until dissolved. [NoTE-May heat up to 50° .
Acceptance criteria: Any secondary spot from the Sample dissolution~] Dissolve the ArginineH n
solution is not larger or more intensethan the principal spot the previously prepared solution of
from the Standard solution. Propylparaben'. Bring to final volume wi
Individual impurities: NMT 0.5% ASSAY
Total impurities: NMT 2.0%
• EDURE
SPECIFIC TESTS 20 mM 'ammonium acetate solution adjusted
• OPTICAL ROTATION, Specific Rotation (781S): +21.4° to w cial acetiC acid 6
+23.6° (t = 20°) Mobile phase: Acetonit, an
Sample solution: 80 mg/mL in 6 N hydrochloric acid through a membrane filter of 0.2
• loss ON DRYING (731): Dry a sampleat 105° for 2 h: it loses Stan sol, n: 0.5 mg/niLof
NMT 0.2% of its weight. SP Argini
• CHLORIDE CONTENT n: ' Transfer'
Sample: 350 mg of Arginine Hydrochloride ~tricflasl<, addappr
Titrimetric system wa f, ' . rtex.Dilute with wat
(See Titrimetry (541).) Chromatographic m. " " . ,.
Mode: Direct titration (See Chromatogfa 621),Sy~telTl Su.itability.)
Titrant: 0.1 N silver nitrate VS M
Endpoint detection: Colorimetric D¢ UV 210 nm
Analysis: Transfer the Sample to a porcelain casserole, and Column:. .6-mm x 15-tm; 5-I.JI11 pacKing L10
add 140 mL of water and 1 mL of dichlorofluorescein TS. Temperatures
Mix and titrate with the Titrant until the silver chloride Autosa~pler:'4°
flocculates and the mixture acquires a faint pink color. Column: 30.°
Calculate the percentage of chloride (CI) in the Sample Flow,rate: 1,mt/min
taken: Injedionvolume:60 ~L

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 USP 43 Official Monographs / Aripiprazole 373

SystelYl suitability add 2 mL of a solution of 0.02% 8-hydroxyquinoline in 3 N

Sa . andardsolution sodium hydroxide, and add 1 mL of 0.1%
[ r~tention timeforargininehydrochloride N-bromosuccinimide solution: an orange color is produced.
is .4 min:] B: It meets the requirements of the tests for Chloride (191).
Su quirements Bacterial Endotoxins Test (85)-lt contains not more than
T or: :NMT 2.0 0.01 USP Endotoxin Unit per mg of arginine hydrochloride.
andarddeviation: .NMT 2.0% for replicate pH (791): between 5.0 and 6.5.
ns Other requirements-It meets the requirements under
Anaysis Injections and Implanted Drug Products (1).
Samples: Standard solutio Assay-
Calculate the percentage Color reagent-Dissolve 28.0 g of potassium hydroxide and
hydrochloride (C6H 14N4 2.0 9 of potassium sodium tartrate in 100 mL of water. Cool,
Solution taken: and add, in the order named, 100 mg of 2,4-dichloro-
1-naphthol, 180 mL of alcohol, and 20.0 mL of 0.475%
Result ;;(rufrs)x(Cs/Cu) x.l og sodium hypochlorite solution. Mix by swirling, and allow to
stand at room temperature for 1 hour before using. This Color
=peak response6f"rginihe hydrochloridefrorn~the reagent may be stored in a glass-stoppered bottle, in a
Sample solution . . refrigerator, for 2 months.
r~ = peak responseof arginine hydrochloride frQm the Standard preparation-Dissolve an accurately weighed
Standard solution quantity of USP Arginine Hydrochloride RS in water, and dilute
=cence . quantitatively and stepwise with water to obtain a solution
in the having a known concentration of about 40 ~g per mL.
= nominal <: e hydrochloride Assay preparation-Pipet into a 1OO-mL volumetric flask a
·il1theSa volume of Injection, equivalent to 200 mg of arginine
hydrochloride, add water to volume, and mix. Pipet 5 mL of
Acceptance.criter.ia: 90.0%::-110~0% this solution into a 250-mL volumetric flask, add water to
volume, and mix.
SPECIFIC TESTS Procedure-Transfer 2.0-mL portions of the Assay
• pH (791~: 5.0-6.0 preparation and the Standardpreparation, respectively, to
AD NAL REQUIRE rS separateflasks, and treat each asfollows. Add 2.0 mL of
potassium iodide solution (3 in 1000), mix, and allow to stand
INC AND.STORA ckage intiglit, light-resistant
s.Store ina r tor or at controlled room for 15 minutes. Add 6.0 mL of Color reagent, mix, and allow
j
reo to stand for 15 minutes. Add 2.0 mL of sodium hypochlorite
SED solution (19 in 10,000), mix, and allow to stand for 15
oun minutes. Concomitantly determine the absorbances of both
solutions in 1-cm cells at the wavelength of maximum
led roo mperature . .. ..
absorbance at about 520 nm, with a suitable
c: Label it to state the Beyond.;Use Date. spectrophotometer, using water as the blank. Calculate the
ERENCE STANDARDS (11)
quantity, in mg, of C6H14N40Z . HCI in each mL of the Injection
inine Hydrochloride RS",,(usp l-May~2020)
taken by the formula:

in which C is the concentration, in ~g per mL, of USP Arginine

Arginine Hydrochloride Injection Hydrochloride RS in the Standard preparation; V is the volume,
in mL, of Injection taken; and A u and A 5 are the absorbances
» Arginine Hydrochloride Injection is a sterile of the solutions from the Assay preparation and the Standard
solution of Arginine Hydrochloride in Water for preparation, respectively.
Injection. It contains not less than 9.5 percent and
not more than 10.5 percent of C6H14N40Z . Hel. It
contains no antimicrobial agents.
rNOTE-The chloride ion content of Arqinine Aripiprazole
Hydrochloride Injection is approximately 475 mEq
per L.]
Packaging and storage-Preserve in single-dose
containers, preferably of Type II glass.
USP Reference standards (11)-
C23H27CI2N30z 448.39
USP Arginine Hydrochloride RS
Labeling-The label statesthe total osmolar concentration in 2(1H)-Quinolinone, 7-[4-[4-(2,3-dichlorophenyl)-1-
piperazinyl]butoxy]-3,4-dihydro-;
mOsmol per L. Where the contents are less than 100 mL, or
where the label states that the Injection is not for direct 7-[4-[4-(2, 3-Dichlorophenyl)- 1-piperazinyl]butoxy]-3,4-
injection but is to be diluted before use, the label alternatively dihydrocarbostyril [129722-12-9].
may state the total osmolar concentration in mOsmol per mL. DEFINITION
Identification- Aripiprazole contains NLT 98.0% and NMT 102.0% of
A:· Transfer 1 mL of the Injection to a 200-mL volumetric aripiprazole (Cz3Hz7C11N302), calculated on the dried basis.
flask, and dilute with water to volume. To 1 mL of this dilution

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374 Aripiprazole / Official Monographs USP 43

IDENTIFICAnON = peak area from the Sample solution

= peak area from the Standard solution
= concentration of USP Aripiprazole RS in the
Standard solution(mg/mL)
• A.:~~ =concentration of Aripiprazole in the Sample
~fiZ~9>i~....... .. solution(mg/mL)
" B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as Acceptance criteria: 98.00/0-102.0% on the dried basis
obtained in the Assay.
IMPURITIES
ASSAY • RESIDUE ON IGNITION (281): NMT 0.1 %
• PROCEDURE • ORGANIC IMPURITIES
Protect the solutions from light. Protect the solutions from light.
Diluent: Acetonitrile, methanol, water, and acetic acid Diluent, Solution A, Solution B, Mobile phase, System
(30:10:60:1 ) suitability solution, Standard solution, Sample solution,
Solution A: Acetonitrile and 0.05% trifluoroacetic acid Chromatographic system, and System suitability:
(10:90) Proceed as directed in the Assay.
Solution B: Acetonitrile and 0.05% trifluoroacetic acid Analysis
(90:10) Sample: Sample solution
Mobile phase: See Table 1. Calculate the percentage of each impurity in the portion of
Aripiprazole taken:
Table 1
Time Solution A Solution B Result = (r ;/r v) x (1 IF) x 100
(min) (%) (%)
0
rj = peak response of each impurity from the Sample
80 20
solution
2 80 20 ru = peak response of Aripiprazole from the Sample
10 65 35
solution
F =relative response factor (see Table 2)
20 10 90
25 10 90
Acceptance criteria: See Table 2.
26 80 20 Table 2
35 80 20 Relative Relative Acceptance
Retention Response Criteria,
Name Time Factor NMT(%)
[NOTE-The gradient was established on an HPLC system
Aripiprazole related com-
with a dwell volume of approximately 650 J.lL.] pound Ga 0.9 0.72 0.10
System suitability solution: 1 J.lg/mL each of USP
Aripiprazole RS and USPAripiprazole Related Compound F Aripiprazole 1.0 - -
RS in Diluent Aripiprazole related com-
Standard solution: 0.1 mg/mL of USP Aripiprazole RS in pound Fb,c 1.1 1.0 0.10
Diluent
Sample solution: 0.1 mg/mL of Aripiprazole in Diluent Aripiprazole 4,4'-dimerd 1.3 1.0 0.10
Chromatographic system . Any other individual im-
(See Chromatography (621), System Suitability.) purity - 1.0 0.10
Mode: LC Total impurities - - 0.50
Detector: UV 254 nm
Column: 4.6-mm x 10-cm; 3-J.lrn packing L1 a 7-(4-[4-(2,3-0ichlorophenyl)piperazin-l-yl]butoxy}quinolin-2(1 H)-one.
Flow rate: 1.2 mL/min b 4-(2,3-Dichlorophenyl)-1-[4-(2-oxo-l,2,3,4-tetrahydroquinolin-7-yloxy)
Injection volume: 20 J.lL butyl]piperazine l-oxide.
System suitability C If possible from the manufacturing process.

Samples: System suitability solution and Standard solution d l,l'-(Ethane-l,l-diyl)bis(2,3-dichloro-4-{4-[3,4-dihydroquinolin-2(1 H)-one-

7-yloxybutyl]piperazin-l-yl}benzene).
[NoTE-The relative retention times for aripiprazole and
aripiprazole related compound Fare 1.0 and 1.1,
respectively. ] SPECIFIC TESTS
Suitability requirements • Loss ON DRYING (731)
Resolution: NLT 2.0 between aripiprazole and Analysis: Dry at 105° for 3 h.
aripiprazole related compound F, System suitability Acceptance criteria: NMT 0.5%
solution ADDITIONAL REQUIREMENTS
Tailing factor: NMT 1.5 for aripiprazole, System • PACKAGING AND STORAGE: Preserve in tight containers.
sUitability solution Store at controlled room temperature.
Relative standard deviation: NMT 1.0%, Standard • USP REFERENCE STANDARDS (11)
solution USP Aripiprazole RS
Analysis USP Aripiprazole Related Compound FRS
Samples: Standard solution and Sample solution 4-(2,3-Dichlorophenyl)-1-[4-(2-oxo-l,2,3,4-
Calculate the percentage of aripiprazole (C23H27CI2N302) in tetrahydroquinolin-7-yloxy)butyl]piperazine l-oxide.
the portion of Aripiprazole taken: C23H27CI2N303 464.38
Result = (r vIr s) x (C sICv) x 100

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 USP 43 OfficialMonographs / Aripiprazole 375

Flow rate: 1 mL/min

Aripiprazole Tablets Injection volume: 10 IJL
DEfiNITION Run time: NLT 2 times the retention time of aripiprazole
Aripiprazole Tablets contain NLT 95.0% and NMT 105.0% of System suitability
the labeled amount of aripiprazole (Cz3Hz7ClzN30z). Sample: Standardsolution
(NoTE-The relative retention times for aripiprazole and
IDENTIFICATION propylparaben are about 1.0 and 1.5, respectively.]
Suitability requirements
Resolution: NLT 8 between aripiprazole and
propylparaben
• A.;~ Tailing factor: NMT 1.7 for aripiprazole and for
$iig¢ < ...< ilY",.) propylparaben
Standard: Add 30 mL of ethyl acetate to 30 mg of USP Relative standard deviation: NMT 2.0% for the peak
Aripiprazole RS. Shake for 10 min, centrifuge for NLT 5 min, response ratio of aripiprazole to propylparaben
and passthe supernatant through a suitable membrane Analysis
filter. To the filtrate add 15 mL of water, shakefor 5 min, Samples: Standardsolution and Sample solution
and centrifuge for NLT 10 min. Transfer 20 mL of the upper Calculate the percentage of the labeled amount of
layer to a container and add anhydrous magnesium aripiprazole (Cz3Hz7ClzN30Z) in the portion of Tablets
sulfate, as needed. Shake well, passthrough a suitable taken:
membrane filter, and evaporate the ethyl acetate on a water
bath under reduced pressure. Use the residue. (NOTE-A Result = (R viR s) x (C siC v) x 100
centrifuge speed of 2000 rpm may be suitable.]
Sample: Grind a suitable number of Tablets and transfer a = peak response ratio of aripiprazole to
suitable portion of the ground Tablets, equivalent to 30 mg propylparaben from the Sample solution
of aripiprazole, to an appropriate container. Add 30 mL of = peak response ratio of aripiprazole to
ethyl acetate, shakefor 10 min, centrifuge for NLT 5 min, propylparaben from the Standardsolution
and passthe supernatant through a suitable membrane = concentration of USP Aripiprazole RS in the
filter. To the filtrate add 15 mL of water, shakefor 5 min, Standardsolution (mg/mL)
and centrifuge for NLT 10 min. Transfer 20 mL of the upper = nominal concentration of aripiprazole in the
layer to a container and add a suitable amount of anhydrous Sample solution (mg/mL)
magnesium sulfate. Shake well, pass through a suitable
membrane filter, and evaporate the ethyl acetate on a water Acceptance criteria: 95.00/0-105.0%
bath under reduced pressure. Use the residue. (NOTE-A PERFORMANCE TESTS
centrifuge speed of 2000 rpm may be suitable.]
• DISSOLUTION (711)
Analysis TEST 1
Samples: Standard and Sample
Medium: pH 1.2 hydrochloric acid buffer (Transfer 250 mL
Acceptance criteria: Meet the requirements . of 14.9 giL of potassium chloride in water to a 1-L
• B. The retention time of the aripiprazole peak of the volumetric flask, add 425 mL of 0.2 N hydrochloric acid,
Sample solution corresponds to that of the Standard and dilute with water to volume. Degas the resulting
solution, as obtained in the Assay. solution or passthe resulting solution through a filter
ASSAY under vacuum.), degassed; 900 mL
• PROCEDURE Apparatus 2: 60 rpm
Solution A: 2.8 giL of anhydrous sodium sulfate in water Time: 30 min
Mobile phase: Acetonitrile, methanol, Solution A, and Procedure: Determine the percentage of the labeled
glacial acetic acid (33:11 :56:1) amount of aripiprazole (CnHz7ClzN30Z) dissolved by using
Internal standard solution: 0.33 mg/mL of USP either the Spectrometric procedure or the Chromatographic
Propylparaben RS in Mobile phase procedure described below.
Standard stock solution: 1 mg/mL of USP Aripiprazole RS Spectrometric procedure
in Mobilephase Standard stock solution: 1 mg/mL of USP Aripiprazole
Standard solution: 0.2 mg/mL of USP Aripiprazole RS RS in alcohol
prepared asfollows. Transfer 10.0 mL of Standard stock Standard solution: (L/900) mg/mL of USP Aripiprazole
solution and 10.0 mL of Internal standard solution to a RS from Standardstock solution in Medium, where Lis
50-mL volumetric flask, and dilute with Mobilephase to the label claim, in mg/Tablet
volume. Sample solution: Pass a portion of the solution under
Sample solution: Nominally 0.2 mg/mL of aripiprazole test through a suitable filter, discarding the first 5 mL
from Tablets prepared as follows. Powder NLT 20 Tablets of filtrate.
and transfer a suitable portion of the powder to an Instrumental conditions
appropriate volumetric flask. Add 40% of the final flask Mode: UV
volume of Mobile phase and 20% of the final flask volume Analytical wavelengths: 249 and 325 nm
of Internal standardsolution. Shakefor 10 min, and dilute Cell length: 1 cm
with Mobilephase to volume. Centrifuge, if necessary, and Blank: Medium
pass the supernatant through a suitable filter of NMT Analysis
0.5-lJm pore size, discard the first 1 mL of filtrate, and use Samples: Standardsolution and Sample solution
the subsequent filtrate. Calculate the percentage of the labeled amount of
Chromatographic system aripiprazole (Cz3Hz7C1zN30z) dissolved:
(See Chromatography (621), System Suitability.)
Mode: LC Result = (A viA s) x C s x V x (1/ L) x 100
Detector: UV 254 nm
Column: 4.6-mm x 25-cm; 5-lJm packing L1

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376 Aripiprazole / Official Monographs USP 43

Au = absorbance at 249 nm minus the absorbance at Buffer: 6.8 giL of monobasic potassium phosphate in
325 nm of the Sample solution water. Adjust with 1 N phosphoric acid TS to a pH of 3.0.
= absorbance at 249 nm minus the absorbance at Mobile phase: Acetonitrile and Buffer(40:60)
325 nm of the Standard solution Standard stock solution: 0.11 mg/mL of USP Aripiprazole
= concentration of USP Aripiprazole RS in the RS in solution prepared as follows. Transfer a suitable
Standardsolution (mg/mL) amount of USP Aripiprazole RS to an appropriate
V = volume of Medium, 900 mL volumetric flask. Add 2% of the flask volume of acetonitrile
L = label claim (mg/Tablet) and 70% of the flask volume of Medium. Sonication may
be used to promote dissolution. Dilute with Medium to
Chromatographic procedure volume.
Solution A: 2.8 giL of anhydrous sodium sulfate Standard solution: (L/900) mg/mL of USP Aripiprazole RS
Solution B: 13.9 giL of glacial acetic acid and 23.9 giL from Standard stock solution in Medium, where L is the
of sodium acetate in water label claim, in mg/Tablet
Mobile phase: Acetonitrile, methanol, Solution A, and Sample solution: Pass a portion of the solution under test
glacial acetic acid (40: 10:50: 1) through a suitable filter, dlscardlnq NLT the first 5 mL of
Diluent: Solution B and methanol (50:50) filtrate.
Internal standard solution: 0.67 IJg/mL of USP Chromatographic system
Propylparaben RS in Diluent (See Chromatography (621), System Suitability.)
Standard stock solution A: 1 mg/mL of USP Mode: LC
Aripiprazole RS in Mobilephase Detector: UV 215 nm
Standard stock solution B: 0.002 mg/mL of USP Column: 4.6-mm x 15-cmi 5-lJm packing L7
Aripiprazole RS from Standard stock solutionA in Column temperature: 40°
Medium passedthrough a suitable filter of NMT 0.5-fJm Flow rate: 1 mL/min
pore size, discarding the first 6 mL of filtrate Injection volume: 5 IJL
Standard solution: 0.001 mg/mL of USP Aripiprazole Run time: NLT 1.6 times the retention time of
RS from Standardstock solution B prepared by aripiprazole
combining 5 mL of Standard stock solutionBand 5 mL System suitability
of Internal standard solution Sample: Standard solution
Sample stock solution: Pass a portion of the solution Suitability requirements
under test through a suitable filter of NMT 0.5-lJm pore Tailing factor: NMT 1.5
size, discarding NLT the first 6 mL of filtrate. Relative standard deviation: NMT 1.0%
Sample solution: Combine 2 mL of Sample stock Analysis
solution with 2 mL of Internal standardsolution. Samples: Standard solution and Sample solution
Chromatographic system: Proceed as directed in the Calculate the percentage of the labeled amount of
Assay except as follows. aripiprazole (C23H27CI2N302) dissolved:
Injection volume: 100 IJL
System suitability Result = (r vir s) x C s x V x (1 I L) x 100
Sample: Standardsolution
[NoTE-The relative retention times for aripiprazole ru = peak response of aripiprazole from the Sample
and propylparaben are about 1.0 and 1.8, solution
respectively.] rs = peak response of aripiprazole from the Standard
Suitability requirements solution
Resolution: NLT 10 between aripiprazole and Cs = concentration of USP Aripiprazole RS in the
propylparaben . Standard solution (mg/mL)
Relative standard deviation: NMT 1.5% for the V =volume of Medium, 900 mL
peak response ratio of aripiprazole to propylparaben L = label claim (mg/Tablet)
Analysis
Samples: Standardsolution and Sample solution Tolerances: NLT 80% (Q) of the labeled amount of
Calculate the percentage of the labeled amount of aripiprazole (C23H27CI2N302) is dissolved.
aripiprazole (C23H27CI2N302) dissolved: • UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
Result = (R viR s) xC s x Vx (lIL) x 100
IMPURITIES
Ru = peak response ratio of aripiprazole to • ORGANIC IMPURITIES
propylparaben from the Sample solution Protect solutions from light.
Rs =peak response ratio of aripiprazole to Buffer: 9.6 giL of dibasic ammonium citrate, 1.6 giL of citric
propylparaben from the Standard solution acid, and 2.9 giL of sodium dodecyl sulfate in water. Adjust
Cs = concentration of USP Aripiprazole
RS in the with 11 giL of dibasic ammonium citrate in water or 9.6 gl
Standardsolution (mg/mL) L of anhydrous citric acid in water to a pH of 4.7, if needed.
V = volume of Medium, 900 mL Mobile phase: Acetonitrile and Buffer (45:55)
L = label claim (mg/Tablet) Diluent: Acetonitrile, water, and glacial acetic acid
(40:60:1)
Tolerances: NLT 75% (Q) of the labeled amount of System suitability solution: 0.5 mg/mL of USPAripiprazole
aripiprazole (C23H27CI2N302) is dissolved. RS, and 0.0005 mg/mL each of USP Aripiprazole Related
TEST 2: If the product complies with this test, the labeling
Compound F RS and USP Aripiprazole Related Compound
indicates that it meets USP Dissolution Test 2. G RS in Diluent
Medium: 0.1 N hydrochloric acid VSi 900 mL Sample solution: Nominally 0.5 mg/mL of arlplprazole
Apparatus 2: 60 rpm from Tablets prepared as follows. Powder NLT 20 Tablets,
Time: 15 min transfer a suitable portion of the powder equivalent to NLT
4 mg of aripiprazole to an appropriate container, and add

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 USP 43 Official Monographs / Aripiprazole 377

a suitable volume of Diluent. Shake for 10 min and

centrifuge, if necessary. Pass the supernatant through a
Aripiprazole Orally Disintegrating
suitable filter of NMT 0.5-~m pore size, discard the first 1 Tablets
mL of filtrate, and use the subsequent filtrate.
Chromatographic system DEFINITION
(See Chromatography (621), System Suitability.) Aripiprazole Orally Disintegrating Tablets contain NLT 90.0%
Mode: LC and NMT 110.0% of the labeled amount of aripiprazole
Detector: UV 254 nm (C23H27C12N302)'
Column: 4.6-mm x 15-cm; 5-~m packing L1
Flow rate: 1 mL/min IDENTIFICATION
Injection volume: 20 ~L
Run time: NLT 2 times the retention time of aripiprazole
System suitability
Sample: System suitability solution • A.
[NoTE-See Table 7 for the relative retention times.] S
Suitability requirements • B.Theretention time of the 'major peak of the Sample
Resolution: NLT 3 between aripiprazole related solution corresponds to that of the Standard solution, as
compound G and aripiprazole obtained in the Assay.
Signal-to-noise ratio: NLT 10 for aripiprazole related ASSAY
compound F and aripiprazole related compound G • PROCEDURE
Analysis Solution A: 2.84 giL of sodium sulfate in water
Sample: Sample solution Buffer: 3.48 giL of dibasic potassium phosphate adjusted
Calculate the percentage of each degradation product in with phosphoric acid to a pH of 8.2
the portion of Tablets taken: Mobile phase: Acetonitrile and Buffer (50:50)
Diluent A: Acetonitrile, methanol, Solution A, and glacial
Result =(r vir r) x 100 acetic acid (33:11 :56:1)
Diluent B: Acetonitrile and 0.1 M hydrochloric acid
rv = peak response of each degradation product (20:80)
from the Sample solution System suitability solution: 0.01 mg/mL each of USP
rt =sum of all the peak responses from the Sample Aripiprazole RS and USPAripiprazole Related Compound G
solution RS in DiluentA. Sonication and shaking may be used to aid
in dissolution.
Acceptance criteria: See Table 7. Disregard peaks that are
Standard solution: 0.25 mg/mL of USP Aripiprazole RS in
less than 0.1 % of the aripiprazole peak. Diluent B. Sonication may be used to aid in dissolution.
Sample solution: Nominally 0.2-0.3 mg/mL of aripiprazole
Table 1
from NLT 5 Orally Disintegrating Tablets prepared as
Relative Acceptance follows. Transfer NLT 5 Orally Disintegrating Tablets to a
Retention Criteria, suitable volumetric flask and dilute with Diluent B to NMT
Name Time NMT (%)
75% of the final flask volume. Sonicate for 5 min and shake
Aripiprazole related compound F 0.54 0.3 for 15 min. Dilute with Diluent B to volume. Pass the
Aripiprazole related compound G 0.81 0.3 resulting solution through a suitable filter and use the
filtrate.
Aripiprazole 1.0 - Chromatographic system '
Any individual unspecified (See Chromatography (621), System Suitability.)
degradation product - 0.2 Mode: LC
Detector: UV 252 nm
Totaldegradation products - 1.0
Column: 4.6-mm x 1O-cm; 3.5-~m packing L1
Flow rate: 1 mL/min
ADDITIONAL REQUIREMENTS Injection volume: 10 ~L
• PACKAGING AND STORAGE: Preserve in tight containers. Run time: NLT 1.4 times the retention time of aripiprazole
Store at controlled room temperature. System suitability
• LABELING: The labeling states the Dissolution test used only Samples: System suitability solution and Standard solution
if Test 7 is not used. [NoTE-The relative retention times of aripiprazole
• USP REFERENCE STANDARDS (11) related compound G and aripiprazole are 0.74 and
USP Aripiprazole RS 1.0, respectively.]
USP Aripiprazole Related Compound F RS Suitability requirements
4-(2,3-Dichlorophenyl)-1-[4-(2-oxo-l,2,3,4- Resolution: NLT 3.0 between aripiprazole related
tetrahyd roq uinolin-7 -yloxy)butyl] pi perazine l-oxide. compound G and aripiprazole, System suitability solution
C23H27CI2N303 464.38 Tailing factor: NMT 1.5, Standard solution
USP Aripiprazole Related Compound G RS Relative standard deviation: NMT 1.0%, Standard
7-{4-[4-(2,3-Dichlorophenyl)piperazin-l-yl]butoxy} solution
quinolln-Ztl H)-one. Analysis
C23H2sCI2N302 446.37 Samples: Standard solution and Sample solution
USP Propylparaben RS Calculate the percentage of the labeled amount of
aripiprazole (C23H27C12N302) in the portion of Orally
Disintegrating Tablets taken:

Result = (rvlrs) x (CslCv) x 100

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378 Aripiprazole / OfficialMonographs USP 43

= peak response from the Sample solution Table 1 (continued)

= peak response from the Standardsolution Time Solution A Solution B
= concentration of USP Aripiprazole RS in the (min) (%) (%)
Standardsolution (mg/mL) 20 70 30
= nominal concentration of aripiprazole in the
Sample solution (mg/mL) 40 42 58

50 10 90
Acceptance criteria: 90.0%-110.0%
55 10 90
PERFORMANCE TESTS
56 90 10
• DISINTEGRATION (701): NMT 60 s
• DISSOLUTION (711) 60 90 10
Medium: pH 4.0 sodium acetate trihydrate buffer (3:0 giL
of sodium acetate prepared asfollows. Transfer a suitable [NOTE-The gradient was established on an HPLC system
quantity of sodium acetate to a suitable container with a dwell volume of approximately 1.0 mL.] .
containing 90% of the final container volume of water. Diluent: Acetonitrile, methanol, Solution C, and glacial
Adjust with glacial acetic acid to a pH of 4.0. Add water to acetic acid (33:11 :56:1)
the final volume.), degassed; 1000 mL System suitability solution: 250 I.Jg/mL of USP
Apparatus 2: 75 rpm Aripiprazole RS and 0.5 I.Jg/mL each of USP Aripiprazole
Related Comp~und F RS and USP Aripiprazole Related .
Time: 30 min
Mobile phase: Acetonitrile and 0.025 M hydrochloric acid Compound G RS in Diluent. Sonication may be used to aid
(40:60) .. in dissolution.
Standard solution: (L/1000) mg/mL of USP Aripiprazole RS Sample solution: Nominally 0.2-0.3 mg/mL of aripiprazole
in Mobile phasewhereL is the label claim in mg/Tablet from NLT 5 Orally Disintegrating Tablets prepared as
Sample solution: Pass a portion of the solution under test follows. Transfer NLT 5 Orally Disintegrating Tablets to a
through a suitable filter, discarding the first few m~. suitable volumetric flask. Add about 70% of the total
Chromatographic system volume of Diluent. Sonicatefor 10 min and shake for 10
(See Chromatography (621), System Suitability.) min. Dilute with Diluent to volume. Pass the resulting
Mode: LC solution through a suitable filter and use the filtrate.
Detector: UV 225 nm Chromatographic system
Column: 4.6-mm x 15-cm; 5-l.Jm packing L1 (See Chromatography (621), System Suitability.)
Flow rate: 1 mL/min Mode: LC
Injection volume: 20 I.JL .. . . Detector: UV 254 nm
Run time: NL7f" 1.5 times the retention time of ariplprazole -Column: 4.6-mm x 15-cm; 3-l.Jm packing L1
System suitability Flow rate: 1 mL/min
Sample: Standardsolution Injection volume: 20 I.JL
Suitability requirements System suitability
Relative standard deviation: NMT 2.0%- Sample: System suitability solution
Analysis [NOTE-See Table 2 for the relative retention times.]
Samples: Standardsolution and Sample solution SUitability requirements
Calculate the percentage of the labeled amount of Resolution: NLT 4.0 between aripiprazole related
aripiprazole (C23H27CI2N302) dissolved: . compound G and aripiprazole; NLT 1.5 between
aripiprazole and aripiprazole related compound F
Result = (rvlrs) x Cs x VX (lIL) x 100 Analysis
Sample: Sample solution
= peak response from the Sample solution Calculate the total peak response for in~ividual impurities
= peak response from the Standardsolution and aripiprazole from the Sample solution:
=concentration of USP Aripiprazole RS in the
Standardsolution (mg/mL) Result =.E[r; x (l/A] + rv
V = volume of Medium, 1000 mL
L = label claim (mg/Tablet) = peak response of each degradation product
from the Sample solution
Tolerances: NLT 80% (Q) of the labeled amount of F = relative response factor (see Table 2)
aripiprazole (C23H27CI2N302) is dissolved. = peak response of aripiprazole from the Sample
• UNIFORMITY OF DOSAGE UNITS (905): Meet the solution
requirements
Calculate the percentage of each degradation product in
IMPURITIES the portion of Orally Disintegrating Tablets taken:
• ORGANIC IMPURITIES
Solution A: Water and trifluoroacetic acid (100: 0.05) Result = (r;/rr) x (1I A x 100
Solution B: Acetonitrile and trifluoroacetic acid (100: 0.05)
Solution C: 2.84 giL of sodium sulfate in water r; = peak response of each degradation product
Mobile phase: See Table 7. from the Sample solution
Table 1
rr =total peak response for individual i.mpurities and
aripiprazole from the Sample solution
Time Solution A Solution B F = relative responsefactor (see Table 2)
(min) (%) (%)
0 90 10 Acceptance criteria: See Table 2. Disregard peaks less than
0.05%.

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 USP 43 Official Monographs / Arsanilic 379

Table 2 65 mg of warm (about 70°to 80°)water, and shakeor sonicate

Relative Relative Acceptance to dissolve. Allow to cool, dilute with water to volume, and
Retention Response Criteria, mix. Dilute a portion of this solution quantitatively and
Name Time Factor NMT (0/0) stepwise with water to obtain a solution having a known
Aripiprazole related concentration of about 0.0012 mg of o-arsanilic acid per mL.
compound G 0.96 0.77 0.3 Test solution-Transfer about 50 mg of Arsanilic Acid,
accuratelyweighed, to a 50-mLvolumetric flask, add about 30
Aripiprazole 1.0 - -
mL of warm water, and shake or sonicate to dissolve. Allow to
Aripiprazole related cool, dilute with water to volume, and mix.
compound F 1.03 1.0 0.3 Chromatographic system (see Chromatography (621»-
Any individual The liquid chromatograph is equipped with a 242-nm
unspecified detector and a 4.6-mm x 15-cm column that contains 5-~m
degradation product - 1.0 0.2 base-deactivated packing L1 and is maintained at a constant
Totaldegradation temperature of about 30°. The flow rate is about 1.5 mL per
products - - 1.0 minute. Chromatograph the Standard solution, and record the
peak responses as directed for Procedure:the capacityfactor,
ADDITIONAL REQUIREMENTS k', for the o-arsanilic acid peak is between 2.8 and 3.8; and the
• PACKAGING AND STORAGE: Preserve in tight containers. relative standard deviation for replicate injections is not more
Store at controlled room temperature. than 2.5%.
• USP REFERENCE STANDARDS (11) Procedure-Separately inject equal volumes (about 20 IJL)
USP Aripiprazole RS of the Standard solution and the Test solution into the
USP Aripiprazole Related Compound F RS chromatograph, record the chromatograms, and measure the
4-(2,3-Dichlorophenyl)-1-[4-(2-oxo-l,2,3,4- areas of the responses for the o-arsanilic acid peaks. Calculate
tetrahydroquinolin-7-yloxy)butyl]piperazine l-oxide. the percentage of o-arsanilic acid in the portion of Arsanilic
C23H27C12N303 464.38 Acid taken by the formula:
USP Aripiprazole Related Compound G RS 5000( CI W)(r vi rs)
7-{4-[4-(2,3-Dichlorophenyl)piperazin-1-yl]butoxy}
quinolin-2(1 H)-one. in which C is the concentration, in mg per mL, of o-arsanilic
C23H2sCI2N302 446.37 acid in the Standard solution; W is the weight, in mg, of
Arsanilic Acid taken to prepare the Test solution; and rv and rs
are the responses of the o-arsanilic acid peaks obtained from
the Test solution and the Standard solution, respectively: not
more than 0.12% is found.
Arsanilic Acid
Limit of aniline-
Mobile phase-Dissolve 7.76 g of monobasic potassium
H\ ; ,o phosphate in 950 mLof water, add 50 mL of methanol, mix,
s-: \
H.N
O::0... I OH and degas. Makeadjustments if necessary (see System
Suitability under Chromatography (621».
Standard solution-Transfer about 176 mg of aniline,
C6H sAsN0 3 217.05 accuratelyweighed, to a 25-mLvolumetric flask, add about 1
p-Aminobenzenearsonic acid [98-50-0]. mL of methanol, swirl, then add about 15 mLof water, and
shake to dissolve. Dilutewith water to volume,and mix. Dilute
» Arsanilic Acid contains not lessthan 98.0 percent a portion of this solution quantitatively and stepwise with
and not more than 102.0 percent of C6H gAsN0 31 water to obtain a solution having a known concentration of
about 0.00045 mg of aniline per mL.
calculated on the dried basis. Test solution-Transfer about 50 mg of Arsanilic Acid,
accuratelyweighed, to a 50-mLvolumetricflask, add about 30
Packaging and storage-Preserve in well-closed mL of warm (about 70° to 80°) water, and shake or sonicate
containers. to dissolve. Allow to cool, dilute with water to volume; and
Labeling-Label it to indicate that it isfor veterinaryuse only. mix.
USP Reference standards (11)- Chromatographic system (see Chromatography (621»-
USP Arsanilic Acid RS The liquid chromatograph is equipped with a 235-nm
detector and a 4.6-mm x 15-cm column that contains 5-~m
base-deactivated packing L1 and is maintained at a constant
temperature of about 30°. The flow rate isabout 1.5 mL per
l~entifi~i1~i.2~t. /cS~[~,·~i~~~~i~~ie. minute. Chromatograph the Standard solution, and record the
(s19t )i'/n(rC!f;gQsSpgg.trt!!$f91)Y: l i? ··.c.. peak responses as directed for Procedure:the capacityfactor,
Loss on drying (731 )-Dry it in vacuum k', for the aniline peak is between 2.3 and 3.3; and the relative
loses not more than 0.5% of its weight. standard deviation for replicate injections is not more than
Limit of o-arsanilic acid- 3.0%.
Mobile phase-Dissolve 4.04 g of monobasic potassium Procedure-Separately inject equal volumes (about 50 ~L)
phosphate in 985 mL of water, add 2 mLof phosphoric acid, of the Standard solution and the Test solution into the
and mix. Add 10 mLof methanol, mix, and degas. Make chromatograph, record the chromatograms, and measure the
adjustments if necessary (see System Suitability under areas of the responses for the aniline peaks. Calculate the
Chromatography (621 ». percentage of aniline in the portion of Arsanllic Acid taken by
the formula:
Standard solution-Transfer about 67 mg of o-arsanilic acid,
accurately weighed, to a 1OO-mL volumetricflask, add about
5000( CI W)(r vi rs)

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 380 Arsanilic / OfficialMonographs USP 43

in which C is the concentration, in mg per mL, of aniline in the ASSAY

Standardsolution; W is the weight, in mg, of Arsanilic Acid • PROCEDURE
taken to prepare the Test solution; and ru and rs are the Sample solution: 250 mg of Articaine Hydrochloride to a
responses of the aniline peaks obtained from the Test solution 250-mL conical flask. Add 5.0 mL of 0.01 M hydrochloric
and the Standardsolution, respectively: not more than 0.045% acid and 50 mL of alcohol. Stir to dissolve.
is found. Analysis: Titrate with 0.1 M sodium hydroxide VS,
Assay-Transfer about 125 mg of Arsanilic Acid, accurately determining the endpoint potentiometrically, using a glass
weighed, to a 50-mL conical flask, and add 10.0 mL of a electrode. Calculate the volume of sodium hydroxide
mixture of sulfuric acid, nitric acid, and perchloric acid consumed by reading the volume added between the two
(1000:50:50) and several glassbeads. Digest on a hot plate for points of inflection. Each mL of 0.1 M sodium hydroxide is
about 1 hour, increasing the temperature of the hot plate in equivalent to 32.08 mg of C13HzoNz03S . HCI.
steps until a ring of sulfuric acid rises into the neck of the flask. Acceptance criteria: 98.5%-101.0% on the dried basis.
Allow to cool, to the colorless solution add about 400 mg of
hydrazine sulfate, and ,heat the flask vigorously on a hot plate IMPURITIES
until a ring of sulfuric acid rises into the neck of the flask. Allow INORGANIC IMPURITIES
to cool, and wash down the rim, neck, and insides of the flask • RESIDUE ON IGNITION (281)
with about 1 mL of water. Heat the flask again until a ring of Sample: 1 g
sulfuric acid rises into the neck of the flask. Allow to cool, and Acceptance criteria: NMT 0.1 %
transfer the colorless solution, with the aid of about 80 mL of ORGANIC IMPURITIES
water, to a 125-mL conical flask. Add 10 mL of hydrochloric • Procedure
acid and several drops of 0.002 M potassium iodide, cool to Buffer solution: 2.02 9 of sodium l-heptanesultonate and
between 0° and 5°, and titrate with 0.1 N potassium 4.08 9 of potassium dihydrogen phosphate in 1 L of water.
permanganate VS to a pale pink endpoint, maintaining the Adjust with phosphoric acid to a pH of 2.0.
temperature between 0° and 5° during the titration. Perform Mobile phase: Acetonitrile and Buffer solution (1 :3)
a blank determination, and make any necessary correction. Standard solution: 2 uq/rn], of USP Articaine Related
Each mL of 0.1 N potassium permanganate is equivalent to Compound A , and 1 ~gfmL each of USp Articaine Related
10.852 mg of C6H aAsN0 3. Compound E RS and USP Articaine Hydrochloride RS in
. Mobilephase. [NOTE-This solution is also used to determine
the reporting threshold limit.]
Sample solution: 1.0 mgfmL of Articaine Hydrochloride in
Mobilephase
Articaine Hydrochloride Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 276 nm
Column: 4.6-mm x 25-cm; packing L1
Temperature: 45°
Flow rate: 1 mLfmin
C13HzoNz03S . HCI 320.84 Injection size: 10 ~L
2-Thiophenecarboxylic acid, 4-methyl-3-[[1-oxo-2- System suitability
(propylamino)propyl]amino]-, methyl ester, Sample: Standard solution
. monohydrochloride; . Suitability requirements
Methyl 4-methyl-3-[2 -(propylam ino)propionamido]-2- Resolution: NLT 1.2 between articaine related compound
thiophenecarboxylate, monohydrochloride [23964-57-0]. A and articaine related compound E
Analysis
DEFINITION Samples: Standard solutionand Sample solution. [NOTE-Run
Articaine Hydrochloride contains NLT 98.5% and NMT time is 5 times the retention time of articaine.]
101.0% of C13HzoNz03S . HCI, calculated on the dried basis. Calculate the percentage of any articaine related compound
IDENTIFICAnON A in the portion of Articaine Hydrochloride taken:

Result = (rufr s) x (CsfCu) x 100

ru = response of articaine related compound A from

• A.<~~~ the Sample solution
$f{(Jc:f:CQ., ·i~ rs = response of articaine related compound A from
Standard solution: 12 mgfmL of USP Articaine RS in the Standardsolution
methylene chloride. Transfer 20 ~L of this solution onto a Cs = concentration of USPArticaine Related
300-mg disk. Compound A RS in the Standard solution
Sample solution: Dissolve 100 mg of Articaine (mgfmL)
Hydrochloride in 5 mL of water. Add 3 mL of a saturated Cu = concentration of Articaine Hydrochloride in the
solution of sodium bicarbonate, and shaketwice with 2 mL Sample solution (mgfmL)
of methylene chloride. Combine the methylene chloride
layers, dilute with methylene chloride to 5.0 mL, and dry Calculate the percentage of each individual impurity in the
over anhydrous sodium sulphate. Transfer 20 ~L of this portion of Articaine Hydrochloride taken:
solution onto a 300-mg disk.
• B. IDENTIFICATION TESTS-GENERAL, Chloride (191) Result = (rufr s) x (CsfCu) x 100

ru = response of each individual impurity from the

Sample solution

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 USP 43 Official Monographs / Articaine 381

= response of articaine hydrochloride from the USP Articaine Related Compound E RS

Standard solution Methyl 3-[2-(isopropylamino) propanamido]-4-
= concentration of USP Articaine Hydrochloride RS methylthiophene-2-carboxylate.
in the Standard solution(mg/mL) C13HzoNz03S 284.37
Cu = concentration of Articaine Hydrochloride in the
Sample solution (mg/mL)

[NoTE-Disregard any peak below 0.05%.]

Acceptance criteria Articaine Hydrochloride and
Individual impurities: See Impurity Table 1. Epinephrine Injection
Total impurities: NMT 0.5%. [NOTE-Excluding articaine
related compound A.] DEFINITION
Articaine Hydrochloride and Epinephrine Injection is a sterile
Impurity Table 1 solution of Articaine Hydrochloride and Epinephrine, in
Relative Acceptance Water for Injection, and contains NLT 95.0% and NMT
Retention Criteria, 105.0% of the labeled amount of articaine hydrochloride
Name Time NMT(%)
(C13HzoNz03S . HCI) and NLT 90.0% and NMT 115.0% of the
Articaine acid" 0.6 0.1 labeled amount of epinephrine (C9H 13N03).
Ethylartlcaine" 0.7 0.1 IDENTIFICATION
Articaine related compound A' 0.8 0.2 • A. The retention times of the articaine and epinephrine
peaks from the Sample solution correspond to those of the
Articaine related compound Ed 0.86 0.1 Standard solution, as obtained in the Assays for Articaine
Articaine acld-proplonarnldes 0.9 0.1 Hydrochloride and Epinephrine, respectively.
Articaine 1.0 - ASSAY
• ARTICAINE HYDROCHLORIDE
Butylartkalne' 1.7 0.1
Buffer: Glacial acetic acid and water (50:930). Adjust with 2
Dlpropylartlcalnes 2.1 0.1 N sodium hydroxide to a pH of 3.4.
Mobile phase: Acetonitrile and Buffer (22:78)
3-Aminoarticaineh 2.6 0.1
Standard stock solution: 40 mg/mL of USP Articaine
Articaine isopropyl ester' 3.6 0.1 Hydrochloride RS in water
Standard solution: 0.8 mg/mL of USP Articaine
Bromo compound' 4.0 0.1
Hydrochloride RS in Mobilephasefrom Standard stock
Any other individual impurity - 0.10 solution
Sample solution: Equivalent to 0.8 mg/mL of articaine
a4-Methyl-3-[[(2RS)-2-(propylamino)propanoyl]amino]thiophene-2-carboxylic hydrochloride in Mobilephase from a portion of Injection
acid. Chromatographic system
b Methyl 3-[[(2RS)-2-( ethylamino)propanoyl]amino]-4-methylthiophene-2-
carboxylate. (See Chromatography (621), System Suitability.)
c Methyl 4-methyl-3-[2-(propylamino)acetamido]thiophene-2-carboxylate. Mode: LC
d Methyl3-[2-(isopropylamino)propanamido]-4-methylthiophene-2- Detector: UV 270 nm
carboxylate. Column: 4.6-mm x 25-cmi 5-~m packing L1
e 4-Methyl-N-propyl-3-[((2RS)-2-(propylamino)propanoyl]amino]thiophene-2- Flow rate: 1 mL/min
carboxamide. Injection size: 10 ~L
f Methyl 3~[[(2RS)-2-(butylamino )propanoyl]amino]-4-methylthiophene-2- Run time: 2.5 times the retention time of articaine
carboxylate.
9 Methyl 3-[[(2RS)-2-(dipropylamino)propanoyl]amino]-4-methylthiophene-2-
System suitability
carboxylate. Sample: Standard solution
h Methyl 3-amino-4-methylthiophene-2-carboxylate. Suitability requirements
i 1-MethylethyI4-methyl-3-[[(2RS)-2-(propylamino)propanoyl]amino] Tailing factor: NMT 2.2
thiophene-2-carboxylate. Relative standard deviation: NMT 1.0%, from six
j Methyl 3-[[(2RS)-2-bromopropanoyl]amino]-4-methylthiophene-2- injections
carboxylate. Analysis
Samples: Standard solution and Sample solution
SPECIFIC TESTS Calculate the percentage of the labeled amount of articaine
• Loss ON DRYING (731): Dry at 105° for 5 h: it loses NMT hydrochloride (C13HzoNz03S . HCI) in the portion of
0.5% of its weight. Injection taken:
• pH (791)
Sample solution: 10 mg/mL Result = (r ulr s) x (C sIC u) x 100
Acceptance criteria: 4.2-5.2
ADDITIONAL REQUIREMENTS ru =peak response from the Sample solution
• PACKAGING AND STORAGE: Preserve in light-resistant rs = peak response from the Standard solution
containers. Cs =concentration of USP Articaine Hydrochloride RS
• USP REFERENCE STANDARDS (11) in the Standard solution (mg/mL)
USP Articaine RS Cu = nominal concentration of articaine hydrochloride
USP Articaine Hydrochloride RS in the Sample solution(mg/mL)
USP Articaine Related Compound A RS
MethyI4-methyl-3-[2-(propylamino) acetamido] Acceptance criteria: 95.0%-105.0%
thiophene-2-carboxylate.
C12H1SNz03S 270.35

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382 Articaine / OfficialMonographs USP 43

• EPINEPHRINE Standard solution: 0.8 mg/mL of USP Articaine

Mobile phase: Mix 50 mL of glacial acetic acid and 930 mL Hydrochloride RS from Standard stock solution and 40
of water. Adjust with 2 N sodium hydroxide to a pH of 3.4. IJg/mL of USP Articaine Related Compound B RS in Mobile
In this solution, dissolve 1.2 9 of sodium phase
1-heptanesulfonate, and add 1.0 mL of 0.1 M edetate System suitability
disodium and 0.298 9 of potassium chloride. Add 150 mL Sample: Standard solution
of methanol. [NOTE-See Table 1 for relative retention times.]
Diluent: 0.5 mg/mL potassium metabisulfite in water Suitability requirements
System suitability solution: 22 IJg/mL of epinephrine from Tailing factor: NMT 2.2 for the articaine peak
USP Epinephrine Bitartrate RS and 20 IJg/mL of Resolution: NLT 1.25 between the articaine related
norepinephrine from USP Norepinephrine Bitartrate RS in compound Band articaine peaks
Diluent Relative standard deviation: NMT 1.0% for the
Standard stock solution: 0.55 mg/mL of epinephrine from articaine peak
USP Epinephrine Bitartrate RS in Diluent Analysis
Standard solution: Dilute a suitable volume of the Standard Samples: Standard solution and Sample solution
stocksolution with Diluent to obtain a final concentration of [Nors-Artlcalne related compounds elute at relative
L mg/mL of epinephrine, where L is the label claim of retention times of NMT 2.0 with respect to the
epinephrine in the Injection. articaine peak.]
Sample solution: Use the Injection directly. Calculate the percentage of articaine related compounds
Chromatographic system and any other individual impurity in the portion of
(See Chromatography (621), System SUitability.) Injection taken:
Mode: LC
Detector: Amperometric electrochemical Result =(r vir s) x (C siC v) x 100
Reference electrode: Silver/silver chloride
Working electrode: Glassy carbon ru =response of each individual impurity from the
Potential: +650 mV Sample solution
Detector temperature: 28 ± 2° r5 = response of articaine from the Standard solution
Column: 4.0-mm x 25-cm; 5-lJm packing L7 Cs = concentration of articaine in the Standard solution
Flow rate: 1 mL/min (mg/mL)
Injection size: 2 IJL Cu =nominal concentration of articaine in the Sample
Run time: 1.7,times the retention time of epinephrine solution(mg/ml)
System suitability
Sample: System suitability solution [NOTE-Disregard any peak below 0.05%.]
[NoTE-The relative retention times for norepinephrine Acceptance criteria: See Table 1.
and epinephrine are 0.90 and 1.0, respectively.]
Suitability requirements Table 1
Resolution: NLT 1.5 between the norepinephrine and Relative Acceptance
epinephrine peaks ' Retention Criteria,
Name Time NMT(%)
Tailing factor: NMT 2.0 for the epinephrine peak
Relative standard deviation: NMT 1.0% for the Articaine related compound B 0.6 0.5
epinephrine peak, from six injections
Analysis Articaine 1.0 -
Samples: Standardsolution and Sample solution Any other individual impurity - 0.2
Calculate the percentage of the labeled amount of
epinephrine (C9H13N03) in the portion of Injection taken: Total impurities - 0.5

Result = (r vir s) x (C siC v) x 100 • ORGANIC IMPURITIES, LIMIT OF EPINEPHRINE RELATED

COMPOUNDS
ru = peak response from the Sample solution Mobile phase, System suitability solution, Standard
rs = peak response from the Standard solution solution, Sample solution, Chromatographic system,
C5 = concentration of epinephrine in the Standard and System suitability: Proceed as directed in the Assay
solution (mg/mL) for Epinephrine.
Cu = nominal concentration of epinephrine in the Analysis
Sample solution (mg/mL) Samples: Standard solution and Sample solution
[NOTE-Epinephrine related compounds elute between
Acceptance criteria: 90.0%-115.0% relative retention times of 0.35 and 1.0, with respect
to the epinephrine peak.]
PERFORMANCE TESTS Calculate the percentage of epinephrine related
• DELIVERABLE VOLUME (698) compounds and any other individual impurity in the
For Articaine Hydrochloride and Epinephrine Injection portion of Injection taken:
packaged in single-dose containers: Meets the
requirements Result = (r vir s) x (C siC v) x 100
IMPURITIES ru =response of each individual impurity from the
• ORGANIC IMPURITIES, LIMIT OF ARTICAINE RELATED Sample solution .
COMPOUNDS r5 =response of epinephrine from the Standard
Mobile phase, Standard stock solution, Sample solution, solution . ,.. .
and Chromatographic system: Proceed asdirected in the C5 =concentration of epinephrine in the Standard
Assay for Articaine Hydrochlori~e. solution(mg/ml)

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 USP 43 OfficialMonographs / Ascorbic 383

cu = nominal concentration of epinephrine in the ASSAY

Sample solution (mg/mL) • PROCEDURE
Sample: 400 mg of Ascorbic Acid
Acceptance criteria: See Table 2. Titrimetric system
(See Titrimetry (541).)
Table 2 Mode: Direct titration
Relative Acceptance Titrant: 0.1 N iodine VS
Retention Criteria, Endpoint detection: Visual
Name Time NMT(%) Blank: 100 mL of water and 25 mL of 2 N sulfuric acid. Add
Epinephrine sulfonate" 0.46 7.5 3 mL of starchTS.
Analysis: Dissolvethe Sample in a mixture of 100 mL of
Specified impurity 0.52 8 water and 25 mL of 2 N sulfuric acid. Add 3 mL of starch
Epinephrine 1.0 - TS, and titrate immediately with Titrant until a persistent
violet-blue color is obtained.
Any other individual impurity - 1 Calculate the percentage of ascorbic acid (C6H B06) in the
Total impurities - 10 portion of Ascorbic Acid taken:

a 1-(3,4-Dihydroxyphenyl)-2-(methylamino)ethanesulfonic acid. Result = [(V- B) x Nx Fx 100]/W

SPECIFICTESTS V = sample titrant volume (mL)

• pH (791): 2.7-5.2 B = blank titrant volume (mL)
• BACTERIAL ENDOTOXINS TEST (85): NMT 0.7 USP Endotoxin N = titrant normality (mEq/mL)
Unit/mg of articaine hydrochloride F = equivalency factor, 88.06 mg/mEq
• STERILITY TESTS (71): It meets the requirements when W = weight of Sample (mg)
tested as directed for Test for Sterilityof the Product to Be
Examined, Membrane Filtration. Acceptance criteria: 99.0%-100.5%
• PARTICULATE MATTER IN INJECTIONS (788): Meets the IIYIPURITIES
requirements • RESIDUE ON IGNITION (281): NMT 0.1%
• OTHER REQUIREMENTS: It meets the requirements under
Injections and ImplantedDrug Products (1). SPECIFIC TESTS
• OPTICAL ROTATION, Specific Rotation (781 S)
ADDITIONAL REQUIREMENTS Sample solution: 100 mg/mL in carbon dioxide-free water.
• PACKAGING AND STORAGE: Preserve in single-dose Perform the test immediately after preparation of the
containers, preferably of Type I glass. Store at controlled Sample solution.
room temperature. Acceptance criteria: +20.5° to +21.5°
• USP REFERENCE STANDARDS (11)
USP Articaine Hydrochloride RS ADDITIONAL REQUIREMENTS
USP Articaine Related Compound B RS • PACKAGING AND STORAGE: Preserve in tight, light-resistant
4-Methyl-3-{[2-(propylamino)propanoyl]amino} containers.
thiophene-2-carboxylic acid. • USP REFERENCE STANDARDS (11)
C12H1BN203S 270.35 USP Ascorbic Acid RS
USP Epinephrine Bitartrate RS
USP Norepinephrine Bitartrate RS

Ascorbic Acid Injection

Ascorbic Acid DEFINITION
Ascorbic Acid Injection is a sterile solution, in Water for
OH
Injection, of Ascorbic Acid prepared with the aid of Sodium
H O••$ H
o Hydroxide, Sodium Carbonate, or Sodium Bicarbonate. It
-0
H-
contains NLT 90.0% and NMT 110.0% of the labeled
HO OH
amount of ascorbic acid (C6H s0 6) .
IDENTIFICATION
C6H B06 176.12
• A.
l-Ascorbic acid [50-81-7]. Analysis: To a volume of Injection, equivalent to 40 mg of
ascorbic acid, add 4 mL of 0.1 N hydrochloric acid, then
DEFINITION
Ascorbic Acid contains NLT 99.0% and NMT 100.5% of add 4 drops of methylene blue TS, and warm to 40°.
Acceptance criteria: The deep blue color becomes
C6H s0 6 • appreciably lighter or is completely discharged within 3
IDENTIFICATION min.
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution,
obtained as directed in the Assay.
• C. The Injection imparts an intense yellow color to a
non luminous flame.

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 384 Ascorbic / OfficialMonographs USP 43

ASSAY
lit PROCEDURE
Ascorbic Acid Oral Solution
Mobile phase: Dissolve 15.6 g of dibasic sodium phosphate
DEFINITION
and 12.2 g of monobasic potassium phosphate in 2000 ml Ascorbic Acid Oral Solution is a solution of Ascorbic Acid in a
of water, and adjust with phosphoric acid to a pH of 2.5 hydroxylic organic solvent or an aqueous mixture thereof. It
±0.05.
contains NlT 90.0% and NMT 110.0% of the labeled
Standard solution: 0.5 mg/ml of USP Ascorbic Acid RS in amount of ascorbic acid (C6H a0 6) .
Mobilephase. [NoTE-Refrigerate and store protected from
light until.use. The solution is stable for at least 24 h. Inject IDENTIFICATION
within 3 h after removal from the refrigerator.] lit A.

Sample solution: Dilute the Injection, if necessary, with Sample solution: A volume of Oral Solution equivalent to
Mobilephase to obtain a solution with a concentration of 40 mg of ascorbic acid
about 0.5 mg/ml. [NOTE-Refrigerate and store protected Analysis: To the Sample solution add 4 ml of 0.1 N
from light until use. The solution is stable for at least 24 h. hydrochloric acid, then 4 drops of methylene blue TS, and
Inject within 3 h after removal from the refrigerator.] warm to 40°.
Chromatographic system Acceptance criteria: The deep blue color becomes
(See Chromatography (621), System Suitability.) appreciably lighter or is completely discharged within 3
Mode: lC min.
Detector: UV 245 nm • B.
Column: 150-cm x 6-mm; packing l39 Sample solution: A volume of Oral Solution equivalent to
Flow rate: 0.6 ml/min 20 mg of ascorbic acid
Injection volume: 4 IJl Analysis: To the Sample solution add 15 ml of trichloroacetic
System suitability acid solution (1 in 20). Add 200 mg of activated charcoal,
Sample: Standardsolution shake the mixture vigorously for 1 min, and pass through a
Suitability requirements small fluted filter, returning the filtrate, if necessary, until
Column efficiency: NlT 3500 theoretical plates clear. To 5 mL of the filtrate add 1 drop of pyrrole, agitate
Tailing factor: NMT 1.6 gently until dissolved, then heat in a bath at 50°.
Relative standard deviation: NMT 1.5% . Acceptance criteria: A blue color develops.
Analysis
Samples: Standardsolution and Sample solution ASSAY
Calculate the percentage of the labeled amount of ascorbic • PROCEDURE
acid (C6H a0 6) in the portion of Injection taken: Sample solution: Transfer a volume of Oral Solution
equivalent to 50 mg of ascorbic acid, previously diluted
Result = (r ulr s) x (C siC u) x 100 with water if necessary, to a 1OO-ml volumetric flask. Add
20 mL of meta phosphoric-acetic acid TS, dilute with water
ru = peak response from the Sample solution to volume, and mix.
rs = peak response from the Standard solution Blank: A mixture of 5.5 ml of metaphosphoric-acetic acid
Cs = concentration of USP Ascorbic Acid RS in the TS and 15 mL of water
Standardsolution (mg/mL) Titrimetric system
Cu = nominal concentration of ascorbic acid in the (See Titrimetry (541 ).)
Sample solution (mg/ml) Mode: Direct titration
Titrant: Standard dichlorophenol-indophenol VS
Acceptance criteria: 90.0%-110.0% Analysis: Transfer a volume of the Sample solution,
equivalent to 2 mg of ascorbic acid, into a 50-mL conical
IMPURITIES flask. Add 5 mL of metaphosphoric-acetic acid TS, and
• LIMIT OF OXALATE titrate with Titrant until a rose-pink color persists for at least
Analysis: Dilute a volume of Injection, equivalent to 50 mg 5 s. Correct for the volume of the Titrant consumed by the
of ascorbic acid, with water to 5 mL. Add 0.2 ml of acetic Blank.
acid and 0.5 ml of calcium chloride TS. Calculate the percentage of ascorbic acid (C6H a0 6) in the
Acceptance criteria: No turbidity is produced in 1 min. portion of Oral Solution taken:
SPECIFIC TESTS
Result = {[(Vs - VB) x F]IW} x 100
• pH (791): 5.5-7.0
• OTHER REQUIREMENTS: It meets the requirements in Vs = Titrant volume consumed by the Sample solution
Injections and Implanted Drug Products (1). (mL)
• BACTERIAL ENDOTOXINS TEST (85): It contains NMT 1.2 USP VB = Titrant volume consumed by the Blank (mL)
Endotoxin Units/mg of ascorbic acid. F = concentration of Titrant in terms of its equivalent
ADDITIONAL REQUIREMENTS of ascorbic acid (mg/mL)
• PACKAGING AND STORAGE: Preserve in light-resistant, W = nominal amount of ascorbic acid taken for
single-dose containers, preferably of Type I or Type II glass. Analysis (mg)
• LABELING: In addition to meeting the requirements in
Labeling (7), Labels and Labeling for Injectable Products, Acceptance criteria: 90.0%-110.0%
fused-seal containers of the Injection in concentrations of OTHER COMPONENTS
250 mg/ml and greater are labeled to indicate that since • ALCOHOL DETERMINATION, MethodI (611) (if present):
pressure may develop on long storage, precautions should 90.00/0-110.0% of the labeled content of alcohol .
be taken to wrap the container in a protective covering (C2H sOH)
while it is being opened.
• USPREFERENCE STANDARDS (11)
USPAscorbic Acid RS

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 USP 43 Official Monographs / Ascorbic 385

ADDITIONAL REQUIREMENTS =peak responseof ascorbic acid from the Standard

• PACKAGING AND STORAGE: Preserve in tight, light-resistant solution
containers. = concentration of USP Ascorbic Acid RS in the
• LABELING: Label Oral Solution that contains alcohol to state Standardsolution (mg/mL)
the alcohol content. = nominal concentration of ascorbic acid in the
Sample solution (mg/mL)

Acceptance criteria: 90.0%-110.0%

Ascorbic Acid Compounded Oral SPECIFIC TESTS
• pH (791): 1.8-2.8
Solution
ADDITIONAL REQUIREMENTS
DEFINITION • PACKAGING AND STORAGE: Package in tight, light-resistant
Ascorbic Acid Compounded Oral Solution contains NLT plastic containers. Store in a refrigerator or at controlled
90.0% and NMT 110.0% of the labeled amount of ascorbic room temperature.
acid (C6H s0 6) . • BEYOND-USE DATE: NMT 90 days after the date on which it
Prepare Ascorbic Acid Compounded Oral Solution, 100 was compounded when stored in a refrigerator; or NMT 60
mg/mL, as follows (see Pharmaceutical Compounding- days after the date on which it was compounded when
Nonsterile Preparations (795»). stored at controlled room temperature
• LABELING: Label it to state the Beyond-Use Date.
Ascorbic Acid
• USP REFERENCE STANDARDS (11)
109
USP Ascorbic Acid RS
Purified Water 50 mL

Ora-Sweet,' a sufficient quantity to

make 100 mL

a Perrigo Laboratories, Allegan, MI. Ascorbic Acid Tablets

Place the Ascorbic Acid into a suitable container. Dissolve the DEFINITION
powder in the Purified Water. Transfer the contents to a Ascorbic Acid Tablets contain ascorbic acid in the form of
calibrated container. Add sufficient Ora-Sweet to bring to ascorbic acid (C6H s0 6) , sodium ascorbate (C6H 7Na06) ,
final volume. Shake to mix well. calcium ascorbate dihydrate (C12H14Ca012 • 2H 20), or their
mixture in an amount equivalent to NLT 90.0% and NMT
ASSAY 110.0% of the labeled amount of ascorbic acid (C6H s0 6) .
• PROCEDURE
Mobile phase: 50 mM monobasic sodium phosphate; IDENTIFICATION
adjusted with phosphoric acid to a pH of 2.5 • A.
Standard solution: 0.2 mg/mL of USP Ascorbic Acid RS in Sample solution: Triturate a quantity of finely powdered
Mobile phase Tablets with diluted alcohol to make a solution of ascorbic
Sample solution: Transfer 0.2 mL of Oral Solution into a acid with a concentration of 20 mg/mL, and filter.
1OO-mL volumetric flask and dilute with Mobilephase to Analysis: Add alkaline cupric tartrate TSto a portion of the
volume. Sample solution.
Chromatographic system Acceptance criteria: The Sample solution reduces alkaline
(See Chromatography (621), System Suitability.) cupric tartrate TS slowly at room temperature but more
Mode: LC readily upon heating.
Detector: UV 220 nm • B.
Column: 4.6-mm x 25-cm; 5-J.lm packing L96 Sample solution: Usethe Sample solutionfrom Identification
Temperatures test A.
Autosampler: 5° Analysis: To 2 ml of the Sample solution add 4 drops of
Column: 10° methylene blue TS, and warm to 40°.
Flow rate: 1.0 ml/min Acceptance criteria: The deep blue color of methylene blue
Injection volume: 10 J.ll becomes appreciably lighter or is completely discharged
System suitability within 3 min.
Sample: Standardsolution • C.
[NoTE-The retention time for ascorbic acid is about 4.4 Sample solution: Usethe Sample solutionfrom Identification
min.] test A.
Suitability requirements Analysis: To 1 ml of the Sample solution add 15 mL of
Tailing factor: NMT 2.0 trichloroacetic acid solution (1 in 20) and 200 mg of
Relative standard deviation: NMT 2.0% for replicate activated charcoal, shakethe mixture Vigorously for 1 min,
injections and passthrough a small fluted filter, returning the filtrate
Analysis if necessary, until clear. To 5 mL of the filtrate add 1 drop
Samples: Standardsolution and Sample solution of pyrrole, agitate gently until dissolved, and then heat in a
Calculate the percentage of the labeled amount of ascorbic bath at 50°.
acid (C6H s0 6) in the portion of Oral Solution taken: Acceptance criteria: A blue color develops.

Result = (rulrs) x (Cs/Cu) x 100

ASSAY
[NOTE-Where more than one assay procedure is given in
ru = peak response of ascorbic acid from the Sample the monograph, the requirements may be met by
solution following anyone of the specified procedures, the

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386 Ascorbic / OfficialMonographs USP43

procedure used being stated in the labeling only if L = label claim of ascorbic acid (mg/Tablet)
Procedure 1 is not used.]
• PROCEDURE 1 For Procedure 2
Sample stock solution: Transfer NLT 20 Tablets to a
1OOO-mL volumetric flask containing 250 mL of Result = (ru/rs) ~ Cs x V x (l/L) x 100
meta phosphoric-acetic acids TS. Insert the stopper in the
flask, and shake by mechanical means for 30 min or until = peak area of ascorbic acid from the Sample
the Tablets have disintegrated completely. Dilute with solution
water to volume. = peak area of ascorbic acid from the Standard
Sample solution: Transfer a portion of the Sample stock solution
solution to a centrifuge tube, and centrifuge until a clear = concentration of USP Ascorbic Acid RS in the
supernatant is obtained. Quantitatively dilute the clear Standard solution (mg/mL)
supernatant with water, if necessary, to obtain a solution V = volume of Medium, 900 mL
containing 0.5 mg/mL of ascorbic acid. L = label claim (mg/Tablet)
Blank: A mixture of 5.5 mL of meta phosphoric-acetic acids
TS and 15 mL of water Tolerances: NLT 75% (Q) of the labeled amount of ascorbic
Titrimetric system acid (C6 Hs0 6) is dissolved.
(See Titrimetry (541 ).) • DISINTEGRATION (701)
Mode: Direct titration [NOTE-Meet this additional test if the label
Titrant: Standard dichlorophenol-indophenol VS recommends to disintegrate the Tablets in the mouth
Endpoint detection: Visual, a rose-pink color that persists before swallowing.]
for at least 5 s Medium: Water
Analysis: Transfer a volume of the Sample solution, Time: NMT 5 min
equivalent to 2 mg of ascorbic acid, to a 50-mLconical flask. Acceptance criteria: Meet the requirements
Add 5 mL of metaphosphoric-acetic acids TS, and titrate • UNIFORMITY OF DOSAGE UNITS (905): Meet the
with Titrant. Correct for the volume of the Titrant requirements
consumed by the Blank. . ADDITIONAL REQUIREMENTS
Calculate the percentage of the labeled amount of ascorbic • PACKAGING AND STORAGE: Preserve in tight, light-resistant
acid (C6 Hs0 6) in the portion of Tablets taken: containers.
• LABELING: The label states the quantity of ascorbic acid in
Result = [(Vs - VB) x F/W] x 100 mg/Tablet, and the chemical form of ascorbic acid present
in the Tablets. The labeling states with which assay
=Titrant volume consumed by the Sample solution procedure the product complies only if Procedure 1 is not
(mL) used. Tablets that are intended to be disintegrated in the
= Titrant volume consumed by the Blank (mL) mouth before swallowing are so labeled.
= concentration of the Titrant in terms of the • USP REFERENCE STANDARDS (11)
equivalent of ascorbic acid (mg/mL) USPAscorbic Acid RS
= nominal weight of ascorbic acid taken for Analysis
(mg)
Acceptance criteria: 90.0%-110.0%
• PROCEDURE 2 . Aspartic Acid
(See Vitamin C Assay (580), Method II-Chromatographic
Method.)
Acceptance criteria: 90.00/0-110.0% . HO~If '(1./'...
'OH

PERFORMANCE TESTS o Nil,

• DISSOLUTION (711) C4 H7N04 133.10

Medium: Water; 900 mL L-Aspartic acid [56-84-8].
Apparatus 2: 50 rpm
Time: 45 min DEFINITION
Sample solution: Withdraw a portion of the solution under Aspartic Acid contains NLT 98.5% and NMT 101.5% of
test, pass through a suitable filter, and use the pooled aspartic acid (C4 H7N04) , calculated on the dried basis.
sample as the test specimen.
Analysis: Proceed as directed in the Assay, Procedure 1 or IDENTIFICATION
Procedure 2, conducting the procedure without delay and
making any necessary modifications.
Calculate the percentage of the labeled amount of ascorbic
acid (C6 Hs0 6) dissolved:
For Procedure 1
ASSAY
Result = [(Vs - VB) x Fx (VM/a)/L] x 100 • PROCEDURE
Sample: 100 mg of Aspartic Acid
=Titrant volume consumed by the Sample solution Titrimetric system
(mL) (See Titrimetry (541 ).)
=Titrant volume consumed by the Blank (mL) Mode: Direct titration
=concentration of the Titrant in terms of the Titrant: O~ 1 N sodium hydroxide VS
equivalent of ascorbic acid (mg/mL) Endpoint detection: Visual
= volume of Medium, 900 mL
=volume of the aliquot taken for Analysis

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USP 43 OfficialMonographs / Aspartic 387

Blank: 50 mL of carbon dioxide-free water. Add 0.1 mL of Sample solution. Disregard this peak in the calculation
bromothymol blue TS. of the impurity.]
Analysis: Transfer the Sample to a 125-mL flask, and dissolve Calculate the percentage of each specified acid in the
in 50 mL of carbon dioxide-free water. Heat slightly if portion of Aspartic Acid taken:
necessary. Cool, add 0.1 mL of bromothymol blue TS, and
titrate with Titrant until the color changes from yellow to Result = (r vIr 5) x (C sICv) x 100
blue. Perform the blank determination.
Calculate the percentage of aspartic acid (C4H 7N04) in the = peak response of maleic acid, malic acid, or
portion of Aspartic Acid taken: fumaric acid from the Sample solution
= peak response of maleic acid, malic acid, or
Result = [(V 5 - V B) x N A X F x 100]/W fumaric acid from the corresponding Standard
solution
V5 = Titrant volume consumed by the Sample (mL) = concentration of USP Maleic Acid RS, USP Malic
VB = Titrant volume consumed by the Blank (mL) Acid RS, or USP Fumaric Acid RS in the
NA =actual normality of the Titrant (rntq/rnt.) corresponding Standard solution (mg/mL)
F = equivalency factor, 133.1 mg/mEq Cu =concentration of Aspartic Acid in the Sample
W = Sample weight (mg) solution(mg/mL)

Acceptance criteria: 98.5%-101.5% on the dried basis Calculate the percentage of any unspecified impurity in the
portion of Aspartic Acid taken:
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1 % Result = (r vIr s) x (C sICu) x 100
• CHLORIDE AND SULFATE (221), Chloride
Sample solution: Dissolve 0.7 9 of Aspartic Acid in 10 mL = peak response of any unspecified impurity from
of diluted nitric acid, and dilute with water to 15 mL. the Sample solution
Acceptance criteria: The Sample solution shows no more =peak response of fumaric acid from the Fumaric
chloride than corresponds to 0.20 mL of 0.020 N acidstandardsolution
hydrochloric acid (NMT 0.02%). =concentration of USP Fumaric Acid RS in the
• CHLORIDE AND SULFATE (221), Sulfate Fumaric acidstandardsolution (mg/mL)
Sample solution: Dissolve 0.8 9 of Aspartic Acid in 4 mL of = concentration of Aspartic Acid in the Sample
hydrochloric acid, and dilute with water to 15 mL. solution(mg/mL)
Acceptance criteria: The Sample solution shows no more
sulfate than corresponds to 0.25 mL of 0.020 N sulfuric acid Acceptance criteria: See Table 7.
(NMT 0.03%). I

• IRON (241): NMT 10 ppm Table 1

• RELATED COMPOUNDS Relative Acceptance
Mobile phase: 0.008 N sulfuric acid Retention Criteria,
System suitability solution: A mixture of 0.1 mg/mL of USP Name Time NMT (%)
Fumaric Acid RS, 0.05 mg/mL of USP Maleic Acid RS, and Maleic acid 0.5 0.05
1.5 mg/mL of USP Malic Acid RS in water
Malicacid 0.6 0.20
Fumaric acid standard solution: 0.1 mg/mL of USP
Fumaric Acid RS in water . Fumaric acid 1.0 0.10
Maleic acid standard solution: 0.05 mg/mL of USP Maleic
Acid RS in water
Aspartic acid Not observed -
Malic acid standard solution: 1.5 mg/mL of USP Malic Acid Anyunspecified impurity - 0.05
RS in water
Sample solution: Transfer 109 of Aspartic Acid to a 100-mL
Total unspecified impurities - 0.10

volumetric flask, add 15-20 mL of 6 N hydrochloric acid,

and mix to dissolve. Dilute with water to volume. SPECIFIC TESTS
Chromatographic system • OPTICAL ROTATION (781 S), Procedures,Specific Rotation
(See Chromatography (621), System Suitability.) Sample solution: 80 mg/mL in 6 N hydrochloric acid
Mode: LC Acceptance criteria: +24.0° to +26.0°, at 20°
Detector: UV 214 nm • Loss ON DRYING (731)
Column: 7.8-mm x 30-cm; 9-lJm packing L17 Analysis: Dry at 105° for 3 h.
Column temperature: 30° Acceptance criteria: NMT 0.5%
Flow rate: 0.6 mL/min ADDITIONAL REQUIREMENTS
Injection volume: 10 IJL • PACKAGING AND STORAGE: Preserve in well-closed
System suitability containers, and store protected from light.
Sample: System sUitability solution . • USP REFERENCE STANDARDS (11)
[NOTE-See Table 7 for the relative retention times.] USP Aspartic Acid RS
Suitability requirements USP Fumaric Acid RS
Resolution: NLT 1.5 between maleic acid and malic acid USP Maleic Acid RS
Relative standard deviation: NMT 10.0% each for USP Malic Acid RS
maleic acid, malic acid, and fumaric acid
Analysis
Samples: Standard solutions and Sample solution
[NOTE-A hydrochloric acid peak at around 6-7 min
may be observed in the chromatogram of the

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 388 Aspirin / OfficialMonographs USP 43

Assay-Place about 1.5 g of Aspirin, accurately weighed, in a

Aspirin flask, add 50.0 ml of 0.5 N sodium hydroxide VS, and boil the
"!lixture gently for 1 ~ minutes. Add phenolphthalein TS, and
titrate the excess sodium hydroxide with 0.5 N sulfuric acid
VS. Perform a blank determination (see Residual Titrations
under Titrimetry(541»). Each ml of 0.5 N sodium hydroxide is
equivalent to 45.04 mg of C9H s0 4 •

C9H s0 4 180.16
Benzoic acid, 2-(acetyloxy)-.
Salicylic acid acetate [50-78-2].
Aspirin Boluses
Aspirin contains not less than 99.5 percent and
» DEFINITION
not more than 100.5 percent of C9H s0 41 calculated Aspirin Boluses contain NlT 90.0% and NMT 110.0% of the
on the dried basis. labeled amount of aspirin (C9 Hs0 4 ) .

Packaging and storage-Preserve in tight containers. IDENTIFICATION .

USP Reference standards (11 )- • A. PROCEDURE
USP Aspirin RS Analysis: Crush 1 Bolus. Boil a portion of the powder,
equivalent to 300 mg of aspirin, with 50 ml of water. Cool
Identification- and add a drop of ferric chloride TS.
A: Heat it with water for several minutes cool and add 1 Acceptance criteria: A violet-red color is produced.
or 2 drops of ferric chloride TS: a violet-red ~olor is produced. • B. Th.e retention time of the aspirin peak of the Sample
solution corresponds to that of the Standardsolution as
obtained in the Assay. '

B: ASSAY
• PROCEDURE
$p~¢t[tJJf9 > i . . Yc ().
Mobile phase: 2 gIL of sodium 1-heptanesulfonate in a
Loss on drying (731 )-Dry it over silica gel for 5 hours: it loses
not more than 0.5% of its weight. mixture of acetonitrile and water (15:85). Adjust with
Readily carbonizable substances (271 )-Dissolve 500 mg in glacial acetic acid to a pH of 3.4.
5 ml of sulfuric acid: the solution has no more color than Diluent: Acetonitrile and formic acid (99:1)
Matching Fluid Q~ Standard solution: 0.4 mg/ml of USP Aspirin RS and 0.01
Residue on ignition (281): not more than 0.05%. mg/ml of USP Salicylic Acid RS in Diluent
Substances insoluble in sodium carbonate TS-A Sample stock solution: Nominally 4 mg/ml of aspirin
solution of 500 mg in 10 ml of warm sodium carbonate TS is prepared as follows. Finelypowder NlT 10 Boluses.Transfer
clear. a port.ion of the powder to an appropriate volumetric flask
Chloride (221 )-Boill.5 g with 75 ml of water for 5 minutes and dilute with Diluent to volume. Stir the solution by
~ool, add sufficient .water to restore the original volume, and mechanical means for 15 min.
filter. A 25-ml portion of the filtrate shows no more chloride Sample solution: Nominally 0.4 mg/ml of aspirin from
Sam~/e stock solution in Diluent. Pass a portion of this
than corresponds to 0.10 ml of 0.020 N hydrochloric acid
(0.014%). . solution through a filter of 0.5-J.Jm or finer pore size. Use
Sulfate -Dissolve 6.0 g in 37 ml of acetone, and add 3 ml the filtrate.
of water. Titrate potentiometrically with 0.02 M lead Chromatographic system
perchlorate, prepared by dissolving 9.20 g of lead perchlorate (See Chromatography (621), System SUitability.)
In water to make 1000 ml of solution, using a pH meter Mode: lC
capable of a mi.nimum ~eproducibility of ±0.1 mV (see pH Detector: UV 254 nm
(791») an?eqUipped with an ~Iectro.de system consisting of a Column: 4.6-mm x 25-cm; 5-J.Jm packing II
lead-specific electrode and a Silver-silver chloride reference Flow rate: 1 ml/min
glass-sleeved electrode containing a solution' of Injection volume: 20 J.JL
tetraethylammonium perchlorate in glacial acetic acid (1 in System suitability
44) (see Titrimetry(541 »): not more than 1.25 ml of 0.02 M Sample: Standardsolution
lead perchlorate is consumed (0.04%). [NoTE-After use rinse [NOTE-The relative retention times for salicylic acid
the lead-specific electrode with water, drain the referen~e and aspirin are 0.6 and 1.0, respectively.]
electrode, flush with water, rinse with methanol, and allow to Suitability requirements
dry.] Resol.ution: NLT 2.0 between salicylicacid and aspirin
Limit of free salicylic acid-Dissolve 2.5 g in sufficient Relative standard deviation: NMT 2.0% for aspirin
alcohol to make 25.0 ml. To each of two matched Analysis
color-comparison tubes add 48 ml of water and 1 ml of a Samples: Standardsolution and Sample solution
freshly prepared, diluted ferric ammonium sulfate solution Calculate t~e percent~ge of the labeled amount of aspirin
(prepared by adding 1 mlof 1 N hydrochloric acid to 2 ml of (C9H s0 4) In the portion of Boluses taken:
ferric ammonium sulfate TS and diluting with water to 100
m~) ..Into one tube pipet 1 ml of a standard solution of salicylic
Result =(rulrs) x (Cs!Cu) x 100
acid In water, containing 0.10 mg of salicylic acid per ml. Into rv = peak response of-aspirinfrom the Sample solution
th~ second tube pipet 1 ml of the 1 in 10 solution of Aspirin.
MIx the contents of each tube: after 30 seconds the color in rs = peak response of aspirin from the Standard
the second tube is not more intense than that i~ the tube solution
containing the salicylic acid (0.1.%). Cs =concentration of USP Aspirin RS in the Standard
solution (mg/mL)

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 USP 43 OfficialMonographs / Aspirin 389

Cv = nominal concentration of aspirin in the Sample Cs =concentration of USP Salicylic Acid RS in the
solution(mg/mL) Salicylic acidstandardsolution (mg/mL)
C =actual concentration of aspirin in the Sample
Acceptance criteria: 90.0%-110.0% solution
PERFORMANCE TESTS Acceptance criteria: NMT 0.3%
• DISSOLUTION (711)
Medium: 0.5 M phosphate buffer (see Reagents, Indicators, ADDITIONAL REQUIREMENTS
and Solutions-Buffer Solutions), pH 7.4; 900 mL • PACKAGING AND STORAGE: Preserve in tight containers.
Apparatus 2: 75 rpm • LABELING: Label Boluses to indicate that they are for
Time: 45 min veterinary use only.
Diluent: Acetonitrile and formic acid (99:1) • USP REFERENCE STANDARDS (11)
Standard solution: USP Aspirin RS in Diluent at a suitable USP Aspirin RS
concentration. [NoTE-Prepare the solution at the time of USP Salicylic Acid RS
use.]
Sample solution: Pass a portion of the solution under test
through a suitable filter. Dilute with Diluent, if necessary.
Instrumental conditions
Mode: UV-Vis Aspirin Capsules
Analytical wavelength: The isosbesticpoint of aspirin and
salicylic acid at 265 ± 2 nm
Analysis » Aspirin Capsules contain not less than 93.0
Samples: Standard solution and Sample solution percent and not more than 107.0 percent of the
Calculate the percentage of the labeled amount of aspirin labeled amount of aspirin (C9H s0 4) .
(C9H s0 4 ) dissolved: [NOTE-Capsules that are enteric-coated or the
contents of which are enteric-coated meet the
Result = (A viAs) x Cs x V x 0 x (1 I L) x 100 requirements for Aspirin Delayed-Release Capsules.
]
Av =absorbance of the Sample solution
As =absorbance of the Standard solution Packaging and storage-Preserve in tight containers.
Cs =concentration of the Standard solution(mg/mL) USPReference standards (11 )-
V =volume of Medium, 900 mL USP Aspirin RS
D =dilution factor of the Sample solution, if necessary Identification-
L = label claim (mg/Bolus)
A: Heat about 100 mg of the Capsule contents with 10 mL
Tolerances: NLT 80% (Q) of the labeled amount of aspirin of water for several minutes, cool, and add 1 drop of ferric
(C9H s04 ) is dissolved. chloride TS: a violet-red color is produced.
8: Shake a quantity of the contents of Capsules, equivalent
• UNIFORMITY OF DOSAGE UNITS (905): Meet the' to about 500 mg of aspirin, with 10 mL of alcohol for several
requirements minutes. Centrifuge the mixture. Pour off the clear
IMPURITIES supernatant and evaporate it to dryness. Dry the residue in
• LIMIT OF SALICYLIC ACID vacuum at 60° for 1 hour: the residue responds to Identification
Mobile phase, Diluent, Sample solution, . - test Identification- under Aspirin.
Chromatographic system, and System suitability: Dissolution (711)-
Proceed as directed in the Assay. . Medium: 0.05 M acetate buffer, prepared by mixing 2.99
Salicylic acid standard solution: 0.01 mg/mL of USP
g of sodium acetate trihydrate and 1 .66 mL of glacial acetic
Salicylic Acid RS in Diluent acid with water to obtain 1000 mL of solution having a pH of
Analysis 4.50 ± 0.05; 500 mL.
Samples: Sample solution and Salicylic acid standard Apparatus 1: 100 rpm.
solution .
Time: 30 minutes.
Calculate the actual concentration (C), in mg/mL, of Procedure-Determine the amount of C9H s04 dissolved
aspirin (C 9H s04) in the Sample solution taken:
from UV absorbances at the wavelength of the isosbestic point
of aspirin and salicylic acid at 265 ± 2 nm of filtered portions
Result = Cv x (Fl100)
of the solution under test, suitably diluted with Medium, if
Cv = nominal concentration of aspirin in the Sample necessary, in comparison with a Standard solution having a
solution (mg/mL) known concentration of USP Aspirin RS in the same Medium.
F = percentage of the labeled amount of aspirin [NOTE-Prepare the Standard solution at the time of use. An
(C 9H s0 4) in the portion of Boluses taken, as amount of alcohol not to exceed 1% of the total volume of the
Standard solution may be used to bring the Reference
determined in the Assay
Standard into solution prior to dilution with Medium.]
Calculate the percentage of salicylic acid (C 7H 60 3) in the Tolerances-Not less than 80% (Q) of the labeled amount
of C9H s0 4 is dissolved in 30 minutes.
portion of Boluses taken:
Uniformity of dosage units (905): meet the requirements.
Result = (rulrs) x (CsIC) x 100 Limit of free salicylic acid-
Ferric chloride-urea reagent-Dissolve by swirling, without
rv = peak response of salicylic acid from the Sample the aid of heat, qO g of urea in a mixture of 8 mL of ferric
solution chloride solution (6 in 10) and 42 mL of 0.05 N hydrochloric
rs = peak response of salicylic acid from the Salicylic acid. Adjust the resulting solution, if necessary, with 6 N
acid standardsolution hydrochloric acid to a pH of 3.2.

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390 Aspirin / Official Monographs USP 43

Standardpreparation-Transfer 75.0 mg of salicylic acid, spectrophotometer, using chloroform as the blank. Calculate
previously dried over silica gel for 3 hours and accurately the quantity, in mg, of aspirin (CgH s0 4) in the portion of
weighed, to a 1OO-mL volumetric flask, add chloroform to Capsules taken by the formula:
volume, and mix. Transfer 10.0 mL of this solution to a second
1OO-mL volumetric flask, dilute with chloroform to volume, C(AuIAs)
and mix. Transfer 10.0 mL of this last solution to a 50-mL
volumetric flask containing 10 mL of methanol, 2 drops of in which C is the concentration, in IJg per mL, of USP Aspirin
hydrochloric acid, and 10 mL of a 1 in 10 solution of glacial RS in the Standardpreparation; and Au and As are the
acetic acid in ether, dilute with chloroform to volume, and absorbances of the Assay preparation and the Standard
mix. preparation, respectively.
Chromatographic column (see Chromatography (621 »)-
Proceed as directed under Column Partition Chromatography,
packing a chromatographic tube with two segments of
packing material. The lower segment is a mixture of 1 g of
SolidSupport and 0.5 mL of 5 M phosphoric acid, and the . Aspirin Delayed-Release Capsules
upper segment is a mixture of 3 g of Solid Support and 2 mL
of freshly prepared Ferric chloride-urea reagent. DEfiNITION
Test preparation-Weigh accurately a portion of the Aspirin Delayed-Release Capsules contain NLT 93.0% and
contents of the Capsules, as determined by the Assay, NMT 107.0% of the labeled amount of aspirin (C9 HS0 4) .
equivalent to 100 mg of aspirin, mix with 10 mL of chloroform IDENTifiCATION
by stirring for 3 minutes, and then transfer to the • A. The retention time of the aspirin peak of the Sample
chromatographic column with the aid of a few mL of solution corresponds to that of the Standardsolution, as
chloroform. Pass 50 mL of chloroform through the column, obtained in the Assay.
rinse the tip of the chromatographic tube with chloroform,
and discard the eluate. Prepare as a receiver a 50-mL
volumetric flask containing 10 mL of methanol and 2 drops of
hydrochloric acid, and elute any salicylic acid from the column
.• B•.~$~~¢tlt.~S~.()~I~c~~~~~.FI~~~I()f\I'fEsTS(197),\ri7frar~a
by passing 10 mL of a 1 in 10 solution of glacial acetic acid in
Spec;t[Q~c;oPY:>l·~?~"'(<:NJ·Mily"2Q;!O)
ether that has been recently saturated with water, followed by Sample: Shake a quantity of the contents of Capsules,
30 mL of chloroform. Dilute the eluate with chloroform to equivalent to about 500 mg of aspirin, with 10 mL of
volume, and mix. alcohol for several minutes. Centrifuge the mixture. Pour
Procedure-eoncomitantly determine the absorbances of off the clear supernatant and evaporate it to dryness. Dry
the solutions in 1-cm cells at the wavelength of maximum the residue under vacuum at 60° for 1 h.
absorbance at about 306 nm, with a suitable Acceptance criteria: Meet the requirements
spectrophotometer, using asthe blank a solvent mixture of the
same composition as that used for the standard preparation: ASSAY
the absorbance of the Test preparation does not exceed that • PROCEDURE
of the Standardpreparation (0.75%, calculated on the labeled Mobile phase: 2 giL of sodium 1-heptanesulfonate in a
aspirin content). mixture of acetonitrile and water (15:85). Adjust with
Assay- [NoTE-In this assay use chloroform recently glacial acetic acid to a pH of 3.4.
saturated with water.] Diluent: Acetonitrile and formic acid (99:1)
Standardpreparation-Transfer about 50 mg of USP Standard solution: 0.5 mg/mL of USP Aspirin RS in Diluent
Aspirin RS, accurately weighed, to a 50-mL volumetric flask, Sample stock solution: Nominally 5 mg/mL of aspirin
add 0.5 mL of glacial acetic acid, add ·chloroform to volume, prepared asfollows. Remove, ascompletely aspossible, the
and mix. Transfer 5.0 mL of this solution to a 100-mL contents of NLT 20 Capsules. Mix the combined contents,
volumetric flask, dilute with a 1 in 100 solution of glacial acetic and transfer a quantity equivalent to about 100 mg of
acid in chloroform to volume, and mix. The concentration of aspirin to a suitable container. Add 20.0 mL of Diluent and
USP Aspirin RS is about 50 IJg per mL. about 10 glass beads. Shake vigorously for about 10 min,
Chromatographic column-Proceed as directed under and centrifuge.
Column PartitionChromatography (see Chromatography (621 »), Sample solution: Nominally 0.5 mg/mL of aspirin in Diluent
packing a chromatographic tube with a mixture of 3 g of Solid from Sample stock solution
Support and 2 mL of freshly prepared sodium bicarbonate Chromatographic system
solution (1 in 12). (See Chromatography (621), System Suitability.)
Assay preparation-Remove, as completely as possible, the Mode: LC
contents of not fewer than 20 Capsules, and weigh accurately. Detector: UV 280 nm
Mix the combined contents, and transfer an accurately Column: 4.0-mm x 30-cm; packing L1
weighed quantity of the powder, equivalent to about 50 mg Flow rate: 2 rnt/rnln
of aspirin, to a 50-mL volumetric flask containing 1 mL of a 1 Injection volume: 10 IJL
in 50 solution of hydrochloric acid in methanol, add System suitability
chloroform to volume, and mix. Transfer 5.0 mL of this Sample: Standardsolution
solution to the column, wash with 5 mL and then with 25 mL Suitability requirements
of chloroform, and discard the washings. Elute into a 100-mL Tailing factor: NMT 2.0
volumetric flask with about 10 mL of a 1 in 10 solution of Relative standard deviation: NMT 2.0%
glacial acetic acid in chloroform and then with about 85 mL Analysis
of a 1 in 100 solution of glacial acetic acid in chloroform, dilute Samples: Standardsolution and Sample solution
with the latter solvent to volume, and mix. Calculate the percentage of the labeled amount of aspirin
Procedure-Without delay, concomitantly determine the (CgH s0 4 ) in the portion of Capsules taken:
absorbances of the solutions in 1-cm cells at the wavelength
of maximum absorbance at about 280 nm, with a suitable Result = (rulr s) x (CslCu) x 100

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 USP 43 Official Monographs / Aspirin 391

=peak response of aspirin from the Sample solution rs = peak response of salicylic acid from the Standard
= peak response of aspirin from the Standard solution
solution Cs = concentration of USP Salicylic Acid RS in the
= concentration of USP Aspirin RS in the Standard Standardsolution (mg/mL)
solution (mg/mL) Cu = nominal concentration of aspirin in the Sample
= nominal concentration of aspirin in the Sample solution (mg/mL)
solution (mg/mL)
Acceptance criteria: NMT 3.0%
Acceptance criteria: 93.0%-107.0%
ADDITIONAL REQUIREMENTS
PERFORMANCE TESTS • PACKAGING AND STORAGE: Preserve in tight containers.
• DISSOLUTION (711), Procedure, Apparatus 7 and Apparatus 2, • LABELING: The label indicates that the Capsules or the
Delayed-Release Dosage Forms, Method A Procedure contents thereof are enteric-coated.
Apparatus 1: 100 rpm • USP REFERENCE STANDARDS (11)
Time: 90 min, for Buffer stage; 2 h, for Acidstage USP Aspirin RS
Diluent: 0.1 N hydrochloric acid and 0.20 M tribasic sodium USP Salicylic Acid RS
phosphate (3:1). Adjust, if necessary, with 2 N hydrochloric
acid or 2 N sodium hydroxide to a pH of 6.8 ± 0.05.
Standard solution: A known concentration of USP Aspirin
RS in a suitable medium
Sample solution: Pass a portion of the solution under test Aspirin Suppositories
through a suitable filter, and dilute with 0.1 N hydrochloric
acid (for analyzing in the Acidstage) and with Diluent (for
analyzing in the Buffer stage), if necessary. » Aspirin Suppositories contain not less than 90.0
Instrumental conditions percent and not more than 110.0 percent of the
Mode: UV labeled amount of aspirin (C9H s0 4) .
Analytical wavelengths
Acid stage: 280 nm Packaging and storage-Preserve in well-closed
Buffer stage: 265 nm containers, in a cool place.
Analysis USP Reference standards (11) -
Samples: Standardsolution and Sample solution USP Aspirin RS
Determine the percentage of the labeled amount of aspirin Identification-Transfer a portion of the melted
(C9H s0 4) dissolved from UV absorbances at the isosbestic Suppositories obtained in the Assay, equivalent to about 1 g
point of aspirin and salicylic acid (about 280 nm in the of aspirin, to a 125-mL conical flask. Add 20 mL of alcohol, and
Acidstage, and about 265 nm in the Buffer stage). warm until completely disintegrated. Cool in an ice bath for 5
Tolerances: The percentages of the labeled amount of minutes, filter, and evaporate the filtrate to dryness: the
aspirin (C9H s0 4) dissolved conform to Dissolution (711), residue responds to Identification- tests A: and B: under
Acceptance Table 3 (Acid stage) and Acceptance Table 4 Aspirin.
(Bufferstage). Limit of free salicylic acid-
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Ferric chloride-urea reagent-To a mixture of 8 mL of ferric
requirements chloride solution (6 in 10) and 42 mL of 0.05 N hydrochloric
IMPURITIES acid add 60 g of urea. Dissolve the urea by swirling and
• LIMIT OF FREE SALICYLIC ACID without the aid of heat, and adjust the resulting solution, if
Mobile phase, Diluent, and Chromatographic system: necessary, by the addition of 6 N hydrochloric acid to a pH of
Proceed as directed in the Assay. 3.2. Prepare on the day of use.
System suitability solution: 0.015 mg/mL of USP Salicylic Procedure-lnsert a small pledget of glass wool above the
Acid RS and 0.5 mg/mL of USP Aspirin RS in Diluent stem constriction of a 20- x 2.5-cm chromatographic tube,
Standard solution: 0.015 mg/mL of USP Salicylic Acid RS in and uniformly pack with a mixture of about 1 g of
Diluent chromatographic siliceous earth and 0.5 mL of 5 M
Sample solution: Use the Sample stock solution prepared as phosphoric acid. Directly above this layer, pack a similar
directed in the Assay. mixture of about 3 g of chromatographic siliceous earth and
System suitability 2 mL of Ferric chloride-urea reagent. Transfer to a small beaker
Samples: System sUitability solution and Standardsolution an accurately weighed portion of the cooled mass from the
[NOTE-The relative retention times for salicylic acid previously melted Suppositories obtained in the Assay,
and aspirin are about 0.7 and 1.0, respectively.] equivalent to 50 mg of aspirin, add 10 mL of chloroform,
Suitability requirements warm slightly, and stir until dissolved. With the aid of 5 mL of
Resolution: NLT 2.0 between salicylic acid and aspirin, chloroform, transfer the mixture to the chromatographic
System suitability solution adsorption column. Pass 50 mL of chloroform in several
Relative standard deviation: NMT 4.0%, Standard portions through the column, rinse the tip of the
solution chromatographic tube with chloroform, and discard the
Analysis eluate. If the purple zone reaches the bottom of the tube,
Samples: Standardsolution and Sample solution discard the column, and repeat the test with a smaller quantity
Calculate the percentage of salicylic acid (C7H 6 0 3) in the of melted Suppositories.
portion of Capsules taken: Elute the adsorbed salicylic acid into a 1OO-mL volumetric
flask-containing 20 mL of methanol and 0.2 mL of hydrochloric
Result = (rulrs) x (CsIC u) x 100 acid by passing two 1O-mL portions of a 1 in 10 solution of
glacial acetic acid in water-saturated ether, and then 30 mL of
to = peak response of salicylic acid from the Sample chloroform, through the column, and dilute the eluate with
solution chloroform to volume. Dissolvea suitable, accurately weighed

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 392 Aspirin / OfficialMonographs USP 43

quantity of salicylic acid in chloroform to obtain a Standard

solut.ion containing 150 ~g of salicylic acid per mL. Pipet 5 mL
of this solution into a 50-mL volumetric flask containing 10 mL
of methanol, 0.1 mL of hydrochloric acid, and 10 mL of a 1 in
10 solution of glacial acetic acid in ether. Add chloroform to Sample: a quantity of finely powdered Tablets,
volume, and mix. Concomitantly determine the absorbances equivalent to about 500 mg of aspirin, with 10 mL of
of both solutions in l-cm cells at the wavelength of maximum alcohol for several min. Centrifuge the mixture. Pour off the
absorbance at about 306 nm, using as the blank a solvent clear supernatant, and evaporate it to dryness. Dry the
mixture of the same composition as that of the Standard residue under vacuum at 60° for 1 h.
solution: the absorbance of the solution from the Acceptance criteria: Meet the requirements
Suppositories does not exceed that of the Standard solution
(3.0%). ASSAY
Assay- [NoTE-In this assay, usechloroform that recently was • PROCEDURE
saturated with water.] Mobile phase: 2 giL of sodium 1-heptanesulfonate in a
Chromatographic column-Uniformly pack a mixture of acetonitrile and water (15:85). Adjust with
chromatographic tube, as described in the test for Limit of free glacial acetic acid to a pH of 3.4.
salicylic acid for Procedure, with a mixture of about 3 g of Diluent: Acetonitrile and formic acid (99: 1)
chromatographic siliceous earth and 2 mL of sodium Standard solution: 0.5 mg/mL of USP Aspirin RS in Diluent
bicarbonate solution (1 in 12) prepared on the day of use. Sample stock solution: Nominally 5 mg/mL of aspirin
Standard preparation-Transfer about 50 mg of USP prepared as follows. Transfer a quantity, equivalent to
Aspirin RS, accurately weighed, to a 50-mL volumetric flask, about 100 mg of aspirin from NLT 20 finely powdered
add 0.5 mL of glacial acetic acid, and add chloroform to Tablets, to a suitable container. Add 20.0 mL of Diluent and
volume. Transfer 5.0 mL of this solution to a 100-mL 10 glass beads. Shakevigorously for about 10 min, and
volumetric flask, add a 1 in 100 solution of glacial acetic acid centrifuge.
in chloroform to volume, and mix. Sample solution: Nominally 0.5 mg/mL of aspirin in Diluent
Assay preparation-Tare a small dish and glass rod, place in from Sample stock solution
the dish not fewer than 5 Suppositories, heat gently on a steam Chromatographic system
bath until melted, then stir, cool while stirring, and weigh. (See Chromatography (621), System SUitability.)
Transfer an accurately weighed portion of the mass,equivalent Mode: LC
to about 50 mg of aspirin, to a 50-mL volumetric flask Detector: UV 280 nm
containing 1 mL of a 1 in 50 solution of hydrochloric acid in Column: 4.0-mm x 30-cm; packing L1
methanol, add 40 mL of chloroform, mix, and add chloroform Flow rate: 2 mL/min
to volume. Injection volume: 10 ~L
Procedure-Pipet 5 mL of the Assay preparation into the System suitability
column, wash with 5 mL and then with 25 mL of chloroform, Sample: Standard solution
and discard the washings. Without delay, elute into a 100-mL SUitability requirements
volumetric flask with about 10 mL of a 1 in 10 solution of Tailing factor: NMT 2.0
glacial acetic acid in chloroform, and then with about 85 mL Relative standard deviation: NMT 2.0%
of a 1 in 100 solution of glacial acetic acid in chloroform, dilute Analysis
with the latter solvent to volume, and mix. Without delay, Samples: Standard solution and Sample solution
concomitantly determine the absorbances of the eluted Assay Calculate the percentage of the labeled amount of aspirin
preparation and the Standardpreparation in l-cm cells at the (C9 Hs0 4 ) in the portion of Tablets taken:
wavelength of maximum absorbance at about 280 nm, with
a suitable spectrophotometer, using chloroform as, the blank. Result = (rufrs) x (CsICu) x 100
Calculate the quantity, in mg, of aspirin (C9H s0 4) in the
portion of Suppositories taken by the formula: =peak responseof aspirin from the Sample solution
= peak response of aspirin from the Standard
C(AuIAs) solution
= concentration of USP Aspirin RS in the Standard
in which C is the concentration, in ~g per mL, of USP Aspirin solution (mg/mL)
RS in the Standard preparation; and Au and As are the Cu = nominal concentration of aspirin in the Sample
absorbances of the Assay preparation and the Standard solution (mg/mL)
preparation, respectively.
Acceptance criteria: 90.00/0-110.0%
PERFORMANCE TESTS
• DISSOLUTION (711)
Medium: 0.05 M acetate buffer, prepared by mixing 2.99 g
Aspirin Tablets of sodium acetate trihydrate and 1.66 mL of glacial acetic
acid with water to obtain a total of 1000 mL of solution
DEFINITION
Aspirin Tablets contain NLT 90.0% and NMT 110.0% of the with a pH of 4.50 ± 0.05; 500 mL
labeled amount of aspirin (C9H s 0 4 ) . Tablets of larger than Apparatus 1: 50 rpm
Time: 30 min
81-mg size contain no sweeteners or other flavors. Standard solution: A known concentration of USP Aspirin
[NOTE-Tablets that are enteric-coated meet the RS in Medium. Prepare the Standard solutionat the time of
requirements for Aspirin Delayed-Release Tablets.] use. [NOTE-A quantity of alcohol not to exceed 1% of the
IDENTIFICATION total volume of the Standard solution may be used to
• A. The retention time of the aspirin peak of the Sample dissolve the Reference Standard prior to dilution with
solution corresponds to that of the Standard solution, as Medium.]
obtained in the Assay.

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 USP 43 Official Monographs / Aspirin 393

Sample solution: Pass a portion of the solution under test

through a suitable filter, and dilute with Medium, if
Buffered Aspirin Tablets
necessary. DEFINITION
Instrumental conditions Buffered Aspirin Tablets contain Aspirin and suitable buffering
Mode: UV agents. Tablets contain NLT 90.0% and NMT 110.0% of the
Analytical wavelength: 265 nm labeled amount of aspirin (C9H 80 4) .
Analysis
Samples: Standard solution and Sample solution IDENTIFICATION
Determine the percentage of the labeled amount of aspirin • A. The retention time of the aspirin peak of the Sample
(C9H 80 4) dissolved from UV absorbances at the isosbestic solution corresponds to that of the Standard solution, as
point of aspirin and salicylic acid at about 265 nm. obtained in the Assay.
Tolerances: NLT 80% (Q) of the labeled amount of aspirin
(C9 H80 4) is dissolved.
• UNIFORMITY OF DOSAGE. UNITS (905): Meet the
requirements
'>< ,'" •••• " . '. . ,~{·fClY;.2Q.20)
IMPURITIES Sample: Shake a quantity of finely powdered Tablets,
• LIMIT OF FREE SALICYLIC ACID equivalent to about 500 mg of aspirin, with 10 mL of
Mobile phase, Diluent, and Chromatographic system: chloroform for several min. Centrifuge the mixture. Pour off
Proceed as directed in the Assay. the clear supernatant, and evaporate it to dryness.
System suitability solution: 0.015 mg/mL of USP Salicylic Acceptance criteria: Meet the requirements
Acid RS and 0.5 mg/mL of USP Aspirin RS in Diluent
Standard solution: 0.015 mg/mL of USP Salicylic Acid RS in ASSAY
Diluent • PROCEDURE
Sample solution: Use the Sample stocksolution prepared as Mobile phase: 2 gIL of sodium l-heptanesulfonate in a
directed in the Assay. mixture of acetonitrile and water (15:85). Adjust with
System suitability glacial acetic acid to a pH of 3.4.
Samples: System suitability solution and Standard solution Diluent: Acetonitrile and formic acid (99:1)
[NoTE-The relative retention times for salicylic acid Standard solution: 0.5 mg/mL of USP Aspirin RS in Diluent
and aspirin are about 0.7 and 1.0, respectively.] Sample stock solution: Nominally 5 mg/mL of aspirin
Suitability requirements prepared as follows. Transfer a quantity, equivalent to
Resolution: NLT 2.0 between salicylic acid and aspirin, about 100 mg of aspirin from NLT 20 finely powdered
System suitability solution Tablets, to a suitable container. Add 20.0 mL of Diluent and
Relative standard deviation: NMT 4.0%, Standard 10 glass beads. Shake vigorously for 10 min, and centrifuge.
solution Sample solution: Nominally 0.5 mg/mL of aspirin in Diluent
Analysis from Sample stock solution
Samples: Standard solution and Sample solution Chromatographic system
Calculate the percentage of salicylic acid (C 7H60 3) in the (See Chromatography (621), System SUitability.)
portion of Tablets taken: Mode: LC
Detector: UV 280 nm
Result = (rulrs) x (CslCu) x 100 Column: 4.0-mm x 30-cm; packing L1
Flow rate: 2 mL/min
ru = peak response of salicylic acid from the Sample Injection volume: 10 IJL
solution System suitability
ts = peak response of salicylic acid from the Standard Sample: Standard solution
solution Suitability requirements
Cs =concentration of USP SalicylicAcid RS in the Tailing factor: NMT 2.0
Standard solution (mg/mL) Relative standard deviation: NMT 2.0%
Cu = nominal concentration of aspirin in the Sample Analysis
solution (mg/mL) Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of aspirin
Acceptance criteria: NMT 0.3%; for coated Tablets: NMT (C9 H80 4) in the portion of Tablets taken:
3.0%
Result = (rulrs) x (CslCu) x 100
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers. = peak response of aspirin from the Sample solution
Preserve flavored or sweetened Tablets of 81-mg size or = peak response of aspirin from the Standard
smaller in containers holding NMT 36 Tablets each. solution
• USP REFERENCE STANDARDS (11) = concentration of USPAspirin RS in the Standard
USP Aspirin RS solution(mg/mL)
USP SalicylicAcid RS = nominal concentration of aspirin in the Sample
solution (mg/mL)

Acceptance criteria: 90.0%-110.0%

PERFORMANCE TESTS
• DISSOLUTION (711)
Medium: 0.05 M acetate buffer, prepared by mixing 2.99 g
of sodium acetate trihydrate and 1.66 mL of glacial acetic

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 394 Aspirin / Official Monographs USP 43

acid with water to obtain a total of 1000 mL of solution ADDITIONAL REQUIREMENTS

with a pH of 4.50 ± 0.05; 500 mL • PACKAGING AND STORAGE: Preserve in tight containers.
Apparatus 2: 75 rpm. [NoTE-Where the Tablet is composed • USP REFERENCE STANDARDS (11)
of multiple layers, a stainless steel wire helix may be used, USPAspirin RS
if needed, to hold the Tablet in proper orientation in the USP SalicylicAcid RS
apparatus.]
Time: 30 min
Standard solution: A known concentration of USP Aspirin
RS in Medium. Prepare the Standardsolution at the time of
use. [NOTE-A quantity of methanol not to exceed 1% of Aspirin Delayed-Release Tablets
the total volume of the Standardsolution may be used to
dissolve the Reference Standard prior to dilution with DEFINITION
Medium.] Aspirin Delayed-Release Tablets contain NLT95.0% and NMT
Sample solution: Pass a portion of the solution under test 105.0% of the labeled amount of aspirin (C9H a0 4) .
through a suitable filter, and dilute with Medium, if IDENTIFICATION
necessary.
• A. The retention time of the aspirin peak of the Sample
Instrumental conditions solution corresponds to that of the Standardsolution, as
Mode: UV
obtained in the Assay.
Analytical wavelength: 265 nm
Analysis
Samples: Standardsolution and Sample solution
Determine the percentage of the labeled amount of aspirin
(C9H a0 4) dissolved from UV absorbances at the isosbestic )
point of aspirin and salicylic acid at about 265 nm. Sample: Shake a quantity 0 finely powdered Tablets,
Tolerances: NLT 80% (Q) of the labeled amount of aspirin equivalent to about 500 mg of aspirin, with 10 mL of
(C9H a0 4) is dissolved. alcohol for several minutes. Centrifuge the mixture. Pour
• UNIFORMITY OF DOSAGE UNITS (905): Meet the off the clear supernatant, and evaporate it to dryness. Dry
requirements the residue under vacuum at 60° for 1 h.
IMPURITIES Acceptance criteria: Meet the requirements
• LIMIT OF FREE SALICYLIC ACID ASSAY
Mobile phase, Diluent, and Chromatographic system: • PROCEDURE
Proceed as directed in the Assay. Mobile phase: 2 gIL of sodium 1-heptanesulfonate in a
System suitabiUty solution: 0.015 mg/mL of USP Salicylic mixture of acetonitrile and water (15:85). Adjust with
Acid RS and 0.5 mg/mL of USP Aspirin RS in Diluent glacial acetic acid to a pH of 3.4.
Standard solution: 0.015 mg/mL of USP SalicylicAcid RS in Diluent: Acetonitrile and formic acid (99:1)
Diluent Standard solution: 0.5 mg/mL of USP Aspirin RS in Diluent
Sample solution: Use the Sample stocksolution prepared as Sample stock solution: Nominally 5 mg/mL of aspirin
directed in the Assay. prepared as follows. Transfer a quantity equivalent to about
System suitability 100 mg of aspirin from NLT 20 finely powdered Tablets to
Samples: System sUitability solution and Standardsolution a suitable container. Add 20.0 mL of Diluent and 10 glass
[NoTE-The relative retention times for salicylic acid beads. Shake vigorously for 10 min, and centrifuge.
and aspirin are about 0.7 and 1.0, respectively.] Sample solution: Nominally 0.5 mg/mL of aspirin in Diluent
SUitability requirements from Sample stocksolution .
Resolution: NLT 2.0 between salicylic acid and aspirin, Chromatographic system
System suitability solution (See Chromatography (621), System Suitability.)
Relative standard deviation: NMT 4.0%, Standard Mode: LC
solution Detector: UV 280 nm
Analysis Column: 4.0-mm x 30-cm; packing L1
Samples: Standardsolution and Sample solution Flow rate: 2 mL/min
Calculate the percentage of salicylic acid (C7H60 3) in the Injection volume: 10 ~L
portion of Tablets taken: System suitability
Sample: Standardsolution
Result = (rulr s) x (CslCu) x 100 Suitability requirements.
Tailing factor: NMT 2.0
tu = peak response of salicylic acid from the Sample Relative standard deviation: NMT 2.0%
solution Analysis
ts = peak response of salicylic acid from the Standard Samples: Standardsolution and Sample solution
solution Calculate the percentage of the labeled amount of aspirin
Cs = concentration of USP SalicylicAcid RS in the (C9H a0 4) in the portion of Tablets taken:
Standardsolution (mg/mL)
Cu = nominal concentration of aspirin in the Sample Result =(rulr s) x (CsICu) x 100
solution (mg/mL)
=peak response of aspirin from the Sample solution
Acceptance criteria: NMT 3.0% = peak response of aspirin from the Standard
SPECIFIC TESTS solution
• ACID-NEUTRALIZING CAPACITY (301): NLT 1.9 mEq of acid = concentration of USP Aspirin RS in the Standard
is consumed for each 325 mg of aspirin in the Tablets. solution (mg/mL)

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 USP 43 Official Monographs / Aspirin 395

Cu = nominal concentration of aspirin in the Sample = peak response of salicylic acid from the Standard
solution (mg/ml) solution
= concentration of USP Salicylic Acid RS in the
Acceptance criteria: 95.0%-105.0% Standardsolution (mg/mL)
PERFORMANCE TESTS Cu =nominal concentration of aspirin in the Sample
solution (mg/mL) .
• DISSOLUTION (711 )," Procedure, Apparatus 1 and Apparatus 2,
Delayed-Release Dosage Forms, Method B Procedure Acceptance criteria: NMT 3.0%
Apparatus 1: 100 rpm
Times ADDITIONAL REQUIREMENTS
Acid stage: 2 h • PACKAGING AND STORAGE: Preserve in tight containers.
Buffer stage: 90 min • USP REFERENCE STANDARDS (11)
Diluent: 0.1 N hydrochloric acid and 0.20 M tribasic sodium USP Aspirin RS
phosphate (3: 1). Adjust, if necessary, with 2 N hydrochloric USP Salicylic Acid RS
acid or 2 N sodium hydroxide to a pH of 6.8 ± 0.05.
Standard solution: USP Aspirin RS of a known
concentration in 0.1 N hydrochloric acid (for analyzing the
Acidstage) and in Diluent (for analyzing the Buffer stage)
Sample solution: Pass a portion of the solution under test Aspirin Effervescent Tablets for Oral
through a suitable filter, diluted, if necessary, with 0.1 N Solution
hydrochloric acid (for analyzing the Acid stage) and with
Diluent (for analyzing the Buffer stage). DEFINITION
Instrumental conditions Aspirin Effervescent Tablets for Oral Solution contain Aspirin
Mode: UV and an effervescent mixture of a suitable organic acid and an
Analytical wavelengths alkali metal bicarbonate and/or carbonate. Tablets contain
Acid stage: 280 nm NLT 90.0% and NMT 110.0% of the labeled amount of
Buffer stage: 265 nm aspirin (C9H a0 4 ) .
Analysis
Samples: Standardsolution and Sample solution IDENTIFICATION
Determine the percentage of the labeled amount of aspirin • A. The retention time of the aspirin peak of the Sample
(C9H a0 4) dissolved by determining UVabsorbances at the solution corresponds to that of the Standardsolution as
isosbestic point of aspirin and salicylic acid (about 280 nm obtained in the Assay. '
in the Acidstage, and about 265 nm in the Buffer stage). • B.
Tolerances . Sample: Y2 Tablet
Acid stage: NMT 10% (Q) of the labeled amount of aspirin Analysis: Add the Sample to 50 ml of water in a flask, and
(C9H a0 4) is dissolved. immediately stopper with a stopper fitted with tubing so
Buffer stage: NLT 75% (Q) of the labeled amount of that the evolved gas passes through calcium hydroxide TS.
aspirin (C9H a0 4) is dissolved. . Acceptance criteria: A white precipitate forms.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the ASSAY
req uirements • PROCEDURE
IMPURITIES Mobile phase: 2 gIL of sodium l-heptanesulfonate in a
• LIMIT OF FREE SALICYLIC ACID mixture of acetonitrile and water (15:85). Adjust with
Mobile phase, Diluent, and Chromatographic system: glacial acetic acid to a pH of 3.4.
Proceed as directed in the Assay. . Diluent: Acetonitrile and formic acid (99:1)
System suitability solution: 0.015 mg/ml of USP Salicylic Standard solution: 0.5 mg/ml of USP Aspirin RS in Diluent
Acid RS and 0.5 mg/ml of USP Aspirin RS in Diluent Sample stock solution: Nominally 5 mg/ml of aspirin
Standard solution: 0.015 mg/mL of USP Salicylic Acid RS in prepared as follows. Transfer a quantity, equivalent to
Diluent about 100 mg of aspirin from NlT 20 finely powdered
Sample solution: Use the Sample stocksolution from the Tablets, to a suitable container. Add 20.0 ml of Diluent and
Assay. 10 glass beads. Shake vigorously for 10 min, and centrifuge.
System suitability Sample solution: Nominally 0.5 mg/ml of aspirin in Diluent
Samples: System suitability solution and Standardsolution from Sample stock solution
[NoTE-The relative retention times for salicylic acid Chromatographic system
and aspirin are about 0.7 and 1.0, respectively.] (See Chromatography (621), System Suitability.)
Suitability requirements Mode: LC
Resolution: NlT 2.0 between salicylic acid and aspirin, Detector: UV280 nm
System sUitabilitysolution Column: 4-mm x 30-cm; packing L1
Relative standard deviation: NMT 4.0%, Standard Flow rate: 2 mL/min
solution Injection volume: 10 IJl
Analysis System suitability
Samples: Standardsolution and Sample solution Sample: Standardsolution
Calculate the percentage of salicylic acid (C 7H 6 0 3) in the Suitability requirements
Tailing factor: NMT 2.0
portion of Tablets taken:
Relative standard deviation: NMT 2.0%
Result = (rulrs) x (Cs/Cu) x 100 Analysis
Samples: Standardsolution and Sample solution
ru = peak response of salicylic acid from the Sample Calculate the percentage of the labeled amount of aspirin
solution . (C9H a0 4 ) in the portion Tablets taken:

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 396 Aspirin / OfficialMonographs USP 43

Result = (ru/rs) x (Cs/C u) x 100

Aspirin Extended-Release Tablets
= peak response of aspirinfrom the Sample solution
DEFINITION
= peak response of aspirin from the Standard
solution Aspirin Extended-Release Tabletscontain NLT 95.0% and
= concentration of USP Aspirin RS in the Standard
NMT 105.0% of the labeled amount of aspirin (C9H s 0 4) .
solution (mg/mL) IDENTIFICATION
= nominal concentration of aspirin in the Sample • A. The retention time of the aspirin peak of the Sa,mple
solution (mg/mL) solution corresponds to that of the Standard solution, as
obtained in the Assay.
Acceptance criteria: 90.00/0-110.0%
PERFORMANCE TESTS
• SOLUTION TIME: NMT 5 min for 2 Tablets completely
dissolved in 180 mL of water at 17.5 ± 2.5°. • B~~~i~
SpggtrQsp
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements Sample: Shake a ~~wde~ed Tablets,
equivalent to mg of aSpirin, with 1.0 mLof
IMPURITIES alcohol for several minutes. Centnfuge the mixture. Pour
• LIMIT OF FREE SALICYLIC ACID off. the clear supernatant, and evaporate it to dryness. Dry
Mobile phase, Diluent, and Chromatographic system: the residue under vacuum at 60° for 1 h.
Proceed as directed in the Assay. . . Acceptance criteria: Meet the requirements
System suitability solution: 0.015 mg/mL of USP Salicylic
ASSAY
Acid RS and 0.5 mg/mL of USP Aspirin RS in pil~ent .
Standard solution: 0.015 mg/mL of USP Salicylic ACid RS • PROCEDURE
in Diluent Mobile phase: 2 gil of sodium 1-heptanesulf?nate .in a
Sample solution: Use the Sample stocksolution prepared as mixture of acetonitrile and water (15:85). Adjustwith
directed in the Assay. glacial acetic acid to a pH of 3.4.
System suitability . Diluent: Acetonitrile and formic acid (99:1)
Samples: System sUitability.solu~ion and Stc:nde;trd s?'utlon Standard solution: 0.5 mg/mL of USP Aspirin RS i~ ,?iluent
[NoTE-The relative retention times for salicylic acid Sample stock solution: Nominally 5 mg/mL of aspmn
and aspirin are 0.7 and 1.0, respectively.] prepared as follows. Transfer a quantity, equivalent to
Suitability requirements about 100 mg of aspirin from NLT 20 finely pow~ered
Resolution: ,NLT 2.0 between salicylic acid and aspirin, Tablets to a suitable container. Add 20.0 mLof DIluent and
System suitability solution 10 glas~ beads. Shake vigorously for about 10 min, and
Relative standard deviation: NMT4.0%, Standard centrifuge. ... .
solution Sample solution: Nominally 0.5 mg/mL of aspirm In Dtluent
~a~ili . from Sample stock solution
Samples: Standard solution and Sample solution Chromatographic system
Calculatethe percentage of salicylic acid (C7H60 3) in the (See Chromatography (621), System Suitability.)
Mode: LC
portion of Tablets taken: Detector: UV 280 nm
Result = (rulrs) x (CslCu) x 100 Column: 4.0-mm x 30-cm; packing L1
Flow rate: 2 mL/min
= peak response of salicylic acid from the Sample Injection volume: 10 IJL
solution System suitability
= peak response of salicylic acid from the Standard Sample: Standard solution
solution Suitability requirements
= concentration of USP Salicylic Acid RS in the Tailing factor: NMT 2.0
Standard solution (mg/mL) Relative standard deviation: NMT 2.0%
= nominal concentration of aspirin in the Sample Analysis
solution (mg/mL) Samples: Standard solution and Sample solution
Calculatethe percentage of the labeled amount of aspirin
Acceptance criteria: NMT 8.0% (C9H s 0 4) in the portion of Tabletstaken:
SPECIFIC TESTS Result = (ru/rs) x (CslCu) x 100
• ACID-NEUTRALIZING CAPACITY (301): NLT 5.0 mEq of acid
is consumed by 1 Tablet. = peak response of aspirin from the Sample solution
ADDITIONAL REQUIREMENTS = peak response of aspirin from the Standard
• PACKAGING AND STORAGE: Preserve in tight containers.
solution
= concentration of USP Aspirin RS in the Standard
• USP REFERENCE STANDARDS (11)
USP Aspirin RS solution (mg/mL)
= nominal concentration of aspirin in the Sample
USP Salicylic Acid RS
solution (mg/mL)
Acceptance criteria: 95.00/0-105.0%
PERFORMANCE TESTS
• DISSOLUTION (711)
Test 1: Ifthe product complie~ With. this test, the labeling
indicates that it meets USP Dissoiution Test 7.

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 USP 43 Official Monographs / Aspirin 397

Medium: 0.1 N hydrochloric acid; 900 mL IMPURITIES

Apparatus 2: 60 rpm • LIMIT OF FREE SALICYLIC ACID
Times: 1 and 4 h Mobile phase, Diluent, and Chromatographic system:
Standard solution: A known concentration of USPAspirin Proceed as directed in the Assay.
RS in Medium System suitability solution: 0.015 mg/mL of USP Salicylic
Sample solution: Pass a portion of the solution under test Acid RS and 0.5 mg/mL of USP Aspirin RS in Diluent
through a suitable filter, and dilute with Medium, if Standard solution: 0.015 mg/mL of USP SalicylicAcid RS in
necessary. Diluent
Instrumental conditions Sample solution: Use the Sample stock solution prepared as
Mode: UV directed in the Assay.
Analytical wavelength: 280 nm System suitability
Analysis Samples: System suitabilitysolution and Standardsolution
Samples: Standard solution and Sample solution [NoTE-The relative retention times for salicylic acid
Determine the percentage of the labeled amount of and aspirin are about 0.7 and 1.0, respectively.]
aspirin (C9 Hs04 ) dissolved from UV absorbances at the Suitability requirements
isosbestic point at about 280 nm. Resolution: NLT 2.0 between salicylic acid and aspirin,
Tolerances: See Table 1. System SUitability solution
Relative standard deviation: NMT 4.0%, Standard
Table 1 solution
Amount Analysis
Time Dissolved Samples: Standard solution and Sample solution
(h) (%) Calculate the percentage of salicylic acid (C7 H603) in the
1 20-55 portion of Tablets taken:
4 NLT 80 Result = (ru/rs) x (Cs/Cu) x 100

The percentages of the labeled amount of aspirin . ru = peak response of salicylic acid from the Sample
(C9 Hs04) dissolved at the times specified conform to solution
Dissolution (711), Acceptance Table 2. rs = peak response of salicylic acid from the Standard
Test 2: If the product complies with this test, the labeling solution
indicates that it meets USP Dissolution Test 2. Cs =concentration of USP SalicylicAcid RS in the
Medium: Water; 1000 mL Standard solution (mg/mL)
Apparatus 2: 30 rpm Cu = nominal concentration of aspirin in the Sample
Times: 1, 2, 4, and 8 h solution (mg/mL)
Standard solution: A known concentration of USP Aspirin
RS in Medium. Prepare the Standard solution at the time Acceptance criteria: NMT 3.0%
of use. [NOTE-A quantity of alcohol not to exceed 5% of ADDITIONAL REQUIREMENTS
the total volume of the Standard solution may be used to • PACKAGING AND STORAGE: Preserve in tight containers.
dissolve the USP Reference Standard prior to dilution with • LABELING: The labeling indicates the Dissolution test with
Medium.] which the product complies.
Sample solutions: Pass a portion of the solution under test • USP REFERENCE STANDARDS (11)
through a suitable filter, and dilute with Medium, if USP Aspirin RS
necessary. USP Salicylic Acid RS
Instrumental conditions
'Mode: UV
Analytical wavelength: 265 nm
Analysis
Samples: Standardsolution and Sample solutions Aspirin, Alumina, and Magnesia Tablets
Determine the percentage of the labeled amount of
aspirin (C9H s0 4) dissolved from UV absorbances at the DEFINITION
isosbestic point at about 265 nm. Aspirin, Alumina, and Magnesia Tablets contain NLT90.0%
Tolerances: See Table 2. and NMT 110.0% of the labeled amount of aspirin (C9H s0 4) ,
the equivalent of NLT 90.0% and NMT 110.0% of the
Table 2 labeled amount of aluminum hydroxide [AI(OH)3], and NLT
Amount 90.0% and NMT 110.0% of the labeled amount of
Time Dissolved magnesium hydroxide [Mg(OH)2]'
(h) (%)
IDENTIFICATION
1 15-40 • A. The retention time of the aspirin peak of the Sample
2 25-60 solution corresponds to that of the Standardsolution, as
obtained in the Assay for Aspirin.
4 35-75 • B. IDENTIFICATION TESTS-GENERAL (191), Magnesium
8 NLT 70 Sample solution: To a 0.7-g portion of finely powdered
Tablets, add 20 mL of 3 N hydrochloric acid and 5 drops of
methyl red TS, heat to boiling, and add 6 N ammonium
The percentages of the labeled amount of aspirin
hydroxide until the color of the solution changes to deep
(C9H s0 4) dissolved at the times specified conform to
yellow. Continue boiling for 2 min, and filter. Use the filtrate
Dissolution (711), Acceptance Table 2. for analysis and use the precipitate in Identification C.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Acceptance criteria: .Meet the requirements
requirements

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398 Aspirin / OfficialMonographs USP43

• c. IDENTIFICATION TESTS-GENERAL (191), Aluminum • ALUMINUM HYDROXIDE

Sample solution: Wash the precipitate obtained in Sample solution: Nominally 1.25 mg/mL. of aluminum
Identification B with a hot solution of ammonium chloride hydroxide prepared asfollows. To a portion of NLT 20
(1 in 50), and dissolve the precipitate in hydrochloric acid. powdered Tablets, equivalent to 250 mg of aluminu.m
Acceptance criteria: Meet the requirements hydroxide in a 150-mL beaker, add 20 mL of water, stir, and
slowly add 30 mL of 3 N hydrochloric acid. Heat gently, if
ASSAV necessary, to aid solution, cool, and .transfer to a ~OO-mL
• ASPIRIN volumetric flask. Wash the beaker with water, adding the
Mobile phase: Dissolve 225 mg of tetramethylammonium washings to the flask, dilute with water to volume, and mix.
hydroxide pentahydrate and 200 mg of sodium Titrimetric system
l-octanesulfonate in 700 mL of water. Add 150 mL of Mode: Residual titration
methanol, 150 mL of acetonitrile, and 1.0 mL of glacial Titrant: 0.05 M edetate disodium VS
acetic acid, and stir. Back titrant: 0.05 M zinc sulfate VS
Diluent: To 2 9 of anhydrous citric acid add 990 mL of. Blank: Water, 50 mL
acetonitrile, 990 mL of chloroform, and 20 mL of formic Endpoint detection: Visual
acid, and stir for about 30 min. Allow to settle, and use the Analysis: To 50 mL of Sample solution add, in the ord~r
decanted clear solution. named and with continuous stirring, 25.0 mL of the Titrant
Internal standard solution: 2 mg/mL of phenacetin in and 20 mL of acetic acid-ammonium acetate buffer TS,and
Diluent heat the solution near the boiling temperature for 5 min.
.Salicylic acid stock solution: 1 mg/mL of USP Salicylic Acid Cool, and add 50 mL of alcohol and 2 mL of dithizone TS.
RS in Diluent Titrate with Back titrant until the color changes from
Standard solution: 6.5 mg/mL of USP Aspirin RS and 0.2 green-violet to rose-pink. Perform a blank determination,
mg/mL each of USP SalicylicAcid RS and phenacetin substituting 50 mL of water for the Sample solution, and
prepared as follows. Transfer, accurately weighed, about make any necessary corrections. Each mL of Titrant
325 mg of USP Aspirin RS to a 50-mL volumetric flask. Add consumed is equivalent to 3.900 mg of aluminum
10.0 mL of Salicylic acidstock solution and 5.0 mL of Internal hydroxide [AI(OH)3]'
standard solution, dilute with Diluent to volume, and mix. . Acceptance criteria: 90.0%-110.0%
Sample solution: Nominally 6.5 mg/mL of aspirin prepared
• MAGNESIUM HYDROXIDE
as follows. Transfer a quantity equivalent to about 325 mg Indicator solution: Dissolve by mixing 200 mg of
of aspirin from NLT 20 finely powdered Tablets to a eriochrome black T in a mixture of 15 mL of triethanolamine
screw-capped, 120-mL bottle. Add.5.0 mL .of Internal. and 5 mL of dehydrated alcohol.
standardsolution and 45.0 mL of Diluent, mix, and sonicate Sample solution: Prepare as directed in the Assay for
for 2-5 min. Centrifuge, and usethe resultant clear
Aluminum Hydroxide.
solution. • Titrimetric system
Chromatographic system Mode: Direct titration
(See Chromatography (621), System Suitability.) Titrant: 0.05 M edetate disodium VS
Mode: LC
Blank: Water, 50 mL
Detector: UV 280 nm
Endpoint detection: Visual
Column: 4-mm x 30-cm; 10-l.Jm packing L1 Analysis: To a volume of Sample solution, equivalent to 80
Flow rate: 2 mL/min mg of magnesium hydroxide, add 200 mL of water, 20 mL
Injection volume: 5 I.JL of triethanolamine, 50 mL of ammonia-ammonium
System suitability chloride buffer TS, and 2 drops of Indicator solution. Cool
Sample: Standardsolution. , . . . the solution to between 3° and 4° by immersion in an ice
[NoTE-The relative retention times for salicylic acid, bath then remove, and titrate with Titrant until the color
aspirin, and phenacetin are about 0.3, 0.6, and 1.0, chan'ges to pure blue. Perform a blank determination,
respectively. Record each chromatogram until the substituting for the Sample solution a volume of water equal
chloroform peak appears at the relative retention to the volume of Sample solution used, and make any
time of about 1.8.] necessary corrections. Each mL of Titrant is equivalent to
Suitability requirements 2.916 mg of magnesium hydroxide [Mg(OH)2]'
Resolution: NLT 2.0 between two adjacent peaksfor
Acceptance criteria: 90.0%-110.0%
salicylic acid, aspirin, and phenacetin
Tailing factor: NMT 2.0 for each peak PERFORMANCE TESTS
Relative standard deviation: NMT 3.0% • DISSOLUTION (711)
Analysis Medium: 0.05 M acetate buffer, prepared by mixing 2.99 9
Samples: Standardsolution and Sample solution of sodium acetate (trihydrate) and 1.66 mL of glacial acetic
Calculate the percentage of the labeled amount of aspirin acid with water to obtain 1000 mL of solution with a pH of
(C9H s0 4) in the portion of Tablets taken: 4.50 ± 0.05; 900 mL
Apparatus 2: 75 rpm
Result = (Ru/R s) x (CslCu) x 100 Time: 45 min
Standard solution: A known concentration of USP Aspirin
= peak response ratio of aspirin to phenacetin RS in Medium. Prepare the Standardsolution at the time of
from the Sample solution use. [NOTE-A quantity of methanol NMT 1% of the total
= peak response ratio of aspirin to phenacetin volume of the Standardsolution may be used to dissolvethe
from the Standardsolution Reference Standard into solution prior to dilution with
= concentration of USP Aspirin RS in the Standard Medium.]
solution (mg/mL) Sample solution: Pass a portio~ of th~ soluti0!1 unger test
= nominal concentration of aspirin in the Sample through a suitable filter, and dilute With Medium, If
solution (mg/mL) necessary.
Acceptance criteria: 90.0%-110.0%

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 USP 43 Official Monographs / Aspirin 399

Instrumental conditions
Mode: UV Aspirin, Alumina, and Magnesium
Analytical wavelength: 265 nm Oxide Tablets
Analysis
Samples: Standard solution and Sample solution DEFINITION
Determine the percentage of the labeled amount of aspirin Aspirin, Alumina, and Magnesium Oxide Tablets contain NLT
(C9H B0 4) dissolved from UV absorbances at the isosbestic 90.0% and NMT 110.0% of the labeled amount of aspirin
point of aspirin and salicylic acid at about 265 nm. (C9HB0 4) , the equivalent of NLT 90.0% and NMT 110.0% of
Tolerances: NLT 75% (Q) of the labeled amount of aspirin the labeled amount of aluminumhydroxide [AI(OH)3], and
(C9H B0 4) is dissolved. NLT 90.0% and NMT 110.0% of the labeled amount of
• UNIFORMITY OF DOSAGE UNITS (905), Weight Variation and magnesium oxide (MgO).
Content Uniformity: Meet the requirements for weight
IDENTIFICATION
variation with respect to aluminum hydroxide and to
SAMPLE: The Sampleisprepared as follows. To a O.7-g portion
magnesium hydroxide. Meet the requirements for content
uniformity with respect to aspirin. of finely powdered Tablets, add 20 mLof 3 N hydrochloric
acid and 5 drops of methyl red TS, heat to boiling, and add
IMPURITIES 6 N ammonium hydroxide until the color of the solution
• LIMIT OF FREE SALICYLIC ACID changes to deep yellow. Continue boilingfor 2 min, and
Mobile phase, Diluent, Internal standard solution, filter. The filtrate is used in Identification test B, and the
Salicylic acid stock solution, Sample solution, and precipitate is used in Identification test C.
Chromatographic system: Proceed as directed in the o A. The retention time of the aspirin peak of the Sample
Assayfor Aspirin. solution corresponds to that of the Standard solution, as
System suitability solution: Transferabout 325 mg of USP obtained in the Assay.
Aspirin RS to a 50-mL volumetric flask. Add 10.0 mL of • B. IDENTIFICATION TESTS-GENERAL, Magnesium (191)
Salicylic acid stock solution and 5.0 mL of Internal standard Sample solution: Sample filtrate
solution, dilute with Diluent to volume, and mix. Acceptance criteria: Meet the requirements
Standard solution: 0.2 mg/mL of USP Salicylic Acid RS • C. IDENTIFICATION TESTS-GENERAL, Aluminum (191)
prepared as follows. Transfer 10.0 mLof Salicylic acid stock Sample solution: Wash the Sample precipitate with a hot
solution and 5.0 mLof Internal standard solution to a 50-mL solution of 20 mg/mL of ammonium chloride, and dissolve
volumetricflask, dilute with Diluent to volume, and mix. the precipitate in hydrochloric acid.
System suitability Acceptance criteria: Meet the requirements
Samples: System suitability solution and Standardsolution • D. PROCEDURE
[NOTE-The relative retention times for salicylic acid, Analysis: Where the Tablets are composed of two layers,
aspirin, and phenacetin are about 0.3, 0.6, and 1.0, scrape a smallamount of each layerinto separate test tubes.
respectively. Record each chromatogram until the Add 2 mLof water and 2 drops of methyl red TS to each
chloroform peak appears at the relative retention tube, and shake for 15 s.
time of about 1.8.] Acceptance criteria: The solutionfrom the
Suitability requirements aspirin-containing layer is red, and the solution from the
Resolution: NLT 2.0 between two adjacent peaksfor buffer-containing layer isyellow.
salicylic acid, aspirin, and phenacetin, System suitability
ASSAY
solution
Tailing factor: NMT 2.0, System suitabjfjty solution • ASPIRIN
Relative standard deviation: NMT 3.0%, Standard Mobile phase: Methanol, phosphoric acid, and water
solution (30:3:70)
Analysis Diluent: Dehydrated alcohol and hydrochloric acid
Samples: Standard solution and Sample solution (2000:20)
Calculate the percentage of salicylic acid in the portion of Aspirin standard stock solution: 5 mg/mL of USP Aspirin
Tablets taken: RS in Diluent prepared by blending at high speed for 1.5
min
Result = (Ru/R s) x (Cs/Cu) x 100 Aspirin standard solution: 0.25 mg/mL of USP Aspirin RS
prepared immediatelyfrom the Aspirinstandard stock
Ru =peak response ratio of salicylic acid to phenacetin solution in dehydrated alcohol. Use these solutions within 1
from the Sample solution h.
Rs = peak response ratio of salicylic acid to phenacetin Salicylic acid standard stock solution: 5 mg/mL of USP
from the Standard solution Salicylic Acid RS in dehydrated alcohol. Transfer 3.0 mLof
Cs = concentration of USP Salicylic Acid RS in the this solution to a 1OO-mL volumetricflask, and dilute with
Standard solution (mg/mL) Diluent to volume.
Cu = nominal concentration of aspirin in the Sample Salicylic acid standard solution: 7.5 pq/rnl, of USP Salicylic
solution (mg/mL) Acid RS from the Salicylic acid standard stock solution in
dehydrated alcohol
Acceptance criteria: NMT 3.0% System suitability solution: Transfer 5.0 mLof the Aspirin
standard stock solution to a 1OO-mL volumetric flask, add
SPECIFIC TESTS 5.0 mLof the Salicylic acidstandardstocksolution, and dilute
• ACID-NEUTRALIZING CAPACITY (301): NLT 1.9 mEq of acid with dehydrated alcohol to volume.
is consumed for each 325 mg of aspirin in the Tablets. Sample solution: Transfer a counted number of Tablets,
ADDITIONAL REQUIREMENTS
equivalent to 2500 mg of aspirin, to a 120-mL blender jar
• PACKAGING AND STORAGE: Preserve in tight containers. containing 100.0 mL of Diluent, and blend at high speed
• USP REFERENCE STANDARDS (11)
for 1.5 min. Immediately filtera portion of the mixture thus
USP Aspirin RS . obtained, and transfer 1.0 mLof the filtrate to a 100-mL
USP Salicylic Acid RS volumetric flask. Immediately dilute with dehydrated

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400 Aspirin / OfficialMonographs, USP 43

alcohol to volume. Promptly inject this Sample solution into add 20 mL of water, stir, and slowly add 30 mL of 3 N
the chromatograph as directed for Analysis. hydrochloric acid. Heat gently, if necessary, to aid solution,
Chromatographic system cool, and transfer to a 200-mL volumetric flask. Washthe
(See Chromatography (621), System Suitability.) beaker with water, adding the washings to the flask, and
Mode: LC add water to volume. .
Detector: UV 205 nm Analysis: To 20 mL of the Sample solution in a 250
Column: 4.6-mm x 3-cm; 5-~m packing L7 mL-beaker, add 20 mL of water; then add, in the order
Flow rate: 3.5 mL/min named and with continuous stirring, 25.0 mL of Edetate
Injection volume: 10 ~L disodium titrant and 20 mL of acetic acid-ammonium
System suitability acetate buffer TS, and heat the solution near the boiling
Samples: Aspirin standard solution, Salicylic acidstandard temperature for 5 min. Cool, and add 50 mL of alcohol and
solution, and System suitability solution 2 mL of dithizone TS. Titrate with 0.05 M zinc sulfate VS
[NoTE-The relative retention times for aspirin and until the color changes from green-violet to rose-pink.
salicylic acid are 0.7 and 1.0, respectively.] Perform a blank determination, substituting 10 mL of water
Suitability requirements for the Sample solution, and make any necessary correction.
Resolution: NLT 2.0 between the aspirin peak and the Each mL of 0.05 M Edetate disodium titrant is equivalent to
salicylic acid peak, System suitability solution 3.900 mg of aluminum hydroxide [AI(OH)3]'
Tailing factor: NMT 2.0 for the aspirin and salicylic acid Acceptance criteria: 90.0%-110.0%
peaks, Aspirin standard solution and Salicylic acid standard • MAGNESIUM OXIDE
solution Sample solution: Prepare as directed in the Assay for
Relative standard deviation: NMT 2.0% for the aspirin Aluminum Hydroxide.
and salicylic acid peaks, Aspirin standard solution and Eriochrome black indicator solution: Dissolve 200 mg of
Salicylic acid standard solution eriochrome black T in a mixture of 15 mL of triethanolamine
Analysis and 5 mL of dehydrated alcohol.
Samples: Aspirin standard solution, Salicylic acidstandard Titrant: 0.05 M edetate disodium VS
solution, and Sample solution Analysis: To a volume of the Sample solution equivalent to
Calculate the percentage of aspirin (C9H s0 4) in each Tablet 40 mg of magnesium oxide in a 400 mL beaker, add, while
taken: mixing, 20 mL of triethanolamine and 200 mL of water.
Cool the solution for 10 min, while stirring, by immersion
Result = (rulrs) x (CsICu) x 100 in an ice bath. Remove the beakerfrom the ice bath, and
add 15 mL of ammonia-ammonium chloride buffer TSand
t» = peak response of aspirin from the Sample solution 2 drops of Eriochrome black indicator solution. Titrate with
rs = peak.response of aspirin from the Standard Titrant to a blue endpoint, allowing 60 s between drops of
solution Titrant as the endpoint is approached (after first color
Cs = concentration of USP Aspirin RS in the Aspirin change is observed). The titration should be completed
standard solution (mg/mL) within 10 min after the addition of the buffer and indicator.
Cu = nominal concentration of aspirin in the Sample If any precipitate is observed prior to titration, the solution
solution (mg/mL) should be discarded, and a new solution prepared. Perform
a blank determination, substituting an equivalent volume
Acceptance criteria: 90.00/0-110.0% of water for the volume of Sample solution used, and make
• ALUMINUM HYDROXIDE any necessary correction. Each mL of Titrant consumed is
Edetate disodium titrant: Dissolve 18.6 g' of edetate equivalent to 2.015 mg of magnesium oxide (MgO).
disodium in water to make 1000 mL, and standardize the Acceptance criteria: 90.0%-110.0%
solution asfollows. Weigh 2 g of aluminum wire, transfer
to a 1OOO-mL volumetric flask, and add 50 mL of a mixture PERFORMANCE TESTS
of hydrochloric acid and water (1:1). Swirl the flask to • DISSOLUTION (711)
ensure contact of the aluminum and the acid, and allow the Medium: 0.05 M acetate buffer, prepared by mixing 2.99 g
reaction to proceed until all of the aluminum has dissolved. of sodium acetate (trihydrate) and 1.66 mL of glacial acetic
Dilute with water to volume. Pipet 10 mL of this solution acid with water to obtain 1000 mL of solution having a pH
into a 250-mL beaker; add, in the order named and with of 4.50 ± 0.05; 900 mL
continuous stirring, 25.0 mL of edetate disodium titrant Apparatus 1 (lO-mesh screen): 100 rpm
and 20 mL of acetic acid-ammonium acetate buffer TS; and Time: 45 min
boil gently for 5 min. Cool, and add 50 mL of alcohol and Determine the amount of aspirin (C9H s 0 4 ) dissolved,
2 mL of dithizone TS. Titrate with 0.05 M zinc sulfate VS to employing the following method.
a bright rose-pink color. Perform a blank determination, Alkaline detergent solution: 30% solution of
substituting 10 mL of water for the aluminum solution, and polyoxyethylene (23) lauryl ether and 1 N sodium
make any necessary correction. hydroxide (0.5: 1000)
Calculate the molarity of the solution taken: pH 4.3 buffer detergent: 12.9 gIL of citric acid
monohydrate and 20.6 gIL of dibasic sodium phosphate
Result = (WI A,) x V heptahydrate in water. Add 0.5 mL of a 30% solution of
polyoxyethylene (23) lauryl ether.
W = weight of aluminum in the portion of the solution Standard solution: 0.45 mg/mL of USP Aspirin RS in
taken (mg) Medium
Ar = atomic weight of aluminum, 26.98 Sample solution: Filtered portion of sample
V = volume of Edetate disodium titrant consumed Analysis: Usean automatic analyzerconsisting of (1) a liquid
(mL) sampler, (2) a proportioning pump, (3) a suitable
fluorometer equipped with a OA-cm flow cell and suitable
Sample solution: To a portion of the powdered Tablets recording devices, and (4) a manifold consisting of the
(NLT 20) equivalent to 600 mg of aluminum hydroxide, components illustrated in Figure 7.

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USP 43 Official Monographs / Aspirin 401

To washI ~ (2.00) pH 4.3 Buffer detergent

receptac e
(0.32) Air
20T (1.40) Alkaline detergent solution
(0.39) Sample*
(0.47) Aux. sample
Waste + - - - - {

*** Sampler

Waste
(0.32) Air Sample rate: 40 per hour

*** (2.00) pH 4.3 Buffer detergent

(0.39) Resample*
(1.00) From flow cell
Fluorometer
Waste
AActive 298 nm
AFluor 425 nm Legend:
-Acld flex pump tube
**SmallID polyethylene
(0.38 mm x 1.09 mm)
*** Polyethylene transmission
tubing (0.86 mm x 1.52 mm)

Figure 1 (Aspirin, Alumina, and Magnesium Oxide Tablets)

With the sample line pumping pH 4.3 bufferdetergent, the Aspirin standard stock solution: 5 mg/mL of USP Aspirin
other lines pumping their respective reagents, and the RS in Diluent prepared by blending at high speed for 1.5
fluorometer set at an excitation wavelength of 298 nm min
and an emission wavelength of 425 nm, adjust the system Aspirin standard solution: 0.25 mg/mL of USP Aspirin RS
until a steady fluorescence baseline has been achieved. prepared immediately from Aspirin standardstocksolution
Start the sampler, and conduct determinations at a rate of in dehydrated alcohol. Use these solutions within 1 h.
40/h, using a ratio of 5:1 for sample and wash time. Salicylic acid standard stock solution: 5 mg/mL of USP
Record the fluorescence values of the Standard solution Salicylic Acid RS in dehydrated alcohol. Transfer 3.0 mL of
and the Sample solution. this solution to a 1OO-mL volumetric flask, and dilute with
Calculate the percentage of aspirin (C9H s0 4) dissolved: Diluent to volume.
Salicylic acid standard solution: 7.5 IJg/mL of USP Salicylic
Result = Cs x (VI L) x (Ful Fs) x 100 Acid RS from Salicylic acid standard stock solution in
dehydrated alcohol
Cs =concentration of USP Aspirin RS in the Standard System suitability solution: Transfer 5.0 mL of the Aspirin
solution (mg/mL) standard stock solution to a 1OO-mL volumetric flask, add
V = volume of medium, 900 mL 5.0 mL of the Salicylic acidstandard stock solution, and dilute
L = label claim (mg/Tablet) with dehydrated alcohol to volume.
Fu = fluorescence values of the solution under test Sample solution: Transfer a counted number of Tablets,
Fs =fluorescence values of the Standard solution equivalent to 2500 mg of aspirin, to a 120-mL blender jar
containing 100.0 mL of Diluent, and blend at high speed
Tolerances: NLT 75% (Q) of the labeled amount of aspirin for 1.5 min. Immediately filter a portion of the mixture thus
(C9H s0 4) is dissolved. obtained, and transfer 1.0 mL of the filtrate to a 100-mL
• UNIFORMITY OF DOSAGE UNITS (905): Meet the volumetric flask. Immediately dilute with dehydrated
requirements for Weight Variation with respect to alcohol to volume. Promptly inject this Sample solution into
aluminum hydroxide and to magnesium oxide, and for the chromatograph as directed for Analysis.
ContentUniformity with respect to aspirin Chromatographic system
(See Chromatography (621), System Suitability.)
IMPURITIES Mode: LC
• ORGANIC IMPURITIES PROCEDURE: LIMIT OF FREE SALICYLIC Detector: UV 205 nm
ACID Column: 4.6-mm x 3-cm; 5-lJm packing L7
[NoTE-The results from the Assay for Aspirin may be Flow rate: 3.5 mL/min
used for this test when calculated as described in the Injection volume: 10 IJL
Analysis section of this test. ] System suitability
Mobile phase: Methanol, phosphoric acid, and water Samples: Aspirin standardsolution, Salicylic acid standard
(30:3:70) solution and System suitabilitysolution
Diluent: Dehydrated alcohol and hydrochloric acid [NOTE-The relative retention times for aspirin and
(2000:20) salicylic acid are 0.7 and 1.0, respectively.]

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 402 Aspirin / OfficialMonographs USP 43

Suitability requirements Time: 45 minutes.

Resolution: NLT 2.0 between the aspirin and salicylic Mobile phase and Chromatographic system-Prepare as
acid peaks, System suitablity solution directed in the Assayand limit of salicylic acid.
Tailing factor: NMT 2.0 for the aspirin and salicylic acid Standard preparation-Prepare a solution in Medium
peaks, Aspirin standard solution and Salicylicacid standard containing known concentrations of about 0.002A mg of USP
solution Aspirin RS, 0.002C mg of USP Caffeine RS, and 0.0020 mg of
Relative standard deviation: NMT 2.0% for aspirin and USP Dihydrocodeine Bitartrate RS per mL, A, C, and 0 being
salicylic acid peaks, Aspirin standard solution and Salicylic the labeled amounts, in mg, of aspirin, caffeine, and
acid standard solution dihydrocodeine bitartrate, respectively, in each Capsule.
Analysis Testpreparation-Filter a portion of the solution under test.
Samples: Aspirin standard solution, Salicylic acid standard Procedure-Separately inject equal volumes (about 10 ~L)
solution, and Sample solution of the Standard preparation and the Test preparation into the
Calculate the percentage of free salicylic acid in the Tablets chromatograph, record the chromatograms, and measure the
taken: responsesfor the major peaks. Calculatethe quantities, in mg,
of aspirin(C9H s04), caffeine (CSH10N402), and dihydrocodeine
Result = (rvlrs) x (CsICv) x 100 bitartrate (ClsH23N03 . C4H 606) dissolved by the same formula:
to =peak response of salicylic acid from the Sample 500C(rvlrs)
solution
ts =peak response from the Salicylic acid standard in which C is the concentration, in mg per mL, of the
solution appropriate USP Reference Standard in the Standard
Cs = concentration of USP Salicylic Acid RS in the preparation; and ru and ts are the peak responses ofthe relevant
Salicylic acid standard solution (~g/mL) analyte obtained from the Testpreparation and the Standard
Cv = nominal concentration of aspirin in the Sample preparation, respectively.
solution (mg/mL) Tolerances-Not lessthan 75% (Q) of the labeled amounts
of C9H s04, CSH lON 402, and ClsH23N03 . C4H606 are dissolved
Acceptance criteria: NMT 3.0%
. in 45 minutes.
SPECIFIC TESTS Uniformity of dosage units (905): meet the requirements.
• ACID-NEUTRALIZING CAPACITY (301): NLT 1.9 mEq of acid Assay and limit of salicylic acid-
is consumed for each 325 mg of aspirin in the Tablets.
Mobile phase-Dissolve 1 g of sodium l-pentanesulfonate
ADDITIONAL REQUIREMENTS and 2.3.g of monobasic ammonium phosphate in 850 mLof
• PACKAGING AND STORAGE: Preserve in tight containers. water. Add 150 mL of acetonitrile, mix,degas, and adjust with
• USP REFERENCE STANDARDS (11) phosphoric acid to a pH of 2.5. Makeadjustments ifnecessary
USP Aspirin RS (see System Suitability under Chromatography (621 ».
USP Salicylic Acid RS Diluent-Prepare a mixture of water and acetonitrile
(53:46), and adjust with phosphoric acid to a pH of 2.5.
Standard preparation-Prepare a solution in Diluent
containing known concentrations of about 0.001 A mg of USP
Aspirin RS, 0.001 C mg of USP Caffeine RS, and 0.0010 mg of
Aspirin, Caffeine, and Dihydrocodeine USP Dihydrocodeine Bitartrate RS per mL, A, C, and 0 being
Bitartrate Capsules . the labeled amounts, in mg, of aspirin, caffeine, and
dihydrocodeine bitartrate, respectively, in each Capsule.
[NOTE-Use this solution within 3 hours.]
» Aspirin, Caffeine, and Dihydrocodeine Bitartrate Standard salicylic acid preparation-Dissolve an accurately
Capsules contain not less than 90.0 percent and weighed quantity of USP Salicylic Acid RS in Diluent to obtain
a solution having a known concentration of about 0.005A ~g
not more than 110.0 percent of the labeled per mL, A being the labeled amount, in mg, of aspirin per
amounts of aspirin (C9H s0 4 ) , caffeine (C SH lON 4 0 2) , Capsule. [NOTE-Use this solution within 3 hours.]
and dihydrocodeine bitartrate (C'SH 23N0 3 • Resolution solution-Prepare a solution in Standard
preparation containing about 0.0001 A mg of USP Salicylic Acid
C4H606) · RS per mL, A being the labeled amount, in mg, of aspirin in
each Capsule. [NOTE-Use this solution within 3 hours.]
Packaging and storage-Preserve in tight containers. Assay preparation-Transfer the contents of 10 Capsules to
USPReference standards (11)- a 500-mLvolumetricflask. Dilute with Diluent to volume, and
USP Aspirin RS mix.Transfer5.0 mL of this mixture to a 1OO-mL volumetric
USP Caffeine RS flask, dilute with Diluent to volume, and mix. Centrifuge a
USP Dihydrocodeine Bitartrate RS portion of this mixture, and use the clear supernatant as the
USP Salicylic Acid RS Assaypreparation. [NOTE-Use this solution within 3 hours.]
Identification-The retention times of the major peaks in Chromatographic system-The liquid chromatograph is
the chromatogram of the Assaypreparation correspond to equipped with a 215-nm detector and a 4.6-mm x 15-cm
those in the chromatogram of the Standard preparation column that contains packing L7. The flow rate is about 2 mL
obtained as directed in the Assay. per minute. Chromatograph the Resolution solution, and
Dissolution, Procedure for a Pooled Sample (711 )- record the responses as directed for Procedure: the relative
Medium: 0.05 M acetate buffer, prepared by mixing 2.99 retention times are about 0.2 for caffeine, 0.3 for
g of sodium acetate trihydrate and 1.66 mL of glacial acetic dihydrocodeine, 0.7 for aspirin, and 1.0 for salicylic acid; and
acid with water to obtain 1000 mLof solution having a pH of the resolution, R, between the caffeine and dihydrocodeine
4.50 ± 0.05; 500 mL. peaks is not less than 2.5, between the dihydrocodeine and
Apparatus 1: 50 rpm. aspirin peaks is not lessthan 1.0, and between the aspirinand

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 USP 43 OfficialMonographs / Aspirin 403

salicylic acid peaks is not lessthan 1.5. Chromatograph the Apparatus 2: 75 rpm.
Standard preparation, and recordthe responsesas directedfor Time: 30 minutes.
Procedure: the relative standard deviation for replicate Determine the amounts of aspirin (C9H s0 4) and codeine
injections is not more than 2.0% for each analyte. phosphate hemihydrate (ClsH21N03' H3P04 • V2H 20)
Procedure-Separately inject equal volumes (about 10 I-IL) dissolved by employingthe following method.
of the Assay preparation, the Standard preparation, and the Mobilephase, Solvent mixture, and Aspirin and codeine
Standard salicylic acid preparation into the chromatograph, phosphate standardpreparation-Prepare as directed in the
record the chromatograms, and measurethe responsesforthe Assay for aspirin and codeine phosphate and limit of free salicylic
major peaks. Calculate the quantities, in mg, of aspirin acid.
(C9H s0 4) , caffeine (CSH lON 40 2), and dihydrocodeinebitartrate Internal standard solution-Dissolve phenacetin in
(ClsH23N03' C4H 606) in each Capsuletaken by the same methanol to obtain a solutionhaving a concentration of about
formula: 0.07 mg per mL.
StandardsolutionA-Prepare a solution of USP Aspirin RS in
Solvent mixture having an accurately known concentration of
about 0.36 mg per mL.
in which C is the concentration, in mg per mL, of the Standardsolution B-Transfer about 12 mg of USP Codeine
appropriate USP Reference Standard in the Standard Phosphate RS and 25 mg of USP Salicylic Acid RS, each
preparation; and ru and ts are the responses of the accuratelyweighed, to a 50-mL volumetric flask, add 2.5 mL
corresponding analyte peaksof the Assay preparation and the of methanol, and mix. Add Medium to volume, and mix. Pipet
Standard preparation, respectively. Calculatethe percentage 10 mL of the resulting solution into a 1OO-mL volumetric
of salicylic acid in the Capsules taken by the formula: flask, add Medium to volume, and mix.
Standardpreparations A and B-Pipet 10 mL of Standard
1OO( CIA)(rvi rs) solution A and 10 mL of Standard solution B into separate
containers, add 3.0 mL ofthe Internalstandardsolutionto each
in which C isthe concentration, in I-Ig per mL, of USP Salicylic container, and mix.
Acid RS in the Standard salicylic acid preparation; A is the Test preparation-Withdraw a portionof the solutionunder
labeled amount, in mg, of aspirin in each Capsuletaken; and test and filter, discarding the few mL of the filtrate. Pipet 10
tu and rs are the salicylic acid peak responses obtained from mL of the filtrate and 3.0 mLof the Internalstandardsolution
the Assay preparation and the Standard salicylic acid into a suitable container, and mix.
preparation, respectively: not more than 3.0% isfound. Chromatographic system-Proceed as directed for
Chromatographic system in the Assay for aspirinand codeine
phosphate and limit of free salicylic acid, except to use only the
Aspirin and codeine phosphate preparation for evaluation of the
suitability of the system.
Aspirin and Codeine Phosphate Tablets Procedure-Proceed as directed in the Assay for aspirin and
codeine phosphate and limit of free salicylic acid, except to inject
about 50 I-IL of the Standard preparations and the Test
» Aspirin and Codeine Phosphate Tablets contain preparation. The relative retention times are 0.3 for salicylic
not less than 90.0 percent and not more than acid, 0.6 for aspirin, 0.8 for codeine phosphate, and 1.0 for
110.0 percent of the labeled amounts of aspirin phenacetin. Calculatethe amount of codeine phosphate
(C9H s0 4 ) and codeine phosphate hemihydrate dissolved by comparison of the relative peak response ratios
for the codeine phosphate peaks, obtained from Standard
(C1sH z1N0 3 • H3P04 • V2H zO). preparation B and the Testpreparation. Calculate the
percentage of aspirin dissolved by the formula:
Packaging and storage-Preserve in well-closed,
light-resistant containers. [0.9C(RvIRs) + 0.9C(R'vIR's)(180.16/138.12)]/3.25
USP Reference standards (11)-
USP Aspirin RS in which C is the concentration, in I-Ig per mL, of USP Aspirin
USP Codeine Phosphate RS RS in Standardsolution A;Rv and Rsare the peak responseratios
USP Salicylic Acid RS for the aspirin component obtained from the Test preparation
Identification-Dissolve a suitable quantity of USP Aspirin and Standardpreparation A, respectively; C is the
RS in the Solvent mixture prepared as directed under Assay for concentration, in I-Ig per mL, of USP Salicylic Acid RS in
aspirin and codeine phosphate and limit of free salicylic acid to Standard solutionB;R'v and R's are the peak response ratiosfor
obtain a Standard aspirin solution containing about 3.3 mg per the salicylic acid component obtained from the Test
mL. Dissolve a suitablequantity of USP Codeine Phosphate RS preparation and Standard preparation B, respectively; and
in the Solvent mixture to obtain a Standard codeine phosphate 180.16 and 138.12 are the molecular weights of aspirin and
solution containing about 1 mg per mL. Chromatograph these salicylic acid, respectively.
solutions as directed for Procedure in the Assay for aspirin and Tolerances--Not less than 75% (Q) of the labeled amounts
codeine phosphate and limit of free salicylic acid. The retention of aspirin (C9H s0 4) and codeine phosphate hemihydrate
times of the major peaks in the chromatogram of the Assay (ClsH21N03' H3P04 • V2H 20) is dissolved in 30 minutes.
preparation, obtained as directed in the Assay for aspirin and
Uniformity of dosage units (905): meetthe requirementsfor
codeine phosphate and limit of free salicylic acid, co.r~espon~ to
those in the chromatrograms of the Standardaspmn solution ContentUniformity with respect to aspirin and codeine
and the Standardcodeine phosphate solution, respectively. phosphate.
Dissolution (711)-
Assay for aspirin and codeine phosphate and limit of
Medium: 0.05 M acetate buffer, prepared by mixing 2.~9 free salicylic acid-
g of sodium acetate trihydrate and 1.66 mLof glacial acetic Mobile phase-Dissolve 225 mg of tetramethylammonium
acid with water to obtain 1000 rnl,: of solution having a pH of hydroxide pentahydrate and 200 mg of sodium
4.50 ± 0.05;900 mL. l-octanesulfonate in 700 mLof water. Add 150 mL of

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404 Aspirin / OfficialMonographs USP 43

methanol, 150 mL of acetonitrile, and 1.0 mL of glacial acetic Aspirin and codeine phosphate standard preparation,
acid, and stir. Pass through a membrane filter, and degas. respectively. Calculate the quantity, in mg, of codeine
[NoTE-The amounts of sodium l-octanesulfonate, methanol, phosphate hemihydrate(ClsHzlN03' H3P04 • V2H zO) in the
and acetonitrile may be varied to obtain acceptable portion of Tablets taken by the formula:
chromatography.]
Solvent mixture-To 15 g of anhydrous citric acid add 200 (406.37/397.37)(50C)(Rui Rs)
mL of methanol and 20 mL of glacial acetic acid, dilute with
chloroform to 1000 mL, and mix until the citric acid is in which 406.37 and 397.37 are the molecular weights of
dissolved. codeine phosphate hemihydrate and anhydrous codeine
Internal standard solution-Dissolve phenacetin in Solvent phosphate, respectively; Cisthe concentration,in mg per mL,
mixture to obtain a solution having a concentration of about of USP Codeine Phosphate RS in the Aspirin and codeine
2 mg per mL. phosphate standard pteparatlon; and Ru and Rs are the ratiosof
Salicylic acid stock standard solution-Dissolve an accurately the peak responses of codeine phosphate and phenacetin
weighed quantity of USP Salicylic Acid RS in Solvent mixture, obtained from the Assay preparation and the Aspirin and
and quantitatively dilutewith Solvent mixture to obtain a codeine phosphate standard preparation, respectively. Calculate
solution havinga known concentrationof about 1 mg per mL. the percentage of freesalicylic acid inthe Tablets taken by the
Salicylic acid standard preparation-Transfer 5.0 mL of formula:
Salicylic acidstock standard solution to a 50-mLvolumetric flask,
add 5.0 mL of Internal standard solution, dilute with Solvent 5000(C/a)(Ru/ Rs)
mixture to volume, and mix.
Codeine phosphate stock standard solution-Transfer about in which C isthe concentration, in mg per mL, of USP Salicylic
325}mg of USP Codeine Phosphate RS, accuratelyweighed, Acid RS in the Salicylic acid standard preparation; a isthe
to a 25-mLvolumetric flask, } being the ratio of the labeled quantity, in mg, of aspirin in the portion of powdered Tablets
amount, in mg, of codeine phosphate to the labeledamount, taken, based on the labeled amount; and Ru and Rs are the
in mg, of aspirin per Tablet. Dissolve in and dilutewith Solvent ratios of the peak responses of salicylic acid and phenacetin
mixture to volume, and mix. obtained from the Assay preparation and the Salicylic acid
Aspirin and codeine phosphate standard preparation- .standard preparation, respectively: not more than 3.0% is
Transfer about 65 mg of USP Aspirin RS, accuratelyweighed, found.
to a 1O-mL volumetric flask. Add 5.0 mL of Codeine phosphate
stock standard solution, 1.0 mL of Salicylic acid stockstandard
solution, and 1.0 mL of Internal standard solution, dilutewith
Solvent mixture to volume, and mix.
Assay preparation-Weigh and finely powder not fewer Aspirin, Codeine Phosphate, Alumina,
than 20 Tablets. Transfer an accurately weighed portionof the and Magnesia Tablets
powder, equivalent to about 325 mg of aspirin, to a
screw-capped, 120-mL bottle, add 5.0 mLof Internal standard
solution and 45.0 mL of Solvent mixture, mix, and sonicate for » Aspirin, Codeine Phosphate, Alumina, and
2 to 5 minutes. Centrifuge, and use a portion of the resultant Magnesia Tablets contain not less than 90.0
clear solution as the Assay preparation. Use on the day
prepared. percent and not more than 110.0 percent of the
Chromatographic system (see Chromatography (621 »- The labeled amounts of aspirin (C9H s04) , codeine
liquid chromatograph is equipped with a 280-nm detector phosphate hemihydrate (ClsH21N03' H3P04 •
and a 3.9-mm x 30-cm column that contains JO-urn packing lhHzO), aluminum hydroxide [AI(OH)3l, and
L1. The flow rate is about 2 mL per minute. Chromatograph
replicate injections of the Salicylic acid standard preparation magnesium hydroxide [Mg(OH)2l.
and the Aspirin and codeine phosphate standard preparation,
and record the peak responses as directed for Procedure: the Packaging and storage-Preserve in well-closed,
relative retention timesfor salicylic acid, aspirin, codeine, and light-resistant containers.
phenacetin are about 0.3,0.5,0.8, and 1.0, respectively; the USP Reference standards (11 )-
resolution, R, between salicylic acid and aspirin, between USP Aspirin RS
aspirin and codeine, and between codeine and phenacetin is USP Codeine Phosphate RS
not less than 2.0; the tailingfactor for each analyte peakis not USP Salicylic Acid RS
more than 2.0; and the relative standard deviationofthe ratios Identification-
of the peak responses of salicylic acid, aspirin, and codeine to A: Tabletsrespond to the Identification-test under Aspirin
the peak response of phenacetin is not more than 3.0%. and Codeine Phosphate Tablets.
Procedure-Separately injectequal volumes(about 5 I.lL) of B: Tablets respond to the /DENTIFICA TlON tests under
the Salicylic acid standard preparation, Aspirin and codeine Alumina and Magnesia Tablets.
phosphate standard preparation, and Assay preparation into the
chromatograph, recordthe chromatograms,and measurethe Dissolution (711 )-
responses for the major peaks. Calculate the quantity, in mg, Medlum: 0.05 M acetate buffer, prepared by mixing 2.99
of aspirin (C9H s04) in the portion of Tablets taken by the g of sodium acetate trihydrateand 1.66 mL of glacial acetic
formula: acid with water to obtain 1000 mL of solution havinq a pH of
4.50 ± 0.05; 900 mL.
Apparatus 2: 75 rpm.
Time: 30 minutes.
in which C isthe concentration, in mg per mL, of USP Aspirin Mobile phase, Internal standard solution, Solvent mixture,
RS in the Aspirin and codeine phosphate standard preparation: Aspirin and codeine phosphate standard preparation, Standard
and Ru and Rs are the ratiosof the peak responses of aspirin solution A, Standard solution 8, Standard preparations A and 8,
and phenacetin obtained from the Assay preparation and the Test preparation, Chromatographic system, and Procedure-

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 USP 43 OfficialMonographs / Atenolol 405

Proceed as directed in the test for Dissolution 771- under from the Assay preparation and the Salicylic acid standard
Aspirin and Codeine Phosphate Tablets. preparation, respectively: not more than 3.0% is found.
Tolerances-Not less than 75% (Q) of the labeled amounts
of aspirin (C9H s04) and codeine phosphate hemihydrate Assay for aluminum hydroxide-
(ClsH21N03' H3P04· VzH 20) are dissolved in 30 minutes.
Edetate disodium titrant-Prepare and standardize as
directed in the Assay under Ammonium Alum.
Uniformity of dosage units (905): meet the requirements Assay preparation-Weigh and finely powder not fewer
for Content Uniformity with respect to aspirin and codeine than 20 Tablets. Transfer an accurately weighed portion of the
phosphate and for Weight Variation with respect to aluminum powder, equivalent to about 600 mg of aluminum hydroxide,
hydroxide and magnesium hydroxide. to a 150-mL beaker, add 20 mL of water, stir, and slowly add
Acid-neutralizing capacity (301): not lessthan 1.9 mEq per 3~ ml o~ 3 N hydrochl~ric a.cid. Heat gently, if necessary, to
Tablet. aid solution, cool, and filter Into a 200-ml volumetric flask.
Assay for aspirin and codeine phosphate and limit of Wash ~he filter with water into the flask, add water to volume,
free salicylic acid- and mix.
Mobile phase, Solvent mixture, Salicylic acid stock standard Procedure-Pipet 10 mL of Assay preparation into a 250-mL
solution, Salicylic acid standard preparation, Aspirin and codeine beaker, add 20 mL of water, then add, in the order named and
phosphate standard preparation, and Chromatographic system with continuous stirring, 25.0 mL of Edetate disodium titrant
-Prepare as directed in the Assay for aspirin and codeine and 20 mL of acetic acid-ammonium acetate buffer TS. Add
phosphate and limit of free salicylic acid under Aspirin and 50 mL of alcohol and 2 mL of dithizone TS, and mix. Titrate
Codeine Phosphate Tablets. with 0.05 M zinc sulfate VS until the color changes from
Assay preparation-Weigh and finely powder not fewer green-violet to rose-pink. Perform a blank determination
than 20 Tablets. Transfer an accurately weighed portion of the substituting 10 ml of water for the Assay preparation, an'd
powder, equivalent to about 325 mg of aspirin, to a make any necessarycorrection. Each mL of 0.05 M Edetate
screw-capped, 120-ml bottle, add 5.0 ml of Internal standard disodium titrant is equivalent to 3.900 mg of AI(OH)3'
solution and 45.0 ml of Solvent mixture, mix, and sonicate for Assay for magnesium hydroxide-
2 to 5 minutes. Centrifuge, and use a portion of the resultant
clear solution as the Assay preparation.
Assay preparation-Prepare as directed in the Assay for
Procedure-Separately inject equal volumes (about 5 IJL) of aluminum oxide.
the Salicylic acid standard preparation, the Aspirin and codeine
Procedure-Pipet a volume of Assay preparation, equivalent
to about 40 mg of magnesium hydroxide, into a 400-mL
phosphate standard preparation, and the Assay preparation into beaker, add 200 mL of water and 20 ml of triethanolamine,
the chromatograph, record the chromatograms, and measure
and stir. Add 10 mL of ammonia-ammonium chloride buffer
the responsesfor the major peaks. The relative retention times
TS and 3 drops of an eriochrome black indicator solution
for salicylic acid, aspirin, codeine, and phenacetin are about
prepared by dissolving 200 mg of eriochrome black T in a
0.3, 0.5, 0.8, and 1.0, respectively. Calculate the quantity, in mixture of 15 ml of triethanolamine and 5 mL of dehydrated
mg, of aspirin (C9H s0 4) in the portion of powdered Tablets
alcohol, and mix. Cool the solution to between 3° and 4° by
taken by the formula: immersion of the beaker in an ice bath, then remove, and
titrate with 0.05 M edetate disodium VS to a blue endpoint.
50C(Ru/Rs) Perform a blank determination, substituting 10 mL of water
for the Assay preparation, and make any necessary correction.
in which C is the concentration, in mg per mL, of USP Aspirin
Each mL of 0.05 M edetate disodium consumed is equivalent
RS in the Aspirin and codeine phosphate standard preparation;
to 2.916 mg of Mg(OH)2' .
and Ru and Rs are the ratios of the peak responses of aspirin
and phenacetin obtained from the Assay preparation and the
Aspirin and codeine phosphate standard preparation, .
respectively. Calculate the quantity, in mg, of codeine
phosphate hemihydrate (ClsH21N03' H3P04· V2H 20), in the Atenolol
portion of powdered Tablets taken by the formula:

(406.37 /397.37)(50C)(R u/Rs)

in which 406.37 and 397.37 are the molecular weights of

codeine phosphate hemihydrate and anhydrous codeine
phosphate, respectively; C is the concentration, in mg per mL, C14H22N203 266.34
of USP Codeine Phosphate RS in the Aspirin and codeine Benzeneacetamide, 4-[2-hydroxy-3-[(1-methylethyl) amino]
phosphate standard preparation; and Ru and Rs are the ratios of propoxy]-;
2-[p-[2-Hydroxy-3-(isopropylamino)propoxy]-phenyl]
the peak responses of codeine phosphate and phenacetin
acetamide [29122-68-7].
obtained from the Assay preparation and the Aspirin and
codeine phosphate Standard preparation, respectively. DEFINITION
Calculate the percentage of free salicylic acid in the Tablets Atenolol contains NLT 98.0% and NMT 102.0% of
taken by the formula: C14H22N203, calculated on the dried basis.

5000(C/a)(R u/Rs) IDENTIFICATION

in whichC is the concentration, in mq per mL, of USP Salicylic

Acid RS in the Salicylic acid standard preparation; a is the
quantity, in mg, of aspirin in the portion of Tablets taken, • A• .t.SPECTROSCOPIC IpENTIFICATION TESTS (197), Infrared
determined as directed above; and Ru and Rs are the ratios of Spectroscopy: 197K~ (eN 1~May.2020)
the peak responses of salicylic acid and phenacetin obtained

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 406 Atenolol / OfficialMonographs USP 43

Calculate the percentage of each impurity in Sample

solution 7:
• B~~~~~f. .~gsf~~~~Yi.~~~. Result = 0.5(r U/rA)
I.JltrC1yi()J~t!l£i~i121~~P~9tD . . •.• ·. . ...+»,
Sample solution: 20 IJg/mL in methanol
ru =peak response of any individual impurity in
ASSAY Sample solution 7
• PROCEDURE rA =peak response of Atenolol in Sample solution 2
Mobile phase: 1.1 g of sodium l-heptanesulfonate and
0.71 g of anhydrous dibasic sodium phosphate in 700 mL Acceptance criteria
of water. Add 2 mL of dibutylamine, and adjust with 0.8 M Individual impurities: NMT 0.25%
phosphoric acid to a pH of 3.0. Add 300 mL of methanol, Total impurities: NMT 0.5%
mix, and pass through a filter having a 0.5-lJm or finer
porosity. Degas this solution before use. SPECIFIC TESTS
Standard solution: 0.01 mq/ml, of USP Atenolol RS in • MELTING RANGE OR TEMPERATURE, Class 1(741): 152°-
Mobilephase 156.5°
Sample solution: 0.01 mg/mL of Atenolol in Mobilephase. • Loss ON DRYING (731): Dry a sample at 105° to constant
Sonicate for 5·min for complete dissolution. weight: it loses NMT 0.5% of its weight.
Chromatographic system ADDITIONAL REQUIREMENTS
(See Chromatography (621), System SUitability.) • PACKAGING AND STORAGE: Preserve in well-closed
Mode: LC containers. Store at room temperature.
Detector: UV 226 nm • USP REFERENCE STANDARDS (11)
Column: 3.9-mm x 30-cm; packing L1 USP Atenolol RS
Flow rate: 0.6 mL/min
Injection size: 10 IJL
System suitability
Sample: Standardsolution
Suitability requirements 'Atenolollnjection
Column efficiency: NLT 5000 theoretical plates
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% » Atenolol Injection is a sterile solution of Atenolol
Analysis in Water for Injection. It contains a suitable
Samples: Standardsolution and Sample solution buffering agent. It contains not less than 90.0
Calculate the percentage of C14H22N203 in the portion of
Atenolol taken: percent and not more than 110.0 percent of the
labeled amount of atenolol (C14HzzNz03)'
Result = (ru/rs) x (Cs/Cu) x 100
Packaging and storage-Preserve in single-dose or in
ru = peak response from the Sample solution multiple-dose containers, preferably of Type I glass, in a cool
rs = peak response from the Standardsolution place or at controlled room temperature, protected from light.
Cs = concentration of USP Atenolol RS in the Standard Avoid freezing.
solution (mg/mL) USP Reference standards (11)-
Cu = concentration of Atenolol in the Sample solution USP Atenolol RS
(mg/mL) Identification-
A: The retention time of the main peak in the
Acceptance criteria: 98.0%-102.0% on the dried basis
chromatogram of the Assay preparation corresponds to that in
IMPURITIES the chromatogram of the Standardpreparation, obtained as
INORGANIC IMPURITIES directed in the Assay.
• Residue on Ignition (281): NMT 0.2%
• Chloride and Sulfate, Chloride (221)
Sample solution: Dissolve 1.0-g in 100 mL of 0.15 N nitric
acid.
Acceptance criteria: Shows no more turbidity with 1 mL of
silver nitrate TS than 1.4 mL of 0.020 N hydrochloric acid in IJg
100 mL of 0.15 N nitric acid (0.1%) Medium: methanol.
ORGANIC IMPURITIES Bacterial Endotoxins Test (85) -It contains not more than
• Procedure 33.3 USP Endotoxin Units per mg of atenolol.
Mobile phase: Prepare as directed in the Assay. Sterility Tests (71) -It meets the requirements when tested
Sample solution 1: 0.1 mg/mL of Atenolol in Mobilephase as directed for Membrane Filtration under Test for Sterilityof the
Sample solution 2: 0.5 IJg/mL of Atenolol, from Sample Product to be Examined.
solution 7 in Mobile phase pH (791): between 5.5 and 6.5.
Chromatographic system: Proceed as directed in the Assay, Particulate Matter in Injections (788): meets the
except use the injection size listed below. requirements for small-volume injections.
Injection size: 50 IJL Assay-
Analysis Citricacid buffer-Transfer 2.5 g of citric acid to a 500-mL
Samples: Sample solution 7 and Sample solution 2 volumetric flask, add 400 mL of water, and swirl to dissolve.
[NOTE-Chromatograph Sample solution 7 for a period of Adjust the solution with 2 N sodium hydroxide to a pH of 6.0,
time that is 6 times the retention time of the atenolol dilute with water to volume, and mix.
peak.]

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 USP 43 Official Monographs / Atenolol 407

Mobilephase-Dissolve 930 mg of sodium octyl sulfate in ADDITIONAL REQUIREMENTS

740 mL of water, add 8 mL of 3.6 N sulfuric acid, mix, and • PACKAGING AND STORAGE: Package in amber, tight
pass through a t-urn or finer porosity filter. To the filtrate add containers, and store at controlled room temperature.
250 mL of acetonitrile, mix, and degas. Make adjustments if • BEYOND-USE DATE: NMT 60 days after the day on which it
necessary (see System Suitability under Chromatography was compounded when stored at controlled room
(621 » temperature
Standard preparation-Transfer about 50 mg of USP • LABELING: Label it to state that it is to be shaken well before
Atenolol RS to a 1OO-mL volumetric flask, add 80 mL of Citric use, and to state the Beyond-Use Date.
acid buffer, and sonicate for about 30 seconds to achieve
dissolution. Dilute with Citricacid buffer to volume, and mix.
Transfer 4.0 mL of this solution to a 1O-mL volumetric flask,
dilute with Citricacid buffer to volume, and mix. This solution
contains about 0.2 mg of USP Atenolol RS per mL. Atenolol Tablets
Assay preparation-Transfer an accurately measured
volume of Injection, equivalent to 2 mg of atenolol, to a 10-mL DEFINITION
volumetric flask, dilute with Citricacid buffer to volume, and Atenolol Tablets contain NLT 90.0% and NMT 110.0% of the
mix. labeled amount of atenolol (C14HzzNz03)'
Chromatographic system (see Chromatography (621 »- IDENTIFICATION
The liquid chromatograph is equipped with a 275-nm
detector and a 4.6-mm x 25-cm column that contains 5-J.Jm
packing L1. The flow rate is about 1.7 mL per minute.
Chromatograph the Standard preparation, and record the
peak responses as directed for Procedure: the tailing factor is )
not more than 2, and the relative standard deviation for a quantity of powdered Tablets, equivalent to
replicate injections is not more than 2.0%. 1 mg of atenolol, with 15 mL of methanol, heat the
Procedure-Separately inject equal volumes (about 10 J.JL) mixture to 50°, and shakefor 5 min. Filter, and evaporate
of the Standard preparation and the Assay preparation into the the filtrate on a water bath to dryness. Add 10 mL of 0.1 N
chromatograph, record the chromatograms, and measurethe hydrochloric acid to the residue, warm the solution, shake,
areas for the major peaks. Calculate the quantity, in mg, of and filter. To the filtrate add sufficient 1 N sodium
C14HzzNz03 in each mL of the Injection taken by the formula: hydroxide to make it alkaline, and extract the solution with
10 mL of chloroform, drying the chloroform extract over
1 O(CIV)(r vir s) anhydrous sodium sulfate. Filter the dried chloroform
solution, evaporate the filtrate on a water bath to dryness,
in which C is the concentration, in mg per mL, of USP Atenolol and dry the residue at 105° for 1 h.
RS in the Standard preparation; V is the volume, in mL, of • B. The retention time of the atenolol peak of the Sample
Injection taken; and r u and r sare the atenolol peak responses solution corresponds to that of the Standard solution, as
obtained from the Assay preparation and the Standard obtained in the Assay.
preparation, respectively.
ASSAY
• PROCEDURE
Mobile phase: 1.1 g of sodium l-heptanesulfonate and .
0.71 g of anhydrous dibasic sodium phosphate in 700 mL
Atenolol Compounded Oral Solution of water. Add 2 mL of dibutylamine, and adjust with 0.8 M
phosphoric acid to a pH of 3.0. Add 300 mL of methanol,
DEFINITION and passthrough a filter having a 0.5-J.Jm or finer porosity.
Atenolol Compounded Oral Solution contains NLT 90.0% and Degas this solution before use.
NMT 110.0% of the labeled amount of atenolol Standard solution: 0.01 mg/mL of USP Atenolol RS in
(C14HnNz03)' Mobilephase
Prepare Atenolol Compounded Oral Solution at a 2-mg/mL Sample stock solution: Transfer 10 Tablets to a 1000-mL
concentration, for example, as follows (see Pharmaceutical volumetric flask. Add 500 mL of Mobilephase, and sonicate
Compounding-Nonsterile Preparations (795». for 15 min to disintegrate the Tablets. Dilute with Mobile
phase to volume.
Atenolol 200 mg Sample solution: Centrifuge a portion of the Sample stock
solution, and dilute a volume of the supernatant with Mobile
Glycerin 5 mL phase to obtain a solution nominally containing 0.01
Vehicle for OralSuspension 45mL mg/mL of atenolol.
Chromatographic system
Vehicle for OralSolution, Sugar Free, a (See Chromatography (621), System Suitability.)
sufficient quantity to make 100 mL
Mode: LC
Detector: UV 226 nm
Calculate the quantity of each ingredient required for the total Column: 3.9-mm x 30-cm; packing L1
volume and atenolol strength to be prepared. Mix the Flow rate: 0.6 mL/min
Atenolol, previously pulverized, and Glycerin to form a Injection size: 10 J.JL
smooth paste. Incorporate the Vehicle for Oral Suspension or System suitability
an equal volume of Vehicle for Oral Solution, Sugar Free. Sample: Standard solution
[NOTE-The Vehicle for Oral Suspension may be omitted.] Suitability requirements
Incorporate sufficient Vehicle for Oral Solution, Sugar Free in Column efficiency: NLT 5000 theoretical plates
increments to bring to volume, and mix well. [NOTE-Do not Tailing factor: NMT 2.0
use a sucrose-containing vehicle for oral solution.] Package, Relative standard deviation: NMT 2.0%
and label.

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 408 Atenolol / OfficialMonographs USP 43

Analysis Prepare Atenolol Compounded Oral Suspension 10 mg/mL as

Samples: Standardsolution and Sample solution follows (see Pharmaceutical Compounding-Nonsterile
Calculate the percentage of C14H22N203 in each Tablet Preparations (795»).
taken:
Atenolol powder 1g
Result =(ru/rs) x (Cs/Cu) x 100
Vehicle: a 1:1 mixtureof Ora-Plus"
ru = peak response from the Sample solution and Ora-Sweet SFa, a sufficient
quantity to make 100 ml
rs = peak response from the Standardsolution
Cs = concentration of USP Atenolol RS in the Standard a Perrigo Pharmaceuticals, Allegan, MI.
solution(mg/mL)
Cu =nominal concentration of atenolol in the Sample Pour Atenololpowderinto a suitable container. Wet the powder
solution(mg/mL) with a small amount of Vehicle, and triturate to make a
smooth paste. Add Vehicle to make the contents pourable.
Acceptance criteria: 90.0%-110.0% Transfer contents stepwise and quantitatively to a calibrated
PERfORMANCE TESTS container using the remainder of Vehicle. Add sufficient
• DISSOLUTION (711) Vehicle to bring to final volume. Shake to mix well.
Medium: 0.1 N acetate buffer, pH 4.6 (prepared by mixing ASSAY
44.9 parts (v/v) of 0.1 N sodium acetate with 55.1 parts (vi • PROCEDURE
v) of 0.1 N acetic acid solution, and adjust with either Solution A: 25 mM sodium phosphate adjusted with
diluted sodium hydroxide or diluted acetic acid to a pH of phosphoric acid to a pH of 3.0
4.6); 900 mL Solution B: Water adjusted with phosphoric acid to a pH of
Apparatus 2: 50 rpm 3.0
Time: 30 min Solution C: Methanol and Solution B (50:50)
Determine the amount of C14H22N203 dissolved by using the Mobile phase: Acetonitrile and Solution A (15:85). Filter,
following method. . and degas.
Mobile phase, Chromatographic system, and System Standard stock solution: 10 mg/mL of USPAtenolol RS in
suitability: Proceed as directed in the Assay under Solution C. Mix well and sonicate for 3 min. Store at 2°_8°.
Atenolol. Standard solution: Transfer 2.0 mL of the Standardstock
Standard solution: 0.01 mg/mL of USPAtenolol RS in solution to a 500-mL volumetric flask, add 0.5 mLof Solution
Mobilephase C, and dilute with Solution B to volume. Mix well.
Sample solution: Pass a portion of the solution under test Centrifuge a portion of the resultant solution for 5 min at
through a suitable 0.45-fJm filter. Quantitatively dilute a 14,000 rpm, and use the supernatant. Protect from light,
measured volume of the filtrate with Mobilephase to obtain and store at 2°_8°.
a solution estimated to contain about 0.01 mg/mL of Sample solution: Shake each bottle of Oral Suspension
atenolol. thoroughly. Transfer 2.0 mL of Oral Suspension into a
Analysis: Proceed as directed in the Assay. 500-mL volumetric flask, and add 0.5 mL of Solution C.
Calculate the percentage of C14H22N203 dissolved: Dilute with Solution B to volume. Mix well. Centrifuge a
portion of the solution for 5 min at 14,000 rpm, and use
Result = (ru/rs) x Cs x V x D x (100/L) the supernatant. Protect from light, and store at 2°_8°.
Chromatographic system
ru = peak response from the Sample solution (See Chromatography (621), System Suitability.)
rs = peak response from the Standard solution Mode: LC
Cs =concentration of USP Atenolol RS in the Standard Detector: UV 227 nm
solution (mg/mL) Column: 4.6-mm x 25-cm; 5-fJm packing L1
V = volume of Medium, 900 mL Temperatures
D = dilution factor for the Sample solution Column: 30°
L =Tablet label claim (mg) Autosampler: 5°
Flow rate: 0.7 mL/min
Tolerances: NLT 80% (Q) of the labeled amount of Injection volume: 20 fJL
C14H22N203 is dissolved. System suitability
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Sample: Standard solution
requirements [NoTE-The retention time for atenolol is about 5.1
min.]
ADDITIONAL REQUIREMENTS Suitability requirements
• PACKAGING AND STORAGE: Preserve in well-closed Tailing factor: NMT 2.0
containers. Relative standard deviation: NMT 2.0% for replicate
• USP REFERENCE STANDARDS (11) injections
USP Atenolol RS Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of atenolol
(C14H22N203) in the portion of Oral Suspension taken:
Atenolol Compounded Oral Suspension Result = (rulrs) x (CsICu) x 100
DEfiNITION
Atenolol Compounded Oral Suspension contains NLT 90.0%
= peak response of atenololfrorn the Sample
solution
and NMT 110.0% of the labeled amount of atenolol = peak response of atenolol from the Standard
(C14H22N203)' solution

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 USP 43 OfficialMonographs / Atenolol 409

Cs = concentration of atenolol in the Standardsolution Veterinary to a 1-L volumetric flask, and add 0.5 mL of
(mg/mL) Solution C. Dilute with Solution B to volume. Centrifuge a
Cu = nominal concentration of atenolol in the Sample portion of the solution for 5 min at 14,000 rpm, and use
solution (mg/mL) the supernatant. Protect from light, and store at 2°_8°.
Chromatographic system
Acceptance criteria: 90.00/0-110.0% (See Chromatography (621), System Suitability.)
Mode: LC
SPECIFIC TESTS
Detector: UV 227 nm
• pH (791): 6.4-7.4 Column: 4.6-mm x 25-cmi 5-pm packing L1
ADDITIONAL REQUIREMENTS Temperatures
• PACKAGING AND STORAGE: Package in tight, light-resistant Column: 30°
containers. Store at 2°_8° or at controlled room Autosampler: 5°
temperature. Flow rate: 0.7 mL/min
• LABELING: Label it to indicate that it is to be well shaken Injection volume: 20 ~L
before use, and to state the Beyond-Use Date. System suitability
• BEYOND-USE DATE: NMT 90 days after the date on which it Sample: Standardsolution
was compounded when stored at 2°_8° or at controlled [NOTE-The retention time for atenolol is about 5.1
room temperature min.]
!D USP REFERENCE STANDARDS (11) Suitability requirements
USP Atenolol RS Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% for replicate
injections
Analysis
Samples: Standardsolution and Sample solution .
Atenolol Compounded Oral Calculate the percentage of the labeled amount of atenolol
Suspension, Veterinary (C14H22N203) in the portion of Oral Suspension,Veterinary
taken:
DEFINITION
Atenolol Compounded Oral Suspension, Veterinary contains Result = (rulrs) x (Cs/Cu) x 100
NLT 90.0% and NMT 110.0% of the labeled amount of
atenolol (C14H22N203)' Prepare Atenolol Compounded Oral ru =peak response of atenolol from the Sample
solution
Suspension, Veterinary 25 mg/mL as follows (see
Pharmaceutical Cofnpounding-Nonsterile Preparations
rs =peak response of atenolol from the Standard
solution
(795».
Cs = concentration of atenolol in the Standardsolution
(mg/mL)
Atenolol powder 2.5 g . Cu = nominal concentration of atenolol in the Sample
Vehicle: a 1:1 mixtureof Ora-Plus- solution (mg/mL)
and Ora-SweetSP, a sufficient
quantity to make 100 mL Acceptance criteria: 90.0%-110.0%
a PerrigoPharmaceuticals, Allegan, MI. SPECIFIC TESTS
• pH (791): 9.1-10.1
Pour the Atenololpowder into a suitable container. Wet the ADDITIONAL REQUIREMENTS
powder with a small amount of Vehicle, and triturate to make • PACKAGING AND STORAGE: Package in tight, light-resistant
a smooth paste. Add the Vehicle to make the contents containers. Store at 2°_8° or at controlled room
pourable. Transfer the contents stepwise and quantitatively temperature.
to a calibrated container using the remainder of the Vehicle. • LABELING: Label it to indicate that it is to be well shaken
Add sufficient Vehicle to bring to final volume. Shake to mix before use, and to state the Beyond-Use Date. Label it to
well. state that it is for veterinary use only.
ASSAY • BEYOND-USE DATE: NMT 90 days after the date on which it
• PROCEDURE was compounded when stored at 2°_8° or controlled
Solution A: 25 mM sodium phosphate adjusted with room temperature
phosphoric acid to a pH of 3.0 • USP REFERENCE STANDARDS (11)
Mobile phase: Acetonitrile and Solution A (15:85). Filter, USP Atenolol RS
and degas.
Solution B: Water adjusted with phosphoric acid to a pH of
3.0
Solution C: Methanol and Solution B (50:50)
Standard stock solution: 25 mg/mL of USP Atenolol RS in Atenolol and Chlorthalidone Tablets
Solution C. Mix well, and sonicate for 3 min. Store at 2°_8°.
Standard solution: Transfer 2.0 mL of the Standardstock » Atenolol and Chlorthalidone Tablets contain not
solution to a 1-L volumetric flask, and add 0.5 mL of Solution
C. Dilute with Solution B to volume, and mix well. less than 90.0 percent and not more than 110.0
Centrifuge a portion of the resultant solution for 5 min at percent of the labeled amounts of atenolol
14,000 rpm, and use the supernatant. Protect from light, (C14H22N203) and chlorthalidone (C14HllCIN204S).
and store at 2°-8°.
Sample solution: Shake thoroughly each bottle of Oral Packaging and storage-Preserve in well-closed
Suspension,Veterinary. Transfer 2.0 mL of Oral Suspension, containers.

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 410 Atenolol / Official Monographs USP 43

USP Reference standards (11)- Procedure for content uniformity-Proceed as directed in the
USP Atenolol RS Assay, except to prepare the Assay preparation as follows.
USP Chlorthalidone RS Transfer1 Tablet to a volumetric flask of such capacity that
Identification- when filled to volume, a concentration of about 0.25 mg of
chlorthalidone per mL isobtained. Add a mixture of water and
A: Shake a quantity of powdered Tablets, equivalent to acetonitrile (1:1) to about halfthe capacity of the flask, and
about 50 mg of chlorthalidone, with 5 mLof methanol for 15 shake by mechanical means for not lessthan 15 minutes to
minutes, and filter. Apply 10 IJL of this test solution, 10 IJL of disintegrate the Tablet. Dilute with water to volume, and mix.
a Standard solution of USP Chlorthalidone RS in methanol Pass a portion of this solutionthrough a filter having a 0.5-lJm
containing 10 mg per mL, and 10 IJL of a second Standard or finer porosity, and use the filtrate as the Assay preparation.
solution of USP Atenolol RS in methanol containing 10j mg Calculatethe quantities, in mg, of atenolol (C14H22N203) and
per mL, j being the ratio of the labeled amount, in mg, of
atenolol to the labeled amount, in mg, of chlorthalidone per chlorthalidone (C14HllClN204S) in the Tablet taken by the
Tablet to a thin-layer chromatographic plate (see formula:
Chromatography (621» coated with a 0.25-mm layerof
chromatographic silica gel mixture.Allow the spots to dry, and
develop the chromatogram in a solvent system consisting of a in which V is the volume, in mL, of the volumetric flask used
mixture of n-butyl alcohol and 1 N ammonium hydroxide prepare the Assay preparation; and the other terms are as
(5:1) until the solvent front has moved about three-fourths of to defined in the Assay.
the length of the plate. Remove the plate from the developing
chamber, and air-dry. Locate the spots on the plate by viewing Assay-
under short-wavelength UV light: the principal spots obtained Mobile phase-Prepare a mixture of 740 mLof water, 250
fromthe test solution correspond in RF value,size,and intensity mL of acetonitrile, 8 mL of 3.6 N sulfuric acid, and 930 mg of
to those obtained from the respective Standard solutions. sodium octyl sulfate. Make adjustments if necessary (see
B: The retention times of the major peaks in the System Suitability under Chromatography (621».
chromatogram of the Assay preparation correspond to those Standard preparation-Dissolve accurately weighed
in the chromatogram of the Standard preparation, as quantities of USP Atenolol RS and USP Chlorthalidone RS in a
obtained in the Assay. -mixture of water and acetonitrile (3:1) to obtain a solution
having known concentrations of about 0.25 mg of USP
Dissolution (711)- Chlorthalidone RS and 0.25}mg of USP Atenolol RS per mL, j
Medium: 0.01 N hydrochloric acid; 900 mL. being the ratio of the labeled amount, in mg, of atenolol to
Apparatus 2: 50 rpm. the labeled amount, in mg, of chlorthalidone per Tablet.
Time: 45 minutes. Assay preparation-Transfer 10 Tabletsto a volumetricflask
Determine the amounts of atenolol (C14H22N203) and of such capacity that when filled to volume, a concentration
chlorthalidone (C14HllClN204S) dissolved by employing the of about 0.5 mg of chlorthalidone per mL is obtained. Add a
following method. mixture of water and acetonitrile (1:1) to about half the
Mobile phase and Chromatographic system-Prepare as capacity of the flask, and shake by mechanical means for not
directed in the Assay. - less than 15 minutes to disintegrate the Tablets. Dilutewith a
Diluent-Prepare a mixture of 1000 mL of acetonitrile and mixture of water and acetonitrile (1:1) to volume, and mix.
32 mL of 3.6 N sulfuric acid. Pass a portion of this stock solution through a filter having a
Standard solvent-Prepare a mixture of water and Diluent 0.5-lJm or finer porosity. Transfer 25.0 mLof the clear filtrate
(750: 225). _ to a 50-mL volumetric flask, dilute with water to volume, and
Standard solution-Dissolve accurately weighed quantities mix.
of USP Atenolol RS and USP Chlorthalidone RS in Standard Chromatographic system (see Chromatography (621»-The
solvent to obtain a solution having known concentrations of liquid chromatograph is equipped with a 275-nm detector
about 0.00085L mg of USP Atenolol RS and 0.00085L' mg of and a 4.6-mm x 25-cm column that contains packing L1. The
USP Chlorthalidone RS per mL, Land L' being the labeled flow rate is about 1.7 mL per minute. Chromatograph the
amounts, in mg, of atenolol and chlorthalidone, respectively, Standard preparation, and record the peak responses as
per Tablet. _ directed for Procedure: the relative retention times are about
Test solution-Mix 10.0 mL of the filtered solution under 0.8 for atenolol and 1.0 for chlorthalidone; the resolution, R,
test and 3.0 mL of Diluent. between the atenolol and chlorthalidonepeaks is not lessthan
Procedure-Separately inject equal volumes (about 10 IJL) 3.0; and the relative standard deviation for replicate injections
of the Standard solution and the Test solution into the is not more than 2.0%.
chromatograph, record the chromatograms, and measure the Procedure-Separately inject equal volumes (about 10 IJL)
areas for the major peaks. Calculatethe quantities, in mg, of of the Assay preparation and the Standard preparation into the
atenolol (C14H22N203) and chlorthalidone (C14Hl1C1N204S) chromatograph, record the chromatograms, and measure the
dissolved by the same formula: areas for the major peaks. Calculate the quantities, in mg, of
atenolol (C14H22N203) and chlorthalidone (C14Hl1CIN204S) in
1170C(ru/rs) each Tablet taken by the formula:
in which C is the concentration, in mg per mL, of the 2C(V/10)(rulrs)
appropriate Reference Standard in the Standard solution; and
ru and ts are the responses of the corresponding analyte in which C isthe concentration, in mg per mL, of the
obtained from the Test solution and the Standard solution, appropriate USP Reference Standard in the Standard
respectively. preparation; Vis the volume,in mL, ofthevolumetric flask used
Tolerances-Not less than 80% (Q) of the labeled amount to prepare the stock solutionfor the Assay preparation; and ru
of atenolol (C14H22N203) is dissolved in 45 minutes, and not and rs are the-responsesfor the corresponding analyte
less than 70% (Q) of the labeled amount of chlorthalidone obtained from the Assay preparation and the Standard
(C14HllCIN204S) is dissolved in 45 minutes. preparation, respectively.
Uniformity of dosage units (905): meet the requirements.

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USP 43 Official Monographs / Atomoxetine 411

[NOTE-Ifa trailing peak or shoulder is observed on the System suitability

chlorthalidone peak with a relative retention time of not Samples: System suitability solution and Standardsolution
more than 1.1 in the chromatograms of both the Standard [NoTE--See Table 1 in Organic Impurities, Procedure 1
preparation and the Assay preparation, sum the areas for for the relative retention times.]
the chlorthalidone peak with the trailing peak or shoulder Suitability requirements
to report the peak responses for chlorthalidone.] Resolution: NLT5.0 between mandelic acid and
atomoxetine related compound A, System suitability
solution
Tailing factor: NMT 1.5 for atornoxetlne, System
suitability solution
Atomoxetine Hydrochloride Relative standard deviation: NMT 0.73%, Standard
solution
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of atomoxetine hydrochloride
(C17Hz1 NO . HCI) in the portion of Atomoxetine
Hydrochloride taken:
C17H21 NO . HCI 291 .82
Benzenepropanamine, N-methyl-y-(2-methylphenoxy)-, Result = (r vir s) x (C siC v) x 100
hydrochloride, (-);
(-)-N-Methyl-3-phenyl-3-(o-tolyloxy)propylamine = peak response from the Sample solution
hydrochloride [82248-59~7]. = peak response from the Standard solution
=concentration of USP Atomoxetine Hydrochloride
DEFINITION RS in the Standardsolution (mg/mL)
Atomoxetine Hydrochloride contains NLT 98.0% and NMT =concentration of Atomoxetine Hydrochloride in
102.0% of atomoxetine hydrochloride (C 17Hz1NO . HCI), the Sample solution (mg/mL)
calculated on the dried basis.
Acceptance criteria: 98.0%-102.0% on the dried basis
IDENTIFICATION
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1%
• ORGANIC IMPURITIES
• A. [NOTE-It is required to perform Organic Impurities,
§P9PY';J Procedure 1 and Organic Impurities, Procedure 2.]
• B. The retention time peak of the Sample Procedure 1
solution corresponds to that of atomoxetine R-isomer Buffer and Mobile phase: Prepare as directed in the Assay.
from the System suitability solution, as obtained in the test System suitability solution: 0.10 mg/mL of USP Mandelic
for Organic Impurities, Procedure 2. Acid RS, 0.15 mg/mL of USP Atomoxetine Related
• C. IDENTIFICATION TESTS-GENERAL (191), Chloride: Meets Compound A RS, and 0.25 mg/mL of USP Atomoxetine
the requirements of test A Hydrochloride RS in Mobile phase
Standard solution: 0.0025 mg/mL of USP Atomoxetine
ASSAY Hydrochloride RS in Mobile phase
• PROCEDURE Sample solution: 2.5 mg/mL of Atomoxetine
Buffer: 2.9 giL of phosphoric acid in water. Adjust with 5 M Hydrochloride in Mobile phase
potassium hydroxide solution to a pH of 2.5. To 1 Lof this Chromatographic system: Proceed as directed in the
solution add 5.9 9 of octanesulfonic acid sodium salt Assay, except for Run time.
monohydrate. Run time: 2.6 times the retention time of atomoxetine
Mobile phase: n-Propanol and Buffer (27:73). [NOTE-The System suitability
ratio of n-propanol in Buffercan be varied between 26:74 Samples: System suitability solution and Standard solution
and 29:71 to meet system suitability requirements.] [NOTE-See Table 1 for the relative retention times.]
System suitability solution: 0.1 mg/mL of USP Mandelic Suitability requirements
Acid RS, 0.15 mg/mL of USP Atomoxetine Related Resolution: NLT 5.0 between mandelic acid and
Compound A RS, and 0.25 mg/mL of USP Atomoxetine atomoxetine related compound A, System sUitability
Hydrochloride RS in Mobile phase solution
Standard solution: 0.25 mg/mL of USPAtomoxetine Tailing factor: NMT 1.5 for atomoxetine, System
Hydrochloride RS in Mobile phase. Sonication may be used sUitability solution
to aid in dissolution. Relative standard deviation: NMT 5% from three
Sample solution: 0.25 mg/mL of Atomoxetine injections, Standardsolution
Hydrochloride in Mobile phase. Sonication may be used to Analysis
aid in dissolution. Samples: Standardsolution and Sample solution
Chromatographic system Calculate the percentage of any individual impurity in the
(See Chromatography (621), System SUitability.) portion of Atomoxetine Hydrochloride taken:
Mode: LC
Detector: UV 215 nm Result =(r vir s) x (C siC v) x 100
Column: 4.6-mm x 15-cm; 3.5-lJm packing L7
Column temperature: 40° ru = peak response of each
individual impurity from
Flow rate: 1 mL/min the Sample solution '
Injection volume: 10 IJL r s = peak response of atomoxetine from the Standard
Run time: 1.3 times the retention time of atomoxetine solution

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412 Atomoxetine / Official Monographs USP 43

= concentration of USP Atomoxetine Hydrochloride Acceptance criteria: See Table 2.

RS in the Standard solution (mg/mL)
= concentration of Atomoxetine Hydrochloride in Table 2
the Sample solution (mg/mL)~ Relative Acceptance
Retention Criteria,
Acceptance criteria: See Table 7. Name Time NMT(%)
AtomoxetineS-isomer" 0.47 0.5
Table 1
Relative Acceptance Atomoxetinerelated com-
Retention Criteria, pound Cb 0.52 0.1
Name Time NMT(%) Atomoxetinerelated com-
Mandelicacid 0.20 0.10 pound B 0.56 0.1

Atomoxetinerelated com- Atomoxetine 1.0 -

pound A 0.27 0.10
a N-Methyl-3-phenyl-3-(o-tolyloxy)propan-l-amine.
Desmethyl atornoxetlnes 0.73 0.3 b N-Methyl-3-phenyl-3-(p-tolyloxy)propan-l-amine.
Atomoxetine 1.0 -
SPECIFIC TESTS
Any individual • Loss ON DRYING (731)
unspecified impurity - 0.10 Analysis: Dry under vacuum at 105° for 2 h.
Total impurities - 0.5 Acceptance criteria: NMT 0.5%

a (R)-N-Methyl-3-phenoxy-3-phenylpropan-l-amine. ADDITIONAL REQUIREMENTS

• PACKAGING AND STORAGE: Preserve in well-closed
Procedure 2 containers. Store at room temperature.
Mobile phase: Isopropyl alcohol, diethylamine, • USP REFERENCE STANDARDS (11)
trifluoroacetic acid, and n-hexane (150: 1.5: 2.0: 846.5) USP Atomoxetine Hydrochloride RS
System suitability solution: 3.5 mg/mL of USP USP Atomoxetine Related Compound A RS
Atomoxetine Hydrochloride RS, 17.5 IJg/mL of USP 3-(Methylamino)-1-phenylpropan-l-ol.
Atomoxetine S-Isomer RS, and 3.5 IJg/mL of USP ClOH 1SNO 165.23
Atomoxetine Related Compound B RS, prepared by first USP Atomoxetine Related Compound B RS
dissolving the Reference Standards in absolute alcohol N-Methyl-3-phenyl-3-(m-tolyloxy)propan-l-amine
using 25% of.the final volume. Dilute with n-hexane t~ hydrochloride.
volume. C17H21 NO . HCI 291.82
Sample solution: 3.5 mg/mL of Atomoxetine USP Atomoxetine S-Isomer RS
Hydrochloride prepared by first dissolving it in absolute (S)-N-Methyl-3-phenyl-3-(o-tolyloxy)propan-l-amine
alcohol, using 25% of the final volume. Dilute with hydrochloride.
n-hexane to volume. C17H21 NO . HCI 291.82
Chromatographic system USP Mandelic Acid RS
(See Chromatography (621), System Suitability.) a.-Hydroxyphenylacetic acid.
Mode: LC CsHs0 3 152.15
Detector: UV 273 nm
Column: 4.6-mm x 25-cm; s-prn packing L40
Flow rate: 1 mL/min .
Injection volume: 10 IJL
Run time: 1.3 times the retention time of atomoxetine Atomoxetine Capsules
System suitability
Sample: System sUitability solution DEFINITION
[NoTE-See Table 2 for the relative retention times.] Atomoxetine Capsulescontain NLT 90.0% and NMT 110.0%
Suitability requirements of the labeled amount of atomoxetine (C17H 21NO).
Resolution: NLT 1.75 between atomoxetine S-isomer IDENTIFICATION
and atomoxetine related compound B
Tailing factor: NMT 1.8 for atomoxetine
Analysis
Sample: Sample solution
Calculate the percentage of atomoxetine related • A.>~~
~P,#cl»()
compound B, atomoxetine related compound C, and Standard: 6 mg/ml of USP Atomoxetir"le Hydrochloride RS
atomoxetine S-isomer in the portion of Atomoxetine in methanol. Dry the solution to a dry powder under an air
Hydrochloride taken: or nitrogen purge for a minimum of 3 h.
Sample: Shakethe contents of a sufficient number of
Result = (ru/r r) x 100
Capsules, equivalent to about 60 mg of atomoxetine, with
ru =peak response of each individual impurity from 10 mL of methanol. Centrifuge at 4000 rpm for 5 min.
the Sample solution Evaporate the solution to a dry powder with the aid of a
rt = sum of all the peak responses of atomoxetine current of air or stream of nitrogen.
related compound B, atomoxetine related Acceptance criteria: The IR spectrum exhibits main bands
at (±2) wavenumbers (crrr") 2955, 2855, 1599-1604,
compound C, atomoxetine S-isomer, and
atomoxetine from the Sample solution 1492, 1048, 1023, and 1010.

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 USP 43 Official Monographs / Atomoxetine 41 3

• B. The retention time of the major peak of the Sample Standard solution: Dilute the Standard stock solution with
solution corresponds to that of the Standard solution, as Medium to obtain a final concentration of (Lll 000) mg/ml,
obtained in the Assay. where L is the Capsule label claim in mg.
Sample solution: Pass a portion of the solution under test
ASSAY
through a suitable filter.
• PROCEDURE Chromatographic system: Proceed as directed in the
Buffer: 5.8 gil of monobasic potassium phosphate in water. Assay.
To each liter of this solution add 3.0 ml of triethylamine, System suitability
and adjust with phosphoric acid to a pH of 2.5. Sample: Standard solution
Mobile phase: Acetonitrile and Buffer (38:62) Suitability requirements
System suitability solution: 0.1 mg/ml of atomoxetine Tailing factor: NMT 2.0
(free base) from USP Atomoxetine Hydrochloride RS and Relative standard deviation: NMT 1.4%
0.02 mg/ml of o-cresol in Mobilephase. Sonicate to aid in Analysis
dissolution. Samples: Standard solution and Sample solution
Standard solution: 0.1 mg/ml of atomoxetine (free base) Calculate the percentage of the labeled amount of
from USP Atomoxetine Hydrochloride RS in Mobilephase. atomoxetine (C17H21 NO) dissolved:
Sonicate to aid in dissolution.
Sample stock solution: From NlT 10 Capsules (including Result = (rulr s) x (CslL) x V x 100
shells) prepared as follows. Add the intact Capsules to a
suitable volumetric flask. Add Mobilephaseto fill.65% of the t» = peak response from the Sample solution
final volume. Allowto stand for at least 10 min, then shake ts = peak response from the Standard solution
for 20 min. Dilute with Mobilephase to volume.
Sample solution: Nominally 0.1 mg/ml of atomoxetine, Cs =concentration of atomoxetine in the Standard
prepared by diluting a suitable volume of Sample stock solution (mg/ml)
solution with Mobilephase L = label claim (mg/Capsule)
Chromatographic system V = volume of Medium (ml)
(See Chromatography (621), System Suitability.) Tolerances: NlT 80% (Q) of the labeled amount of
Mode: lC atomoxetine (C17H 21 NO) is dissolved.
Detector: UV 220 nm
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
Column: 4.6-mm x 7.5-cm; 3.5-~m packing l7
Column temperature: 35° requirements
Flow rate: 1.5 ml/min IMPURITIES
Injection volume: 10 ut, • ORGANIC IMPURITIES
Run time: 1.7 times the retention time of atomoxetine Buffer: Dissolve4.9 g of sodium 1-decanesulfonate and 6.9
System suitability 9 of monobasic potassium phosphate in 1 l of water. Adjust
Samples: System suitability solution and Standard solution with phosphoric acid to a pH of 3.1.
[NOTE-The relative retention times for atornoxetlne Mobile phase: Acetonitrile and Buffer (41 :59)
and o-cresol are 1.0 and 1.3, respectively.] Sensitivity solution: 0.1 ~g/ml of atomoxetine in Mobile
Suitability requirements phase
Resolution: NlT 3.5 between atomoxetine and o-cresol, System suitability solution: 1 mg/ml of atomoxetine
System suitability solution containing atomoxetine N-amide prepared as follows.
Tailing factor: NMT 2.0 for atomoxetine, System Weigh equal amounts of USP Atomoxetine Hydrochloride
suitability solution RS and urea, and place in a volumetric flask. Add water to
Relative standard deviation: NMT 1.0% for fill 10% of the final volume. Sonicate for 3 min. Place the
atomoxetine, Standardsolution flask in an 85° oven for 40 min. Allowthe solution to cool
Analysis to room temperature. Dilute with Mobilephase to volume.
Samples: Standard solution and Sample solution [NOTE-Theoven temperature and time in the oven can be
Calculate the percentage of the labeled amount of adjusted to give a suitable level of atomoxetine N-amide
atomoxetine (C17H 21 NO) in the portion of Capsules taken: peak.]
Sample solution: 1 mg/mL of atomoxetine in Mobile
Result = (rulr s) x (CslCu) x 100 phase, from the contents of NlT 5 Capsules prepared as
follows. Transfer the Capsule contents to a suitable
= peak response from the Sample solution volumetric flask. Fill 50% of the final volume with Mobile
= peak response from the Standard solution phase. Swirl, and let stand for 15 min. Dilute with Mobile
= concentration of atomoxetine in theStandard phase to volume.
solution (mg/ml) Chromatographic system
= nominal concentration of atomoxetine in the (See Chromatography (621), System Suitability.)
Sample solution (mg/ml) Mode: LC
Detector: UV 215 nm
Acceptance criteria: 90.0%-110.0% Column: 4.6-mm x 15-cm; 3.5-~m packing l7
Column temperature: 30°
PERFORMANCE TESTS
Flow rate: 1 ml/min
• DISSOLUTION (711)
Injection volume: 10 ul,
Medium: 0.1 N hydrochloric acid; 1000 ml, deaerated Run time: 2.3 times the retention time of atomoxetine
Apparatus 2: 50 rpm, with three-prong sinker System suitability .
Time: 30 min Samples: Sensitivity solution and System suitabilitysolution
Buffer and Mobile phase: Prepare as directed in the Assay. [NOTE-See Table 1 for the relative retention times.]
Standard stock solution: 0.1 mg/ml of atomoxetine (free
base) from USPAtomoxetine Hydrochloride RS in Medium.
Sonicate to aid in dissolution.

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414 Atomoxetine / OfficialMonographs USP 43

Suitability requirements Anhydrous [134523-03-8].

Resolution: NLT 2.6 between atomoxetine and C66H6SCaF2N401O·3H20 1209.41
atomoxetine N-amide, System suitability solution
Relative standard deviation: NMT 5%, Sensitivity Trihydrate [344423-98-9].
solution C66H6SCaF2N4010 . C3H s0 2
Analysis Propylene glycol solvate 1231.46
Sample: Sample solution
Calculate the percentage of each individual impurity in the DEFINITION
portion of Capsules taken: Atorvastatin Calcium contains NLT 98.0% and NMT 102.0%
of atorvastatin calcium(C66H6SCaF2N4010), calculated on the
Result= (rv/rr) x 100 anhydrous and solvent-free basis. If labeled as a propylene
glycol solvate, it contains NLT 98.0% and NMT 102.0% of
tu = peak response of each individual impurityfrom atorvastatin calcium (C66H6SCaF2N4010), calculated on the
the Sample solution anhydrous, propylene glycol-free, and solvent-free basis. It
rr = sum of all the peak responses from the Sample may contain a suitable antioxidant.
solution
IDENTIFICATION
Acceptance criteria: See Table 1.
Table 1
Relative Acceptance
Retention Criteria,
Name Time NMT(%) a appears IR spectra of the analyte
and the standard, separatelydissolve equal portions of the
Desmethyl atornoxetlne" 0.76 0.3 sample specimen and the USP Reference Standard in
Atomoxetine 1.0 - equal volumes of methanol, evaporate the solution to
dryness in similar containers under identical conditions,
Atomoxetine and repeat the test on the residues.]
N-amide b 1.2 0.2
• B. CALCIUM
Any individual Diluent: Methanol, water, and hydrochloric acid (75:25:2)
unspecified degradation - Sample solution: 0.05 mg/mL of Atorvastatin Calcium in
product 0.2
Diluent
Total impurities I - 1.0 Blank: Diluent
Analysis
a (R)-N-Methyl-3-phenoxy-3-phenylpropan-l-amine. Samples: Sample solution and Blank
b (R)-1-Methyl-1-[3-phenyl-3-( o-tolyloxy)propyl]urea. Instrumental conditions
(See AtomicAbsorption Spectroscopy (852).)
ADDITIONAL REQUIREMENTS Mode: Atomic absorption spectrophotometry
• PACKAGING AND STORAGE: Preserve in well-closed Analytical wavelength: Calcium emission line at 422.7
containers. Store at controlled room temperature. nm
• USP REFERENCE STANDARDS (11) Flame: Air-acetylene
USP Atomoxetine Hydrochloride RS Acceptance criteria: The Sample solution exhibits a
significantabsorption at the calcium emission lineat 422.7
nm.
ASSAY
Atorvastatin Calcium • PROCEDURE
Buffer: 3.9 giL of ammonium acetate in water. Adjustwith
glacial acetic acid to a pH of 5.0 ± 0.1.
Solution A: Acetonitrile, stabilizer-free tetrahydrofuran, and
Buffer(21:12:67)
Solution B: Acetonitrile, stabilizer-free tetrahydrofuran, and
Buffer(61:12:27)
Mobile phase: See Table 1. [NOTE-If necessary, adjust the
Mobilephase by increasingor decreasing the percentage of
acetonitrile or the pH of the ammonium acetate solution to
achieve a retention time of 26-34 min for the atorvastatin
peak.]
C66H6SCaF2N401O 1155.36 Table 1
1 H-pyrrole-1-heptanoic acid, Time Solution A Solution B
2-(4-fluorophenyl)-~,8-dihydroxy-5-(1-methylethyl)­ (min) (%) (%)
3-phenyl-4-[(phenylami no)carbonyl]-, calcium salt (2:1), [R-
(R*,R*)]-; 0 100 0
Calciurn (~R,8 R)-2-(p-fl uorophenyli-B, 8-dihydroxy- 40 100 0
5-isopropyl-3-phenyl-4-(phenylcarbamoyl)pyrrole-
1-heptanoate (1:2); 70 20 80
[(3R,5R)-7-[3-(Phenylcarbamoyl)-5-(4-fluorophenyl)-2- 85 0 100
isopropyl-4-phenyl-1 H-pyrrol-1-yl]-3,5-dihydroxyheptanoic
100 0 100
acid, calcium salt]

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 USP 43 Official Monographs / Atorvastatin 415

Table 1 (continued) Detector: Flame ionization

Time Solution A Solution B Column: 0.53-mm x 75-mi 3-l.Im coating of G43
(min) (%) (%) Temperatures
105 100 0
Injection port: 230 0
Detector: 250 0
115 100 0 Column: See Table 2.

Diluent: N,N-dimethylformamide Table 2

System sultablltty solution: 0.05 mg/mL of USP Hold Time at Fi·
Atorvastatin Calcium RS and 0.06 mg/mL of USP Initial Temperature Final nal
Temperature Ramp Temperature Temperature
Atorvastatin Related Compound B RS in Diluent e) ("/min) (0) (min)
Standard solution: 0.4 mg/mL of USPAtorvastatin Calcium
RS in Diluent. [NoTE-Use sonication if necessary.] 100 0 100 1
Sample solution: 0.4 mg/mL of Atorvastatin Calcium in 100 10 140 5
Diluent. [NoTE-Use sonication if necessary.]
Chromatographic system 140 30 225 3
(See Chromatography (621), System Suitability.)
[NOTE-If significant fronting of the peaks for atorvastatin Carrier gas: Helium
related compound Band atorvastatin is observed, use Flow rate: 6.0 mL/min
the following diluent to prepare the Sample solution, Injection volume: 1 I.IL
the Standard solution, and the System suitability Injection type: Splitless, using a suitable inlet liner
solution: acetonitrile, stabilizer-free tetrahydrofuran, System suitability
and water (1:1 :2).] Sample: Standard solution
Mode: LC Suitability requirements
Detector: UV 244 nm Tailing factor: NMT 2.0
Column: 4.6-mm x 25-cm; 5-l.Im packing L7 Relative standard deviation: NMT 5.0%
Column temperature: 35 0 Analysis
Flow rate: 1.5 mL/min Samples: Standard solution and Sample solution
Injection volume: 20 I.IL Calculate the percentage of propylene glycol in the portion
System suitability of Atorvastatin Calcium as propylene glycol solvate taken:
Samples: System suitability solution and Standard solution
Suitability requirements Result = (r vir s) x (C siC v) x 100
Resolution: NLT 1.5 between the peaks for atorvastatin
related compound Band atorvastatin, System suitability ru =peak response of propylene glycol from the
solution Sample solution
Tailing factor: NMT 1.6, Standard solution rs = peak response of propylene glycol from the
Relative standard deviation: NMT 0.6%, Standard Standard solution
solution Cs = concentration of propylene glycol in the Standard
Analysis solution(mg/mL)
Samples: Standard solution and Sample solution Cu = concentration of Atorvastatin Calcium (as
Calculate the percentage of atorvastatin calcium (C66H68 propylene glycol solvate) in the Sample solution
CaF2N40 1o) in the portion of Atorvastatin Calcium taken: (mg/mL)

Result =(r vir s)x (C siC v) x 100 Acceptance criteria: 5.4%-7.3%

IMPURITIES
ru =peak response from the Sample solution • ORGANIC IMPURITIES, PROCEDURE 1: [NOTE-On the basis
rs = peak response from the Standard solution of the synthetic route or of the solid state nature of the drug
Cs =concentration of USPAtorvastatin Calcium RS in substance, perform either Procedure 1 or Procedure 2.
the Standard solution (mg/mL) Procedure 2 may be suitable when atorvastatin lactone,
Cu =concentration of Atorvastatin Calcium in the atorvastatin epoxy tetrahydrofuran analog, and
Sample solution (mg/mL) atorvastatin acetonide are possible related compounds, and
it may be suitable for an amorphous form of the drug
Acceptance criteria: 98.0%-102.0% on the anhydrous and substance.]
solvent-free basis. If labeled as a propylene glycol solvate, Buffer, Solution A, Solution B, Mobile phase, Diluent,
98.0%-102.0% on the anhydrous, propylene glycol-free, System suitability solution, and Chromatographic
and solvent-free basis. system: Proceed as directed in the Assay.
OTHER COMPONENTS Standard solution: 1.5 I.Ig/mL each of USP Atorvastatin
• CONTENT OF PROPYLENE GLYCOL (if labeled as a propylene Related Compound A RS, USPAtorvastatin Related
glycol solvate) Compound B RS, USP Atorvastatin Related Compound C
Diluent: Dimethylsulfoxide RS, and USP Atorvastatin Related Compound D RS in Diluent
Standard solution: 0.125 mg/mL of propylene glycol in Sample solution: 1 mg/mL of Atorvastatin Calcium in
Diluent Diluent. [NoTE-Use sonication if necessary.]
Sample solution: 2.5 mg/mL of Atorvastatin Calcium (as System suitability
propylene glycol solvate) in Diluent. Use sonication as Sample: System suitabilitysolution
needed to achieve a complete dissolution. Suitability requirements
Chromatographic system Resolution: NLT 1.5 between the peaks for atorvastatin
(See Chromatography (621), System Suitability.) related compound Band atorvastatin
Mode: GC

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 416 Atorvastatin / Official Monographs USP 43

Analysis ammonium acetate, and dissolve in 950 mL of water. Adjust

Samples: Standardsolution and Sample solution with 20% formic acid to a pH of 5.0, and dilute with water
Chromatograph the Standardsolution, and identify the to 1 L.
components based on their relative retention times, Solution A: Acetonitrile and Buffer(33:67)
given in Table 3. Solution B: Acetonitrile
Calculate the percentage of each of the atorvastatin related Solution C: Stabilizer-free tetrahydrofuran
compounds A, B, C, and D in the portion of Atorvastatin Mobile phase: See Table 4. Return to original conditions,
Calcium taken: and re-equilibrate the system.
Result =(r vir s) x (C siC v) x 100 Table 4
Time Solution A Solution B Solution C
=peak response of the relevant atorvastatin related (min) (0/0) (%) (%)
compound from the Sample solution
=peak response of the relevant atorvastatin related 0 91 0 9
compound from the Standardsolution 15 91 6 3
= concentration of the relevant atorvastatin related 82 16 2
compound in the Standardsolution (mg/mL) 20
=concentration of Atorvastatin Calcium in the 25 82 16 2
Sample solution (mg/mL)
50 32 66 2
Calculate the percentage of any other individual impurity 55 32 66 2
in the portion of ~torvastatin Calcium taken:
Diluent: Acetonitrile, stabilizer-free tetrahydrofuran, and
Result = (r vir T) x 100
Buffer (60:5:35)
ru =peak response of any other individual impurity Peak identification solution: 0.5 mg/mL of USP
from the Sample solution Atorvastatin Calcium RS and 2.5 ~g/mL each of USP
rT = sum of all the peak responses from the Sample Atorvastatin Related Compound A RS, USP Atorvastatin
solution Related Compound B RS, USP Atorvastatin Related
Compound H RS, and USPAtorvastatin Related Compound
Acceptance criteria: See Table 3. Disregard any peak I RS in Diluent
observed in the blank; the reporting level for impurities is Sample solution: 0.5 mg/mL of Atorvastatin Calcium in
0.05%. Diluent. Use sonication to dissolve. [NOTE-The solution is
stable for 3 h at room temperature and for 24 h when stored
Table 3 at 2°_8°, protected from light.]
Chromatographic system .
Relative Acceptance
Retention Criteria, (See Chromatography (621 ~ System Suitability.)
Name Time NMT(%) Mode: LC
Detector: UV 254 nm
Atorvastatin related com- Column: 4.6-mm x 25-cm; 4-~m packing L11
pound Aa 0.8 0.3
Temperatures
Atorvastatin related com- Column: 40°
pound Bb 0.9 0.3 Autosampler: 4°
Atorvastatin 1.0 - Flow rate: 1.1 mL/min
Injection volume: 15 ~L
Atorvastatin related com- System suitability
pound CC 1.2 0.3
Sample: Peak identification solution
Atorvastatin related com- Suitability requirements
pound Dd,e 2.1 0.2 Peak-to-valley ratio: NLT 2 between the peaks for
Anyother individual atorvastatin related compound Band atorvastatin
impurity - 0.1 Analysis
Sample: Sample solution
Total Impurities' - 1.0
Calculate the percentage of each impurity in the portion of
a Desfluoro impurity. Atorvastatin Calcium taken:
b 3S,5R Isomer.
C Difluoro impurity.
Result = (r vir T) x (1IF) x 100
d Epoxide impurity.
eAtorvastatin related compound D may undergo a conversion to its cyclic ru = peak response of the impurity from the Sample
hemiketal, which is a specifiedimpuritylistedin Table 5 in Organic Impurities, solution
Procedure 2, as "atorvastatinepoxy tetrahydrofuran analog". The cyclic
hemiketal of atorvastatin related compound D elutesabout 1-2 min before
rT =sum of all the peak responses, each divided by the
atorvastatinrelated compound D. Usethe sum of the areasof the two peaksas
corresponding value of the relative response
a peak responsefor atorvastatin related compound D in the Standard solution factor from Table 5
and the Sample solution. F = relative response factor for the impurity (see Table
f Thistotal does not includeatorvastatinrelated compound E, as determined in 5)
the Enantiomeric Purity test.
Acceptance criteria: See Table 5. Disregard any peak
• ORGANIC IMPURITIES, PROCEDURE 2 eluting before 2 min and any peak observed in the blank;
Buffer: pH 5.0 mixture of 0.045 M ammonium formate and the reporting level for impurities is 0.05%.
0:0045 M ammonium acetate solutions, prepared as
follows. Weigh 2.84 g of ammonium formate and 0.35 g of

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 USP 43 Official Monographs / Atorvastatin 41 7

Table 5 Flow rate: 1.0 mL/min

Relative Relative Acceptance Injection volume: 20 IJL
Retention Response Criteria, System suitability
Name Time Factor NMT(%) Sample: System suitability solution
Atorvastatin [NOTE-The elution order of the peaks is atorvastatin
diamino' 0.58 0.74 0.15 related compound Efollowed by atorvastatin.]
Resolution: NLT 2.0 between the peaks for atorvastatin
Atorvastatin related com-
pound Ab 0.86 1.0 0.3 related compound E and atorvastatin
Analysis
Atorvastatin related com- Sample: Sample solution
pound Be 0.94 1.0 0.3 Calculate the percentage of atorvastatin related compound
Atorvastatin 1.0 - - E in the portion of Atorvastatin Calcium taken:
Atorvastatin related com- Result = (rvIr T) x 100
pound Cd (if present) 1.1 1.0 0.3
Atorvastatin 3-deoxy- ru = peak response for atorvastatin related
hept-2-enoic aclds 1.45 1.0 0.10 compound E
Atorvastatin related com- rT = sum of the peak responses for atorvastatin related
pound HI 1.90 1.0 0.15 I compound E and atorvastatin
Atorvastatin epoxy tetra- Acceptance criteria: NMT 0.3% of atorvastatin related
hydrofuran analoqs 2.00 0.71 0.15
compound E
Atorvastatin ethyl ester" 2.08 1.0 0.15
SPECIFIC TESTS
Atorvastatin related com- • WATER DETERMINATION, Method la (921): 3.5%-5.5% for
pound 0' 2.18 1.3 0.15 the trihydrate form. If labeled as amorphous or as
Atorvastatin related com- semicrystalline, NMT 6.0%. Iflabeled as a propylene glycol
pound II 2.75 1.0 0.15 solvate, NMT 1.0%.
Anyother individual im- ADDITIONAL REQUIREMENTS
purity - 1.0 0.10 • PACKAGING AND STORAGE: Preserve the trihydrate form in
Total lmpuritles'' - - 1.0 well-closed containers, and store at room temperature. If
labeled as amorphous or semicrystalline or as a propylene
a (3 R,5R)-7 -{(3R,5R)-7-~2-(4-Fluorophenyl)-5-isopropyl-3-phenyl-4- glycol solvate, store as per labeling instructions. Possible
(phenylcarbamoyl)-l H-pyrrol-l-yl]-3,5-dihydroxyheptanamido}-3,5- packaging and storage conditions could include the
dihydroxyheptanoic acid.
b Oesfluoro impurity.
followinq: Preserve in well-closed containers protected
c 3S,5R Isomer.
from light and moisture, or in tight containers; store at
d Oifluoroimpurity.
room temperature, at controlled room temperature, or at
e (5, E)-7-[2-(4-Fluorophenyl)-5-isopropyl-3-phenyl-4-(phenylcarbamoyl)·1 H- 2°_8°; store under nitrogen atmosphere or packed with an
wrrol-l-yl]-5-hydroxyhept-2.enoic acid. oxygen absorber; and store under nitrogen atmosphere,
Lactone impurity. packed with silica gel and an oxygen absorber.
9 4-(4-Fluorophenyl)-2,4-dihydroxy-2·isopropyl-N,5-dipherWI-3,6- • LABELING: Where it is an amorphous form, the label so
dioxabicyclo[3.1.0]hexane-l-carboxamide. indicates. Where it is a semicrystalline form, the label so
h (3R,5R)-Ethyl 7-(2-(4-fluorophenyl)-5-isopropyl-3-phenyl-4- indicates. Where it is a propylene glycol solvate form, the
(phenylcarbamoyl)-l H-pyrrol-l-yl)-3,5·dihydroxyheptanoate.
label so indicates. If a test for OrganicImpurities other than
i Epoxide impurity.
j Acetonide impurity.
Procedure 7 is used, the labeling states the test with which
kThistotal does not include atorvastatin related compound E,as determined in the article complies. Label it to indicate the name and
the Enantiomeric Purity test. quantity of any added antioxidant.

• ENANTIOMERIC PURITY
Mobile phase: Hexane, dehydrated alcohol, and
trifluoroacetic acid (940:60:1) • USP REFERENCE STANDARDS (11)
System suitability stock solution: 5 mg/mL of USP USP Atorvastatin Calcium RS
Atorvastatin Calcium RS and 37.5 IJg/mL of USP USPAtorvastatin Related Compou_nd A RS .
Atorvastatin Related Compound E RS in methanol. A • 3R,5R)-7-[2-isopropyl-4,5-diphehyl-3:-
[NOTE-Atorvastatin related compound E is the 3S,5S rbamoyl)-1 H-pyrrol-l-yl]~'
enantiomer of atorvastatin.] ,3,. roxyheptanoate (1 :2);
System suitability solution: Transfer 2.0 mL of the System Also known as.... (ERR l-MaY-2019) Desfluoro impurity, or (3R,
suitability stock solution to a 1O-mL volumetric flask, add 2.0 5R)-7 -[3-(phenylcarbamoyl)-2-isopropyl-4, 5-d iphenyl-
mL of dehydrated alcohol, and dilute with hexane to 1H-pyrrol-l-yl]-3,5-dihydroxyheptanoicacid, calcium
volume. salt.
Sample solution: Transfer 10 mg of Atorvastatin Calcium to C66H70CaN4010 1119.38
a 1O-mL volumetric flask, dissolve in 2.0 mL of methanol, USP Atorvastatin Related Compound B RS
add 2.0 mL of dehydrated alcohol, and dilute with hexane 3S,5R Isomer, or (3S,5R)-7-[3-(phenylcarbamoyl)-5-(4-
to volume. fluorophenyl)-2-isopropyl-4-phenyl-l H-pyrrol-l-yl]-3,5-
Chromatographic system dihydroxyheptanoic acid, calcium salt.
(See Chromatography (621), System Suitability.) C66H6sCaF2N4010 1155.34
Mode: LC
Detector: UV 244 nm
Column: 4.6-mm x 25-cm; packing L51

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418 Atorvastatin / OfficialMonographs USP 43

USP Atorvastatin Related Compound C RS Diluent to volume. Centrifuge or passthrough a suitable

Difluoro impurity, or (3R,5R)-7-[3-(phenylcarbamoyl)- filter of 0.45-lJm pore size.
4,5-bis(4-fluorophenyl)-2-isopropyl-l H-pyrrol-l-yl]-3,5- Sample solution: Nominally equivalent to 0.1 mg/mlof
dihydroxyheptanoic acid, calcium salt. atorvastatin in Diluent from the Sample stock solution
C66H66F4N4010 1191.34 Chromatographic system '
USP Atorvastatin Related Compound D RS (See Chromatography (621), System Suitability.)
Epoxide impurity, or 3-(4-fluorobenzoyl)-2-isobutyryl-3- Mode: lC
phenyl-oxirane-2-carboxylic acid phenylamide. Detector
CZ6HzzFN04 431.46 Assay: UV 244 nm
USP Atorvastatin Related Cornpound E RS Identification A: Diode array; UV 200-400 nm
3S,5S Enantiomer, or (3S,5S)-7-[3-(phenylcarbamoyl)-5- Column: 4.6-mm x 25-cmi 5-lJm packing II
(4-fluorophenyl)-2-isopropyl-4-phenyl-l H-pyrrol-l-yl]- Column temperature: 30°
3,5-dihydroxyheptanoic acid, calcium salt. Flow rate: 1.5 ml/min
C66H6SCaFzN401O 1155.34 Injection volume: 20 IJl
USP Atorvastatin Related Compound H RS (lactone System suitability
impurity) Samples: System suitability solution and Standard solution
5-(4-Fluorophenyl)-1-{2-[(2R,4R)-4-hydroxy-6- Suitability requirements
oxotetrahydro-2H-pyran-2-yl]ethyl}-2-isopropyl-N,4- Resolution: NlT 5.0 between atorvastatin and
diphenyl-l H-pyrrole-3-carboxamide. atorvastatin related compound H, System suitability
C33H33FNz04 540.62 solution
USP Atorvastatin Related Compound I RS (acetonide Tailing factor: NMT 1.5 for atorvastatin, System
impurity) suitability solution
tert-Butyl 2-« 4R,6R)-6-{2-[2-(4-fluorophenyl)-5- Relative standard deviation: NMT 1.0%, Standard
isopropyl-3-phenyl-4-(phenylcarbamoyl)-1 H-pyrrol-l- solution
yl]ethyl}-2,2-dimethyl-l,3-dioxan-4-yl)acetate. Analysis
C4oH47FNzOs 654.81 Samples: Standardsolution and Sample solution
Calculatethe percentage of the labeled amount of
atorvastatin (C33H3SFNzOs) in the portion ofTabletstaken:
Result = (rulrs) x (CslCu) x [M x (M rdMr2 ) ] x 100
Atorvastatin Calcium Tablets
t» = peak response of atorvastatin from the Sample
DEFINITION solution
Atorvastatin Calcium Tablets contain an amount of rs = peak response of atorvastatin from the Standard
atorvastatin calcium [(C33H34FNzOs)zCa], equivalent to NlT solution
94.5% and NMT 105.0% of the labeled amount of Cs = concentration of USP Atorvastatin Calcium RS in
atorvastatin. the Standardsolution (mg/ml)
Cu = nominal concentration of atorvastatin in the
IDENTIFICATION Sample solution (mg/ml)
• A. The UV absorption spectrum of the major peak of the M = number of moles of atorvastatin per mole of
Sample solution corresponds to that of the Standard atorvastatin calcium, 2
solution, as obtained in the Assay. Mr1 = molecularweight of atorvastatin, 558.64
• B. The retention time of the major peak of the Sample Mr2 = molecularweight of atorvastatin calcium,
solution corresponds to that of the Standardsolution, as 1155.34
obtained in the Assay.
ASSAY Acceptance criteria: 94.5%-105.0%
• PROCEDURE PERFORMANCE TESTS
Buffer: 0.05 M ammonium citrate buffer pH 4,0 prepared
as follows. Dissolve 9.62 9 of anhydrous citric acid in 950
ml ofwater, adjust with ammonium hydroxide to a pH of
4.0, and dilute with water to 1000 ml. • DISSOLUTION (711)
Mobile phase: Acetonitrile, stabilizer-free tetrahydrofuran, Test 1
and Buffer(27:20:53) Buffer: 0.05 M phosphate buffer prepared as follows.
Solution A: Dissolve 9.62 9 of anhydrous citric acid in 900 Dissolve 6.8 9 of monobasic potassium phosphate in 900
ml of water, adjust with ammonium hydroxide to a pH of ml of water. Adjustwith 6 N sodium hydroxide to a pH
7.4, and dilute with water to 1000 ml. of 6.8 and dilute with water to 1 l.
Diluent: Acetonitrile and Solution A (1:1) Medium: Buffer; 900 ml
System suitability solution. 0.1 mg/ml of USP Atorvastatin Apparatus 2: 75 rpm
Calcium RS and 0.01 mg/ml of USP Atorvastatin Related Time: 15 min
Compound H RS in Diluent. Shake mechanicallyfor 30 min Diluent: Acetonitrile and water (50:50)
or until dissolved. Standard stock solution: 1 mg/ml of USP Atorvastatin
Standard solution: 0.1 mg/ml of USP Atorvastatin Calcium Calcium RS in Diluent. Shake mechanically for 10 min or
RS in Diluent. Shake mechanically for 15 min or until until dissolved.
dissolved. Standard solution: (LI900) mg/ml in Medium from
Sample stock solution: Prepare a known nominal Standardstocksolution, where L isthe label claim in mgl
concentration ofatorvastatin by transferring NlT 10 Tablets Tablet
to an appropriate volumetricflask. Add Diluent to about Sample solution: Pass a portion of the solution under test
50% of the final volume of the flask, and shake the mixture through a suitable filter or centrifuge prior to analysis.
mechanically for 15 min or until dissolved. Dilute with

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USP 43 OfficialMonographs / Atorvastatin 419

Instrumental conditions Table 2

(See Ultraviolet-Visible Spectroscopy (857).) Time Solution A Solution B
Mode: UV (min) (%) (%)
Analytical wavelength: 244 nm 0.00 30 70
Cell: See Table 1 or make appropriate dilutions of the
solutions with Medium to be within the validated 0.69 30 70
linearity range of the suitable spectrophotometer. 0.74 0 100

Table 1 2.73 0 100

Label Claim Cell 2.77 30 70
(mg/Tablet) (em)
5.00 30 70
10 1.0
20 and 40 0.5 Medium: Solution C and Buffer (6:94); 900 mL
80 0.2 r' Apparatus 2: 75 rpm
Time: 30 min
Standard stock solution: 0.96 mg/ml of USP Atorvastatin
Blank: Medium Calcium RS in methanol
Analysis Standard solution: Dilute the Standard stock solution with
Samples: Standardsolution and Sample solution Medium to obtain a final concentration of (LI900) mg/ml,
Calculate the percentage of the labeled amount of where L is the label claim in mg/Tablet.
atorvastatin (C33H3SFN20S) dissolved: Sample solution: Pass a portion of the solution under test
through a suitable filter of 0.45-lJm pore size.
(AulAs) x Cs x V x 0 x [M x (M,dM'2)] x (lIL) x 100 Chromatographic system
(See Chromatography (621), System Suitability.)
Au = absorbance of the Sample solution Mode: LC
As =absorbance of the Standardsolution Detector: UV 248 nm
Cs = concentration of USP Atorvastatin Calcium RS in Column: 2.1-mm x 5-cm; 2.6-lJm packing L1
the Standardsolution (mg/ml) Column temperature: 40°
V = volume of Medium, 900 ml Flow rate: 0.7 mL/min
D = dilution factor for the Sample solution, if Injection volume: 2 IJL
applicable System suitability
M = number of moles of atorvastatin per mole of Sample: Standard solution
atorvastatin calcium, 2 Suitability requirements
M'I = molecular weight of atorvastatin, 558.64 Tailing factor: NMT 1.5
M'2 = molecular weight of atorvastatin calcium, Relative standard deviation: NMT 2.0%
1155.34 Analysis
L =label claim (mg/Tablet) Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of
Tolerances: NlT 80% (Q) of the labeled amount of atorvastatin (C33H3SFN20S) dissolved:
atorvastatin (C33H3SFN20S) is dissolved. .
Test 2: If the product complies with this test, the labeling (rulrs) x c, x V x [M x (Mr1IM,2)] x (l/L) x 100
indicates that it meets USP Dissolution Test 2. Dissolution
Test 2 is suitable for products labeled to contain 80 mg of t» = peak response of atorvastatin from the Sample
atorvastatin. solution
Medium and Apparatus 2: Proceed as directed in Test 1. rs = peak response of atorvastatin from the Standard
Time: 30 min solution
Diluent, Standard solution, Sample solution, Cs =concentration of USP Atorvastatin Calcium RS in
Instrumental conditions, and Blank: Proceed as the Standard solution (mg/ml)
directed in Test 1. V =volume of Medium, 900 mL
Tolerances: NlT 85% (Q) of the labeled amount of M = number of moles of atorvastatin per mole of
atorvastatin (CnH3sFN20S) is dissolved. atorvastatin calcium, 2
Test 3: Ifthe product complies with this test, the labeling M'I = molecular weight of atorvastatin, 558.64
indicates that it meets USP Dissolution Test 3. M'2 = molecular weight of atorvastatln calcium,
Buffer: Combine 250 ml of 0.2 M monobasic potassium 1155.34
phosphate, 112 ml of 0.2 N sodium hydroxide, and 638 L = label claim (mg/Tablet)
ml of water. Adjust with either 0.02 N sodium hydroxide
or phosphoric acid to a pH of 6.8. Tolerances: NLT 80% (Q) of the labeled amount of
Solution A: Acetonitrile, methanol, and 0.1 % atorvastatin (C33H3SFN20S) is dissolved.
trifluoroacetic acid (5:5:90) Test 4: Ifthe product complies with this test, the labeling
Solution B: Acetonitrile, methanol, and 0.1% indicates that it meets USP Dissolution Test 4.
trifluoroacetic acid (45:45:10) Medium: Dissolve 6.8 g of monobasic potassium
Solution C: Dissolve 50 g of Tween 80 in 1 l of Buffer. phosphate and 0.89 g of sodium hydroxide in 1 lofwater.
Mobile phase: See Table 2. Adjust with either 1 N sodium hydroxide or phosphoric
acid to a pH of 6.8; 900 mL.
Apparatus 2: 75 rpm
Time: 15 min

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420 Atorvastatin / Official Monographs USP 43

Buffer: Dissolve about 6.8 9 of monobasic potassium 0.45-~m pore size and discard the first few mifliliters of the
phosphate in 1000 mL of water. Adjust with 0.5 N filtrate.:
potassium hydroxide solution to a pH of 6.0. Sample solution: Pass a portion of the.solutionunder test
Mobile phase: Acetonitrile and Buffer (55:45) through a suitable ·filter of 0.45-Jlm pore size anq discard
Standard stock solution: 0.225 mg/mL of atorvastatin the first few milliliters of the filtrate.
from USPAtorvastatin Calcium RS prepared as follows. To Chromato.graphic system. .. ' . .
a suitable amount of USP Atorvastatin Calcium RS, add 5% (See Chromatography.(621), System Suitability.)
of total volume of methanol, sonicate to dissolve, and Mode: LC
cool. Dilute with Medium to volume. Detector: UV 244 nm
Standard solution: Dilute the Standardstock solution with Column: 4.6-mm x l5-cm; 5-llm packing (l
Medium to obtain a final concentration of (L/900) mg/mL, Temperatu es
where L is the label claim in mg/Tablet. Autos ler: .10°
Sample solution: Pass a portion of the solution under test Co . 0°
through a suitable filter of 0.45-Jlm pore size. Flo e: 1.5 mL/min
Chromatographic system
Injection volume: 50 J.l.L .
(See Chromatography (621), System Suitability.)
Run time: NLT2 times the retention time of atorvastatin
Mode: LC System suitability .. . ..,
Detector: UV 248 nm
Column: 4.6-mm x 25-cm; 5-Jlm packing L1 Sample: Standardsolution
Column temperature: 30° Suitability requirements
flow rate: 1 mL/min Tailing factor:. NMT 2.0
Injection volume: 20 JlL Relative standard deviation: NMT 2.0%.
System suitability Analysis' .' " . ,.
Sample: Standardsolution Samples: .Standardsolution and Sample solution
Suitability requirements Calculate the percentage of the labeled amountof
Tailing factor: NMT 2.0 atorvastatil) (C33H3SFNzOs) dissolved:
Relative standard deviation: NMT 2.0%
Analysis Result = (rulrs) x Cs x Vx[M x (MrdMdlx (l/L) x 100
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of = peak response of atorvastatin from the Sample
atorvastatin (C33H3SFNzOs) dissolved: solution .
=peak response of atorvastatlnfrom the Standard
(ru/r s) x c, x Vx [M x (M,dM'2)] x (l/L) x 100 solution
Cs =concentration of USP Aiorvastatin CalciumRS in
ru = peak response of atorvastatin from the Sample the Standardsolution (mg/mL) . .
solution V =volume of Medium, 900 mL
rs = peak response of atorvastatin from the Standard M = number of moles of atorvastatin per mole of
solution atorvastatin calcium, 2
Cs = concentration of USP Atorvastatin Calcium RS in = molecular weight of atorvastatin, 558~64
the Standardsolution (mg/mL) ::: molecular weight ofatorvastatin calcium,
V =volume of Medium, 900 mL 1155.34
M = number of moles of atorvastatin per mole of L = label claim (mg/Tablet)
atorvastatin calcium, 2
M'I = molecular weight of atorvastatin, 558.64 Tolerances: NLT80%(Q) of the labeled arnourWof
M'2 = molecular weight of atorvastatin calcium, atorvastatin (C33H3SFNzOs) is dissolveq ..... (RB1.Feb.2019)
1155.34 • UNIFORMITY OF DOSAGE UNITS (905): Meet the
L = label claim (mg/Tablet) requirements
Tolerances: NLT80% (Q) of the labeled amount of IMPURITIES
atorvastatin (C33H3SFNzOs) is dissolved. • ORGANIC IMPURITIES
""'TestS: If theproductcomplies w s test, the labeling Rinse glassware with Diluent before preparing solutions
indicates that it meets . Dissol st5: containing atorvastatin calcium.
Medium:' Dissolve 6.8 monobasi . sium Buffer: 5.75 giL of monobasic ammonium phosphate in
phosphate and 0.9 9 ium hydro n lL of water~ water. Adjust with dilute acetic acid (10% v/v) or dilute
Adjust with either so ium ydroxide or p osphoric .acid ammonium hydroxide (10% v/v) to it pH of 4.3 ± 0.05.
to a pH of 6.8; 900.mL Solution A: Acetonitrile and stabilizer-free tetrahydrofuran
Apparatus 2: 75 rpm (925:75)
Time: 20 min Solution B: Solution A and Buffer (42:58)
Buffer: 'Dissolve 10.5 g of citric acid in 1000 mL ofwater. Solution C: Methanol, Solution A, and Buffer(60:20:20)
Adjust with ammonium hydroxide to a pHof 4.0. Diluent: N,N-Dimethylformamide
System suitability solution: 60 I-Ig/mL of USP Atorvastatin
Mobile phase:. Acetonitrile, tetrahY9rofuran, and Buffer
Calcium RS, 50 I-Ig/mL of USP Atorvastatin Related
(50:10:40) Compound B RS, 10 I-Ig/mL of USP Atorvastatin Related
Diluent: Acetonitrile'an er (50:50) Compound H RS, and 0.5 I-Ig/mL of USP Atorvastatin
Standard stock solution. " 25 mg/mL of USP Related Compound D RS in Diluent
Atorvastatin Calcium RS in Diluent. Sonicate to dissolve. Standard solution: 5 I-Ig/mL of USP Atorvastatin Calcium RS
Standard solution: (L/900) mg/mL in Medium from in Diluent. Sonication may be necessary for complete
Standardstocksolution, wh~re l. is,the label' claim in mg/ dissolution.
Tablet. Pass the solution through a suitable filter of

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USP 43 Official Monographs / Atorvastatin 421

Sample solution: Nominally equivalent to 1 mg/mL of M = number of moles of atorvastatin per mole of
atorvastatin, prepared as follows. Crush and finely powder atorvastatin calcium, 2
NLT20 Tablets. Transfer the amount of powder, equivalent Mr1 = molecular weight of atorvastatin, 558.64
to about 50 mg of atorvastatin, to a 50-mL volumetric flask. M'2 = molecular weight of atorvastatin calcium,
Add 30 mL of Diluent and shake mechanically for 15 min. 1155.34
Dilute with Diluentto volume and pass the solution through F = relative response factor (see Table 4)
a suitable filter of 0.45-lJm pore size, discarding the first few
mL of the filtrate. Acceptance criteria: See Table 4.
Mobile phase: See Table 3.
Table 4
Table 3 Relative Relative Acceptance
Time Solution B Solution C Flow Rate Retention Response Criteria,
(min) (%) (%) (ml/min) Name Time Factor NMT(%)

0 100 0 1.8 Atorvastatin arnldevb 0.44 - -

30 0 1.8 Atorvastatin related
100
compound Ab, c 0.84 - -
45 25 75 1.5
Atorvastatin
50 25 75 1.5 pyrrolidone analoq" 0.88 0.68 0.5
55 20 80 1.5 Atorvastatin related
compound Bb. e 0.94 - -
58 100 0 1.8
Atorvastatin 1.00 - -
65 100 0 1.8
Atorvastatin related
compound Cb.f 1.09 - -
For the Standard solution, the run time is only 30 min. For
the System suitabilitysolution and Sample solution, the run Atorvastatin
. pyrrolidone lactone" 9 1.62
- -
time is 65 min.
Chromatographic system Atorvastatin related
(See Chromatography (621), System Suitability.) compound Hh 1.00 1.18 1.0
Mode: LC Atorvastatin epoxy
Detector: UV 244 nm pyrrolooxazin
Column: 4.6-mm x 25-cm; 5-lJm packing L1 6-hydroxy analogi 1.06 0.53 0.5
Temperatures Atorvastatin methyl
Autosampler: 10° esters j 1.12 - -
Column: 30°
Flow rate: See Table 3. Atorvastatin epoxy
pyrrolooxazin
Injection volume: 20 IJL 7-hydroxy analog, if
System suitability present" 1.14 0.53 0.5
Sample: System suitability solution
Atorvastatin epoxy THF
[NOTE-The relative retention times of all peaks eluting analog~m 1.20 1.12 1.0
before atorvastatin related compound H as given in
Table 4 are calculated with respect to the atorvastatin Atorvastatin related
peak. The relative retention times for all peaks eluting compound on 1.27 1.12 0.5
after atorvastatin related compound H are calculated Atorvastatin tert-butyl
with respect to atorvastatin related compound H.] esterb·o 1.49 - -
Suitability requirements Any other unspecified
Resolution: NLT 1.4 between atorvastatin related degradation product - 1.00 0.2
compound Band atorvastatin
Tailing factor: NMT 1.5 for the atorvastatin peak
Relative standard deviation: NMT 5% for the
atorvastatin peak
Signal-to-noise ratio: NLT 10 for atorvastatin related
compound D
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of each impurity in the portion of
Tablets taken:
Result = (rulrs) x (CsICu) x [M x (M,dM'2)] x (l/A x 100
= peak response of each impurity from the Sample
solution
= peak response of atorvastatin from the Standard
solution
= concentration of USPAtorvastatin Calcium RS in
the Standard solution (mg/mL)
Cu = nominal concentration of atorvastatin in the
Sample solution (mgimL)

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422 Atorvastatin / Official Monographs USP 43

Table 4 (continued)
Relative Relative Acceptance
Atovaquone
Retention Response Criteria,
Name Time Factor NMT(%)
Totaldegradation products - - 4.0

a (3 R,S R)-7-{(3R,SR)-7 -[2-(4-Fluorophenyl)-S-isopropyl-3-phenyl-4-

(phenylcarbamoyl)-l H-pyrrol-l-yl]-3,S-dihydroxyheptanamido}-3,S-
dihydroxyheptanoic acid.
b Process impurityincludedinthe tableforidentification only.Process impurities
are controlledin the drug substance, and are not to be reported or includedin C2i H19C103 366.84
the total impurities for the drug product. 1,4-Naphthalenedione, 2-[4-(4-chlorophenyl)cyclohexyl]-3-
c (3R,SR)-7-[2-lsopropyl-4,S-diphenyl-3-(phenylcarbamoyl)-1 H-pyrrol-1-yl]- hydroxy-, trans-;
3,S-dihydroxyheptanoic acid. 2-[trans-4-(p-Chlorophenyl)cyclohexyl]-3-hydroxy-l,4-
d (3R,S R)-7-[S-(4-Fluorophenyl)-3-isopropyl-2-oxo-4-phenyl-3- naphthoquinone [95233-18-4].
(phenylcarbamoyl)-2,3-dihydro-1 H-pyrrol-1-yl]-3,S-dihydroxyheptanoic acid.
e (35,SR)-7-[2-(4-Fluorophenyl)-S-isopropyl-3-phenyl-4-(phenylcarbamoyl)- DEFINITION
1H-pyrrol-1-yIJ-3,S-dihydroxyheptanoic acid. Atovaquone contains NLT 97.5% and NMT 101.5% of
f (3R,SR)-7 -[2,3-Bis(4-fluorophenyl)-S-isopropyl-4-(phenylcarbamoyl)-1 H-
pyrrol-1-yIJ-3,S-dihydroxyheptanoic acid. atovaquone (C22H,9CI03), calculated on the anhydrous and
9 S-(4-Fluorophenyl)-1-{2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl] organic solvent-free basis.
ethyl}-3-isopropyl-2-oxo-N,4-diphenyl-2,3-dihydro-1 H-pyrrole-3-
carboxamide. IDENTIFICATION
h S-(4-Fluorophenyl)-1-{2-[(2R,4R)-4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl]
ethyl}-2-isopropyl-N,4-diphenyl-1 H-pyrrole-3-carboxamide.
i 4-{6-(4-Fluorophenyl)-7,8-epoxy-6-hydroxy-8a-isopropyl-7-phenyl-8-
(phenylcarbamoyl)hexahydro-2H-pyrrolo[2, 1-b][1 ,3]oxazin-2-yl}-3-
hydroxybutanoic acid.
j (3R,SR)-Methyl 7-(2-(4-fluorophenyl)-S-isopropyl-3-phenyl-4-
(phenylcarbamoyl)-l H-pyrrol-1-yl)-3,S-dihydroxyheptanoate. • B.' The retention time of the major peak of theSample
k(3R)-4-(1 b-(4-Fluorophenyl)-7-hydroxy-7-isopropyl-1 a-phenyl-7a- solution corresponds to that of the Standard solution, as
(phenylcarbamoyl)hexahydro-l aH-oxireno[2',3':3,4]pyrrolo[2,l-b][l,3 [oxazln- obtained in the Assay.
3-yl)-3-hydroxybutanoic acid.
'4-(4-Fluorophenyl)-2,4-dihydroxy-2-isopropyl-N,S-diphenyl-3,6- ASSAY
dioxabicyclo[3.1.0]hexane-1-carboxamide. • PROCEDURE
mAtorvastatin related compound D can undergo transformation equilibrium to Mobile phase: Acetonitrile, methanol, water, and
the atorvastatinepoxy, THF analog.Theequilibrium can be shiftedunder slightly phosphoric acid (525:175:300:5)
acidicconditionsand thereforesome products could have a combined
specification reported under atorvastatinrelated compound D. Diluent: Acetonitrile and water (80:20)
n 3-(4-Fluorobenzoyl)-2-isobutyryl-N,3-diphenyloxirane-2-carboxamide. System suitability solution: 0.25 mg/mL of USP
o (3R,SR)-tert-Butyl 7-(2-(4-fluorophenyl)-S-isopropyl-3-phenyl-4- Atovaquone RS and 0.02 mg/mL of USP Atovaquone
(phenylcarbamoyl)-l H-pyrrol-l-yl)-3,5-dihydroxyheptanoate. Related Compound A RS in Diluent. Store in a low-actinic
glass container.
ADDITIONAL REQUIREMENTS Standard solution: 0.25 mg/mL of USP Atovaquone RS in
• PACKAGING AND STORAGE: Preserve in tight containers, and Diluent
store at controlled room temperature. . Sample solution: 0.25 mg/mL of Atovaquone in Diluent.
• LABELING: When more than one Dissolution test is given, the Use a low-actinic volumetric flask.
labeling states the test used, only if Test 1 is not used. Chromatographic system
• USP REFERENCE STANDARDS (11) (See Chromatography (621), System Suitability.)
USPAtorvastatin Calcium RS Mode: LC
USP Atorvastatin Related Compound B RS Detector: UV 220 nm
Calcium (3S,5R)-7 -[2-(4-fluorophenyl)-5-isopropyl-3- Column: 4.6-mm x 25-cm; packing L1
phenyl-4-(phenylcarbamoyl)-1 H-pyrrol-l-yl]- Flow rate: 3 mL/min
3,5-dihydroxyheptanoate (1:2). Injection volume: 20 J.JL
C66H68CaF2N401O 1155.34 System suitability
USPAtorvastatin Related Compound D RS Samples: System suitability solution and Standardsolution
3-(4-Fluorobenzoyl)-2-isobutyryl-N,3-diphenyl oxirane-2- [NoTE-The relative retention times for atovaquone
carboxamide. related compound A and atovaquone are about 0.85
C26H22FN04 431.46 and 1.0, respectively.]
USP Atorvastatin Related Compound H RS Suitability requirements
5-(4-Fluorophenyl)~1-{2-[(2R,4R)-4-hydroxy-6- Resolution: NLT 4 between atovaquone related
oxotetrahydro-2H-pyran-2-yl]ethyl}-2-isopropyl-N,4- compound A and atovaquone, System suitability solution
diphenyl-l H-pyrrole-3-carboxamide. Column efficiency: NLT 9000 theoretical plates,
C33H33FN204 540.62 Standardsolution
Tailing factor: NMT 1.5, Standard solution
Relative standard deviation: NMT 2%, Standard
solution
Analysis
Samples: Standard solution and Sample solution .
Calculate the percentage of atovaquone (C22H19CI03) in the
portion of Atovaquone taken:
Result = (r vir s) x (C siC v) x 100

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 USP 43 OfficialMonographs / Atovaquone 423

=peak response from the Sample solution Atovaquone Oral Suspension

= peak response from the Standardsolution
=concentration of USP Atovaquone RS in the DEfINITION
Standardsolution (mg/mL) Atovaquone Oral Suspension contains NLT 90.0% and NMT
= concentration of Atovaquone in the Sample 110.0% of the labeled amount of atovaquone (C22H19CI03)'
solution (mg/mL)
IDENTIfiCATION
Acceptance criteria: 9i5%-1 01.5% on the anhydrous and
organic solvent-free basis
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1%
• RELATED COMPOUNDS Me~alllm: 1\J10Tn~lnnl and water (1:1) -
System suitability solution and Sample solution: Prepare Standard solution: Dilute 5 mL of Standard solution from
as directed in the Assay. the Assay with Medium to 50 rnl.,
Analysis Sample solution: Dilute 5 mL of Sample solution from the
Samples: System suitability solution and Sample solution Assay with Medium to 50 mL.
Using the chromatograms of the Sample solution and the Acceptance criteria: Meets the requirements
System suitability solution, calculate the percentage of • B. The retention time of the major peak of the Sample
atovaquone related compounds in the portion of solution corresponds to that of the Standardsolution, as
Atovaquone taken: obtained in the Assay.
Result =(r vir r) x 100 ASSAY
• PROCEDURE
ru = peak response of each impurity from the Sample Mobile phase: Acetonitrile, methanol, water, and
solution phosphoric acid (480:160:360:5)
r T =sum of all the peak responses from the Sample System suitability solution: 0.09 mg/mL of USP
solution Atovaquone RS and 0.01 mg/mL of USP Atovaquone
Related Compound A RS in 0.1 M methanolic sodium
Acceptance criteria: See Table 7. hydroxide, Store in a low-actinic glass container.
Standard stock solution: 3 mg/mL of USP Atovaquone RS
Table 1 in a low-actinic, appropriately sized volumetric flask. Add
I Relative Acceptance 20% water and 60% 0.1 M methanolic sodium hydroxide.
Retention Criteria, Sonicate for 5 min or until the material has dissolved. Allow
Name Time NMT(%) to cool, and dilute with 0.1 M methanolic sodium
Indene isomer" 0.63 0.5 hydroxide to volume.
Standard solution: 0.09 mg/mL of USP Atovaquone RS
Atovaquone related compound A 0.85 1.0
from Standardstock solution. Transfer to an appropriately
Dldehydrcatovaquone" 0.89 0.3 sized, low-actinic volumetric flask in.a mixture of methanol
and water (1:1). Minimize exposure of this solution to light.
Atovaquone 1.0 - Sample stock solution: Nominally 3 mg/mL from a known
O-Methyl atovaquone" 1.8 0.5 volume of well-mixed Oral Suspension NLT 750 mg of
atovaquone prepared as follows. In an appropriately sized,
Any other individual,
unidentified impurity - 0.2 low-actinic volumetric flask, add 20% volume of water,
swirl for 5 min, add 60% volume of 0.1 M methanolic
Sum of all other individual sodium hydroxide, and sonicate for 15 min. Allow to cool,
impurities
- 1.0
and dilute with 0.1 M methanolic sodium hydroxide to
Total impurities - 1.5 volume. Immediately filter a 20-mL portion, discarding the
first 5 mL of the filtrate.
a trans-2-[4-( 4-Chlorophenyl)cyclohexyl]-1-oxo-1 H-indene-3-carboxylic acid. Sample solution: 0.09 mg/mL of atovaquone from the clear
b (RS)-2-[4-( 4-Chlorophenyl)cyclohex-3-enyl]-3-hydroxy-1 A-naphthoquinone. filtrate of the Sample stock solution. Transfer to an
c trans-2-[4-(4-Chlorophenyl)cyclohexyl]-3-methoxy-1 A-naphthoquinone. appropriately sized, low-actinic volumetric flask, and dilute
with a mixture of methanol and water (1:1) to volume.
SPECIFIC TESTS Minimize exposure of this solution to light.
• WATER DETERMINATION, Method I (921): NMT 1.0% Chromatographic system
ADDITIONAL REQUIREMENTS (See Chromatography (621), System Suitability.)
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Mode: LC
containers. Detector: UV 220 nm
Column: 4.6-mm x 12.5-cm; packing L1
• USP REFERENCE STANDARDS (11)
USP Atovaquone RS Flow rate: 3 mL/min
USP Atovaquone Related Compound A RS Injection volume: 20 IJL
cis-2-[4-(4-Chlorophenyl)cyclohexyl]-3-hydroxy-l,4- System suitability
naphthoquinone. Samples: System suitability solution and Standardsolution
[NoTE-The relative retention times for atovaquone
C22H19CI03 366.84
related compound A and atovaquone are 0.86 and
1.0, respectively.]
Suitability requirements
Tailing factor: NMT 1.5
Relative standard deviation: NMT 2.0%

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424 Atovaquone / OfficialMonographs USP 43

Analysis Table 1 (continued)

Samples: Standard solution and Sample solution Relative Relative Acceptance
Calculate the percentage of the labeled amount of Retention Response Criteria,
atovaquone (C22H19CI03) in the portion of Oral Name Time Factor NMT(%)
Suspension taken: Total impurities - - 2.0

Result =(rulrs) x (Cs/C u) x 100 a Disregard any peak having a relative retention time of 0.3, which is due to
photodegradation during preparation of the Sample solution.
ru = peak response of atovaquone from the Sample
solution SPECIFIC TESTS
rs = peak response of atovaquone from the Standard • pH (791): 3.5-7.0
solution • SEDIMENTATION
Cs =concentration of USP Atovaquone RS in the For oral suspension packaged in multiple-unit containers
Standard solution (mg/mL) Analysis: Transfer 50 mL of well-mixed Oral Suspension to
Cu = nominal concentration of atovaquone in the a glass-stoppered graduated cylinder, and allow to stand
Sample solution (mg/mL) for 16 h. Measure the volume, if any, of clear liquid
observed in the cylinder.
Acceptance criteria: 90.0%-110.0% Acceptance criteria: NMT 1 mL of clear liquid
PERFORMANCE TESTS ADDITIONAL REQUIREMENTS
• UNIFORMITY OF DOSAGE UNITS (905): Meets the • PACKAGING AND STORAGE: Preserve in tight, light-resistant
requirements for oral suspension packaged in single-unit containers.
containers • USP REFERENCE STANDARDS (11)
• DELIVERABLE VOLUME (698): Meets the requirements for USPAtovaquone RS
oral suspension packaged in multiple-unit containers USPAtovaquone Related Compound A RS
cis-2-[4-(4-Chlorophenyl)cycJohexyl]-3-hydroxy-1,4-
IMPURITIES naphthoquinone.
• ORGANIC IMPURITIES C22H19CI03 366.84
Mobile phase, System suitability solution, Standard
solution, Sample solution, Chromatographic system,
and System suitability: Proceed as directed in the Assay.
Analysis
Samples: System suitability solution, Standard solution, and Atracurium Besylate
Sample solution
Using the chromatograms of the System suitability solution
and the Sample solution, calculate the percentage of
atovaquone related compounds in the portion of Oral
Suspension taken: .
Result = (rulrs) x (Cs/Cu) x (1IF) x 100
to = individual peak response of an atovaquone
related compound, if any, from the Sample
solution C6sHs2N20'SS2 1243.48
rs = peak response of atovaquone from the·Standard Isoquinolinium, 2,2'-[1,5-pentanediylbis[oxy(3-oxo-3,l-
solution propanediyl)]]bis[1-[(3,4-dimethoxyphenyl)methyl]-
C, =concentration of USP Atovaquone RS in the 1,2, 3,4-tetrahydro-6,7-dimethoxy-2-methyl-,
Standard solution (mg/mL) dibenzenesulfonate;
Cu = nominal concentration of Oral Suspension inthe 2-(2-Carboxyethyl)-1 ,2, 3,4-tetrahydro-6, 7-dimeth oxy-2-
Sample solution (mg/mL) methyl-l-veratrylisoquinolinium benzenesulfonate,
F = relative response factor of an individual pentamethylene ester [64228-81-5].
atovaquone related compound relative to the
response of atovaquone (see Table 1) DEFINITION
Atracurium Besylate contains NLT 96.0% and NMT 102.0%
Acceptance criteria: See Table 1. of C6sHs2N20'SS2, calculated on the anhydrous basis. It
contains NLT 5.0% and NMT 6.5% of the trans-trans isomer,
Table 1 NLT 34.5% and NMT 38.5% of the cis-trans isomer, and NLT
Relative Relative Acceptance 55.0% and NMT 60.0% of the cis-cis isomer.
Retention Response Criteria,
Name Time Factor NMT(%) IDENTIFICATION
Photodegradation peaks 0.3 - -
Atovaquone impurity 0.65 1.08 0.5
• A.A5PE
Atovaquone related com- SpectrosCopy: 1 ~
pound A 0.86 0.85 1.0
• B. The retention times of the three main isomeric peaks of
Atovaquone impurity 0.88 1.0 0.3 the Sample solution correspond to those of the Standard
solution, as obtained in the Assay.
Atovaquone 1.0 1.0 -
Any other atovaquone relat-
ed compound - 1.0 0.2

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USP 43 .OfficialMonographs / Atracurium 425

ASSAY cis-trans isomer, and NLT 55.0% and NMT 60.0% of the
• PROCEDURE cis-cis isomer.
Buffer: 10.2 9 of monobasic potassium phosphate in a
1OOO-mL volumetric flask. Dissolve in 950 mL of water. IMPURITIES
While stirring, adjust with phosphoric acid to a pH of 3.1 • RESIDUE ON IGNITION (281): NMT 0.2%
and dilute with water to volume. ' • ORGANIC IMPURITIES
Solution A: Acetonitrile, methanol, and Buffer(20:5:75) Buffer, Solution A, Solution B, Mobile phase,
Solution B: Acetonitrile, methanol, and Buffer (20:30:50) Chromatographic system, and Sample solution:
Mobile phase: See Table 7. Proceed as directed in the Assay.
Standard solution: 0.01 mg/mL of USP Atracurium Besylate
Table 1 RS in Solution A
System suitability solution: 1 mg/mL of USP Atracurium
Time Solution A Solution B
(min) (%) (%)
Besylate RS in Solution A
System suitability
0 80 20 Sample: System sUitability solution
5 80 20 Suitability requirements
Resolution: NLT 1.5 between the atracurium trans-trans
15 40 60 isomer and the cis-trans isomer peaks; NLT 1.5 between
25 40 60 the atracurium cis-trans isomer and the cis-cis isomer
peaks
30 0 100 Analysis
45 0 100 Samples: Standardsolution and Sample solution
Record the chromatograms, and measure all of the peak
50 80 20 responses, except the three main isomeric peaks.
Calculate the percentage of each impurity in the portion of
Standard solution: 1 mg/mL of USP Atracurium Besylate RS Atracurium Besylate taken:
in Solution A
Sample solution: 1 mg/mL of Atracurium Besylate in Result = (r vir r) x (C siC v) x (1 IF) x 100
Solution A
Chromatographic system = peak response for each impurity from the
(See Chromatography, (621) System Suitability.) Sample solution
Mode: LC =sum of peak responses due to the atracurium
Detector: UV 280 nm cis-cis, trans-trans, and cis-trans isomers from the
Column: 4.6-mm x 25-cm; 5-~m base-deactivated Standardsolution
packing Ll = concentration of USP Atracurium Besylate RS in
Flow rate: 1 mL/min the Standardsolution (mg/mL)
Injection size: 20 ~L =concentration of Atracurium Besylate in the
System suitability Sample solution (mg/mL)
Sample: Standard solution F = relative response factor (see Table 2)
[NoTE-Refer to Table 2 for relative retention times.]
Suitability requirements Acceptance criteria: See Table 2. [NOTE-Disregard any
Resolution: NLT 1.5 between the atracurium trans-trans peak less than 0.05%.]
isomer and the cis-trans isomer peaks; NLT 1.5 between
the atracurium cis-trans isomer and the cis-cis isomer Table 2
peaks Relative Relative Acceptance
Relative standard deviation: NMT 2.0%,·for the cis-cis Retention Response Criteria,
Name Time Factor NMT (%)
isomer peak
Analysis Impurity E" 0.2 1.0 1.5
Samples: Standard solution and Sample solution Impurity Fb 0.25 1.0 1.0
Calculate the percentage of atracurium besylate
(C6sH8zNz018SZ) in the portion of Atracurium Besylate Impurity G
taken: (laudanosine)' 0.3 2.0 1.0
Impurity 0 0.45 d and 0.5" 1.0 1.5P
Result =(r vir s) x (C siC v) x 100
Atracurium
trans-trans isomer - -
=sum of the peak responses for the trans-trans 0.8
isomer, the trans-cis isomer, and the cis-cis isomer Atracurium
from the Sample solution cis-trans isomer 0.9 - -
= sum of the peak responses for the trans-trans Atracurium
isomer, the trans-cis isomer, and the cis-cis isomer cis-cis isomer 1.0 - -
from the Standard solution
= concentration of USP Atracurium Besylate RS in Impurity A 1.04f and 1.08 1 1.0 1.5P
the Standard solution (mg/mL) Impurity I 1.079 and 1.12 k 1.0 LOP
Cu = concentration of Atracurium Besylate in the
Impurity H 1.07h and 1.12' 1.0 LOP
Sample solution (mg/mL)
Impurity KI 1.09 and 1.12 1.0 1.0P
Acceptance criteria: 96.0%-102.0%, calculated on the
Impurity Bm 1.15 1.0 0.1
anhydrous basis. It contains NLT 5.0% and NMT 6.5% of
the trans-trans isomer, NLT34.5% and NMT 38.5% of the Impurity C 1.2" and 1.3° 1.0 LOP

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426 Atracurium / OfficialMonographs USP 43

Table 2 (continued) Chromatographic system

Relative Relative Acceptance Mode: LC
Retention Response Criteria, Detector: UV 217 nm
Name Time Factor NMT(%) Column: 4.6-mm x 25-cm; 5-J.Im base-deactivated
Anyindividual packing L1
impurity - 1.0 0.1 Flow rate: 1 mL/min
Injection size: 100 J.IL
Total impurities - - 3.5
Analysis
a 3-[l-(3,4-Dimethoxybenzyl)-6, 7-dimethoxy-2-methyl-l ,2,3,4- Samples: Standardsolution and Sample solution
tetrahydroisoquinolinio]propanoate. Measure the responses for the Impurity J (methyl
b 1-(3,4-Dimethoxybenzyl)-6,7-dimethoxy-2,2-dimethyl-1 ,2,3,4- benzenesulfonate) peaks.
tetrahydroisoquinolinium. Acceptance criteria: NMT 0.01%, the peak response of the
c 1-(3,4-Dimethoxybenzyl)-6, 7-dimethoxy-2-methyl-l ,2,3,4- Sample solution being NMT that of the Standardsolution
tetrahydroisoquinoline.
d trans Isomerof 1-(3,4-dimethoxybenzyl)-2-[3-[(5-hydroxypentyl)oxy]-3- SPECIFIC TESTS
oxopropyl]-6, 7-dimethoxy-2-methyl-l,2,3,4-tetrahydroisoquinolinium. • WATER DETERMINATION, Method 1(921): NMT 5.0%
e cisIsomerof 1-(3,4-dimethoxybenzyl)-2-[3-[(5-hydroxypentyl)oxy]-3-
oxopropyl]-6, 7-dimethoxy-2-methyl-l,2,3,4-tetrahydroisoquinolinium. ADDITIONAL REQUIREMENTS
f cis-trans Isomerof 1-(3,4-dimethoxybenzyl)-2-[13-(1-(3,4-dimethoxybenzyl)- • PACKAGING AND STORAGE: Preserve in tight, light-resistant
6,7-dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl]-3, ll-dioxo-4,10- containers, in a cold place. [Nors-Atracurlurn Besylate is
dioxatridecyl]-6,7-dimethoxy-2-methyl-l ,2,3,4-tetrahydroisoquinolinium.
9 cis-trans Isomerof 2,2'-[(3-methylpentane-l,5)-diylbis[oxy(3-oxopropane- unstable at room temperature.]
1,3-diyl)]Jbis[l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl-l ,2,3,4- • USP REfERENCE STANDARDS (11)
tetrahydroisoquinolinium]. USP Atracurium Besylate RS
h cis-trans Isomerof 2,2'-[hexane-l ,6-diylbis[oxy(3-oxopropane-l ,3-diyl)]J
bis[l-(3,4-dimethoxybenzyl)-6, 7-dimethoxy-2-methyl-l ,2,3,4-
tetrahydroisoquinolinium].
i cis-cis Isomerof 1-(3,4-dimethoxybenzyl)-2-[13-[l-(3,4-dimethoxybenzyl)-6, 7-
dimethoxy-3,4-dihydroisoquinolin-2(1 H)-yl]-3, ll-dioxo-4,1O-dioxatridecyl]-
6,7-dimethoxy-2-methyl-l,2,3,4-tetrahydroisoquinolinium. Atracurium Besylate Injection
j 2,2'-[(Hexane-l,5)-diylbis(3-oxopropane-l ,3-diyl)]]bis(1-(3,4- .
dimethoxybenzyl)-6,7-dimethoxy-2-methyl-l ,2,3,4-tetrahydroisoquinolinium]. DEFINITION
k cis-cis Isomerof 2,2'-[(3-methylpentane-l,5)-diylbis[oxy(3-oxopropane-l ,3- Atracurium Besylate Injection is a sterilesolution containing
diyl)]]bis[l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl-l ,2,3,4- NLT 90.0% and NMT 115.0% of the labeled amount of
tetrahydroisoquinolinium].
Icis-cis Isomerof 2,2'-[hexane-l ,6-diylbis[oxy(3-oxopropane-1 ,3-diyl)]]bis[l- atracurium besylate (C6SHS2N201SS2)' It contains an amount
(3,4-dimethoxybenzyt)-6, 7-dimethoxy-2-methyl-l ,2,3,4-tetrahydro- of the trans-trans isomer equivalent to NLT 5.0% and NMT
isoquinolinium]. 6.5% of the labeled amount of atracurium besylate, an
mPentane-l ,5-diyl bis[3-[l-(3,4-dimethoxybenzyl)-6,7-dimethoxy-3,4- amount of the cis-trans isomerequivalent to NLT, 34.5% and
dihydroisoquinolin-2(1 H)-yl)propanoate]. NMT 38.5% of the labeled amount of atracurium besylate,
n trans Isomerof 1-(3,4-dimethoxybenzyl)-2-(3,ll-dioxo-4,10-dioxatridec-12-
enyl)-6,7-dimethoxy-2-methyl-l,2,3,4-tetrahydroisoquinolinium and an amount of the cis-cis isomerequivalentto NLT55.0%
benzenesulfonate. and NMT 60.0% of the labeled amount of atracurium
o cisIsomerof 1-(3,4-dimethoxybenzyl)-2-(3,ll-dioxo-4, 10-dioxatridec-12- besylate.
enyl)-6,7-dimethoxy-2-methyl-1 ,2,3,4-tetrahydroisoquinolinium [NOTE-The Injection is unstable at room temperature.
benzenesulfonate. Store all samples in the refrigerator. Analyze all
PImpurityconsistsof two isomers that are separated under these conditions; preparations as soon as possible, or use a refrigerated
integrate both peaksfor the impuritycalculations.
injector.]
• LIMIT OF IMPURITY J (Methyl Benzenesulfonate) IDENTIFICATION
Buffer, Solution A, and Solution B: Prepare as directed in • A. The retention times of the peaks of the three atracurium
the Assay. besylate isomersfrom the Sample solution correspond to
Standard stock solution: 0.2 mg/mL of Impurity J (methyl those from the Standardsolution, as obtained in the Assay.
benzenesulfonate) in acetonitrile
Standard solution: 1 J.Ig/mL of Impurity J (methyl ASSAY
benzenesulfonate) in Solution A from Standardstock solution • PROCEDURE
Sample solution: 10 mg/mL of Atracurium Besylate in Buffer: 10.2 g of monobasic potassium phosphate in a
Solution A 1OOO-mL volumetricflask. Dissolve in 950 mLof water.
Mobile phase: See Table 3. While stirring, adjust with phosphoric acid to a pH of 3.1,
and dilute with water to volume.
Table 3 Solution A: Acetonitrile, methanol, and Buffer (20:5:75)
Solution B: Acetonitrile, methanol, and Buffer (20:30:50)
Time Solution A Solution B
(min) (%) (%) Mobile phase: See Table 1.
0 80 20 Table 1
5 80 20 Time Solution A Solution B
(min) (%) (%)
15 75 25
0 80 20
25 75 25
5 80 20
30 55 45
15 40 60
38 0 100
25 40 60
45 0 100
30 0 100

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USP 43 Official Monographs / Atracurium 427

Table 1 (continued) Suitability requirements

Time Solution A Solution B Resolution: NLT 1.5 between the atracurium trans-trans
(min) (%) (%) isomer and the cis-trans isomer peaks; NLT 1.5 between
45 100
the atracurium cis-trans isomer and the cis-cis isomer
0
peaks
50 80 20 Analysis
Samples: Standardsolution and Sample solution
Standard solution: 1 mg/mL of USPAtracurium Besylate RS Calculate the percentage of each impurity in the portion of
in Solution A Sample solution taken:
Sample solution: Nominally equivalent to 1 mg/mL of
atracurium besylate from Injection in Solution A Result =(ru/rr) x (CsICu) x (1 IF) x 100
Chromatographic system
(See Chromatography (621), System Suitability.) = peak response for each impurity from the
Mode: LC Sample solution
Detector: UV 280 nm =sum of all the peak responses from the Standard
solution
Column: 4.6-mm x 25-cm; 5-l..Im base-deactivated
packing Ll =concentration of USPAtracurium Besylate RS in
Flow rate: 1 mL/min the Standardsolution (mg/mL)
= nominal concentration of atracurium besylate in
Injection size: 20I..lL
System suitability the Sample solution (mg/mL)
F = relative response factor (see Table 2)
Sample: Standard solution
[NoTE-Refer to Table 2 under Organic Impurities for Acceptance criteria: See Table 2.
relative retention times.]
Suitability requirements Table 2
Resolution: NLT 1.5 between the atracurium trans-trans
isomer and the cis-trans isomer peaks; NLT 1.5 between Relative Relative Acceptance
Retention Response Criteria,
the atracurium cis-trans isomer and the cis-cis isomer Name Time Factor NMT (0/0)
peaks
Relative standard deviation: NMT 2.0%, for the cis-cis Benzenesulfonic
isomer peak acid" 0.08 - -
Analysis Acidic compound 0.22 1.0 6.0
Samples: Standard solution and Sample solution
Impurity G
Measure the responses for the three atracurium besylate (laudanosine) 0.29 2.0 3.0
isomer peaks.
Calculate the percentage of the labeled amount of cis- and trans-isomers of b
0.44 and
atracurium besylate (C6sHs2N201SS2) in each mL of the the hydroxycompound 0.50c 1.0 6.01
Injection taken: . Atracurium trans-trans
isomer 0.8 - -
Result = (rulr s) x (CsICu) x 100 Atracurium cis-trans
ru = sum of the peak responses for the trans-trans
isomer 0.9 - -
isomer, the trans-cis isomer, and the cis-cis Atracurium cis-cis
isomer from the Sample solution
isomer 1.0 - -
ts =sum of the peak responses for the trans-trans cis- and trans-isomers of d
1.28 and
isomer, the trans-cis isomer, and the cis-cis the monoacrylate 1.33e 1.0 3.01
isomer from the Standardsolution Any individual unspecl-
Cs = concentration of USPAtracurium Besylate RS in fied degradation prod-
the Standardsolution (mg/mL) uct - 1.0 0.1
Cu = nominal concentration of atracurium besylate in Total impurities - - 15.0
the Sample solution (mg/mL)
a Foridentification purposesonly.
Acceptance criteria: 90.0%-115.0% of the labeled amount b trans isomer of the hydroxycompound.
of atracurium besylate (C6sHs2N201SS2)' It contains NLT C cis isomer of the hydroxy compound.

5.0% and NMT 6.5% of the trans-trans isomer, NLT 34.5% d trans isomer of the monoacrylate.
and NMT 38.5% of the cis-trans isomer, and NLT 55.0% e cis isomerof the monoacrylate.
and NMT 60.0% of the cis-cis isomer. f Impurity consistsof two isomersthat are separated under these conditions;
integrate both peaksfor the impurity calculations.
IMPURITIES
• ORGANIC IMPURITIES SPECIFIC TESTS
Buffer, Solution A, Solution B, Mobile phase, Sample • pH (791): 3.00-3.65
solution, and Chromatographic system: Proceed as • STERILITY TESTS (71): It meets the requirements when
directed in the Assay. tested as directed for Test for Sterility of the Product to Be
Standard stock solution: 1 mg/mL of USP Atracurium Examined, Membrane Filtration.
Besylate RS in Solution A • BACTERIAL ENDOTOXINS TEST (85): It contains NMT 5.56
Standard solution: 0.02 mg/mL of USPAtracurium Besylate USP Endotoxin Units/mg of atracurium besylate.
RS in Solution A, from Standardstocksolution • INJECTIONS AND IMPLANTED DRUG PRODUCTS (1): Meets the
System suitability requirements
Sample: Standardsolution

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428 Atracurium / Official Monographs USP 43

ADDITIONAL REQUIREMENTS IMPURITIES

• PACKAGING AND STORAGE: Preserve in single-dose or • RESIDUE ON IGNITION (281): NMT 0.1%
multiple-dose containers, preferably of Type I glass, in a • LIMIT OF FOREIGN ALKALOIDS AND OTHER IMPURITIES
refrigerator, and protect from freezing. Protect from light. Standard solution: 24 mg/ml of USPAtropine Sulfate RS in
• USP REFERENCE STANDARDS (11) methanol
USP Atracurium Besylate RS Sample solution A: 20 mg/ml of Atropine in methanol
Sample solution B: 1 mg/ml of Atropine in methanol
Chromatographic system .
(See Chromatography (621), Thin-Layer Chromatography.)
Mode: TlC
Atropine Adsorbent: 0.5-mm layer of chromatographic silica gel
Application volume: See Analysis.
Developing solvent system: Chloroform, acetone, and
diethylamine (5:4:1)
Spray reagent: Potassium iodoplatinate TS
Analysis
Samples: Standard solution, 5 ~l; Sample solution A, 25 ~l;
Sample solution B, 1 ~L
Apply the Samples to the TLC plate. Allow the spots to dry,
and develop the chromatogram in the Developing
C17H 23N03 289.37 solvent system until the solvent front has moved
Benzeneacetic acid, a-(hydroxymethyl)-8-methyl-8- three-fourths of the length of the plate. Allow the solvent
azabicyclo[3.2.1 ]oct-3-yl ester, endo-(f)-; to evaporate. Locate the spots on the plate by spraying
1aH,5aH-Tropan-3a-ol (f)-tropate (ester) [51-55-8]. with Spray reagent.
Acceptance criteria: NMT 0.2%; the RFvalue of the
DEFINITION principal spot of each Sample solution corresponds to that
Atropine contains NlT 99.0% and NMT 100.5% of atropine of the Standard solution; no secondary spot of Sample
(C17H 23N03), calculated on the anhydrous basis. solution A exhibits intensity equal to or greater than the
[CAUTIoN-Handle Atropine with exceptional care, because principal spot of Sample solution B. .
it is highly potent.] • READILY CARBONIZABLE SUBSTANCES TEST (271)
IDENTIFICATION Sample solution: 200 mg in 5 mL of 2 N sulfuric acid
Acceptance criteria: The solution has no more color than
• A. Matching Fluid A, and the solution is colored no more than
Standard: 36 mg of USP Atropine Sulfate RS
Sample: 30 mg light yellow upon the addition of 0.2 mL of nitric acid.
Analysis: Dissolve the Standard and Sample in individual SPECIFIC TESTS
60-ml separators with the aid of 5-ml portions of water. • OPTICAL ROTATION, AngularRotation (781)
To each separator add 1.5 ml of 1 N sodium hydroxide Sample solution: 1 g, previously dried at 105° for 1 h, in
solution and 10 ml of chloroform. Shake for 1 min, allow sufficient 50% alcohol (w/w) to obtain a volume of 20 mL
the layers to separate, and pass the chloroform extracts at 25° (using a 200-mm tube)
through separate filters of 2 g of anhydrous granular Acceptance criteria: -0.70° to +0.05° (limit of
sodium sulfate supported on pledgets of glass wool. Extract hyoscyamine)
each aqueous layer with two additional 1O-ml portions of • MELTING RANGE OR TEMPERATURE (741): 114°-118°
chloroform, filtering and combining with the respective • WATER DETERMINATION, Method I (921): NMT 0.2%
main extracts. Evaporate the chloroform solutions under
reduced pressure to dryness, and dissolve each residue in ADDITIONAL REQUIREMENTS
10 ml of carbon disulfide. • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Acceptance criteria: The IR absorption spectrum, containers.
determined in a 1-mm cell, of the solution of the Sample • USP REFERENCE STANDARDS (11)
exhibits maxima only at the same wavelengths as that of USP Atropine Sulfate RS
the solution of the Standard.
• B.
Sample solution: A solution (1 in 50) in 3 N hydrochloric
acid
Analysis: Add gold chloride TS to the Sample solution. Atropine Sulfate
Acceptance criteria: .A lusterless precipitate is formed
(distinction from hyoscyamine, which, similarly treated,
yields a lustrous precipitate). .
ASSAY
• PROCEDURE
Sample: 400 mg of Atropine
Analysis: Dissolve the Sample in 50 ml of glacial acetic acid.
Titrate with 0.1 N perchloric acid VS to a green endpoint,
using 1 drop of crystal violet TS. Perform a blank
determination (see Titrimetry (541». Each ml of 0.1 N
perchloric acid is equivalent to 28.94 mg of atropine 694.83
(C17H23N03)'
Acceptance criteria: 99.0%-100.5% on the anhydrous
basis

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USP 43 Official Monographs / Atropine 429

Anhydrous Injection volume: 5 ~L

Benzeneacetic acid, a-(hydroxymethyl)-, X_rnPl"nllll_X_ System suitability
azabicyclo[3.2.1 ]oct-3-yl ester, endo-(±)-, S~;~eJ.~.~. :)Ks.~~~~.~i~g~!!if~.~g~~t{9n, Sensitivity solution,
monohydrate; ~~ngy.~t(;1.lf1~qti:ly~()Iqtj.QlJ . . . v4S_(USp-4i )
1aH,5aH-Tropan-3-a-ol (±)-tropate (ester), sulfate (2:1) (salt) [NoTE-See Table 2 for the relative retention times.]
monohydrate [5908-99-6]. Suitability requirements
Anhydrous [55-48-1]. Resolution: NLT 1.4 between hyoscyamine related
DEFINITION compound A and atropine, System suitability solution
Atropine Sulfate contains NLT 98.0% and NMT 102.0% of Tailing factor: 0.8-1.8 for atropine, System suitability
solution
atropine sulfate [(C17H23N03) 2 • H2S04] , calculated on the
Relative standard deviation:
anhydrous basis.
[CAUTIoN-Handle atropine sulfate with exceptional care, ~Qly;t{gjjftli~t(f~~41vi .
because it is highly potent.] Signal-to-noise ratio: NLT 10 for atropine, Sensitivity
solution
IDENTIFICATION Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of atropine sulfate [(C17H23N03) 2
. H2S04] in the portion of Atropine Sulfatetaken:
• A.,~~~~~~~~~~~
Spggt[()§q:>P}/':?"Q,......,.Qr." .: Result = (rulrs) x (CslCu) x 100
• B. IDENTIFICATION TESTS-GENERAL (191), Chemical
Identification Tests, Sulfate ru " = peak response of atropine from the Sample
Sample solution: 50 mg/mL solution
Acceptance criteria: Meets the requirements fs = peak response of atropine from the Standard
• C. The retention time of the major peak of the Sample solution
solution corresponds to that of the System suitability c, = concentration of~~E~tr8pi~iS~I!~~.e RS in the
solution, as obtained in the Assay. Standard solution~,(mgXmt:.)"igs(U5P4n
ASSAY Cu = concen~r~~ie.~e!;~~~eeI~~F?ulfate in the Sample
solution;~(m9XJ1T3t:.)"'Y?s(QsPtlt)
Acceptance criteria: 98.0%-102.0% on the anhydrous
• PROCEDURE , basis
Buffer: 1.8 giL of monobasic potassium phosphate and 2.5 IMPURITIES
giL of sodium 1-pentanesulfonate, adjusted with • RESIDUE ON IGNITION (281): NMT 0.2%
phosphoric acid to a pH of 2.5
Diluent: Acetonitrile and Buffer (20:80)
Solution A: Acetonitrile and Buffer (5:95)
Solution B: Acetonitrile and Buffer (80:20) • ORGANIC IMPURITIES
Mobile phase: See Table 1. Buffer, Diluent, Solution A, Solution B, Mobile phase,
Table 1
~r.s~~IJl~~i~~~il!tr~~!~~!~~i~••~ensitivity solution,
~~t~n~~,~g.i~Qll.ltiQO!:';4s(lJSPtlJ) Sample solution,
Time Solution A Solution B Chromatographic system, and System SUitability:
(min) (%) (%)- Proceed as directed in the Assay.
0 92 8 Analysis
Sample: Sample solution
11 79 21
Calculate the percentage of each impurity in the portion of
15 46 54 Atropine Sulfatetaken:
15.1 92 8 Result =(rulrT) x (1 IF) x 100
20 92 8
fu = peak response of each impurityfrom the Sample
solution
[NOTE-The gradient was established on an HPLC system rT =sum of all the peak responsesfrom the Sample
with a dwellvolume of approximately 0.8 mL.] solution
System suitability solution: 1 ~g/mL of USP Hyoscyamine F = relative response factor for each impurity (see
Related Compound A RS and 0.5 mg/mL of USP Atropine Table 2)
Sulfate RS in Diluent
Sensitivity solution: 0.25 ~g/mL of USP Atropine SulfateRS Ac;.ceptance_~~itel'ia: See Table 2.
in Diluent is0.05%.,.. 25 (USN1)
Standard solution: 0.5 mg/mL of USP Atropine Sulfate RS
in Diluent Table 2
Sample solution: 0.5 mg/mL of Atropine Sulfate in Diluent
Chromatographic system Relative Relative Acceptance
Retention Response Criteria,
(See Chromatography (621), System Suitability.) Name Time Factor NMT (%)
Mode: LC
Detector: UV 210 nm Tropic acid" 0.56 2.1 0.2
Column: 4.6-mm x 15-cm; 3-lJm packing L1 7-Hydroxyhyoscyarnlne'' 0.66 1.0 0.2
Column temperature: 50°
Flow rate: 2 mL/min Scopolamine' 0.72 1.0 0.2

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 430 Atropine / OfficialMonographs USP 43

Table 2 (continued) ASSAY

Relative Relative Acceptance • PROCEDURE
Retention Response Criteria, Buffer: Dissolve4.1 9 of anhydrous sodium acetate and 2.9
Name Time Factor NMT(%) mL ofgladal acetic add in 1 L of water.
6-Hydroxyhyoscyamined 0.75 1.0 0.2 Mobile phase: Transfer 5.1 9 of tetrabutylammonium
hydrogen sulfate to a 1-L volumetric flask. Add 50 mL of
Hyoscyamine related com- acetonitrile, and dilute with Buffer to volume. Adjust with 5
pound A 0.97 1.2 0.3
N sodium hydroxide to a pH of 5.5.
Atropine 1.0 1.0 - System suitability solution: 0.5 ~g/mL of
p-hydroxybenzoic acid and 64 ~g/mL of USP Atropine
Llttorlne" 1.13 1.2 0.2
Sulfate RS in water
Apoatroplne' 1.60 2.0 0.2 Standard solution: 80 ~g/mL of USP Atropine Sulfate RS
Any individual,
Sample solution: Nominally equivalent to 80 ~g/mL of
unspecified impurity - 1.0 0.1 atropine sulfate in water, from a volume of the Injection
containing an amount equivalent to 2 mg of atropine
Totalimpurities - - 0.5 sulfate
Chromatographic system
a 3-Hydroxy-2-phenylpropanoic acid; also known as (2RS)-3-Hydroxy-2-
phenylpropanoic acid. (See Chromatography (621)/ System Suitability.)
b (1S,3R,5S)-6-Hydroxy-8-methyl-8-azabicyclo[3.2.1 ]oct-3-yl (S)-3-hydroxy-2- Mode: LC
phenylpropanoate;also known as (1S,3R,5S,6RS)-6-Hydroxy-8-methyl-8- Detector: UV 254 nm
azabicyclo[3.2.1 ]oct-3-yl (2S)-3-hydroxy-2-phenylpropanoate. Column: 30-cm x 3.9-mm; packing L1
c (S)-(1 R,2R,4S,5S,7s)-9-Methyl-3-oxa-9-azatricyclo[3.3.1.0 2,4]no nan-7-yl 3- . Flow rate: 2 mL/min
hydroxy-2-phenylpropanoate; also known as (S)-(1 R,2R,4S,5S,7s)-9-Methyl-3- Injection volume: 100 ~L
oxa-9-azatricyclo[3.3.1.02.4]non-7-yl (2S)-3-hydroxy-2-phenylpropanoate.
d (1R,3S,5R)-6-Hydroxy-8-methyl-8-azabicyclo[3.2. 1]octan-3-yl (S)-3-hydroxy- System suitability
. 2-phenylpropanoate;alsoknown as (1R,3S,5R,6RS)-6-Hydroxy-8-methyl-8- Samples: System suitability solution and Standardsolution
azabicyclo[3.2.1 ]oct-3-yl (2S)-3-hydroxy-2-phenylpropanoate. [NOTE-The relative retention times of atropine and
e (1 R,3r,5S)-8-Methyl-8-azabicyclo[3.2.1]octan-3-yl 2-hydroxy-3- p-hydroxybenzoic acid are 1.0 and 1.6, respectively.
phenylpropanoate;also known as (1R,3r,5S)-8-Methyl-8-azabicyclo[3.2.1 ]oct- ]
3-yl (2RS)-2-hydroxy-3-phenylpropanoate.
f (1R,3r,5S)-8-Methyl-8-azabicyclo[3.2.1 ]octan-3-yl 2-phenylacrylate; also
Suitability requirements
known as (1R,3r,5S)-8-Methyl-8-azabicyclo[3'.2.1 ]oct-3-yl 2- Resolution: NLT 2.2 between p-hydroxybenzoic add
phenylpropenoate. and atropine, System suitability solution
Relative standard deviation: NMT 1.5%, Standard
SPECIFICTESTS. solution
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of atropine
• OPTICAL D ....' ,.A,....~ .. IJ~/ sulfate monohydrate [(C17 H23N0 3)2' H2S04 , H20] in the
Rotation portion of the Injection taken:
S~~pl.~~«:)I~~ic:>. ~:Q.l g/mL of Atropine Sulfate in water
~~~~~~r . '../
9B* Result = (r vir s) x (C siC v) x (M ,riM (2) x 100
Path • len r/·~*Qjjg~B~i~~·(Q$~~1.)
Acceptance criteria: Between -0.50° and +0.05° ru = peak response from the Sample solution
• WATER DETERMINATION (921), Method I: NMT 4.0% rs = peak responsefrom the Standardsolution
ADDITIONAL REQUIREMENTS
Cs =concentration of USP Atropine Sulfate RS in the
Standardsolution (mg/mL)
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Cv =nominal concentration of atropine sulfate in the
containers.
Sample solution (mg/mL)
• USP REFERENCE STANDARDS (11) M r1 =molecular weight of atropine sulfate
USP Atropine Sulfate RS monohydrate, 694.85
USP Hyoscyamine Related Compound A RS M r2 = molecular weight of anhydrous atropine sulfate,
Norhyoscyamine sulfate; (1 R,3r,5S)-8-Azabicyclo[3.2.1]
676.83
octan- 3-yl (5)-3-hyd roxy-2 -phenyl propanoate suIfate
(2:1). Acceptance criteria: 93.0%-107.0%
(C,6H2,N03)2' H2S04 648.77
SPECIFIC TESTS
• pH (791): 3.0-6.5
• BACTERIAL ENDOTOXINS TEST (85): NMT 55.6 USP
Endotoxin Units/mg of atropine sulfate
Atropine Sulfate Injection • OTHER REQUIREMENTS: Meets the requirements in Injections
and Implanted Drug Products (1)
DEFINITION
Atropine Sulfate Injection is a sterile solution of Atropine ADDITIONAL REQUIREMENTS
Sulfate in Water for Injection. It contains NLT 93.0% and • PACKAGING AND STORAGE: Preserve in single-dose or
NMT 107.0% of the labeled amount of atropine sulfate multiple-dose containers, preferably of Type I glass. Store
monohydrate [(C17H23N03h . H2S04 , H20]. at controlled room temperature.
• USP REFERENCE STANDARDS (11)
IDENTIFICATION USP Atropine Sulfate RS
• A. The retention time of the major peak of the Sample
solution corresponds to that of the Standardsolution, as
obtained in the Assay.

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USP 43 OfficialMonographs / Atropine 431

Suitability requirements
Atropine Sulfate Ophthalmic Ointment Resolution: NLT 2.0 between atropine and Iittorine,
DEFINITION System'suitability solution
Atropine Sulfate Ophthalmic Ointment is Atropine Sulfate in a Tailing factor: NMT 1.5, Standardsolution
suitable ophthalmic ointment base. It contains NLT 90.0% Relative standard deviation: NMT 1.0%, Standard
and NMT 110.0% of the labeled amount of atropine sulfate solution
monohydrate [(C17H23N03) 2 • H2S04 , H20]. It is sterile. Analysis
Samples: Standardsolution and Sample solution
IDENTIFICATION Calculate the percentage of the labeled amount of atropine
• A. The retention time of the major peak of the Sample sulfate monohydrate [(C17H23N0 3) 2 • H2S04 , H20 ] in the
solution corresponds to that of the Standardsolution, as portion of Ophthalmic Ointment taken:
obtained in the Assay.
• B. IDENTIFICATION TESTs-GENERAL (191), Chemical Result =(rulrs) x (Cs/Cu) x (M rdMr2) x 100
Identification Tests, Sulfate
Sample solution: Transfer 5 g of Ophthalmic Ointment to ru = peak response of atropine from the Sample
a separator, dissolve in 50 mL of ether, and extract with 20 solution
mL of water. rs = peak response of atropine from the Standard
Acceptance criteria: Meets the requirements solution
Cs = concentration of USP Atropine Sulfate RS in the
ASSAY Standardsolution (mg/mL)
• PROCEDURE Cu = nominal concentration of atropine sulfate in the
Solution A: Stronger ammonia water and water (1:100), Sample solution (mg/mL)
adjusted with perchloricacid to a pH of 3.0 M r, = molecular weight of atropine sulfate
Solution B: Acetonitrile monohydrate, 694.83
Mobile phase: See Table 1. Mr2 = molecular weight of anhydrous atropine sulfate,
676.82
Table 1
Time Solution A Solution B Acceptance criteria: 90.0%-110.0%
(min) (%) (%)
IMPURITIES
0 95 5 • ORGANIC IMPURITIES
3 95 5 Solution A, Solution B, Mobile phase, Diluent, System
suitability solution, and Chromatographic system:
14 I
75 25 Proceed as directed in the Assay.
19 60 40 Standard solution: 1.0 ~g/mL of USP Atropine Sulfate RS in
Diluent
21 60 40 Sample solution: Nominally 500 ~g/mL of atropine sulfate
22 95 5 from a portion of Ophthalmic Ointment prepared as
follows. Transfer a portion of Ophthalmic Ointment
25 95 5 equivalent to 25 mg of atropine sulfate to a stoppered
250-mL flask, add 50 mL of Diluent, and shake at 40 0 for 60
Diluent: Acetonitrile and Solution A (25:75) . min. Centrifuge and use the lower layer clear solution.
System suitability solution: 1.0 ~g/mL each of USP [NoTE-The use of a centrifuge speed of 4000 rpm for 10
Atropine Sulfate RS and Iittorine hydrochloride in Oiluent min may be suitable.]
Standard solution: 0.1 mg/mL of USP Atropine Sulfate RS System suitability
in Diluent Samples: System suitability solution and Standardsolution
Sample stock solution: Nominally 0.5 mg/mL of atropine [NOTE-The relative retention times for atropine and
sulfate from a portion of Ophthalmic Ointment prepared as littorine are 1.0 and 1.04, respectively.]
follows. Transfer 10 mg of atropine sulfate from a portion Suitability requirements
of Ophthalmic Ointment to a stoppered 250-mL flask, add Resolution: NLT 2.0 between atropine and Iittorine,
20 mL of Diluent, and shake at 40 0 for 60 min. Centrifuge System SUitability solution
and use the lower layer clear solution. [NOTE-The use of a Relative standard deviation: NMT 5.0%, Standard
centrifuge speed of 4000 rpm for 10 min may be suitable.] solution
Sample solution: Nominally 0.1 mg/mL of atropine sulfate Analysis
from the Sample stock solution in Diluent Samples: Standardsolution and Sample solution
Chromatographic system Calculate the percentage of each specified and any
unspecified degradation product in the portion of
(See Chromatography (621), System Suitability.) Ophthalmic Ointment taken:
[NoTE-Rinse the system with water after each set of
analysis.] Result = (rulrs) x (CsICu) x (1 IF) x 100
Mode: LC
Detector: UV 210 nm rv = peak response of each specified and any
Column: 2.1-mm x 10-cm; 1 .8-~m packing L96 unspecified degradation product from the
0
Column temperature: 30 Sample solution
Flow rate: 0.4 mL/min rs = peak response of atropine from the Standard
Injection volume: 3 ~L solution
System suitability Cs =concentration of USP Atropine Sulfate RS in the
Samples: System suitability solution and Standardsolution Standardsolution (~g/mL)
[NoTE-The relative retention times for atropine and Cv = nominal concentration of atropine sulfate in the
Iittorine are 1.0 and 1.04, respectively.] Sample solution (~g/mL)

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432 Atropine / Official Monographs USP 43

F = relative response factor (see Table2) Table 1

Time Solution A Solution B
Acceptance criteria: See Table 2. Disregard peaks less than (min) (%) (%)
0.1%.
0 95 5
Table 2 3 95 5
Relative Relative Acceptance 14 75 25
Retention Response Criteria,
Name Time Factor NMT(%) 19 60 40
Tropic acid" 0.35 2.1 0.2 21 60 40
Scopolarnlne'' 0.74 1.0 0.2 22 95 5
Hyoscyamine related com- 25 95 5
pound N 0.92 1.0 0.3
Atropine 1.0 - - Diluent: Acetonitrile and SolutionA (25:75)
Apoatroplne" 1.46 2.1 0.2
System suitability solution: 1.0 IJg/mL each of USP
Atropine Sulfate RS and Iittorine hydrochloride in Diluent
Any individual Standard solution: 0.1 mg/mL of USP Atropine Sulfate RS
unspecified - in Diluent
degradation product 1.0 0.2
Sample solution: Nominally 0.1 mg/mL of atropine sulfate
a 3-Hydroxy-2-phenylpropanoic acid. from a volume of Ophthalmic Solution in Diluent
b(S)-(l R,2R,4S,5S,7s)-9-Methyl-3-oxa-9-azatricyclo[3.3.1.0 2•4]n onan-7-yl 3- Chromatographic system
hydroxy-2-phenylpropanoate. (See Chromatography (621), System Suitability.)
c (1 R,3r,5S)-8-Azabicyclo[3.2.1 ]octan-3-yl (S)-3-hydroxy-2-phenylpropanoate. [NOTE-Rinse the system with water after each set of
d (1 R,3r,5S)-8-Methyl-8-azabicyclo[3.2.1 ]octan-3-yl 2-phenylacrylate. - analysis.]
Mode: LC
SPECIFIC TESTS Detector: UV 21 0 nm
• STERILITY TESTS (71): Meets the requirements Column: 2.1-mm x 10-cm; 1 .s-urn packing L96
• OTHER REQUIREMENTS: It meets the requirements for Column temperature: 30°
Particulateand Foreign Matter in Ophthalmic Products- Flow rate: 0.4 mL/min
Quality Tests (771), Drug Product Quality, Universal Tests, Injection volume: 3 IJL
Particulateand,Foreign Matter. System suitability
ADDITIONAL REQUIREMENTS
Samples: System suitability solution and Standardsolution
[NOTE-The relative retention times for atropine and
• PACKAGING AND STORAGE: Preserve in collapsible
ophthalmic ointment tubes. Store at controlled room Iittorine are 1.0 and 1.04, respectively.]
temperature. Suitability requirements
Resolution: NLT2.0 between atropine and Iittorine,
• USP REFERENCE STANDARDS (11)
System suitability solution
USP Atropine Sulfate RS
Tailing factor: NMT 1.5, Standardsolution
Relative standard deviation: NMT 1.0%, Standard
solution
Analysis
Atropine Sulfate Ophthalmic Solution Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of atropine
DEFINITION sulfate monohydrate [(C17H23N03)2' H2S0 4 , H20] in the
Atropine Sulfate Ophthalmic Solution is a sterile, aqueous portion of Ophthalmic Solution taken:
solution of Atropine Sulfate. It contains NLT93.0% and NMT
107.0% of the labeled amount of atropine sulfate Result = (rvlrs) x (CsICv) x (Mr tlMr2 ) x 100
monohydrate [(C17H 23N0 3)2' H2S0 4 , H20]. It may contain
suitable stabilizers and antimicrobial agents. rv = peak response of atropine from the Sample
solution
IDENTIFICATION rs = peak response of atropine from the Standard
• A. The retention time of the major peak of the Sample solution .
solution corresponds to that of the Standardsolution, as Cs = concentration of USP Atropine Sulfate RS in the
obtained in the Assay. Standard solution (mg/mL)
• B. IDENTIFICATION TESTS-GENERAL (191), Chemical Cu = nominal concentration of atropine sulfate in the
Identification Tests, Sulfate Sample solution (mg/mL)
Sample solution: Evaporate to dryness a quantity of Mr1 = molecular weight of atropine sulfate
Ophthalmic Solution. Prepare a solution from the residue monohydrate, 694.83
that contains the equivalent of 50 mg of atropine sulfate/ Mr2 = molecular weight of anhydrous atropine sulfate,
mL. 676.82
Acceptance criteria: Meets the requirements
Acceptance criteria: 93.00/0-107.0%
ASSAY
• PROCEDURE IMPURITIES
Solution A: Stronger ammonia water and water (1:100), • ORGANIC IMPURITIES
adjusted with perchloric acid to a pH of 3.0 Solution A, Solution B, Mobile phase, Diluent, System
Solution B: Acetonitrile suitability solution, and Chromatographic system:
Mobile phase: See Table 7. Proceed as directed in the Assay. '

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 USP 43 Official Monographs / Attapulgite 433

Standard solution: 1.0 JJg/mLof USPAtropine Sulfate RS in

Diluent Activated Attapulgite
Sample solution: Nominally 500 JJg/mLof atropine sulfate
from a volume of Ophthalmic Solution in Diluent » Activated Attapulgite is a highly heat-treated,
System suitability
Samples: System sUitabilitysolution and Standardsolution processed, native magnesium aluminum silicate.
[~OTE-:-The relative retention times for atropine and
littorine are 1.0 and 1.04, respectively.] Packaging and storage-Preserve in well-closed
Suitability requirements containers.
Resolution: N~! 2.0 between atropine and Iittorine, Iden~~fic~tion-Activated ~ttapul~ite responds to the
System swtablltty solution Identlflcat!o'!- test for Colloido! Activated Attapulgite, the
Relative standard deviation: NMT 5.0%, Standard characteristic peak, however, being much less intense.
solution Loss on drying (731)-Dry it at 105° to constant weight: it
Analysis loses not more than 4.0% of its weight.
Samples: Standardsolution and Sample solution Loss on ignition (733)-When ignited at 1000° for 1 hour, it
Calculat~ ~he percenta~e of each s~ecified and any
loses between 4.0% and 12.0% of its weight.
unspecified degradation product In the portion of Volatile matter-When ignited at 600° for 1 hour, it loses
Ophthalmic Solution taken: between 3.0% and 7.5% of its weight on the dried basis.
Powder fineness-Proceed as directed in the test for Powder
Result = (rulrs) x (CsIC u) x (lIP) x 100 fineness- under Colloidal ActivatedAttapulgite. The dry weight
of the residue is not more than 0.10% of the weight of the
= peak response of each specified and any specimen taken.
unspecified degradation product from the Acid-soluble matter-Boil 2.0 g with 100 mL of 0.2 N
Sample solution hydrochloric acid for 5 minutes, and cool. Add water to adjust
= peak response of atropine from the Standard the volume to 100 mL, and filter. Evaporate 50 mL of the
solution filtrate so obtained to dryness, and ignite the residue at 600°:
= concentration of USP Atropine Sulfate RS in the not more than 0.25 g is found (25%).
Standardsolution (lJg/mL) Other requirements-It meets the requirements of the
= nominal concentrationof atropine sulfate in the te~ts for Mi~robial enumeration tests 61 and Tests for specified
Sample solution (JJg/mL) tmcroorqamsms 62-, pH 791-, Carbonate-, Arsenic and
F = relative response factor (see Table 2) Lead-, and Adsorptive capacity- under Colloidal Activated
Attapulgite.
Acceptance criteria: See Table 2. Disregard peaks less than
0.1%.

Table 2
Relative Relative Acceptance
Colloidal Activated Attapulgite
Retention Response Criteria,
Name Time Factor NMT(%)
»Colloidal Activated Attapulgite isa purified native
Tropicacid" 0.35 2.1 0.2 magnesium aluminum silicate.
Scopolerntne" 0.74 1.0 0.2
Packaging and storage-Preserve in well-closed
Hyoscyamine related com- containers.
pound AC 0.92 1.0 . 0.3
Identification-Add 2 g in small portions to 100 mL of
Atropine 1.0 - - water, with vigorous agitation. Allow to stand for at least 12
Apoatroplne" 1.46 2.1 0.2
hours to ensure complete hydration. Place 2 mL of the
resulting mixture on a suitable glass slide, and allow to air-dry
Anyindividual at room temperature to produce a uniform film. Place the slide
unspecified - in a vacuum desiccator over a free surface of ethylene glycol.
degradation product 1.0 0.2
Evacuate the desiccator, and close the stopcock so that the
a 3-Hydroxy-2-phenylpropanoic acid. ethylene glycol saturates the desiccator chamber. Allowto
b (5)-(1
R,2R,4S,5S,7s)-9-Methyl-3-oxa-9-azatricyclo[3.3.1.02.4]nonan-7-yl 3- stand for 12 hours. Record the X-ray diffraction pattern (see
hydroxy-2-phenylpropanoate. X-Ray Diffraction (941»), and calculate the d values: several
c(1R,3r,55)-8-Azabicyclo[3.2.1 ]octan-3-yl (5)-3-hydroxy-2-phenylpropanoate. peaks are observed; the characteristic peak corresponds to a d
d (1R,3r,55)-8-Methyl-8-azabicyclo[3.2.1 ]octan-3-yl 2-phenylacrylate. value between 10.3 and 10.7 Angstrom units.
Microbial enumeration tests (61) and Tests for specified
SPECIFIC TESTS microorganisms (62)-lt meets the requirements of the test
• pH (791): 3.5-6.0 for absence of Escherichia coli.
• STERILITY TESTS (71): Meets the requirements pH (791 )-Disperse 1.0 g in 10 mL of carbon dioxide-free
water, and mix: the pH of the mixed dispersion so obtained is
ADDITIONAL REQUIREMENTS between 7.0 and 9.5.
• PACKAGING AND STORAGE: Preserve in tight containers, and Loss on drying (731)-Dry it at 105° to constant weight: it
store at controlled room temperature. loses between 5.0% and 17.0% of its weight.
• USP REFERENCE STANDARDS (11) Loss on ignition (733)-When ignited at 1000° for 1 hour, it
USP Atropine Sulfate RS loses between 17.0% and 27.0% of its weight.
Volatile matter-When ignited at 600° for 1 hour, it loses
between 7.5% and 12.5% of its weight on the dried basis.
Powder fineness-Add 50 g to 450 mL of water containing
5 g of sodium pyrophosphate, and stir for 10 minutes. Pour

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434 Attapulgite / OfficialMonographs USP 43

the resulting dispersion slowly through a No. 325 standard ut,ofan aqueous Standardsolution of USP Aurothioglucose RS
sieve (see Particle Size Distribution Estimation by Analytical containing 4 mg per mLto a suitable thin-layer
Sieving (786», and carefully wash the residue untilclean. Dry chromatographic glass microfilament sheet (see
the residueat 105° to constant weight: the dry weight of the Chromatography (621» impregnated with silicic acid and a
residueso obtained is not more than 0.30% of the weight of suitablefluorescing substance. Allow the spots to dry, and
the specimen taken. developthe chromatogram in a solventsystemconsisting of a
Add-soluble matter-Boil 2.0 g with 100 mL of 0.2 N mixtureof n-propyl alcohol, water, and ethyl acetate (3:3:1)
hydrochloric acidfor 5 minutes, and cool. Addwater to adjust until the solventfront has moved about three-fourths of the
the volume to 100 mL, and filter. Evaporate 50 mL of the length of the plate. Remove the sheet from the developing
filtrate so obtained to dryness, and ignite the residue at 600°: chamber, markthe solventfront, and allow the solventto
not more than 0.15 g is found (15%). evaporate. locate the spots on the plate byexamination under
Carbonate-Mix 1.0 g with 15 mL of 0.5 N sulfuric acid: no short-wavelength UV light: the RF value of the principal spot
effervescence occurs. obtained from the solution under test correspondsto that
Arsenic and lead-To 5.0 g add 50 mL of 1 N nitric acid, obtained from the Standard solution.
and boilfor 30 minutes, adding 1 N nitricacid at times to B: To a portion of the filtrate obtained in the Assay add
maintain the volume. Filter into a 1OO-mL volumetric flask, barium chlorideTS: a heavy, white precipitate isformed.
washthe filter withwater, and dilutethe combinedfiltrate and Specific rotation (781 S): between +65° and +75°.
washings with water to volume. Test solution: 10 mg per mL, in water.
Arsenic-Determine the arsenic in the solution by atomic Loss on drying (731 )-Dry it over phosphorus pentoxide for
absorption spectrometry (see Atomic Absorption Spectroscopy 24 hours: it loses not more than 1.0% of its weight.
(852», using a graphite furnace to volatilize the arsenic, as Assay-Accurately weigh about 1 g of Aurothioglucose, and
directed by the manufacturer of the instrument used, and dissolve in 100 mL of water in a 300-mL Kjeldahl flask. Slowly
measuring the absorbance at 189.0 nm against a standard: not add 10 ml of nitric acid, and when the reaction has subsided,
more than 2 ppm isfound. boil the mixturefor 5 minutes. Filter, wash well the separated
Lead-Determine the lead in the solution by atomic gold with hot water, dry, and igniteto constant weight. The
absorption spectrometry (see Atomic Absorption Spectroscopy weight of the gold so obtained, multiplied by 1.991,
(852», using a graphite furnace to volatilize the lead, as .representsthe vyeight of C6H ll AuOsS in the portion of
directed by the manufacturer of the instrument used, and Aurothiogluro~ taken.
measuringthe absorbance at 283.3 nm againsta standard: not
more than 0.001% isfound.
Adsorptive capadty-To 10 mL of a 1 in 10 suspension of
the specimen in water add 80 mL of methylene bluesolution
(1 in 1000), and shake.Add 10 mL of bariumchloridesolution
(1 in 50), and shake. Allow to stand for 15 minutes. Transfer
40 mL of the supernatant to a 50-mL centrifuge tube, and
centrifuge. To 5 mL of the clear supernatant add 495 mLof
water, and mix: the color of the solutionso obtained is not
deeper than that of a solution containing 1.5 ~g of methylene
blue per mL.

Aurothioglucose

C6H 11AuOsS 392.18

Gold, (l-thio-D-glucopyranosato)-.
(l-Thio-D-glucopyranosato)gold [12192-57-3].
» Aurothioglucose contains not less than 95.0
percent and not more than 105.0 percent of
C6H llAuOsS, calculated on the dried basis. It is
stabilized by the addition of a small amount of
Sodium Acetate.
Packaging and storage-Preserve in tight, light-resistant
containers. Store at room temperature.
USP Reference standards (11 )-
USP Aurothioglucose RS
Identification-
A: Dissolve a suitable quantity in water to obtain a solution
containing 4 mg per mL. Apply 10 ~L of this solution and 10

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 USP 43 Official Monographs / Azaperone 435

Carrier gas: Helium

Injection volume: 1 J.JL
Split ratio: 50:1
System suitability
Sample: Standard solution
.Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of avobenzone (C2oH2203) in the
portion of Avobenzone taken:
Result = (rulr s) x (CslCu) x 100
tu = peak response from the Sample solution
Avobenzone rs = peak response from the Standard solution
Cs = concentration of USP Avobenzone RS in the
Standard solution (mg/mL)

~
O O ,;:::
Cu =concentration of Avobenzone in the Sample
H,C ~ I I h solution (mg/mL)
OCH,
H,C
CH,
Acceptance criteria: 95.0%-105.0% on the dried basis
C2oH2203 310.40 IMPURITIES
l,3-Propanedione, 1-[4-(l,l-dimethylethyl)phenyl]-3-(4- • ORGANIC IMPURITIES
methoxyphenyl)-; Sample solution, Chromatographic system, and System
1-(p-tert-Butylphenyl)-3-(p-methoxyphenyl)-l,3- . suitability: Proceed as directed in the Assay.
propanedione [70356-09-1]. Analysis
DEFINITION Sample: Sample solution
Avobenzone contains NLT 95.0% and NMT 105.0% of Calculate the percentage of each impurity in the portion of
avobenzone (C2oH2203), calculated on the dried basis. Avobenzone taken:

IDENTifiCATION I Result = (rulrr) x 100

ru = peak response for each impurity from the
Sample solution
rr =sum of all peak responses from the Sample solution
Acceptance criteria
Each individual impurity: NMT 3.0%
Total impurities: NMT 4.5%
SPECifiC TESTS
• Loss ON DRYING (731)
Analysis: Dry a sample under vacuum at 70 0 for 4 h.
Acceptance criteria: NMT 0.5%
ADDITIONAL REQUIREMENTS
ASSAY • PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
• PROCEDURE • USP REFERENCE STANDARDS (11)
Standard solution: 50 mg/mL of USP Avobenzone RS in USP Avobenzone RS
acetone
Sample solution: 50 mg/mL of Avobenzone in acetone
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC Azaperone
Detector: Flame ionization
Column: 0.32-mm x 25-m fused silica capillary coated
with phase G1
Temperatures
Injection port: 200 0
Detector: 280 0 C,9H22FN30 327.40
Column: See Table 7. 1-Butanone, 1-(4-fluorophenyl)-4-[4-(2-pyridinyl)-1-
piperazinyl]-.
Table 1
4'-Fluoro-4-[ 4-(2-pyridyl)-1-piperazinyl]butyrophenone
Hold Time at Fi- [1649-18-9].
Initial Temperature Final nal
Temperature Ramp Temperature Temperature
e) elm in) e) (min)
200 4 280 -

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 436 Azaperone / Official Monographs USP 43

» Azaperone contains not less than 98.0 percent Packaging and storage-Preserve in single-dose or in
and not more than 102.0 percent of C19H22FN30, multiple-dose containers, preferably of TypeIglass, protected
from light.
calculated on the dried basis. Labeling-Label it to indicatethat it is forveterinary useonly.
USPReference standards (11)-
Packaging and storage-Preserve in well-closed USP Azaperone RS
containers, protected from light. Store at room temperature. Identification-The chromatogram ofthe Assay preparation
Labeling-label it to indicatethat it isfor veterinaryuseonly. obtained as directed in the Assay exhibits a majorpeak for
USPReference standards (11 )- azaperone, the retention time of which corresponds to that
USP Azaperone RS exhibited in the chromatogram of the Standardpreparation,
obtained as directed in the Assay.
pH (791): between 4.0 and 5.6.
Other requirements-It meets the requirements under
Injections and ImplantedDrug Products (1).
Assay-
Melting range (741): between 92° and 95°. Mobilephase-Prepare a filtered and degassed mixture
Loss on drying (731 )-Dry it in vacuum at 60° for 4 hours: it containing 6 volumesof acetonitrile and 4 volumes of 0.01 M
loses not more than 0.5% of its weight. dibasic potassium phosphate, and adjust by the addition of
Residue on ignition (281): not more than 0.1%. dilute phosphoricacid (1 in 10) to a pHof 7.8 ± 0.1. Make
Chromatographic purity-Dissolve an accuratelyweighed adjustments if necessary (see System Suitability under
quantity of USP Azaperone RS in a mixture of acetone and Chromatography (621».
methylene chloride(5:1) to obtain a solution having a Internal standard solution-Prepare a solutionof
concentration of 0.50 mg per mL (StandardsolutionA). benzophenone in methanol containingabout 0.5 mg per mL.
Quantitatively dilute a volume of StandardsolutionA with the Standardpreparation-Dissolve an accurately weighed
same solvent mixtureto obtain a solution having a quantity of USP Azaperone RS in methanol, and dilute
concentrationof 0.25 mg per mL (StandardsolutionB). Prepare quantitatively with methanol to obtain a solution havinga
a test solution by dissolving an accuratelyweighed quantity of .known concentration of about 0.5 mg per mL. Combine 2.5
Azaperone in a mixtureof acetone and methylene chloride mL of this solution with 2.5 mL of Internal standardsolution,
(5:1) to obtain a solution containing 50 mg per mL. Separately dilute quantitatively with methanol to 10.0 mL, and mix.
apply 1 IJL each of StandardsolutionA, StandardsolutionB, and Assay preparation-Dilute an accurately measured volume
the test solution to a thin-layer chromatographic plate (see of Injection quantitatively with methanol to obtain a solution
Chromatograp hr.(621» coated with a 0.2-mm layerof containing about 0.5 mg of azaperone per mL. Combine 2.5
chromatographic silica gel mixture with chemically bonded mL of this solution with 2.5 mL of Internal standardsolution,
amino groups, and allow the spots to dry. Develop the dilute quantitatively with methanol to 10.0 mL, and mix.
chromatograms in a solventsystem consisting of a mixtureof Chromatographic system (see Chromatography (621 »-
cyclohexane, acetone, and methanol (65:30:5) until the The liquid chromatograph is equipped with 243-nrn detector
solventfront has moved about three-fourths'ofthe length of and a 4.6-mm x 25-cm column that contains packing L1. The
the plate. Remove the plate from the chromatographic flow rate is about 2 mL per minute. Chromatograph the
chamber, and allowthe plate to air-dry. Examine the plate Standardpreparation, and record the peak responsesas
under short-wavelength UV light, and compare the intensities directed for Procedure: the resolution, R, between the
of any secondary spots, other than any spot at the origin, azaperone and internalstandard peaksisnot less than 2.7; and
observed in the chromatogram of the test solutionwith those the relative standard deviation for replicate injections is not
of the principal spots in the chromatograms of Standard more than 2.0%.
solutionA and StandardsolutionB: the sum of the intensities of Procedure-Separately inject equal volumes (about 10 IJL)
the secondaryspots obtained from the test solution of the Standardpreparation and the Assay preparation into the
corresponds to not more than the intensityof the principal chromatograph, record the chromatograms,and measurethe
spot in the chromatogram of StandardsolutionA (1 .0%). responses for the major peaks. Calculate the quantity, in mg,
Assay-Dissolve about 120 mg of Azaperone, accurately of azaperone (C'9 H22 FN30 ) in each mL of the Injection taken
weighed, in 50 mL of a mixture of methyl ethyl ketone and by the formula:
glacial acetic acid(7:1).Add 3 drops ofp-naphtholbenzeinTS,
and titrate with 0.1 N perchloric acid VS. Perform a blank (C)(L/ D)(Ru/ Rs)
determination, and make any necessary correction. Each mL
of 0.1 N perchloric acid is equivalent to 16.37 mg of in which C is the concentration, in mg per ml, of USP
C19H 2l N30. Azaperone RS in the Standardpreparation; L is the labeled
quantity, in mg, of azaperone in each ml of the Injection; D
isthe concentration, in mg per mL, of azaperone in the Assay
preparation, based on the volume of Injection taken and the
extent ofdilution; and Ru and Rs are the ratios of the azaperone
Azaperone Injection peak to the benzophenone peak obtained from the Assay
preparation and the Standardpreparation, respectively.
» Azaperone Injection is a sterile solution of
Azaperone in Water for Injection, prepared with
the aid of Tartaric Acid. It may contain a suitable
preservative and a stabilizing agent. It contains not
less than 90.0 percent and not more than 110.0
percent of the labeled amount of C19H22FN30.

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USP 43 OfficialMonographs / Azatadine 437

Re~ult'~-(f~/~~fx{t.ilC(J~;-l-·~.~

sporise of 'azata'cUrJ~ fromlh-eS(1mp/~

-rise of ~zafifdiDefr9m ttie'Standgrd

Table 1
Time
(min)
Sob.ltionA
(%r- ~ql~~~;Ii
0 80 2.0

7.0 70 3"0.
12.0 60 40
andard
VotO so SQ
16·0 30 70 Cs
!j8:0 ~O ZQ
]8;1' 80 .20
20.0 80 ~~

ion:O.5:mg{mlof LJSI"AZatadfn~-Mareate

L .2U~zat~din~~M~al~a~e:llJ~ateri

;'SHt~nl/Sylt?f~lJ]li,~

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 438 Azatadine / Official Monographs USP 43

Delete the foUlowing: a 50':ITlLflask fitted with a glassstopper; Ad·

N hydrochloric acid, insertthe stopper,
by mechanical means for about 30 min
intoa suitableglass-stoppered vessel, disc
of the filtrate.
»Azatadine Maleate Table Procedure-Separately transfer1.5.0 mL of theStandar~
preparation, 15.0 mL of th say preparation, tof
90.0 percent and not mar 0.1 N hydrochloric acid to videt hree
the labeled amount ofC 20 H22 50~mL centrifugetubes fitte with gl ,h
centrifugetube add 10.0 mL of 1. ide and
Packaging and storag~Preservein well~d()sed 20 mL of solvent hexane, insertt - -
containers. _ .. centrifugetu for about 15 minu es
USPReference Standards ( 11.)·. - supernata ane phas .
USP Azatadine Maleate RS separate s es
Identification-Transfer 1S.O mL of the S{andciid centrifuge
preparation and 15.0 mLofthe Assay re . spectively, with 10 mL
prepared as directed in the A . aqueous ph IC
centrifugetubes fitted with gl removed. Insert.thestoppe
tube add 10.0 mL of 1.0 N sodi minutes, and centrifuge.Tr
solventhexane, insertthe stopp respective supernatant prev. lIect
for about 15 minutes,· and.cen Nhydrochloric acid iritoeach centiifug
hexane .extracts (upper phase) combined supernatants, inse-rt t
separate 50-mL conical ftasks fi e forabout15 minutes,an .
Evaporate the solvent hexane extra supernatants. Conco .
a stream of nitrogen to dryness, . the solutions in. l-cm c
into each flask, insertthe stopper absorbance at about 28 n .e
mixer(or equivalent) untilthe re , spectrophotometer zeroed WIth 0.1 Y rochl ng
these solutions as the Standard the prepared reagent blank. Cal ate th (of
respectively. Apply sepa· 1 CzoHzzNz : 2C4H4 0 4 in the porti f Tab
and the Standard sofuti formula:
chromatographic pl 25C(Au/As)
with a 0.25-mmlay
Allow the spots to d in which C is the concentration, in mg per m
solvent system cons . ft Azatadine MaleateRS in the Standard a' nd
diethylamine (10:10:1) untilthe As arethe absorbances of the solutio
about three-fourths of the lengt
plate from the developingcha preparation and the Standardprepara on,
and allow the plate to air-dry..E respectively.... (USP l-May-2020)
short-wavelength UV light: the R
principal spot in the chromatogr
correspond to those obtained fromth
Standard solution. Azathioprine
Dissolution (711)-
Medium: 0.01 Nhydrochloric acid;·500 mL.
Apparatus 2: 50 rpm. . .
Time: 30 minutes.
Procedure-Determine the amount Cii404
dissolved by employing UV absorpti ngth of
maximumabsorbance at about ·rtionsof C9H 7N 70ZS 277.26
the solution under test, diluted essary, hi 1H-Purine, 6-[(1-methyl-4-nitro-l H-imidazol-5-yl)thio]-;
comparison with a Standard so n 6-[(1-Methyl-4-nitroimidazol-5-yl)thio]purine [446-86-6].
concentration of USP Azatadine -Maleate RS i same
Medium. DEFINITION
Toler.ances-Not less than 80% (Q) I led ~afllount Azathioprine contains NLT 98.0% and NMT 102.0% of
of CzoHzzNz: 2C4H40 4 is dissolved in-3 .. utes. azathioprine (C9H7N 70 ZS), calculated on the dried basis.
. Uniformity of Dosage Units (. 905 '> : meet the IDENTIFICATION
requirements.
Assay- Change to read:
Standard preparation--:-Dissolve an acclmlt • A. "'SPECTROSCOPIC IDENTIFICATiON TESTS:(197), Infrared
quantity of USP Azatadine Maleate RS i . Spectroscopy: 197K... (eN l-May-2020) .,. -
acid,'and dilute quantitatively, and ste • B. The retention time of the major peak of the Sample
0.1.N hydrochloric acid to obt solution corresponds to that of the Standard solution as
concentration of aboutO. obtained in the Assay. '
Assaypreparation:--,Weig
than 20 Tablets.Transfer anaccuratelyw e
powder, equivalentto about 1.5mg of azatadine , 0

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USP 43 Official Monographs / Azathioprine 439

ASSAY Diluent: 0.8 giL of sodium hydroxide in water

• PROCEDURE System suitability stock solution A: 0.2 mg/mL each of
Solution A: 1.6 giL of sodium 1-heptanesulfonate in water USP Azathioprine Related Compound A RS and USP
Mobile phase: Methanol and Solution A (30: 70). Adjust with Mercaptopurine RS prepared as follows. Transfer USP
1 N hydrochloric acid to a pH of 3.5 ± 0.1. Azathioprine Related Compound A RS and USP
Standard stock solution: 0.5 mg/mL of USP Azathioprine Mercaptopurine RS to a suitable volumetric flask. Add 35%
RS prepared asfollows. Transfer USP Azathioprine RS to a of the flask volume of Diluent, and dilute with Bufferto
suitable volumetric flask. Add 25% of the flask volume of volume.
methanol and 1% of ammonium hydroxide to the flask. System suitability stock solution B: 0.1 mg/mL each of USP
Swirl, and sonicate for 2 min or until dissolved. Dilute with Azathioprine RelatedCompound G RS and USP
methanol to volume. Azathioprine RS prepared asfollows. Transfer USP
Standard solution: 0.1 mg/mL of USP Azathioprine RS in Azathioprine Related Compound G RS and USP
water from the Standardstock solution Azathioprine RS to a suitable volumetric flask. Add 35% of
Sample solution: 0.1 mg/mL of Azathioprine prepared as the flaskvolume of Diluent, and dilute with Bufferto volume.
follows. Transfer 50 mg of sample to a 1OO-mL volumetric System suitability solution: 0.002 mg/mL each of USP
flask. Add 25 mL of methanol and 1.0 mL of ammonium Azathioprine Related Compound A RS, USP
hydroxide to the flask, swirl, and sonicate for 2 min or until Mercaptopurine RS, USP Azathioprine Related Compound
dissolved. Dilute with methanol to volume. Transfer 10.0 G RS, and USP Azathioprine RS prepared asfollows. Transfer
mL of this solution into a 50-mL volumetric flask, and dilute 1 mL of System suitabilitystock solutionA and 2 mL of System
with water to volume. suitabilitystock solutionB to a 1OO-mL volumetric flask. Add
Chromatographic system 35 mL of Diluent, and dilute with Buffer to volume.
(See Chromatography (621), System Suitability.) Standard stock solution: 0.1 mg/mL of USP Azathioprine
Mode: LC RS prepared as follows. Transfer USP Azathioprine RS to a
Detector: UV 254 nm suitable volumetric flask. Add 35% of the flask volume of
Column: 3.9-mm x 30-cm; 1O-urn packing L1 Diluent, and dilute with Bufferto volume.
Flow rate: 1.8 mL/min Standard solution: 0.1 uq/rnl, of USP Azathioprine RS in
Injection volume: 10 ~L Buffer from Standard stock solution
System suitability Sample solution: 0.1 mg/mL of Azathioprine prepared as
Sample: Standardsolution follows. Transfer Azathioprine to a suitable volumetric flask.
Suitability requirements Add 35% of the flask volume with Diluent, and dilute with
Tailing factor: NMT 2 Buffer to volume.
Relative standard deviation: NMT 0.73% Chromatographic system
Analysis . (See Chromatography (621), System Suitability.)
Samples: Standardsolution and Sample solution Mode: LC
Calculate the percentage of azathioprine (C9H 7N 70 2S) in Detector: UV 240 nm
the portion of Azathioprine taken: Column: 4.6-mm x 15-cm; 5-~m packing L11
Column temperature: 30°
=
Result (rulrs) x (CsICu) x 100 Flow rate: 1.0 mL/min
Injection volume: 20 ~L
ru = peak response of azathioprine from the Sample System suitability
solution Sample: System suitability solution
r, = peak response of azathioprine from" the Standard Suitability requirements
solution Resolution: NLT 2 between azathioprine related
Cs =concentration of USP Azathioprine RS in the compound A and mercaptopurine; NLT 2 between
Standardsolution (mg/mL) azathioprine related compound G and azathioprine
Cu =concentration of Azathioprine in the Sample Analysis
solution (mg/mL) Samples: Standardsolution and Sample solution
Calculate the percentage of each impurity in the portion of
Acceptance criteria: 98.0%-102.0% on the dried basis Azathioprine taken:
IMPURITIES Result = (rulrs) x (CsICu) x 100
• RESIDUE ON IGNITION (281): NMT 0.1%
• ORGANIC IMPURITIES
Buffer: 2.76 giL of monobasic sodium phosphate. Adjust
tu = peak response of each impurity from the Sample
solution
with phosphoric acid to a pH of 2.5. ts = peak response of azathioprine from the Standard
Solution A: Methanol and Buffer (5:95) solution
Solution B: Methanol and Buffer(60:40) Cs = concentration of USP Azathioprine RS in the
Mobile phase: See Table 7. Return to original conditions, Standardsolution (mg/mL)
and re-equilibrate the system. Cu = concentration of Azathioprine in the Sample
solution (mg/mL)
Table 1
Time Solution A Solution 8 Acceptance criteria: See Table 2. Disregard any impurity
(min) (%) (%) peaks lessthan 0.05%.
a 100 a
5 100 a
15 a 100
20 a 100

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440 Azathioprine / OfficialMonographs USP 43

Table 2 ASSAY
Relative Acceptance • PROCEDURE
Retention Criteria, Mobile phase: Dissolve 1.1 g of sodium-1-heptanesulfonate
Name Time NMT (%) in 700 mL of water, and add 300 mL of methanol. Adjust
Azathioprine related with 1 N hydrochloric acid to a pH of 3.5.
compound A" 0.3 0.15 Standard solution: Transfer 25 mg of USP Azathioprine RS
to a'50-mL volumetric flask. Add 15 mL of methanol and
Mercaptopurine" 0.4 0.15
0.5 mL of ammonium hydroxide to the flask, swirl, and
Azathioprine related sonicate for 2 min. Dilute with methanol to volume.
compound GC 0.97 0.10 Transfer 10 mL of this solution to a 50-mL volumetric flask,
Azathioprine 1.0 - and dilute with water to volume.
Sample solution: Agitate the container of Oral Suspension
Any other unspecified impurity - 0.10 for 30 min on a rotating mixer, remove a 5-mL sample, and
Totalimpurities - 0.5 store in a clear glass vial at -70 0 until analyzed. At the time
of analysis, remove the sample from the freezer, allow it to
a 1-Methyl-4-nitro-l H-imidazol-5-amine. reach room temperature, and mix with a vortex mixer for
b 9H-Purine-6.thiol. 30 s. Pipet 1.0 mL of the sample into a 1OO-mL volumetric
c 6-[(1-Methyl-4-nitro-l H-imidazol·5-yl)thio]-9H-purin-2-amlne. flask, and dilute with Mobilephase to volume.
Chromatographic system
SPECifiC TESTS (See Chromatography (621), System Suitability.)
• Loss ON DRYING (731) Mode: LC
Analysis: Dry under vacuum at 105 0 for 5 h. Detector: UV 254 nm
Acceptance criteria: NMT 1.0% Column: 4.6-mm x 25-cmj 5-JJm packing L1
Flow rate: 2 mL/min
ADDITIONAL REQUIREMENTS Injection volume: 20 JJL
• PACKAGING AND STORAGE: Preserve in tight, light-resistant System suitability
containers. Sample: Standard solution
• USP REFERENCE STANDARDS (11) [NOTE-The retention time for the azathioprine peak is
USP Azathioprine RS about 4 min.]
USP Azathioprine Related Compound A RS Suitability requirements
1-Methyl-4-nitro-1 H-imidazol-5-amine. Relative standard deviation: NMT 1.3% for replicate
C4H6N40 Z 142.12 injections
USP Azathioprine Related Compound G RS Analysis
6-[(1-Methyl-4-nitro-1 H-imidazol-5-yl)thio]-9H-purin-2- Samples: Standardsolution and Sample solution
amine. Calculate the percentage of the labeled amount of
C9H aNaOzS 292.28 azathioprine (C9H 7N 70 ZS) in the portion of Oral
USP Mercaptopurine RS Suspension taken:

Result =(rufrs) x (CslCu) x 100

tu = peak response from the Sample solution

Azathioprine Compounded Oral rs = peak response from the Standard solution
Suspension Cs = concentration of USPAzathioprine RS in the
Standard solution (mg/mL)
DEFINITION . Cu = nominal concentration of azathioprine in the
Azathioprine Compounded Oral Suspension contains NLT Sample solution (mg/mL)
90.0% and NMT 110.0% of the labeled amount of
azathioprine (C 9H 7N 70 ZS). Acceptance criteria: 90.0%-110.0%
Prepare Azathioprine Compounded Oral Suspension 50
SPECIFIC TESTS
mg/mL asfollows (see Pharmaceutical Compounding-
Nonsterile Preparations (795». • pH (791): 3.8-4.8
ADDITIONAL REQUIREMENTS
Azathioprine 5g • PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store at room temperature, or in a refrigerator.
Vehicle: a 1:1 mixture ofVehicle for Oral Solution, (regularor
sugar-free), NFand Vehicle for • BEYOND-USE DATE: NMT 60 days after the day on which it
Oral Suspension, NF, a sufficient quantity to make 100 mL was compounded when stored at room temperature, or in
a refrigerator
• LABELING: Label it to state that it is to be well shaken before
If using tablets, comminute them to a fine powder in a suitable
use, and to state the Beyond-Use Date.
mortar, or add Azathioprine powder to the mortar. Add
• USP REFERENCE STANDARDS (11)
about 10 mL of the Vehicle, and mix to a uniform paste. Add
USP Azathioprine RS
the Vehicle in small portions almost to volume, and mix
thoroughly after each addition. Transfer the contents of the
mortar, stepwise and quantitatively, to a calibrated bottle.
Add sufficient Vehicle to bring to final volume, and mix well.
[CAUTION-Avoid skin contact or inhalation of
azathioprine by using protective gloves and a fume hood
or surgical mask.]

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USP 43 Official Monographs / Azathioprine 441

Calculate the percentage of the labeled amount of

Azathioprine Tablets azathioprine (C9H 7N 70 2S) in the portion of Tablets taken:
DEFINITION
Azathioprine Tablets contain NLT 93.0% and NMT 107.0% of Result =(rulrs) x (CsICu) x 100
the labeled amount of azathioprine (C9 H7 N702S).
= peak response of azathioprine from the Sample
IDENTIFICATION solution
= peak response of azathioprine from the Standard
solution
= concentration of USP Azathioprine RS in the
Standard solution (mg/mL)
=nominal concentration of azathioprine in the
Sample solution (mg/mL)
Acceptance criteria: 93.0%-107.0%
PERFORMANCETESTS
s
obtained in the Assay. A
ASSAY • DISSOLUTION (711)
Medium: Water; 900 mL
Apparatus 2: 50 rpm
Change to read: _
Time: 30 min
• PROCEDURE Standard solution: USPAzathioprineRS in Medium
Mobile phase: Dissolve 1.1 9 of sodium 1-heptanesulfonate Samp.le. solutions:;,t;Passportions' oHhe solUtion ljnde'r test
in 700 mL of water, and add 300 mL of methanol. Adjust through a suitable A(USP l-May.2020) filter and dilute with .
the solution with 1 N hydrochloric acid to a pH of 3.5. Medium, if necessary, to a concentration similar to that of
A
A (USP1-May-2020)
-
. the Standard solution.
Standard stock solution: 0.5 mg/mL of USPAzathioprine Instrumental conditions
RS prepared as follows. Transfer USP Azathioprine RS to a Mode: UV
suitable volumetric flask. Add methanol equivalent to 30% Analytical wavelength: Maximum absorbance at about
of the flask volume and ammonium hydroxide equivalent 280 nm
to 1% of the flask volume, swirl, and sonicate for 2 min. r
Dilute with methanol to volume.
Standard solution: 0.1 mg/mL of USP Azathioprine RS in
water prepared ,Afrom the Standard stoCk
solution A (USP1-May-2020) .. _......
Sample stock solution: Nominally 0.5 mg/mL of Result,.; (A~/As) x Csx Vxp x.~1{[rxl0()
azathioprine prepared as follows.· A . .
Tabletsandtransfef rtiono' Au ~?pbsorbance ofthe5Clmples91ution
volumetric flask.- As = absorbance of the Standard solution
flask . -and Cs -,,' ," _ . 'tliOprinein the Standard
of blu"
with _, hanoi to·volume. Allow t V
settle. A(USP 1-May-2020) . D
Sample solution: Nominally 0.1 m
Afrom the Ie stock solutio L
the solutio ugh a suitabl
size.A (USP ·1- -2020) Tolerances: NLT 75% (Q) of the labeled amount of
Chromatographic system azathioprine (C9H 7N 70 2S) is dissolved.
(See Chromatography (621), System Suitability.) • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Mode: LC requirements
Detector: UV 254 nm. A' IMPURITIES
array detector in the i:
Column: 4-mm x 30-cm;
Ll
Flow rate: 2 mL/min
Injection volume: 10 IJL
System suitability
Sample: Standard solution
Suitability requirements
A'A CUSP 1.M~y~2020)
Tailing factor: NMT 1.5
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution

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442 Azathioprine / OfficialMonographs USP 43

A RS, and chloromethylnitrolmida oleprepar table 1: (continued)

Transfer a suitable amount·ofUSP . ri . Relative
suitablevolumetric flask. Add me .i Retention
of the flask volume andsonicafe t . .Ive. Add.. . Ie Time
amount of the System suitability stock solution and djfut~ . 0.7~
with Mobile phase to yolume;
Standard stock solutio . Chloromethylnttroiirii7
daiol.!=a,c
RS prepared as follows
Azathioprine RS to a }\zathi6prine 1.0
methanolequivalen
to dissolve, and dilu
Standard solution:
Mobile phasefromt
Total degrad~tion prod~
Sensitivitysolution: ~.. m ucts
in Mobilephase from the Stand
Sample solution: Nominally O.
prepared as follows. sfe'
NLT 20 powdered T 15,
Add 20% of the flask volume 0 0
min with intermittent shakin
phase to volume. sapo
suitablefilter 0 IJrn· pore
ADDITIONAL REQUIREMENTS
Chromatographic system
(See Chromatography·(621), System Jui,tability) Change tOl'ead
Mode: LC • PACKAGING AND STORAGE: . Protectfrom light. ."Stq:r~at
Detector:' UV 220 nm controlled room. temperature.'i (U~Pl~MaY~2040)
Column: 3.9 mm x30tmi 10-lJm packing L1
Column temperature: 30°
Flowrate: 1.1.mL/min
Injection volume: 25 IJL • USP REFERENCE STANDARDS (11)
System suitability . . ... .. .. '. . USP Azathioprine RS
Samples: Syst~m SUitability solution, Standard solution, and . . us .... .S
Sensitivitysolution ,1-
Suitability req .
Resolution:
compound Aand ri
chloromethylnitroimidazole and aza
suitability solution ...
Relativestandard deviation: NMT5.0%, Standard
solution
Signal-to-noise ratio: NLT10, Sensitivity solution
Analysis Azathioprine Sodium for Injection
Samples: Standard s
Calculate the perce radation DEFINITION
product in the po Azathioprine Sodium for Injection isa sterile solid prepared by
the freeze-drying of an aqueous solution of Azathioprine and
Result = .(r~/rs)' x (CslCu)x 100 SodiumHydroxide. ItcontainsNLT 93.0% and NMT 107.0%
of the labeled amount of azathioprine (C9H 7N 70 zS).
=peak respo e chdegradation ,product IDENTIFICATION
from theS u·
• A. The principal spot from the Sample solution shows the
= peak response a prine fr:om the Standard same R F value as that obtained from Standard solution A in
solution the test for Limit of Mercaptopurine.
= concentration of USP Azathioprine~S inthe • B. The retention time of the major peak of the Sample
Standard solution (m L) solution corresponds to that of the Standard solution, as
=nominal concentrati f azathioprine Inthe obtained in the Assay.
Sample solution(mg/
ASSAY
Acceptance criteria: .See Table 1. • PROCEDURE
Solution A: 1.6 giL of sodium 1-heptanesulfonatein water
l'abl"e 1 Mobile phase: Methanol and Solution A (30:70).Adjust with
1 N hydrochloric acid to a pH of 3.5 ± 0.1.
Reten Standard stock solution: 0.5 mg/mL of USP Azathioprine
Name Time RS prepared as follows. Transfer USP Azathioprine RS to a
Azathioprine related
suitable volumetric flask. Add 25% of the flask volume of
compoundN OA7 methanol and 1% of ammonium hydroxide to the flask,
swirl, and sonicate for 2 min or until dissolved. Dilute with
Merc;aptopurine 0.52· 0:5 methanol to volume.

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USP 43 OfficialMonographs / Azelastine 443

Standard solution: 0.1 mg/mL of USP Azathioprine RS in SPECIFIC TESTS

water from the Standard stock solution • pH (791)
Sample solution: Nominally equivalent to 0.1 mg/mL of Sample solution: The contents of one container dissolved
azathioprine in water from Azathioprine Sodium for in 10 mL of water
Injection Acceptance criteria: 9.8-11.0
Chromatographic system • BACTERIAL ENDOTOXINS TEST(85): It contains NMT 1.0 USP
(See Chromatography (621), System Suitability.) Endotoxin Unit/mg of azathioprine.
Mode: LC • WATER DETERMINATION (921), MethodI: NMT 7.0%, when
Detector: UV 254 nm the Test Preparation is prepared asdirected for a
Column: 3.9-mm x 30-cm; 10-l.Im packing L1 hygroscopic specimen
Flow rate: 1.8 mL/min • OTHER REQUIREMENTS: It meets the requirements in
Injection volume: 10 I.IL Injections and ImplantedDrug Products (1).
System suitability
ADDITIONAL REQUIREMENTS
Sample: Standard solution
Suitability requirements • PACKAGING AND STORAGE: Preserve as described in
Tailing factor: NMT 2 Packaging and Storage Requirements (659), Injection
Relative standard deviation: NMT 1.0% Packaging, Packaging for constitution, at controlled room
Analysis temperature.
Samples: Standard solution and Sample solution • USP REFERENCE STANDARDS (11)
Calculate the percentage of the labeled amount of USP Azathioprine RS
azathioprine (C9H 7N 70zS) in the portion of Azathioprine USP Mercaptopurine RS
Sodium for Injection taken:

Result = (r ir s) x (C siC u) x 100

= peak response of azathioprine from the Sample

Azelastlne Hydrochloride
solution
= peak response of azathioprine from the Standard
solution
= concentration of USP Azathioprine RS in the • HCl
Standard solution (mg/mL)
= nominal concentration of azathioprine in the
Sample soiution (mg/mL)

Acceptance criteria: 93.0%-107.0% CzzHz4CIN30· HCI 418.36

1(2H)-Phthalazinone, 4-[( 4-chlorophenyl)methyl]-2-
PERFORMANCE TESTS (hexahydro-1-methyl-l H-azepin-4-yl), monohydrochloride;
• UNIFORMITY OF DOSAGE UNITS (905): Meets the 4-(p-Chlorobenzyl)-2-(hexahydro-l-methyl-1 H-azepin-4-yl)-
requirements 1(2H)-phthalazinone monohydrochloride [79307-93-0].
IMPURITIES DEFINITION
• LIMIT OF MERCAPTOPURINE Azelastine Hydrochloride contains NLT 99.0% and NMT
Standard solution A: 10 mg/mL of USP Azathioprine RS in 101.0% of azelastine hydrochloride (CzzHz4CIN30 . HCI),
dimethylformamide
calculated on the dried basis.
Standard solution B: 100 I.Ig/mL of USP Mercaptopurine RS
in dimethylformamide IDENTIFICATION
Sample solution: 10 mg/mL of Azathioprine Sodium for.
Injection in dimethylformamide
Chromatographic system
(See Chromatography (621), Thin-Layer Chromatography.) • A.~S
Mode: TLC $p~9te9$ )
Adsorbent: 0.25-mm layer of microcrystalline cellulose • B. The retention time of the major peak of the Sample
Application volume: 5 I.IL for Standard solution A and the solution corresponds to that of the Identification solution, as
Sample solution, and 15 I.IL for Standardsolution B obtained in the test for Organic Impurities.
Developing solvent system: Butyl alcohol saturated with • C. IDENTIFICATION TESTS-GENERAL (191), Chloride: Meets
5 N ammonium hydroxide the requirements
Analysis
Samples: Standard solution A, Standard solution B, and ASSAY
Sample solution • PROCEDURE
Proceed as directed in the chapter. Apply the Samples at Sample: 300 mg
points 2 cm from the bottom edge of a TLC plate. Allow Blank: 5 mL of anhydrous formic acid and 30 mL of acetic
the spots to dry, and develop the chromatogram in a anhydride
suitable chamber until the solvent front has moved 15 cm Titrimetric system
from the point of application. Remove the plate, air-dry, (See Titrimetry (541).)
and locate the spots by viewing under short- and Mode: Direct titration
long-wavelength UV light. Titrant: 0.1 N perchloric acid VS
Acceptance criteria: 3.0%; any spot from the Sample Endpoint detection: Potentiometric
solution, other than the principal spot, is not more intense Analysis: In order to avoid overheating in the reaction
than the spot from Standard solution B. medium, mix thoroughly throughout and stop the titration
immediately after the endpoint has been reached.

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444 Azelastine / OfficialMonographs USP 43

Dissolve the Sample in 5 mL of anhydrous formic acid, and = peak area of each impurityfrom the Sample
add 30 mL of acetic anhydride. Titrate with Titrant. solution
Calculatethe percentage of azelastine hydrochloride = peak area of azelastinefrom the Standardsolution
(CzzHz4CIN30 . HCI) in the portion of Azelastine = concentration of USP Azelastine Hydrochloride RS
Hydrochloride taken: in the Standard solution (mg/mL)
= concentration of Azelastine Hydrochloride in the
Result = {[(Vs- VB) x N x AIWl x 100 Sample solution (mg/mL)
F = relative response factor (see Table 7)
VS = Titrant volume consumed by the Sample (mL)
VB = Titrant volume consumed by the Blank (mL) Acceptance criteria: See Table 7. [NOTE-Disregard peaks
N = actual normality of the Titrant (mEq/mL) that are less than 0.05% of the azelastine peak.]
F = equivalency factor, 41.84 mg/mEq
W = weight of the Sample (mg) Table 1
Relative Relative Acceptance
Acceptance criteria: 99.0%-101.0% on the dried basis Retention Response Criteria,
Name Time Factor NMT (%)
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.2% Benzohydrazide 0.2 0.38 0.1
• ORGANIC IMPURITIES Azelastine related com-
Dilute phosphoric acid; 115 giL of phosphoric acid in pound Ba 0.3 0.22 0.1
water Chlorophenylacetyl-
Buffer: 2.92 giL of octanesulfonicacid sodium salt and 0.92 benzoic acld'' 0.4 0.45 0.1
giL of monobasic potassium phosphate in water. Adjust
with Dilutephosphoric acid to a pH of 3.0-3.1. Azelastine related com-
pound DC 0.6 1.2 0.1
Mobile phase: Acetonitrile and Buffer (260:740)
Diluent: Acetonitrile and water (45:55) Azelastine 1.0 1.0 -
Identification solution: 2.5 mg/mL of USP Azelastine Azelastinerelated com-
Hydrochloride RS in Diluent. [Nora-This solution isused for pound Ed 1.4 0.48 0.1
Identification test B.]
System suitability stock solution: 0.5 mg/mL each of USP Anyindividual
unspecified -
Azelastine Related Compound BRS, USP Azelastine Related impurity 1.0 0.10
Compound DRS,and USP Azelastine Related Compound E
RS in acetonitrile Total impurities - - 0.2
System suitability solution: 50 ~g/mL each of USP
Azelastine Related Compound BRS, USP Azelastine Related a N'-(1-Methylazepan-4-yl)benzohydrazide; also known as 1-Benzoyl-2-[(4RS)-
1-methylhexahydro-l H-azepin-4-yl]diazane.
Compound DRS, and USP Azelastine Related Compound E b 2-[2-(4-Chlorophenyl)acetyl]benzoic acid.
RS from the System suitability stock solution and 2.5 mg/mL c 4-(4-Chforobenzyl)phthalazin-l (2H)-one.
of USP Azelastine Hydrochloride RS in Diluent d 3-(4-Chlorobenzylidene)isobenzofuran-l (3H)-one.
Standard stock solution: 50 ~g/mL of USP Azelastine
Hydrochloride RS in acetonitrile SPECIFIC TESTS
Standard solution: 2.5 ~g/mL of USP Azelastine • Loss ON DRYING (731)
Hydrochloride RS in Diluent . Analysis: Dryat 105° to constant weight.
Sample solution: 2.5 mg/mL of Azelastine Hydrochloride in Acceptance criteria: NMT 1.0%
Diluent • ACIDITY OR ALKALINITY
Chromatographic system Sample solution: 10 mg/mL of Azelastine Hydrochloride
(See Chromatography (621), System Suitability.) in water
Mode: LC Analysis: Add 0.2 mL of bromothymol blue TS to 10 mLof
Detector: UV 210 nm the Sample solution.
Column: 4.6-mm x 25-cm; 1O-~m packing L10 Acceptance criteria: NMT 0.1 mLof 0.01 Mhydrochloric
Column temperature: 30° acid or 0.01 M sodium hydroxide is required to produce a
Flow rate: 2 mL/min color change.
Injection volume: 10 ~L
Run time: 2.4 times the retention time of azelastine ADDITIONAL REQUIREMENTS
System suitability • PACKAGING AND STORAGE: Preserve in well-closed
Samples: System suitability solution and Standard solution containers. Protect from light and moisture. Store at
Suitability requirements controlled room temperature.
Resolution: NLT 4.0 between azelastinerelated • USP REFERENCE STANDARDS (11)
compound Band azelastine related compound D; NLT USP Azelastine Hydrochloride RS
1.5 between azelastine related compound D and USP Azelastine Related Compound B RS
azelastine; NLT 1.5 between azelastineand azelastine N'-(1-Methylazepan-4-yl)benzohydrazide.
related compound E, System suitability solution C'4Hz,N30 247.34
Relative standard deviation: NMT 5.0%, Standard USP Az.elastine Related Compound D RS
solution 4-(4-Chlorobenzyl)phthalazin-l (2H)-one.
Analysis C,sH llCIN zO 270.71
Samples: Identificationsolution, Standardsolution, and USP Azelastine Related Compound ERS .
Sample solution 3-(4-Chlorobenzylidene)isobenzofuran-l (3H)-one.
Calculatethe percentage of each impurityin the portion of C,sH 9CIOz . 256.68
Azelastine Hydrochloride taken:
Result = (r vir s) x (C siC u) x (1IF) x 100

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USP 43 Official Monographs / Azelastine 445

Relative standarddeviation:l\JMt~2.0%,' Stanc/ard

.solution· . . . _. . .. . - -
is

Ta61el
Tillle } SolLltionA $q:liitl(i[i~
(n:do) (0/9) . (Wo)
o j~()(j 0
20 85 IS,
e e
35 tS.5 3~ n.]
45 45 ,5~ tanaaraCJe\llatioll.:.·NMt~.Q%~.st(il'ldiiiii

60 45 $5 ;'Iloise.ratio: :. NLt"'0, SensitivitY':~QIi.itiQri

Result;:;:. (r vi r~)x (CsfCu) x (l/Ffx:,J OQ

~ ~

'~s :~~

Cv =
F
Accepfancecriteria; $ee" fabfe·2,
Tilb'e'2

~ame

Benzobydrazidea
~elastine relatedcompound aa,b 0.1.7

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446 Azelastine / Official Monographs USP 43

(continued) 1-0xa-6-azacyclopentadecan-15-one, 13-[(2,6-dideoxy-3.-C-

methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-
Relatl~ 3,4,10-trihydroxy-3,5;6,8,lO,12,14-heptamethyl-ll-
Retention [[3,4,6-trideoxy-3-(dimethylamino)-~-D-xylo­
Name , Time hexopyranosyl]oxy]-, [2R-(2R*,35*,4R*,5R*,8R*, 1OR*,ll R*,
~hlol"()ph~nyJac~tylbenzoic 125* 135* 14R*)]'
acida• c (2R,35,4R,5'R,8R,16R,11 R,12S, 135,14R)-13-[(2,6-Dideoxy-3-
C-methyl-3-O-methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-
AZelastine re.lateclcompound ()d b.7 1.20 0.5 3,4,10-trihydroxy-3,5,6,8, 10,12,14-heptamethyl-l1-
[[3,4,6-trideoxy':'3-(dimethylamino)-~-D-xylo­
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one;
9-Deoxo-9a-aza-9a-methyl-9a-homoerythromycin A
Anhydrous [83905-01-5].
Monohydrate [121470-24-4].
Dihydrate [11 7772-70-0].
DEFINITION
Azithromycin is anhydrous or contains 1 or 2 molecules of
water of hydration. It contains the equivalent of NLT 945
~g/mg and NMT 1030 ~g/mg of azithromycin
(C3sHnN2012), calculated on the anhydrous basis.
IDENTIFICATION

• A· . . ~i~~~~tij~~~~i~~~2!.yyy.; y(
5pectroscopy;197.K.1.(cN Ifa r1itt.......nr..
• 0):
the IR spectra of the analyte and the Standard,
equal portions of the test specimen and the USP Reference
Standard in equal volumes of methanol. Evaporate the
solutions to dryness on a water bath, and dry at 80 for 30
0

min under vacuum. Perform the test on the residues.

• B. The retention time of the azithromycin peak of the
Sample solution corresponds to that of the Standard
solution, as obtained in the Assay.
ASSAY
• PROCEDURE
Solution A: 10M potassium hydroxide
Solution B: 6.7 giL of dibasic potassium phosphate,
adjusted with Solution A to a pH of 11.0
Solution C: 6.7 giL of dibasic potassium phosphate,
adjusted with phosphoric acid to a pH of 8.0
Mobile phase: Acetonitrile and Solution B (60:40)
Diluent: Acetonitrile and Solution C (60:40)
System suitability solution: 0.5 mg/mL each of USP
Azithromycin RS and USP Azaerythromycin A RS prepared
as follows. Dissolve USP Azithromycin RS and USP
Azithromycin Azaerythromycin A RS first in acetonitrile, using 5% of the
final volume, and then dilute with Diluent to volume.
Standard solution: 0.53 mg/mL of USP Azithromycin RS
prepared as follows. Dissolve USP Azithromycin RS first in
acetonitrile, using 2% of the final volume, and then dilute
with Diluent to volume.
Sample solution: 0.53 mg/mL of Azithromycin prepared as
• xH,O
follows. Dissolve Azithromycin firstin acetonitrile, using 2%
of the final volume, and then dilute with Diluent to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 210 nm
C3sH72N2012 748.98 Column: 4.6-mm x 25-cm; 5-~m packing L67
C3sH72N2012 . H20 767.00 Column temperature: 40 0

Flow rate: 1 mL/min

C3sH72N2012 . 2H20 785.02 Injection volume: 10 ~L
System suitability
Samples: System suitability solution and Standard solution

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USP 43 Official Monographs / Azithromycin 447

[NOTE-The relative retention times for

azaerythromycin A and azithromycin are 0.7 and 1.0,
respectively.]
Suitability requirements
Resolution: NLT 3.0 between azaerythromycin A and
azithromycin, System suitability solution
Tailing factor: 0.8-1.5 for azithromycin, Standard
solution
Relative standard deviation: NMT 1.10% for
azithromycin, Standard solution ~~nazithromydn ~nd
Analysis
Samples: Standard solution and Sample solution ·rai:lthr()mycin; NMI2...5 ·fqr
Calculate the quantity, in ~g, ofazithromycin (C38HnN2012)
in each milligram of Azithromycin taken: 10.()% fQr
Result = (rulrs) x (CsICu) x P
ru = peak response from the Sample solution
ts = peak responsefrom the Standard solution
Cs = concentration of USP Azithromycin RS in the
Standard solution
Cu = concentration of Azithromycin in the Sample
solution
P = potency of USP Azithromycin RS (~g/mg of
azithromycin)
Acceptance criteria: 945-1 030 ~g/mg on the anhydrous ResuIF=(rdrs) x (CsICu) xF xl 00
basis
tl)e relevantanalyte from tneSample
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.3%, moistening the
charred residue with 2 mL of nitric acid and 5 drops of
sulfuric acid nce

cJ
iF ;;C:onversfi>rdactor,'O.OOf mg/pg
Calculate r relatedsubstances inthe
portion,o
R~sylt='(rulrs) x (CslCu.}x Fx.l 00
ru h'additionalJmpurity from the
f s z i t h r o m y c i n peak from the
Cs Azithr()myc:in RS in the
L
Cu lesolution(mg/mL)
F ~conversionfactor,O:OO"mg/~g

Acceptancecriteria: SeeTable 7~
Tablet
Relative' Acceptance
Retention Criteria,
Name Time NMT(%)

EiYthromycinAimlnoethe~ 0.19 0.5

Desosamjnyl~ithrorTiyclnQ 0:29 0.3
037 0.5
N-Oemethylazithromycind 0.49 0]

0.80 1.0
1~0

233 1.0

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 448 Azithromycin / OfficialMonographs USP 43

Suitability requirements
Peak-to-valley ratio: NLT 1.4, System suitability solution
Calculate the peak-to-valley ratio:
Result = HplH v

H, = height above the baseline of the

desosaminylazithromycin peak
Hv = height above the baseline of the lowest point of
the curve separating the
desosaminylazithromycin and azithromycin
related compound F peaks
Tailing factor: 0.8-1 .5, Standardsolution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of each impurity in the portion of
Azithromycin taken:
Result = (r ulrs) x (CsiCu) x P X F7 x (1001F2 )
= peak response of each impurity from the Sample
solution
=peak response of azlthromycln from the Standard
solution
= concentration of USP Azithromycin RS in the
Standardsolution (mg/mL)
• ORGANIC IMPURITIES =concentration of Azithromycin in the Sample
~(~~(Q$~;1:~~~~·~~i~) solution (mg/mL)
Solution A: 1.8 mg/mL of anhydrous dibasic sodium P = potency of USPAzithromycin RS (lJg/mg of
phosphate in water. Adjust with 1 N sodium hydroxide or azithromycin)
10% phosphoric acid to a pH of 8.9. =conversion factor, 0.001 mg/lJg
Solution B: Acetonitrile and methanol (3:1) = relative response factor (see Table 2)
Solution C: 1.73 mg/mL of monobasic ammonium
phosphate. Adjust with ammonia TSto a pH of 10.0 ± 0.05. Acceptance criteria: See Table 2. Disregard peaks eluting
Solution D: Methanol, acetonitrile, and Solution C (7:6:7) before azithromycin N-oxide and after
Mobile phase: See Table 1. 3-deoxyazithromycin (azithromycin B). Disregard peaks
with a response less than 0.1 times the response of the
Table 1 azithromycin peak in the Standardsolution (0.1 %).
Time Solution A Solution B SPECIFIC TESTS
(min) (%) (%) • OPTICAL ROTATION (781 S), Procedures, Specific Rotation
Sample solution: 20 mg/mL in dehydrated alcohol
0 50 50
Acceptance criteria: -45° to -49°, at 20°
25 45 55 • CRYSTALLINITY (695): Meets the requirements, except
30 60
where it is labeled as amorphous, most of the particles do
40
not exhibit birefringence and extinction positions
80 25 75 • pH (791) .
81 50 50 Sample stocksolution: 4 mg/mL in methanol
Sample solution: 2 mg/mL obtained by mixing equal
93 50 50 volumes of Sample stock solution and water
Acceptance criteria: 9.0-11.0
System suitability solution: 0.0165 mg/mL of USP • WATER DETERMINATION (921), Method I
Azithromycin Related Compound F RS and 0.027 mg/mL Where it is labeled as anhydrous: NMT 2.0%
of USP Desosaminylazithromycin RS in Solution D Where it is labeled as the dihydrate: 4.00/0-5.0%
Standard solution: 86 IJg/mL of USP Azithromycin RS in Where it is labeled as the monohydrate: 1.80/0-4.0%,
Solution D except that it may be 4.00/0-6.5% when the requirements
Sample solution: 8.6 mg/mL of Azithromycin in Solution D of the Loss on Drying test are met,
Chromatographic system • Loss ON DRYING: Where it is labeled as Azithromycin
(See Chromatography (621), System Suitability.) monohydrate and has a water content of 4.0%-6.5% (see
Mode: LC Thermal Analysis (891) ,
Detector: UV 210 nm [NoTE-The quantity taken for this procedure may be
Column: 4.6-mm x 25-cm; 5-lJm packing L1 adjusted, if necessary, for instrument sensitivity.]
Column temperature: 60° Analysis: Determine the percentage of volatile substances
Flow rate: 1 mL/min by thermogravimetric analysis in an appropriately
Injection volume: 50 IJl calibrated instrument, using about 10 mg of Azithromycin.
.> System suitability Heat the specimen at the rate of 10°Imin between ambient
Samples: System sUitabilitysolution and Standard solution temperature and 150° in an atmosphere of nitrogen at a
constant flow rate of about 35 mL/min. From the
thermogram plot the derivatives of the loss ondrylnq

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 USP 43 Official Monographs / Azithromycin 449

Table 2 (Azithromvcin)
Relative Relative Acceptance
Retention Response Criteria,
Name Time Factor NMT(%)
Azithromycin N-oxide a 0.Z9 0.43 0.5
3'~("';~."';~~i~~methyl)-3'-N-formylazithromycinb,~~l'
(UsrJ~May,~OI9) 0.37 1.7 0.5
,,..;
"'1
'C

.", ,oJ '."_I1'_'\.!I::LIII::I 0;42 no ():~~~tis~"ijSM~i0iijj;j

3'-(N,N-Didemethyl)azithromycin(aminoazithromy.
cin)e 0.43 1.0 0.5
Azithromycin related compound Fe, f 0.51 3.8 0.5
Desosaminylazithromycin 9 0.54 1.0 0.3
3'-N-{[4-(Acetylamino)phenyl]sulfonyl}-3',3'-dideme-
thylazlthrornycln" 0.55 1Z 0.15
N-Demethylazithromycini 0.61 1.0 0.7
~~rYt"roI"O.Yc:irliA()xrl'l1el 0.64 1.0 i;':5S "<

Azithromycin C (3"-O-demethylazithromycin)k 0.73 1.0 0.5

3'-De(dimethylamino)-3'-oxoazlthromycln' 0.76 1.5 0.5
3'-N-{[4-(Acetylamino)phenyl]sulfonyl}-3'-demethyla-
zlthrornycln'" 0.79 10 O.S
Azaerythromycin An 0.83 1.0 O.S
Azithromycin impurity po 0.9Z 1.0 O.Z
Azithromycin 1.0 - -
Z-Desethyl-Z-propylazithromycin P 1.23 1.0 O.S
3'-N-Demethyl-3'-N-[(4-methylphenyl)sulfonyl]azi-
thromydns I 1.Z6 5 0.5
3.Deoxyazithromycin (azithromycin By 1.31 1.0 1.0
Any individual, unidentified impurity - 1.0 O.Z
Total impurities - - 3.0

a (ZR,3S,4R,SR,8R,lOR,ll R,lZS, 13S,14R)-13-[(Z,6-Dideoxy-3-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4,10-trihydroxy-3,5,6,8,l O,lZ,14-

heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylazinoyl)-~-o-xy/o-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
b (ZR, 3S,4R,SR,8R,l OR,ll R,1ZS,13S,14R)-13-[(Z,6-Dideoxy-~-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4, 10-trihydroxy-3,5,6,8, 1O,lZ,14-
heptamethyl-11-[[3-formamido-3,4,6-trideoxy-~-o-xy/o-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.

C The.syste.m rnayr~s~lv~ f:\.v~ . r~t~I11~~~~> zclt)~~-~~!t.~;t\Al~r~t~~.;rs·~~~$~ij·~~y·~gI~)}> . . . . .••.. . "'»"'/c/ C

d(3Rj4R,.?$,Q.B,~~{J9~j1>1.si1~~I~E~~iWS:~,·' .1 •... 'Prrxr'f'3!,,~x~pyral'l8syl]p"'Y]S§'7thylf~,SSC!j9ygr'PxYfl()-r(2i6fg ide9Jiy..30C-

methyl"3"Q7IT1etbyl.;~+ribOfbexopYril!JC)syl) joxa~2~i:l,Zal:>igy!=IC)[lJ.Z.l ]hexa<:fec,;J~~I"l"a-c)rle.
e (ZR,3S,4R,SR,8R,l OR,ll R,lZS, 13S,14R)-13-[(Z,6-Dideoxy-3-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4,l 0-trihydroxy-3,5,6,8,10,12,14-
heptamethyl-11-[[3-amino-3,4,6-trideoxy-~-o-xy/o-hexopyranosyl]oxy]-1-oxa-6.aza!=yclopentadecan-15-one.
f 3'-N-Demethyl-3'-N-formylazithromycin; (ZR,3S,4R,5R,8R,lOR, 11R,lZS,13S,14R)-13-[(Z,6-Dideoxy-3-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-
ethyl-3,4,l 0-trihydroxy-3,5,6,8,1O,lZ,14-heptamethyl-11.[[3-(N-methyl)formamido-3,4,6-trideoxy-p-o-xy/o-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-
15-one.
9 (ZR,3S,4R,5R,8R,1OR,ll R,lZS,13S, 14R)-Z-Ethyl-3,4,l 0,13-tetrahydroxy-3,5,6,8,10, 1Z,14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethylamino-~-D-xy/O­
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
h (ZR,3S,4R,5R,8R,1OR,ll R,lZS,13S,14R)-13-[(Z,6-Dideoxy-3-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4,10-trihydroxy-3,5,6,8,1O,lZ,14-
heptamethyl-11-[[3-[ N-(4-acetamidophenylsulfonyl)amino]-3,4,6-trideoxy-~-o-xy/o-hexopyranosyl]oxy ]-1-oxa-6-azacyclopentadecan-1S-one.
i (ZR,3S,4R,SR,8R,l OR, 11R,1ZS,13S,14R)-13-[(Z,6-Dideoxy-3-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4,l 0-trihydroxy-3,5,6,8,10,1Z,14-
h7pt~rn~t9yl.s11. :[[~,4.!§.;tri~~8"'Ys3;~;~bx,!~~ino-p-o-x /o-hexopyranos I ox -1_.;8~~;~-a~~~Y518~~p~~?~~an.s1. ~:~p~:
l.(?61~~;?§;9~i7B,.Q.~ill§,l~I3~1~~'cl;~~;@7~7[[~; .):~:I:?:*¥l9:.I"i~~8PX.r8n~sXI]g~yJT~~:~tH~I~~fi~;i~7tHb~aI'9~Y"4~[(g;~2aia~§*Y2~£C2f
lT1ethyl-,3~Q,methyl-a,l-ripo-hel(6pyriln()sy1)()l(Y ;"<,f •Y. r()~. .. ,.l~ffJ~/<~m~~l)yl()><a~y~l()t~tril<:f~@nf-?f()I"I~;
k (ZR,3S,4R,SR,8R,1OR,ll R,lZS, 13S,14R)-13-[(Z,6-Dideoxy-3-C-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4,10-trihydroxy-3,S,6,8,1O,lZ,14-heptamethyl-11-
[[3,4,6-trideoxy-3-dimethylamino-p-o-xy/o-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-1S-one.
I (ZR,3S,4R,5R,8R,l OR,ll R,1ZS,13S, 14R)-13-[(Z,6-Dideoxy-3,3-dimethyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4,l 0-trihydroxy-3,S,6,8,10,1Z,14-heptamethyl-
11-[[3,4,6-trideoxy-3-oxo-p-o-xy/o-hexopyranosyl]oxYJ-1-oxa-6-azacyclopentadecan-1S-one.
m (ZR,3S,4R,5R,8R,l OR,ll R,1ZS,13S, 14R)-13-[(Z,6-Dideoxy-3-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4,10-trihydroxy-3,5,6,8,l O,lZ,14-
heptamethyl-11-[[3-[N-(4-acetamidophenylsulfonyl)-N-methylamino]-3,4,6-trideoxy-p-o"xy/o-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-1S-one.
n 9-Deoxo-9a-aza-9a-homoerythromycin A; 6-Demethylazithromycin.
o Specifiedunidentified impurity.
p (ZR,3S,4R,SR,8R,1OR,ll R,lZS,13S,14R)-13-[(Z,6-Dideoxy-3-C-methyl.3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-propyl-3,4,l 0-trihydroxy-3,5,6,8,l 0,lZ,14-
heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-p-D-Xy/o-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
q (ZR,3S,4R,5R,8R,1OR,ll R,lZS, 13S,14R)-13-[(Z,6-Dideoxy-3-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy]-Z-ethyl-3,4,l O-trihydroxy-3,5,6,8,l O,lZ,14-
heptamethyl-11-[[3-[N-(4-methylphenylsulfonyl)-N-methylamino]-3,4,6-trideoxy-p-D-xy/o-hexopyranosyl]oxy]-1-oxa-6-azacydopentadecan-15-one.
r (ZR,3 R,4S,SR,8R,1OR, 11R,1ZS,13S,14R)·13-[(Z,6-Dideoxy-3-C-methyl-3-0-methyl-a-l-ribo-hexopyranosyl)oxy].2-ethyl-4,l 0-dihydroxy-3,5,6,8,l 0,lZ,14-
heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-P-D-xy/o-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.

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 450 Azithromycin / OfficialMonographs USP 43

(percentage loss/min), and identifythe inflection points of Standard stock solution: 0.165 mg/mL of USP
the two weight loss steps atabout 70° and 130°. Azithromycin RS in acetonitrile. Swirl, and sonicate as
Acceptance criteria: NMT 4.5% between ambient necessary. ..
temperature and the inflection point at about 70°, and Standard solution: 3.3 J..Ig/mL of USP Azlthrornycin RS
1.80/0-2.6% between the inflection point at about 70° and from the Standard stock solution in Mobile phase
the inflection point at about 130°. System suitability stock solution: 0.16 mg/mL of USP
Azaerythromycin A RS in acetonitrileand Mobile phase
ADDITIONAL REQUIREMENTS (1 :9). Dissolve first in a~etonitril~, using 1~% of ~he final.
• PACKAGING AND STORAGE: Preserve in tight containers.
volume. Swirl, and sonicate to dissolve. Dilute With MobIle
phase to volume. .
System suitability solution: 3.2 J..Ig/mL of azaerythrornycin
• LABELING: Label it to indicate whether it is anhydrous, the Afrom the System suitability stock solution an~ 3.~ J..Ig/m.L
monohydrate, or the dihydrate. The amorphous form is so of azithromycinfrom the Standard stock solution In Mobtle
labeled. Where the quantity of azithromycin is indicated in phase
Sample stock solution: Remove, as completely as possible,
the labeling of any preparation containing Azithromycin, the contents of NLT 20 Capsules. Prepare a l-mg/mL
this shall be understood to b~Hi.~;~;~~~i;;~f;:.~hydrous solution of anhydrous azithromycin in acetonitrile. Dissolve
azithromycin (C3sH72N2012).~",,;(U§P/l,M~Y?~Ol~) a portion of the mixed.C~psule contents first in 70~ of the
final volume of acetonitrile, and shake by mechanical
means for 30 min. Dilute with acetonitrileto volume. Place
40 mL of the resultingsuspension in a centrifuge tube, and
• USP REFERENCE STANDARDS (11) centrifuge. Usethe supernatant to prepare the Sample
USP Azaerythromycin A RS solution.
9-Deoxo-9a-aza-9a-homoerythromycin Ai Sample solution: 3.2 J..Ig/mL of azithromycin from the
6-Demethylazithromycin. Sample stock solution in Mobile phase
C37H70N2012 734.96 Chromatographic system
USP Azithromycin RS (See Chromatography (621), System Suitability.)
USP Azithromycin Related Compound F RS Mode: LC
3'-N-Demethyl-3'-N-formylazithromycini Detector: Amperometric electrochemical detector
(2R,3S,4R,5R,8R,1OR,ll R,12S,13S,14R)-13-[(2,6- Electrode: Dual glassy carbon electrodes
Dideoxy-3-C-methyl-3-O-methyl-a-L-ribo- Mode: Oxidativescreen mode
hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy- Electrode 1: +0.70 ± 0.05 V
3,5,6,8,10,12,14-heptamethyl-l1-[[3-(N-methyl) Electrode 2: +0.82 ± 0.05 V
formamido-3,4,6-trideoxY-~-D-xylo-hexopyranosyl] Background current: 85 ± 15 nanoampheres
oxy]-1-oxa-6-azacydopentadecan-15-one. Columns
C3sH7oN20n 762.97 Guard: 4.6-mm x 5-cmi 5-J..Im packing L29
'~
. .·..•.•.S;A(USP1·M~y-;g()19)
.i;. ii.;;"'./.;?·';;;;·
Analytical: 4.6-mm x 15-cmi 5-J..Im packing L29 or 3-J..Im
USP Desosaminylazithromycin RS packing L49 without the guard column
(2R,3S,4R,5R,8R,l OR, 11 R,12S,13S,14R)-2-Ethyl- Flow rate: 1.5 mL/min
3,4,1O,13-tetrahydroxy-3,5,6,8,1O,12,14-heptamethyl- Injection size: 50 J..IL
11-[[3,4,6-trideoxy-3-dimethylamino-~-D-xylo­ System suitability
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15- Samples: Standard solution and System suitability solution
one. [NOTE"':'"The relative retention times for
C30HssN209 590.79 azaerythromycin Aand azithromycin with the L29
column are 0.7 and 1.0, respectively; the relative
retention times for azaerythromycin A and
azithromycin with the L49 column are 0.8 and 1.0,
respectively.]
Azithromycin Capsules Suitability requirements
Resolution: NLT 2.5 between azaerythromycin A and
DEFINITION azithromycin, System suitability solution
Azithromycin Capsules contain the equivalent of NLT 90.0% Column efficiency: NLT 1000 theoretical plates,
and NMT 110.0% of the labeled amount of azithromycin Standard solution
(C3sH72N2012)' Tailing factor: 0.9-1.5, Standard solution
IDENTIFICATION Relative standard deviation: NMT 2.0%, Standard
• A. The retention time of the azithromycin peak of the solution
Sample solution corresponds to that of the Standard Analysis
solution, as obtained in the Assay. Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
ASSAY azithromycin (C3sH72N2012) in the portion of Capsules
• PROCEDURE taken:
[NOTE-Use water that has a resistivity of NLT 18
Mohm-cm.] Result = (ru/rs) x (Cs/Cu) x P x F x 100
Mobile phase: Dissolve 5.8 g of monobasic potassium
phosphate in 2130 mL of water, and add 870 mLof = peak response from the Sample solution
acetonitrile. Adjustwith about 6 mLof 10 N potassium = peak response from the Standard solution
hydroxide to a pHof 11.0 ± 0.1, and passthrough a suitable =concentration of USP Azithromycin RS in the
filter. Standard solution (J..Ig/mL)

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 USP 43 Official Monographs / Azithromycin 451

=nominal concentration of azithromycin in the

Azithromycin for Injection
Sample solution (~g/mL)
P = potency of azithromycin in USP Azithromycin RS DEFINITION
(~g/mg)
Azithromycin for Injection is a sterile, dry mixture of .
F = conversion factor, 0.001 mg/~g azithromycin and a suitable stabilizing agent. It contains
NLT 90.0% and NMT 110.0% of the labeled amount of
Acceptance criteria: 90.0%-110.0%
azithromycin (C3sHnNz012)'
PERFORMANCE TESTS
• DISSOLUTION (711)
IDENTIFICATION
[NoTE-Use water that has a resistivity of NLT 18 • A. The retention time of the major peak of the Sample
Mohm-cm.] solution corresponds to that of the Standardsolution, as
Medium: pH 6.0 sodium phosphate buffer (Prepare 6L of obtained in the Assay.
0.1 M dibasic sodium phosphate. Adjust with about 40 mL
of hydrochloric acid to a pH of 6.0 ± 0.05, and add 600 mg
of trypsin); 900 mL
Apparatus 2: 100 rpm
Time: 45 min
Mobile phase, Chromatographic system, and System
suitability: Proceed as directed in the Assay. . .
Standard stock solution: 0.3 mg/mL of USP Azlthrornycln
RS in Medium. Sonicate briefly to dissolve.
Standard solution: 3.84 ~g/mL of azithromycin from the
Standardstock solution in" Mobilephase
Sample solution: Pass a portion of the solution under test
through a suitable filter of 0.5-~m or finer pore size.Transfer
2.0 mL of the filtrate to a 25-mL volumetric flask, and
dilute with Mobilephase to volume. !ransfer 4.0 f!lL of t~is
solution to a second 25-mL volumetric flask, and dilute With
Mobile phase to volume.
Analysis
Samples: Standardsolution and Sample solution
Determine the amount of azithromycin (C3sHnNzOlz)
ASSAY
• PROCEDURE
dissolved using the procedure in the Assay, making any Buffer: 6.7 mg/mL of dibasic potassium phosphate in water
necessary modifications. Mobile phase: Acetonitrile and Buffer(52:48). Adjust with
Calculate the percentage of azithromycin (C3sHnNzOlz) 10 N potassium hydroxide to a pH of 11.0 ± 0.1.
dissolved: Diluent: Acetonitrile and water (52:48)
System suitability solution: 1 mg/mL each of USP
Result =(ru/rs) x (Cs/L) x D x Vx 100 Azaerythromycin ':- RS and USP Azithromyci~ RS in ~ .
mixture of acetonitrile and water (52:48). Dissolve first In
= peak responsefrom the Sample solution
acetonitrile, and then dilute with water to volume.
= peak response from the Standardsolution Standard solution: 1 mg/mL of USP Azithromycin RS in a
= concentration of USP Azithromycin RS in the mixture of acetonitrile and water (52:48). Dissolvefirst in
Standardsolution (mg/mL) acetonitrile, and dilute with water to volume.
L = label claim (mg/Capsule) Sample solution: No~inally e9uivalen~ to.l m.g/m.L of
D = dilution factor of the Sample solution azithromycin from Azlthrornycln for Injection In Diluent
V = volume of Medium, 900 mL prepared asfollows. Reconstitute 3 vials individually as
directed in the labeling. Mix the contents of all the
Tolerances: NLT 75% (Q) of the labeled amount of reconstituted vials. Dilute a portion of the mixture with
azithromycin (C3sHnNzOd is dissolved. Diluent.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Chromatographic system
requirements (See Chromatography (621), System Suitability.)
SPECIFIC TESTS Mode: LC
• WATER DETERMINATION, Method I (921): NMT 5.0% Detector: UV 215 nm
Columns
ADDITIONAL REQUIREMENTS Guard: 4.6-mm x l-cm; 5-~m packing L67
• PACKAGING AND STORAGE: Preserve in well-closed Analytical: 4.6-mm x 15-cm; 5-~m packing L67
containers. Where packaged in unit-of-use containers, each Temperatures
container contains six 250-mg Capsules, and the label Autosampler: 15°
indicates the intended sequential day of usefor each Column: 40°
Capsule. Flow rate: 1 mL/min
• USP REFERENCE STANDARDS (11) Injection volume: 15 ~L
USP Azaerythromycin A RS System suitability
USP Azithromycin RS Samples: System suitability solution and Standardsolution
[NoTE-The relative retention times for
azaerythromycin A and azithromycin are 0.68 and
1.0, respectively.]

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452 Azithromycin / OfficialMonographs USP 43

Suitability requirements Electrode 2: +0.82 ± 0.05 V

Resolution: NLT 2.5 between azaerythromycin A and +.~r0$~',1"~Jg"2Q19)
azithromycin, System sUitability solution Column; 4.6-mm x 15-cm; 3-lJm packing L49
Tailing factor: NLT 0.9 and NMT 1.5, Standard solution Autosampler temperature: 5°
Relative standard deviation: NMT 2%, Standard Flow rate: 1 mL/min
solution Injection volume: 50 IJL
Analysis System suitability
Samples: Standard solution and Sample solution Sample: Standard solution
Calculate the percentage of the labeled amount of [NOTE-See Table 7 for relative retention times.]
azithromycin (C3SH72N2012) in the portion of Suitability requirements
Azithromycin for Injection taken: Tailing factor: NMT 2.0 for azithromycin and NMT 2.6
for N-demethylazithromycin
Result = (ru/rs) x (Cs/Cu) x P x F x 100 Relative standard deviation: NMT 10.0% for
azithromycin N-oxide, desosaminylazithromycin,
to = peak response from the Sample solution N-demethylazithromycin, and azithromycin
rs = peak response from the Standard solution Analysis
Cs =concentration of USP Azithromycin RS in the Samples: Standard solution and Sample solution
Standard solution (mg/mL) Calculate the percentage of each specified impurity in the
Cu = nominal concentration of azlthrornycin in the portion of Azithromycin for Injection taken:
Sample solution (mg/mL)
P = potency of USP Azithromycin RS (lJg/mg) Result = (ru/rs) x (Cs/Cu) x P x 100
F =conversion factor, 0.001 mg/lJg
tu = peak response of each specified impurity from the
Acceptance criteria: so.oss.u 0.0% Sample solution
rs = peak response of each specified impurity from the
PERFORMANCE TESTS Standard solution
• UNIFORMITY OF DOSAGE UNITS (905): Meets the Cs = concentration of the relevant impurity Reference
requirements Standard in the Standard solution (mg/mL)
IMPURITIES Cu = nominal concentration of azithromycin in the
[NoTE-The test for Limit of Azithromycin N-Oxide, Sample solution (mg/mL)
Desosaminylazithromycin, and N-Demethylazithromycin P = potency of the relevant Reference Standard (mg/
does not quantify aminoazithromycin, formamido mg)
analog, methylformamido analog, and
3'-de(dimethylamino)-3'-oxoazithromycin. If these Acceptance criteria:. See Table 7. The reporting
impurities are part of the impurity profile, the Limit of ·threshold. (USP 1~A~9-2019) is 0.05%.
Aminoazithromycin, Formamido Analog,
Methylformamido Analog, and 3'-De(dimethylpmino)- Table 1
3'-oxoazithromycin test is recommended in addition to Relative Acceptance
the test for Limit of Azithromycin N-Oxide, Retention Criteria,
Name TIme NMT (%)
Desosaminylazithromycin, and N-
Demethylazithromycin.] Azithromycin N-oxidea 0.17 1.0
Desosamtnylazlthromydn'' 0.27 0.3
Erythromycin Aoxime- d 0.35 -
• LIMIT OF AZITHROMYCIN N-OXIDE,
DESOSAMINYLAZITHROMYCIN, AND N-Oemethylazithromycin e 0.50 1.0
N-DEMETHYLAZITHROMYCIN Azaerythromycin N. f 0.85 -
Buffer: 3.5 gil of dibasic potassium phosphate
Mobile phase: Acetonitrile and Buffer (23:77). Adjust with Azithromycin 1.00 -
5 N potassium hydroxide to a pH of 10.55 ± 0.05.
a (2R,35,4R,SR,8R, lOR,11R, 125,135,14R)-13-[(2,6-0ideoxy-3-C-methyl-3-0-
Standard stock solution: 0.05 mg/mL of USP Azithromycin methyl-c-t.-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy-
N-oxide RS, 45 IJg/mL of USP Desosaminylazithromycin 3,5,6,8,10,12,14-heptamethyl-l1-[[3,4,6-trideoxy-3-(dimethylazinoyl)-p-o-
RS, and 160 IJg/mL each of USP N-Demethylazithromycin xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-1S-0 ne.
RS and USP Azithromycin RS in acetonitrile. Sonicate if b (2R,35,4R,SR,8R, lOR,11R,l zs,
135,14R)-2-Ethyl-3,4,10,13-tetrahydroxy-
3,5,6,8,10,12,14-heptamethyl-ll-[[3,4,6-trideoxy-3-dimethylamino-p-o-xylo-
necessary to dissolve. hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-1S-one.
Standard solution: 0.001 mg/mL of azithromycin N-oxide, C Process impuritiesthat are controlled in the drug substance are not to be
0.9 IJg/mL of desosaminylazithromycin, and 3.2 IJg/mL reported. They are listed here for informationonly.
each of N-demethylazithromycin and azithromycin from d (3R,4S,S5,6R,7R,9R, 115,12R,135,14R,E)-6-[[3,4,6-Trideoxy-3-(dimeth-
Standard stock solution in Mobilephase ylamino)-p-o-xylo-hexopyranosyl]oxy]-14-ethyl-7, 12,13-trihydroxy-4-[(2,6-
Sample solution: Nominally equivalent to 0.3 mg/mL of dideoxy-3-C-methyl-3-O-methyl-a-L-ribo-h exopyranosyl)oxy]-l 0-
(hydroxyimino)-3,S, 7,9,11,13-hexamethyloxacyclotetradecan-2-one.
azithromycin in Mobilephasefrom Azithromycin for e (2R,3S,4R,SR,8R, lOR,11R, 125,135,14R)-13-[(2,6-0ideoxy-3-C-methyl-3-0-
Injection methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4, 1O-trihydroxy-
Chromatographic system 3,5,6,8,10,12,14-heptamethyl-l1-[[3,4,6-trideoxy-3-methylamino-p-o-xyl0-
(See Chromatography (621), System Suitability.) hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-1S-one.
Mode: LC f 9-0eoxo-9a-aza-9a-homoerythromycin A.

Detector: Amperometric electrochemical

Electrodes: Dual series glassy carbon
Mode: Oxidative screen
Electrode 1: +0.70 ± 0.05 V

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USP 43 Official Monographs / Azithromycin 453

Acceptance criteria: See Table 2.

• LIMIT OF AMINOAZITHROMYCIN, FORMAMIDO ANALOG, Table 2

METHYLFORMAMIDO ANALOG, AND Relative Acceptance
3'-DE(DIMETHYLAMINO)-3'-OXOAZITHROMYCIN (if present) Retention Criteria,
Buffer: 3.5 giL of dibasic potassium phosphate in water Name Time NMT(%)
Mobile phase: Acetonitrile and Buffer (46:54). Adjust with Erythromycin A lrnlnoether- b 0.20 -
10 N potassium hydroxide to a pH of 11.0 ± 0.1.
Diluent: Acetonitrile and water (46:54) 3'-(N,N"Didemethyl)azith-
romycin (aminoazithromy-
Standard stock solution: 0.09 mg/mL of USP cin)C + 3'-(N,N.didemethyl)-
Desosaminylazithromycin RS, 0.21 mg/mL of USP 3'-N-formylazithrom1cin
N-Demethylazithromycin RS, and 0.30 mg/mL of USP (formamido analog) 0.25 1.0
Azithromycin RS in acetonitrile
Standard solution: 0.0018 mg/mL of
Azithromycin p. e 0.30 -
desosaminylazithromycin, 0.0042 mg/mL of Desosaminylazithromycinf. 9 0.31 -
N-demethylazithromycin, and 0.006 mg/mL of
3'-N-Demethyl-3'-N-formyla-
azithromycin in Diluent zithromycin
Sample solution: Nominally equivalent to 0.6 mg/mL of (methylformamido analog)" 0.32 1.0
azithromycin from Azithromycin for Injection in Diluent.
Reconstitute 3 vials individually, asdirected in the labeling. N-Demethylazithromycinf• 1 0.35 -
Mix the contents of all the reconstituted vials. Dilute a Erythromycin A oxlmevl 0.42 -
portion of the mixture with Diluent. The Sample solution
must be. injected immediately after preparation.
Azaerythromycin Aa, k 0.63 -
Blank: Use the Diluent. 3'-De(dimethylamino)-3'-
Chromatographic system oxoazithromycin' 0.72 1.0
(See Chromatography (621), System Suitability.) 3'-N-Demethyl-
Mode: LC 3'-N-[(4-methylphenyl)sul-
Detector: Amperometric electrochemical fonyl]- -
Electrodes: Dual series glassy carbon azlthromyclnv m 0.85
Mode: Oxidative screen Azithromycin 1.00 -
Electrode 1: +0.70 ± 0.05 V
Electrode 2: +0.82 ± 0.05 V Azithromycin B
(3.deoxyazithromycin)a. n 1.64 -
~~(tJsfli3All~iirii9) ,
Columns Any other unspecified
-
impurity 0.2
Guard: 4.6-mm x l-cm; 5-l.lm packing L67
Analytical: 4.6-mm x 25-cm; 5-l.lm packing L67
Temperatures
Autosampler: 150
Column: 40 0
Flow rate: 1 mL/min
Injection volume: 25 I.lL
System suitability
Sample: Standardsolution
[NoTE-See Table 2 for relative retention times.]
Suitability requirements
Resolution: NLT 1.5 between desosaminylazithromycin
and N-demethylazithromycin
Tailing factor: NMT 1.5 for azithromycin
Relative standard deviation: NMT 5% for azithromycin
Analysis
Samples: Standard solution, Sample solution, and Blank
Disregard any peaks corresponding to those obtained
from the Blank.
Calculate the percentage of each impurity in the portion of
Azithromycin for Injection taken:

Result =(rulrs) x (CsICu) x Px Fx 100

ru = peak response of each impurity from the Sample

solution
rs = peak response of azithromycin from the Standard
solution
Cs = concentration of USP Azithromycin RS in the
Standardsolution (mg/mL)
Cu = nominal concentration of azithromycin in the
Sample solution (mg/mL)
P = potency of USP Azithromycin RS (I.lg/mg)
F = conversion factor, 0.001 mg/l.lg

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454 Azithromycin / Official Monograph5 USP 43

Table 2 (continued) ADDITIONAL REQUIREMENTS

Relative Acceptance • PACKAGING AND STORAGE: Preserve as described under
Retention Criteria, Packaging and Storage Requirements (659), Injection
Name Time NMT (%) Packaging, Packaging for Constitution. Store at controlled
Total lmpurltles'' - 3.0 room temperature.
• LABELING: It meets the requirements for Labeling (7), Labels
a Process impuritiesthat are controlled in the drug substance are not to be and Labeling for Injectable Products.
reported. They are listed here for information only. • USP REFERENCE STANDARDS (11)
b (3R,4R,55,6R,9R, 105,115,12R,135,15R,Z}-12-[[3,4,6-Trideoxy-3- USP Azaerythromycin A RS
(dimethylamino)-p-o-xylo-hexopyranosyl]oxy]-6-ethyl-4,5-dihydroxy-1 0-[(2,6- 9-Deoxo-9a-aza-9a-homoerythromycin A.
dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)0xy]-3,5,9,11,13,15-
hexamethyl-7,16-dioxa-2-azabicyclo[11.2.1 ]hexadec-1-en-8-one. C37H70N2012 734.96
c (2R,35,4R,5R,8R, lOR,11R,125,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0- USP Azithromycin RS
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy- USP Azithromycin N-oxide RS
3,5,6,8,10,12,14-heptamethyl-11-[[3-amino-3,4;6-trideoxy-p-o-xylo- (2R,3S,4R,5R,8R,l OR, 11 R, 125,135/14R)-13-[(2,6-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
d (2R,35,4R,5 R,8R, lOR,11 R,125,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0- Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy- hexopyranosyl)oxy]-2-ethyl-3,4/10-trihydroxy-
3,5,6,8,10,12,14-heptamethyl-11-[[3-formamido-3,4,6-trideoxy-p-o-xyl0- 3/5,6,8/10,12,14-heptamethyl-l1-[[3,4/6-trideoxy-3-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. (dimethylazinoyl)-~-D-xylo-hexopyranosyl]oxy]- l-oxa-
e (2R,35,4R,5R,8R,1 OR, 11R,125,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0- 6-azacyclopentadecan-15-one.
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12-
hexamethyl-14-hydroxymethyl-11-[[3-dimethylamino-3,4,6-trideoxy-p-o-xylo- C38H72N2013 764.98 .
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. USP N-Demethylazithromycin RS
fThese impuritiesare controlled using the Limitof Azithromycin N-Oxide, (2R,35/4R,5R,8R,l OR,ll R, 125/135,14R)-13-[(2,6-
Desosaminylazithromycin, and N-Demethylazithromycin test. Theyare listedhere Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-
for informationonly.
9 (2R,35,4R,5R,8R, lOR,11R,125,135,14R)-2-Ethyl-3,4,10,13-tetrahydroxy-
hexopyranosyl)oxy]-2-ethyl-3,4/10-trihydroxy-
3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethylamino-p-o-xylo- 3/5,6/8/10/12,14-heptamethyl-ll-[[3,4,6-trideoxy-3-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. methylamino-~-D-xylo-hexopyranosyl]oxy]-1-oxa-6­
h (2R,35,4R,5R,8R,1 OR, 11R,125,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3.0- azacyclopentadecan-15-one.
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy- C37H70N2012 734.96
3,5,6,8,10,12,14-heptamethyl-11-[[3-(N-methyl)formamido-3,4,6.tridf'oxy-p-
o-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. USP Desosaminylazithromycin RS
i (2R,35,4R,5R,8R, lOR,11 R,125,135,14R)-13-[(2,6-Dideoxy·3-C-methyl-3-0- (2R,35,4R,5R,8R,l OR, 11 R,12S, 135,14R)-2-Ethyl-
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl.3,4,1O-trihydroxy- 3,4,10,1 3-tetrahydroxy-3,5,6/8,l 0,12,14-heptamethyl-
3,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-methylamino-p-o-xy10- 11-[[3,4,6-trideoxy-3-dimethylamino-~-D-xylo­
hexopyranosyl]oxyl-t-oxa-e-azacydopentadecan-l s-one. hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-
j (3R,45,55,6R,7R,9R, 115,12R,135,14R,E)-6-[[3,4,6-Trideoxy-3-(dimeth-
ylamino)-p-o-xylo-hexopyranosyl]oxy]-14-ethyl-7, 12,13-trihydroxy-4·[(2,6- one.
dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl)oxy]-10· C30H58N209 590.79
(hydroxyimino)-3,5,7,9,11,13-hexamethyloxacyclotetradecan.2-one.
k9-Deoxo-9a-aza-9a-homberythromycin A. .
I(2R,35,4R,5R,8R, lOR,11R,125,135,14R)-13-[(2,6-Dideoxy-3,3-dimethyl-a-L-
ribo-hexopyranosyl)oxy]-2-ethyl-3,4, 10-trihydroxy-3,5,6,8,10,12,14-
heptamethyl-11-[[3,4,6-trideoxy-3-oxo-p-o-xylo-hexopyranosyl]oxy]-1-oxa-6-
azacyclopentadecan-15-one. Azithromycin for Oral Suspension
m (2R,35,4R,5R,8R, lOR,11R,125,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trthydroxy- DEFINITION
3,5,6,8,10,12,14-heptamethyl-11-[[3-[N-(4-acetamidophenylsulfonyl)-N- Azithromycin for Oral Suspension is a dry mixture of
methylamino]-3,4,6-trideoxy-p-o-xylo-hexopyranosyl]oxy]-1-oxa-6- Azithromycin and one or more buffers, sweeteners,
azacyclopentadecan-l Svone. .
n (2R,3R,45,5R,8R, lOR,11 R,125,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-
diluents, anticaking agents, and flavors. It contains NlT
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-4,10-dihydroxy-3,5,6,8,1 0,12,14- 90.0% and NMT 110.0% of the labeled amount of
heptamethyl-l1-[[3,4,6-trideoxy-3-(dimethylamino)-p-o-xylo-hexopyranosyl] azithromycin (C38HnN2012)'
oxy]-1-oxa-6-azacyclopentadecan-15-one.
o Total impuritiesinclude desosaminylazithromycin and N- IDENTIFICATION
demethylazithromycin. • A. The retention time of the azithromycin peak of the
Sample solution corresponds to that of the Standard
SPECIFIC TESTS solution, as obtained in the Assay.
ASSAY

• BACTERIAL ENDOTOXINS TEST (85):

requirements A(~JSP 1·Aug-201!)

• S.!~~~LI!! . !~.S1"~ •. ~"~}.'.):.i~~~~~the

tl:!q i.J ir~m~!1f$A(lJSP1:AlJg-20i~) .
• PARTICULATE MATTER IN INJECTIONS (788): Meets the
requirements
• pH (791): 6.4-6.8, determined in a solution constituted as
directed in the labeling
• WATER DETERMINATION (921), Method I: NMT2.0%
• OTHER REQUIREMENTS: It meets the requirements under
Injections and Implanted Drug Products (1).

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 USP 43 OfficialMonographs / Azithromycin 455

Sample solution: Nominai 6-mg/mLof azithrQmycinin

Diluent prepared as foil sfer. "n.accurately
measuredp . of t uted"'-
suitable volu nc flas lIuen
volume oUheflask,' and, .' tefor
in cool water. Dilute with Diluent to v
of this solution through a siJitabl~filterof ntalii
size. _ S.i~
Chromatographic system . ' . ., . . Ion
(See Chromatography (621), System Su{tabilify.) por:tion·ofthesolutlon,..urider test
Mode: LC i ter~
Detector: .UV210nm stem
Column: 4;6-mmx25-cm; 5-I.m1packlng l} hy'(621 )~·SY5tem.SlJitabillty;)
Temperatures. ,
Autosampler:'10°
Column: 30°
Flow rate: 2mL/rhin
InjeCtion volume: .so.
pt " __ . '.'_ " •... _. ,"
Run ti,me: .NLT2 times the retention time ot;azitbromycin'
System suitability
Sample: Standard solution,
Suitability re· ents , nes tl}~e re"tentiontime of azithrolnycln
Tailing f a c t . O . .., , ,._ .. ,
Relativesta iation: NMT 2.0%
Analysis, , '. ',' . , - .
Samples: Standard solution and
Calculate the percentage ~efative'sta
~zithromycin (C38H72N2 Analysis
Azithromycin for Oral S Samples: ~St
Calculate the Ilfol
Result = (ru/rs) x (C~/Cu) x, P-xf ><foq aZithromycin (
ru ::: peak respqnseof aZithromyCinfrom' !tie,'~arnj:J/~ Result ~(rulrs)x(?:.s/ L) .~. D .~(dlW)'x V x J(j~
solution
rs = peak response of azithromydn from:tne Stanaclrd ru ='peak response of azlthromyeinfromthe SiJmp7e
solution . solution
= concentration of US'P Azithromydri RS i.ri the rs = peak response of azithroiny~in fr<?mth'e Standard
Standard solutio g/mL) . ., '. .' - so .
= nominal conceh 'on of azlthr6mycifl jnth-e =.co
Sample solution ,( /mL), ", S
P = potency of USPAzithromycin RS mg)" L =1
F == conversion factor, 0.001 mg/lJg. ,"May:2019)
D
Acceptance criteria: 90.00/0-110.0%
PERFORMANCE TESTS d
• DELIVERABLE VOLUME (698): Meets the requirements
W
• UNIFORMITY OF DOSAGE UNITS (905)
(g)
For single-unit containers: Meets the requirements V f::volum,e of Medilim, 900 mL
Tolerances: N
Add the following:
azithromycin - 19)
•• DISSOLUTION .(711) . . .' .. _. . . .. IMPURITIES
[NOTE"':"Solutionscontalning azithromycin are stabfe up t()
12 h at 10°.] .. '. .'. . .,. ',.. ..". .. , . . Addth
Medh,IITi: Sodium phosphatel:>uffer at apHof 6.0;9Qo-,mL
Apparatus 2: .50 rpm
Time: 30 min diurTl'J1ydrogen phosphate
$Qlution A: Dissolve.8.7 g. of dipotassium fly an9adjustwith ~:liIute
phosphate anhydrous in 1000 mL of water a
potassium hydroxideor~dilute orthcopbospOor..
pH of 8.2.
SolutionB: Acetonitrile
Mobile' phase:· Solution A cll1d 'Soli,ti6 . Ta61el
Standard stock solution: O. in Time $oIQtioil'A 'SolutionB
RS prepared as follows. Tran , (min) (%) "(%)
amount ofUspAzithromycin RSto a. ". .
flask. Add acetonitrile to fjJI5% of theyoJL!m 0 56 SO

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456 Azithromycin / OfficialMonographs USP 43

Table 1 (continued) Table 2

Tirri~ SQlUl:ionA SoUi' B Relative Relative Acceptance
(mill) -(0/0)' ,- Retention Response Criteria;
25 45 ~.~
Name Time Fadol: NMT(%)

.0.29 0.43 ().So

30 :\f0 !5Q
80 25, 75 0;37 1.7 0;50
81 50 59 3'·(N
myc 0.43 1.0 0.50
90 50 ~o
Azithromycin retat~d
compound Fd 0.51 3.8 0.50

Des.osaminylazithroinyCfne 0.54 1.0 0.30

1.0 0.50
Azithromycin C(3":'O~deine·
~hylazithromycin)g, h 0.73
,
3'.De( hyl~miino).3}~
Qxoazi ycinl 0.76 1.5 0.50
AzaerythrorriydnAh, J 0.83
ed,unidentiffed
k 0.92
Azithrorriycin 1.0
,2-Desethyl-2-propylaZlthro·
mycinh,l 1.23

L31

1:0 0.20

entionjime'qf~ithiorriycin

'Ity ;so1!-!'tiQ'1<arld~itC1.iJ,(JairJ solt.ition

rtiorlof

Resuli-;'; (rulrs) x'(Cs/Cu)><'Px F/~(lIF~rxo)JO~

nseof :~ach-implU:itYfrc)rrftheCSarnpte
rs onseof ~;dthi9'mycrnfforptf-re'5tanCJard

p ~g7rT1gJ
Fj,
f2 =:relativerespon'se fci<:tor (se,e'Table 2)
Acceptimc
relative re

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 USP 43 OfficialMonographs / Azithromycin 457

Azithromycin Tablets
DEFINITION
Azithromycin Tablets contain NLT 90.0% and NMT 110.0%
of the labeled amount of azithromycin (C38HnN2012)'
IDENTIFICATION
• ·A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in the Assay.

ASSAY
• PROCEDURE
Buffer: Dissolve 4.6 g of monobasic potassium phosphate
anhydrous in 900 mL of water. Adjust with 1 N sodium
hydroxide to a pH of 7.5, and dilute with water to 1 L.
Mobile phase: Acetonitrile and Buffer (65:35)
Standard solution: 1 mg/mL of USP Azithromycin RS in
SPECIFIC TESTS Mobilephase. Sonicate and shake as needed to dissolve.
• pH (791) Sample solution: Nominally 1 mg/mL of azithromycin in
For a solid packaged in single-unit containers Mobilephase from NLT 20 Tablets, finely powdered.
Sample: The suspension constituted as directed in the Sonicate and shake as needed to dissolve.
labeling Chromatographic system
Acceptance criteria: 9.0-11.0 (See Chromatography (621), System SUitability.)
For a solid packaged in multiple-unit containers Mode: LC
Sample: The suspension constituted as directed in the Detector: UV 210 nm
labeling Column: 4.6-mm x 25-cm; 5-lJm packing L1
Acceptance criteria: 8.5-11.0 Column temperature: 50°
ADDITIONAL REQUIREMENTS Flow rate: 2 mL/min
• PACKAGING AND STORAGE: Preserve in tight containers. Injection volume: 100 IJL
System suitability
Sample: Standardsolution
Suitability requirements
• USP REFERENCE STANDARDS (11) Tailing factor: NMT 2.0
:~l (IJs';;i'M~~6~~) Relative standard deviation: NMT 2.0%
USP Azithromycin RS

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458 Azithromycin / Official Monographs USP 43

Analysis = peak response of azithromycin from the Sample

Samples: Standard solution and Sample solution solution
Calculate the percentage of the labeled amount of = peak response of azithromycin from the Standard
azithromycin (C3sHnN2012) in the portion of Tablets solution
taken: = concentration of USP Azithromycin RS in the
Standard solution (mg/mL)
Result =(ru/r s) x (CslCu) x P x F x 100 L = label claim (mg/Tablet)
V of tv1i?dium,9Q9.mh,c., . . ,
ru = peak response of azithromycin from the Sample ..D f . 'grpplgi§9}i:!ti9t1'i.lt
solution
ts = peak response of azithromycin from the Standard
solution Tolerances: NLT80% (Q) of the labeled amount of
C, =concentration of USP Azithromycin RS in the azithromycin (C3sHnN2012) is dissolved.
Standard solution (mg/mL) • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Cu = nominal concentration of azithromycin in the requirements
Sample solution (mg/mL)
P = potency of USP Azithromycin RS (~g/mg) IMPURITIES
F =conversion factor, 0.001 mg/~g
Acceptance criteria: 90.0%-110.0%
• ORGANIC IMPURITIES
PERFORMANCE TESTS Protect all solutions containing azithromycin from light.
Refrigerate the Standardsolution and the Sample solution
after preparation and during analysis, using a refrigerated
autosampler set at 4°. The solutions must be analyzed
• DISSOLUTION (711 ) within 24 h of preparation.
Medium: pH 6.0 phosphate buffer; 900 mL Solution A: Water and ammonium hydroxide (2000: 1.2).
Apparatus 2: 75 rpm The pH of this solution is about 10.5.
Time: 30 min Solution B: Acetonitrile, methanol, and ammonium
Solution A: 4.4 mg/mL of dibasic potassium phosphate and hydroxide (1800: 200: 1.2)
0.5 mg/mL of sodium l-octanesulfonate; adjusted with Mobile phase: See Table 1.
phosphoric acid to a pH of 8.20 ± 0.05
Mobile phase: Acetonitrile, methanol, and Solution A Table 1
(9:3:8) ,
Time Solution A Solution B
Diluent: 17.5 mg/mL of dibasic potassium phosphate. (min) (%) (%)
Adjust with phosphoric acid to a pH of 8.00 ± 0.05. Prepare
a mixture of this solution and acetonitrile (80:20). 0 54 46
Standard stock solution: Dissolve USP Azithr.omycin RS in 20 54 46
Medium to obtain a solution with a known concentration of
about (L/l 000) mg/mL, where L is the label claim in mgl 35 10 90
Tablet. 35.1 54 46
Standard solution: Dilute the Standardstocksolution with
Diluent to obtain a solution with a known concentration of ,(lJSp·;D"'.Z019) 54 46
about (L/2000) mg/mL, where L is the label claim in mgl
Tablet. Buffer: 1.7 giL of monobasic ammonium phosphate in
Sample solution: Pass a portion of the solution under test water. Adjust with ammonium hydroxide to a pH of 10
through a suitable filter of 0.45-~m pore size. Dilute a ....± 0.05..... (USP 1~Dec~2019)
portion of the filtrate with Diluent to obtain a solution with Diluent A: Methanol, acetonitrile, and Buffer
a theoretical concentration of about (L/2000) mg/mL, (350:300:350). . ... '
where L is the label claim in mgjTablet, assuming complete mixing the organ .' .
dissolution.
Diluent B: Methanol and Buffer (1:1)
Chromatographic system
System suitability stock solution: 0.1 mg/mL each of USP
(See Chromatography (621), System Suitability.)
Mode: LC Desosaminylazithromycin RS, USPAzithromycinRelated
Detector: UV 210 nm Compound.F RS, and .... USPN.Demethyla,zi~hromyCin
Column: 4.6-mm x 15-cm; 5-~m packing L1 RS .... (USP1-Dec-io19) in acetonitrile
Column temperature: 50° System suitability solution: 0.028 mg/mL each of USP
Flow rate: 1.5 mL/min Desosaminylazithromycin RS, USPAzithromycin Related
Injection volume: 50 ~L Compound F.RS, and .... USP N-Dernethylazithromycin
System suitability RS .... (USP l-Dec-2Q19) from the System suitability stock solution in
Sample: Standard solution Diluent A
Suitability requirements .... Peak identification solution'
Tailing factor: NMT 2.0 Desosaminylazithromycin R
Relative standard deviation: NMT 2.0% Compound FRS, and USP N- _.
Analysis from the System suitability solu '. . 019)
Samples: Standard solution and Sample solution Standard stock solution: 0.4 mg/mL of USP Azithromycin
Calculate the percentage of the labeled amount of RS in acetonitrile. Sonicate and shake as needed to dissolve.
azithromycin (C3sHnN2012) dissolved: Standard solution: 0.02 mg/mL of azithromycin from the
Standard stock solution in Diluent A
Result = (rufr s) x (CsiL) x V x , n~~'''n1lm X 100

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USP 43 Official Monographs / Azithromycin 459

Sensitivity solution: 0.004 mg/mL of azlthrornycin from Table 2 (continued)

the Standard solution in Diluent A Accept-
Sample stock solution: Nominally 14.3 mg/mL of Relative Relative ance
azithromycin prepared asfollows. Transfer nominally 1430 Retention Response Criteria,
Name Time Factor NMT(%)
mg of azithromycin, from finely powdered Tablets (NLT
20), to a 1OO-mL volumetric flask. Add 75 mL of 3'-(N,N-Didemeth- 0.29
acetonitrile, and sonicate for NLT 15 min. Shake by yl)-3'-N~forrnyl~zi-
mechanical means for NLT 15 min. Allow the solution to ~~~~.~.~;i~~:,~;~f~l
WS~;l'"P~~;Z(;"l~) -
.. 1.0
0.30 1.7
equilibrate to room temperature, dilute with acetonitrile to
volume, and mix. 3'-(N,N-
Sample solution: Nominally 4 mg/mL of azithromycin Didemethyl)azi-
prepared asfollows. Centrifuge an aliquot of the Sample thromycin (amino-
azlthrornycln)" 0.34 ~Q;44jjf 0.5
stock solution for NLT 15 min. Transfer 7.0 mL of the
supernatant to a 25-mL volumetric flask, and dilute with
Diluent B to volume. (G~~4;6l:~;;Z619)
Azithromycin relat-
Blank: Diluent A
Chromatographic system F~P~""f'i' .... /, 'p 'i,"
\u><~
iRiE p,;
...,.... ,LYH' . .:5;~'~i(lj,sef~ib!!o;.iu.i~) 1.0
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 210 nm
Desosaminylazithro-
rnydn' ;;:';~£~i...;".Jt,,(
,,.,,'x _"";6V'. 1.1 0.5
Column: 4.6-mm x 15-cm; 3.5-~m packing L1 N-Demethylazithro-
Temperatures ff· :,ii"
mycin 9 0.50 tPi4Z, 0.7
Autosampler: 4°
3'-De(dimethylami-
Column: 50° no)-3' -oxoazlthro-
Flow rate: 1.2 mL/min rnydn" 0.87 iJr.;'(USP iJ6~.~~j;) 1.0
Injection volume: 100 ~L
Azaerythromycin
System suitability Ai,j 0.94 - -
Samples: System suitability solution, Standardsolution, and
Sensitivity solution Azithromycin 1.0 - -
Suitability requirements 2-Desethyl-2-
Resolution: NLT 1.0 between desosaminylazithromycin propylazithro- - -
and azithromycin related compound F, System suitability mycin~k 1.10
solution I
3'-N-Demethyl-
Relative standard deviation: NMT 2.0%, Standard 3'-N-[(4-methyl-
solution phenyl)sulfonyl] - -
Signal-to-noise ratio: NLT 10, Sensitivity solution azithro-
mycinl,1 1.11
Analysis .•... , . " ., . . •. . . '7 ',.7'.7<7 .
Samples: .~StClf1(j(:lrd.§()1u,ti()l],.J.(USPFPe<:~2Q19)Sample 3-Deoxyazithromy-
solution, and Blank cin (azithromycin - -
B)i,m 1.14
Calculate the percentage of each impurity in the portion of
Tablets taken: Any individual
unspecified -
Result = (rulrs) x (CsICu) x P x F] x (1IF2 ) x 100 impurity' 1.0 0.2

ru = peak response of each impurity from the Sample

solution
rs =peak response of azithromycin from the Standard
solution
Cs =concentration of USP Azithromycin RS in the
Standardsolution (mg/mL)
Cu = nominal concentration of azithromycin in the
Sample solution (mg/mL)
P = potency of USP Azithromycin RS (~g/mg)
F, = conversion factor, 0.001 mg/~g
F2 = relative response factor (see Table 2)
Acceptance criteria: See Table 2. The reporting threshold is
0.1%. Disregard any peaks in the Sample solution that
correspond to peaks in the Blank.

Table 2
Accept-
Relative Relative ance
Retention Response Criteria,
Name Time Factor NMT(%)

Azithromycin
"0'''' .i",·e:'C2CS''-''3.i.
N-oxide a 0.20 0". 1.0
"

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460 Azithromycin / Official Monographs USP 43

Table 2 (continued)
Accept-
Relative Relative ance
Retention Response Criteria,
Name Time Factor NMT (%)
Total impurities' - - 5.0

a (2R,35,4R,5R,8R, lOR,11 R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-

methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy- <:?~
3,5,6,8,10,12, 14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylazinoyl)-~-0­ USP Desosaminylazithromycin RS
xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one. (2R, 35,4R,5 R,8R, 1OR, 11 R, 125,135,14R)-2-Ethyl-
b (2R,35,4R,5R,8R, lOR,11 R,12S,13S,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0- 3,4,10,13-tetrahydroxy-3,5,6,8,10,12,14-heptamethyl-
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy- 11-[[3,4,6-trideoxy-3-dimethylamino-~-D-xylo­
3,5/6,8,10,12/ 14-heptamethyl-11-[[3-formamido-3/4,6-trideoxY-~-D-xylo-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-
h~x8R~r~.~.os~IJ?~r]:1.:?~~:6-~~.~•.~~.~I~p~Dt~.g~~~. ~:1 .~ -8n~·
ITT.l'le$YstE!ITI'.mpyresqlvE!·twPfPtpmE!r$;mJ"tE!:.Iim'iti$:fpr 2tl1ec$l.iroYqf.th~·twP one.
rotamers; C30HsgNz09 590.79
d (2R,35,4R, 5R,8R, lOR,11 R,12S,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy-
3,5,6,8,10,12/ 14-heptamethyl-11-[[3-amino-3,4,6-trideoxY-~-D-xylo­
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
e 3'-(N-Demethyl)-3'-N-formylazithromycin; (2R,35/4R,5R,8R,1 OR, 11R,125,
135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-methyl-a-L-ribo-hexopyranosyl) Aztreonam
oxy]-2-ethyl-3,4,1 0-trihydroxy-3/5,6/8,10,12,14-heptamethyl-11-[[3-(N-
methyl)formamido-3,4,6-trideoxy-~-D-xylo-hexopyranosyl]oxy]-1-oxa-6­
azacyclopentadecan-15-one.
f (2R,35,4R,5R,8R, lOR,11R,125,13S,14R)-2-Ethyl-3/4,10/13-tetrahydroxy-
3,5,6/8,10/12/ 14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethylamino-~-D-xylo­
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
9 (2R,35,4R,5R,8R, lOR,11 R, 125/135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0-
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy-
3,5,6/8,10,12/ 14-heptamethyl-11-[[3/4,6-trideoxy-3-methylamino-~-D-XY10­
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one.
h (2R,35,4R,5R,8R,1 OR, 11 R, 125/135/14R)-13-[(2,6-Dideoxy-3,3-dimethyl-a-L-
ribo-hexopyranosyl)oxy]-2-ethyl-3,4, 10-trihydroxy-3/5,6,8,10/12/14- C13H17NsOgSz 435.43
heptamethyl-11-[[3,4,6-trideoxy-3-oxo-~-D-xylo-hexopyranosyl]oxy]-1-oxa-6­ Propanoic acid, 2-[[[1-(2-amino-4-thiazolyl)-2-[(2-methyl-4-
azacyclopentadecan-15-one.
i Process impurities that are controlled in the drug substance are not to be
oxo-1-sulfo-3-azetidinyl)amino]-2-oxoethylidene]amino]
reported. They are listed here for informationonly.The unspecified impurities oxy]-2-methyl-, [25-[2a,3~(Z)]]-;
and total impurities limitsdo not include these impurities. (Z)-2-({[(2-Amino-4-thiazolyl){[(25,3S)-2-methyl-4-oxo-1-
j 9-Deoxo-9a-aza-9a-homoerythromycin A. sulfo-3-azetidinyl]carbamoyl}methylene]amino}oxy)-2-
k(2R,35,4R,5R,8R, lOR,11 R, 125,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0- methylpropionic acid [78110-38-0].
methyl-a-L-ribo-hexopyranosyl)oxy]-2-propyl-3,4/1O-trlhydrcxy-
3,5,6,8,10,12, 14-heptamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-~-0­ DEFINITION
xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one dihydrate. Aztreonam, which may be anhydrous or hydrated, contains
I (2R,35,4R,5R,8R, lOR,11 R,125/135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0- NLT 92.0% and NMT 105.0% of aztreonam
methyl-c-t,-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,1O-trihydroxy-
3,5/6,8,10,12,14-heptamethyl-11-[[3-[N-(4-methylphenylsulfonyl)-N- (C13H17NsOgSz), calculated on the anhydrous and
methylamino]-3,4,6-trideoxY-~-D-xylo-hexopyranosyl]oxy]"1-oxa-6- solvent-free basis.
azacyclopentadecan-15-one.
m (2R,3R,45,5R,8R, lOR,11R, 125,135,14R)-13-[(2,6-Dideoxy-3-C-methyl-3-0- IDENTIFICATION
methyl-a-L-ribo-hexopyranosyl)oxy]-2-ethyl-4,10-dihydroxy-3,5,6,8,10,12,14-
heptamethyl-11-[[3,4/6-trideoxy-3-(dimethylaminoH-D-xylo-hexopyranosyl]
oxy]-1-oxa-6-azacyclopentadecan-15-one.

ADDITIONAL REQUIREMENTS • A••~~~~~~~g~

Spggt~g~g(JP~ ~ ~: a appears
• PACKAGING AND STORAGE: Preserve in tight containers.
Store at controlled room temperature. the IR spectra of the analyte and the standard, dissolve
equal portions of the test specimen and the Reference
Standard in equal volumesof methanol. [NOTE-To achieve
a complete dissolution, it issuggested to use about 25 mL
• USP REFERENCE STANDARDS (11) of methanol for each 50 mg of material, and stir the mixture
USP Azithromycin RS for 40 min at room temperature.]
USP Azithromycin Related Compound F RS Evaporatethe solutionsto dryness under vacuum, and dry at
3'-(N-Demethyl)-3'-N-formylazithromycin; 40° for 4 h under vacuum. Perform the test on the
(2R,35,4R,5R,8R,1 OR, 11 R, 125,135,14R)-13-[(2,6- residues.
Dideoxy-3-C-methyl-3-O-methyl-a-L-rioo- • lB. The retention time of the major peak of the Sample
hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydroxy- solution corresponds to that of the Standard solution, as
3,5,6,8,10,12,14-heptamethyl-11-[[3-(N-methyl) obtained in the Assay.
formamido-3,4,6-trideoxy-~-D-xylo-hexopyranosyl]
ASSAY
oxy]-1-oxa-6-azacyclopentadecan-15-one. • PROCEDURE
C3gH70Nz013 762.97 [NOTE-Store the System suitability solution, Standard
solution, and Sample solution at 5°, and protect from
light to prevent lsornerlzatlon of aztreonam Z-isomer
to aztreonam E-isomer.]
Buffer: 6.8 mg/mL of monobasic potassium phosphate in
water. Adjustwith 1 M phosphoric acid to a pH of 3.0.

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USP 43 OfficialMonographs / Aztreonam 461

Mobile phase: Methanol and Buffer (1:4) Cu =concentration of Aztreonam in the Sample
System suitability solution: 1 mg/mlof USPAztreonam RS solution (mg/ml)
and 1 mg/mL of USP Aztreonam E-Isomer RS in Mobile P = potency of USP Aztreonam RS (I.lg/mg)
phase F = unit conversion factor, 0.001 mg/l.lg
Standard solution: 1 mg/ml of USPAztreonam RS in Mobile
phase Acceptance criteria: See Table 7.
Sample solution: 1 mg/ml of Aztreonam in Mobile phase
Chromatographic system Table 1
(See Chromatography (621), System Suitability.) Relative Acceptance
Mode: lC Retention Criteria,
Detector: UV 254 nm Name Time NMT(%)
Column: 3.9-mm x 30-cm; to-urn packing 11 Open-ringaztreonarn- and open-ring desul-
Flow rate: 1.5 ml/min fated aztreonam'v c 0.55 1.0
Injection volume: 10 I.ll
Aztreonam (Z-isomer) 1.0 -
System suitability
Samples: System sUitabilitysolution and Standard solution Desulfated aztreonarn" 1.6 1.5
[NOTE-The relative retention times foraztreonam and
Aztreonam E-isomere 1.8 0.5
aztreonam E-isomer are 1.0 and 1.8, respectively.]
Suitability requirements Aztreonam ethyl ester f
3.9 1.5
Resolution: NlT 2.0 between aztreonam and aztreonam
E-isomer, System suitability solution
Anyindividual unspecified impurity - 0.1
Tailing factor: NMT 2 for aztreonam, System suitability Total impurities - 3.0
solution
Relative standard deviation: NMT 2.0%, Standard a(2S,3S)-2-(Z)-2-[2-Aminothiazol-4-yl]-2-[2-carboxypropan-t-yloxyimino]
acetamido}-3-(sulfoamino)butanoic acid.
solution
b (2S,3S)-3-Amino-2-(Z)-2-[2-aminothiazol-4-yl]-2-[2-carboxypropan-2-
Analysis yloxyimino]acetamido}butanoic acid.
Samples: Standardsolution and Sample solution cOpen-ring aztreonam and open-ring desulfatedaztreonam coelute. The limit
Calculate the percentage of aztreonam (CnH 17NsOsSJ in isfor the sum of these two impurities.
the portion of Aztreonam taken: d (Z)-2-({[(2-Amino-4-thiazolyl){[(2S,3S)-2-methyl-4-oxo-3-azetidinyl]
carbamoyl}methylene]amino}oxy)-2-methylpropionic acid.
Result = (r vir 5) x (C siC v) x P x F x 100 e(E)-2-({[(2-Amino-4-thiazolyl){[(2S,3S)-2-methyl-4.oxo-l-sulfo-3-azetidinyl]
carbamoyl}methylene]amino}oxy)-2-methylpropionic acid.
f Ethyl (Z)-2-({[(2-amino-4-thiazolyl){[(2S,3S)-2-methyl-4-oxo-l-sulfo-3-
ru = peak response from the Sample solution azetidinyl]carbamoyl}methylene]amino}oxy)-2-methylpropionate.
r5 = peak response from the Standard solution
C5 = concentration of USP Aztreonam RS in the SPECIFIC TESTS
Standardsolution (mg/mL) . • STERILITY TESTS (71), Test for Sterilityof the Product to Be
Cu = concentration of Aztreonam in the Sample Examined, MembraneFiltration: Where the label states that
solution (mg/mL) Aztreonam is sterile, it meets the requirements using Fluid
P = potency of USP Aztreonam RS (I.lg/mg) A, to which 23.4 9 of sterile arginine has been added to
F = unit conversion factor, 0.001 mg/l.lg each 1000 ml.
• WATER DETERMINATION (921), Method I: NMT 2.0%; if
Acceptance criteria: 92.0%-105.0% on the anhydrous and labeled as the hydrated form: 12.00/0-18.0%. [NOTE-The
solvent-free basis term "hydrated form" refers to the a-form of Aztreonam,
IMPURITIES
which is not a stoichiometric hydrate.]
• BACTERIAL ENDOTOXINS TEST (85): Where the label states
• RESIDUE ON IGNITION(281): NMT0.1 %, the charred residue
being moistened with 2 ml of nitric acid and 5 drops of that Aztreonam is sterile or must be subjected to further
sulfuric acid processing during the preparation of injectable dosage
forms, it contains NMT 0.1 7 USP Endotoxin Units/mg of
• ORGANIC IMPURITIES
[NOTE-Store the System suitability solution, Standard aztreonam.
solution, and Sample solution at 5°, and protect from • LIMIT OF ALCOHOL
light to prevent isomerization of aztreonam Z-isomer [NOTE-This test is to be performed if alcohol is used
to aztreonam E-isomer.] while manufacturing Aztreonam.]
Mobile phase, System suitability solution, Standard Standard solution: 0.004 ml/mL of alcohol from USP
solution, Sample solution, Chromatographic system, Alcohol Determination-Alcohol RS and 0.004 ml/mL of
and System suitability: Proceed as directed in the Assay. acetonitrile from USP Alcohol Determination-Acetonitrile
RS in dimethylformamide. [NOTE-The Standardsolution
Analysis
Samples: Standardsolution and Sample solution contains 0.4% alcohol and 0.4% acetonitrile.]
Calculate the percentage of each impurity in the portion of Sample solution: 80 mg/ml of Aztreonam and 0.004
Aztreonam taken: mL/mL of acetonitrile in dimethylformamide.
[NOTE-Dissolve Aztreonam in dimethylformamide using
Result =(r vir 5) x (C 51 C v) x P x F x 100 20% of the final volume. Add a suitable aliquot of USP
Alcohol Determination-Acetonitrile RS, and dilute with
ru = peak response of each impurity from the Sample dimethylformamide to volume. The concentration of
solution acetonitrile in the Sample solution is 0.4%.]
r5 = peak response of aztreonam from the Standard Chromatographic system
(See Chromatography(621), System Suitability.)
solution
C5 = concentration of USP Aztreonam RS in the Mode: GC
Standardsolution (mg/ml) Detector: Flame ionization
Column: 0.53-mm x 30-m; phase G43

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462 Aztreonam / Official Monographs USP43

Film thickness: 3.0 urn

Temperatures Aztreonam Injection
Injector: 210 0
DEFINITION
Detector: 280 0 Aztreonam Injection is a sterile solution of Aztreonam and
Column: See Table 2. Arginine and a suitable osmolality-adjusting substance in
Water for Injection. It contains NLT 90.0% and NMT 120.0%
Table 2
of the labeled amount of aztreonam (CnH17NsOsS2)'
Hold Time
Initial Temperature Final at Final IDENTIFICATION
Temperature Ramp Temperature Temperature • A. The retention times of the major peaks of the Sample
e) e/min) (0) (min)
solution correspond to those of the Standard solution, as
50 0 50 5 obtained in the Assay.
50 10 200 4 ASSAY
• PROCEDURE
Carrier gas: Helium Buffer: 1.15 giL of monobasic ammonium phosphate in
Linear velocity: 35 cm/s water. Before final dilution, adjust with phosphoric acid to
Injection mode: Split a pH of 2.0 ± 0.1.
Injection volume: 0.5 ul, Mobile phase: Acetonitrile and Buffer (75:25)
Injection type: Split ratio, 5:1 System suitability solution: 0.2 mg/mL each of USP
System suitability Aztreonam RS and USP Open Ring Aztreonam RS in Mobile
Sample: Standard solution phase
[NoTE-The relative retention times for alcohol and Standard solution: 0.2 mg/mL each of USP Aztreonam RS
acetonitrile are 1.0 and 1.3, respectively.] and USP L-Arginine RS in Mobile phase
Suitability requirements Sample solution: Nominally 0.2 mg/mL of aztreonam from
Resolution: NLT 2.0 between alcohol and acetonitrile Injection in Mobile phase
Tailing factor: NMT 1.5 Chromatographic system
Relative standard deviation: NMT 2.0% . (See Chromatography (621), System Suitability.)
Analysis Mode: LC
Samples: Standard solution and Sample solution Detector: UV 206 nm
Calculate the percentage of alcohol in the portion of Column: 4-mm x 25-cm; 5- to 10-J.lm packing L20
Aztreonam taken: Flow rate: 1 mL/min
Injection volume: 20 ul,
Result ~ (R viR 5) x [C 5 x (DIC v)] x Fx 100 System suitability
Sample: System suitability solution
Ru =peak response ratio of alcohol to acetonitrile [NoTE-The relative retention times for aztreonam and
from the Sample solution . open ring aztreonam are 0.8 and 1.0, respectively.
R5 = peak response ratio of alcohol to acetonitrile The relative retention times for aztreonam and
from the Standard solution arginine are 0.3 and 1.0, respectively.]
C5 = concentration of alcohol in the Standard solution Suitability requirements
(mL/mL) Resolution: NLT 2.0 between aztreonam and open ring
o =density of alcohol (g/mL) aztreonam
Cu = concentration of Aztreonam in the Sample Tailing factor: NMT 2.0 for the aztreonam peak
solution (mg/mL) . Relative standard deviation: NMT 2.0% for the
F = unit conversion factor, 1000 mglg aztreonam peak
Analysis
Acceptance criteria: NMT 4% Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
ADDITIONAL REQUIREMENTS aztreonam (CnH17NsOsS2) in the portion of Injection
• PACKAGING AND STORAGE: Preserve in tight containers.
taken:
• LABELING: Where it is intended for use in preparing
injectable dosage forms, the label states that it is sterile or Result = (r vir 5) x (C 51 C v) x P x F x 100
must be subjected to further processing during the
preparation of injectable dosage forms. Where it is the ru = peak response of aztreonam from the Sample
hydrated form, the label so indicates. solution
• USP REFERENCE STANDARDS (11) r5 = peak response of aztreonam from the Standard
USP Alcohol Determination-Acetonitrile RS solution
C2H 3N 41.05 Cs =concentration of USP Aztreonam RS in the
USP Alcohol Determination-Alcohol RS Standard solution (mg/mL)
C2HsOH 46.07 Cu = nominal concentration of aztreonam in the
USP Aztreonam RS Sample solution (mg/mL)
USP Aztreonam E-Isomer RS P = potency of aztreonam in USP Aztreonam RS
(E)-2-({[(2-Amino-4-thiazolyl){[(2S,3S)-2-methyl-4-oxo- (Ilg/mg)
l-sulfo-3-azetidinyl]carbamoyl}methylene]amino}oxy)- F = conversion factor, 0.001 mg/J.lg
2-methylpropionic acid.
CnH17NsOsS2 435.43 Acceptance criteria: 90.0%-120.0%
SPECIFICTEStS
• BACTERIAL ENDOTOXINS TEST (85): NMT 0.25 USP
Endotoxin Unit/mg of aztreonam

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USP 43 Official Monographs / Aztreonam 463

• STERILITY TESTS (71): It meets the requirements when the container with Mobilephase to obtain the final
tested as directed for Test for Sterility of the Product to Be concentration ..
Examined, Membrane Filtration. Chromatographic system .
• pH (791): 4.5-7.5 (See Chromatography (621), System Suitability.)
• PARTICULATE MATTER IN INJECTIONS (788): It meets the Mode: LC
requirements for small-volume injections. Detector: UV 206 nm
Column: 4-mm x 25-cm; 5- to 10-lJm packing l20
ADDITIONAL REQUIREMENTS Flow rate: 1 ml/min
• PACKAGING AND STORAGE: Preserve as described in
Injection volume: 20 IJl
Packaging and Storage Requirements (659), Injection System suitability
Packaging, Packaging for constitution. Maintain in the Sample: System suitability solution
frozen state. [NoTE-The relative retention times for aztreonam and
• LABELING: It meets the requirements for Labeling (7), Labels
open ring aztreonam are about 0.8 and 1.0,
and Labeling for Injectable Products. The label states that the respectively. The relative retention times for
Injection is to be thawed just prior to use, describes aztreonam and arginine are 0.3 and 1.0,
conditions for proper storage of the resultant solution, and respectively.]
directs that the solution is not to be refrozen. Suitability requirements
• USP REFERENCE STANDARDS (11) Resolution: NlT 2.0 between aztreonam and open ring
USP L-Arginine RS aztreonam
USP Aztreonam RS Tailing factor: NMT 2.0 for the aztreonam peak
USP Open Ring Aztreonam RS Relative standard deviation: NMT 2.0% for the
(2S,3S)-2-{(Z)-2-[2-Aminothiazol-4-yl]-2-[2- aztreonam peak
carboxypropan-2-yloxyi mi no]acetam ido}-3- Analysis
(sulfoamino)butanoic acid. Samples: Standardsolution, Sample solution 1, and Sample
C13H19Ns09SZ 453.45 solution2
Calculate the percentage of the labeled amount of
aztreonam (C13H17NsOsSz) in the portion of Aztreonam for
Injection taken:
Aztreonam for Injection Result = (r vir s) x (C siC v) x P x Fx 100
DEFINITION
Aztreonam for Injection is a dry mixture of sterile Aztreonam rv =peak response for aztreonam from Sample
and Arginine. It contains NLT 90.0% and NMT 105.0% of
solution 7
aztreonam (C13H17NsOsSz), calculated on the anhydrous and
rs = peak response for aztreonam from the Standard
solution
arginine-free basis. Each container contains NLT 90.0% and
NMT 120.0% of the labeled amount of aztreonam
Cs = concentration of USP Aztreonam RS in the
Standardsolution (mg/ml)
(C13H17NsOsSz)· Cu = nominal concentration of Aztreonam for
IDENTIFICATION Injection in Sample solution 7 (mg/mL), corrected
• A. The retention times of the major peaks of the Sample for water and arginine content (see Content of
solution correspond to those of the Standardsolution, as Arginine)
obtained in the Assay. . P = potency of aztreonam in USP Aztreonam RS
(lJg/mg)
ASSAY F =conversion factor, 0.001 mg/lJg
• PROCEDURE
Buffer: 1.15 giL of monobasic ammonium phosphate in Acceptance criteria: 90.00/0-105.0% on the anhydrous and
water. Before final dilution, adjust with phosphoric acid to arginine-free basis
a pH of 2.0 ± 0.1. Calculate the percentage of the labeled amount of
Mobile phase: Acetonitrile and Buffer (75:25) aztreonam (C13H17NsOsSz) in each container of Aztreonam
System suitability solution: 0.2 mg/mL each of USP for Injection taken:
Aztreonam RS and USP Open Ring Aztreonam RS in Mobile
phase Result = (r vir s) x (C siC v) x P x F x 100
Standard solution: 0.2 mg/mL each of USP Aztreonam RS
and USP L-Arginine RS in Mobile phase ru = peak response for aztreonam from Sample
Sample solution 1: Nominally 0.2 mg/mL of aztreonam in solution 2
Mobile phase from Aztreonam for Injection prepared as rs = peak response for aztreonam from the Standard
follows. Weigh one container of Aztreonam for Injection, solution
transfer the contents to a suitable container, and dilute with Cs =concentration of USP Aztreonam RS in the
Mobile phase to the appropriate volume. Weigh the empty Standardsolution (mg/mL)
container, and calculate the weight, in mg, of Aztreonam Cu = nominal concentration of aztreonam in Sample
for Injection used. solution 2 (mg/ml)
Sample solution 2: Nominally 0.2 mg/mL of aztreonam P =potency of aztreonam in USP Aztreonam RS
from Aztreonam for Injection constituted asdirected below (lJg/mg)
and diluted with Mobile phase. F = conversion factor, 0.001 mg/lJg
Where the vial hasa capacity of less than 100 mL, constitute
with water using the volume of solvent specified in the Acceptance criteria: 90.00/0-120.0%
labeling.
Where the vial capacity is 2:100 mL, constitute with 10 mL
of water and dilute the entire withdrawable contents of

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464 Aztreonam I OfficialMonographs USP 43

OTHER COMPONENTS
III CONTENT OF ARGININE
Bacitracin
Use the result of this test to calculate, on the anhydrous and
arginine-free basis, the Assayresult from Sample solution
1, obtained as directed in the Assay.
Buffer, Mobile phase, System suitability solution,
Standard solution, Sample solution 1, Chromatographic
system, and System suitability: Proceed as directed in the
Assay.
Analysis
Sample: Sample solution 7
Calculatethe percentage of arginine (C6H14N40Z) in the
portion of Aztreonam for Injection taken:
Result = (r vir s) x (C siC v) x 100 Bacitracin [1405-87-4].
= peak responsefor arginine from Sample solution 7 DEFINITION
= peak response for arginine from the Standard Bacitracin is a mixture of polypeptides produced by the
solution growth of an organism of the licheniformis group of Bacillus
= concentration of USP L-Arginine RS in the subtilis (Fam. Bacillacaea), the main components being
Standardsolution (mg/mL) bacitracins A, B1, B2, and B3. It has a potency of NLT 65
= concentration of Aztreonam for Injection in Bacitracin units/mg, calculated on the dried basis.
Sample solution 7 (mg/mL) IDENTIFICATION
• A. Meets the requirements of the test for Composition of
PERFORMANCE TESTS Bacitracin
• UNIFORMITY OF DOSAGE UNITS (905): Meets the
requirements
SPECIFIC TESTS
• CONSTITUTED SOLUTION: At the time of use, it meets the • B.
requirements for Injections and Implanted Drug Products (1), Sample: 0.2 g
Specific Tests, Completeness and clarity of solutions. Analysis: Ignite the Sample. Allow to cool. Dissolve the
• BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.17 residue in 0.1 mLof cJil~!~~x~r8shI8riS~ci~<'.~.~~.. ~ . .!:Rb of
USP Endotoxin' Unit/mg of aztreonam. water and 0.2 mL of~~qcJJYmHPyqr(;)c~ig~21"~ii~*:i4~(UfiR~1)
• STERILITY TESTS (71): It meets the requirements when Acceptance criteria: No white precipitate isformed.
tested as directed for Test for Sterility of the Product to Be ASSAY
Examined, Membrane Filtration. • PROCEDURE
• pH (791) (See Antibiotics-Microbial Assays (81 ).)
Sample solution: 100 mg/mL of aztreonam Analysis: Proceed as directed in the chapter.
Acceptance criteria: 4.5-7.5 Acceptance criteria: NLT 65 Bacitracin units/mg on the
• WATER DETERMINATION, Method I (921): NMT 2.0% dried basis
• PARTICULATE MATTER IN INJECTIONS (788):. It meets the
requirements for small-volume injections. IMPURITIES
• OTHER REQUIREMENTS: It meets the requirements for • RESIDUE ON IGNITION (281): NMT 0.5%
Labeling (7), Labels and Labeling for InjectableProducts.
SPECIFIC TESTS
ADDITIONAL REQUIREMENTS • COMPOSITION 'OF BACITRACIN
• PACKAGING AND STORAGE: Preserve as described in Diluent: 40 giL of edetate disodium in water adjusted with
Packaging and Storage Requirements (659), Injection 8 N sodium hydroxide to a pH of 7.0
Packaging, Packaging for constitution. Solution A: 34.8 giL of dibasic potassium phosphate in
• USP REFERENCE STANDARDS (11) water
USP L-Arginine RS Solution B: 27.2 giL of monobasic potassium phosphate in
USP Aztreonam RS water
USP Open Ring Aztreonam RS Solution C: SolutionA and Solution B (2:9). The pH of the
(2S,3S)-2-{(Z)-2-[2-Aminothiazol-4-yl]-2-[2- mixture is about 6.
carboxypropan-2-yloxyimino]acetamido}-3- Solution D: 0.1 mM edetate disodium in a mixture of
(sulfoamino)butanoicacid. Solution C and water (1 :3)
C13H19Ns09SZ 453.45 Solution E: Methanol and acetonitrile(27:2)
Mobile phase: Solution 0 and Solution E(37:63)
System suitability solution: 2 mg/mL of USP Bacitracin
Zinc RS in Diluent
Reporting threshold solution: 0.01 mg/mL of USP
Bacitracin Zinc RS from System suitability solution in water
Peak identification solution: 2 mg/mL of USP Bacitracin
Zinc RS in Diluent. Heat in a boiling water bath for 30 min,
and cool to room temperature. .
Sample solution: 2 mg/mL of Bacitracin in Mobile phase
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC

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USP 43 OfficialMonographs / Bacitracin 465

Detector: UV 254 and 300 nm. Quantitative analysis is r82 = peak area of bacitracin B2 from the Sample
performed at 254 nrn; 300 nm isonly used to identifythe solution
location of bacitracin F. r83 = peak area of bacitracin B3 from the Sample
Column: 4.6-mm x 25-cmi end-capped 5-l..Jm packing L1 solution
Flow rate: 1 mL/min rr =sum of all peak areas above the reporting
Injection volume: 100 I..JL threshold from the Sample solution
System suitability
Samples: System suitability solution and Peak identification Limitof early-eluting peptides
solution Calculatethe percentage of early-eluting peptides (peaks
Analyze the Peak identification solution at 300 nm. Identify eluting before bacitracin B1) in the portion of Bacitracin
bacitracin F, a known impurity,usingthe relativeretention taken:
time provided in Table 1. Analyze the System suitability
solution at 254 nm. Identifythe peaks of the most active Result =(rp/rr) x 100
components of bacitracin (bacitracinsA, B1, B2, and B3),
early-eluting peptides (those eluting before the bacitracin r, =sum of peak areas for all peaks before bacitracin
B1 peak), and the impurity (bacitracin F) using the relative B1 from the Sample solution
retention times in Table 1. (r =sum of all peak areas above the reporting
threshold from the Sample solution
Table 1
Limitof bacitracin F
Relative
Nature of Retention Calculatethe percentage of bacitracin F in the portion of
Name Component Time Bacitracin taken:
Bacitracin C1 0.5 Result = (rJ rA) x 100
Bacitracin C2 Early-eluting peptides 0.6
rF = peak area of bacitracin Ffrom the Sample solution
Bacitracin C3 0.6
rA = peak area of bacitracinAfrom the Sample solution
Bacitracin B1 0.7
-'--- Acceptance criteria: See Table 2. Disregard any peaksfrom
Bacitracin B2 0.7
Active bacitracin the Sample solution that are observed in the Diluent
Bacitracin B3 0.8 chromatogram. Disregard any peaks from the Sample
Bacitracin A 1.0
solution that have a peak area lessthan bacitracin Ain the
, Reporting threshold solution.
Bacitracin F Impurity 2.4
Table 2
Suitability requirements Acceptance
Peak-to-valley ratio: NLT 1.2, System suitability solution Criteria,
Name (%)
The Peak-to-valley ratio is calculated as follows:
Content of bacitracin A NLT 40.0
Result = Hp/H v
Content of active bacitracin NLT 70.0
Hp = height above the baseline of the peak due to Limit of early-eluting peptides NMT 20.0
bacitracin B1
Limit of bacitracin F NMT 6.0
Hv = height above the baseline of the lowest point of
the curve separating the bacitracin B1 peak from
the bacitracin B2 peak • pH (791)
Sample solution: 10,000 Bacitracin units/mL in water
Analysis Acceptance criteria: 5.5-7.5
Samples: Diluent, Reporting threshold solution, and Sample • Loss ON DRYING (731)
solution Sample: 100 mg
Content of bacitracin A Analysis: Drythe Sample in a capillary-stoppered bottle
Calculate the percentage of bacitracin A in the portion of under vacuum at a pressure not exceeding 5 mm of
Bacitracin taken: mercury at 60° for 3 h.
Acceptance criteria: NMT 5.0%
Result = (rA/rr) x 100 • STERILITY TESTS (71): Where the label states that the
Bacitracin is sterile, it meets the requirements.
rA =peak area of bacitracin Afrom the Sample solution
rr = sum of all peak areas above the reporting
threshold from the Sample solution
Content of active bacitracin
Calculate the percentage of active bacitracin (bacitracin
A, B1, B2, and B3) in the portion of Bacitracin taken:
Result = [(rA + r81 + r82 + r83)/rr] x 100
rA = peak area of bacitracin Afrom the Sample solution
r81 = peak area of bacitracin B1 from the Sample
solution

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466 Bacitracin / Official Monographs USP 43

ADDITIONAL REQUIREMENTS SPECIFIC TESTS

• PACKAGING AND STORAGE: Preserve in tight containers and • CONSTITUTED SOLUTION: At the time of use, it meets the
store below 8°. ' requirements in Injections and ImplantedDrug Products (1),
Specific Tests, Completeness and clarity of solutions.
• pH (791)
Sample solution: A solution containing 10,000 Bacitracin
• LABELING: vyhere it is packaged for prescription Units/mL
compounding, label it to indicate that it is not sterile and Acceptance criteria: 5.5-7.5
that the potency cannot be assured for than 60 days • Loss ON DRYING (731)
after Units/ Analysis: Dry 100 mg in a capillary-stoppered bottle under
vacuum at a pressure of NMT 5 mm of mercury at 60° for
3 h.
Acceptance criteria: NMT 5.0%
• STERILITY TESTS (71): It meets the requirements when
tested as directed in Test for Sterilityof the Product to Be
Examined, Membrane Filtration.
• BACTERIAL ENDOTOXINS TEST (85): It contains NMT 0.01
• USP REFERENCE STANDARDS (11) USP Endotoxin Unit/Bacitracin Unit.
USP Bacitracin Zinc RS • OTHER REQUIREMENTS: Meets the requirements in Injections
44(<:NlirV1~Y12()ls) and ImplantedDrug Products (1)
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve as described in
Packaging and Storage Requirements (659), Injection
Bacitracin for Injection Packaging, Packaging for constitution, and store in a cool
place.
DEFINITION • USP REFERENCE STANDARDS (11)
Bacitracin for Injection has a potency of NLT 50 Bacitracin " USP Bacitracin Zinc RS
Units/mg. It contains NLT 90.0% and NMT 115.0% of the
labeled amount of bacitracin.
IDENTIFICATION
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Bacitracin Ointment
(201 BNP): Meets the requirements
DEFINITION
ASSAY Bacitracin Ointment is Bacitracin in an anhydrous ointment
• PROCEDURE base.It contains NLT 90.0% and NMT 140.0% of the labeled
(See Antibiotics-Microbial Assays (81).) amount of bacitracin. It may contain a suitable anesthetic.
Sample solution 1: Nominally 100 Bacitracin" Units/mL,
prepared as follows. Constitute one container of Bacitracin IDENTIFICATION
for Injection as directed in the labeling. Using a suitable • A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
hypodermic needle and syringe, withdraw the contents of (201 BNP): Meets the requirements
the container, and dilute with Buffer B. 1 (see the chapter)
ASSAY
to a suitable volume.
Sample solution 2 (where the label states the number of • PROCEDURE
(See Antibiotics-Microbial Assays (81).)
Bacit~acin Units in a. giv~n vol~me of constituted solution):
Sample solution: Use a portion of Ointment shaken with
Noml~ally 100 Bacltracin Unlts/rnl, prepared asfollows.
about 50 mL of ether in a separator and extracted with four
Constitute one container of Bacitracin for Injection as
20-mL portions of Buffer B. 1 (seethe chapter). Combine the
directed in the labeling. Dilute a suitable aliquot of the
buffer extracts, and dilute with Buffer B. 1 to a suitable
constituted solution with Buffer B.1 (see the chapter) to a
volume.
suitable final volume.
Analysis: Proceed as directed in the chapter. Add sufficient
Analysis
0.01 .N hydrochloric acid to a suitable aliquot of the Sample
Samples: Sample solution 1 or Sample solution2
so.'ut~on ~o that the am<;>unt of hydrochloric acid in the Test
Proceed as directed in the chapter. Add sufficient 0.01 N
Dilution IS the same as In the median level of the standard.
hydrochloric acid to the Sample solution so that the
DiI~te ~ith Buffer B.1.to obtai.n a Te~t Dilution having a
amount of hydrochloric acid in the Test Dilution is the
bacitracln concentration that IS nominally equivalent to the
same as in the median level of the standard. Dilute with
median level of the standard.
Buffer B. 1 to obtain a Test Dilution having a bacitracin
Acceptance criteria: 90.0%-140.0%
concentration that is nominally equivalent to the median
level of the standard. SPECIFIC TESTS
Acceptance criteria: 90.0%-115.0% • WATER DETERMINATION, Method I (921)
Analysis: Use 20 mL of a mixture of toluene and methanol
PERFORMANCE TESTS
(7:3) in place of methanol in the titration vessel.
• UNIFORMITY OF DOSAGE UNITS (905): Meets the
Acceptance criteria: NMT 0.5%
requirements "
• MINIMUM FILL (755): Meets the requirements
IMPURITIES
ADDITIONAL REQUIREMENTS
• RESIDUE ON IGNITION (281)
• PACKAGING AND STORAGE: Preserve in well-closed
Analysis: Moisten the charred residue with 2 mL of nitric
containers containing NMT 60 g, unless labeled solely for
acid and 5 drops of sulfuric acid.
hospital use, preferably at controlled room temperature.
Acceptance criteria: NMT 3.0%

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 USP43 Official Monographs / Bacitracin 467

• USP REFERENCE STANDARDS (11) bacitracin assumed to be equal to the median dose level of
USP Bacitracin Zinc RS the Standard. [NoTE-The amount of hydrochloric acid in
the Test dilution should be the same as that in the median
dose level of the Standard.]
Analysis: Proceed as directed for Bacitracin in Antibiotics-
MicrobialAssays (81).
Bacitracin Ophthalmic Ointment Acceptance criteria: NLT 8 Bacitracin Units/mg on the
dried basis
DEFINITION
Bacitracin Ophthalmic Ointment is a sterile preparation of SPECIFIC TESTS
Bacitracin in an anhydrous ointment base. It contains NLT • pH (791): 8.0-9.5, in a 25 mg/mL solution
90.0% and NMT 140.0% of the labeled amount of • Loss ON DRYING (731): Dry 100 mg in a capillary-stoppered
bacitracin. bottle in vacuum at a pressure not exceeding 5 mm of
mercury at 60° for 3 h: it loses NMT 8.5% of its weight.
IDENTIFICATION
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST ADDITIONAL REQUIREMENTS
(201 BNP): Meets the requirements • PACKAGING AND STORAGE: Preserve in well-closed
containers.
ASSAY • LABELING: Label it to indicate that it isfor veterinary use
• PROCEDURE only.
(See Antibiotics-Microbial Assays (81).) • USP REFERENCE STANDARDS (11)
Sample solution: Use a portion of Ophthalmic Ointment USP Bacitracin Zinc RS
shaken with about 50 mLof ether in a separator and
extracted with four 20-mL portions of Buffer B.1 (see the
chapter). Combine the buffer extracts, and dilute with
Buffer B. 1 to a suitable volume.
Analysis: Proceed as directed in the chapter. Add sufficient Bacitracin Methylenedisalicylate
0.01 N hydrochloric acid to a suitable aliquot of the Sample
solution so that the amount of hydrochloricacid in the Test Soluble Powder
Dilution is the same as in the median level of the standard.
DEFINITION
Dilute with Buffer B.1 to obtain a Test Dilution having a
bacitracinconcentration that is nominallyequivalentto the Bacitracin Methylenedisalicylate Soluble Powder contains NLT
median level of the standard. 90.0% and NMT 120.0% of the labeled amount of
Acceptance criteria: 90.00/0-140.0% bacitracin.
ASSAY
SPECIFIC TESTS
• ANTIBIOTiCS-MICROBIAL ASSAYS (81)
• STERILITY TESTS (71): Meets the requirements
• OTHER' REQUIREMENTS: It meets the requirements for
Diluent: 20 giL of sodium bicarbonate
Particulate and Foreign Matter in OphthalmicProducts- Sample stock solution: Transfer a suitable amount of
Quality Tests (771), Drug Product Quality, Universal Tests, Bacitracin Methylenedisalicylate Soluble Powder to a
Particulate and Foreign Matter. high-speed glass blender jar, add 99.0 mLof Diluent and
1.0 mLof polysorbate 80, and blend for 3 min.
ADDITIONAL REQUIREMENTS Test dilution: To a suitable aliquot of the Sample stock
• PACKAGING AND STORAGE: Preserve in collapsible solution, add a suitable volume of 0.01 N hydrochloric acid
ophthalmic ointment tubes. Store at controlled room and dilute with Buffer B. 1 to obtain a concentration of
. temperature. bacitracin assumed to be equal to the median dose level of
• USP REFERENCE STANDARDS (11) the Standard. [NoTE-The amount of hydrochloric acid in
USP Bacitracin Zinc RS the Test dilution should be the same as that in the median
dose level of the Standard.]
Analysis: Proceed as directed for Bacitracin in Antibiotics-
MicrobialAssays (81).
Acceptance criteria: 90.0%-120.0%
Soluble Bacitracin
SPECIFIC TESTS .
Methylenedisalicylate • pH (791): 8.0-9.5 in a 50 mg/mL solution
• Loss ON DRYING (731): Dry 100 mg in a capillary-stoppered
DEFINITION bottle in a vacuum at a pressure not exceeding 5 mm of
Soluble Bacitracin Methylenedisalicylate is a mixture of mercury at 60° for 3 h: it loses NMT 8.5% of its weight.
Bacitracin Methylenedisalicylate and Sodium Bicarbonate. It
has a potency of NLT 8 Bacitracin Units/mg, calculated on ADDITIONAL REQUIREMENTS
the dried basis. . • PACKAGING AND STORAGE: Preserve in tight containers.
• LABELING: Label it to indicate that it isfor veterinary use
ASSAY only. Label it to state the content of bacitracin in terms of
• ANTIBIOTics-MICROBIAL ASSAYS (81) grams per pound, each gram of bacitracin being equivalent
Diluent: 20 giL of sodium bicarbonate to 42,000 Bacitracin Units.
Sample stock solution: Transfera suitable amount of • USP REFERENCE STANDARDS (11)
Soluble Bacitracin Methylenedisalicylate to a high-speed USP Bacitracin Zinc RS
glass blender jar, add 99.0 mLof Diluent and 1.0 mL of
polysorbate 80, and blend for 3 min.
Test dilution: To a suitable aliquot of the Sample stock
solution, add a suitable volume of 0.01 N hydrochloric acid,
and dilute with Buffer B. 1 to obtain a concentration of

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468 Bacitracin / Official Monographs USP 43

ADDITIONAL REQUIREMENTS
Bacitracin and Polymyxin B Sulfate • PACKAGING AND STORAGE: Preserve in pressurized
Topical Aerosol containers, and avoid exposure to excessive heat.
• USP REFERENCE STANDARDS (11)
DEfiNITION USP Bacitracin Zinc RS
Bacitracin and Polymyxin BSulfate Topical Aerosol is a USP Polymyxin BSulfate RS
suspension of Bacitracin and Polymyxin BSulfate in a suitable
vehicle, packaged in a pressurized container with a suitable
inert propellant. It contains NLT 90.0% and NMT 130.0% of
the labeled amounts of bacitracin and polymyxin B. It may
contain a suitable local anesthetic. Bacitracin Zinc
Prepare the specimen for the following tests and assays as
follows. Maintain the container in the inverted position
throughout this procedure. Store the container in a freezer
at -70 for 16-24 h. Remove the container from the freezer,
0

promptly puncture the container, and allow the propellant

to volatilize. Open the container, and mix the contents.
IDENTifiCATION
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201 BNP)
Sample: Prepare as directed above.
Analysis: Test as directed in the section ForCreams, Lotions, Bacitracins, zinc complex;
and Ointments in the chapter. Bacitracin zinc complex [1405-89-6].
Acceptance criteria: Meets the requirements
DEfiNITION
ASSAY Bacitracin Zinc is the zinc complex of bacitracin, which
• BACITRACIN . consistsof a mixture of antimicrobial polypeptides, the main
(See Antibiotics-Microbial Assays (81).) components being bacitracins A, Bl, B2, and B3. It has a
Sample solution: Use a portion of the contents of one potency of NLT 65 Bacitracin Units/mg, calculated on the
container, containing nominally500 USP Bacitracin Units, dried basis. Itcontains NLT 4.0% and NMT 6.0% of zinc(Zn),
prepared as directed above. Transferto a suitableseparator calculated on the dried basis.
containing 50 mLof ether, and extract with three 25-mL
portions of Buffer B. 1 (see the chapter). Combine the buffer IDENTIFICATION
extracts in a 1OO-mL volumetricflask, dilute with Buffer B. 1 • A. Meets the requirements of the test for Composition of
to volume, and mix. Bacitracin
Analysis: Proceed as directed in the chapter. Add sufficient • B. Meets the requirements of the test for Zinc Content
0.01 N hydrochloric acid to this solutionso that the amount
of hydrochloric acid in the Test Dilution isthe same as in the ASSAY
median level of the standard. Dilute with Buffer B. 1 to • PROCEDURE
obtain a Test Dilution having a bacitracinconcentration that (See Antibiotics-Microbial Assays (81 ).)
is nominally equivalent to the median level of the standard. Analysis: Proceed as directed in the chapter.
Acceptance criteria: 90.0%-130.0% . Acceptance criteria: NlT 65 Bacitracin Units/mg on the
• POLYMYXIN B dried basis
(See Antibiotics-Microbial Assays (81).) SPECIFIC TESTS
Sample solution: Use a portion of the contents of one • COMPOSITION OF BACITRACIN
container, containing nominally5000 USP Polymyxin B Diluent: 40 giL of edetate disodium in water adjusted with
Units, prepared as directed above. Transferto a suitable 8 N sodium hydroxide to a pH of 7.0
separator containing 50 mLof ether, and extract with three Solution A: 34.8 giL of dibasic potassium phosphate in
25-mLportions of Buffer B. 6 (see the chapter). Combine the water
bufferextracts in a 1OO-mL volumetricflask, dilute with Solution B: 27.2 giL of monobasic potassium phosphate
Buffer B.6 to volume, and mix. in water
Analysis: Proceed as directed in the chapter. Dilute a Solution C: Solution B and Solution A (9:2). The pH of the
suitable aliquot of the Sample solution with Buffer B.6 to mixture is about 6.
obtain a Test Dilution having a polymyxin Bconcentration Solution D: 0.1 mM edetate disodium in a mixture of
that is nominallyequivalent to the median level of the Solution C and water (1 :3)
standard. Solution E: Methanol and acetonitrile (27:2)
Acceptance criteria: 90.0%-130.0% Mobile phase: Solution E and Solution 0 (63:37)
SPECifiC TESTS System suitability solution: 2 mg/mL of USP Bacitracin
• WATER DETERMINATION, Method I (921) Zinc RS in Diluent
Analysis: Use a portion of the contents of one container, Reporting threshold solution: 0.01 mg/mL of USP
prepared as directed above, and 20 mLof a mixture of Bacitracin Zinc RS from System suitability solution in water
toluene and methanol (7:3) in place of methanol in the Peak identification solution: 2 mg/mL of USP Bacitracin
titration vessel. Zinc RS in Diluent. Heat in a boilingwater bath for 30 min,
Acceptance criteria: NMT 0.5% and cool to room temperature.
• OTHER REQUIREMENTS: It meets the requirementsfor Topical Sample solution: 2 mg/mL of Bacitracin Zinc in Diluent
Aerosols (603), in the sections Pressure Test, Minimum Fill, Chromatographic system
and Leakage Test. (See Chromatography (621), System Suitability.)
Mode: LC

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USP 43 OfficialMonographs / Bacitracin 469

Detector: UV 254 nm and 300 nm. Quantitative analysis rBI = peak area of bacitracin B1 from the Sample
is performed at 254 nm; 300 nm is only used to identify solution
the location of bacitracin F. rB2 = peak area of bacitracin B2 from the Sample
Column: 4.6-mm x 25-cm; end-capped 5-lJm packing L1 solution
Flow rate: 1 mL/min rB3 = peak area of bacitracin B3 from the Sample
Injection volume: 100 IJL solution
Run time: NLT 3 times the retention time of bacitracin A rr = sum of all peak areas above the reporting
System suitability threshold from the Sample solution
Samples: System suitability solution and Peak identification
solution Limit of early eluting peptides
Analyze the Peak identification solution at 300 nm. Identify Calculate the percentage of early eluting peptides (peaks
bacitracin F,a known impurity, using the relative retention eluting before bacitracin B1) in the portion of Bacitracin
time provided in Table 1. Analyze the System suitability Zinc taken:
solution at 254 nm. Identify the peaks of the most active
components of bacitracin (bacitracins A, B1, B2, and B3), Result = (rplrT) x 100
early eluting peptides (those eluting before the peak due
to bacitracin B1), and the impurity (bacitracin F)using the r, = sum of peak areas for all peaks before bacitracin
relative retention time values in Table 1. B1 from the Sample solution
rr = sum of all peak areas above the reporting
Table 1 threshold from the Sample solution
Relative
Nature of Retention Limit of bacitracin F
Name Component Time Calculate the percentage of bacitracin F in the portion of
Bacitracin Zinc taken:
Bacitracin C1 0.5

Bacitracin C2 Early eluting peptides 0.6 Result = (rJrA) x 100

Bacitracin C3 0.6 .rF = peak area of bacitracin Ffrom the Sample solution
Bacitracin B1 0.7 rA = peak area of bacitracin A from the Sample solution
Bacitracin B2 0.7
Active bacitracin Acceptance criteria: See Table 2. Disregard any peaksfrom
Bacitracin B3 0.8 the Sample solution that are observed in the Diluent
Bacitracin A I 1.0
chromatogram. Disregard any peaksfrom the Sample
solution having a peak area less than bacitracin A in the
Bacitracin F Impurity 2.4 Reporting thresholdsolution.

Suitability requirements Table 2

Peak-to-valley ratio: NLT 1.2 Acceptance
The Peak-to-valley ratio is calculated asfollows: Criteria
(%)
Result = Hpl Hv Content of bacitracin A NLT 40.0

Hp = height above the baseline of the peak due to Content of active bacitracin NLT 70.0
bacitracin B1 . Limit of early eluting peptides NMT 20.0
Hv = height above the baseline of the lowest point of
Limit of bacitracin F NMT 6.0
the curve separating the bacitracin B1 peak from
the bacitracin B2 peak
• ZINC CONTENT
Analysis [NOTE-The Standardsolutions and the Sample solution
Samples: Diluent, Reporting threshold solution, and Sample may be quantitatively diluted with 1 mM
solution hydrochloric acid, if necessary, to obtain solutions of
Quantitative analysis is performed at 254 nm. suitable concentrations, adaptable to the linear or
Content of bacitracin A working range of the instrument.]
Calculate the percentage of bacitracin A in the portion of Standard stock solution: 10 mg/mL of zinc from zinc oxide
Bacitracin Zinc taken: in 1 N hydrochloric acid. Prepare as follows. Transfer a
suitable amount of zinc oxide to a suitable volumetric flask,
Result = (rAlrT) x 100 add 1 N hydrochloric acid using 32% of the final volume,
warm to dissolve, cool and dilute with water to volume.
rA = peak area of bacitracin A from the Sample solution Standard solutions: 0.5, 1.5, and 2.5 IJg/ml of zinc from
rr = sum of all peak areas above the reporting Standardstock solution in 0.001 N hydrochloric acid
threshold from the Sample solution Sample stock solution: 2 mg/mL of Bacitracin Zinc in 0.01
N hydrochloric acid
Content of active bacitracin Sample solution: 0.02 mg/mL of Bacitracin Zinc from
Calculate the percentage of active bacitracin (bacitracin Sample stocksolution in 0.001 N hydrochloric acid
A, B1, B2, and B3) in the portion of Bacitracin Zinc Instrumental conditions
taken: (See Atomic AbsorptionSpectroscopy (852).)
Mode: Atomic absorption spectrophotometry
Result = [erA + rBI + rB2 + rB3)1rT] x 100 Analytical wavelength: 213.8 nm
lamp: Zinc hollow-cathode
rA = peak area of bacitracin A from the Sample solution Flame: Air-acetylene

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470 Bacitracin / OfficialMonographs USP43

Blank: 0.001 N hydrochloric acid hydrochloric acid to obtain the same concentration of
Analysis hydrochloric acid as in the Test Dilution of Ointment.
Samples: Standardsolutions, Sample solution, and Blank Sample solution: Use a portion of Ointment shaken with
Plot the absorbances of the Standardsolutions versus about 50 mL of ether in a separator and extracted with four
concentration, in IJg/mL, of zinc, and draw the straight 20-mL portions of 0.01 N hydrochloric acid. Combine the
line best fitting the three plotted points. From the graph, acid extracts, and dilute with 0.01 N hydrochloric acid to a
determine the concentration, in IJg/mL, of zinc in the suitable volume.
Sample solution. Analysis: Proceed as directed in the chapter. Dilute the
Calculate the percentage of zinc in the portion of Bacitracin Sample solution with Buffer B.1 to obtain a Test Dilution
Zinc taken: having a bacitracin concentration that is nominally
equivalent to the median level of the standard. Add
Result = Cx D x (V/W) x Fx 100 sufficient hydrochloric acid to each Test Dilution of the
standard to obtain the same concentration of hydrochloric
C = concentration of zinc in the Sample solution acid as in the Test Dilution of the sample.
obtained from the curve (lJg/mL) Acceptance criteria: 90.00/0-140.0%
D = dilution factor for the Sample solution, 100
mL/mL SPECIFIC TESTS
V = volume of Sample stock solution (mL) • WATER DETERMINATION, Method 1(921)
W = weight of Bacitracin Zinc used to prepare the Analysis: Use 20 mL of a mixture of toluene and methanol
Sample stocksolution (mg) (7:3) in place of methanol in the titration vessel.
F = conversion factor, 0.001 mg/lJg Acceptance criteria: NMT 0.5%
• MINIMUM FILL (755): Meets the requirements
Acceptance criteria: 4.00/0-6.0% on the dried basis
·pH(791) ADDITIONAL REQUIREMENTS
Sample solution: A saturated solution in water containing • PACKAGING AND STORAGE: Preserve in well-closed
containers containing NMT 60 g, unless labeled solely for
about 100 mg/mL
hospital use, preferably at controlled room temperature.
Acceptance criteria: 6.0-7.5
~ USP REFERENCE STANDARDS (11)
• Loss ON DRYING (731)
USP Bacitracin Zinc RS
Sample: 100 mg
Analysis: Dry the Sample in a capillary-stoppered bottle
under vacuum at 60° for 3 h.
Acceptance criteria: NMT 5.0%
• STERILITY TESTS (71): Where the label states that it is sterile, Bacitracin Zinc Soluble Powder
it meets the requirements of the chapter. If the membrane
filtration test is used, add 20 g of edetate disodium to each
L of FluidA. » Bacitracin Zinc Soluble Powder is a mixture of
ADDITIONAL REQUIREMENTS Bacitracin Zinc and zinc proteinates. It contains not
• PACKAGING AND STORAGE: Preserve in tight containers, and less than 90.0 percent and not more than 120.0
store below 25°. percent of the labeled amount of bacitracin.
• LABELING: Label it to indicate that it is to be used in the
manufacture of nonparenteral drugs only. Where it is Packaging and storage-Preserve in tight containers.
packaged for prescription compounding, label it to indicate Labeling-Label it to indicate that it is for veterinary use only.
that it is not sterile and that the potency cannot be assured Label it to state the content of bacitracin in terms of grams per
for longer than 60 days after opening, and to state the pound, each gram of bacitracin being equivalent to 42,000
number of Bacitracin Units/mg. Where it is intended for use Bacitracin Units.
in preparing sterile dosage forms, the label states that it is USP Reference standards (11)-
sterile or must be subjected to further processing during the USP Bacitracin Zinc RS
preparation of sterile dosage forms. Loss on drying (731)-Dry about 100 mg, accurately
• USP REFERENCE STANDARDS (11) weighed, in a capillary-stoppered bottle in vacuum at a
USP Bacitracin Zinc RS pressure not exceeding 5 mm of mercury at 60° for 3 hours:
it loses not more than 5.0% of its weight.
Zinc content-Using Powder, proceed as directed for Zinc
Content under Bacitracin Zinc. Calculate the zinc content, in g,
in relation to each 42,000 Bacitracin Units in the specimen
Bacitracin Zinc Ointment taken by the formula:
DEFINITION 280,000C/WA
Bacitracin Zinc Ointment is Bacitracin Zinc in an anhydrous
ointment base. It contains NLT 90.0% and NMT 140.0% of in which A is the bacitracin content of the specimen, in
the labeled amount of bacitracin. Bacitracin Units per g, and the other terms are as defined
IDENTIFICATION therein: it contains not more than 2.0 g for each 42,000
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Bacitracin Units.
(201 BNP): Meets the requirements Assay-Dissolve an accurately weighed quantity of Powder
quantitatively in 0.01 N hydrochloric acid to obtain a stock
ASSAY solution containing about 100 Bacitracin Units per mL.
• PROCEDURE Proceed as directed under Antibiotics-Microbial Assays (81),
(See Antibiotics-Microbial Assays (81).) using an accurately measured volume of this stock solution
Standard solution: Proceed as directed in the chapter. To diluted quantitatively and stepwise with Buffer B. 1 to obtain a
each Test Dilution of the standard add sufficient Test Dilution having a concentration assumed to be equal to

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 USP 43 Official Monographs / Baclofen 471

the median dose level of the Standard. In preparing each test

dilution of the Standard, add additional hydrochloric acid to
Bacitracin Zinc and Polymyxin B Sulfate
each to obtain the same concentration of hydrochloric acid as Ophthalmic Ointment
in the Test Dilution.
DEFINITION
Bacitracin Zinc and Polymyxin B Sulfate Ophthalmic Ointment
contains the equivalent of NLT 90.0% and NMT 130.0% of
the labeled amounts of bacitracin and polymyxin B.
Bacitracin Zinc and Polymyxin B
IDENTIFICATION
Sulfate Ointment • A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
(201 BNP): Meets the requirements
DEFINITION
Bacitracin Zinc and Polymyxin B Sulfate Ointment contains the ASSAY
equivalent of NLT 90.0% and NMT 130.0% of the labeled • BACITRACIN
amounts of bacitracin and polymyxin B. It may contain a (See Antibiotics-Mic;:robial Assays (81).)
suitable local anesthetic. Standard solution: Proceed as directed in the chapter. To
each Test Dilution of the standard add sufficient
IDENTIFICATION hydrochloric acid to obtain the same concentration of
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST hydrochloric acid as in the Test Dilution of Ophthalmic
(201 BNP): Meets the requirements Ointment.
ASSAY Sample solution: Use a portion of Ophthalmic Ointment
• BACITRACIN shaken with about 50 mL of ether in a separator and
(See Antibiotics-Microbial Assays (81». extracted with four 20-mL portions of 0.01 N hydrochloric
acid. Combine the acid extracts, and dilute with 0.01 N
Standard solution: Proceed as directed in the chapter. To
each Test Dilution of the standard add sufficient hydrochloric acid to a suitable volume.
hydrochloric acid to obtain the same concentration of Analysis: Proceed as directed in the chapter. Dilute the
hydrochloric acid as in the Test Dilution of Ointment. Sample solution with Buffer B. 7 to obtain a Test Dilution
Sample solution: Shake a portion of Ointment with about having a bacitracin concentration that is nominally
50 mL of ether in a separator, and extract with four 20-mL equivalent to the median level of the standard.
portions of 0.01 N hydrochloric acid. Combine the acid Acceptance criteria: 90.00/0-130.0%
extracts, and dilute with 0.01 N hydrochloric acid to a • POLYMYXIN B
suitable volume. (See Antibiotics-Microbial Assays (81).)
Analysis: Proceed'as directed in the chapter. Dilute the Sample solution: Shake a portion of Ophthalmic Ointment
Sample solution with Buffer 8. 7 to obtain a Test Dilution with about 50 mL of ether in a separator, and extract with
having a bacitracin concentration that is nominally four 20-mL portions of Buffer B.6 (see the chapter).
equivalent to the median level of the standard. Add Combine the buffer extracts, and dilute with Buffer B.6 to a
sufficient hydrochloric acid to each Test Dilution of the suitable volume.
standard to obtain the same concentration of hydrochloric Analysis: Proceed as directed in the chapter. Dilute the
acid as in the Test Dilution of the sample. Sample solution with Buffer B.6 to obtain a Test Dilution
Acceptance criteria: 90.0%-130.0% having a polymyxin B concentration that is nominally
• POLYMYXIN B equivalent to the median level of the standard.
(See Antibiotics-Microbial Assays (81 » .. Acceptance criteria: 90.0%-130.0%
Sample solution: Shake a portion of Ointment with about SPECIFIC TESTS
50 mL of ether in a separator, and extract with four 20-mL • STERILITY TESTS (71): Meets the requirements
portions of Buffer B.6 (seethe chapter). Combine the buffer • OTHER REQUIREMENTS: It meets the requirements for
extracts, and dilute with BufferB.6 to a suitable volume. Particulate and Foreign Matter and Container Contents in
Analysis: Proceed as directed in the chapter. Dilute the Ophthalmic Products-Quality Tests (771), Drug Product
Sample solution with Buffer 8.6 to obtain a Test Dilution Quality, Universal Tests, Particulate and Foreign Matter and
having a polymyxin B concentration that is nominally ContainerContents. '.
equivalent to the median level of the standard.
Acceptance criteria: 90.00/0-1 30.0% ADDITIONAL REQUIREMENTS
• PACKAGI~G AND STORAGE: Preserve in collapsible
SPECIFIC TESTS ophthalmic ointment tubes. Store at controlled room
• WATER DETERMINATION, Method I (921) temperature.
Analysis: Use 20 mL of a mixture of toluene and methanol • USP REFERENCE STANDARDS (11)
(7:3) in place of methanol in the titration vessel. USP Bacitracin Zinc RS
Acceptance criteria: NMT 0.5% USP Polymyxin B Sulfate RS
• MINIMUM FILL (755): Meets the requirements
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed,
light-resistant containers. Baclofen
• USP REFERENCE STANDARDS (11)
USP Bacitracin Zinc RS
USP Polymyxin B Sulfate RS

213.66

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472 Baclofen / Official Monographs USP 43

Butanoic acid, 4-amino-3-(4-chlorophenyl)-; IMPURITIES

p-(Aminomethyl)-p-chlorohydrocinnamic acid [1134-47-0]. • RESIDUE ON IGNITION (281): NMT 0.3%
DEFINITION • ORGANIC IMPURIT8ES
Solution A, Solution B, Diluent, Mobile phase, and
Baclofen contains NLT 98.0% and NMT 102.0% of baclofen Chromatographic system: Proceed as directed in the
(C1oH 12CIN02) , calculated on the anhydrous basis. Assay.
IDENTIFICATION Standard solution: 0.0015 mg/mL of USP Baclofen RS and
0.003 mg/mL of USP Baclofen Related Compound A RS in
Diluent
Sample solution: 0.3 mg/mL ofBaclofen in Diluent
• A •.••~• ~.~~~~ System suitability
SpgctcP§Goj:f._... • . _ 0) Sample: Standard solution
• B. The retention time of the Sample solution corresponds to [NoTE-See Table 2 for relative retention times.]
that of the Standard solution, as obtained in the Assay. SUitability requirements
Tailing factor: NMT 1.5 for baclofen
ASSAY Relative standard deviation: NMT 5.0% for both
• PROCEDURE baclofen and baclofen related compound A
Solution A: Dissolve 1.38 9 of potassium dihydrogen Analysis
phosphate and 1.74 9 of sodium-l-pentanesulfonate in 1 L Samples: Standard solution and Sample solution
of water. Adjust with dilute phosphoric acid to a pH of 3.0. Calculate the percentage of baclofen related compound
Solution B: Acetonitrile and methanol (1:1) A in the portion of the Baclofen taken:
Diluent: Solution A and Solution B (65:35)
Mobile phase: See Table 1. Result = (r vir s) x (C siC v) x 100

Table 1 = peak response of baclofen related compound A

Time Solution A Solution B from the Sample solution
(min) (%) (%) = peak response of baclofen related compound A
from the Standard solution
0 65 35
= concentration of USP Baclofen Related
5 65 35 Compound A RS in the Standard solution
15 45 55
(mg/mL)
= concentration of Baclofen in the Sample solution
25 45 55 (mg/mL)
27 65 35
Calculate the percentage of any unspecified impurity in
30 65 35 the portion of the Baclofen taken:

Standard solution: 0.2 mg/mL of USP Baclofen RS in Diluent Result = (r vir s) x (C siC v) x 100
Sample solution: 0.2 mg/mL of Baclofenin Diluent
Chromatographic system ru = peak response of any unspecified impurity from
the Sample solution
(See Chromatography (621), System Suitability.)
Mode: LC . rs = peak response of baclofen from the Standard
Detector: UV 225 nm solution
Column: 4.6-mm x 25.0-cm; 5-l-Im packing Ll
Cs = concentration of USP Baclofen RS in the Standard
Column temperature: 35° solution(mg/mL)
Cu = concentration of Baclofen in the Sample solution
Flow rate: 0.8 mL/min
Injection volume: 10 I-IL
(mg/mL)
System suitability
Acceptance criteria: See Table 2.
Sample: Standard solution
SUitability requirements Table 2
Tailing factor: NMT 1.5
Relative standard deviation: NMT 1.0% Relative Acceptance
Retention Criteria,
Analysis Name Time NMT(%)
Samples: Standard solution and Sample solution
Calculate the percentage of baclofen (ClO H12CIN02) in the Baclofen 1.0 -
portion of the Baclofen taken: Baclofen related compound A 2.3 1.0

Result = (r vir s) x (C siC v) x 100 Any individual unspecified impurity - 0.10

Total impurities -, 2.0
ru = peak response from the Sample solution
rs = peak response from the Standard solution SPECIFIC TESTS
Cs =concentration of USP Baclofen RS in the Standard • WATER DETERMINATION, Method I (921): NMT 3.0%
solution (mg/mL)
Cu = concentration of Baclofen in the Sample solution ADDITIONAL REQUIREMENTS
(mg/mL) • PACKAGING AND STORAGE: Preserve in tight containers.
Store at room temperature.
Acceptance criteria: 98.0%-102.0% on the anhydrous • USP REFERENCE STANDARDS (11)
basis USP Baclofen RS

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USP 43 OfficialMonographs / Baclofen 473

USP Baclofen Related Compound A RS Acceptance criteria: 90.0%-110.0%

4-( 4-Chlorophenyl)-2-pyrrolidinone.
ClOHlOCINO 195.65 SPECIFIC TESTS
• pH (791): 4.2-5.2
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Package in tight, light-resistant
containers. Store in a refrigerator.
Bac.ofen Compounded Oral Suspension lit BEYOND-USE DATE: NMT 35 days after the day on which it
was compounded when stored in a refrigerator
DEFINITION lit LABELING: Label it to state that it is to be well shaken, and
Baclofen Compounded Oral Suspension contains NLT 90.0% to state the Beyond-Use Date.
and NMT 110.0% of the labeled amount of baclofen • USP REFERENCE STANDARDS (11)
(CloH12CIN02)' USP Baclofen RS
Prepare Baclofen Compounded Oral Suspension 5 mg/mL as
follows (see Pharmaceutical Compounding-Nonsterile
Preparations (795»).

Baclofen 500mg Baclofen Tablets

Vehicle: a 1:1 mixture of Vehicle for Oral DEFINITION
Solution (regular or sugar-free), NF, and Vehicle for
Oral Suspension, NF, a sufficient quantity to make 100 ml Baclofen Tablets contain NLT 90.0% and NMT 110.0% of the
labeled amount of baclofen (C1oH 12CIN02).
If using Baclofen Tablets, place the Tablets in a suitable mortar IDENTIFICATION
and comminute to a fine powder, or add Bac/ofen powder. • A. The retention time of the Sample solution corresponds to
Add 5 mL of the Vehicle to wet the powder, and triturate the that of the Standardsolution, as obtained in the Assay.
powder to form a fine paste. Add the Vehicle in small portions lit B. The UV spectrum of the major peak of the Sample

almost to volume, and mix thoroughly after each addition. . solution corresponds to that of the Standardsolution, as
Transfer, stepwise and quantitatively, the contents of the obtained in the Assay.
mortar to a calibrated bottle. Add sufficient Vehicle to bring
to final volume, and mix well. ASSAY
• PROCEDURE
ASSAY Solution A: 1.4 giL of monobasic sodium phosphate and
• PROCEDURE I 1.7 giL of sodium 1-pentanesulfonate in water. Adjust with
Mobile phase: Acetonitrile and 0.05 M monobasic sodium 1.5 M phosphoric acid TS to a pH of 3.0.
phosphate (20:80). Adjust with phosphoric acid to a pH of Solution B: Acetonitrile and methanol (50:50)
3.5. Mobile phase: See Table}.
Standard solution: 5 IJg/mL of USP Baclofen RS in water
Sample solution: Shake thoroughly by hand each bottle of Table 1
Oral Suspension. Pipet 0.5 mL of Oral Suspensionfrom each Time Solution A Solution B
bottle to a 500-mL volumetric flask, dilute with water to (min) (%) (%)
volume to obtain a concentration of 5 IJg/mL, and pass
0 65 35
through a 0.22-lJm polyvinylidene fluoride (PVDF) filter.
Chromatographic system 5 65 35
(See Chromatography (621), System Suitability.)
15 45 55
Mode: LC
Detector: UV 220 nm 25 45 55
Column: 4.6-mm x 25-cm; 5-lJm packing L1 27 65 35
Flow rate: 1 mL/min
Injection volume: 15 IJL 35 65 35
System suitability
Sample: Standardsolution Diluent: Solution A and Solution B (65:35)
[NoTE-The retention time of baclofen is about 5.5 Standard solution: 200 IJg/mL of USP Baclofen RS in Diluent
min.] Sample solution: Nominally 200 IJg/mL of baclofen
Suitability requirements prepared as follows. Finely powder NLT 20 Tablets and
Relative standard deviation: NMT 2.0% for replicate transfer a portion of the powder to an appropriate
injections volumetric flask. Add Diluent to about 80% of the flask
Analysis volume, sonicate for 10 min, and shake by mechanical
Samples: Standardsolution and Sample solution means for 30 min. Dilute with Diluentto volume. Centrifuge
Calculate the percentage of the labeled amount of baclofen a portion of this solution and use the supernatant.
(Cl0H12CIN02) in the portion of Oral Suspension taken: Chromatographic system
(See Chromatography (621); System Suitability.)
Result = (rulrs) x (CsICu) x 100 Mode: LC
Detector: UV 225 nm. For Identification B, use a diode array
= peak response from the Sample solution detector in the range of 200-400 nm.
= peak response from the Standardsolution Column: 4.6-mm x 25-cm; 5-lJm packing L1
= concentration of USP Baclofen RS in the Standard Column temperature: 35°
solution (lJg/mL) Flow rate: 0.8 mL/min
= nominal concentration of baclofen in the Sample Injection volume: 10 IJL
solution (lJg/mL)

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474 Baclofen / Official Monographs USP 43

System suitability IMPURITIES

Sample: Standardsolution • ORGANIC IMPURITIES
Suitability requirements Solution A, Solution B, Mobile phase, Diluent, Sample
Tailing factor: NMT 2.0 solution, and Chromatographic system: Proceed as
Relative standard deviation: NMT 1.0% directed in the Assay.
Analysis Standard solution: 0.4 IJg/mL of USP Baclofen RS and 8 IJgI
Samples: Standardsolution and Sample solution mL of USP Baclofen Related Compound A RS in Diluent
Calculate the percentage of the labeled amount of baclofen System suitability
(ClOH12CINOz) in the portion of Tablets taken: Sample: Standard solution
[NoTE-See Table 2 for the relative retention times.]
Result = (r vir s) x (C siC v) x 100 Suitability requirements
Tailing factor: NMT 1.5 for baclofen
ru = peak response from the Sample solution Relative standard deviation: NMT 5.0% each for
rs = peak response from the Standardsolution baclofen and baclofen related compound A
Cs = concentration of USP Baclofen RS in the Standard Analysis
solution (pq/rnt) Samples: Standard solution and Sample solution
Cu = nominal concentration of baclofen in the Sample Calculate the percentage of baclofen related compound A
solution (lJg/mL) in the portion of Tablets taken:

Acceptance criteria: 90.0%-110.0% Result = (r vir s) x (C siC u) x 100

PERFORMANCE TESTS ru = peak response of baclofen related compound A
• DISSOLUTION (711), Procedure, Apparatus 7 and Apparatus 2, from the Sample solution
Immediate-Release Dosage Forms, Procedure for a pooled rs =peak response of baclofen related compound A
sample for immediate-release dosage forms from the Standard solution
Medium: 0.01 N hydrochloric acid; 500 mL for Tablets Cs =concentration of USP Baclofen Related
containing NMT 10 mg of baclofen; 1000 mL for Tablets Compound A RS in the Standard solution
containing more than 10 mg of baclofen (lJg/mL)
Apparatus 2: 50 rpm Cu = nominal concentration of baclofen in the Sample
Time: 30 min solution (lJg/mL)
Solution A: 62.7 giL of sodium l-pentanesulfonate in water
Mobile phase: Methanol, 0.3 N acetic acid, and Solution A Calculate the percentage of any individual degradation
(44:55:2) product in the portion of Tablets taken:
Standard solution: USP Baclofen RS in Medium
Sample solution: Use a portion of the solution under test. Result =(r vir s) x (C siC v) x 100
Chromatographic system
(See Chromatography (621), System Suitability.) ru = peak response of any individual degradation
Mode: LC product from the Sample solution
Detector: UV 265 nm rs = peak response of baclofen from the Standard
Column: 3.9-mm x 30-cm; 10-lJm packing Ll solution
Flow rate: 0.6 mL/min Cs = concentration of USP Baclofen RS in the Standard
Injection volume: 190 IJL solution (uq/rnl)
Run time: NLT2 times the retention time of the baclofen Cu = nominal concentration of baclofen in the Sample
peak solution (lJg/mL)
System suitability
Sample: Standardsolution Acceptance criteria: See Table 2.
Suitability requirements
Relative standard deviation: NMT 2.0% Table 2
Analysis Relative Acceptance
Samples: Standardsolution and Sample solution Retention Criteria,
Calculate the percentage of the labeled amount of baclofen Name Time NMT(%)
(C,oH,zCINOz) dissolved: Baclofen 1.0 -
Result =(r vir s) xC s x Vx (lIL) x 100 Baclofen related com-
pound A 3.0 4.0
= peak response from the Sample solution Anyindividual
= peak response from the Standard solution degradation
product
-
0.2
=concentration of USP Baclofen RS in the Standard
solution (mg/mL) Total degradation prod-
ucts - 1.0'
V = volume of Medium; 500 or 1000 mL
L = label claim (mg/Tablet) a Baclofen relatedcompound Aisnot includedinthe total degradation products.
Tolerances: NLT75% (Q) ofthe labeled amount of baclofen
(C,oH12CINOz) is dissolved.
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
containers. Store at controlled room temperature.
requirements • USP REFERENCE STANDARDS (11)
USP Baclofen RS

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 USP 43 OfficialMonographs / Balsalazide 475

USP Baclofen Related Compound A RS of the USP Reference Standard to calculatethe

4-(4-Chlorophenyl)-2-pyrrolidinone. concentration, as appropriate.]
C1oH lOCINO 195.65 Sample solution: 1 mg/mL of Balsalazide Disodium in Diluent
Mobile phase: See the gradient table below.

Time Solution A Solution B

(min) (%) (%)
Balsalazide Disodium
0 90 10
4 90 10
• 2H,O 40 75 25
47 75 25
55 50 50
C17H13N3Naz06·2HzO 437.31 60 50 50
Benzoic acid, 5-[[4-[[(2-carboxyethyl)amino]carbonyl]
phenyl]azo]-2-hydroxy-, disodium salt, dihydrate, (E)-; 60.1 90 10
(E)-5-[[p-[(2-Carboxyethyl)carbamoyl]phenyl]azo]salicylic 70 90 10
acid, disodium salt, dihydrate [150399-21-6].
DEFINITION Chromatographic system
Balsalazide Disodium contains NLT 98.0% and NMT 102.0% (See Chromatography (621), System Suitability.)
of C17H13N3Naz06 . 2HzO, calculated on the as-is basis. Mode: LC
IDENTIFICATION
Detector: UV 238 nm
Column: 4.6-mm x 25-cm; 5-J,Jm packing L1
Column temperature: 45°
Flow rate: 1 mL/min
Injection size: 30 J,JL
System suitability
Sample: Standardsolution
Suitability requirements
Resolution: NLT 5 between balsalazide and balsalazide
related compound B
• B~. •. ~1~~~~~g~~9~~,f!~~~!I.~~~~!g,~'~!~~~?$~.i~~~' Relative standard deviation: NMT 5% for each peak
'l.lltCgVlglgt7'~l~tPlg/§pg,fJCg~q.9PX:il~?""A'(CN·.·.l,.M~Y·~Q20) Tailing factor: NMT 1.8 for the balsalazide peak
Sample solution: 10 J,Jg/mL in water
• C. IDENTIFICATION TESTS-GENERAL, Sodium (191) Analysis
Samples: Standardsolution and Sample solution
ASSAY Calculatethe percentage of each individual impurity in the
• PROCEDURE portion of Balsalazide Disodium taken:
Sample: 219 mg
Analysis: Add 80 mLof glacial acetic acid to the Sample, Result = (ru/rs) x (Cs/C u) x 100
sonicate to dissolve, and titrate with 0.1 N perchloric acid
VS. Perform a blankdetermination, and make any necessary ru = peak response for each individual impurityfrom
correction (see Titrimetry (541 ». Each mLof 0.1 N . the Sample solution
perchloric acid is equivalent to 21.87 mg of rs = peak response for the corresponding impurity
C17H13N3Naz06 . 2HzO. from the Standardsolution. [NoTE-For unspecified
Acceptance criteria: 98.0%-102.0% on the as-is basis impurities, rs is the peak response for the
balsalazide peak from the Standard solution.]
IMPURITIES Cs = concentration of the corresponding impurity in
ORGANIC IMPURITIES the Standardsolution (mg/mL). [NoTE-For
[NOTE-On the basis of the synthetic route, perform unspecified impurities, Cs isthe concentration of
either Procedure 7 or Procedure 2. Procedure 2 is balsalazide disodium in the Standard solution.]
recommended when impurities 1, 2, and 3 listed in Cu = concentration of Balsalazide Disodium in the
Impurity Table 2 may be present.] Sample solution (mg/mL)
• Procedure 1
Solution A: Dissolve 2.7 9 of monobasic potassium Acceptance criteria
phosphate in 1000 mLof water, and adjust with 10% Individual impurities: See Impurity Table 7.
potassium hydroxide solution to a pH of 6.00±0.1. Reporting level for impurities: 0.03%
Diluent: Use Solution A. Total impurities: NMT 1.0%
Solution B: Use acetonitrile.
Standard solution: 0.5 J,Jg/mL of USP Balsalazide Disodium Impurity Table 1
RS, 0.5 J,Jg/mL of USP Balsalazide Related Compound A RS, Relative Acceptance
0.5 J,Jg/mL of USP Balsalazide Related Compound B RS, and Retention Criteria,
0.5 J,Jg/mL of USP Salicylic Acid RS in Diluent. Ifneeded, a Name Time NMT (%)
small amount of acetonitrile may be added to facilitate Salicylic acid 0.37 0.05
dissolution. [NOTE-USP Balsalazide Related Compound A RS
is the disodium salt of (E)-5-[(p-carboxyphenyl)azo]- Balsalazide related
2-salicylic acid. Use the correction factor stated on the label compound A" 0.70 0.05
Balsalazide 1.00 -

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 476 Balsalazide / OfficialMonographs USP 43

Impurity Table 1 (continued) Calculate the percentage of each individual impurity in the
Relative Acceptance portion of Balsalazide Disodium taken:
Retention Criteria,
Name Time NMT (%) Result =(ru/rs) x (Cs/Cu) x (1 IF) x 100
Balsalazide related
compound Bb 1.2 0.05 ru = peak response for each individual impurity from
the Sample solution
Any other individual unspeci- - 0.05 rs = peak response for balsalazide from the Standard
fied impurity
solution
a (E)-5-[(p-Carboxyphenyl)azo]-2-salicylic acid. Cs =concentration of USP Balsalazide Disodium RS in
b (E)-5-({m-[(2-Carboxyethyl)carbamoyl]phenyl}azo)-2-salicylic acid. the Standard solution
Cu = concentration of Balsalazide Disodium in the
• Procedure 2 Sample solution (mg/mL) .
Solution A: Prepare 50 mM monobasic potassium phosphate F = relative response factor (see Impurity Table 2)
buffer as follows: Dissolve 6.8 9 of monobasic potassium
phosphate in 1000 mL of water, and adjust with 2 N Acceptance criteria
potassium hydroxide solution to a pH of 6.8-7.0. [NOTE-To Individual impurities: See Impurity Table 2.
ensure proper identification of impurity 1, the pH must be Reporting level for impurities: 0.03%
maintained between 6.8 and 7.0.] Total impurities: NMT 0.50%
Solution B: Acetonitrile, methanol and Solution A (5:1 :14)
Solution C: Acetonitrile, methanol and Solution A (9:1:10) Impurity Table 2
Diluent: Use Solution B. Relative Relative Acceptance
Mobile phase: See the gradient table below. Retention Response Criteria,
Name Time Factor NMT(%)

Time Solution A Solution B Solution C N-(4-Aminobenzoyl)-P

(min) (%) (%) (%) -alanlnes 0.29 1.8 0.05

a 100 a a Salicylic acid 0.55 1.4 0.05

-- Balsalazide related 0.88 1.4 0.05
37 a 100 0
compound Ab
60 a a 100
Impurity l C 0.91 1.9 0.05
75 100 a a Impurity 2 d 0.92 1.4 0.05
85
, 100 a a Impurity 3e 0.94 0.83 0.05

Standard solution: 0.075 mg/mL of USP Balsalazide Balsalazide 1.00 -

Disodium RS in Diluent. [NoTE-Use sonication to dissolve.] Impurity 41 1.35 2.1 0.05
Sensitivity solution: 0.375 I-Ig/mL in Diluent,'from Standard
solution Impurity 59 1.77 0.91 0.05
System suitability solution: 1.5 mg/mL of USP Balsalazide Any other individual - - 0.05
Disodium RS and 1.5 I-Ig/mL of USP Balsalazide Related unspecified impurity
Compound A RS in Diluent
Sample solution: 1.5 mg/mL of Balsalazide Disodium in aThis impurity may be present as two peaks.Use the sum of the two peaks to
determine compliance.
Diluent. [NoTE-Use sonication to dissolve.] .
b (E)-5-[(p-Carboxyphenyl)azo]-2-salicylic acid.
Chromatographic system c (E,E)-3,5-di-[4-(2-Carboxyethylcarbamoyl) phenylazo]-salicylic acid.
(See Chromatography (621), System Suitability.) d (E)-3-[4-(2-Carboxyethylcarbamoyl) phenylazo]-salicylic acid.
Mode: LC e (E, E)-5-{{2-[4-( 2-Carboxyethylcarbamoyl)phenylazo]-4-[2-carboxyethyl-
Detector: UV 300 nm carbamoyl]}phenylazo}-salicylic acid.
Column: 4.6-mm x 15-cm; 3-l-Im packing L1 f (E)-2-[4-(2-Carboxyethylcarbamoyl)phenoxy]-5-{[4-(2-carboxyethyl-
Column temperature: 2SO-27°. [NOTE-To ensure proper carbamoyl)]phenylazo}-benzoic acid
identification of impurity 1, the column temperature must 9 (E)-2-Hydroxy-5-[[4-[[(3-isopropoxy-3-oxopropyl)amino]carbonyl]
phenyl]azo]benzoic acid
be maintained between 2SO and 2r.]
Flow rate: 1 mL/min
Injection size: 10 I-IL SPECIFIC TESTS
System suitability • WATER DETERMINATION, Method la (921): 7.80/0-9.0%
Samples: Standard solution, Sensitivity solution, and System ADDITIONAL REQUIREMENTS
suitability solution • PACKAGING AND STORAGE: Preserve in tight containers.
Suitability requirements Store at controlled room temperature.
Resolution: NLT 8.5 between balsalazide related • LABELING: Ifa test for Organic Impurities other than Test 1 is
compound A and balsalazide, from the System SUitability used, the labeling states the test with which the article
solution complies. .
Tailing factor: NMT 3.4 for the balsalazide peak, from the • USP REFERENCE STANDARDS (11)
System suitability solution USP Balsalazide Disodium RS
Signal-to-noise ratio: NLT 10, from the Sensitivity solution USP Balsalazide Related Compound A RS
Relative standard deviation: NMT 2.0%, from the (E)-5-[(p-Carboxyphenyl)azo]-2-salicylic acid, disodium
Standard solution salt.
Analysis C'4HaN20sNa2 330.12
Samples: Standard solution and Sample solution

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USP 43 .Official Monographs / Balsalazide 477

USP Balsalazide Related Compound B RS Cu = nominal concentration of balsalazide disodium

(£)-5-({m-[(2-Carboxyethyl)carbamoyl]phenyl}azo)-2- in the Sample solution (mg/mL)
salicylic acid.
C17H1SN306 357.17 Acceptance criteria: 90.0%-110.0%
USP Salicylic Acid RS IMPURITIES
ORGANIC IMPURITIES
• Procedure
Buffer: Dissolve 2.7 9 of monobasic potassium phosphate in
Balsalazide Disodium Capsules 1000 mL ofwater, and adjust with 10% potassium hydroxide
solution to a pH of 6.00 ± 0.1.
DEfiNITION Diluent: Water
Balsalazide Disodium Capsules contain NLT 90.0% and NMT Solution A: Buffer
110.0% of the labeled amount of balsalazide disodium Solution B: Acetonitrile
(C17H13N3Na206·2H20). Sample solution: Transferan amount of finely crushed
powder equivalent to 100 mg of balsalazide disodium to a
IDENTifiCATION 1OO-mL volumetricflask, add 70 mLof Diluent, sonicate for
• A. The retention time of the major peak of the Sample 5 min, and dilute with Diluent to volume. Passa portion of
solution corresponds to that of the Standardsolution, as this solution through a PVDF filterof 0.45-lJm pore size.
obtained in the Assay. Standard solution: 1.0 IJg/mL of USP Balsalazide Disodium
RS in Diluent
System suitability solution: 1.0 pg/mL of USP Balsalazide
Disodium RS, 1.5 IJg/mL of USP Balsalazide Related .
c Compound A RS, 0.5 IJg/mL of USP Balsalazide Related
'" ,",",>,~, _"Q)
trz:""", ..."JPe",pgt:€""",>"" Compound B RS, and 0.5 IJg/mL of USP Salicylic Acid RS in
Using a 0.2-cm cell, record the UV spectrum of the Sample Diluent
solution, obtained in the Assay, in the range of 200-400 Mobile phase: See the gradient table below.
nm: it exhibits maxima at about 261 nm and 357 nm.
ASSAY Time Solution A Solution B
(min) (%) (%)
• PROCEDURE
Buffer: Add 5 mL of triethylamine to 1000 mLof water, and 0 90 10
adjust with phosphoric acid to a pH of 6.00 ± 0.1.
Mobile phase: Acetonitrile and Buffer (1 :4) 4 90 10
Diluent: Water 40 75 25
Standard solution: 60 IJg/mL of USP Balsalazide Disodium
47 75 25
RS in Diluent. [NoTE-Use sonication as necessary.]
Sample stock solution: Transferan equivalent to 150 mg of 55 50 50
balsalazide disodium, from the Capsulescontents, to a 60 50 50
1OO-mL volumetricflask, add 70 mLof Diluent, sonicate for
5 min, and dilute with Diluent to volume. 60.1 90 10
Sample solution: 60 IJg/mL of balsalazide disodium, from 70 90 10
the Sample stock solution, in Diluent. Pass a portion of this
solution through a suitable filter, discarding the first 3 mL.
Chromatographic system Chromatographic system
(See Chromatography (621), System Suitability.) (See Chromatography (621), System Suitability.)
Mode: LC Mode: LC
Detector: UV 360 nm Detector: UV 238 nm
Column: 4.6-mm x 25-cm; 5-lJm packing L1 Column: 4.6-mm x 25-cm; 5-lJm packing L1
Column temperature: 30° Column temperature: 45°
Flow rate: 1 mL/min Flow rate: 1 mL/min
Injection size: 10 IJL Injection size: 30 IJL
System suitability System suitability
Sample: Standardsolution Samples: Standardsolution and System suitability solution
Suitability requirements Suitability requirements
Column efficiency: NLT 10,000 theoretical plates Resolution: NLT 5 between balsalazide and balsalazide
Tailing factor: NMT 2.0 related compound B, System suitability solution
Relative standard deviation: NMT 2.0% Relative standard deviation: NMT 5.0%, Standard
Analysis solution
Sample: Standardsolution and Sample solution Tailing factor: NMT 1.5, Standardsolution
Calculate the percentage of the labeled amount of Analysis
C17H13N3Na206' 2H20 in the portion of Capsules taken: Samples: Standardsolution and Sample solution
Calculate the percentage of each impurity in the portion of
Result =(ru/rs) x (Cs/C u) x 100 Capsules taken:

= peak response from the Sample solution Result = (ru/r s) x (Cs/Cu) x (1 IF) x 100
= peak response from the Standardsolution = peak response for each individual impurityfrom
=concentration of USP Balsalazide Disodium RS in the Sample solution
the Standardsolution (mg/mL) = peak response for the balsalazide peak from the
Standardsolution

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478 Balsalazide / OfficialMonographs USP 43

C, = concentration of USP Balsalazide Disodium RS in • USP REFERENCE STANDARDS (11)

the Standard solution (mg/mL) USP Balsalazide Disodium RS
Cu = nominal concentration of balsalazide disodium USP Balsalazide Related Compound A RS
in the Sample solution, based on the label claim (E)-5-[(p-Carboxyphenyl)azo]-2-salicylic acid, disodium
(mg/mL) salt.
F = relative response factor (see Impurity Table 1) C14H8N20sNa2 330.12
USP Balsalazide Related Compound BRS
Acceptance criteria (E)-5-({m-[(2-Carboxyethyl)carbamoyl]phenyl}azo)-2-
Individual impurities: See Impurity Table 1. salicylic acid.
Reporting level for impurities: 0.05% C17H1SN306 357.17
Total impurities: NMT 1.0%. [NOTE-When reporting USP Salicylic Acid RS
resultsfor Individual impurities and Totalimpurities,
disregard peaks corresponding to salicylic acid and
balsalazide related compound B, as these impurities are
controlled in the drug substance only.]
Adhesive Bandage
Impurity Table 1
Relative Relative Acceptance
Retention Response Criteria, » Adhesive Bandage consists of a compress of four
Name Time Factor NMT (0/0) layers of Type IAbsorbent Gauze,or other suitable
Salicylic acid 0.37 - - material, affixed to a film or fabric coated with a
Balsalazide related com- pressure-sensitive adhesive substance. It is sterile.
0.70 1.3 0.15
pound A" The compress may contain a suitable antimicrobial
Balsalazide 1.00 - - agent and may contain one or more suitable colors.
Balsalazide related corn- The adhesive surface is protected by a suitable
pound Bb 1.2 - - removable covering.
Anyother individual
unspecified impurity - 1.0 0.10 Packaging and storage-Package Adhesive Bandage that
does not exceed 15 cm (6 inches) in width individually in such
a (E)-5-[(p-Carboxyphenyl)azo]-2-salicylic acid. manner that sterility ismaintained untilthe individual package
b (E)-5-({m-[(2-Carboxyethyl)carbamoyl]phenyl}azo)-2-salicylic acid. isopened. Packageindividual packages in a second protective
container.
PERFORMANCE TESTS Labeling-The labelof the second protectivecontainer bears
• DISSOLUTION (711) a statement that the contents may not be sterile if the
Medium: pH 6.8 phosphate buffer; 900 mL individual package has been damaged or previously opened,
Apparatus 2: 50 rpm, with stainless steel wire helix sinkers and it bears the names of any added antimicrobial agents.
Time: 30 min Each individual package is labeled to indicate the dimensions
Detector: UV 357 nm, with background correction at 590 of the compress and the name of the manufacturer, packer, or
nm distributor, and each protective container indicates also the
Path length: 0.02-cm flow cell address of the manufacturer, packer, or distributor.
Blank: Medium Sterility Tests (71): meets the requirements.
Standard solution: 0.83 mg/mL of USP Balsalazide
Disodium RS in Medium .
Sample solution: Passa portion of the solution under test
through a suitable filter of 20-l.lm pore size.
Analysis Gauze Bandage
Samples: Standard solution and Sample solution
Calculatethe percentage of C17H13N3Na206 . 2H20
dissolved: » Gauze Bandage is Type IAbsorbent Gauze. Its
length is not less than 98.0 percent of that declared
Result = (Au/As) x Cs x V x (100/L) on the label, and itsaverage width isnot more than
1.6 mm less than the declared width. It contains
Au = absorbance of the Sample solution
no dye or other additives.
As = absorbance of the Standard solution
C, = concentration of USP Balsalazide Disodium RS in Packaging and storage-Gauze Bandage that has been
the Standard solution (mg/mL) rendered sterile isso packaged that the sterility of the contents·
V = volume of Medium (mL), 900 of the package is maintained until the package is opened for
L = Capsule label claim (mg) use.
Labeling-The width and length ofthe Bandage,the number
Tolerances: NLT 70% (Q) of the labeled amount of of pieces contained, and the name of the manufacturer,
C17H13N3Na206' 2H20 is dissolved. packer, or distributor, are stated on the package. The
• UNIFORMITY OF DOSAGE UNITS (905): Meet the designation "non-sterilized" or "not sterilized" appears
requirements prominently on the package unless the Gauze Bandage has
ADDITIONAL REQUIREMENTS been rendered sterile, in which case it may be labeled to
• PACKAGING AND STORAGE: Preserve in tight containers, and indicate that it is sterile and that the contents may not be
store at controlled room temperature. sterile ifthe package bears evidence of damage or if the
package has been previously opened.

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 USP 43 OfficialMonographs / Barium 479

[NOTE-Before determining the thread count, dimensions, 40 standard-mesh sieve. NMT 7.0% is retained on the
and weight, hold the Bandage, unrolled, for not less than coarse-mesh sieve, and NMT 15.0% passes through the
4 hours in a standard atmosphere of 65 ± 2% relative fine-mesh sieve designated on the label.
humidity at 21 ± 1.1° C (70 ± 2°F).] • Loss ON DRYING (731)
Thread count-Count the number of warp and filling Sample: 10 g
threads of it in areas of 1.27 cm (V2 inch) square at 5 points Analysis: Weigh the Sample in a tared weighing bottle, and
evenly spread along the center line of the Bandage, no point dry at 105° for 2 h.
being within 30.5 cm (12 inches) of either end of the Bandage, Acceptance criteria: 11.0%-16.0%
and calculate the average number of threads per 2.54 em (1 • HARDNESS
inch) in each direction. A variation of not more than 3 threads Sample: 200 g
per inch is allowed in either warp or filling, provided that the Analysis: Screen the Sample on a mechanical sieve shaker
combined variations do not exceed 5 threads per square inch. (see (786») with a frequency of oscillation of 285 ± 3 cycles/
Width-Measure its width at each of the 5 points selected for min, for 3 min, to remove granules coarser than 4-mesh
the determination of the thread count: the average of 5 and finer than 8-mesh. Weigh 50 g of the granules retained
measurements is not more than 1.6 mm (1/16 inch) less than on the screen, and place them in a hardness pan that has a
the labeled width of the Bandage. diameter of 200 mm and a concave brass bottom. The
Length-Measure the length of the unrolled Gauze Bandage, bottom of the pan is 7.9 mm thick at the circumference and
smoothed without tension, along the center line of the Gauze 3.2 mm thick at the center and has an inside spherical
Bandage: the length is not less than 98.0% of the labeled radius of curvature of 109 cm. Add 15 steel balls of 7.9-mm
length of the Bandage. diameter, and shake on a mechanical sieve shaker for 30
Weight-Weigh the entire Bandage: the calculated weight in min. Remove the steel balls, brush the contents of the
g per 0.894 square meter (1 linear yard Type I gauze), using hardness pan onto a sieve of the fine-mesh size designated
the measurements obtained as described in the two on the label, shake for 3 min on the mechanical sieve
paragraphs just preceding, is not less than 39.2 g. shaker, and weigh.
Absorbency-Hold a rolled Gauze Bandage horizontal to and Acceptance criteria: The percentage of Barium Hydroxide
almost in contact with the surface of water at 25°, and allow Lime retained on the screen is NLT 75.0% and represents
it to drop lightly upon the water: complete submersion takes the hardness.
place in not more than 30 seconds. • CARBON DIOXIDE ABSORBENCY
Sterility Tests (71): Gauze Bandage that has been rendered Analysis: Fill the lower transverse section of aU-shaped
sterile meets the requirements. drying tube of about 15-mm internal diameter and 15-cm
Other requirements-It meets the requirements of the height with loosely packed glass wool. Place in one arm of
tests for Ignited residue, Acid or alkali, and Dextrin or starch, in the tube about 5 g of anhydrous calcium chloride, and
water extract, Residue on ignition, Fattymatter, and weigh the tube and the contents. Into the other arm of the
Alcohol-soluble dyes under Absorbent Gauze tube, place 9.5-10.5 g of Barium Hydroxide Lime, and
again weigh. Insert stoppers in the open arms of the U-tube,
and connect the side tube of the arm filled with Barium
Hydroxide Lime to a calcium chloride drying tube, which in
turn is connected to a suitable supply source of carbon
Barium Hydroxide lime dioxide. Pass the carbon dioxide through the U-tube at a
rate of 75 mL/min for 20 min, timed. Disconnect the
DEFINITION U-tube, cool to room temperature, remove the stoppers,
Barium Hydroxide Lime is a mixture of barium hydroxide and weigh.
octahydrate and Calcium Hydroxide. It may also contain Acceptance criteria: The increase in weight is NLT 19.0%
Potassium Hydroxide and may contain an indicator that is of the weight of Barium Hydroxide Lime used for the test.
inert toward anesthetic gases such as Ether, Cyclopropane,
and Nitrous Oxide and that changes color when the Barium ADDITIONAL REQUIREMENTS
Hydroxide Lime no longer can absorb carbon dioxide. • PACKAGING AND STORAGE: Preserve in tight containers.
[CAUTIoN-Because Barium Hydroxide Lime contains a • LABELING: If an indicator has been added, the name and
soluble form of barium, it is toxic if swallowed.] color change of such indicator are stated on the container
label. The container label indicates also the mesh size in
IDENTIFICATION terms of standard-mesh sieve sizes (see Powder Fineness
• A. (811 »).
Analysis: Placea granule of it on a piece of moistened red
litmus paper.
Acceptance criteria: The paper turns blue immediately.
• B. IDENTIFICATION TESTS-GENERAL (191), Barium
Sample solution: 100 mg/mL in 6 N acetic acid Barium Sulfate
Acceptance criteria: Meets the requirements
• C. IDENTIFICATION TESTS-GENERAL (191), Calcium BaS04 233.39
Sample solution: 100 mg/mL in 6 N acetic acid Sulfuric acid, barium salt (1: 1);
Acceptance criteria: Meets the requirements Barium sulfate (1:1) [7727-43-7].
SPECIFIC TESTS DEFINITION
• PARTICLE SIZE DISTRIBUTION ESTIMATION BY ANALVTICAl Barium Sulfate contains NLT 97.5% and NMT 100.5% of
SIEVING (786) barium sulfate (BaS0 4) .
Sample: 100 g
Analysis: Screen the Sample for 5 min as directed in the IDENTIFICATION
chapter, using a mechanical shaker. • A. IDENTIFICATION TESTS-GENERAL, Sulfate (191)
Acceptance criteria: It passes completely through a No.2 Sample solution: Mix 0.5 g of Barium Sulfate with 2 g each
standard-mesh sieve, and NMT 2.0% passes through a No. of anhydrous sodium carbonate and anhydrous potassium
carbonate, heat the mixture in a crucible until fusion is

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480 Barium / OfficialMonographs USP 43

complete, treat the resulting fused mass with hot water, hydrochloricacid and 90 mLof water, returning the first
and filter. portions, if necessary, to obtain a clearfiltrate.
Acceptance criteria: The filtrate, acidified with hydrochloric Analysis: Evaporate 50 mL of the filtrate on a steam bath to
acid, meets the requirements. dryness, and add 2 drops of hydrochloric acid and 10 mL
• B. IDENTIFICATION TESTS-GENERAL, Barium (191) of hot water. Filter again through acid-washed paper,
Sample solution: Dissolve a portion of the well-washed prepared as directed above. Wash the filter with 10 mL of
residuefrom Identification test A in 6 N acetic acid. hot water, and evaporate the combined filtrate and
Acceptance criteria: The solution meets the requirements. washings in a tared dish on a steam bath to dryness. Dry
the residue at 105° for 1 h. .
ASSAY
Acceptance criteria: NMT 0.3%; the residue weighs NMT
• PROCEDURE 15 mg.
Sample: 0.58-0.62 g, weighed in a tared platinum crucible • LIMIT OF SOLUBLE BARIUM SALTS
Analysis: Add 109 of anhydrous sodium carbonate to the Sample: The residue obtained in the test for Limit of
crucible, and mix by rotating the crucible. Fuseover a blast Acid-Soluble Substances
burner until a clear melt is obtained, and heat for an Control: 10 mLof water containing 0.5 mL of 2 N sulfuric
additional 30 min. Cool, place the crucible in a 400-mL acid and 50 j.lg of barium
beaker,add 250 mLof water, stir with a glass rod, and heat Analysis: Treat the Sample with 10 mLof water, pass the
to dislodge the melt. Remove the cruciblefrom the beaker, solution through a filter previously washed with 100 mL of
and wash with water, collectingthe washings inthe beaker. 0.3 N hydrochloric acid, and add 0.5 mL of 2 Nsulfuric acid.
Rinse the inside of the crucible with 2 mLof 6 N acetic acid Acceptance criteria: NMT 0.001%; any turbidityformed in
and then with water, again collecting the washings in the the Sample within 30 min is NMT that produced in the
beaker, and continue heating and stirring until the melt is similarly treated Control.
disintegrated. Cool the beaker in an ice bath until the
precipitatesettles, and decant the clear liquidthrough filter SPECIFIC TESTS
paper (Whatman No. 40, or equivalent), taking care to • pH (791): 3.5-10.0, in a 10% (w/w) aqueous suspension
transfer as little precipitate as possibleto the paper.
ADDITIONAL REQUIREMENTS
Wash twice by decantation as follows. Wash down the
AND STORAGE: Preserve in well-closed
insideof the beaker with 10 mLof cold sodium carbonate .• PACKAGING
containers.
solution (1 in 50), swirl the contents of the beaker, allow
the precipitate to settle, and decant the supernatant
through the same filter paper as before, transferring as
little precipitate as possible to the paper. Placethe beaker
containing the bulk of the barium carbonate precipitate Barium Sulfate Paste
under the funnel, wash the filter paper with five l-mL
portions of 3 N hydrochloric acid, and wash the paper DEFINITION
with water. [NoTE-The solution may be slightly hazy.] Barium Sulfate Paste.is a semisolid formulation of finely divided
Add 100 mLof water, 5.0 mLof hydrochloric acid, 10.0 mL particlesof Barium Sulfate in a suitable base. It contains NLT
of ammonium acetate solution (2 in 5), 25 mLof potassium 90.0% and NMT 110.0% of the labeled amount of barium
dichromate solution (1 in 10), and 10.0 g of urea. Cover sulfate (BaS04) . It may contain one or more suitable colors,
the beaker with a watch glass, .and digest at 80°-85° for flavors, suspending or dispersing agents, and preservatives.
NLT 16 h. Filter while hot through a tared, fine-porosity,
sintered-glass crucible, transferring all of the precipitate IDENTIFICATION
with the aid of a rubber-tipped stirring rod. Wash the • A. IDENTIFICATION TESTS-GENERAL, Sulfate (191)
precipitate with potassium dichromate solution (1 in 200), Sample: Ignite a quantity of Pasteequivalent to 0.5 g of
and finally with 20 mL of water. Dryat 105° for 2 h, cool, barium sulfate to constant weight.
and weigh. The weight of the barium chromate so Analysis: Mix 0.5 g of the ignited Sample with 2 g each of
obtained, multiplied by 0.9213, represents the weight of anhydrous sodium carbonate and anhydrous potassium
barium sulfate (BaS04) . carbonate, heat the mixture in a crucible until fusion is
Acceptance criteria: 97.5%-100.5% complete, treat the resultingfused mass with hot water,
and filter. Proceed as directed in the chapter.
IMPURITIES Acceptance criteria: Thefiltrate, acidified with hydrochloric
• LIMIT OF SULFIDE acid, meets the requirements.
Sample solution: Transfer 10 g to a 500-mL conical flask. • B. IDENTIFICATION TESTS-GENERAL, Barium (191)
Add 100 mLof 0.3 N hydrochloric acid. Sample solution: Dissolve a portion of the well-washed
Control solution: 100 mL of 0.3 N hydrochloric acid residue from Identification test A in 6 N acetic acid.
containing 5 j.lg of sulfide in a 500-mL conicalflask Acceptance criteria: The solution meets the requirements.
Analysis: Coverthe mouth of both conicalflasks with a circle
of filter paper that has been moistened at the area over the ASSAY
mouth of the flask with 0.15 mL of lead acetate TS, the • PROCEDURE
paper being held in place with a string tied around the neck Sample: Barium Sulfate Paste, equivalentto 0.60 g of
of the flask. Boil each mixture gently for 10 min, taking care barium sulfate, weighed in a tared platinum crucible
to avoid spattering the paper. Analysis: Ignite the Sample over a low flame until any
Acceptance criteria: NMT 0.5 j.lg/g; any darkening of the organic matter is thoroughly carbonized. Cool, cautiously
paper by the Sample solution is not greater than that add 0.5 mL of nitric acid and 0.5 mLof sulfuric acid, and
produced by the similarly treated Controlsolution. continue the ignition over a lowflame until the residue
• LIMIT OF ACID-SOLUBLE SUBSTANCES becomes gray in color, then ignite over the full heat of a
Sample solution: Cool the mixture obtained in the test for blast burner. Allow the contents of the crucibleto cool to
Limit of Sulfide, add water to restore approximatelythe room temperature.
original volume, and filter it through paper that previously [NOTE-If the specimen contains a silicate, such as
has been washed with a mixture of 10 mL of 3 N bentonite, proceed as follows. Add 10 mLof water

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 USP 43 OfficialMonographs / Barium 481

and 1 mLofsulfuric acid to the residuein the crucible, exceed 102 cfu/g. It meets the requirements of the tests for
mix,and add 10 mL of hydrofluoric acid. Heat gently absence of Salmonella species, Escherichia coli,
over a lowflame untilfumes of sulfurtrioxide appear. Staphylococcus aureus, and Pseudomonas aeruginosa. The
Add 5 mL more of hydrofluoric acid, heat again over total enterobacterial count does not exceed 101 cfu/g.
a low flame to the appearance of dense fumes, and • pH (791): 3.0-10.0
continue heating until the sulfuric acid has been
completely volatilized. Allow the contents of the ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers,
crucible to cool.]
[NOTE-If the specimen does not contain a silicate, omit protected from freezing and from excessive heat.
the treatment of the specimen with hydrofluoric and
sulfuric acids.]
Addto the treated or untreated specimen in the platinum
crucible 10 g of anhydrous sodium carbonate, fuse over a Barium Sulfate Suspension
blast burner until a clear melt is obtained, and heat for an
additional 30 min. Cool, place the crucible in a 400-mL DEFINITION
beaker, add 250 mLof water, stir with a glass rod, and Barium Sulfate Suspension contains NLT 90.0% and NMT
heat to dislodge the melt. Remove the crucible from the 110.0% of the labeled amount of barium sulfate (BaS04). It
beaker, and wash with water, collectingthe washings in contains suitable dispersing and/or suspending agents so
the beaker. Rinse the inside of the crucible with 2 mL of 6 that when mixed as directed in the labeling, it yields a
N acetic acid and then with water, again collecting the uniformly dispersed suspension. It may contain one or more
washings in the beaker, and continue heating and stirring suitable colors, flavors, fluidizing agents, and preservatives.
until the melt is disintegrated. Cool the beaker in an ice
bath until the precipitate settles, and decant the clear IDENTIFICATION
liquid through filter paper (Whatman No. 40, or • A. IDENTIFICATION TESTS-GENERAL, Sulfate (191)
equivalent), taking care to transfer as little precipitate as Sample: Shake the Suspension, and transfer a volume
possibleto the paper. equivalentto 0.5 g of barium sulfateto a suitable container.
Wash twice by decantation as follows. Wash down the Ignite to constant weight.
insideof the beaker with 10 mLof cold sodium carbonate Analysis: Mix 0.5 g of the ignited Sample with 2 g each of
solution (1 in 50), swirl the contents of the beaker, allow anhydrous sodium carbonate and anhydrous potassium
the precipitate to settle, and decant the supernatant carbonate, heat the mixture in a crucible until fusion is
through the same filter paper as before, transferring as complete, treat the resultingfused mass with hot water,
littleprecipitate as possibleto the paper. Placethe beaker and filter. Proceed as directed in the chapter.
containing the bulk of the barium carbonate precipitate Acceptance criteria: Thefiltrate, acidified with hydrochloric
under the funnel, wash the filter paper with five 1-mL acid, meets the requirements.
portions of 3 N hydrochloric acid, and wash the paper • B. IDENTIFICATION TESTs-GENERAL, Barium (191)
with water. [NOTE-The solution may be slightly hazy.] Sample solution: Dissolve a portion of the well-washed
Add 100 mLof water, 5.0 mLof hydrochloric acid, 10.0 mL residue from Identification test A in 6 N acetic acid.
of ammonium acetate solution (2 in 5), 25 mLof Acceptance criteria: The solution meets the requirements.
potassium dichromate solution (1 in 10), and 10.0 g of
urea. Cover the beaker with a watch glass, and digest at ASSAY
80°-85° for NLT 16 h. Filter while hot through a tared, • PROCEDURE
fine-porosity, sintered-glasscrucible, transferringallof the Sample: Avolume of Suspension, previously well shaken in
precipitate with the aid of a rubber-tipped stirring rod. itsoriginal container, equivalent to 0.60 g of barium sulfate,
Wash the precipitate with potassium dichromate s.olution in a tared platinum crucible
(1 in 200), and finally with 20 mLof water. Dryat 105° Analysis: Ignite over a lowflame until any organic matter is
for 2 h, cool, and weigh. The weight of the barium thoroughly carbonized. Cool, cautiously add 0.5 mLof
chromate so obtained, multiplied by 0.9213, represents nitric acid and 0.5 mLof sulfuric acid, and continue the
the weight of barium sulfate (BaS04)' ignition over a low flame until the residue becomes gray in
color, then ignite over the full heat of a blast burner. Allow
Acceptance criteria: 90.0%-110.0% the contents of the crucible to cool to room temperature.
SPECIFIC TESTS [NOTE-If the specimen contains a silicate, such as
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR bentonite, proceed as follows. Add 10 mLof water
SPECIFIED MICROORGANISMS (62) and 1 mL of sulfuric acid to the residuein the crucible,
For products labeled for oral administration only: The mix,and add 10 mLof hydrofluoric acid. Heat gently
total aerobic microbial count does not exceed 102 cfu/g. over a lowflame untilfumes of sulfurtrioxide appear.
The total combined molds and yeasts count does not Add 5 mL more of hydrofluoric acid, heat again over
exceed 10 1 cfu/g. It meets the requirements of the tests for a lowflame to the appearance of dense fumes, and
absence of Salmonella species, Escherichia coli, continue heating until the sulfuric acid has been
Staphylococcus aureus, and Pseudomonas aeruginosa. The completely volatilized. Allow the contents of the
total enterobacterial count does not exceed 10 1 cfu/g. crucible to cool.]
For products labeled for oral administration and rectal [NOTE-If the specimen does not contain a silicate, omit
administration: The total aerobic microbial count does the treatment of the specimen with hydrofluoric and
not exceed 102 cfu/g. The total combined molds and yeasts sulfuric acids.]
count does not exceed 101 cfu/g. It meets the requirements Add to the treated or untreated specimen in the platinum
of the tests for absence of Salmonella species, Escherichia crucible10 g of anhydrous sodium carbonate, fuse over a
coli, Staphylococcus aureus, and Pseudomonas aeruginosa. blast burner until a clear melt isobtained, and heat for an
The total enterobacterial count does not exceed 101 cfu/g. additional 30 min. Cool, place the crucible in a 400-mL
For products labeled for rectal administration only: The beaker, add 250 mL of water, stir with a glass rod, and
total aerobic microbial count does not exceed 103 cfu/g. heat to dislodge the melt. Remove the cruciblefrom the
The total combined molds and yeasts count does not beaker, and wash with water, collecting the washings in

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482 Barium / Official Monographs USP 43

the beaker. Rinse the inside of the crucible with 2 ml of 6 III B. IDENTIFICATION TESTS-GENERAL, Barium (191)
N ac~tic a.cid and then with water, again collecting the Sample solution: Dissolve a portion of the well-washed
wa~hlngs In th~ b~~ker, and continue heating and stirring residue from Identification test A in 6 N acetic acid.
until the melt IS disintegrated. Cool the beaker in an ice Acceptance criteria: The solution meets the requirements.
bath until the precipitate settles, and decant the clear
liquid through filter paper (Whatman No. 40, or ASSAY
equivalent), taking care to transfer as little precipitate as • PROCEDURE
possible to the paper. Sample: Barium Sulfate for Suspension, equivalent to 0.60
Wash twice by decantation as follows. Wash down the g of barium sulfate, weighed in a tared platinum crucible
inside of the beaker with 10 ml of cold sodium carbonate Analysis: Ignite over a low flame until any organic matter is
solution (1 in 50), swirl the contents of the beaker, allow thoroughly carbonized. Cool, cautiously add 0.5 ml of
the precipitate to settle, and decant the supernatant nitric acid and 0.5 ml of sulfuric acid, and continue the
through the same filter paper as before, transferring as ignition over a low flame until the residue becomes gray in
little precipitate as possible to the paper. Place the beaker color, then ignite over the full heat of a blast burner. Allow
containing the bulk of the barium carbonate precipitate the contents of the crucible to cool to room temperature.
under the funnel, wash the filter paper with five 1-ml [NOTE-If the specimen contains a silicate, such as
p<:>rtions of 3 N hydrochloric .acid, and wash the paper bentonite, proceed as follows. Add 10 ml of water
with water. [NOTE-The solution may be slightly hazy.] and 1 ml of sulfuric acid to the residue in the crucible,
Add 100 ml of water, 5.0 ml of hydrochloric acid, 10.0 ml mix, and add 10 ml of hydrofluoric acid. Heat gently
of ammonium acetate solution (2 in 5), 25 ml of over a low flame until fumes of sulfur trioxide appear.
potassium dichromate solution (1 in 10), and 10.0 g of Add 5 ml more of hydrofluoric acid, heat again over
urea. Cover the beaker with a watch glass, and digest at a low flame to the appearance of dense fumes, and
80°-85° for NlT 16 h. Filter while hot through a tared, continue heating until the sulfuric acid has been
fine-porosity, sintered-glass crucible, transferring all of the completely volatilized. Allow the contents of the
precipitate with the aid of a rubber-tipped stirring rod. crucible to cool.]
Wash the precipitate with potassium dichromate solution [NOTE-If the specimen does not contain a silicate, omit
(1 in 200), and finally with 20 ml of water. Dry at 105° the treatment of the specimen with hydrofluoric and
for 2 h, cool, and weigh. The weight of the barium sulfuric acids.]
chromate so obtained, multiplied by 0.9213, represents Add to the treated or untreated specimen in the platinum
the weight of barium sulfate (BaS0 4). crucible 10 g of anhydrous sodium carbonate, fuse over a
blast burner until a clear melt is obtained, and heat for an
Acceptance criteria: 90.00/0-110.0%
additional 30 min. Cool, place the crucible in a 400-ml
SPECIFIC TESTS beaker, add 250 ml of water, stir with a glass rod, and
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR heat to dislodge the melt. Remove the crucible from the
SPECIFIED MICROORGANISMS (62): The total bacterial beaker, and wash with water, collecting the washings in
count does not exceed 10 2 cfu/ml, the total combined the beaker. Rinse the inside of the crucible with 2 ml of 6
molds and yeasts count does not exceed 10 1 cfu/ml, and N acetic acid and then with water, again collecting the
it meets the requirements of the tests for absence of washings in the beaker, and continue heating and stirring
Salmonella species, Staphylococcus aureus, and until the melt is disintegrated. Cool the beaker in an ice
Pseudomonas aeruginosa. bath until the precipitate settles, and decant the clear
• pH (791): 3.5-10.0 liquid through filter paper (Whatman No. 40, or
equivalent), taking care to transfer as little precipitate as
ADDITIONAL REQUIREMENTS possible to the paper.
• PACKAGING AND STORAGE: Preserve in tight containers, and Wash twice by decantation as follows. Wash down the
avoid freezing. inside of the beaker with 10 ml of cold sodium carbonate
solution (1 in 50), swirl the contents of the beaker, allow
the precipitate to settle, and decant the supernatant
through the same filter paper as before, transferring as
little precipitate as possible to the paper. Place the beaker
Barium Sulfate for Suspension containing the bulk of the barium carbonate precipitate
DEFINITION under the funnel, wash the filter paper with five 1-ml
Barium Sulfate for Suspension is a dry mixture of Barium portions of 3 N hydrochloric acid, and wash the paper
Sulfate and one or more suitable dispersing and/or with water. [NOTE-The solution may be slightly hazy.]
suspending agents. It contains NlT 90.0% and NMT 110.0% Add 100 ml of water, 5.0 ml of hydrochloric acid, 10.0 ml
of the labeled amount of barium sulfate (BaS0 4). It may of ammonium acetate solution (2 in 5), 25 ml of
potassium dichromate solution (1 in 10), and 10.0 g of
contain one o~ more suitable colors, flavors, fluidizing agents,
urea. Cover the beaker with a watch glass, and digest at
and preservatives. 80°-85° for NlT 16 h. Filter while hot through a tared,
IDENTIFICATION fine-porosity, sintered-glass crucible, transferring all of the
• A. IDENTIFICATION TESTS-GENERAL, Sulfate (191) precipitate with the aid of a rubber-tipped stirring rod.
Sample: Ignite 1g to constant weight. Wash the precipitate with potassium dichromate solution
Analysis: Mix 0.5 g of the ignited Sample with 2 g each of (1 in 200), and finally with 20 ml of water. Dry at 105°
anhydrous sodium carbonate and anhydrous potassium for 2 h, cool, and weigh. The weight of the barium
carbonate, heat the mixture in a crucible until fusion is chromate so obtained, multiplied by 0.9213, represents
complete, treat the resulting fused mass with hot water, the weight of barium sulfate (BaS04)'
and filter. Acceptance criteria: 90.0%-110.0% .
Acceptance criteria: The filtrate, acidified with hydrochloric
SPECIFIC TESTS
acid, meets the requirements.
• LOSS ON DRYING (731)
Analysis: Dry at 105° for 4 h.

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USP 43 OfficialMonographs / Beclomethasone 483

Acceptance criteria: NMT 1.0% for 2 h, cool, and weigh. The weight of the barium
• pH (791): 3.5-10.0, in a 60% (w/w) aqueous suspension, chromate so obtained, multiplied by 0.9213, represents
or constituted for its intended use as directed inthe labeling the weight of barium sulfate (BaS04)'
ADDITIONAL REQUIREMENTS Acceptance criteria: 90.0%-110.0%
• PACKAGING AND STORAGE: Preserve in well-closed PERFORMANCE TESTS
containers. • DISINTEGRATION (701): NLT 10 min and NMT 30 min
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
ADDITIONAL REQUIREMENTS
Barium Sulfate Tablets • PACKAGING AND STORAGE: Preserve in well-closed
containers.
DEFINITION
Barium Sulfate Tablets are flat-sided disks between 11.5 mm
and 13.5 mm in diameter and contain NLT 90.0% and NMT
110.0% of the labeled amount of barium sulfate (BaS04) .
Beclomethasone Dipropionate
IDENTIFICATION
• A. IDENTIFICATION TESTS-GENERAL, Sulfate (191)
Sample: A portion of powdered Tablets equivalent to 0.6 g
of barium sulfate
Analysis: Mix the Sample with 2 g each of anhydrous
sodium carbonate and anhydrous potassium carbonate,
heat the mixture in a crucible untilfusion iscomplete, treat
the resultingfused mass with hot water, and filter. Proceed
as directed in the chapter. C2sH37C107' H20 539.07
Acceptance criteria: The filtrate, acidified with hydrochloric C2sH37CI07 521.04
acid, meets the requirements.
• B. IDENTIFICATION TESTS-GENERAL, Barium (191) Pregna-l,4-diene-3,20-dione, 9-chloro-ll-hydroxy-16-
Sample solution: Dissolve a portion of the well-washed methyl-17,21-bis(1-oxopropoxy)-, (11~, 16~)-;
9-Chloro-ll~, 17,21-trihydroxy-16~-methylpregna-l ,4-diene-
residue from Identification test A in 6 N acetic acid. 3,20-dione 17,21-dipropionate [5534-09-8].
Acceptance criteria: The solution meets the requirements.
ASSAY DEFINITION
• PROCEDURE
Beclomethasone Dipropionate is anhydrous or contains one
Sample: Aportion of powdered Tablets, equivalent to 0.6 g molecule of water of hydration. It contains NLT 97.0% and
of barium sulfate, weighed in a tared platinum crucible NMT 103.0% of beclomethasone dipropionate (C2sH37C107),
Analysis: Add 109 of anhydrous sodium carbonate to the calculated on the dried basis.
crucible,and mix by rotating the crucible. Fuse over a blast IDENTIFICATION
burner until a clear melt is obtained, and heat for an
additional 30 min. Cool, place the crucible in a 400-mL
beaker, add 250 mLof water, stirwith a glass rod, and heat
to dislodge the melt. Remove the cruciblefrom the beaker,
and wash with water, collectingthe washings in the beaker.
Rinse the inside of the cruciblewith 2 mLof 6 N acetic acid
and then with water, again collecting the washings in the ASSAY
beaker, and continue heating and stirring until the melt is • PROCEDURE
disintegrated. Cool the beaker in an ice bath until the Mobile phase: Acetonitrile and water (60:40) such that the
precipitate settles, and decant the clear liquidthrough filter retention times for beclomethasone dipropionate and
paper (Whatman No. 40, or equivalent), taking care to testost~rone propionate are approximately 6 and 10 min,
transfer as little precipitate as possibleto the paper. respectively
Wash twice by decantation as follows. Wash down the Internal standard solution: 1.2 mg/mL of USP
inside of the beaker with 10 mL of cold sodium carbonate Testosterone Propionate RS in methanol
solution (1 in 50), swirl the contents of the beaker, allow Standard stock solution: 1.4 mg/mL of USP
the precipitate to settle, and decant the supernatant Beclomethasone Dipropionate RS in methanol
through the same filter paper as before, transferring as Standard solution: 0.7 mg/mL of USP Beclomethasone
little precipitate as possibleto the paper. Placethe beaker Dipropionate RS and 0.6 mg/mL of USP Testosterone
containing the bulk of the barium carbonate precipitate Propionate RS prepared as follows. Transfer 4.0 mLof
under the funnel, wash the filter paper with five 1-mL Standardstocksolution to a suitable vial, and add 4.0 mL of
portions of 3 N hydrochloricacid, and wash the paper Internal standard solution.
with water. [NOTE-The solution may be slightly hazy.] Sample stock solution: 1.4 mg/mL of Beclomethasone
Add 100 mLof water, 5.0 mLof hydrochloric acid, 10.0 mL Dipropionate in methanol
of ammonium acetate solution (2 in 5), 25 mLof Sample solution: 0.7 mg/mL of Beclomethasone
potassium dichromate solution (1 in 10), and 10.0 g of Dipropionate and 0.6 mg/mL of USP Testosterone
urea. Cover the beaker with a watch glass, and digest at Propionate RS prepared as follows. Transfer 4.0 mL of
~oo_85° for NLT 16 h. Filter while hot through a tared, Sample stocksolution to a suitable vial, and add 4.0 mL of
fine-porosity, sintered-glasscrucible,transferringallof the Internal standard solution.
precipitate with the aid of a rubber-tipped stirring rod. Chromatographic system
Wash the precipitate with potassium dichromate solution (See Chromatography (621), System Suitability.)
(1 in 200), and finally with 20 mL of water. Dry at 105° Mode: LC

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484 Beclomethasone / Official Monographs USP43

Detector: UV 254 nm and triturate to make a smooth paste. Add the Corn Oil to
Column: 4-mm x 30-cm; packing L1 make the contents pourable. Transfer contents stepwise and
Injection volume: 5-25 IJL quantitatively to a calibrated container using the Corn Oi/.
System suitability Add sufficient Corn Oil to bring to final volume. Place on a
Sample: Standardsolution shaker until dissolved. [NQTE-Maytake up to 24 h to .
Suitability requirements dissolve.]
Relative standard deviation: NMT 3.0% for five
replicate injections ASSAY
Analysis • PROCEDURE
Samples: Standardsolution and Sample solution Mobile phase: Acetonitrile and water (65:35)
Calculate the percentage of beclomethasone dipropionate Standard stock solution: 0.5 mg/mL of beclomethasone
dipropionate prepared from USP Beclomethasone
(C2sH37C/07) in the portion of Beclomethasone
Dipropionate RS in ethanol. Sonicate and mix well.
Dipropionate taken: Standard solution: 0.02 mg/mL of beclomethasone
Result = (RuIR s) x (CslCu) x 100 diproprionate prepared from Standard stock solution and
ethanol
Ru = peak height ratio of beclomethasone Sample solution: Transfer 1.0 mL of Oral Solution to a
dipropionate to the internal standard from the 25-mL volumetric flask, add approximately 20 mL of
Sample solution ethanol, vortex for 30 s, and warm under running water
Rs = peak height ratio of beclomethasone until dissolved. Dilute with ethanol to volume.
dipropionate to the internal standard from the Chromatographic system
Standardsolution (See Chromatography (621), System Suitability.)
Cs = concentration of USP Beclomethasone Mode: LC
Dipropionate RS in the Standardsolution Detector: UV-Vis 240 nm
(mg/mL) Column: 2.0-mm x 10-cm; 2.5-lJm packing L1
Cu =concentration of Beclomethasone Dipropionate Column temperature: 35 0
in the Sample solution (mg/mL) Flow rate: 0.35 mL/min
Injection volume: 5 IJL
Acceptance criteria: 97.00/0-103.0% on the dried basis System suitability
Sample: Standardsolution
IMPURITIES [NoTE-The retention time for beclomethasone
• RESIDUE ON IGNITION (281): NMT 0.1 % dipropionate is about 3.2 min.]
Suitability requirements
SPECIFIC TEST~ Tailing factor: NMT 2.0
• OPTICAL ROTATION, Specific Rotation (781 S) Relative standard deviation: NMT 2.0% for replicate
Sample solution: 10 mg/mL in dioxane
Acceptance criteria: +88 0 to +94 0 injections
Analysis
• Loss ON DRYING (731) Samples: Standardsolution and Sample solution
Analysis: Dry a sample at 105 0 for 3 h.
Calculate the percentage of the labeled amount of
Acceptance criteria: NMT 0.5% for the anhydrous form;
bedornethasone dipropionate (C2sH37CI07) in the portion
2.80/0-3.8% for the monohydrate form
of Oral Solution taken:
ADDITIONAL REQUIREMENTS .
• PACKAGING AND STORAGE: Preserve in well-closed Result = (rulrs) x (CsICu) x 100
containers.
• USP REFERENCE STANDARDS (11) to = peak response of beclomethasone dipropionate
USP Beclomethasone Dipropionate RS from the Sample solution
USP Testosterone Propionate RS rs = peak response of beclomethasone dipropionate
from the Standardsolution
Cs =concentration of USP Beclomethasone
Dipropionate RS in the Standard solution
(mg/mL)
Beclomethasone Dipropionate Cu = nominal concentration of beclomethasone
dipropionate in the Sample solution (mg/mL)
Compounded Oral Solution
DEFINITION Acceptance criteria: 90.0%-110.0%
Beclomethasone Dipropionate Compounded Oral Solution ADDITIONAL REQUIREMENTS
contains NLT 90.0% and NMT 110.0% of the labeled • PACKAGING AND STORAGE: Package in tight, light-resistant,
amount of beclomethasone dipropionate (C2sH37CI07)' plastic containers. Store at controlled room temperature.
Prepare Beclomethasone Dipropionate Compounded Oral • BEYOND-USE DATE: NMT 90 days after the date on which it
Solution 0.5 mg/mL as follows (see Pharmaceutical was compounded when stored at controlled room
Compounding-Nonsterile Preparations (795». temperature.
• LABELING: Label it to be well-shaken before use, and to state
Beclomethasone Dipropionatepow- the Beyond-Use Date.
der 50mg • USP REFERENCE STANDARDS (11)
USP Beclomethasone Dipropionate RS
Corn Oil, NF, a sufficient
quantity to make 100 ml

Pour the Beclomethasone Dipropionate powder into a suitable

container. Wet the powder with a small amount of Corn Oil

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USP43 OfficialMonographs / Belladonna 485

Belladonna leaf Add another 15 mLof chloroform, and extract again,

discardingthe chloroform phase. Add 15 mLof Phosphate
DEFINITION buffer and sufficient 1 N sodium hydroxide to yield a final
Belladonna Leaf consists of the dried leaf and flowering or pH between 9.0 and 9.5. Add 15 mLof chloroform, shake
fruitingtop of Atropabelladonna L. or of itsvariety acuminata vigorously, and allow the layers to separate. Filter the
Royle ex Lindley (Fam. Solanaceae). Belladonna Leaf yields organic phase through 109 of anhydrous sodium sulfate
NLT 0.35% of the alkaloids of belladonna leaf. (see Reagents, Indicators, and Solutions-Sodium Sulfate,
Anhydrous, Suitability for AlkaloidAssays), previously
ASSAY washed with chloroform and supported in a funnel with
• PROCEDURE a small pledget of glasswool, into a suitable container.
Phosphate buffer: 34.8 g of dibasic potassium phosphate Extract again with two 15-mLportions of chloroform,
in 900 mL of water. Adjust to a pH of 9.5 by the addition again collectingthe clarified organic phase. Wash the
of 3 N hydrochloric acid or sodium hydroxide, with mixing. sodium sulfate and the.tip of the funnel with 5 mL of
Diluent: Dilute sulfuric acid (1 in 350) chloroform. Evaporate the combined organic phases
Internal standard solution: 0.8 mg/mL of USP under reduced pressure at a temperature below 45°, add
Homatropine Hydrobromide RS in Diluent. [Nets-Prepare 1 mLof chloroform, and mix to dissolve the alkaloids,
fresh on the day of use.] taking care to wet the sides of the container.
Standard stock solution A: 1.0 mg/mL of USP Scopolamine Chromatographic system
Hydrobromide RS in Diluent (See Chromatography (621 >, System Suitability.)
Standard stock solution B: Dissolve 20 mg of USP Atropine Mode: GC
Sulfate RS in 25 mL of Diluent in a 50-mLvolumetricflask, Detector: Flame ionization
add 2.0 mL of Standardstocksolution A, and mix. Add Column: 1.2-m x 4-mm glass; packed with 3% G3 on
Diluent to volume. [Note-Prepare fresh on the day of Sl AB. [NoTE-The column may be cured and conditioned
use. ] as specified under Chromatography (621 >, General
Standard solutions: Pipet into three separate 60-mL Procedures, Gas Chromatography.]
separators 1.0-, 2.0-, and 3.0-mL portions, respectively, of Temperatures
Standard stock solution A, and add 9.0, 8.0, and 7.0 mL, . Injection port: 240°
respectively, of Diluent. Add 1.0 mLof Internal standard Detector: 240°
solution, then add 15 mLof chloroform, shake vigorously, Column: 215°
allowthe layers to separate, and discard the chloroform Carrier gas: Dry helium
layer. Flow rate: 65 mL/min
Sample solution: Moisten 109, previously reduced to a Injection volume: 5 J.lL
moderately coarse powder, with a mixture of 8 mL of System suitability
ammonium hydroxide, 10 mL of alcohol, and 20 mL of Sample: Sample solution
ether, and extract the alkaloids by either Method I or Suitability requirements
Method" as follows. Ifnecessary, reduce the volume of the Resolution: NLT 3.0 between atropine and
extract to 100 mLby evaporation on a steam bath. homatropine peaks
Extraction blank: Place 10 mL of Diluent in a 60-mL Tailing factor: NMT 2.0 for atropine peak
separator. Prepare as directed under Standardsolutions, Relative standard deviation: The analytical system is
beginning with "then add 15 mLof chloroform". The blank suitablefor conducting this Assay ifthe relativestandard
chromatogram contains no significant interferences at the deviation for the ratio, RAJ is NMT 2.0%, for the atropine
locusof atropine, scopolamine, or homatropine. peak in repeated injections.
Method I: Place the moistened drug in a Analysis
continuous-extraction thimble, and allow maceration to Samples: Standardsolutions and Sample solution
proceed overnight, then extract with etherfor 3 h, or longer Measure the areas, aA, aH, and as, of the atropine,
if necessary, to effect complete extraction. homatropine, and scopolamine peaks, respectively, in
Method II: Place the moistened drug in a small percolator, each chromatogram of Standardsolution, and calculate
and allow maceration to proceed overnight. Percolate the ratios AA and As:
slowly with a mixture of three volumes of ether and one
volume of chloroform. Continue the percolation until the AA = aiaH
residue from 3-4 mLof percolate last passed, when
dissolved in dilute sulfuric acid (1 in 70) and treated with As = as/aH
mercuric iodide TS, shows NMTa faint turbidity.
Transfer the extract from Method I or Method /I to a Plotthe standard curvesof the values of RA and Rs against
separator with the aid of ether. Extractwith five 15-mL the amounts, in milligrams, of atropine and scopolamine
portions of dilute sulfuric acid (1 in 70), filtering each in the solutions. (The ratio of the molecularweight of
portion drawn off into a 1OO-mL volumetric flask. Wash atropine to that of anhydrous atropine sulfate is 0.8551,
the filterwith dilute sulfuric acid (1 in 70), and collectthe and the ratio of the molecularweight of scopolamine to
washings in the flask. Add dilute sulfuric acid (1 in 70) to that of anhydrous scopolamine hydrobromide is
volume, and mix. Dilute 20.0 mLof the resultingsolution 0.7894.) Inject a portion of the Sample solution into the
with the same dilute acid to 100.0 mL. Pipet 10 mL of this chromatograph, measure the peak areas, and calculate
solution into a 60-mL separator. Add 1.0 mL of Internal the area ratios, as with the Standardsolutions. Record
standardsolution, then add 15 mLof chloroform, shake from the standard curvesthe quantities, in milligrams, of
vigorously, allow the layers to separate, and discard the atropine and scopolamine in the weight of the specimen
chloroform layer.[NOTE-If emulsions areformed, a mixed taken. Add the quantity, in milligrams, of atropine and
solvent consisting of chloroform-isopropyl alcohol (10:3) scopolamine, and multiply by 50 to obtain the weight,
may be substituted for chloroform throughout the in milligrams, of alkaloids in the portion of Belladonna
extraction procedure.] . Leaf taken.

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486 Belladonna / OfficialMonographs USP43

Acceptance criteria: NLT 0.35% of the alkaloids of Powdered Belladonna leaf: Lightolive-brown to
belladonna leaf moderate olive-green in color. The following are among
the elements of identification: the separate microcrystals,
CONTAMINANTS
the dark gray crystal cells, th~ cuti~ular. striping of the.
• ARTICLES Of BOTANICAL ORIGIN (561), Methods of Analysis, epidermal cells, the vessels With ellipsoidal bordered pits,
Acid-Insoluble Ash: NMT 3.0% the fibers of the stem, and occasional hairs and pollen
• ARTICLES Of BOTANICAL ORIGIN (561), Pesticide Residue grains. Rosette aggregates of calcium oxalate and
Analysis: Meets the requirements fragments of the seed occur when the drug contains
• BIELLADONNA STEMS:The proportion of belladonna stems belladonna fruits. Examine Belladonna Leaf for hairs
over 10 mm in diameter does not exceed 3.0%. having a papillose cuticle and.for raphides of calcium
SPECIFIC TESTS oxalate: their presence indicates adulteration.
• BOTANICAL CHARACTERISTICS ADDITIONAL REQUIREMENTS
Macroscopic: Usually partly matted together, crumpled or • PACKAGING AND STORAGE: Preserve in well-closed
broken leaves, together with some smallerstems and a containers and avoid long exposure to direct sunlight.
number offlowers and fruits. The leaves are thin and brittle, Preserve powdered Belladonna Leaf in light-resistant
mostly light green to moderate olive-green. The lamina is containers.
mostly 5-25 em in length and 4-12 em in width and • USP REFERENCE STANDARDS (11)
possesses an ovate-lanceolate to broadly ovate outline, an USP Atropine Sulfate RS
acute to acuminate apex, an entire margin, an acute to USP Homatropine Hydrobromide RS
somewhat decurrent base and slightly hairysurface, the USP Scopolamine Hydrobromide RS
hairs being more abundant along the veins; when broken
transversely, it shows numerous light-colored dots (crystal
cells) visible with a lens. The petiole isslender and usually
up to 4 em in length. The flowers possess a campanulate
corollawith five small, reflexed lobes, purplishto yellowish Belladonna Extract
purple, becoming faded to brown or duskyyellow or
yellow; a green, five-lobed calyx; fiveepipetalous stamens;
and a superior, bilocular ovary with numerous ovules. The » Belladonna Extract contains, in each 100 g, not
fruit issubglobular, darkyellowto yellowish brown to dusky less than 1.15 g and not more than 1.35 g of the
red or black, up to 12 mm in width, and sometimes alkaloids of belladonna leaf.
subtended by the persistent calyx and containing
numerous flattened, somewhat reniform seeds, the latter PILULAR BELLADONNA EXTRACT
up to 2 mm in, width. The stems are more or lessflattened Prepare the extract by percolating 1000 g of
and hollow and finely hairywhen young. Belladonna Leaf, using a mixture of 3 volumes of
Microscopic alcohol and 1 volume of water as the menstruum.
leaf: The epidermis of the lamina possesses wavyanticlinal Maceratethe drug for 16 hours, and then percolate
walls and a distinctly striated cuticle. Stom.ata are more
numerous in the lower epidermis and are surrounded by it at a moderate rate, Evaporate the percolate
three or four neighboring cells, one of which issmaller under reduced pressure and at a temperature not
than the others. The nonglandular hairsare uniseriateand exceeding 60° to a pilular consistency, and adjust
up to six-celled. Short club-shaped glandular hairs with a the remaining extract, after assaying, by dilution
one-celled stalkand multicellular head and long glandular
hairs with a uniseriatestalk and unicellular head occur on with liquid glucose so that the finished Extract will
both epidermises.The mesophyll consistsof a single layer contain, in each 100 g, 1.25 g of the alkaloids of
of palisade parenchyma beneath which occurs spongy belladonna leaf.
parenchyma, the latter with scattered cells filled with POWDERED BELLADONNA EXTRACT
microcrystals. The midrib contains an arc of bicollateral Prepare the extract by percolating 1000 g of
bundles, collenchyma beneath upper epidermis, and
scattered parenchyma cells with microcrystals. Belladonna Leaf, using alcohol as the menstruum.
Stem: The stem shows an epidermis with striated cuticle Maceratethe drug for 16 hours, and then percolate
and few hairs;a distinctendoderm is; small strands of long, it slowly. Evaporate the percolate under reduced
thin-walled, slightlylignified pericyclic fibers; and a circle pressure and at a temperature not exceeding 60°
of bicollateral bundles. The parenchyma of the cortex and to a soft extract, add 50 g of dry starch, and
pith is interspersed with crystal cells.
Flower: The calyx possessesnumerous glandular hairswith continue the evaporation, at the same
uniseriatestalksand one- to three-celledglandular heads. temperature, until the product is dry. Powder the
The corollashows a papilloseinnerepidermisand an outer residue. The extract may be deprived of its fat by
epidermis with glandular hairs similar to those of the treating either the soft extract first obtained, or the
calyx. The pollengrains,when mounted in chloralhydrate dry and powdered extract, as directed under
solution, are subspherical, 40 IJm in diameter, tricolpate,
having three germinal furrows and rows of pits between Extracts (see Pharmaceutical Dosage Forms (1151».
the ridges on the exine. Assay the powdered residue, and add sufficient
Fruit: The epicarp exhibits polygonal epidermal cells with starch, previously dried at 100°, to obtain a finished
a striated cuticle and stomata. The mesocarp consistsof Extract containing 1.25 g of the alkaloids of
large pulp cellssome of which contain rosette aggregate
crystals of calcium oxalate. belladonna leaf in each 100 g. Mix the powders,
Seed: The seed ischaracterized by an epidermis of large, and pass the Extract through a fine sieve.
wavy-walled cells with prominent ridges over the
anticlinal walls.

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 USP 43 Official Monographs / Belladonna 487

Packaging and stora~e-Preservein tight containers, at a . Chromatograp~ic system-Under typical condition, the
temperature not exceeding 30°. Instrument contains a 1.2-m x 4-mm glass column packed
USP Reference standards (11)- with 3% G3 on SlAB. The column may be cured and
USP Atropine Sulfate RS conditioned as specified under Gas Chromatography (see
USP Homatropine Hydrobromide RS ».
Chromatography (621 The column is maintained at a
USP Scopolamine Hydrobromide RS temperature of about 215°, and the injection port and
det~ctor block at about 240°, and dry helium is used as a
Assay- earner gas at a flow rate of about 65 mL per minute.
pH 9.5 Phosphate buffer-Dissolve 34.8 g of dibasic System suitability-Chromatograph six to ten injections of
potassium phosphate in 900 mL of water, and adjust to a pH the Assay preparation, and record peak areas as directed for
of 9.5, determined electrometrically, by the addition of 3 N
Proce~ure. The a~alytical system i.s s.uitable for conducting this
hydrochloric acid or sodium hydroxide, with mixing. assay If the relative standard deviation for the ratio, RAt
Internal standardsolution-Dissolve about 40 mg of USP
calculated by the formula:
Homatropine Hydrobromide RS, accurately weighed, in about
25 mL of dilute sulfuric acid (1 in 350) in a 50-mL volumetric 100 x (standard deviation/mean ratio)
flask, add the same dilute acid to volume, and mix. Prepare
fresh on the day of use. does not exceed 2.0%; the resolution, R, between aH and aA is
Standa~d preparation-Dissolve about 10 mg of USP
not less than 3; and the tailing factor (the sum of the distances
Scopolamme Hydrobromide RS, accurately weighed, in about from peak center to the leading edge and to the tailing edge
5 mL of dilute sulfuric acid (1 in 350) in a 1O-mL volumetric divided by twice the distance from peak center to the leading
flask, add the same dilute acid to volume, and mix (Solution edge), measured at 5% of the peak height of aAt does not
A). Dissolve a~out 2~ mg of USP Atropine Sulfate RS, exceed 2.0.
accurately weighed, In about 25 mL of dilute sulfuric acid (1
Procedure-Inject a portion (about 5 IJL) of each Standard
in 350) in a 50-mL volumetric flask, add 2.0 mL of Solution A,
solutio~ i~to ~ suitable gas chromatograph equipped with a
and mix. Add dilute sulfuric acid (1 in 350) to volume and
flarne-ionlzation detector. Measure the areas, aAt aH, and as, of
mix. Prepare fresh on the day of use. '
Extraction blank-Place about 10 mL of dilute sulfuric acid the atropine, homatropine, and scopolamine peaks
(1 in 350) in a 60-mL separator. Proceed as directed under respectively, in each chromatogram, and calculate the ratios
Assay preparation, beginning with "then add 15 mL of AA and As by the formulas:
~hloroform." The blank chromatogram contains no significant
mterferen~es at the locus of atropine, scopolamine, or
a.ja; and as/aH•
homatropme.
Assay preparation-Weigh accurately about 0.5 g of Extract, Plot the Standard curves of the values of RAand Rs against the
transfer to a 125-mL 'Conical flask, and add 40 mL of dilute amounts, in mg, of atropine and scopolamine in the solutions.
sulfuric acid (1 in 350). Heat to a temperature not above 45°, (The ratio of the molecular weight of atropine to that of
and stir to hasten solution. Filter the solution through filter anhydrous atropine sulfate is 0.8551, and the ratio of the
paper into a 1OO-mL volumetric flask. Wash the flask and the molecular weight of scopolamine to that of anhydrous
filter with two 20-mL portions of warmed dilute sulfuric acid scopolamine hydrobromide is 0.7894.) Inject a portion of the
(1 in 350), and collect the washings in the 1OO-mL volumetric Assay preparation into the chromatograph, obtain the
flask. Add dilute sulfuric acid (1 in 350) to volume, and mix. chromatogram area ratios, measure the peak areas, and
Pipet 10 mL of this solution into a 60-mL separator. To the calculate the area ratios, as with the Standard solutions. Record
separator add 1.0 mL of Internal standard solution, then add from the Standardcurve the quantities, in mg, of atropine and
15 mL of chloroform, shake vigorously, allow the layers to scopolamine in the volume of specimen taken. Add the
separate, and discard the chloroform layer. (If emulsions are quantity, in mg, of atropine and scopolamine, and multiply by
formed, a mixedsolventconsisting of chloroform and isopropyl 10 to obtain the weight, in mg, of alkaloids in the portion of
alcohol (10:3) may be substituted for chloroform throughout Extract taken.
, the extraction procedure.) Add another 15 mL of chloroform
and extract again, discarding the chloroform phase. Add 15 '
mL of pH 9.5 Phosphate buffer and sufficient 1 N sodium
hydroxide to yield a final pH between 9.0 and 9.5. Add 15 mL
of chloroform, shake vigorously, and allow the layersto Belladonna Extract Tablets
separate. Filter the organic phase through 109 of anhydrous
sodium sulfate (see Suitability for alkaloid assays under Sodium » Belladonna Extract Tablets contain not less than
Sulfate, Anhydrous, in the section Reagents, Indicators, and
~olutions), pr~viously washed with chloroform and supported
90.0 percent and not more than 110.0 percent of
m a funnel With a small pledget of glass wool, into a suitable the labeled amount of the alkaloids of belladonna
container. Extract again with two 15-mL portions of leaf.
chloroform, again collecting the clarified organic phase. Wash
the sodium sulfate and the tip of the funnel with 5 mL of Pack~ging and storage-Preserve in tight, light-resistant
chloroform. Evaporate the combined organic phases under containers.
reduced pressure, at a temperature below 45°, add 1 mL of USPReference standards (11)-
chloroform, and mix to dissolve the alkaloids, taking care to USP Atropine Sulfate RS
wet the sides of the container. USP Homatropine Hydrobromide RS
Standardcurve-Prepare three Standardsolutions asfollows. USP Scopolamine Hydrobromide RS
Pipet into three separate.60-mL separators 1.0-,· 2.0-, and Ide~tification-Macerate a quantity of powdered Tablets,
3.0-mL portions, respectively, of Standardpreparation, and equivalent to about 5 mg of the alkaloids of belladonna
add 9.0, 8.0, and 7.0 mL, respectively, of dilute sulfuric acid extract, with 20 mL of water, and transfer to a separator.
(1 in 350). Proceed as directed under Assay preparation, Render the solution al.kalin~ with 6 N ammonium hydroxide,
beginning with "add 1.0 mL of Internal standard solution." and extract the alkaloids With 50 mL of chloroform. Filter the
chloroform layer, divide it into two equal portions, and

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488 Belladonna / OfficialMonographs USP 43

evaporate to dryness: the residue responds to the following Assay-

tests. pH 9.5 Phosphate buffer , Internalstandardsolution,
A: To one portion of the dry residue add 2 drops of nitric Standardpreparation, Extraction blank, Standardcurve,
acid, evaporate on a steam bath to dryness, and add a few Chromatographic system, and System suitability-Proceed as
drops of alcoholic potassium hydroxide TS: a violet color is directed in the Assay under Belladonna Extract.
produced. Assay preparation-Proceed with Tincture as directed in the
B: Dissolve the other portion of the residue in 1 mLof dilute Assay under Belladonna Leaf but pipet 2 mL of Tincture (in
hydrochloric acid (1 in 120), and add gold chloride TS, place of "10 mL of this solution") into a 60-mL separator
dropwise with shaking, until a definite precipitate separates. containing 10 mL of dilute sulfuric acid (1 in 350).
Slowly heat until the precipitate dissolves, and allow the Procedure-Proceed as directed in the Assay under
solution to cool: a lusterless precipitate is produced. Belladonna Leaf. Record from the Standard curvethe quantities,
Disintegration (701): 30 minutes. in mg, of atropine and scopolamine in the specimen. Add the
Uniformity of dosage units (905): meet the requirements. quantity, in mg, of atropine and scopolamine, and multiply by
50 to obtain the weight, in mg, of alkaloids per 100 mL.
Assay-
pH 9.5 Phosphate buffer, Internalstandardsolution, Standard
preparation, Extraction blank, Standard curve, Chromatographic
system, and System suitability-Proceed as directed in the
Assay under Belladonna Extract Benazepril Hydrochloride
Assay preparation-Weigh and finely powder not fewer
than 20 Tablets. Transfer an accurately weighed portion of the
powder, equivalent to about 600 IJg of atropine and
scopolamine, to a 60-mL separator, add 10.0 mL of dilute
sulfuric acid (1 in 350), and sonicate to dissolve as much as • Hel
possible of the specimen. Proceed as directed for Assay
preparation in the Assay under Belladonna Leaf, beginning with
"add 1.0 mL of Internal standardsolution."
Procedure-Proceed as directed for Procedure- in the Assay

the quantities, in mg, of atropine and scopolamine in the

°
- under Belladonna Extract. Record from the Standard curves C 24 H 28 N 2 5 • HCI 460.95
1H-l-Benzazepine-l-acetic acid, 3-[[1-(ethoxycarbonyl)-3-
weight of specimen taken. phenylpropyl]amino]-2,3,4,5-tetrahydro-2-oxo-,
monohydrochloride, [S-(R*,R*)]-.
(35)-3-[[(15)-1-Carboxy-3-phenylpropyl]amino]-2,3,4,5-
tetrahydro-2-oxo-l H-l-benzazepine-l-acetic acid, 3-ethyl
ester, monohydrochloride [86541-74-4].
Belladonna Tincture
» Benazepril Hydrochloride contains not less than
» Belladonna Tincture yields, from each 100 mL, 98.0 percent and not more than 102.0 percent of
not less than 27 mg and not more than 33 mg of C 24 H 28 N 2 0 5 • Hel, calculated on the dried basis.
the alkaloids of belladonna leaf. Packaging and storage-Preserve in well-closed
containers, and store at a temperature below 30°, preferably
Belladonna Leaf, in moderately between 15° and 30°.
coarse powder . . 100 9 USP Reference standards (11)-
----- USP Benazepril Hydrochloride RS
To make about . 1000 mL USP Benazepril Related Compound A RS
(3R) 3-[[(1 R) 1-(Ethoxycarbonyl)-3-phenylpropyl]
amino]-2,3,4,5-tetrahydro-2-oxo-1 H-l-benzazepine-l-
Prepare a tincture by Process P as modified for acetic acid, monohydrochloride.
assayed Tinctures (see Pharmaceutical Dosage Forms C24H28N20S' HCI 460.95
(1151 »,using a mixture of 3 volumes of alcohol USP Benazepril Related Compound B RS
(35) 3-[[(1 R) 1-(Ethoxycarbonyl)-3-phenylpropyl]
and 1 volume of water as the menstruum. Finally
amino]-2,3,4,5-tetrahydro-2-oxo-l H-1-benzazepine-l-
adjust the Tincture to contain, in each 100 mL, 30 acetic acid, monohydrochloride.
mg of the alkaloids of belladonna leaf. C24H28N20S' HCI 460.95
USP Benazepril Related Compound C RS
Packaging and storage-Preserve in tight, light-resistant 3-(1-Carboxy-3-phenyl-l (5)-propyl)amino-2,3,4,5-
containers, and avoid exposure to direct sunlight and to tetrahydro-2-oxo-1 H-1-(3S)-benzazepine-l-acetic acid.
excessive heat. C22H24N20S 396.44
USP Reference standards (11)-
USP Benazepril Related Compound D RS
USP Atropine Sulfate RS (3-(1-Ethoxycarbonyl-3-cyclohexyl-(1 5)-propyl)amino-
USP Homatropine Hydrobromide RS 2,3,4,5-tetrahydro-2-oxo-1 H-1-(3S)-benzazepine)-1-
USP Scopolamine Hydrobromide RS
acetic acid, monohydrochloride.
Alcohol Determination, Method /I (611): between 65.0%
and 70.0% of C2H sOH, determined by the gas-liquid C24H34N20S' HCI 467.00
USP Benazepril Related Compound E RS
chromatographic procedure, acetone being used as the 3-Amino~2,3,4,5-tetrahydro-2-oxo-1 H-1-(3S)-
internal standard.
benzazepine-1-acetic acid.

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 USP43 Official Monographs / Benazepril 489

USP Benazepril Related Compound F RS is maintained at 30°. Chromatograph the Resolution solution
tert-Butyl-3-amino-2,3,4,5-tetrahydro-2-oxo-l H-1-(35)- and record the peak responses as directed for Procedure: th~
benzazepine-l-acetic acid. resolution, R, between benazepril hydrochloride and
USP Benazepril Related Compound G RS benazepril related compound A is not less than 2.0.
(3-(1-Ethoxycarbonyl-3-phenyl-(1 5)-propyl)amino- Chromatograph the Dilute standard solution, and record the
2,3,4,5-tetrahydro-2-oxo-l H-l-(35)-benzazepine)-1- peak responses as directed for Procedure: the signal-to-noise
acetic acid ethyl ester. ratio .is not less t~an 10:1. Chromatograph the Standard
solution: the relative standard deviation for replicate injections
Identification- determined from the benazepril related compound A peak is
not more than 10%.
Procedure-Separately inject equal volumes (about 50 IJL) of
the Standard solution and the Test solution into the
chromatograph, record the chromatograms, and measurethe
lIY~Q)'
area for the benazepril related compound A peak. Calculate
of the major peak in the
the percentage of benazepril related compound A in the
chromatogram of the Assay preparation corresponds to that in
portion of Benazepril Hydrochloride taken by the formula:
the chromatogram of the Standard preparation, as obtained
in the Assay. 1OO(C siC r)(r vir 5)
C: It responds to the test for Chloride (191).
Absorbance of solution-The absorbance of a 1 in 100 in which C s is the concentration, in mg per mL, of USP
solution of it in methanol, determined in a 1-cm cell at 420 Benazepril Related Compound A RS in the Standard solution'
nm, is not more than 0.015, methanol being used asthe blank. C t is the concentration, in mg per mL, of Benazepril '
Absorptivity- Hydrochloride in the Test solution; r v is the peak responsefor
Test preparation-Dissolve an accurately weighed quantity benazepril related compound A obtained from the Test
of Benazepril Hydrochloride in methanol, and dilute solution; and r s is the peak response for benazepril related
quantitatively, and stepwise if necessary, to obtain a solution compound A obtained from the Standard solution: The limit of
having a known concentration of about 0.025 mg per mL. benazepril related compound A is given in the table below.
Procedure-Proceed as directed under Ultraviolet-Visible
Spectroscopy (857), and measure the absorbance at 238 nm: Relative
the absorptivity is between 21.0 and 23.2. Benazepril Related Retention
Loss on drying (731)-Dry it at 105° for 3 hours: it loses not Compound Time Limit (%)
more than 1.5% of its weight.
2.3 0.1
Residue on ignition (281 )-Ignite at 600°. Not more than
0.1% residue is found. 1 «3R)-3-[[(1R)-1.(ethoxycarbonyl)-3-phenylpropyl]amino]-2,3,4,5-
tetrahydro-z-oxo-t H-1·benzazepine·1·acetic acid, monohydrochloride
Related compounds-
TEST 1 (FOR BENAZEPRIL RELATED COMPOUND A)-
TEST 2 (FOR BENAZEPRILRELATED COMPOUNDS B, C, 0, E, F, AND G)
pH 6.0. Phosphate buffer-Dissolve 9.66 g of monobasic
potassium phosphate and 2.68 g of dibasic sodium
phosphate, heptahydrate in about 900 mL of water, and dilute Te~rab.~tylamm.onium bromide solution, Mobile phase, System
with water to 1000 mL. sUitability solution, and Chromatographic system-Proceed as
Mobile phase-Prepare a filtered and degassed mixture of pH directed in the Assay.
6.0 Phosphate buffer and methanol (80:20). Make adjustments Standard solution-Dissolve accurately weighed quantities of
if necessary (see System Suitability under Chromatography USP Benazepril Hydrochloride RS, USP Benazepril Related
(621» Compound B RS, USP Benazepril Related Compound C RS
USP Benazepril Related Compound DRS, USP Benazepril '
Resolution solution-Dissolve accurately weighed quantities of
USP Benazepril Hydrochloride RS and USP Benazepril Related Related Compound E RS, USP Benazepril Related Compound
FRS, and USP Benazepril Related Compound G RS in Mobile
Comp.ou~d A RS in Mob~/e phase~ and dilute quantitatively, and
stepwise If necessary, With Mobile phase to obtain a solution phase to obtain a solution having known concentrations of
having known concentrations of about 1.0 mg per mL and about 1 IJg of USP Benazepril Hydrochloride RS per mL and 10
0.005 mg per mL, respectively. J.lg of each related compound per mL.
Standqrd stock solution-,?issolve an accurately weighed Test solutio0-Transfer about.50 mg of Benazepril
quantity of USP Benazepnl Related Compound A RS in Mobile Hydrochloride, accurately weighed, to a 50-mL volumetric
phase to obtain a solution having a known concentration of fla.sk, dissolve in and dilute with Mobile phase to volume, and
about 0.05 mg per mL. mix.
Standard solution-Dilute a suitable portion of Standard stock Procedure-Separately inject equal volumes (about 25 IJL) of
solution, accurately measured, with Mobile phase to obtain a the Standard solution and the Test solution into the
s~lution having a known concentration of about 5 IJg per mL.
chromatograph, record the chromatograms, and measurethe
Dilute standard solution-Dilute a suitable portion of Standard areasfor all the peaks. Calculate the percentage of benazepril
related compounds in the portion of Benazepril Hydrochloride
stoc~ solution,. accura~ely measured, with Mobile phase to
obtain a solution having a known concentration of about 1 IJg taken by the formula:
per mL.
1OO(C siC r)(r vir 5)
Test solution-Transfer about 50 mg of Benazepril
Hydrochloride, accurately weighed, to a 50-mL volumetric in which C s is the concentration, in mg per mL, of the relevant
flask, dissolve in and dilute with Mobile phase to volume, and
USp Reference Standard in the Standard solution; C r is the
mix.
Chromatographic system (see Chromatography (621»-The concentration, in mg per mL, of benazepril hydrochloride in
liquid chromatograph is equipped with a 240-nm detector the Test solution; r u is the peak response for the relevant
and a 4.0-mm x 1O-cmcolumn that contains packing L41. The benazepril related compound obtained from the Test solution'
flow rate is about 0.9 mL per minute. The column temperature and r s is the peak response for the relevant benazepril related

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 490 Benazepril / OfficialMonographs USP 43

compound obtained from the Standard solution (see Table 1 Procedure-Separately inject equal volumes (about 25 ~L)
for values). of the Standard preparation and the Assay preparation into the
chromatograph, record the chromatograms, and measure the
Table 1 responses for all the peaks. Calculate the quantity, in mg, of C
Relative 24 H 28 N 20 S • HCI in the portion of Benazepril Hydrochloride
Benazepril Related Retention taken by the formula:
Compound Time Limit (%)
El 0.4 0.2 250C(r ulr s)
F2 0.5 0.2 in which C is the concentration, in mg per mL, of USP
C3 0.6 0.3 Benazepril Hydrochloride RS in the Standard preparation; and
B4
r u and r s are the peak responses obtained from the Assay
1.5 0.5
preparation and the Standard preparation, respectively.
05 1.7 0.2
G6 2.0 0.2

1 3-Amino-2,3,4,5-tetrahydro-2-oxo-l H-l-(3S)-benzazepine-1-acetic acid

2 t-Butyl-3-amino-2,3,4,5-tetrahydro-2-oxo-1 H-l-(3S)-benzazepine-1-acetic
Benazepril Hydrochloride Compounded
acid
3 3-(1-Carboxy-3-phenyl-(1 S)-propyl)amino-2,3,4,5-tetrahydro-2-oxo-1 H-1-
Oral Suspension, Veterinary
(3S)-benzazepine)-1-acetic acid
4 Mixture of diastereoisomers (3-(1-ethoxycarbonyl-3-phenyl-(1 R)-propyl)
DEFINITION ,
amino-2,3,4,5-tetrahydro-2-oxo-1 H-1-(3S)-benzazepine)-1-acetic acid and (3- Benazepril Hydrochloride Compounded Oral Suspension,
(1-ethoxycarbonyl-3-phenyl-(1 S)-propyl)amino-2,3,4,5-tetrahydro-2-oxo-1 H- Veterinary contains NLT 90.0% and NMT 110.0% of the
1-(3R)-benzazepine)-1-acetic acid labeled amount of benazepril hydrochloride (C24H28N20s .
5 3-(1-Ethoxycarbonyl-3-cyclohexyl-(1 S)-propyl)amino-2,3,4,5-tetrahydro-2- HCI).
oxo-1 H-1-(3S)-benzazepine)-1-acetic acid monohydrochloride
Prepare Benazepril Hydrochloride Compounded Oral
6 3-(1-Ethoxycarbonyl-3-phenyl-(1 S)-propyl)amino-2,3,4,5-tetrahydro-2-oxo-
1H-1-(3S)-benzazepine)-1-acetic acid ethyl ester Suspension, Veterinary, 5 mg/mL, as follows (see
Pharmaceutical Compounding-NonsterilePreparations
In addition to not exceeding the limits for benazepril related (795».
compounds in Table 1 not more than 0.1% of any other single
impurity is found; [NOTE-For calculating any other single Benazepril Hydrochloride
unspecified impurity, C s is the concentration of the USP powder 500mg
Benazepril Hydrochloride RS in the Standard solution.] and not Vehicle: a 1:1 mixtureof Ora-Plus'
more than 2.0% of total impurities (excluding benazepril and Ora-Sweet" r a sufficient quantity
related compound A from Test 1) is found. to make 100 mL

Assay- a Perrigo Pharmaceuticals, Allegan, MI.

Tetrabutylammonium bromidesolution-Dissolve 0.81 g of
tetrabutylammonium bromide in 360 mL of water containing Pour the Benazepril Hydrochloride powder into a suitable
0.2 mL of glacial acetic acid. container. Wet the powder with a small amount of Vehicle,
Mobilephase-Prepare a filtered and degassed mixture of and triturate to make a smooth paste. Add the Vehicle to
methanol and Tetrabutylammonium bromide solution (64:36). make the contents pourable. Transfer the contents, stepwise
Make adjustments if necessary (see System Suitability under and quantitatively, to a calibrated container using the
Chromatography (621 ». . remainder of the Vehicle. Add sufficient Vehicle to bring to
System suitability solution-Dissolve accurately weighed final volume. Shake to mix well.
quantities of USP Benazepril Hydrochloride RS and USP
Benazepril Related Compound B RS in Mobilephase to obtain ASSAY
a solution having known concentrations of about 0.4 mg of • PROCEDURE
each per mL. Solution A: 25 mM sodium phosphate adjusted with
Standard preparation-Dissolve an accurately weighed phosphoric acid to a pH of 3.0. Pass through a nylon filter
quantity of USP Benazepril Hydrochloride RS in Mobilephase of 0.45-~m pore size.
to obtain a solution having a known concentration of about Mobile phase: Acetonitrile and Solution A (40:60)
0.2 mg per mL. Diluent: Water adjusted with phosphoric acid to a pH of 3.0
Assay preparation-Transfer about 10.0 mL of the Test Standard stock solution: 5 mg/mL of USP Benazepril
solution (from either Test 1 or Test 2), prepared as directed in Hydrochloride RS in Diluent. Sonicate for 3 min. Mix well,
the test for Related compounds, to a 50-mL volumetric flask, and store at 2°-8°.
dilute with Mobilephase to volume, and mix. Standard solution: O.Olmg/mL of benazepril
Chromatographic system (see Chromatography (621 The »- hydrochloride prepared with Standard stock solution and
Diluent. Centrifuge for 5 min at 14,000 rpm, and use the
liquid chromatograph is equipped with a 240-nm detector
and a 4.6-mm x 3-cm guard column that contains packing L1 supernatant. Protect from light, and store at 2°_8°.
connected to a 3.9-mm x 30-cm column that contains packing Sample solution: Shakethoroughly each bottle of Oral
L1. The flow rate is about 1 mL per minute. Chromatograph Suspension, Veterinary. Transfer 2.0 mL of the Oral
the System suitability solution, and record the peak responses Suspension, Veterinary into a l-L volumetric flask, and
asdirected for Procedure: the resolution, R, between benazepril dilute with Diluent to volume. Mix well. Centrifuge for 5 min
hydrochloride and benazepril related compound B is not less at 14,000 rpm, and usethe supernatant. Protect from light,
than 1.7; and the relative standard deviation for replicate and store at 2°-8°.
injections determined from benazepril hydrochloride and Chromatographic system
benazepril related compound Bis not more than 2.0% for (See Chromatography (621), System Suitability.)
each. Mode: LC

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 USP 43 Official Monographs / Benazepril 491

Detector: UV 210 nm an' aliquot of the supernatant through a suitable filter,

Column: 4.6-mm x 25-cm; 5-l..Im packing L1 discarding the first 6mLof the filtrate.]
Temperatures Application v()lume: 20 1..11.:" "
Column: 30° Developing solvent system: Etnyl~ acetate,methqnol,' and
Autosampler: 5° arn,_moriium hydroxide (8,0:20:1,5)4(USJll:Ai.;9-z019) ,
Flow rate: 1.2 mL/min
Injection volume: 251..1L ~4dthefollowing:
System suitability
Sample: Standard solution .... he UVspec~rum of themajbrpeak of the Sample
[NoTE-The retention time for benazepril sou Ion correspondstp thatof the Standard solution, as
hydrochloride is about 6.5 min.] obtained intheAssaY'4 (USP 1~Aug~z019)
Suitability requirements - B. The retention time of the major peak of the Sample
Tailing factor: NMT 2.0 solution corresponds to that of the Standard solution, as
Relative standard deviation: NMT 2.0% for replicate obtained in the Assay.
injections
Analysis ASSAY
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of Change to read:
benazepril hydrochloride (Cz4HzsNzOs . HCI) in the
- PROCEDURE
portion of Oral Suspension, Veterinary taken:
Solution A: Dissolve 0.81 g of tetrabutylammonium
Result =(rufrs) x (Cs/Cu) x 100 bromide in 360 mL of water containing 0.2 mL of acetic
acid, glacial.
ru =peak response of benazepril hydrochloride from Mobile phase: Methanol and Solution A (64:36)
the Sample solution System suitability solution: 0.4 mg/mL each of USP
rs = peak response of benazepril hydrochloride from Benazepril Hydrochloride RS and USP Benazepril Related
the Standard solution Compound B RS in Mobilephase
Cs = concentration of benazepril hydrochloride in the Standard solution: 0.2 mg/mL of USP Benazepril
Standard solution (mg/mL) Hydrochloride RS in Mobile phase
Cu = nominal concentration of benazepril Sample solution: .i.No . ally 0.2 mg/mL ofber'lazepril
hydrochloride in the Sample solution (mg/mL) hydrochloride in ,, . hoseprepared as
follows..i.(USP1-A~g- 9) Transfer a portion from NLT 20 finely
Acceptance criter!a: 90.0%-110.0% powdered Tablets, equivalent to 50 mg of benazepril
hydrochloride, to a 250-mL volumetric flask. Add
SPECIFIC TESTS A.i. (USP1-Au9-z019) 150 mL of Mobilephase, and shake by
- pH (791): 3.8-4.8 mechanical means for 30 min. Dilute with Mobilephase to
ADDITIONAL REQUIREMENTS volume, mix, and centrifuge. Pass an aliquot of the
• PACKAGING AND STORAGE: Package in tight, light-resistant supernatant through a suitable filter, discarding the first 6
containers. Store at 2°_8° or at controlled room mL of the filtrate.
temperature. Chromatographic system
- LABELING: Label it to indicate that it is to be well-shaken (See Chromatography (621), System Suitability.)
before use, and to state the Beyond-Use Date: Label it to Mode: LC
state that it is for veterinary use only. Detector: UV240 nm . .i.For Identification A, use a diode
- BEYOND-USE DATE: NMT 90 days after the date on which it array detector in the range of 200-400 nrr..i.(USP l-Aug-Z019)
was compounded when stored at 2°_8° or at controlled Columns
room temperature Guard: 4.6-mm x 3-cm; .i.7-l..Im.i.(uSP 1:Aug-Z019) packing
- USP REFERENCE STANDARDS (11) .i.L7.i.(USP ,.~Aug-2019)
USP Benazepril Hydrochloride RS Analytical: 3.9-mm x 30-cm; .i.1 O-I.lm.i. (USP l-Aug-Z019)
packing L1
Flow rate: 1 mL/min
Injection volume: 25 I..IL
Benazepril Hydrochloride Tablets .i.Run time: NLT 3 times .the retention time of
benazepril:" (USP l-Aug~Z019) "
DEFINITION System suitability
Benazepril Hydrochloride Tablets contain NLT 90.0% and Sample: System suitabilitysolution
NMT 110.0% of the labeled amount of benazepril .i.[NOTE-The relative retention times for benazepril and
hydrochloride (Cz4HzsNzOs . HCI). benazepril related compound Bpeaks are about l.O
and 1.5, respectively.].i. (USP 1:Aug-2019)
IDENTIFICATION Suitability requirements
Resolution: NLT 42.0.i.(USP':Aug-Z019) between benazepril
Delete the following: ...'(USP l-Aug-Z019) and benazepril related compound B
A_A. THIN~LAYER CHROMATOGRAPH'le IDENTIFICATioN-TEsT
peaks
(201)' , '" ' Relative standard deviation: NMT 2.0% for both
Sample solution: ,Equivalent to . benazepril ~.i..i. (USP 1.Aug-Z019) and benazepril related
hydrochloride from p compound B peaks
methanol. [NoTE""':'Sha Analysis
Dilute with methanol to volu Samples: Standard solution and Sample solution

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492 Benazepril / OfficialMonographs USP 43

CalculGlte the percentage of AtO<rlciBelecr-llmoumt Tolerances: NlT 70% (Q) of the labeled amount of
ofA(USP1-Aug,2019) benazepril hydrochloride (C24H28N20s . benazepril hydrochloride (C24H28N20s . HCI) is dissolved.
HC!) in the portion of Tablets taken:
Chcinge .to r,ead:
Result =(rvlrs) x (CsICv) x 100
• UNIFORMITY OF DOSAGE UNITS (90S): Meet the
rv = peak response of benazeprilfrom the Sample requirements
solution ... A' (USP, l-Aug-2019)
rs = peak response of benazepril from the Standard
IMPURITIES
solution
Cs = concentration of USP Benazepril Hydrochloride
RS in the Standard solution (mg/ml) Change to read:
Cv = nominal concentration of benazepril • ORGANIC IMPURITIES
hydrochloride in the Sample solution (mg/ml) Solution A, Mobile phase, System suitability solution,
Sample solution, and System suitability: Proceed as
Acceptance criteria: 90.0%-110.0% directed in the Assay.
PERFORMANCE TESTS Standard solution: 0.006 mg/ml of USP Benazepril Related
Compound C RS in Mobile phase
,Change to read: Chromatographic systel11: Proceed as directed in the
Assay, Aexceptfor the.fnjection volume. A (USP 1-Aug-2019)
• DISSOLUTION (711) Injection volume: ....25IJLfor System ,suitability . "
Test 1 solution; ... (USP 1.A~9-2019)80 IJli "'for Standard solution and
Medium: Water; 500 ml Sample solution... (USP1~Aug-201'9)
Apparatus 2: 50 rpm Analysis
Time: 30 min Samples: Sample solution and Standard solution
Solution A, Mobile phase, System suitability solution, Calculate the percentage of benazepril related compound
...... «(lsi> 1-Aug-2019) and System suitability:Proceed as C in the portion of Tabletstaken:
directed in the Assay.
Standard solution: ~o.o2~ Result = (rulrs) x (CslCv) x 100
Benazepril Hydrochloride R
Sam Iesolution: I
= peak response of benazepril related compound C
from the Sample solution
= peak response of benazepril related compound C
from the Standard solution
Ch = concentration of USP Benazepril Related
Assay, except fo Compound C RS in the Standard solution
Injection volume: (mg/ml)
= nominal concentration of benazepril
solution; A ( ug.2019)60 IJl"'fo hydrochloride in the Sample solution (mg/ml)
Samplesolu ... (USP1.Aug-2019)
Analysis Calculatethe percentage of any unspecified impurityin the
Samples: Standard solution and Sample solution portion of Tablets taken:
Calculatethe percentage of :"the labeled amount
of... (USP 1.Aug-2019) benazepril hydrochloride (C24H28N20s . Result =(rvlrr) x 100
HCI) dissolved:
rv = peak response of each impurityfrom the Sample
"'Result;:;' (r~/rs)x(ciL)x Vxl0p solution
rr = sum of the responses of all the peaks including
=peak responseof benazeprilfroridhe Sample benazepril related compound C from the Sample
solution solution
~ peak response of benazepril from the Standard
solution Acceptance criteria: See Table 7.
=concentrati oride
RS in the St Table 1
L = label im "'Relative
Retention
Acceptance
V :::: volu of Criteria,
Name Time... (UsP).A~g.2019) NMT (%)
Tolerances: NlT 80% (Q) of the labeled amount of Benazepril related
benazepril hydrochloride (C24H28N20s' HC!) is dissolved. compound C ":0.6.: (USP 1.A~9:20i~) 3.0
Test 2: Ifthe product complies to this test, the labeling Benazepril .l1.0Jo.(&SP1~u9-20;9) -
indicates that the prodLJ5~. Il1~~~,~~P.iPissolution Test 2.
Medium, Apparatus~2,jli(lJ$P,il:Aug-29i9) Standard solution, 4 $enazep'ril.related
+Jo.(U~p··;-Au~.iti19)
Sample solution, Chromatographic system, and cbmpoondBa,b J.5
Analysis:Proceed as directed in Dissolution Test 7. Anyunspecified impurity - AO.2A·(lfSP1,.A~g'2019)
Solution A, Mobile phase, System suitability solution,
and System suitability: Proceed as directed in the Assay.
Time: 45 min

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 USP 43 Official Monographs / Bendamustine 493

Table 1 (continued) DEFINITION

Acceptance Bendamustine Hydrochloride is anhydrous or contains one
Criteria, molecule of hydration. The anhydrous form contains NLT
Name NMT (%) 98.0% and NMT 102.0% of bendamustine hydrochloride
Total impurities' 2.0 (C16H21C12N302' HCI), calculated on the as-is basis. The
monohydrate form contains NLT 98.0% and NMT 102.0%
of bendamustine hydrochloride (C16H21ClzN30Z' HCI),
calculated on the anhydrous and solvent-free basis.
IDENTIFICAnON

ADDITIONAL REQUIREMENTS

, Q)
time of t e major peak of the Sample
I"of",onf"inn

solution corresponds to that of the Standardsolution, as

.~
obtained in the Assay.
more than one Dissolution test is given, the • C. IDENTIFICATION TESTS-GENERAL (191), Chemical
labeling states the Dissolution test used only if Test 7 is not Identification Tests, Chloride
used. ASSAY
• PROCEDURE
Solution A: 0.1% (v/v) trifluoroacetic acid in water
Solution B: 0.1% (v/v) trifluoroacetic acid in acetonitrile
• USP REFERENCE STANDARDS (11) Mobile phase: See Table 7.
USP Benazepril Hydrochloride RS
USP. Ben_~zepril Related<:C?rTlP()~n<:l~c~~ Table 1
~:~~~. . .
Time Solution A Solution B
(min) (%) (%)
0 93 7
5 93 7
13 73 27
16 73 27
25 43 57
26 10 90
31 10 90
40 93 7
45 93 7

Diluent: 1-Methyl-2-pyrrolidone and Solution A (1:1)

Standard solution: 4.2 mg/mL of USP Bendamustine
Hydrochloride RS in Diluent
Bendamustine Hydrochloride Sample solution: 4.2 mg/mL of Bendamustine
Hydrochloride in Diluent
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Hel
Detector: UV 254 nm
Column: 4.6-mm x 15-cm; 5-lJm packing L60
Temperatures
Autosampler: 2°-8°
C16HzlC12N302' HCI 394.72 Column: 30°
1H-Benzimidazole-2-butanoic acid, 5-[bis(2-chloroethyl) Flow rate: 1 mL/min
amino]-l-methyl-, monohydrochloride; Injection volume: 2 IJL
4-{5-[Bis(2-chloroethyl)amino]-1-methyl-1 H-benzimidazole- Analysis time: 25 min
2-yl}butanoic acid monohydrochloride [3543-75-7]. System suitability
Bendamustine (free base) [NOTE-The slower syringe draw rate and higher detector
C16H21CI2N30Z 358.26 sampling rate can be applied in order to improve the
[16506-27-7]. precision.]
Monohydrate Sample: Standardsolution
Suitability requirements
C16H21C12N302' HCI· H20 412.74 Tailing factor: NMT 2.0
[1374784-02-7]. Relative standard deviation: NMT 1.0%

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494 Bendamustine / OfficialMonographs USP 43

Analysis Table 2
Samples: Standardsolution and Sample solution Relative Relative Acceptance
Calculate the percentage of bendamustine hydrochloride Retention Response Criteria,
(C16HzlC1zN30Z' HC!) in the portion of Bendamustine Name Time Factor NMT (0/0)
Hydrochloride taken: Bendamustine related
compound A 0.25 0.76 0.25
Result = (ru/r s) x (Cs/Cu) x 100
Bendamustine related
compound C 0.60 0.83 0.20
= peak response from the Sample solution
= peak response from the Standard solution Bendamustine related
compound D 0.69 0.93 iJ.·F·;~C(
= concentration of USP Bendamustine
Hydrochloride RS in the Standardsolution Bendamustine related
(mg/mL) compound E 0.73 1.2 0.45
=concentration of Bendamustine Hydrochloride Bendamustine related
in the Sample solution (mg/mL) compound G 0.90 3.1 0.35

Acceptance criteria: 98.0%-102.0% on the as-is basisfor

Bendamustlne 1.0 - -
the anhydrous form; 98.00/0-1 02.0% on the anhydrous and Bendamustine related
solvent-free basisfor the monohydrate form compound H 1.15 0.98 0.30
Bendamustine related
IMPURITIES 1.20 1.1 0.40
compound I
• RESIDUE ON IGNITION (281): NMT 0.1%
Any individual
unspecified -
impurity 1.0 0.10
• ORGANIC IMPURITIES Total impurities - - 1.0
Mobile phase, Diluent, Standard solution, Sample
solution, and Chromatographic system: Proceed as 'SPECIFIC TESTS
directed in the Assay. • WATER DETERMINATION (921), Method I, Method la: NMT
System suitability solution: 4.2 mg/mL of USP 1.0% for the anhydrous form; 3.00/0-5.5% for the
Bendamustine Hydrochloride RS, and 0.02 mg/mL each of monohydrate form
USP Bendamustine Related Compound A RS, USP • BACTERIAL ENDOTOXINS TEST (85): Meets the requirements
Bendamustine Related Compound C RS, USP • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
BendarnustlneRelated Compound DRS, USP SPECIFIED MICROORGANISMS (62): The total aerobic
Bendamustine Related Compound ERS, USP Bendamustine microbial count is NMT 10 3 du/g. The total combined
Related Compound G RS, USP Bendamustine Related molds and yeasts count is NMT 10z du/g.
Compound H RS, and USP Bendamustine Related
Compound I RS in Diluent . ADDITIONAL REQUIREMENTS
Sensitivity solution: 2 IJg/mL of USP Bendamustine • PACKAGING AND STORAGE: Preserve in well-closed
Hydrochloride RS in Diluent, from the Standardsolution containers. Store at room temperature.
System suitability • USP REFERENCE STANDARDS (11)
Samples: System suitability solution and S~nsitivity solution USP Bendamustine Hydrochloride RS
Suitability requirements USP Bendamustine Related Compound A RS
Resolution: NLT 5 between the bendamustine related 4-{5-[Bis(2-hydroxyethyl)amino]-1-methyl-l H-
compound G and bendamustine peaks; NLT 4 between benzimidazol-2-yl}butanoic acid.
the bendamustine related compound Hand C16Hz3N304 321.38
bendamustine related compound I peaks, System USP Bendamustine Related Compound C RS
suitability solution Ethyl 4-{5-[bis(2-hydroxyethyl)amino]-1-methyl-l H-
Signal-to-noise ratio: NLT 10, Sensitivity solution benzim idazol-2 -yl}butanoate.
~~yili . ClsHz7N304 349.43
Sample: Sample solution USP Bendamustine Related Compound D RS
Calculate the percentage of each impurity in the portion of 4-{5-[(2-Chloroethyl)amino]-1-methyl-l H-benzimidazol-
Bendarnustlne Hydrochloride taken: 2-yl}butanoic acid.
C14H1SCIN30Z 295.77
Result = (ru/{L[ru x (1/f)] + rsD x (1/f) x 100 USP Bendamustine Related Compound E RS
4-{5-[(2-Chloroethyl)(2-hydroxyethyl)amino]-l-methyl-
rv = peak area of each impurity from the Sample 1H-benzimidazol-2-yl}butanoic acid.
solution
C16HzzCIN303 339.82
F = relative response factor for each impurity (see
USP Bendamustine Related Compound G RS
Table 2)
4-[6-(2-Chloroethyl)-3,6,7,8-tetrahydro-3-
ts = peak area of bendamustine from the Sample methylim idazo[4 ,5-h ][1 /4]benzothiazin-2-yl] butanoic
solution
acid.
Acceptance criteria: See Table 2. The reporting threshold is C16HzoCIN30ZS 353.86
0.05%. USP Bendamustine Related Compound H RS
4-[5-({2-[(4-{5-[Bis(2-chloroethyl)amino]-l-methyl-l H-
benzimidazol-2-yl}butanoyl)oxy]ethyl}(2-chloroethyl)
amino)-l-methyl-l H-benzimidazol-2-yl]butanoic acid.
C3zH41CI3N604 680.07

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USP 43 Official Monographs / Bendamustine 495

USP Bendamustine Related Compound I RS Temperatures

Ethyl 4-{5-[bis(2-chloroethyl)amino]-1-methyl-1 H- 'Autosam I :2°_8°
benzimidazol-2-yl}butanoate. Collini
ClsH2sCl2N302 386.32 F
I

dard solution
,eqlJi obi
eslo ringe 'draw rate and higher
g r(;lte can be applied in order to
precision.]
Tailing ac or: NMT 2.0
ION Relative ~tan~ard deviation:' NMT 1.0%
stine,Hydrochloride Analysis' ," , , ' " - " ..
lyophilized mixture of Ben and Samples: Standard solation and Sample solution ,
Mannitol. It contains (\jLT9 of the Calculate the percentage of the labeled amount of ,' ,
labeied amountof,bendamustlne bendamustine hydrochloride (C16H21C12N302 ~, Hel) in the
(C}6H21 Cl2N302 . HC!)~ portioI') of Bendamustine Hydrochloride for Injection '
~~! '
IDENTIFICATION
• ,A. The retention tim theScimple Result =(rv/fs)x(CslCv)x1 00
solution corr d solutIon" as
obtained in t rv = lJe~krespons~ from the Sample solution
ts = peak'response trom the Standard solution
, Cs = concentration of USP Bendamustlne
Hydrochloride RS in the Standard solution
(mglmL)
Cu == nominal concentration of benda'mustine
hydrochloride in the Sample solution (mg/mL)

Acceptance criteria: 90.0%-110.0%

ASSAY p" FORMANCE TESTS

;. IFORMITV OF DOSJ\<iE UNITS (905):' Meets the
• PROCEDURE
requirements
Solution A: 0.1% (v/v) trifluoroacetic acid in water
Solution 8:0.1 % (v/v) trifluoroacetic acid inacetonitdle
Mobile phase: See Table 7.

Table 1
Time Solution A SolutionS
(min) (%) (%)
0 93 7
5 93 7
13 73 27
16 73 27

25 43 57
26 10 90
31 10 90
40 93 7

45 93 7

Diluent: 1,:,Methyl~2~pyrroliaone and Solution 1:1)

Standard solution: 4.2 mg/mL of USP Benda e
Hydrochloride RS in piluent
Sample solution: Nom Iyequivalerit'to 4:2 mg!mL'of ben
bendamustine hydr() Ide in Diluent, from suit .ity, lution
Bendamustine Hydroc,. oride for Injection Sigh ~noiseratio: NLT1 0,'Sensltivitysolution
chromatographic' system, , " 7. ""
~rialysis,
(See Chromatography:(621'>, System Suitability.) Sample:'S
Mode:",LC Calculatef ortion~of
Detector:UV 254nm BeridamustJn
Column: 4.6-mm x 15-cm; 5-l..Impackirig'L60

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496 Bendamustine / OfficialMonographs USP 43

Result=: (ru/{~[t'u x~(ll f)J+rs}) x(ljf) x 1()O USPBendamustine Related CompoundS,RS . ..

4-(1-Methyl-5-rnorpholino-l H-benzimidazol-:2-yl)
=peak area of each.impurityfrorritfieSample butanoic acid.
solution C16H21N303 303.3.6
::; relative response factorforeach irT)purity (see USP Bendamustine,Related Compound C RS, ' ..
Table 2) Ethyl 4-{5-[bis(2~hydroxyethyl)amino ]-l-methyl-:lH:.
=.peak area of bendamustinefr()rTl the ~ample benzimidazol"2"xl}butanoate.
solution .ClsH27N304349.43.
USP Bendamustine Relate
Acceptance criteria: .SeeTable2. The reporting threshold is 4-(5-[(2:-Chloroethyl)allli
0.1%. .
2-yl}butanoic acid.
Table 2 C14H1SCIN302 .295.77
asp Bendamustine Related C
Relative REH~dve ACceptance
Retehtion Response Criteria, 4i{~~~;~~~~~~~~~J.,.~'~;l~::.. oF1~me!nyl:':
Name Time FCido.r NMT(%)
C16H22CIN303 82
BEmdamustine related USP Bendamust lated C
compdundA 0.25 0:76 (j.3
Mannitol-l-yl 4-{5-[bis(2-cho]:1-~methyl~';
Bendamustine related 0.2 1H-benzimidazol-2-yl}butanoa e.
compound B 0.57 0.84 C22H33CI2N307 . 522.42
Beridamustihe related USP Sendamustine Rei
---;
compoundC a 0.60 0~83 4-[6-(2-:-Chloroethyl
methylimidazo[4, - Jb.iltans>!c
~I:~~ttlne related acid.
c und 0 0.69 0.93 0.6
C16H20CIN302S 353.86
BE!ridamusfine related
compound E 0.73 1.2 1.5 USP Bendamustine Rela
.- 4-[5-({2-[(4-{5-[Bis(2-c
Bendamustine related benzimidazol- .
compoundF 0.88 0.61 0.5
amino)-l-met
Bendamustine'related C32H41CI 3N604
compound 0 ~ 0.90 3., -
USP Bendamustin
Sendamustine 1.0 - >
Ethyl +·{5-[bis(2-ch
benzimidazol-2-yl}b
Bendamustine,related
compound H 1.15 0;98 O.~
ClsH2SCI2N302 38632..:.
Beridamustine related
compoundJ~ 1.20 1.1 -
Any individual
unspeqifi,ed - Bendroflumethiazide
implll'lty 1.0 0;2
Totarlmpurities - - 3.5

aThisprocessimpurity iscontrolledin the drug substanc raph. It is

includedin,the table for identification only,and it is not orted in the
total impurities:
ClsH14F3N304S2 421.41
2H-1,2,4-Benzothiadiazine-7-sulfonamide, 3,4-dihydro-3-
(phenylmethyl)-6-(trifluoromethyl)-, 1,1-dioxide,(±)-;
(±)-3-Benzyl-3,4-dihydro-6-(trifluoromethyl)-2H-1,2,4-
benzothiadiazine-7-sulfonamide l,l-dioxide [73-48-3].
DEFINITION
Bendroflumethiazide contains NLT 98.0% and NMT 102.0%
of bendroflumethiazide (ClsH14F3N304S2), calculatedon the
anhydrous basis.
IDENTIFICATION

Change to read:

Packa • A. ·SPECTROSCOPIC IDE

• lABELING ( : Meets Spectroscopy: 197A or 19 .
• USP REFERENCE S • B. The retention time of the major peak of the Sample
USP Bendamusti ydrochlo,rideRS solution corresponds to that of the Standard solution, as
USP Bendamustine Related Compound A RS obtained in the Assay.
4-{5-[Bis(2~hydroxyethyl)amino} 1-methyl-:1 H-
benzimidazol-2-yl}butanoi~'acid.
C16H23N304 321.38

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 USP 43 Official Monographs / Bendroflumethiazide 497

ASSAY Mode: LC
• PROCEDURE Detector: UV 270 nm
Use low-actinic glasswarefor the Standardsolution and the Column: 4.6-mm x 3D-em; packing L11
Sample solution, and use the solutions within 30 min of Column temperature: 35°
preparation. Flow rate: 1.5 mL/min
Diluent: Methanol and water (80:20) Injection volume: 20 ~L
Mobile phase: Dissolve 5.62 9 of sodium chloride and System suitability
1.97 g of anhydrous sodium sulfate in 1000 mL of water in Sample: Standardsolution
a 2-L volumetric flask. Add 4.0 mL of glacial acetic acid and Suitability requirements
800 mL of methanol, and dilute with water to volume. Resolution: NLT 1.4 between the methanol and
Standard solution: 0.05 mg/mL of USP 2,4-disulfamyl-5-trifluoromethylaniline peaks
Bendroflumethiazide RS in Diluent Relative standard deviation: NMT 3.0% for 5 replicate
Sample solution: 0.05 mg/mL of Bendroflumethiazide in injections
Diluent Analysis
Chromatographic system Samples: Standardsolution and Sample solution
(See Chromatography (621), System Suitability.) Calculate the percentage of 2,4-disulfamyl-
Mode: LC 5-trifluoromethylaniline in the portion of
Detector: UV 270 nm Bendroflumethiazide taken:
Column: 4.6-mm x 15-cm; 5-~m packing L11
Column temperature: 3SO Result = (ru/rs) x (CsICu) x 100
Flow rate: 1.5 mL/min
Injection volume: 20 ~L =peak response of 2,4-disulfamyl-
Run time: NLT 2 times the retention time of 5-trifluoromethylaniline from the Sample solution
bendroflumethiazide =peak response of 2,4-disulfamyl-
System suitability 5-trifluoromethylaniline from the Standard
Sample: Standardsolution solution
Suitability requirements = concentration of USP 2,4-Disulfamyl-
Tailing factor: NMT 2.0 5-trifluoromethylaniline RS in the Standard
Relative standard deviation: NMT 0.73% solution (uq/rnt)
Analysis = concentration of Bendroflumethiazide in the
Samples: Standardsolution and Sample solution Sample solution (~g/mL)
Calculate the percentage of bendroflumethiazide
(ClsH14F3N304S2) in the portion of Bendroflumethiazide Acceptance criteria: NMT 1.5%
taken: SPECIFIC TESTS
• WATER DETERMINATION (921), Method I: NMT 0.5%
Result =(rulrs) x (CsICu) x 100
ADDITIONAL REQUIREMENTS
ru = peak response of bendroflurnethlazlde from the • PACKAGING AND STORAGE: Preserve in tight containers.
Sample solution • USP REFERENCE STANDARDS (11)
rs = peak response of bendroflumethiazide from the USP Bendroflumethiazide RS
Standardsolution USP 2,4-Disulfamyl-5-trifluoromethylaniline RS
Cs = concentration of USP Bendroflumethiazide RS in 4-Amino-6-(trifluoromethyl)-benzene-1,3-disulfonamide.
the Standardsolution (mg/mL) C7HsF3N304S2 319.29
Cu = concentration of Bendroflumethiazide in the
Sample solution (mg/mL)

Acceptance criteria: 98.0%-102.0% on the anhydrous

basis Bendroflumethiazide Tablets
IMPURITIES DEFINITION
• RESIDUE ON IGNITION (281): NMT 0.2% Bendroflumethiazide Tablets contain NLT 90.0% and NMT
• SELENIUM (291) 110.0% of the labeled amount of bendroflumethiazide
Sample: 100 mg of Bendroflumethiazide and 100 mg of (ClsH14F3N304S2)'
magnesium oxide
Acceptance criteria: The absorbance from the Test Solution IDENTIFICATION
is NMT one-half that from the StandardSolution (NMT 30 • The retention time of the major peak of the Sample solution
ppm). corresponds to that of the Standardsolution, asobtained in
• LIMIT OF 2,4-DISULFAMYL-5-TRIFLUOROMETHYLANILINE the Assay.
Use low-actinic glassware for the Standardsolution and the
Sample solution. ASSAY
Mobile phase: Dissolve 5.62 9 of sodium chloride and • PROCEDURE
1.97 g of anhydrous sodium sulfate in lobo mL of water in [NOTE-Use low-actinic glasswarefor the Sample
a 2-L volumetric flask. Add 4.0 mL of glacial acetic acid and solution and the Standard solution.]
800 mL of methanol, and dilute with water to volume. Mobile phase: Dissolve5.62 g of sodium chloride and 1.97
Standard solution: 0.75 ~g/mL of USP 2,4-Disulfamyl- 9 of anhydrous sodium sulfate in 1000 mL of water in a 2-L
5-trifluoromethylaniline RS in methanol volumetric flask. Add 4.0 mL of glacial acetic acid and 800
Sample solution: 50 ~g/mL of Bendroflumethiazide in mL of methanol, and dilute with water to volume.
methanol Standard solution: 50 ~g/mL of USP Bendroflumethiazide
Chromatographic system . RS in methanol
(See Chromatography (621), System Suitability.)

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498 Bendroflumethiazide / OfficialMonographs USP43

Sample solution: Nominally equivalent to 50 ~g/mL from

finely powdered Tablets (NLT 20). Initially add methanol Benoxinate Hydrochloride
(70% of the volume of the flask) and sonicate for 15 min
with occasional shaking. Dilute further with methanol to
the required concentration, and centrifuge for 15 min. • HCI

Chromatographic system
(See Chromatography (621)/ System Suitability.)
Mode: LC C17H2SN203 . HCI , 344.88
Detector: UV 270 nm Benzoic acid, 4-amino-3-butoxy-, 2-(diethylamino)ethyl ester,
Column: 4.6-mm x 30-cm; packing L11 monohydrochloride;
Temperature: 35 ± 5° 2-(Diethylamino)ethyl 4-amino-3-butoxybenzoate
Flow rate: 1.5 mL/min monohydrochloride [5987-82-6].
Injection size: 20 ~L
System suitability DEFINITION
Sample: Standardsolution Benoxinate Hydrochloride contains NLT 98.5% and NMT
Suitability requirements 101.5% of benoxinate hydrochloride (C17H2SN203 . HCI),
Tailing factor: NMT 2.0 calculated on the dried basis.
Relative standard deviation: NMT 3.0% for five
replicate injections IDENTIFICATION
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of ClsH14F3N304S2 in the portion
of Tablets taken:

Result =(ru/rs) x (Cs/Cu) x 100

= peak response from the Sample solution

= peak responsefrom the Standardsolution
= concentration of USP Bendroflumethiazide RS in
the Standardsolution (uq/rnl)
= nominal concentration of bendroflumethiazide
in the Sample solution (l..lg/mL) Acceptance criteria: the requirements
I
• C. TESTS-GENERAL, Chloride (191)
Acceptance criteria: 90.00/0-110.0% Sample solution: 10 mg/mL
PERFORMANCE TESTS Acceptance criteria: Meets the requirements
• DISSOLUTION (711) ASSAY
[NoTE-Protect solutions from light throughout this
• PROCEDURE
test.] Sample solution: Dissolve 250 mg of Benoxinate
Medium: 0.01 N hydrochloric acid; 900 mL Hydrochloride in a mixture of 20 mL of glacial acetic acid
Apparatus 2: 50 rpm and 20 mL of acetic anhydride.
Time: 45 min Titrimetric system
Detector: UV 271 nm Mode: Direct titration
Sample solution: Sample per Dissolution (711). , Titrant: 0.1 N perchloric acid VS
Standard solution: Prepare a stock solution of USP Endpoint detection: Potentiometric
Bendroflumethiazide RS in an appropriate organic solvent, Analysis: Titrate, perform a blank determination, and make
and dilute this solution with Medium to obtain a final any necessary correction. Each mL of 0.1 N perchloric acid
concentration similar to the one expected in the Sample is equivalent to 34.49 mg of benoxinate hydrochloride
solution.
(C17H2sN203' HCI).
Analysis: Determine the amount of C15H14F3N304S2
Acceptance criteria: 98.5%-101.5% on the dried basis
dissolved by using UV absorption on filtered portions of the
Sample solution, suitably diluted with water, if necessary, in IMPURITIES
comparison with a Standardsolution having a known • RESIDUE ON IGNITION (281): NMT 0.2%
concentration of USP Bendroflumethiazide RS. • ORDINARY IMPURITIES (466)
Tolerances: NLT 75% (Q) of the labeled amount of Standard solution: Methanol
C15H14F3N304S2 is dissolved. Sample solution: Methanol
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Eluant: A mixture of chloroform, cyclohexane, and
requirements diethylamine (5:4:1)
Visualization: 12
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers. SPECIFIC TESTS
• USP REFERENCE STANDARDS (11) • pH (791)
USP Bendroflumethiazide RS Sample solution: 10 mg/mL
Acceptance criteria: 5.0-6.0
• Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 3 h.
Acceptance criteria: NMT 1.0%

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USP 43 Official Monographs / Benzethonium 499

ADDITIONAL REQUIREMENTS SPECIFIC TESTS

CD PACKAGING AND STORAGE: Preserve in well-closed • STERIUTY TESTS (71): Meets the requirements
containers. • pH (791): 3.0-6.0
• USP REFERENCE STANDARDS (11)
USP Benoxinate Hydrochloride RS ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers.
• USP REFERENCE STANDARDS (11)
USP Benoxinate Hydrochloride RS

Benoxinate Hydrochloride Ophthalmic

Solution
Benzethonium Chloride
DEFINITION
Benoxinate Hydrochloride Ophthalmic Solution is a sterile
solution of Benoxinate Hydrochloride in water. It contains H,C
"=
H'\lH,
o~0 ~N~
0
W
H'
NLT 95.0% and NMT 105.0% of the labeled amount of H,C I + cf"
h-
benoxinate hydrochloride (C17H2SN203 . HCI).
H,C CH,
IDENTIFICATION
• A. IDENTIFICATION-ORGANIC NITROGENOUS BASES (181) C27H42CIN02 448.08
Sample solution: Nominally 2 mg/mL of benoxinate Benzenemethanaminium, N,N-dimethyl-N-[2-[2-[4-(l,l,3,3-
hydrochloride, prepared as follows. Dilute a volume of tetramethylbutyl)phenoxy]ethoxy]ethyl]-, chloride;
solution, equivalent to 50 mg of benoxinate hydrochloride, Benzyldimethyl[2-[2-[p-(l, l,3,3-tetramethylbutyl)phenoxy]
with 0.01 N hydrochloric acid to 25 mL. ethoxy]ethyl]ammonium chloride [121-54-0].
Analysis: Proceed asdirected in the chapter, beginning with
"Transfer the liquid to a separator". DEFINITION
Acceptance criteria: The solution meets the requirements. Benzethonium Chloride contains NLT 97.0% and NMT
-103.0% of benzethonium chloride (C27H42CIN02),
ASSAY calculated on the dried basis.
• PROCEDUR-=
Standard solution: 400 pq/rnl, of USP Benoxinate IDENTIFICATION
Hydrochloride RS in 0.1 N hydrochloric acid • A.
Sample solution: Nominally 400 ~g/mL of benoxinate Sample solution: 10 mg/mL
hydrochloride, prepared as follows. Transfer a volume of Analysis: Add 2 mL of alcohol, 0.5 mL of 2 N nitric acid, and
Ophthalmic Solution, equivalent to 20 mg of benoxinate 1 ml of silver nitrate TS to 1 mL of the Sample solution.
hydrochloride, to a separator containing 15 mL of water. Acceptance criteria: A white precipitate, which is insoluble
Add 1 mL of ammonium hydroxide, and extract with five in 2 N nitric acid but soluble in 6 N ammonium
20-mL portions of ether. Wash the combined ether extracts hydroxide, is formed.
with 10 ml of water, extract the water washing with 10 mL
of ether, and add this ether extract to the main extract.
Extract the ether solution with three 5-mL portions of 0.1 • B.A
N hydrochloric acid, collect the acid extracts. in a 50-mL
volumetric flask, and dilute with 0.1 N hydrochloric acid to mg· )
volume. • C. The retention time of the major peak of the Sample
Instrumental conditions solution corresponds to that of the Standard solution, as
Mode: UV obtained in the Assay.
Analytical wavelength: About 308 nm ASSAY
Cell: 1 cm • PROCEDURE
Blank: 0.1 N hydrochloric acid Buffer: Dilute 20 mL of triethylamine with water to 1000
Analysis mL, and adjust with phosphoric acid to a pH of 3.0.
Samples: Standard solution, Sample solution, and Blank Mobile phase: Acetonitrile and Buffer (42:58)
Transfer 5.0 mL of each sample to separate 200-ml Diluent: Acetonitrile and water (42:58)
volumetric flasks. Dilute the contents of each flask with System suitability solution: 0.15 mg/mL each of USP
water to volume. Concomitantly determine the Benzethonium Chloride RS and USP Methylbenzethonium
absorbances of the solutions, using the Blank to set the Chloride RS in Diluent
instrument. - Standard solution: 0.15 mg/mL of USP Benzethonium
Calculate the percentage of the labeled amount of Chloride RS in Diluent
benoxinate hydrochloride (C17H2sN203 . HCI) in each mL Sample solution: 0.15 mg/mL of Benzethonium Chloride in
of the Ophthalmic Solution taken: Diluent
Chromatographic system
Result = (Au/As) x (Cs/Cu) x 100 (See Chromatography (621), System Suitability.)
Mode: LC
= absorbance of the Sample solution Detector: UV 225 nm
= absorbance of the Standardsolution Column: 4.6-mm x 15-cm; 5-~m packing L7
= concentration of USP Benoxinate Hydrochloride Column temperature: 40°
RS in the Standard solution (~g/mL) Flow rate: 1 mL/min
= nominal concentration of benoxinate Injection volume: 10 ~L
hydrochloride in the Sample solution (mg/mL) Run time: 1.5 times the retention time of the
methylbenzethonium peak
Acceptance criteria: 95.0%-105.0%

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500 Benzethonium / OfficialMonographs USP 43

System suitability SPECIFIC TESTS

Sample: System suitability solution • Loss ON DRYING (731)
[NOTE-The relative retention times for benzethonium Analysis: Dry at 105° for 4 h.
and methylbenzethonium are 0.7 and 1.0, Acceptance criteria: NMT 5.0%
respectively.] 0

Suitability requirements ADDITIONAL REQUIREMENTS

• PACKAGING AND STORAGE: Preserve in tight, light-resistant
Resolution: NLT 7.0 between the benzethonium and
methylbenzethonium peaks containers.
Tailing factor: NMT 2.0 for the benzethonium peak • USP REFERENCE STANDARDS (11)
Relative standard deviation: NMT 1 .0% for the USP Benzethonium Chloride RS
benzethonium peak USP Methylbenzethonium Chloride RS
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of benzethonium chloride
(C27H42CIN02) in the portion of Benzethonium Chloride Benzethonium Chloride Concentrate
taken:
DEFINITION
Result = (rvlrs) x (CsICv) x 100 Benzethonium Chloride Concentrate contains NLT 94.0% and
NMT 106.0% of the labeled amount of benzethonium
rv = peak response of benzethonium from the chloride (C27H42CIN02)'
Sample solution
ts = peak response of benzethonium from the IDENTIFICAnON
Standard solution • A.
Cs =concentration of USP Benzethonium Chloride RS Sample: Evaporatea volume of the Concentrate, equivalent
in the Standard solution (mg/mL) to 200 mg of benzethonium chloride, on a steam bath.
Cv = concentration of Benzethonium Chloride in the Analysis: To the residue add 2 mL of alcohol, 0.5 mL of 2 N
Sample solution (mg/mL) nitric acid, and 1 mL of silver nitrate TS.
oAcceptance criteria: A white precipitate, which is insoluble
Acceptance criteria: 97.0%-103.0% on the dried basis in 2 N nitric acid but soluble in 6 N ammonium hydroxide,
is formed.
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1% • B.
Sample: Evaporate a volume of the Concentrate, equivalent
to 200 mg of benzethonium chloride, on a steam bath.
Analysis: To the residue add 0.1 g of potassium nitrate, and
• ORGANIC IMPURITIES
heat on a steam bath for 3 min. Cautiously dilute the
Buffer, Mobile phase, Diluent, System suitability solution, solution with water to 10 mL, add 0.5 g of granulated zinc,
and Chromatographic system: Proceed as directed in the and warm the mixture for 10 min. Cool. Add 0.2 g of
Assay. sodium nitrite to 1 mL of the clear liquid, and add this
Standard solution: 1 pg/mL of USP Benzethonium Chloride mixture to 20 mg of naphthol dipotassium disulfonate or
RS in Diluent naphthol disodium disulfonate in 1 mL of ammonium
Sample solution: 1 mg/mL of Benzethonium Chloride in hydroxide.
D~~t . Acceptance criteria: The solution turns orange-red, and a
System suitability brown precipitate may be formed.
Samples: System suitability solution and Standard solution ASSAY
Suitability requirements: Proceed as directed in the • PROCEDURE
Assay, except for Relative standard deviation. Sample solution: Equivalent to 200 mg of benzethonium
Relative standard deviation: NMT 5.0%, Standard chloride from a volume of Concentrate, in a
solution glass-stoppered flask
Analysis Analysis: Add 0.4 mL of bromophenol blue solution (1 in
Samples: Standard solution and Sample solution 2000), 10 mL of chloroform, and 1 mL of 1 N sodium
Calculate the percentage of any individual impurity in the hydroxide. Titrate with 0.02 M sodium tetraphenylboron
portion of Benzethonium Chloride taken: VS until the blue color disappearsfrom the chloroform
layer. Add the last portions of the sodium tetra phenyl boron
Result = (rvlrs) x (CslCv) x 100 solution dropwise, agitating vigorously after each addition.
Each mL of 0.02 M sodium tetraphenylboron is equivalent
tu = peak responseof any individual impurity from the to 8.962 mg of benzethonium chloride (C27H42CIN02)'
Sample solution Acceptance criteria: 94.0%-106.0% of the labeled amount
rs = peak response of benzethonium from the
of benzethonium chloride
Standard solution
Cs = concentration of USP Benzethonium Chloride RS IMPURITIES
in the Standard solution (mg/mL) • LIMIT OF NITRITES
Cv = concentration of Benzethonium Chloride in the Sample: One drop of Concentrate on a spot plate
Sample solution (mg/mL) Analysis: To the Sample add one drop each of glacial acetic
acid, sulfanilic acid in acetic acid solution (1 in 100), and
Acceptance criteria 0_ _ •
l-naphthylamine-acetic acid solution (prepared by boiling
Total impurities: ~NMTi. (ERRHeb.2019) 1.0% 30 mg of l-naphthylamine in 70 mL of water, decanting
the colorless solution from the blue-violet residue, and
mixing with 30 mL of glacial acetic acid).

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USP 43 OfficialMonographs / Benzethonium 501

Acceptance criteria: No red color develops in the resulting IMPURITIES

solution within 10 min. • ORGANIC IMPURITIES, LIMIT OF NITRITES
Sample: One drop of Topical Solution on a spot plate
SPECIFIC TESTS Analysis: To the Sample add one drop each of glacial acetic
• OXIDIZING SUBSTANCES acid, sulfanilic acid in acetic acid (1 in 100), and
Sample: 5 mL 1-naphthylamine-acetic acid solution (prepared by boiling
Analysis: To the Sample add 0.5 mL of potassium iodide TS 30 mg of 1-naphthylamine in 70 mL of water, decanting
and a few drops of 3 N hydrochloric acid. the colorless solution from the blue-violet residue, and
Acceptance criteria: The solution does not acquire a yellow mixing with 30 mL of glacial acetic acid).
color. Acceptance criteria: No red color develops in the resulting
ADDITIONAL REQUIREMENTS solution within 10 min.
• PACKAGING AND STORAGE: Preserve in tight, light-resistant SPECIFIC TESTS
containers. Store at room temperature. • OXIDIZING SUBSTANCES
• LABELING: The label states that this article is not intended Sample: 5 mL
for direct administration to humans or animals. Analysis: To the Sample add 0.5 mL of potassium iodide TS
and a few drops of 3 N hydrochloric acid.
Acceptance criteria: The solution does not acquire a yellow
color.
Benzethonium Chloride Topical ADDITIONAL REQUIREMENTS
Solution • PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
DEFINITION
Benzethonium Chloride Topical Solution contains NLT 95.0%
and NMT 105.0% of the labeled amount of benzethonium
chloride (Cz7H4zCINOz)'
Benzethonium Chloride Tincture
IDENTIFICATION
• A. DEFINITION
Sample solution: Evaporate a volume of Topical Solution, Benzethonium Chloride Tincture contains, in each 100 mL,
equivalent to 200 mg of benzethonium chloride, on a NLT 190 mg and NMT 210 mg of benzethonium chloride
steam bath. (Cz7H4zCINOz)·
Analysis: To the residue add 2 mL of alcohol, 0.5 mL of 2 N Prepare Benzethonium Chloride Tincture 2 mg/mL as
nitric acid, and 1 mL of silver nitrate TS. follows.
Acceptance criteria: A white precipitate, which is insoluble
in 2 N nitric acid but soluble in 6 N ammonium hydroxide, Benzethonium Chloride 29
is formed.
Alcohol 685 mL
• B.
Sample solution: Evaporate a volume of Topical Solution, Acetone 100mL
equivalent to 200 mg of benzethonium chloride, on a
Purified Water, a sufficient quantity to make 1000 mL
steam bath.
Analysis: To the residue add 0.1 g of potassium nitrate, and
heat on a steam bath for 3 min. Cautiously dilute the Dissolvethe Benzethonium Chloride in a mixture of Alcoholand
solution with water to 10 mL, add 0.5 g of granulated zinc, Acetone. Add sufficient Purified Water to make 1000 mL.
and warm the mixture for 10 min. Cool. Add 0.2 g of [NoTE-Benzethonium Chloride Tincture may be colored by
sodium nitrite to 1 mL of the clear liquid, and add this the addition of any suitable color or combination of colors
mixture to 20 mg of naphthol dipotassium disulfonate or certified by the FDA for use in drugs.]
naphthol disodium disulfonate in 1 mL of ammonium IDENTifiCATION
hydroxide.
• A. PROCEDURE
Acceptance criteria: The solution turns orange-red, and a Sample: 50 mL
brown precipitate may be formed. Analysis: To the residue obtained by evaporating the
ASSAY Sample on a steam bath, add 2 mL of alcohol, 0.5 mL of 2
• PROCEDURE N nitric acid, and 1 mL of silver nitrate TS.
Sample solution: Equivalent to 200 mg of benzethonium Acceptance criteria: A white precipitate, which is insoluble
chloride from a volume of Topical Solution, in a in 2 N nitric acid but soluble in 6 N ammonium hydroxide,
glass-stoppered flask is formed.
Analysis: Add 0.4 mL of bromophenol blue solution (1 in • B. PROCEDURE
2000), 10 mL of chloroform, and 1 mL of 1 N sodium Sample: 50 mL
hydroxide. Titrate with 0.02 M sodium tetraphenylboron Analysis: Evaporate the Sample on a steam bath.
VS until the blue color disappears from the chloroform Acceptance criteria: The residue obtained forms
layer. Add the last portions of the sodium tetraphenylboron precipitates with 2 N nitric acid and with mercuric chloride
solution dropwise, agitating vigorously after each addition. TS, both of which dissolve upon the addition of alcohol.
Each mL of 0.02 M sodium tetra phenyl boron is equivalent
ASSAY
to 8.962 mg of benzethonium chloride (Cz7H42CINOz)'
• PROCEDURE
Acceptance criteria: 95.0%-105.0% of the labeled amount Sample: 50 mL
of benzethonium chloride Analysis: Transfer the Sample to a 150-mL beaker, and add,
with continuous stirring, 10 mL of 25 mg/mL of sodium
tetraphenylboron solution. Cover, and allow to stand for 16

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502 Benzethonium / OfficialMonographs USP 43

h. Decant the supernatant into a tared sintered-glass Acetone (C3H60): 9.00/0-11.0%

crucible, applying vacuum filtration. Suspend the
precipitate in 20 mL of water. Transfer the precipitate to the SPECIFIC TESTS
crucible, washing well with water. Dry the precipitate and • SPECIFIC GRAVITY (841): 0.868-0.876
the crucible at 105 0 for 1 h, cool, and weigh. The weight of ADDITIONAL REQUIREMENTS
the precipitate so obtained, multiplied by 0.6122, • PACKAGING AND STORAGE: Package in tight, light-resistant
represents its equivalent of benzethonium chloride containers.
(C27H42C1N02)'
Acceptance criteria: 190-210 mg
OTHER COMPONENTS
• ALCOHOL AND ACETONE CONTENT
Standard solution A (alcohol low standard solution): Add
Benzocaine
5.0 mL of methanol and 11.0 mL of dehydrated alcohol to o

~"'
a 1OO-mL volumetric flask, and dilute with water to volume.
Standard solution B (alcohol high standard solution):
Add 5.0 mL of methanol and 14.0 mL of dehydrated alcohol H,N

to a 1OO-mL volumetric flask, and dilute with water to

volume. C9H ll N0 2 165.19
Standard solution C (acetone low standard solution): Benzoic acid, 4-amino-, ethyl ester;
Add 5.0 mL of methanol and 1.7 mL of acetone to a 100-mL Ethyl p-aminobenzoate [94-09-7].
volumetric flask, and dilute with water to volume. DEFINITION
Standard solution D (acetone high standard solution): Benzocaine, dried over phosphorus pentoxide for 3 h, contains
Add 5.0 mL of methanol and 2.2 mL of acetone to a 100-mL NLT 98.0% and NMT 102.0% of benzocaine (C9H llN0 2).
volumetric flask, and dilute with water to volume.
Sample solution: Transfer 20 mL of Tincture to a 100-mL IDENTIFICATION
volumetric flask, add 5.0 mL of methanol as the internal
standard, and dilute with water to volume.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: GC
Detector: Flame ionization
Column: 120-cm x 4-mm packed with a suitable type of h
support, such' as 80- to 100-mesh S3 Acceptance criteria: Meets the requirements
Carrier gas: Dry helium • B. The retention time of the major peak of the Sample
Temperature solution corresponds to that of the Standard solution, as
Injector port: 240 0 obtained in the Assay.
Detector block: 240 0
Column: 120 0 ASSAY
Flow rate: 90 mL/min • PROCEDURE
Injection size: 0.8 J..IL Solution A: Acetic acid, triethylamine, and water
Analysis (20:1 :980). The pH should be between 2.95 and 3.0 (adjust
as needed).
Samples: Standard solutions A, B, C/ and 0, and Sample
solution Mobile phase: Methanol and Solution A (40:60)
From the respective chromatograms obtained as "described Standard solution: 0.024 mg/mL of USP Benzocaine RS in
previously, calculate the ratios of peak areas for alcohol to Mobile phase
internal standard and for acetone to internal standard. Sample solution: 0.024 mg/mL of Benzocaine in Mobile
Calculate the percentage of alcohol and of acetone in the phase
portion of Tincture taken: Chromatographic system
(See Chromatography (621), System Suitability.)
Result = [A(Y - Z) + B(Z - X)]/(Y - X) Mode: LC
Detector: UV 285 nm
A = percentage of alcohol, or of acetone, in Standard Column: 2.0-mm x 15-cm; 5-J..Im packing L11
solution A and Standard solution C, respectively Flow rate: 0.2 mL/min
Y = ratio of the alcohol peak areas, or the acetone Injection volume: 10 J..IL
peak areas, to the internal standard peak areas for System suitability
Standard solution Band Standard solution 0, Sample: Standard solution
respectively Suitability requirements
z = ratio of the alcohol peak areas, or the acetone Tailing factor: NMT 2.0
peak areas, to the internal standard peak areas Relative standard deviation: NMT 2.0%
for the Sample solution Analysis
B = percentage of alcohol, or of acetone, in Standard Samples: Standard solution and Sample solution
solution B and Standard solution 0, respectively Calculate the percentage of benzocaine (C9H llN0 2) in the
= ratio of the alcohol peak areas, or the acetone portion of Benzocaine taken:
peak areas, to the internal standard peak areas for
Standard solution A and Standard solution C, Result = (r vir s) x (C siC v) x 100
respectively
ru = peak response from the Sample solution
Acceptance criteria rs = peak response from the Standard solution
Alcohol (C 2H sOH): 62.0%-68.0%

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USP43 OfficialMonographs / Benzocaine 503

= concentration of USP Benzocaine RS in the Cu = concentration of Benzocaine in the Sample

Standardsolution (mg/mL) solution (mg/mL)
= concentration of Benzocaine in the Sample
solution (mg/mL) Calculate 'the percentage of any other individual
unspecified impurity in the portion of Benzocaine taken:
Acceptance criteria: 98.0%-102.0% on the previously
dried basis Result = (r vir s) x (C siC u) x 100
IMPURITIES ru =peak response of any other individual impurity
• RESIDUE ON IGNITION (281): NMT 0.1 % from the Sample solution
• CHLORIDE rs = peak response of benzocaine from the Standard
Analysis: To a solution of 200 mg in 5 mL of alcohol, solution
previously acidified with a few drops of diluted nitric acid, Cs =concentration of USP Benzocaine RS in the
add a few drops of silver nitrate TS. Standardsolution (mg/mL)
Acceptance criteria: No turbidity is produced immediately. Cu = concentration of Benzocaine in the Sample
• ORGANIC IMPURITIES solution (mg/mL)
Solution A: Add 1.0 mL of trifluoroacetic acid in 1 L of water.
Solution B: Acetonitrile Acceptance criteria: See Table 2. Disregard peaks less than
Mobile phase: See Table 7. 0.05%.

Table 1 Table 2
Time Solution A Solution B Relative Acceptance
(min) (%) (%) Retention Criteria,
Name Time NMT (%)
0 90 10
Aminobenzoic acid 0.29 0.10
34 50 50
35 90 10
Benzocaine 1.0 -
Ethyl 4-nitrobenzoate 2.1 0.10
38 90 10
Anyother unspecified impur-
ity - 0.10
Diluent: Solution A and Solution B (1:1)
Standard stock solution: 0.1 mg/mL each of USP Total impurities - 1.0
Benzocaine RS, USP Aminobenzoic Acid RS, and USP Ethyl
4-nitrobenzoate RS in Diluent. Sonicate for 2-5 min to SPECIFIC TESTS
dissolve before diluting to final volume. • Loss ON DRYING (731)
Standard solution: 1 IJg/mL each of USP Benzocaine RS, Analysis: Dry over phosphorus pentoxide for 3 h.
USP Aminobenzoic Acid RS, and USP Ethyl 4-nitrobenzoate Acceptance criteria: NMT 1.0%
RS in Diluent from the Standardstock solution
Sample solution: 1 mg/mL of Benzocaine in Diluent. ADDITIONAL REQUIREMENTS
Sonicate for 2-5 min to assist in dissolution as needed • PACKAGING AND STORAGE: Preserve in well-closed
before diluting to final volume. containers.
Chromatographic system • USP REFERENCE STANDARDS (11 )
(See Chromatography (621), System Suitability.) USP Aminobenzoic Acid RS
Mode: LC Benzoic acid, 4-amino.
Detector: UV 280 nm C7H7NOz 137.14
Column: 4.6-mm x 25-cm; 5-lJm packing L7 USP Benzocaine RS
Flow rate: 1.5 mL/min USP Ethyl4-nitrobenzoate RS
Injection volume: 20 IJL Benzoic acid, 4-nitro·, ethyl ester.
System suitability C9H9N04 195.17
Sample: Standardsolution
Suitability requirements
Resolution: NLT 10 from any two peaks
Relative standard deviation: NMT 2.0% for each peak
corresponding to benzocaine, aminobenzoic acid, and Benzocaine Topical Aerosol
ethyl4-nitrobenzoate
Analysis DEFINITION
Samples: Standardsolution and Sample solution Benzocaine Topical Aerosol is a solution of Benzocaine in a
Calculate the percentage of aminobenzoic acid and ethyl pressurized container. It contains NLT 90.0% and NMT
4-nitrobenzoate in the portion of Benzocaine taken: 110.0% of the labeled amount of benzocaine (C9H l1NO z).
IDENTIFICATION
Result = (r vir s) x (C siC v) x 100 • A. The UV spectrum of the major peak of the Sample
solution corresponds to that of the Standardsolution, as
ru = peak response of aminobenzoic acid or ethyl obtained in the Assay.
4-nitrobenzoate from the Sample solution
= peak response of corresponding reference
• B. The retention time of the major peak of the Sample
rs solution corresponds to that of the Standardsolution, as
standard from the Standardsolution
obtained in the Assay.
= concentration of USP Aminobenzoic Acid RS or
USP Ethyl 4-nitrobenzoate RS in the Standard
solution (mg/mL) .

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504 Benzocaine / OfficialMonographs USP43

ASSAY IMPURITIES
• PROCEDURE • ORGANIC IMPURITIES
Solution A: 0.1% Trifluoroacetic acid, prepared by diluting Solution A: 0.1% Trifluoroacetic acid, prepared by diluting
1.0 mL of trifluoroacetic acid with water to 1 L 1.0 mL of trifluoroacetic acid with water to 1 L
Solution B: Acetonitrile Solution B: Acetonitrile
Mobile phase: See Table 1. Mobile phase: See Table 1.
Diluent: Solution A and Solution 8 (1:1)
Table 1 Standard solution: 1 j.Jg/mL each of USP Benzocaine RS,
Time Solution A Solution B USP Aminobenzoic Acid RS, and USP Ethyl4-Nitrobenzoate
(min) (%) (%) RS in Diluent
0 90
Sample composite: Spray a portion of the Topical Aerosol
10
into a beaker or a glass tube, and heat on a steam bath or
34 50 50 a heating module at 100° for a few min to expel residual
35 90 10
propellant. Usethe resulting benzocaine solution.
Sample solution: Nominally 0.5 mg/mL of benzocaine in
38 90 10 Diluent prepared as follows. Transfer 50 mg of benzocaine
from a portion of Sample composite to a 1OO-mL volumetric
Diluent: Solution A and Solution 8 (1 :1) flask, dissolve, and dilute with Diluent to volume.
System suitability solution: 1 j.Jg/mL each of USP Chromatographic system
Benzocaine RS, USP Aminobenzoic Acid RS, and USP Ethyl (See Chromatography (621), System Suitability.)
4-Nitrobenzoate RS in Diluent Mode: LC
Standard solution: 0.1 mg/mL of USP Benzocaine RS in Detector: UV 280 nm
Diluent . Column: 4.6-mm x 25-cm; 5-j.Jm packing L7
Sample composite: Spray a portion of Topical Aerosol into Flow rate: 1.5 mL/min
a beaker or glasstube, and heat on a steam bath or a Injection volume: 20 j.JL
heating module at 100° for a few min to expel residual System suitability
propellant. Use the resulting benzocaine solution. Sample: Standard solution
Saf'!'lple solution: Nominally 0.1 mg/mL of benzocaine in [NoTE-See Table 2 for relative retention times.]
Diluent prepared as follows. Transfer a known quantity of Suitability requirements
benzocaine solution from a portion of Sample composite to Resolution: NLT 6 between aminobenzoic acid and
an appropriate volumetric flask, dissolve, and dilute with benzocaine; NLT 6 between benzocaine and ethyl
Diluent to volume. 4-nitrobenzoate
Chromatographic system Relative standard deviation: NMT 3% for each peak
(See Chromatography (621), System Suitability.) corresponding to benzocaine, aminobenzoic acid, and
Mode: LC ethyl4-nitrobenzoate
Detector: UV 280 nm. For Identification test A, use a diode Analysis
array detector in the range of 200-400 nm. Samples: Standard solution and Sample solution
Column: 4.6-mm x 25-cm; 5-j.Jm packing L7 Calculate the percentage of aminobenzoic acid and ethyl
Flow rate: 1.5 mL/min 4-nitrobenzoate in the portion of Topical Aerosol taken:
Injection volume: 20 j.JL
System suitability . Result =(rulrs) x (CsICu) x 100
Samples: System suitability solution and Standardsolution
[NoTE-See Table 2 for relative retention times.] ru = peak response of aminobenzoic acid or ethyl
Suitability requirements 4-nitrobenzoate from the Sample solution
Resolution: NLT 6 between aminobenzoic acid and rs = peak response of aminobenzoic acid or ethyl
benzocaine; NLT 6 between benzocaine and ethyl 4-nitrobenzoate from the Standardsolution
4-nitrobenzoate, System suitability solution Cs = concentration of USP Aminobenzoic Acid RS or
Tailing factor: NMT 1.5, Standardsolution USP Ethyl 4-Nitrobenzoate RS in the Standard
Relative standard deviation: NMT 1.0%, Standard solution (mg/mL)
solution Cu = nominal concentration of benzocaine in the
Analysis Sample solution (mg/mL)
Samples: Standardsolution and Sample solution
Calculate the percentage of any other individual,
Calculate the percentage of the labeled amount of
unspecified impurity in the portion of Topical Aerosol
benzocaine (C9 H" N0 2) in the portion of Topical Aerosol
taken:
taken:
Result = (rulrs) x (CsICu) x 100
Result = (rulr s) x (CslCu) x 100
= peak response of any other individual, unspecified
= peak response of benzocaine from the Sample impurity from the Sample solution
solution = peak response of benzocaine from the Standard
= peak response of benzocaine from the Standard solution
solution = concentration of USP Benzocaine RS in the
= concentration of USP Benzocaine RS in the Standard solution (mg/mL)
Standardsolution (mg/mL) = nominal concentration of benzocaine in the
= nominal concentration of benzocaine in the Sample solution (mg/mL)
Sample solution (mg/mL)
Acceptance criteria: See Table 2. Disregard peaks less than
Acceptance criteria: 90.0%-110.0% 0.05%.

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 USP 43 Official Monographs / Benzocaine 505

Table 2 Standard solution: 0.1 mg/mL of USP Benzocaine RS in

Relative Acceptance Diluent. Sonicate to dissolve, if necessary.
Retention Criteria, Sample solution: Nominally equivalent to 0.1 mg/mL of
Name Time NMT (%) benzocaine in Diluent prepared as follows. Transfer a
Aminobenzoic acid 0.3 0.20 portion of Cream, nominally equivalent to 10 mg of
benzocaine, into a 1OO-mL volumetric flask, and dissolve it
Benzocaine 1.0 - in about 2% of the final volume of tetrahydrofuran. Dilute
Ethyl 4-nitrobenzoate 2.1 0.20 with Diluent to volume, and pass through a suitable filter of
0.45-lJm pore size, discarding the first 2-3 mL of filtrate.
Anyother individual, - Chromatographic system
unspecified impurity 0.10
(See Chromatography (621), System Suitability.)
Total impurities - 1.0 Mode: LC
Detector: UV 280 nm. For Identification test A, use a diode
SPECIFIC TESTS array detector in the range of 200-400 nm.
• TOPICAL AEROSOLS, Pressure Test (603): Meets the Column: 4.6-mm x 25-cm; 5-lJm packing L7
requirements Flow rate: 1.5 mL/min
• LEAK RATE (604): Meets the requirements Injection volume: 20 IJL
• MINIMUM FILL (755): Meets the requirements System.suitability
Samples: System suitability solution and Standardsolution
ADDITIONAL REQUIREMENTS Suitability requirements
• PACKAGING AND STORAGE: Preserve in pressurized Resolution: NLT 10 between aminobenzoic acid and
containers, and avoid exposure to excessive heat. benzocaine, and between benzocaine and ethyl
• USP REFERENCE STANDARDS (11) 4-nitrobenzoate, System suitability solution
USP Aminobenzoic Acid RS Tailing factor: NMT 1.5, Standardsolution
Benzoic acid, 4-amino. Relative standard deviation: NMT 1.0%, Standard
C7H7NO z 137.14 solution
USP Benzocaine RS Analysis
USP Ethyl 4-Nitrobenzoate RS Samples: Standardsolution and Sample solution
. Benzoic acid, 4-nitro-, ethyl ester. Calculate the percentage of the labeled amount of
C9H9N04 195.17 benzocaine (C9H llNO z) in the portion of Cream taken:

Result =(ru/rs) x (Cs/Cu) x 100

ru = peak response of benzocaine from the Sample

Benzocaine Cream solution
ts = peak responseof benzocaine from the Standard
DEFINITION solution
Benzocaine Cream contains NLT 90.0% and NMT 110.0% of Cs = concentration of USP Benzocaine RS in the
the labeled amount of benzocaine (C9H11NOz) in a suitable Standardsolution (mg/mL)
cream base. Cu = nominal concentration of benzocaine in the
IDENTIFICATION Sample solution (mg/mL)
• A. The UV spectrum of the major peak of the Sample Acceptance criteria: 90.0%-110.0%
solution corresponds to that of the Standardsolution, as
obtained in the Assay. . PERFORMANCE TESTS
• B. The retention time of the major peak of the Sample • MINIMUM FILL (755): Meets the requirements
solution corresponds to that of the Standardsolution, as
obtained in the Assay. IMPURITIES
• ORGANIC IMPURITIES
ASSAY Solution A: 0.1% Trifluoroacetic acid, prepared by diluting
• PROCEDURE 1.0 mL of trifluoroacetic acid with water to 1 L
Solution A: 0.1% Trifluoroacetic acid, prepared by diluting Solution B: Acetonitrile
1.0 mL of trifluoroacetic acid with water to 1 L Mobile phase: See Table 1 in the Assay.
Solution B: Acetonitrile Diluent: Solution A and Solution B (1:1)
Mobile phase: See Table 1. Standard solution: 1 IJg/mL of USP Benzocaine RS and 2
IJg/mL each of USP Aminobenzoic Acid RS and USP Ethyl
Table 1 4-nitrobenzoate RS in Diluent
Time Solution A Solution B Sample solution: Nominally equivalent to 1 mg/mL of
(min) (%) (%) benzocaine in Diluent prepared as follows. Transfer a
90 10
portion of Cream, nominally equivalent to 50 mg of
0
benzocaine, into a volumetric flask, and dissolve in 10% of
34 50 50 the final volume of tetrahydrofuran, with the aid of
35 90 10 sonication as needed. Dilute with Diluent to volume, and
pass through a suitable filter of 0.45-lJm pore size,
38 90 10 discarding the first 2-3 mL of filtrate.
Chromatographic system
Diluent: Solution A and Solution B (1:1) (See Chromatography (621), System Suitability.)
System suitability solution: 1 IJg/mL of USP BenzocaineRS Mode: LC
and 2 IJg/mL each of USP Aminobenzoic Acid RS and USP Detector: UV 280 nm
Ethyl 4-nitrobenzoate RS in Diluent Column: 4.6-mm x 25-cm; 5-lJm packing L7

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506 Benzocaine / Official Monographs USP 43

Flow rate: 1.5 mL/min • USP REFERENCE STANDARDS (11 )

Injection volume: 20 J-lL USP Aminobenzoic Acid RS
System suitability Benzoic acid, 4-amino.
Sample: Standard solution C7H 7NOz 137.14
SUitability requirements USP Benzocaine RS
Resolution: NLT 10 between aminobenzoic acid and USP Ethyl 4-nitrobenioate RS
benzocaine, and between benzocaine and ethyl Benzoic acid, 4-nitro-, ethyl ester.
4-nitrobenzoate CgH g N0 4 195.17
Relative standard deviation: NMT 2.0% for each peak
corresponding to benzocaine, aminobenzoic acid, and
ethyl4-nitrobenzoate
Analysis
Samples: Standard solution and Sample solution Benzocaine Gel
Calculate the percentage of aminobenzoic acid and ethyl
4-nitrobenzoate in the portion of Cream taken: DEFINITION
Benzocaine Gel contains NLT 90.0% and NMT 110.0% of the
Result = (ru/rs) x (CslCu) x 100 labeled amount of benzocaine (C9HllNO z)'

to = peak response of aminobenzoic acid or ethyl IDENTIFICATION

4-nitrobenzoate from the Sample solution • A. The UV spectrum of the major peak of the Sample
ts = peak response of the corresponding Reference solution corresponds to that of the Standard solution, as
Standard from the Standard solution obtained in the Assay.
Cs =concentration of USP Aminobenzoic Acid RS or • B. The retention time of the major peak of the Sample
USP Ethyl 4-nitrobenzoate RS in the Standard solution corresponds to that of the Standard solution, as
solution (mg/mL) obtained in the Assay. .
Cu =nominal concentration of benzocaine in the ASSAY
Sample solution (mg/mL) • PROCEDURE
.Solution A: 0.1 % Trifluoroacetic acid, prepared by diluting
Calculate the percentage of any other individual 1.0 mL of trifluoroacetic acid with water to 1 L
unspecified impurity in the portion of Cream taken: Solution B: Acetonitrile
Mobile phase: See Table 7.
Result = (ru/rs) x (Cs/Cu) x 100
Table 1
=peak response of any other individual unspecified
impurity from the Sample solution Time Solution A Solution B
(min) (%) (%)
= peak response of benzocaine from the Standard
solution 0 90 10
=concentration of USP Benzocaine RS in the 34 50 50
Standard solution (mg/mL)
= nominal concentration of benzocaine in the 35 90 10
Sample solution (mg/mL) 38 90 10
Acceptance criteria: See Table 2. Disregard peaks less than
0.05%. Diluent: Solution A and Solution B (1:1)
System suitability solution: 1 J-lg/mL of USP Benzocaine RS
Table 2 and 2 uq/rnl, each of USP Aminobenzoic Acid RS and USP
Relative Acceptance . Ethyl 4-nitrobenzoate RS in Diluent
Retention Criteria, Standard solution: 0.1 mg/mL of USP Benzocaine RS in
Name Time NMT(%) Diluent. Sonicate to dissolve, if necessary.
Aminobenzoic acid 0.29 0.20 Sample solution: Nominally 0.1 mg/mL of benzocaine in
Diluent prepared asfollows. Transfer a portion of Gel,
Benzocaine 1.0 - equivalent to 10 mg of benzocaine, into a 100-mL
Ethyl 4-nitrobenzoate 2.1 0.20 volumetric flask and dissolve it in Diluent. Pass through a
suitable filter of 0.45-J-lm pore size, discarding the first 2-3
Any other individual un- mL of filtrate.
specified impurity - 0.10 Chromatographic system
Total impurities - 1.0 (See Chromatography (621), System Suitability.)
Mode: LC
SPECIFIC TESTS Detector: UV 280 nm. For Identification test A, use a diode
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR array detector in the range of 200-400 nm.
SPECIFIED MICROORGANISMS (62): It meets the Column: 4.6-mm x 25-cm; 5-J-lm packing L7
requirements of the tests for absence of Staphylococcus Flow rate: 1.5 mL/min
aureus and Pseudomonas aeruginosa. Injection volume: 20 J-lL
System suitability
ADDITIONAL REQUIREMENTS Samples: System sUitability solution and Standard solution
• PACKAGING AND STORAGE: Preserve in tight containers, Suitability requirements
protected from light, and avoid prolonged exposure to Resolution: NLT 10 between aminobenzoic acid and
temperatures exceeding 30°. benzocaine, and between benzocaine and ethyl
4-nitrobenzoate, System suitability solution
Tailing factor: NMT 1.5, Standard solution

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USP 43 OfficialMonographs / Benzocaine 507

Relative standard deviation: NMT 1.0%, Standard Cu =nominal concentration of benzocaine in the
solution Sample solution (mg/mL)
Analysis
Samples: Standardsolution and Sample solution Calculate the percentage of any other individual
Calculate the percentage of the labeled amount of unspecified impurity in the portion of Gel taken:
benzocaine (C9H"NOz) in the portion of Gel taken:
Result = (rulr s) x (CsICu) x 100
Result = (rulrs) x (CsICu) x 100
to =peak response of any other individual unspecified
t» = peak response of benzocaine from the Sample impurity from the Sample solution
solution rs =peak response of benzocaine from the Standard
rs =peak response of benzocaine from the Standard solution
solution Cs = concentration of USP Benzocaine RS in the
Cs = concentration of USP Benzocaine RS in the Standardsolution (mg/mL)
Standardsolution(mg/mL) Cu = nominal concentration of benzocaine in the
Cu =nominal concentration of benzocaine in the Sample solution (mg/mL)
Sample solution (mg/mL)
Acceptance criteria: See Table 2. Disregard peaks lessthan
Acceptance criteria: 90.0%-110.0% 0.05%.
PERFORMANCE TESTS Table 2
• MINIMUM FILL (755): Meets the requirements
Relative Acceptance
IMPURITIES Retention Criteria,
Name Time NMT (%)
• ORGANIC IMPURITIES
Solution A: 0.1 % Trifluoroacetic acid, prepared by diluting Aminobenzoic acid 0.29 0.20
1.0 mL of trifluoroacetic acid with water to 1 L
Solution B: Acetonitrile
Benzocaine 1.0 -
Mobile phase: See Table 7 in the Assay. Ethyl 4-nitrobenzoate 2.1 0.20
Diluent: Solution A and Solution B (1:1) Anyother individual un-
Standard solution: 1 IJg/mL of USP Benzocaine RS and 2 specified impurity - 0.10
IJg/mL each of USP Aminobenzoic Acid RS and USP Ethyl
4-nitrobenzoate RS in Diluent Totalimpurities - 1.0
Sample solutiom Nominally 1 mg/mL of benzocaine in
Diluent prepared asfollows. Transfer a portion of Gel, SPECIFIC TESTS
equivalent to 50 mg of benzocaine, into a 50-mL • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
volumetric flask, and dissolve in Diluent. Pass through a SPECIFIED MICROORGANISMS (62): It meets the
suitable filter of 0.45-lJm pore size, discarding the first 2-3 requirements of the tests for absence of Staphylococcus
mL of filtrate. aureus and Pseudomonas aeruginosa.
Chromatographic system
(See Chromatography (621), System Suitability.) ADDITIONAL REQUIREMENTS
Mode: LC • PACKAGING AND STORAGE: Preserve in well-closed
Detector: UV 280 nm containers.
Column: 4.6-mm x 25-cm; 5-lJm packing L7 • USP REFERENCE STANDARDS (11)
Flow rate: 1.5 mL/min USP Aminobenzoic Acid RS
Injection volume: 20 IJL Benzoic acid, 4-amino.
System suitability C7H 7NO z 137.14
Sample: Standardsolution USP Benzocaine RS
Suitability requirements USP Ethyl 4-nitrobenzoate RS
Resolution: NLT 10 between aminobenzoic acid and Benzoic acid, 4-nitro-, ethyl ester.
benzocaine, and between benzocaine and ethyl C9H9N04 195.17
4-n itrobenzoate
Relative standard deviation: NMT 2.0% for each peak
corresponding to benzocaine, aminobenzoic acid, and
ethyl4-nitrobenzoate
Analysis
Benzocaine Lozenges
Samples: Standardsolution and Sample solution DEFINITION
Calculate the percentage of aminobenzoic acid and ethyl Benzocaine Lozenges contain NLT 85.0% and NMT 120.0%
4-nitrobenzoate in the portion of Gel taken: of the labeled amount of benzocaine (C9H"NO z) .
Result = (rulrs) x (CsICu) x 100 IDENTIFICATION
• A. The UV spectrum of the major peak of the Sample
= peak response of aminobenzoic acid or ethyl solution corresponds to that of the Standardsolution, as
4-nitrobenzoate from the Sample solution obtained in the Assay.
= peak response of the corresponding Reference • B. The retention time of the major peak of the Sample
Standard from the Standardsolution solution corresponds to that of the Standardsolution, as
= concentration of USP Aminobenzoic Acid RS or obtained in the Assay.
USP Ethyl4-nitrobenzoate RS in the Standard
solution (mg/mL)

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508 Benzocaine / Official Monographs USP 43

ASSAY IMPURITIES
• PROCIEDURE • ORGANIC IMPURITIIES
Buffer: 1.0 M monobasic potassium phosphate, adjusted Solution A: Dissolve 9.1 g of monobasic potassium
with phosphoric acid to a pH of 3.0 phosphate in 1000 mL of water. Adjust with phosphoric
Mobile phase: Acetonitrile, water, and Buffer (250:700:50) acid to a pH of 3.0.
Diluent A: 0.1 N hydrochloric acid Solution B: Acetonitrile
Diluent B: Acetonitrile and water (1:1) Mobile phase: See Table 7.
Standard solution A: 0.01 mg/mL of USP Benzocaine RS in
DiluentA Table 1
Standard solution B: O.Olmg/mL of USP Benzocaine RS in Time Solution A Solution B
Diluent B (min) (%) (%)
Sample stock solution A: Transfer the equivalent of 40 mg
0 60 40
of benzocaine from powdered Lozenges (NLT 20) to a
200-mL volumetric flask. Add 150 mL of DiluentA, and stir 10 45 55
for NLT 2 h. Dilute with DiluentA to volume.
10.1 60 40
Sample stock solution B: Transfer the equivalent of 40 mg
of benzocaine from powdered Lozenges (NLT 20) to a 13 60 40
200-mL volumetric flask. Add 150 mL of Diluent B, and stir
for NLT 30 min. Dilute with Diluent B to volume. Diluent: Acetonitrile and water (10:90)
Sample solution A: Nominally 0.01 mg/mL of benzocaine Standard stock solution: 0.03 mg/mL each of USP
in DiluentA from Sample stocksolutionA Benzocaine RS, USP Aminobenzoic Acid RS, and USP Ethyl
Sample solution B: Nominally 0.01 mg/mL of benzocaine 4-Nitrobenzoate RS in Diluent. Sonicate for 2-5 min to
in Diluent B from Sample stocksolution B dissolve before diluting to final volume.
Chromatographic system Standard solution: 0.3 IJg/mL each of USP Benzocaine RS,
(See Chromatography (621), System Suitability.) USP Aminobenzoic Acid RS, and USP Ethyl4-Nitrobenzoate
Mode: LC RS in Diluent from the Standardstock solution
Detector: UV 280 nm. For Identification test A, usea diode , Sample solution: Nominally 150 IJg/mL of benzocaine in
array detector in the range of 200-400 nm. Diluent prepared as follows. Transfer 10 Lozenges to an
Column: 4.6-mm x 25-cm; packing L7 appropriate volumetric flask to obtain a nominal
Flow rate: 1.5 mL/min benzocaine concentration of 0.15 mg/mL. Dissolve
Injection volume: 20 IJL Lozenges in Diluent and dilute with Diluent to volume.
System suitability Chromatographic system
Samples: StandardsolutionA and Standardsolution B (See Chromatography (621), System Suitability.)
Suitability requirements Mode: LC
Tailing factor: NMT 1.5 Detector: UV 280 nm
Relative standard deviation: NMT 2.0% Column: 4.6-mm x 25-cm; 5-lJm packing L7
AM~ili . Flow rate: 1.5 mL/min
Samples: StandardsolutionA, Standardsolution B, Sample Injection volume: 100 IJL
solutionA, and Sample solution B System suitability
Calculate the percentage of the total labeled amount of Sample: Standardsolution
benzocaine (C9H ll N02) in the portion of Lozenges taken: Suitability requirements
Resolution: NLT 10 between benzocaine and
Result = (ru/rs) x (Cs/Cu) x 100 aminobenzoic acid; NLT 10 between ethyl
4-nitrobenzoate and benzocaine
rv = peak responsefrom Sample solution A Relative standard deviation: NMT 2.0% for each peak
ts = peak responsefrom Standardsolution A corresponding to benzocaine, aminobenzoic acid, and
Cs = concentration of USP Benzocaine RS in Standard ethyl 4-nitrobenzoate
solutionA (mg/mL) Analysis
Cv = nominal concentration of benzocaine in Sample Samples: Standardsolution and Sample solution
solutionA (mg/mL) Calculate the percentage of aminobenzoic acid and ethyl
4-nitrobenzoate in the portion of Lozenges taken:
Calculate the percentage of free benzocaine (C9H" N02 ) in
the portion of Lozenges taken: Result = (ru/rs) x (Cs/Cu) x 100

Result = (ru/rs) x (Cs/Cu) x 100 = peak response of aminobenzoic acid or ethyl

4-nitrobenzoate from the Sample solution
= peak responsefrom Sample solution B = peak response of the corresponding Reference
= peak responsefrom Standardsolution B Standard from the Standard solution
= concentration of USP Benzocaine RS in Standard = concentration of USP Aminobenzoic Acid RS or
solution B (mg/mL) USP Ethyl 4-Nitrobenzoate RS in the Standard
= nominal concentration of benzocaine in Sample solution (lJg/mL)
solution B (mg/mL) = nominal concentration of benzocaine in the
Sample solution (lJg/mL)
Acceptance criteria
Total labeled amount of benzocaine: 85.0%-120.0% Calculate the percentage of each unspecified degradation
Free benzocaine: 85.0%-120.0% product inthe portion of Lozenges taken:

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 USP 43 Official Monographs / Benzocaine 509

=peak response of each unspecified degradation Table 1 (continued)

product from the Sample solution Time Solution A Solution B
= peak response of benzocaine from the Standard (min) (%) (%)
solution 35 90 10
=concentration of USP Benzocaine RS in the
Standardsolution (~g/mL) 38 90 10
= nominal concentration of benzocaine in the
Sample solution (~g/mL) Diluent: Solution A and Solution B (1:1)
Standard solution: 0.1 mg/mL of USP Benzocaine RS in
Acceptance criteria: See Table 2. Disregard peaks less than Diluent. Sonication may be needed to aid in the dissolution.
0.05%. Sample solution: Nominally 0.1 mg/mL of benzocaine in
Diluent prepared as follows
Table 2 Ointments having water-soluble bases: Transfer a
Relative Acceptance portion of Ointment, equivalent to 10 mg of benzocaine,
Retention Criteria, into a volumetric flask, and dissolve in Diluent.
Name Time NMT(%)
Ointments having water-insoluble bases: Transfer a
Aminobenzoic acid 0.46 0.2 portion of Ointment, equivalent to 10 mg of benzocaine,
into a volumetric flask, and dissolve in tetrahydrofuran,
Benzocaine 1.00 -
using about 2% of the final volume, then dilute with
Ethyl4-nitrobenzoate 1.86 0.2 Diluent to volume. Pass through a suitable filter of
Any unspecified 0.45-~m pore size, discarding the first 2-3 mL of the
degradation product - 0.1 filtrate.
Chromatographic system
Total degradation
products - 2.0 (See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 280 nm. For Identification test B, use a diode
ADDITIONAL REQUIREMENTS array detector in the range of 200-400 nm.
• PACKAGING AND STORAGE: Preserve in well-closed Column: 4.6-mm x 25-cm; 5-~m packing L7
containers, and store at controlled room temperature. Flow rate: 1.5 mL/min
• USP REFERENCE STANDARDS (11) Injection volume: 20 ~L
USP Aminobenzoic Acid RS System suitability
Benzoic acid, 4-amino. Sample: Standardsolution
C7H7N02 137.14 Suitability requirements
USP Benzocaine RS Tailing factor: NMT 1.5
USP Ethyl 4-Nitrobenzoate RS Relative standard deviation: NMT 0.73%
Benzoic acid, 4-nitro-, ethyl ester. Analysis
C9H9N04 195.17 Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of
benzocaine (C9 H11N02) in the portion of Ointment taken:

Result = (ru/rs) x (Cs/Cu) x 100

Benzocaine Ointment
=peak response of benzocaine from the Sample
DEFINITION solution
Benzocaine Ointment contains NLT 90.0% and NMT 110.0% = peak response of benzocaine from the Standard
of the labeled amount of benzocaine (C9 H11 N02) in a suitable solution
ointment base. = concentration of USP Benzocaine RS in the
IDENTifiCATION Standardsolution (mg/mL)
• A. The retention time of the major peak of the Sample = nominal concentration of benzocaine in the
solution corresponds to that of the Standardsolution, as Sample solution (mg/mL)
obtained in the Assay.
Acceptance criteria: 90.0%-110.0%
• B. The UV spectrum of the major peak of the Sample
solution corresponds to that of the Standardsolution, as IMPURITIES
obtained in the Assay. • ORGANIC IMPURITIES
Solution A: 0.1% Trifluoroacetic acid, prepared by adding
ASSAY 1.0 mL of trifluoroacetic acid to 1 L of water
• PROCEDURE Solution B: Acetonitrile
Solution A: 0.1% Trifluoroacetic acid, prepared by adding
Mobile phase: See Table 1 in the Assay.
1.0 mL of trifluoroacetic acid to 1 L of water
Diluent: Solution A and Solution B (1 :1)
Solution B: Acetonitrile
System suitability solution: 0.2 mg/mL of USP
Mobile phase: See Table 1.
Benzocaine RS and 0.01 mg/mL each of USPAminobenzoic
Table 1 Acid RS and USP Ethyl4-Nitrobenzoate RS in Diluent
Standard solution: 1 ~g/mL each of USP Benzocaine RS,
Time Solution A Solution B USP Aminobenzoic Acid RS, and USP Ethyl4-Nitrobenzoate
(min) (%) (%)
RS in Diluent
0 90 10 Sample solution: Nominally 1 mg/mL of benzocaine in
50 50
Diluent prepared as follows
34

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510 Benzocaine / OfficialMonographs USP43

Ointments having water-soluble bases: Transfer a Table 2 (continued)

portion of Ointment, equivalent to 50 mg of benzocaine, Relative Acceptance
into a volumetric flask, and dissolve in Diluent. Retention Criteria,
Ointments having water-insoluble bases: Transfer a Name Time NMT (%)
portion of Ointment, equivalent to 50 mg of benzocaine, Total impurities - 1.0
into a volumetric flask, and dissolve in about 10% of final
volume of tetrahydrofuran, then dilute with Diluent to
volume. Pass through a suitable filter of 0.45-l..Im pore size, PERfORMANCE TESTS
discarding the first 2-3 mL of the filtrate. • MINIMUM IFILL (755): Meets the requirements
Chromatographic system SPECifiC TESTS
(See Chromatography (621), System Suitability.) • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Mode: LC SPECIFIED MICROORGANISMS (62): It meets the
Detector: UV 280 nm requirements of the tests for absence of Staphylococcus
Column: 4.6-mm x 25-cm; 5-lJm packing L7 aureus and Pseudomonas aeruginosa.
Flow rate: 1.5 mL/min
Injection volume: 20 I..IL ADDITIONAL REQUIREMENTS
System suitability • PACKAGING AND STORAGE: Preserve in tight containers,
Samples: System suitability solution and Standard solution protected from light, and store at room temperature at
Suitability requirements 150 - 2 5 0 •
Resolution: NLT 10 between aminobenzoic acid and • USP REFERENCE STANDARDS (11)
benzocaine, and between benzocaine and ethyl USP Aminobenzoic Acid RS
4-nitrobenzoate, System suitability solution Benzoic acid, 4-amino.
Relative standard deviation: NMT 2.0% for each peak C7H 7N02 137.14
corresponding to benzocaine, aminobenzoic acid, and USP Benzocaine RS
ethyl 4-nitrobenzoate, Standard solution USP Ethyl4-Nitrobenzoate RS
Analysis Benzoic acid, 4-nitro-, ethyl ester.
Samples: Standard solution and Sample solution C9H9N04 195.17
Calculate the percentage of aminobenzoic acid and ethyl
4-nitrobenzoate in the portion of Ointment taken:

Result = (ru/rs) x (CslCu) x 100

Benzocaine Otic Solution
= peak response of aminobenzoic acid or ethyl
4-nitrobenzoate from the Sample solution DEfiNITION
= peak response of the corresponding Reference Benzocaine Otic Solution contains NLT 90.0% and NMT
Standard from the Standard solution 110.0% of the labeled amount of benzocaine (C9H" N02 ) .
= concentration of USP Aminobenzoic Acid RS or
USP Ethyl 4-Nitrobenzoate RS in the Standard IDENTifiCATION
solution (mg/mL) • A. The retention time of the major peak of the Sample
= nominal concentration of benzocaine in the solution corresponds to that of the Standard solution, as
Sample solution (mg/mL) obtained in the Assay.
• B. The UV spectrum of the major peak of the Sample
Calculate the percentage of any other individual solution corresponds to that of the Standard solution, as
unspecified impurity in the portion of Ointment taken: obtained in the Assay.
ASSAY
Result = (ru/rs) x (CslCu) x 100 • PROCEDURE
Solution A: 0.1% Trifluoroacetic acid, prepared by diluting
= peak response for any other individual 1.0 mL of trifluoroacetic acid with water to 1 L
unspecified impurity from the Sample solution Solution B: Acetonitrile
= peak response of benzocaine from the Standard Mobile phase: See Table 1.
solution
= concentration of USP Benzocaine RS in the Table 1
Standard solution (mg/mL)
Time Solution A Solution B
= nominal concentration of benzocaine in the (min) (%) (%)
Sample solution (mg/mL)
0 90 10
Acceptance criteria: See Table 2. Disregard peaks less than 34 50 50
0.05%.
35 90 10
Table 2 38 90 10
Relative Acceptance
Retention Criteria,
Name Time NMT (%) Diluent: Solution A and Solution 8 (1:1)
System suitability solution: 1 I..Ig/mL each of USP
Aminobenzoic acid 0.29 0.10
Benzocaine RS, USP Aminobenzoic Acid RS, and USP Ethyl
Benzocaine 1.0 - 4-Nitrobenzoate RS in Diluent. Sonicate to dissolve, if
necessary.
Ethyl 4-nitrobenzoate 2.1 0.10
Standard solution: 0.1 mg/mL of USP Benzocaine RS in
Any other individual
- Diluent. Sonicate to dissolve, if necessary.
unspecified impurity 0.10

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USP43 Official Monographs / Benzocaine 511

Sample solution: Nominally 0.1 mg/mL of benzocaine in tu =peak response of aminobenzoic acid or ethyl
Diluent prepared as follows. Transfer a portion of Otic 4-nitrobenzoate from the Sample solution
Solution, equivalent to 10 mg of benzocaine, into a 100-mL rs = peak response of the corresponding Reference
volumetric flask and dissolve it in Diluent. Standard from the Standardsolution
Chromatographic system Cs = concentration of USP Aminobenzoic Acid RS or
(See Chromatography (621), System Suitability.) USP Ethyl 4-Nitrobenzoate RS in the Standard
Mode: LC solution (mg/mL)
Detector: UV 280 nm. For Identification test 8, use a diode Cu = nominal concentration of benzocaine in the
array detector in the range of 200-400 nm. Sample solution(mg/mL)
Column: 4.6-mm x 25-cm; 5-J.Im packing L7
Flow rate: 1.5 mL/min Calculate the percentage of any individual unspecified
Injection volume: 20 J.IL degradation product in the portion of Otic Solution taken:
System suitability
Samples: System suitability solution and Standardsolution Result = (rulrs) x (CsICv) x 100
Suitability requirements
Resolution: NLT 6 between aminobenzoic acid and ru =peak response of any individual unspecified
benzocaine, and between benzocaine and ethyl degradation product from the Sample solution
4-nitrobenzoate, System sUitabilitysolution rs = peak response of benzocaine from the Standard
Tailing factor: NMT 1.5, Standardsolution solution
Relative standard deviation: NMT 1.0%, Standard Cs = concentration of USP Benzocaine RS in the
solution Standardsolution (mg/mL)
Analysis Cu = nominal concentration of benzocaine in the
Samples: Standardsolution and Sample solution Sample solution (mg/mL)
Calculate the percentage of the labeled amount of
benzocaine (C9H,lN02) in the portion of Otic Solution Acceptance criteria: See Table 2. Disregard peaks less than
taken: 0.05%.

Result =(rvlrs) x (CslCv) x 100 Table 2

Relative Acceptance
rv = peak response of benzocaine from the Sample Retention Criteria,
Name Time NMT (%)
solution
rs =peak response of benzocaine from the Standard Aminobenzoic acid 0.3 0.20
solution,
Cs =concentration of USP Benzocaine RS in the Benzocaine 1.0 -
Standardsolution (mg/mL) Ethyl 4·nitrobenzoate 2.1 0.20
Cu = nominal concentration of benzocaine in the
Any individual
Sample solution (mg/mL) unspecified deqrada- -
tion product 0.20
Acceptance criteria: 90.0%-110.0%
Total impurities - 1.0
IMPURITIES
• ORGANIC IMPURITIES SPECIFIC TESTS
Solution A, Solution B, Mobile phase, Diluent, and • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Chromatographic system: Proceed as directed in the SPECIFIED MICROORGANISMS (62): It meets the
Assay. . requirements of the tests for absence of Salmonella species
Standard solution: 1 J.Ig/mL each of USP Benzocaine RS, and Escherichia coli and for absence of Staphylococcus
USP Aminobenzoic Acid RS, and USP Ethyl 4-Nitrobenzoate aureus and Pseudomonas aeruginosa. The total aerobic
RS in Diluent. Sonicate to dissolve, if necessary. microbial count is less than 10 2 cfu/mL.
Sample solution: Nominally 0.5 mg/mL of benzocaine in
Diluent prepared as follows. Transfer a portion of Otic ADDITIONAL REQUIREMENTS
Solution, equivalent to 50 mg of benzocaine, into a 100-mL • PACKAGING AND STORAGE: Preserve in tight, light-resistant
volumetric flask, dissolve, and dilute with Diluent to volume. containers.
System suitability • USP REFERENCE STANDARDS (11)
Sample: Standardsolution USP Aminobenzoic Acid RS
[NoTE-See Table 2 for relative retention times.] Benzoic acid, 4-amino.
Suitability requirements C7H 7N0 2 137.14
Resolution: NLT 6 between aminobenzoic acid and USP Benzocaine RS
benzocaine, and between benzocaine and ethyl USP Ethyl 4-Nitrobenzoate RS
4-n itrobenzoate Benzoic acid, 4-nitro-, ethyl ester.
Relative standard deviation: NMT 3.0% for each peak C9H9N0 4 195.17
corresponding to benzocaine, aminobenzoic acid, and
ethyl4-nitrobenzoate
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of aminobenzoic acid and ethyl Benzocaine Topical Solution
4-nitrobenzoate in the portion of Otic Solution taken:
DEFINITION
Result =(rvlrs) x (CsICv) x 100 Benzocaine Topical Solution is a solution of Benzocaine in a
suitable solvent. It contains NLT90.0% and NMT 110.0% of

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512 Benzocaine / OfficialMonographs USP 43

the labeled amount of benzocaine (C9H llN02) . It contains a = peak response of benzocaine from the Standard
suitable antimicrobial agent. solution
= concentration of USP Benzocaine RS in the
IDENTIFICATION Standard solution (mg/mL)
• A. The UV spectrum of the major peak of the Sample = nominal concentration of benzocaine in the
solution corresponds to that of the Standard solution, as Sample solution (mg/mL)
obtained in the Assay.
• B. The retention time of the major peak of the Sample Acceptance criteria: 90.00/0-110.0%
solution corresponds to that of the Standard solution, as
obtained in the Assay. IMPURITIES
• ORGANIC IMPURITIES
ASSAY Solution A: 0.1% Trifluoroacetic acid, prepared by diluting
• PROCEDURE 1.0 mL of trifluoroacetic acid with water to 1 L
Solution A: 0.1% Trifluoroacetic acid, prepared by diluting Solution B: Acetonitrile
1.0 mL of trifluoroacetic acid with water to 1 L Mobile phase: See Table 2.
Solution B: Acetonitrile
Mobile phase: See Table 1. Table 2
Time Solution A Solution B
Table 1 (min) (%) (%)
Time Solution A Solution B
(min) (%) (%) 0 85 15

0 90 10 34 55 45

34 50 50 35 85 15

35 90 10 38 85 15

38 90 10
Diluent: Solution A and Solution B (1: 1)
.Standard solution: 1 ~g/mL of USP Benzocaine RS and 2
Diluent: Solution A and Solution B (1:1) ~g/mL each USP Aminobenzoic Acid RS and USP Ethyl
System suitability solution: 1 ~g/mL of USP Benzocaine RS 4-Nitrobenzoate RS in Diluent
and 2 ~g/mL each of USP Aminobenzoic Acid RS and USP Sample solution: Nominally 1 mg/mL of benzocaine in.
Ethyl 4-Nitrobenzoate RS in Diluent Diluent prepared as follows. Transfer 50 mg of benzocaine
Standard solution: 0.1 mg/mL of USP Benzocaine RS in from a portion of Topical Solution to a volumetric flask and
Diluent I
dissolve with aid of sonication as needed, then dilute with
Sample solution: Nominally 0.1 mg/mL of benzocaine in Diluent to volume. Pass through a suitable filter of 0.45-~m
Diluent prepared asfollows. Transfer 10 mg of benzocaine pore size as needed, discarding the first 2-3 mL of filtrate.
from a portion of Topical Solution to a 1OO-mL volumetric Chromatographic system
flask, and dilute with Diluent to volume. Pass through a (See Chromatography (621), System Suitability.)
suitable filter of 0.45-~m pore size as needed, discarding Mode: LC
the first 2-3 mL of filtrate. Detector: UV 280 nm
Chromatographic system Column: 4.6-mm x 25-cm; 5-~m packing L7
(See Chromatography (621), System Suitability.) Flow rate: 1.5 mLlmin
Mode: LC Injection volume: 20 ~L
Detector: UV 280 nm. For Identification test A, use a diode System suitability
array detector in the range of 200-400 nm. . Sample: Standard solution
Column: 4.6-mm x 25-cm; 5-~m packing L7 [NoTE-See Table 3 for relative retention times.]
Flow rate: 1.5 mL/min Suitability requirements
Injection volume: 20 ~L Resolution: NLT 6 between aminobenzoic acid and
System suitability benzocaine; and between benzocaine and ethyl
Samples: System suitability solution and Standard solution 4-nitrobenzoate
[NoTE~The relative retention times for aminobenzoic Relative standard deviation: NMT 2.0% for each peak
acid, benzocaine, and ethyl 4-nitrobenzoate are 0.3, corresponding to benzocaine, aminobenzoic acid, and
1.0, and 2.1, respectively.] ethyl4-nitrobenzoate
Suitability requirements Analysis
Resolution: NLT 6 between aminobenzoic acid and Samples: Standard solution and Sample solution
benzocaine; and between benzocaine and ethyl Calculate the percentage of aminobenzoic acid and ethyl
4-nitrobenzoate, System suitability solution 4-nitrobenzoate in the portion of Topical Solution taken:
Tailing factor: NMT 1.5, Standard solution
Relative standard deviation: NMT 1.0%, Standard Result = (ru/rs) x (Cs/Cu) x 100
solution
Analysis =peak response of aminobenzoic acid or ethyl
Samples: Standard solution and Sample solution 4-nitrobenzoate from the Sample solution
Calculate the percentage of the labeled amount of =peak response of aminobenzoic acid or ethyl
benzocaine (C9H llN02) in the portion of Topical Solution 4-nitrobenzoate from the Standard solution
taken: = concentration of USP Aminobenzoic Acid RS or
USP Ethyl 4-Nitrobenzoate RS in the Standard
Result = (ru/rs) x (Cs/Cu) x 100 solution (mg/mL)
= nominal concentration of benzocaine in the
=peak response of benzocaine from the Sample Sample solution (mg/mL)
solution

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 USP 43 OfficialMonographs / Benzocaine 51 3

Calculate the percentage of any other individual • B. The UV spectrum of the major peaks of the Sample
unspecified impurity in the portion of Topical Solution solution corresponds to that of the Standardsolution, as
taken: obtained· in the Assay.

Result = (rvlrs) x (CslCv) x 100 ASSAY

• PROCEDURE
= peak response of any other individual unspecified Solution A: 0.1% formic acid in water
impurity from the Sample solution Solution B: 0.1% formic acid in acetonitrile
= peak response of benzocaine from the Standard Mobile phase: See Table 1.
solution
= concentration of USP Benzocaine RS in the Table 1
Standardsolution (mg/mL) Time Solution A Solution B
Cv = nominal concentration of benzocaine in the (min) (0/0) (0/0)
Sample solution (mg/mL) 0 91 9

Acceptance criteria: See Table 3. Disregard peaks less than 2.5 50 50

0.05%. 3.9 50 50

Table 3 4 91 9
Relative Acceptance 5 91 9
Retention Criteria,
Name Time NMT (0/0)
Diluent: Acetonitrile and water (10:90)
Aminobenzoic acid 0.27 0.20 Standard stock solution A: 1750 ~g/mL of USP Benzocaine
Benzocaine 1.0 - RS prepared as follows. Transfer a suitable amount of USP
Benzocaine RS to a suitable volumetric flask and dissolve in
Ethyl 4-nitrobenzoate 2.5 0.20 10% of the total volume of acetonitrile. Dilute with water
Anyother individual 'to volume.
unspecified impurity - 0.10 Standard stock solution B: 250 ~g/mL each of USP
Total impurities - 1.0 Butamben RS and USP Tetracaine Hydrochloride RS
prepared as follows. Transfer a suitable amount of USP
Butamben RS and USP Tetracaine Hydrochloride RS to a
SPECIFIC TESTS suitable volumetric flask and dissolve in 10% of the total
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR volume of acetonitrile. Dilute with water to volume.
SPECIFIED MICROORGANISMS (62): It meets the Standard solution: 175 ~g/mL of USP Benzocaine RS from
requirements of the tests for absence of Staphylococcus Standardstock solution A and 25 ~g/mL each of USP
aureus and Pseudomonas aeruginosa. Butamben RS and USP Tetracaine Hydrochloride RS from
ADDITIONAL REQUIREMENTS Standardstock solution 8 diluted in Diluent
• PACKAGING AND STORAGE: Preserve in tight containers, Sample solution: Nominally 175 ~g/mL of benzocaine and
protected from light, and avoid prolonged exposure to 25 uq/rnl, each of butamben and tetracaine hydrochloride,
temperatures exceeding 30°. prepared asfollows. Accurately weigh about 125 mg of the
• USP REFERENCE STANDARDS (11) evaporated sample into a 1OO-mL volumetric flask. Dissolve
USP Aminobenzoic Acid RS in 50 mL of methanol and dilute with Diluent to volume.
Benzoic acid, 4-amino. Chromatographic system
C7H7N02 137.14 (See Chromatography (621), System Suitability.)
USP Benzocaine RS Mode: LC
Detector: UV 300 nm. For Identification 8, usea diode array
USP Ethyl 4-Nitrobenzoate RS
detector in the range of 200-400 nm.
Benzoic acid, 4-nitro-, ethyl ester.
C9H9N04 195.17 Column: 2.1-mm x 5-cm; 'l.Z-urn packing L1
Flow rate: 0.6 mL/min
Injection volume: 1 ~L
System suitability
Sample: Standardsolution
Benzocaine, Butamben, and Tetracaine [NOTE-The relative retention times for benzocaine,
tetracaine, and butamben are about 0.71, 0.74, and
Hydrochloride Topical Aerosol 1.0, respectively.]
SUitability requirements
DEFINITION Resolution: NLT 2 between benzocaine and tetracaine
Benzocaine, Butamben, and Tetracaine Hydrochloride Topical Relative standard deviation: NMT 2.0% for each of the
Aerosol is Benzocaine, Butamben, and Tetracaine three analyte peaks
Hydrochloride Topical Solution packaged in a pressurized Analysis
container with a suitable inert propellant. It contains NLT Samples: Standardsolution and Sample solution
90.0% and NMT 110.0% of the labeled amount of Calculate the percentage of the labeled amount of
benzocaine (C9H1,N02 ) , butamben (C"H,sN0 2) , and benzocaine (C9H ll N0 2) , butamben (C" HlSN02) , and
tetracaine hydrochloride (C,sH24N 202 • HCI). tetracaine hydrochloride (C,sH24N 202 • HCI) in the portion
IDENTIFICATION of Topical Aerosol taken:
• A. The retention times of the major peaks of the Sample
solution correspond to those of.the Standardsolution, as Result = (rvlrs) x (CsICv) x 100
obtained in the Assay.

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514 Benzocaine / Official Monographs USP 43

tu = peak response of the corresponding analyte Calculate the percentage of tetracaine related compound B
from the Sample solution and any individual unspecified degradation product in the
rs = peak response of the corresponding analyte portion of Topical Aerosol taken:
from the Standardsolution
Cs = concentration of the corresponding Reference Result = (rulr s) x (CsICv) x (1 IF) x 100
Standard in the Standardsolution (~g/mL)
Cv = nominal concentration of the corresponding rv = peak response of tetracaine related compound B
analyte in the Sample solution (~g/mL) or any individual unspecified degradation
product from the Sample solution
Acceptance criteria: 90.0%-110.0% rs = peak response of tetracaine from the Standard
solution
PERFORMANCE TESTS Cs = concentration of USP Tetracaine Hydrochloride
• MINIMUM FILL (755): Meets the requirements RS in the Standardsolution (~g/mL)
IMPURITIES Cv = nominal concentration of tetracaine
• ORGANIC IMPURITIES hydrochloride in the Sample solution (~g/mL)
Mobile phase, Diluent, and Chromatographic system: F = relative response factor (see Table 2)
Proceed as directed in the Assay.
System suitability solution: 10 ~g/mL each of USP Acceptance criteria: See Table 2. Disregard any impurity
Benzocaine RS, USP Tetracaine Hydrochloride RS, USP peaks less than 0.05%.
Butamben RS, and USP Ethyl 4-Nitrobenzoate RS in Diluent
Standard solution: 3.4 ~g/mL each of USP Benzocaine RS Table 2
and USP Ethyl 4-Nitrobenzoate RS and 1 ~g/mL of USP Relative Relative Acceptance
Tetracaine Hydrochloride RS in Diluent Retention Response Criteria,
Name Time Factor NMT (0/0)
Sample solution: Nominally 1.68 mg/mL of benzocaine,
0.24 mg/mL of butamben, and 0.24 mg/mL of tetracaine 4-Aminobenzoic acid 0.15 1.3 0.3
prepared as follows. Accurately weigh about 600 mg of Benzocaine 0.70 - -
evaporated sample into a 50-mLvolumetricflask, dissolve
with 25 mLof methanol, and dilute with Diluent to volume. Tetracaine 0.74 - -
[NOTE-Sonication for about 1 min may be necessary.] Tetracaine related
System suitability compound Ba 0.93 0.6 0.4
Samples: System suitability solution and Standardsolution
[NOTE-See Table 2 for relative retention times.] Butamben 1.0 - -
Suitability requirements Ethyl 4-nitrobenzoate 1.04 - 0.2
Resolution: NLT 2 between butamben and ethyl
Any individual
4-nitrobenzoate; NLT 2 between benzocaine and unspecified -
tetracaine, System suitability solution degradation product 1.0 0.4
Relative standard deviation: NMT 5.0% for each of the
analyte peaks, Standardsolution Total degradation products - - 2.0
Analysis a 4-(Butylamino)benzoic acid.
Samples: Standard solution and Sample solution
Calculate the percentage of 4-aminobenzojc acid in the SPECIFICTESTS
portion of Topical Aerosol taken: • TOPICAL AEROSOLS (603), Pressure Test: Meets the
Result = (rulr s) x (CslCu) x (1 IF) x 100 . requirements
• LEAK RATE (604): Meets the requirements
rv = peak response of 4-aminobenzoic acid from the ADDITIONAL REQUIREMENTS
Sample solution • PACKAGING AND STORAGE: Preserve in pressurized
rs = peak response of benzocaine from the Standard containers, and avoid exposure to excessive heat.
solution • USP REFERENCE STANDARDS (11)
Cs =concentration of USP Benzocaine RS in the USP Benzocaine RS
Standardsolution (~g/mL) USP Butamben RS
Cv = nominal concentration of benzocaine in the USP Ethyl 4-Nitrobenzoate RS
Sample solution (uq/rnt) Ethyl p-nitrobenzoate.
F = relative response factor (see Table 2) C9H9N04 195.17
USP Tetracaine Hydrochloride RS
Calculate the percentage of ethyl 4-nitrobenzoate in the
portion of Topical Aerosol taken:
Result =(rvlrs) x (CslCv) x 100
= peak response of ethyl 4-nitrobenzoate from the
Benzocaine, Butamben, and Tetracaine
Sample solution Hydrochloride. Gel
= peak response of ethyl 4-nitrobenzoate from the
Standardsolution DEFINITION
=concentration of USP Ethyl 4-Nitrobenzoate RS in Benzocaine, Butamben, and Tetracaine Hydrochloride Gel is
the Standard solution (~g/mL) Benzocaine, Butamben, and Tetracaine Hydrochloride in a
= nominal concentration of benzocaine in the suitable gel base. It contains NLT 90.0% and NMT 110.0%
Sample solution (~g/mL) of the labeled amount of benzocaine (C9H l1 N02) ,

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USP 43 Official Monographs / Benzocaine 515

butamben (Cll H1SN02), and tetracaine hydrochloride ts = peak response of the corresponding analyte
(C15H24N202 . HCI). from the Standardsolution
Cs = concentration of the corresponding Reference
IDENTIFICATION Standard in the Standardsolution (J.Jg/mL)
• A. The retention times of the major peaks of the Sample Cu = nominal concentration of the corresponding
solution correspond to those of the Standard solution, as analyte in the Sample solution (J.Jg/mL)
obtained in the Assay.
• B. The UV spectrum of the major peaks of the Sample Acceptance criteria: 90.0%-110.0%
solution corresponds to that of the Standardsolution, as
obtained in the Assay. PERFORMANCE TESTS
• MINIMUM FILL (755): Meets the requirements
ASSAY
• PROCEDURE IMPURITIES
Solution A: 0.1% formic acid in water • ORGANIC IMPURITIES
Solution B: 0.1% formic acid in acetonitrile Mobile phase, Diluent, and Chromatographic system:
Mobile phase: See Table 7. Proceed as directed in the Assay.
System suitability solution: 10 J.Jg/mL each of USP
Table 1 Benzocaine RS, USP Tetracaine Hydrochloride RS, USP
Time Solution A Solution B
Butamben RS, and USP Ethyl 4-Nitrobenzoate RS prepared
(min) (%) (%) as follows. Transfera suitable amount of USP Benzocaine
RS, USP Tetracaine Hydrochloride RS, USP Butamben RS,
0 91 9 and USP Ethyl 4-Nitrobenzoate RS to a suitable volumetric
2.5 50 50 flask and dissolve in 20% of the total volume of
acetonitrile. Dilutewith water to volume.
3.9 50 50 Standard solution: 3.4 J.Jg/mL each of USP Benzocaine RS
4 91 9 and USP Ethyl 4-Nitrobenzoate RS and 1 J.Jg/mL of USP
Tetracaine Hydrochloride RS prepared as follows. Transfer
5 91 9 .a suitable amount of USP Benzocaine RS, USP Tetracaine
Hydrochloride RS, and USP Ethyl4-Nitrobenzoate RS to a
Diluent: Acetonitrile and water (20:80) suitable volumetric flask and dissolve in 20% of the total
Standard solution: 175 J.Jg/mL of USP Benzocaine. RS and volume of acetonitrile. Dilute with water to volume.
25 J.Jg/mL each of USP Butamben RS and USP Tetracaine Sample solution: Nominally 1.68 mg/mL of benzocaine,
Hydrochloride RS prepared as follows. Transfera suitable 0.24 mg/mL of butamben, and 0.24 mg/mL of tetracaine
amount of USP BenzocaineRS, USP Butamben RS, and USP prepared as follows. Transfera suitable quantity of Gel into
Tetracaine Hydrochloride RS to a suitable volumetric flask a suitable volumetric flask and dissolve in 20% of the total
and dissolve in 20% of the total volume of acetonitrile. volume of acetonitrile. Dilutewith water to volume.
Dilute with water to volume. [NOTE-Sonication for about 1 min may be necessary.]
Sample solution: Nominally175 J.Jg/mL of benzocaine and System suitability
25 J.Jg/mL each of butamben and tetracaine hydrochloride, Samples: System sUitability solution and Standardsolution
prepared as follows. Transfer a suitable quantity of Gel into [NOTE-See Table 2 for relative retention times.]
a suitable volumetric flask and dissolve in 20% of the total Suitability requirements
volume of acetonitrile. [NOTE-Sonication for.about 1 min Resolution: NLT 2 between butamben and ethyl
may be necessary.] Dilute with water to volume. 4-nitrobenzoate; NLT 2 between benzocaine and
Chromatographic system tetracaine, System suitability solution
(See Chromatography (621), System Suitability.) Relative standard deviation: NMT 5.0% for each of the
Mode: LC analyte peaks, Standardsolution
Detector: UV 300 nm. For Identification B, use a diode array Analysis
detector in the range of 200-400 nm. Samples: Standardsolution and Sample solution
Column: 2.1-mm x 5-cm; l.7-J.Jm packing L1 Calculate the percentage of 4-aminobenzoic acid in the
Flow rate: 0.6 mL/min portion of Gel taken:
Injection volume: 1 J.JL
System suitability Result =(rulr s) x (CslCu) x (l/A x 100
Sample: Standardsolution
[NOTE-See Table 2 for relative retention times.] ru = peak response of 4-aminobenzoic acid from the
Suitability requirements Sample solution
Resolution: NLT 2 between benzocaine and tetracaine rs = peak response of benzocaine from the Standard
Relative standard deviation: NMT 2.0% for each of the solution
three analyte peaks Cs = concentration of USP Benzocaine RS in the
Analysis Standardsolution (J.Jg/mL)
Samples: Standardsolution and Sample solution Cu = nominal concentration of benzocaine in the
Calculate the percentage of the labeled amount of Sample solution (J.Jg/mL)
benzocaine (C9H ll N0 2), butamben (C11H,sN0 2), and F = relative response factor (see Table 2)
tetracaine hydrochloride (C1sH24N202 . HCI) in the portion
of Gel taken: Calculate the percentage of ethyl 4-nitrobenzoate in the
portion of Gel taken:
Resuit = (rulrs) x (CsICu) x 100
Result =(rulrs) x (CslCu) x 100
= peak response of the corresponding analyte = peak response of ethyl 4-nitrobenzoate from the
from the Sample solution
Sample solution

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516 Benzocaine / OfficialMonographs USP43

rs = peak response of ethyl 4-nitrobenzoate from the

Standardsolution
Benzocaine, Butamben, and Tetracaine
Cs = concentration of USP Ethyl4-Nitrobenzoate RS in Hydrochloride Ointment
the Standardsolution (~g/mL)
Cu = nominal concentration of benzocaine in the DEFINITION
Sample solution (~g/mL) Benzocaine, Butamben, and Tetracaine Hydrochloride
Ointment is Benzocaine, Butamben, and Tetracaine
Calculatethe percentage of tetracaine related compound B Hydrochloride in a suitable ointment base. It contains NLT
and any individual unspecified degradation product in the 90.0% and NMT 110.0% of the labeled amounts of
portion of Gel taken: benzocaine (C9H llN02) , butamben (C"H,sN02) , and
tetracaine hydrochloride(C,sH 24N202 • He/).
Result = (rulrs) x (CsICu) x (1IF) x 100
IDENTifiCATION
to = peak response of tetracaine related compound B • A. The retention times of the major peaks of the Sample
or any individual unspecified degradation solution correspond to those of the Standardsolution, as
product from the Sample solution obtained in the Assay.
ts = peak response of tetracaine from the Standard
ASSAY
solution
Cs = concentration of USP Tetracaine Hydrochloride • PROCEDURE
RS in the Standardsolution (uq/rnl.) Diluent: Methanol and water (60:40)
Cu = nominal concentration of tetracaine Mobile phase: Methanol, water, and 0.25 M sodium
hydrochloride in the Sample solution (lJg/mL) l-heptanesulfonate (500:500:20)
F = relative response factor (see Table 2)
Standard solution: .Transfer 140 mg of USP Benzocaine RS
to a 1OO-mL volumetricflask with the aid of 25 mL of
Acceptance criteria: See Table 2. Disregard any impurity methanol, and swirl. Transfer 140/ mg of USP Butamben RS
peaks lessthan 0.05%. to the same volumetricflask with the aid of 25 mL of water,
Jbeing the ratio of the labeled amount, as a percentage, of
Table 2 butamben to the labeled amount, as a percentage, of
benzocaine in the Ointment. Transfer 140}'mg of USP
Relative Relative Acceptance Tetracaine Hydrochloride RS to the same volumetricflask
Retention Response Criteria,
Name Time Factor NMT (%) with the aid of 25 mLof water, J' being the ratio of the
labeled amount, as a percentage, of tetracaine
4-Aminobenzoic acid 0.23 1.3 0.3 hydrochlorideto the labeled amount, as a percentage, of
, benzocaine in the Ointment. Sonicatefor about 1 min, and
Benzocaine 1.0 - -
dilute with Diluent to volume.
Tetracaine 1.07 - - Sample stock solution: Nominally 14 mg/mL of
Tetracaine related benzocaine, prepared as follows. Transfer a portion of
compound Ba 1.34 0.6 0.4 Ointment, equivalent to 1400 mg of benzocaine, to a
Butamben 1,45 - -
1OO-ml volumetric flask. Add 75 mL of methanol, and mix.
Sonicate for about 1 min, and dilute with methanol to
Ethyl 4-nitrobenzoate 1.50 - 0.2 volume.
Any individual Sample solution: Nominally 1.4 mg/mL of benzocaine in
unspecified - Diluent from the Sample stock solution
degradation product 1.0 0.4 Chromatographic system
Total degradation
(See Chromatography (621), System Suitability.)
products - - 2.0 Mode: LC
Detector: UV 313 nm
a 4-(Butylamino)benzoic acid. Column: 3.9-mm x 30-cm; packing L1
Flow rate: 2 mL/min
SPECIFIC TESTS Injection volume: 10 IJL
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR System suitability
SPECIFIED MICROORGANISMS (62): The total aerobic Sample: Standard solution
microbial count is NMT 102 cfu/g. The total combined [NoTE-The relative retention times for benzocaine,
molds and yeasts count is NMT 10' cfu/g. It meets the butamben, and tetracaine are about 0.3, 0.8, and
requirements of the tests for absence of Staphylococcus 1.0, respectively.]
aureus and Pseudomonas aeruginosa. Suitability requirements
Resolution: NLT 2 between benzocaine and butamben,
ADDITIONAL REQUIREMENTS and between butamben and tetracaine
• PACKAGING AND STORAGE: Preserve in tight containers in a Relative standard deviation: NMT 2.0% for each of the
cool dry place and avoid freezing. three analyte peaks
• USP REFERENCE STANDARDS (11) Analysis
USP Benzocaine RS Samples: Standardsolution and Sample solution
USP Butamben RS Calculatethe percentage of the labeled amounts of
USP Ethyl 4-Nitrobenzoate RS benzocaine (C9H"N02) , butamben (C"H,sN02) , and
Ethyl p-nitrobenzoate. tetracaine hydrochloride(C,sH 24N202 • HC/) in the portion
C9H9N04 195.17
of Ointment taken:
USP Tetracaine Hydrochloride RS
Result « (rulrs) x (CsICu) x 100

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USP 43 Official Monographs / Benzocaine 51 7

ru = peak response of the corresponding analyte Butamben RS and USP Tetracaine Hydrochloride RS to a
from the Sample solution suitable volumetric flask and dissolve in 80% of the total
ts = peak response of the corresponding analyte volume of Diluent. [NoTE-Sonication for about 10 min may
from the Standardsolution be necessary.] Dilute with Diluent to volume.
Cs = concentration of the corresponding Reference Standard solution: 175 J.lg/mLof USP Benzocaine RS from
Standard in the Standardsolution (mg/mL) Standardstock solution A and 25 IJg/mL each of USP
Cu = nominal concentration of the corresponding Butamben RS and USP Tetracaine Hydrochloride RS from
analyte in the Sample solution (mg/mL) Standardstock solution B diluted in Diluent
Sample solution: Nominally 175 IJg/mL of benzocaine and
Acceptance criteria: 90.0%-110.0% 25 IJg/mL each of butamben and tetracaine hydrochloride,
prepared asfollows. Transfer a suitable portion of Topical
PERFORMANCE TESTS Solution to a suitable volumetric flask and dissolve in 80%
• MINIMUM FILL (755): Meets the requirements
of the total volume of Diluent. [NoTE-Sonication for about
ADDITIONAL REQUIREMENTS 10 min may be necessary.] Dilute with Diluent to volume.
• PACKAGING AND STORAGE: Preserve in tight containers, and Chromatographic system
avoid freezing. (See Chromatography (621), System Suitability.)
• USP REfERENCE STANDARDS (11) Mode: LC
USP Benzocaine RS Detector: UV 300 nm. For Identification B, usea diode array
USP Butamben RS detector in the range of 200-400 nm.
USP Tetracaine Hydrochloride RS Column: 2.1-mm x 5-cm; 1.7-J.lm packing L1
Flow rate: 0.6 mL/min
Injection volume: 1 IJL
System suitability
Sample: Standardsolution
Benzocaine, Butamben, and Tetracaine [NOTE-The relative retention times for benzocaine,
Hydrochloride Topical Solution tetracaine, and butamben are about 0.71,0.74, and
. 1.0, respectively.]
DEFINITION Suitability requirements
Benzocaine, Butamben, and Tetracaine Hydrochloride Topical Resolution: NLT 2.0 between benzocaine and tetracaine
Solution contains NLT 90.0% and NMT 110.0% of the Relative standard deviation: NMT 2.0% for each of the
labeled amount of benzocaine (C 9H 11NOz), butamben three analyte peaks
Analysis
(CllH1SNOz), and tetracaine hydrochloride (ClsHz4NzOz'
Samples: Standardsolution and Sample solution
HCI). '
Calculate the percentage of the labeled amount of
IDENTIFICATION benzocaine (C 9H nNOz), butamben (CnH 1sNOz), and
• A. The retention times of the major peaks of the Sample tetracaine hydrochloride (ClsHz4NzOz . HCI) in the portion
solution correspond to those of the Standardsolution, as of Topical Solution taken:
obtained in the Assay.
• B. The UV spectrum of the major peaks of the Sample Result = (rufrs) x (Cs/Cu) x 100
solution corresponds to that of the Standardsolution, as
obtained in the Assay. tu = peak response of the corresponding analyte
from the Sample solution
ASSAY rs = peak response of the corresponding analyte
• PROCEDURE from the Standardsolution
Solution A: 0.1% formic acid in water Cs =concentration of the corresponding Reference
Solution B: 0.1% formic acid in acetonitrile Standard in the Standardsolution (J.lg/mL)
Mobile phase: See Table 1. Cu = nominal concentration of the corresponding
analyte in the Sample solution (lJg/mL)
Table 1
Time Solution A Solution B Acceptance criteria: 90.0%-110.0%
(min) (%) (%)
PERFORMANCE TESTS
0 91 9 • MINIMUM Fill (755): Meets the requirements
2.5 50 50
IMPURITIES
3.9 50 50 • ORGANIC IMPURITIES
Mobile phase, Diluent, and Chromatographic system:
4 91 9
Proceed as directed in the Assay.
5 91 9 System suitability solution: 10 IJg/mL each of USP
Benzocaine RS i USP Tetracaine Hydrochloride RS, USP
Diluent: Acetonitrile and water (10:90) Butamben RS, and USP Ethyl4-Nitrobenzoate RS in Diluent
Standard stock solution A: 1750 IJg/mL of USP Benzocaine Standard solution: 3.4 IJg/mL each of USP Benzocaine RS
RS prepared as follows. Transfer a suitable amount of USP and USP Ethyl 4-Nitrobenzoate RS and 1 IJg/mL of USP
Benzocaine RS to a suitable volumetric flask and dissolve in Tetracaine Hydrochloride RS in Diluent
80% of the total volume of Diluent. [NoTE-Sonication for Sample solution: Nominally 1.68 mg/mL of benzocaine,
about 5 min may be necessary.] Dilute with Diluent to 0.24 mg/mL of butamben, and 0.24 mg/mL of tetracaine
volume. prepared asfollows. Accurately weigh a suitable quantity of
Standard stock solution B: 125 IJg/mL each of USP Topical Solution into a suitable volumetric flask, dissolve
Butamben RS and USP Tetracaine Hydrochloride RS with 20% of the total volume of acetonitrile, and dilute with
prepared as follows. Transfer a suitable amount of USP

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518 Benzocaine / OfficialMonographs USP 43

water to volume. [NoTE-Sonication for about 1 min may Table 2 (continued)

be necessary.] Relative Relative Acceptance
System suitability Retention Response Criteria,
Samples: System sUitabilitysolution and Standardsolution Name Time Factor NMT(%)
[NoTE-See Table 2 for relative retention times.] Tetracaine 0.74 - -
Suitability requirements
Resolution: NLT 2 between butamben and ethyl Tetracaine related
compound Ba 0.93 0.6 0.4
4-nitrobenzoate; NLT 2 between benzocaine and
tetracaine, System suitability solution Butarnben 1.0 - -
Relative standard deviation: NMT 5.0% for each of the
analyte peaks, Standardsolution Ethyl4-nitrobenzoate 1.04 - 0.2
Analysis Any individual
Samples: Standardsolution and Sample solution unspecified -
degradation product 1.0 0.4
Calculate the percentage of 4-aminobenzoic acid in the
portion of Topical Solution taken: Total degradation
products - - 2.0
Result = (rulr s) x (CsICu) x (1 IF) x 100
a 4-(Butylamino)benzoic acid.
= peak response of 4-aminobenzoic acid from the
Sample solution ADDITIONAL REQUIREMENTS
=peak response of benzocaine from the Standard • PACKAGING AND STORAGE: Preserve in tight containers, and
solution avoid freezing.
= concentration of USP Benzocaine RS in the • USP REFERENCE STANDARDS (11)
Standardsolution (~g/mL) USP Benzocaine RS
= nominal concentration of benzocaine in the USP Butamben RS
Sample solution (~g/mL) USP Ethyl 4-Nitrobenzoate RS
F = relative response factor (see Table 2) Ethyl p-nitrobenzoate.
C9H 9 N0 4 195.17
Calculate the percentage of ethyl 4-nitrobenzoate in the USP Tetracaine Hydrochloride RS
portion of Topical Solution taken:

Result =(rulr s) x (CsICu) x 100

ru = peak response of ethyl4-nitrobenzoate from the Benzocaine and Menthol Topical

Sample solution Aerosol
rs = peak response of ethyl 4-nitrobenzoate from the
Standardsolution DEfiNITION
Cs =concentration of USP Ethyl4-Nitrobenzoate RS in Benzocaine and Menthol Topical Aerosol is a solution of
the Standardsolution (~g/mL) Benzocaine and Menthol with suitable propellants in a
Cu =nominal concentration of benzocaine in the pressurized container. It contains NLT 90.0% and NMT
Sample solution (uq/rnt) 110.0% of the labeled amounts of benzocaine (C9H11N02)
and menthol (ClOH200).
Calculate the percentage of tetracaine related compound B
and any individual unspecified degradation product in the IDENTifiCATION
portion of Topical Solution taken: . • A. The UV spectrum of the major peak of the Sample
solution corresponds to that of the Standardsolution, as
Result = (rulr s) x (Cs/Cu) x (1 IF) x 100 obtained in the Assay.
• B. The retention time of the major peak for benzocaine of
ru = peak response of tetracaine related compound B the Sample solution corresponds to that of the Standard
or any individual unspecified degradation solution, as obtained in the Assay.
product from the Sample solution • C. The retention time of the major peak for menthol of the
rs = peak response of tetracaine from the. Standard Sample solution corresponds to that of the Standard
solution solution, as obtained in the Assay.
Cs = concentration of USP Tetracaine Hydrochloride
RS in the Standardsolution (~g/mL) ASSAY
Cu = nominal concentration of tetracaine • BENZOCAINE
hydrochloride in the Sample solution (~g/mL) Solution A: Dilute 1 .0 mL of trifluoroacetic acid with water
F = relative response factor (see Table 2) to 1 L.
Solution B: Acetonitrile
Acceptance criteria: See Table 2. Disregard any impurity Mobile phase: See Table 1.
peaks less than 0.05%.
Table 1
Table 2 Time Solution A Solution B
Relative Relative Acceptance (min) (%) (%)
Retention Response Criteria,
Name Time Factor NMT(%) 0 90 10

4-Aminobenzoic acid 0.15 1.3 0.3 15 72 28

18 50 50
Benzocaine 0.70 - -
18.1 90 10

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USP 43 OfficialMonographs / Benzocaine 519

Table 1 (continued) Temperatures

Time Solution A Solution B Injection port: 250 0
(min) (%) (%) Detector: 250 0
20 90 10
Column: See Table 2.

Table 2
Diluent: Solution A and Solution B (1:1) Hold Time
Standard solution: 0.1 mg/mL of USP Benzocaine RS in Initial Tempera- Final at Final
Diluent. Sonicate for 2-5 min to dissolve before diluting to Tempera- Hold Time at ture Tempera- Tempera-
final volume. ture 130° Ramp ture ture
Sample solution: Nominally 0.1 mg/mL of benzocaine in e) (min) (o/min) e) (min)
Diluent prepared as follows. Spray the contents of the 130 7.5 35 240 1.0
Topical Aerosol into a flask with a stopper. Heat and stir the
sprayed Topical Aerosol at 1000 for 30 min in an oil bath to Flow rate: 10 mL/min
obtain a viscous liquid sample. Cool the sample to room Injection volume: 1 IJL
temperature. Transfer an amount of Topical Aerosol Injection type: Split, split ratio 10:1
equivalent to 10 mg of benzocaine to a 1OO-mL volumetric System suitability
flask. Dissolve the sample in Diluent and dilute with Diluent Sample: Standard solution
to volume. [NOTE-The relative retention times for menthol and
Chromatographic system decanol are 1.0 and 1.6, respectively.]
(See Chromatography (621), System Suitability.) Suitability requirements
Mode: LC Resolution: NLT 2.5 between the menthol and decanol
Detector: UV 280 nm. For Identification test A use a diode peaks
array detector in the range of 200-400 nm. ' Relative standard deviation: NMT 1.0% of the ratio of
Column: 4.6-mm x 25-cm; 5-lJm packing L7 the peak response of menthol to that of decanol
Flow rate: 1.5 mL/min Analysis
Injection volume: 20 IJL .Samples: Standardsolution and Sample solution
System suitability Calculate the percentage of the labeled amount of menthol
Sample: Standardsolution (ClOHzoO) in the portion of Topical Aerosol taken:
Suitability requirements
Tailing factor: NMT 1.5 Result = (RuIRs) x (CsICu) x 100
Relative standard deviation: NMT 2.0%
Analysis , = peak response ratio of menthol to decanol from
Samples: Standardsolution and Sample solution the Sample solution
Calculate the percentage of the labeled amount of = peak response ratio of menthol to decanol from
benzocaine (C9H ll NO z) in the portion of Topical Aerosol the Standardsolution
taken: =concentration of USP Menthol RS in the Standard
solution (mg/mL)
Result = (rulr s) x (CsICu) x 100 = nominal concentration of menthol in the Sample
solution (mg/mL)
= peak response from the Sample solution
= peak response from the Standardsolution Acceptance criteria: 90.0%-110.0%
= concentration of USP Benzocaine RS in the
Standardsolution (mg/mL) . PERFORMANCE TESTS
= nominal concentration of benzocaine in the • MINIMUM FILL (755): Meets the requirements
Sample solution (mg/mL) IMPURITIES
• ORGANIC IMPURITIES
Acceptance criteria: 90.0%-110.0% Solution A: Dilute 1.0 mL of trifluoroacetic acid with water
• MENTHOL to 1 L.
Internal standard solution: 1 mg/mL of decanol in Solution B: Acetonitrile
isopropyl alcohol Mobile phase: See Table 3.
Standard stock solution: 1 mg/mL of USP Menthol RS in
isopropyl alcohol Table 3
Standard solution: 0.5 mg/mL each of USP Menthol RS and
decanol in isopropyl alcohol from Internal standard solution Time Solution A Solution B
(min) (%) (%)
and Standardstock solution
Sample solution: Nominally 0.5 mg/mL of menthol 0 90 10
prepare~ as follows. Spray the contents of the Topical 34 50 50
Aerosol Into a flask. Transfer an amount of Topical Aerosol
equivalent to 5 mg of menthol to a 1O-mL volumetric flask 35 90 10
and add 5.0 mL of Internal standardsolution. Dilute with 38 90 10
isopropyl alcohol to volume.
Chromatographic system
(See Chromatography (621), System Suitability.) Diluent: Solution A and Solution B (1:1)
Mode: GC Standard.stock solution: 0.1 mg/mL each of USP
Detector: Flame ionization Ben~ocalne RS, USP ~mi~obenzoic Acid RS, and USP Ethyl
Column: 0.53-mm x 30-m; fused-silica capillary column 4-Nltrobenzoate RS In Diluent. Sonicate for about 5 min to
bonded with a 1.O-um film of phase G16 dissolve before diluting to final volume.
Carrier gas: Hydrogen Standard solution: 0.001 mg/mL each of USP Benzocaine
RS, USP Aminobenzoic Acid RS, and USP Ethyl

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520 Benzocaine / OfficialMonographs USP 43

4-Nitrobenzoate RS in Diluent from the Standard stock Table 4 (continued)

solution Relative Acceptance
Sample solution: Nominally 0.5 mg/ml of benzocaine in Retention Criteria,
Diluent prepared as follows. Spray the contents of the Name Time NMT(%)
Topical Aerosol into a flask. Heat and stir the sprayed Totaldegradation
Topical Aerosol at 100° for 30 min in an oil bath to obtain products - 2.0
a viscous liquid sample. Cool the sample to room
temperature. Transfer an amount of Topical Aerosol SPECifiC TESTS
equivalent to 50 mg of benzocaine to a 1OO-ml volumetric • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
flask. Dissolve the sample in Diluent and dilute with Diluent SPECIFIED MICROORGANISMS (62): It meets the
to volume. requirements for the absence of Staphylococcus aureus and
Chromatographic system
(See Chromatography (621), System Suitability.)
Pseudomonas aeruginosa.
• PRESSURE TEST: It meets the requirements in Topical Aerosols
Mode: lC (603).
Detector: UV 280 nm • LEAKAGE TEST: It meets the requirements in Leak Rate (604).
Column: 4.6-mm x 25-cm; 5-lJm packing l7
Flow rate: 1.5 ml/min ADDITIONAL REQUIREMENTS
Injection volume: 20 IJl • PACKAGING AND STORAGE: Preserve in tight, pressurized
System suitability containers, and avoid exposure to excessive heat.
Sample: Standard solution • USP REFERENCE STANDARDS (11)
Suitability requirements USP Aminobenzoic Acid RS
Relative standard deviation: NMT 2.0% for each peak Benzoic acid, 4-amino.
corresponding to benzocaine, aminobenzoic acid, and C7H 7NO z 137.14
ethyl4-nitrobenzoate USP Benzocaine RS
Analysis USP Ethyl 4-Nitrobenzoate RS
Samples: Standard solution and Sample solution Benzoic acid, 4-nitro-, ethyl ester.
Calculate the percentage of aminobenzoic acid and ethyl C9H9N04 195.17
4-nitrobenzoate in the portion of Topical Aerosol taken: USP Menthol RS
Result = (rulrs) x (CsICu) x 100
= peak response of aminobenzoic acid or ethyl
4-nitrobenzoate from the Sample solution Benzoic Acid
= peak response of the corresponding Reference
Standard from the Standard solution
= concentration of USP Aminobenzoic Acid RS or
USP Ethyl 4-Nitrobenzoate RS in the Standard
solution (mg/ml)
= nominal concentration of benzocaine in the C7H 60Z 122.12
Sample solution (mg/ml) Benzoic acid [65-85-0].
Calculate the percentage of each unspecified degradation DEfiNITION
product in the portion of Topical Aerosol taken: Benzoic Acid contains NlT 99.5% and NMT 100.5% of
benzoic acid (C 7H 60Z) , calculated on the anhydrous basis.
Result = (rulrs) x (CsICu) x 100
IDENTifiCATION
tu = peak response of each unspecified degradation • A.
product from the Sample solution Sample solution: Prepare a saturated solution of Benzoic
rs = peak response of benzocaine from the Standard Acid in water, and filter twice.
solution Analysis 1: To one portion of the filtrate add ferric chloride
Cs = concentration of USP Benzocaine RS in the TS.
Standard solution (mg/ml) Acceptance criteria 1: A salmon-colored precipitate is
Cu = nominal concentration of benzocaine in the formed.
Sample solution (mg/mL) Analysis 2: To a separate 1O-ml portion of the filtrate add 1
mL of 7 N sulfuric acid, and cool the mixture.
Acceptance criteria: See Table 4. Disregard peaks less than Acceptance criteria 2: A white precipitate forms in 10 min.
0.05%. This precipitate is soluble in ether.
ASSAY
Table 4 • PROCEDURE
Relative Acceptance Sample: 500 mg of Benzoic Acid
Retention Criteria, Analysis: Dissolve the Sample in 25 ml of diluted alcohol
Name Time NMT (%)
that previously has been neutralized with 0.1 N sodium
Aminobenzoic acid 0.3 0.2 hydroxide. Add phenolphthalein TS, and titrate with 0.1 N
sodium hydroxide VS to a pink color. Each ml of 0.1 N
Benzocaine 1.0 - sodium hydroxide is equivalent to 12.21 mg of benzoic acid
Ethyl 4-nitrobenzoate 2.1 0.2 (C7H 60Z) '
Any other unspecified Acceptance criteria: 99.5%-100.5% on the anhydrous
degradation product - 0.1 basis

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USP 43 Official Monographs / Benzoic 521

IMPURITIES ASSAY
• RESIDUE ON IGNITION (281): NMT 0.05% • PROCEDURE
Ferric chloride-urea reagent: On the day of use, dissolve,
SPECIFIC TESTS
without heating, 18 g of urea in a mixture of 2.5 mL offerric
• CONGEALING TEMPERATURE (651): 121°-123°
chloride solution (6 in 10) and 12.5 mL of 0.05 N
• WATER DETERMINATION, Method I (921) hydrochloric acid.
Sample solution: A 1-in-2 solution of methanol in pyridine Column A: Insert a small pledget of glass wool above the
is used as the solvent. stem constriction of a 20- x 2.5-cm chromatographic tube.
Acceptance criteria: NMT 0.7% Mix 1 g of chromatographic siliceous earth with 0.5 mL of
• READILY CARBONIZABLE SUBSTANCES TEST (271)
dilute phosphoric acid (3 in 10) to form a uniform, fluffy
Sample solution: 500 mg in 5 mL of sulfuric acid mixture; transfer to the chromatographic tube; and pack
Acceptance criteria: The solution has no more color than
evenly over the glasswool, exerting~e~~le pressure. .
Matching Fluid Q. Similarly, mix 4 g of chromatographIC slllceous earth With
• READilY OXIDIZABLE SUBSTANCES
3 mL of Ferric chloride-urea reagent, and pack uniformly
Sample solution: Add 1.5 mL of sulfuric acid to 100 mL of over the first layer. Cover the column with a pad of glass
water. Heat to boiling, and add 0.1 N potassium wool.
permanganate, dropwise, ~ntil t.he.pinkcolor persi.sts for 30 Column B: Insert a small pledget of glass wool above the
s. Dissolve 1.00 g of Benzoic ACid In the hot solution. stem constriction of a second 20- x 2.5-cm
Analysis: Titrate with 0.1 N potassium permanganate VS to chromatographic tube. Mix 4 g of chromatographic
a pink color that persists for 15 s. . siliceous earth with 2 mL of sodium bicarbonate solution (1
Acceptance criteria: NMT 0.50 mL of 0.10 N potassium in 12), prepared just before use,to a uniform, fluffy mixtu~e;
permanganate is consumed. and pack evenly over the glass wool. Cover the column With
ADDITIONAL REQUIREMENTS a pad of glasswool.
• PACKAGING AND STORAGE: Preserve in well-closed Diluent: Glacial acetic acid in chloroform (3 in 100)
containers. Standard solution A: 20 ~g/mL of USP Salicylic Acid RS in
Diluent
Standard solution B: 40 ~g/mL of USP Benzoic Acid RS in
Diluent
Sample solution: Transfer an a!TI0u.nt of the Ointmen~, .
Benzoic and Salicylic Acids Ointment equivalent to 100 mg of benzolc acid and 50 mg of salicylic
acid to a 250-mL volumetric flask, and dissolve in 150 mL
DEFINITION of chloroform by warming on a steam bath. Cool. Dilute
Benzoic and Salicylic; Acids Ointment is Benzoic Acid and with chloroform to volume to obtain a solution having a
Salicylic Acid, present in a ratio of 2:1, in a suitable ointment nominal concentration of 200 ~g/mL of salicylic acid and
base. It contains NLT 90.0% and NMT 110.0% of the labeled 400 ~g/mL of benzoic acid.
amounts of benzoic acid (C7H60 Z) and salicylic acid (C7H 60 3) . Analysis
Samples: Standard solution A, Standardsolution B, and
IDENTIFICATION Sample solution
• A. THIN-LAYER CHROMATOGRAPHY Mount Column A directly over Column B, then pipet 10 mL
Diluent: Mixture of chloroform and methanol (1:1) of Sample solution onto Column A, and allow it to pass
Standard solution A: 2.4 mg/mL of USP Benzoic Acid RS in into the column. Wash the columns with two 40-mL
Diluent. . portions of chloroform, allowing the first portion to
Standard solution B: 1.2 mg/mL of USP SalicylicAcid RS in recede to the top of each column before adding the
Diluent second portion. Discard the eluates, and separate the
Sample solution: Equivalent to 60 mg of benzoic acid. and columns.
30 mg of salicylic acid from Ointment, in 25 mL of DIluent Salicylic acid content: Elute ~olumn A with 95 mL .of
Chromatographic system Diluent, collecting the eluate In a 1OO-mL volumetric flask.
(See Chromatography (621), Thin-Layer Chromatography.) Dilute the contents of the flask with Diluent to volume,
Mode: TLC and mix. Concomitantly determine the absorbances of
Adsorbent: 0.25-mm layer of chromatographic silica gel the eluate and Standard solutionA in 1-cm cells at the
mixture wavelength of maximum absorbance at 311 nm, with a
Application volume: 5 ~L of each solution at separate suitable spectrophotometer, using Diluent as the blank.
points 2.5 cm from the bottom edge of a 20- x 20-cm Calculate the percentage of the labeled amount of salicylic
thin-layer chromatographic plate acid (C 7H 60 3) in the portion of Ointment taken:
Developing solvent system: Chloroform, acetone,
isopropyl alcohol, methanol, and ammonium hydroxide Result = (Au/As) x (Cs/ Cu) x F x 100
(30:30:15:15:10)
Analysis
Samples: Standard solution A, Standard solution B, and
=absorbance of the diluted eluate from Column A
Sample solution
=absorbance of Standard solutionA
Develop the chromatogram in the Developing solvent
=concentration of USP Salicylic Acid RS in Standard
solution A (~g/mL)
system until the solvent front has moved three-fourths of
= nominal concentration of the salicylic acid in the
the length of the plate. Remove the plate from the
Sample solution (uq/rnl)
chromatographic chamber, mark the solvent front, and
allow the solvent to evaporate. View the chromatogram
F =sample dilution factor, 10
under short-wavelength (254 nm) UV radiation. Acceptance criteria: 90.0%-110.0%
Acceptance criteria: The two m~jor fluoresc~nt spots from Benzoic acid content: Elute Column B with 95 mL of
the Sample solution correspond In color and In RF ~alue to Diluent collecting the eluate in a 1OO-mL volumetric flask.
those from Standard solution A and Standard solution B. Dilute the contents of the flask with Diluent to volume,

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522 Benzoic / Official Monographs USP 43

and mix. Concomitantly determine the absorbances of Analysis: On a separate portion of Benzoin, determine the
eluate and Standardsolution B in 1-cm cells at the water content as directed for WaterDetermination (921),
wavelength of maximum absorbance at 275 nm, with a Method II. Calculate the weight of water in the quantity of
suitable spectrophotometer, using Diluent as the blank. the Benzoin taken for the Assay, and subtract it from the
Calculate the percentage of the labeled amount of original weight of the Benzoin taken. The difference
benzoic acid (C 7H6 0 z) in the portion of Ointment taken: between this result and the weight of the residue in the
extraction thimble represents the alcohol-soluble
Result =(Au/As) x (Cs/Cu) x F x 100 extractive.
Acceptance criteria: The alcohol-soluble extractive is NLT
Au = absorbance of the diluted eluate from Column B 75.0% for Sumatra Benzoin and NLT 90.0% for Siam
As = absorbance of Standardsolution B Benzoin.
Cs = concentration of USPBenzoic Acid RS in Standard
OTHER COMPONENTS
solution B (lJg/mL)
• CONTENT OF BENZOIC ACID
Cu = nominal concentration of benzoic acid in the
Analysis: Treat 1 g of powdered Benzoin with 15 mL of
Sample solution (lJg/mL) warm carbon disulfide. Filter through a small pledget of
F = sample dilution factor, 10
cotton, wash the cotton with an additional 5 mL of carbon
disulfide, and allow the filtrate to evaporate spontaneously.
Acceptance criteria: 90.00/0-110.0%
Acceptance criteria: The weight of the residue is NLT 6.0%
PERFORMANCE TESTS of the weight of Benzoin taken for Sumatra Benzoin and
• MINIMUM Flu (755): Meets the requirements NLT 12.0% for Siam Benzoin. This residue meets the
requirements for Identification Tests-General (191),
ADDITIONAL REQUIREMENTS Benzoate.
• PACKAGING AND STORAGE: Preserve in well-closed
containers, and avoid exposure to temperatures exceeding IMPURITIES
30°. INORGANIC IMPURITIES
• LABELING: Label Ointment to indicate the concentrations of • Articles of Botanical Origin, Acid-Insoluble Ash (561): NMT
Benzoic Acid and Salicylic Acid and to indicate whether the 1.0% in Sumatra Benzoin; NMT 0.5% in Siam Benzoin
ointment base is water-soluble or water-insoluble. ORGANIC IMPURITIES
• USP REFERENCE STANDARDS (11) • Procedure: Articles of Botanical Origin, Foreign Organic
USP Benzoic Acid RS Matter(561): NMT 1.0% in Siam Benzoin
USP Salicylic Acid RS
SPECIFIC TESTS
• BOTANIC CHARACTERISTICS
Sumatra Benzoin: Blocks or lumps of varying size, made up
of tears, compacted together, with a reddish brown,
Benzoin reddish gray, or grayish brown resinous mass; the tears are
externally yellowish or rusty brown, milky white on fresh
DEFINITION fracture; hard and brittle at ordinary temperatures, but
Benzoin is the balsamic resin obtained from Styrax benzoin softened by heat.
Dryand. or Styrax paralleloneurus Perkins, known in Siam Benzoin: Pebble-like tears of variable size and shape,
commerce as Sumatra Benzoin, or from Styrax tonkinensis compressed, yellowish brown to rusty brown externally,
(Pierre) Craib ex Hartwich, or other species'of the Section milky white on fracture, separate or very slightly
Anthostyrax of the genus Styrax, known in commerce as Siam agglutinated; hard and brittle at ordinary temperatures, but
Benzoin (Fam. Styraceae). softened by heat.
Sumatra Benzoin yields NLT 75.0% of alcohol-soluble ADDITIONAL REQUIREMENTS
extractive, and Siam Benzoin yields NLT 90.0% of • PACKAGING AND STORAGE: Preserve in well-closed
alcohol-soluble extractive. containers.
IDENTIFICATION • LABELING: Label it to indicate whether it is Sumatra Benzoin
• A. A solution in alcohol becomes milky upon the addition or Siam Benzoin.
of water, and the mixture is acid to litmus paper.
• B. IDENTIFICATION OF ARTICLES OF BOTANICAL ORIGIN (563)
Analysis: Heat a few fragments in a test tube.
Acceptance criteria: Sumatra Benzoin evolves a sublimate
consisting of plates and small, rod-like crystals of cinnamic Compound Benzoin Tincture
acid and its esters that strongly polarize light. Siam Benzoin
DEFINITION
evolves a sublimate directly above the melted mass,
Prepare Compound Benzoin Tincture as follows.
consisting of numerous long, rod-shaped crystals of
benzoic acid that do not strongly polarize light.
Benzoin, in moderately coarse powder 100 g
ASSAY
Aloe, in moderately coarse powder 20g
• PROCEDURE
Sample: Place 2 g of Benzoin in a tared extraction thimble, Storax 80g
and insert the thimble in a continuous-extraction
Tolu Balsam 40g
apparatus. Place 100 mg of sodium hydroxide in the
receiving flask of the apparatus, and extract the Benzoin Alcohol, a sufficient quantity to make 1000 mL
with alcohol for 5 h, or until completely extracted. Dry the
extraction thimble containing the insoluble residue at 105° Macerate the ingredients with 750 mL of AlcohoJin a container
for 2 h. that can be closed, and put in a warm place. Agitate it
frequently during 3 days or until the soluble matter is

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 USP 43 OfficialMonographs / Benzonatate 523

dissolved. Transfer the mixture to a filter. When most of the produced by 0.10 mL of 0.020 N hydrochloric acid
liquid has drained away, wash the residue on the filter with (0.0035%).
a sufficient quantity of Alcohol, combining the filtrates to Sulfate (221 )-Mix 5 mL of a solution (1 in 20) with 5 mL of
produce 1000 mL of Tincture, and mix. water and 1 mL of 3 N hydrochloric acid, shakefor 1 hour, and
allow to stand for 1 hour. Pass through afilter having a porosity
OTHER COMPONENTS of 0.2 J.Jm, and to the filtrate add 1 mL of barium chloride TS.
• ALCOHOL DETERMINATION (611), Method II: 74.0%-80.0% Mix, and allow to stand for 10 minutes: the turbidity does not
of alcohol (C2H sOH), the dilution made with methanol exceed that produced by 0.10 mL of 0.020 N sulfuric acid
instead of with water, to approximately 2% alcohol' (0.04%).
SPECIFIC TESTS Assay-Weigh accurately about 5 g of Benzonatate, and
• SPECIFIC GRAVITY (841): 0.870-0.885 reflux with 25.0 mL of 0.5 N sodium hydroxide VSfor 1 hour.
lit LIMIT OF NONVOLATILE RESIDUE
Cool, add 25 mL of water and 10 drops of bromothymol blue
Analysis: Evaporate 3 mL of Tincture in a suitable tared dish TS, and titrate the excess alkali with 0.5 N hydrochloric acid
on a steam bath, and dry the residue at 100° for 2 h. VS. Perform a blank determination (see Residual Titrations
Acceptance criteria: The weight of the residue is 525-675 under Titrimetry (541»). Each mL of 0.5 N sodium hydroxide is
mg. equivalent to 301.5 mg of C30Hs3NOll'

ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Package in tight, light-resistant
containers, and avoid exposure to direct sunlight and to
excessive heat. Benzonatate Capsules
• LABELING: Label it to indicate that it is flammable.
DEFINITION
Benzonatate Capsules contain NLT 90.0% and NMT 110.0%
of the labeled amount of benzonatate [C30Hs3NOll (av.)].

Benzonatate IDENTIFICATION

• A·.. •~·.~~~~~~g~f~.~i~);~~~~~I~!~~~.IO~.t~$I~>(~g~)~; i)li1f~q,.~q

~pg.£t[q~tr.()PY;l~tF;#'(C;NJ "MaY-202(» . .
Sample: The contents of Capsules
Acceptance criteria: Meets the requirements. If a difference
C30Hs3NOll (av.) 603.74 (average) is observed, or if excipients are present, use an amount of
Benzoic acid, 4-(butylamino)-, 2,5,8,11,14,17,20,23,26- the contents of Capsules equivalent to about 100 mg of
nonaoxaoctacos-28-yl ester. benzonatate. Mix with 25 mL of 0.01 N hydrochloric acid,
2,5,8,11,14,1 7,20,23,26-Nonaoxaoctacosan-28-yl p- and proceed as directed in Identification-Organic
(butylamino)benzoate [104-31-4]. Nitrogenous Bases (181), beginning with "Transfer the
liquid to a separator".
» Benzonatate contains not less than 95.0 percent
and not more than 105.0 percent of C30Hs3NOll'
Packaging and storage-Preserve in tight, light-resistant
containers.
USP Reference standards (11)-
.·
• B.~~~~~"'ij~S~•.~~I~>I~~~~I~~~~"'I~ij • • J:~.~~~ ~J~~~~
Q1t(C1.'{i()lgt-.vl§iblg.·$pgtr.t(()§g()PY;/1~7'~A(CNJ''MaY"2029)
Sample solution: Nominally 15 1l9/mL of benzonatate from
USP Benzonatate RS thecontents of Capsules
Acceptance criteria: Meet the requirements
Identification-
ASSAY
• PROCEDURE
Standard solution: 500 Ilg/mL of USP Benzonatate RS
Sample stock solution: Nominally 5 mg/mL of benzonatate
in chloroform, prepared as follows. Mix a number of
Capsules,equivalent to about 500 mg of benzonatate, with
40 mL of chloroform in a suitable high-speed blender, and
dilute with chloroform to 100.0 mL.
Sample solution: Nominally 500 1l9/mL of benzonatate
prepared as follows. Transfer 10.0 mL of Sample stock
J.Jg per solution into a 1OO-mL volumetric flask. Evaporate the
Medium: water. chloroform on a steam bath with the aid of a current of air.
Refractive index (831): between 1.509 and 1.511 at 20°. Dissolve the residue in water and dilute with water to
Water Determination, Method I (921): not more than 0.3%. volume.
Residue. on ignition (281): not more than 0.1%. Instrumental conditions
Chloride (221 )-Mix 20 mL of a solution (1 in 10) with 20 mL Mode: Vis
of water and 1 mL of nitric acid, shake for 1 hour, and allow Analytical wavelength: 500 nm
to stand for 1 hour. Pass through a filter having a porosity of Cell: 1 cm .
0.2 J.Jm, and to the filtrate add 1 mL of silver nitrate TS. Dilute Blank: Water
with water to 50 mL, mix, and allow to stand protected from Analysis
light for 10 minutes: the turbidity does not exceed that Samples: Standard solution, Sample solution, and Blank

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524 Benzonatate / Official Monographs USP 43

Transfer 4.0 mL each of the Standard solution, Sample

solution, and Blank to separate test tubes. To each tube
add in succession 1.0 mL of 1 M hydroxylamine
hydrochloride and 1.0 mL of 3.5 N sodium hydroxide,
mixing after each addition. Allow to stand for 10 min,
accurately timed, then add 1.0 mL of 3.5 N hydrochloric
acid, mix, add 1.0 mL of an 80-mg/mL ferric chloride
solution, and mix. Allow to stand for 30 min, accurately
timed. Gently swirl the tubes for 1 min to remove any gas
bubbles present, then concomitantly determine the
absorbances of the solutions.
Calculate the percentage of the labeled amount of
benzonatate [C30Hs3NOll (av.)] in the Capsules taken:

Result = (AulAs) x (CsICu) x 100

U
= absorbance of the Sample solution ital wavelength: c31 0 om.
= absorbance of the Standard solution Analysis
= concentration of USP Benzonatate RS in the ,Samp Standard sol tion.
Standard solution (~g/mL) Calcul he percenta punto~
= nominal concentration of benzonatate in the benz natate [C 30Hs3NO" (
Sample solution (~g/mL)
JlL x 1°9
C

Result = (A~/As) ~Cscx V x D ><

Acceptance criteria: 90.00/0-110.0%
~~ ab~orbal1ce of the Sample solution
PERFORMANCE TESTS = cibsorhance·oftne' Standard solution
== concentrati ' .natate RS ri1 the
Stcmdard so .
•.. DIS~OL~TIO~ (711)
v = volume of
.l:l"est~.I:(RB 1-)UI-2018)
o =dilution fac
Medium: Water; 900 mL L = label claim
Apparatus 2: 50 rpm
Time: 30 min'
Mobile phase: Acetonitrile and 0.04 M monobasic
potassium phosphate (75:25)
Standard solution: 0.1 mg/mL of USP Benzonatate RS.
Sonicate to dissolve, if needed.
Sample solution: Pass a portion of the solution under test
through a suitable filter of 0.45-f.lm pore size.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC .
Detector: UV 310 nm
Column: 3.9-mm x 30-cm; packing L1
Flow rate: 1.5 mL/min
Injection volume: 15 IJL
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 2.0%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
benzonatate [C30Hs3NOll (av.)] dissolved:

Result = (rulrs) x Cs x Vx llL x 100

to = peak response of benzonatate fromthe Sample

solution
rs = peak response of benzonatate from the Standard
solution
Cs = concentration of USP Benzonatate RS in the
Standard solution(mg/mL)
V = volume of Medium, 900 mL
L = label claim (mg/Capsule)
Tolerances: NLT 80% (Q) of the labeled amount of
benzonatate [C30Hs3NOll (av.)] is dissolved. Analysis . ..c. .• _ _ _c _ . . c 'c ,_.

Samples: Standard solution and Sample solution

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 USP 43 OfficialMonographs / Benzoyl 525

Analysis
Samples: Standard solution and Sample solution
Placethe plate in a developing chamber containing and
equilibrated with the Developing solvent system. Develop
the chromatogram until the solvent front has moved
TV three-fourths of the length of the plate. Remove the
plate, and allow the solvent to evaporate. Observe the
plate under short-wavelength UV light.
Acceptance criteria: The RF value of the principal spot of the
Sample solution corresponds to that of the Standard
solution.
V • B. The Sample solution in the test for Organic Impurities
D exhibits a major peak for benzoyl peroxide, the retention
L time of which corresponds to that exhibited by the
Standard solution.
ASSAY
• PROCEDURE
• UNIFORMITY
Sample: 300 mg of previously mixed Hydrous Benzoyl
Peroxide in a conical flask fitted with a ground-glass
ADDITIONAL REQUIREMENTS stopper. Weigh again to obtain the weight of the Sample.
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Analysis: Add 30 mL of glacial acetic acid, previously
containers. sparged with carbon dioxide for NLT 2 min just before use,
and swirl the flask gently to dissolve. Add 5 mL of potassium
iodide solution (1 in 2), and mix. Allow the solution to stand
for 1 min. Titrate the liberated iodine with 0.1 N sodium
thiosulfate VS. As the endpoint is approached, add 1 drop
'of starch iodide paste TS, or equivalent, and continue the
titration to the discharge of the blue color. Perform a blank
REFERENCE STANDARDS (11) determination, and make any necessarycorrection (see
USP Benzonatate RS ».
Titrimetry (541 Each mL of 0.1 N sodium thiosulfate is
equivalent to 12.11 mg of C14H lO 0 4•
Acceptance criteria: 90.0%-110.0% of the labeled amount
IMPURITIES
Hydrous Benzoyl Peroxide ORGANIC IMPURITIES
• Procedure
Solution A: Preparea mixture of acetonitrile and glacial acetic
acid (1000:1).
Solution B: Prepare a mixture of water and glacial acetic acid
(1000:1 ).
Mobile phase: Seethe gradient table below.
C14HlO 0 4 (anhydrous) 242.23
Peroxide, dibenzoyl; Time Solution A Solution B
Benzoyl peroxide [94-36-0]. (min) (%) (%)

DEFINITION 0 18 82
Hydrous Benzoyl Peroxide contains NLT 90.0% and NMT 20 60 40
110.0% of the labeled amount of C14H 10 0 4 • It contains a
minimum of 20% of water for the purpose of reducing 30 60 40
flammability and shock sensitivity.
[CAUTION-Hydrous Benzoyl Peroxide may explode at System suitability solution: 100 IJg/mL of benzoic acid and
temperatures higher than 60° or causefires in the 60 IJg/mL of methylparaben in acetonitrile
presence of reducing substances. Store it in the original Standard solution: Dissolve a quantity of Hydrous Benzoyl
container, treated to reduce static charges.] Peroxide, previously subjected to the Assay, in acetonitrile to
obtain a solution containing 0.32 mg/mL.
IDENTIFICATION Sample solution: 0.32 mg/mL of benzoyl peroxide in
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST acetonitrile
(201) Chromatographic system
Standard solution: 10 mg/mL of Hydrous Benzoyl (See Chromatography (621), System Suitability.)
Peroxide, previously subjected to the Assay, in methanol Mode: LC
Sample solution: 10 mg/mL of benzoyl peroxide in Detector: UV 235 nm
methanol Column: 4.6-mm x 25-cm; packing L1
Mode: TLC Flow rate: 1.2 mL/min
Adsorbent: 0.25-mm layer of chromatographic silica gel Injection size: 10 IJL
mixture System suitability
Application volume: 5 IJL Sample: System suitability solution
Developing solvent system: Toluene, dichloromethane, Suitability requirements
and glacial acetic acid (50:2: 1) . Resolution: NLT 2.0 between benzoic acid and
methylparaben

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526 Benzoyl/Official Monographs USP 43

Tailing factors: NMT 2.0 for the benzoic acid and Column: 3.9-mm x 30-cm; packing L1
methylparaben peaks Flow rate: 1 mL/min
Analysis Injection volume: 10 ~L
Samples: Standard solution and Sample solution System suitability
Calculate the area, as a percentage, of each peak in the Sample: Standard solution (three replicate injections)
chromatogram of the Sample solution: [NOTE-The retention times for ethyl benzoate and
benzoyl peroxide are 7 and 14 min, respectively.]
Result = (rU/rT) x 100 Suitability requirements
Resolution: NLT 2.0 between ethyl benzoate and
ru = peak response for any individual peak other than benzoyl peroxide
the principal peak in the Sample solution Tailing factors: NMT 2.0 for the ethyl benzoate and
rT =sum of the peak responses of all the individual benzoyl peroxide peaks .
peaks including the principal peak in the Sample Peak response ratios: The lowest and highest peak
solution response ratios (Rs) agree within 2.0%.
Analysis
Acceptance criteria: The area of any individual peak other Samples: Standard solution and Sample solution
than the principal peak is NMT 1.5% of the total area. The Calculate the percentage of the labeled amount of benzoyl
sum of the areas of all peaks other than the principal peak is peroxide (C14H lO0 4) in the portion of Gel taken:
NMT 2.0% of the total area.
ADDITIONAL REQUIREMENTS Result = (RulR s) x (Cs/Cu) x 100
• PACKAGING AND STORAGE: Store in the original container,
at room temperature. [NOTE-Do not transfer Hydrous = peak response ratio of benzoyl peroxide to ethyl
Benzoyl Peroxide to metal or glass containers fitted with benzoate from the Sample solution
friction tops. Do not return unused material to its original = peak response ratio of benzoyl peroxide to ethyl
container, but destroy it by treatment with sodium benzoate from the Standard solution
hydroxide solution (1 in 10) until addition of a crystal of =concentration of benzoyl peroxide in the
potassium iodide results in no release of free iodine.] Standard solution (mg/mL)
=nominal concentration of benzoyl peroxide in the
Sample solution (mg/mL)
Acceptance criteria: 90.00/0-125.0%
Benzoyl Peroxide Gel IMPURITIES
• ORGANIC IMPURITIES
DEFINITION
Solution A: Acetonitrile and glacial acetic acid (1000:1)
Benzoyl Peroxide Gel is benzoyl peroxide in a suitable gel base.
Solution B: Water and glacial acetic acid (1000:1)
It contains NLT 90.0% and NMT 125.0% of the labeled
amount of benzoyl peroxide (C14H w0 4) . Mobile phase: See Table 7.

IDENTIFICATION Table 1
A. The retention time of the major peak for benzoyl peroxide Time Solution A Solution B
of the Sample solution corresponds to that of the Standard (min) (0/0) (0/0)
solution, as obtained in the Assay. 0 18 82
ASSAY 20 60 40
• PROCEDURE
Mobile phase: Acetonitrile in water (5 in 10) 30 60 40
Internal standard solution: 3.6 mg/mL of ethyl benzoate
in acetonitrile System suitability solution: 100 ~g/mL of benzoic acid and
Standard stock solution: 0.8 mg/mL of benzoyl peroxide 60 ~g/mL of methylparaben in acetonitrile
prepared as follows. Transfer a suitable quantity of benzoyl Standard solution A: 500 ~g/mL of benzoic acid in
peroxide, recently subjected to the Assayin Hydrous acetonitrile
Benzoyl Peroxide, into a weighed conical flask fitted with a Standard solution B: 20 ~g/mL of ethyl benzoate in
glass stopper. Weigh again to obtain the weight of the acetonitrile
specimen, and quantitatively dissolve in acetonitrile. Standard solution C: 20 ~g/mL of benzaldehyde in
Standard solution: 0.32 mg/mL of benzoyl peroxide acetonitrile -
prepared as follows. Mix 10 mL of Standard stocksolution Standard solution D: Equivalent to 40 pg/mL of anhydrous
and 5 mL of Internal standard solution, and dilute with benzoyl peroxide in acetonitrile, prepared from hydrous
acetonitrile to 25 mL. benzoyl peroxide, which has been analyzed as follows.
Sample stock solution: Transfer an equivalent to 40 mg of Place 300 mg of previously mixed hydrous benzoyl
benzoyl peroxide from Gel into a 50-mL volumetric flask, peroxide in a conical flask fitted with a ground-glass
add 40 mL of acetonitrile, and shake until the material is stopper. Weigh again to obtain the weight of the sample.
thoroughly dispersed. Sonicate the mixture for 5 min, dilute Add 30 mL of glacial acetic acid, previously sparged with
with acetonitrile to volume, mix, and filter. carbon dioxide for NLT 2 min just before use, and swirl the
Sample solution: 10 mL of Sample stock solution and 5 mL flask gently to dissolve. Add 5 mL of potassium iodide
of Internal standard solution; dilute with acetonitrile to 25 solution (1 in 2), and mix. Allow the solution to stand for 1
mL. min. Titrate the liberated iodine with 0.1 N sodium
Chromatographic system thiosulfate VS. As the endpoint is approached, add 1 drop
(See Chromatography (621), System Suitability.) of starch iodide paste TS, or equivalent, and continue the
Mode: LC titration to the discharge of the blue color. Perform a blank
Detector: UV 254 nm determination, and make any necessary correction (see

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 USP43 OfficialMonographs / Benzoyl 527

Titrimetry (541». Each mL of 0.1 N sodium thiosulfate is Standard solution: 10 mL of Standard stock solution and 5
equivalent to 12.11 mg of anhydrous benzoyl peroxide mL of Internal standard solution. Dilute with acetonitrile to
(C'4H'004)' 25 mL. This Standard solution contains 0.32 mg/mL of
Sample solution: Transfer an amount of Gel equivalent to benzoyl peroxide.
·100 mg of benzoyl peroxide to a 50-mL volumetric flask, Sample stock solution: Transfer the equivalent to 40 mg of
and add 25 mL of acetonitrile. Shakevigorously to disperse benzoyl peroxide from Lotion in a 50-mL volumetric flask,
the specimen, sonicate for 5 min, dilute with acetonitrile to and add 40 mL of acetonitrile. Shake Vigorously until the
volume, and filter. material is thoroughly dispersed. Sonicate the mixture for
Chromatographic system 5 min, .dilute with acetonitrile to volume, mix, and filter.
(See Chromatography (621), System Suitability.) Sample solution: 10 mL of Sample stock solution and 5 mL
Mode: LC of Internal standard solution. Dilute with acetonitrile to 25
Detector: UV 235 nm mL.
Column: 4.6-mm x 25-cm; packing L1 Chromatographic system
Flow rate: 1.2 mL/min (See Chromatography (621), System Suitability.)
Injection volume: 10 IJL Mode: LC
System suitability Detector: UV 254 nm
Sample: System suitability solution Column: 3.9-mm x 30-cm; packing L1
Suitability requirements Flow rate: 1 mL/min
Resolution: NLT 2.0 between benzoic acid and Injection volume: 10 IJL
methylparaben System suitability
Tailing factor: NMT 2.0 for the benzoic acid and Sample: Standard solution (three replicate injections)
methylparaben peaks [NOTE-The retention times for ethyl benzoate and
Analysis benzoyl peroxide are 7 and 14 min, respectively.]
Samples: Standard solution and Sample solution Suitability requirements
Acceptance criteria: The responses of any peaks from the Resolution: NLT 2.0 between ethyl benzoate and
Sample solution corresponding to benzoic acid, ethyl benzoyl peroxide
benzoate, and benzaldehyde are NMT those of the main Tailing factor: NMT 2.0 for the ethyl benzoate and
peaks from Standard solution A (25%), Standard solution B benzoyl peroxide peaks
(1%), and Standard solution C (1%), respectively. The Peak response ratios: The lowest and highest peak
response of any other impurity peak from the Sample response ratios (Rs) agree within 2.0%.
solution-other than the main benzoyl peroxide peak, any Analysis
benzoic acid, ethyl benzoate, benzaldehyde, Samples: Standard solution and Sample solution
methylparaben, or propylparaben peak, and any solvent Calculate the percentage of the labeled amount of benzoyl
peak-is NMT that from Standard solution D (2%); and the peroxide (C14Hl004) in the portion of Lotion taken:
sum of the responses of all the impurity peaks-other than
those of benzoic acid, ethyl benzoate, and benzaldehyde- Result = (Ru/R s) x (CslC u) x 100
is NMT that from Standard solution D (2%).
SPECIFIC TESTS
Ru =peak response ratio of benzoyl peroxide to ethyl
benzoate from the Sample solution
• pH (791): 2.8-6.6 Rs = peak response ratio of benzoyl peroxide to ethyl
ADDITIONAL REQUIREMENTS . benzoate from the Standard solution
• PACKAGING AND STORAGE: Preserve in tight containers. Cs =concentration of benzoyl peroxide in the
Standard solution (mg/mL)
Cu =nominal concentration of benzoyl peroxide in the
Sample solution (mg/mL)
Benzoyl Peroxide Lotion Acceptance criteria: 90.0%-110.0%

DEFINITION IMPURITIES
• ORGANIC IMPURITIES
Benzoyl Peroxide Lotion is benzoyl peroxide in a suitable lotion
base. It contains NLT 90.0% and NMT 110.0% of the labeled Solution A: Acetonitrile and glacial acetic acid (1000:1)
Solution B: Glacial acetic acid and water (1:1000)
amount of benzoyl peroxide (C'4H'004)'
Mobile phase: See Table 1.
IDENTIFICATION
• A. The retention time of the major peak of the Sample Table 1
solution corresponds to that of the Standard solution, as Time Solution A Solution B
obtained in the Assay. (min) (%) (%)

ASSAY 0 18 82
• PROCEDURE 20 60 40
Mobile phase: Acetonitrile in water (5 in 10)
Internal standard solution: 3.6 mg/mL of ethyl benzoate 30 60 40
in acetonitrile
Standard stock solution: Transfer a suitable quantity of System suitability solution: 100 IJg/mL of benzoic acid and
benzoyl peroxide, recently subjected to the Assayunder 60 IJg/mL of methylparaben in acetonitrile
Hydrous Benzoyl Peroxide, in a weighed conical flask fitted Standard solution A: 500 IJg/mL of benzoic acid in
with a glass stopper. Weigh again to obtain the weight of acetonitrile
the specimen, and quantitatively dissolve in acetonitrile to Standard solution B: 20 IJg/mL of ethyl benzoate in
obtain a solution containing 0.8 mg/mL. acetonitrile

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 528 Benzoyl/Official Monographs USP43

Standard solution C: 20 IJg/mL of benzaldehyde in IOIENTlFI(ATIQN

acetonitrile
Standard solution D: Prepare a solution of hydrous benzoyl
peroxide, previously subjected to the Assay under Hydrous
Benzoyl Peroxide, in acetonitrile containing the equivalent
of 40 IJg/mL of anhydrous benzoyl peroxide.
Sample solution: Equivalent to 100 mg of benzoyl peroxide
from Lotion. In a 50-mL volumetric flask add 25 mL of
acetonitrile, and shake vigorously to disperse the specimen.
Sonicate for 5 min, dilute with acetonitrile to volume, mix,
and filter.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC RI:
Detector: UV 235 nm .01 Mmo-nobasic sodiurn-phos' , l~water
Column: 4.6-mm x 25-cm; packing L1 So A:,AtetonitrlIe arid Buffer(10:9
Flow rate: 1.2 mL/min Solu Ion B:,Acetonitrile and water (70:30)
Injection volume: 10 IJL Mobile phase: See Tablet.
System suitability
Sample: System suitability solution Table 1
Suitability requirements
Resolution: NLT 2.0 between benzoic acid and
Time
~mln)
Solu(ion.'A
(0/0>,
Sol~~~Q'&
methylparaben
0 100 0
Tailing factor: NMT 2.0 for the benzoic acid and
methylparaben peaks 5 100 0
Analysis
~o 70 30
Samples: Standard solution and Sample solution
Acceptance criteria: The responses of any peaks from the 35 60 40
Sample solution corresponding to benzoic acid, ethyl ,10
40 ~9,
benzoate, and benzaldehyde are NMT those of the main
peaks from Standardsolution A (25%), Standardsolution B 115 0 '100
(1%), and Standardsolution C (1%), respectively. The
47 100 ,0
response of any other impurity peak from the Sample
solution, other than the main benzoyl peroxide peak, any 55 100 0
benzoic acid, ethyl benzoate, benzaldehyde,
methylparaben, or propylparaben peak, and any solvent :50)
peak, is NMT that from Standardsolution D ,(2%); and the USP Benzphetiimlne
sum of the responses of all the impurity peaks, other than
those of benzoic acid, ethyl benzoate, and benzaldehyde,
is NMT that from Standardsolution D (2%). . mg/mL pf Benzphetamine
i1uent
SPECIFIC TESTS C " ,ra . system
• pH (791): 2.8-6.6 (See Chromatography (621), System SUitability.)
ADDITIONAL REQUIREMENTS Mode: LC
Detector: U 0'7 nm
• PACKAGING AND STORAGE: Preserve in tight containers.
-em; 5-lJm packing L1
0°

Add the followi"g:

C17H21 N . HCI 275.82

(+)-N~Ben I~N,:a~dinie ocl]forfde;:
(S)-N~~eI-N-methyl-
hydrocide [5411:-2:- • Result ::(ru/rs) x'(Cs/Cu) x 1ob
DEFINITION e,of benzphetamineJrom the
Benzphetamine Hyaroctll6riCie contaInS n
102.0% of benzphetami, ydrochlo = f beniphetamine from the
calculated on the'dried,b sis. n
nofUSPBenzphe e
ide RS in the Stand lution

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USP 43 OfficialMonographs / Benzphetamine 529

= concentration ofBEmzphetamine Hydrocnloride F =relative respon'se factor. of each individual

in th.e Sample solution (mg/mL) ImpLJrity (see Table 3)
Acc:eptcmce criteria: 98.00/0-1 02.00/0 on'the dried basi~ Acceptance criteri,,:-~See 'Table 3:
IMp' RillES 1:al1le3
• UE 0 ION (281): NMT02%
• 'NIC. TIES Relative Relative
Retention Response
Sol ,~SolutionBJand Dllueni::~Prepaie' as Name Time Fa.c~or
dire d i , , Assay.
Mobile phase: See Table 2. Pseudoephedrine
hydrochloride 0.22 0.62 0.10
Methamphetamine
hydrochloride 0.37 0.65 0.10
Time
(min)
Solution A
(%)
Sq!~~~!;i'>~
Berizyl,alcohol 0.57 1.18 0.10
0 100 0 Berizphetamine related
compound E 0.87 O.9j 0.10
5 100 0
Benzphetamine
30 70 30 hydrochloride LOO 1.00
35 60 40 , roe related
F - Lr1 p.50 0.10
45 55 4-$
60 1.5 85 ndividuC!1
LOO 0.10
62 100 0
Totalimpurities· 0.5
70 100 0
a Benzphetamine related compound A. monitoredin the EnantiomeriC Puritytest
is not includedin the total impurities.

S u er(50:50)
r (50:50) . . .
S gmL of USP Benzyl ChlorideRS
S

• UV 207 nm
6-m -cm; 5-lJm packing 11
empe. . . 30°
te: 1.0 mL/min
In. n volume: 20 IJL
Runtime: NLT 1.4 timesthe retentioritime of benzyl
chi .' e
billty _ .
dard solution
ion, . _ ., _ . ._ .' . equirements
,o-noise ratio: NlT 1O( Sensitivity solution or:· NMT 1.5
andard deviation: NMT 5:0%
e SlJIUf/On Ana yss , ' __ ' , . ,.
riM! in l~he-p()rtion of
Samples: Standardsolution and.Sample solution
Calculate the percentage of benzyl chloride in. the portion
of Benzphetamrn~ Hydrochloride taken:
Result = (rU/rS) X (CSICu) x ( 1/ F) x 10 q
Result= (rurrsrx:<C;~Cu)x:1 00
ru onse of each impUritY-fromthe.Sclrri'ple
ru = peaK respon~e ()fbE!nzyl chloride from the
r~ onse of.benzpnetamine from the. Sample solution. _. _ _.. _.,. .__
Ii s tiori rs = peak response of benzyl chloride froin the
f-USP Benzphetamine Standard solution
.H .. S in the Standard solution = conc;entrationofUSP B-enzyt ChloridecRS intQ~
(rng/ml) .' .' - _. _'. - , Std .
Cu = concentration of Benzphetamine' Hydrochloride =con amin~fHydrochloriae
in the Sample solution (mgfmL) in the Samp e ution(mg/mL)

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530 Benzphetamine / OfficialMonographs USP 43

min~

5~cm; 5~~mp·~ckil)g lBO

in
SIiL
titnesthe retentfontillleof

Result= (ru/rs) x(CiCu)x·' O:q

= peal<·
tom
= peak
comp
=~onc
Compoun
(mg/mL)
= concentration of Be
in the Sample so/uti
Acceptance criteria:. See Table 4<
Table 4
Relative
Retentio-"
Nam~ . Time
Benzphetamine LOO
Benzphetamine related
compound A 1.16 o·r~

. 20 mg!ml·of Beniphetamine
water
a: .4.0':~:O

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USP 43 Official Monographs / Benzphetamine 531

Corumn: 40.° Calculate the perceritageof the'Ii\llJeled ,amount of

Flow rate: 1.0 rnL/min benzph~tamine hyqrochloride (C17Hz1 N . HC!) dissolve_~:
Injection volume': ,10 IJL ._
Run time: NLT 2.1 times the Jetention time .of Result ~(ru/rs) x Cs >< Vx(l/L) x 100
, benzphetamine
System suitability ru =peak response of benzphetqmine from the
Sample: Standardsolution Sample -solution
Suitability requirements rs =peak response of benzphetaminefrom the
Tailing fador:NMT2.0 Standardsolution
Relative standard deviation: ,NMT2:0% 'Cs =' concen .of USP Benzphetamine .
Analysis . . - . - Hydroc RS in ,the Stimdard solution
Sa andard$ollitibn a )
percentage of th V
..e hydrochloride. L
lets taken: ..
LT80<Ml(Q) of the labeled amount of.
Result = (ru/rs)x(CsICu) x 100 'ne hydrochloride (C17Hz1N· HCI) is dissolved.

== peak response'of bel1zphetiunirle from th~ - DOSAGE'UNITS (905): Meet.the

Sample solution ,. _., , " , ' ,.

= peak respo'nse of benzphetamine from the IMPURITIES
-ORGANI " PURITIES
Standard solution
= concentration ofL1 Benzphetamine Sol' 1.38 9 of monobasiC sodium phosphate
HydrochlorideR' e Standard solution di e in 1000 mL of water
(mg/mL) Sol : Acetonitrile and water (80:20)
='nominal concentration of oenzphef Mobile phase: See Table 1.
hydrochloride in the Sample solutio. /mL) Table· 1
Acceptance criteria: 93.0%..;.105.0% Time Solution A SolutionB
(min) (0/0) (0/0)
0 80 .20
5 75 25
10 60 40
15 40 60
20 15 85
22 15 85
23 80 20
30 80 20

Diluent: Methanoland water (50:50)

System suitability solution: 0.002 mg/mL each ofUSP
Benzphetamine Hydrochloride RS, USP Benzphetamine
Related Compound E,RS,and USPMethamphetamine
HY,drochl.<>ride RS.in Diluent. Sonicate to. dissolve if
, ary.·
ity solution: 0;0001 mglmL of USP Benzpnetamine
hloride RSin Diluent
solution:0~002 mg/mL of USP Beniphetamine
loride RS in Diluent: Sonicate to dissolve if

-cm;'5-tJm packing'll S ution: N

ra u 5° ridein Oil
mL/min amount
e: ,10 pL Inelyp
.8 times the reteiltion time of abou
_ peine sha ,oiously" sperse ,let
Sy suitability for an additional NLT 60min with intermedia mg.
Sample: Standard solution Cool to room temperature and dilute'with Oil, to
Suitability tequir~ments . Pass a portion through a suitable filter with a.
Taili g faCtor: ,N 2.0,. _,_ ' _._ _... pore size~ Discardthe firstfew miIHliters offUtrClte.
ve-,standar ,viation:NMT2.0% graphic system
Ana ysis . " _ ,_, " " ' - . . ' _ . ,',. ,_ . matography(621), SystefrJS~itdbiIitY.)
Samples:- 'Standardsolutfonand 'Sample' solution : LC
De eetor:·UV 207 nrn

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532 Benzphetamine / OfficialMonographs USP43

Column: 4.6~mmx 2S,:,cm;5-!.Jmpackirigll

Te~perature~
Benztropine Mesylate
Autosa" '1 S°,
Calum .. _.
Flowr
Injecti
Sy~tem .
Sampl
Stan
Suitab , " irements
Re.solution: NLT3,0 b'etWeerrbe
C21H2S NO · CH403 S 403.53
compound E and benzphetami
8-Azabicyclo[3.2.1]octane, 3-(diphenylmethoxy)-N-methyl-,
solution
endo-, methanesulfonate;
Relative standard,aeviation: ·.NMT5;O%rStandard 3a-(Diphenylmethoxy)-1 aH,5aH-tropane methanesulfonate
solution . .
[132-17-2].
,~ignal-t~-noise ratio:. NLT1 0, SensitivitysoJutiorl
Analysis DEFINITION
Samples: StandardsolUtion ' pie solution Benztropine Mesylate contains NLT 98.0% and NMT 100.5%
Calculate the percentage of e purity in the portion of of benztropine mesylate (C21 H2S NO . CH4 0 3 S), calculated on
Tablets taken: . the dried basis.

~esult ~. (rulrs) x (C$I fu) ~.(] l F) .>5IfJ9

IDENTIFICATION

=peak resporis~ ofeadlirriP~rityfr9·mtne·Scjrnple

solution • A. ·IONtESTS:(l97)~· fnfrared
= peak
i

response ofbenzph eta mille frc>m the

Standard solution Spec • 20)

=concentration amine ASSAY

Hydrochloride rdsQlution • PROCEDURE
. (mg/mL). Sample: 60 mg of Benztropine Mesylate
Cu == no . I c Analysis: Dissolve the Sample in 25 mL of water, add 5 mL
hy ,/0 of sodium carbonate TS, and extract with four 10-mL
F =rela Ive re portions of chloroform. Wash the combined chloroform
impurity (see extracts with about 10 mL of water, and extract the wash
solution with 5 mL of chloroform. Filter the combined
Acceptance criteria: .See·Table 2. chloroform extracts through a tightly packed pledget of
cotton, and wash the cotton with about 5 mL of
Table 2 chloroform. Add methyl red TS, and titrate the chloroform
Relative Relative Acceptance solution with 0.01 N perchloric acid in dioxane VS. Perform
Retentioii Response <i:riteria, a blank determination, and make any necessary correction
Name Time Factor NMT(%) (see Titrimetry (541 »). Each mL of 0.01 N perchloric acid is
equivalent to 4.035 mg of benztropine mesylate (C 21 H2S NO
Methamphetamine
hydrochloride 0,35 0:68 O.fO . CH403S).
Acceptance criteria: 98.0%-100.5% on the dried basis
Benzphet'llnine related.
compoundE 0.90 0.93 (j.J~ IMPURITIES
Benzphetamine • RESIDUE ON IGNITION (281): NMT 0.1 %
hydrochloride 1.00 1.00 -
SPECIFIC TESTS
Anyunspecified • MELTING RANGE OR TEMPERATURE (741): 141°-148°
~
impurity 1;()0 0.10 • Loss ON DRYING (731)
Analysis: Dry a sample at 105° for 2 h.
Totalimpurities - - 1.0
Acceptance criteria: NMT 5.0%
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers.
• USP REFERENCE STANDARDS (11)
USP Benztropine Mesylate RS

C
Benztropine Mesylate Injection
asp Meth' e RS DEFINITION
(5)-"N-Meth ine hydrochlorfde. Benztropine Mesylate Injection is a sterile solution of
C,oH 1SN· HCI 19) Benztropine Mesylate in Water for Injection. It contains NLT
90.0% and NMT 110.0% of the labeled amount of
benztropine mesylate (C21 H2S NO . CH403 S).

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USP 43 OfficialMonographs / Benztropine 533

IDENTIFICATION rs = peak response from the Standardsolution

• A. Cs = concentration of USP Benztropine Mesylate RS in
Standard stock solution: 0.2 mg/mL of USP Benztropine the Standardsolution (mg/mL)
Mesylate RS Cu = nominal concentration of benztropine mesylate
Standard solution: In a separator containing the Standard in the Sample solution (mg/mL)
stock solution add 2 mL of 1 N sodium hydroxide. Extract
with three 1O-mL portions of chloroform, collecting the Acceptance criteria: 90.0%-110.0%
chloroform extracts to a 50-mL beaker. Evaporate the
chloroform extracts with the aid of gentle heat and a SPECIFIC TESTS
current of air to dryness, and dissolve the residue in 1 mL • BACTERIAL ENDOTOXINS TEST (85): NMT 55.6 USP
of chloroform. Endotoxin Units/mg of benztropine mesylate
Sample stock solution: Dilute a volume of Injection, • pH (791): 5.0-8.0
equivalent to 10 mg of benztropine mesylate, in a separator • OTHER REQUIREMENTS: Meets the requirements in Injections
to 50 mL with water (0.2 mg/mL). and Implanted Drug Products (1)
Sample solution: In a separator containing the Sample stock ADDITIONAL REQUIREMENTS
solution add 2 mL of 1 N sodium hydroxide. Extract with • PACKAGING AND STORAGE: Preserve in single-dose or in
three 1O-mL portions of chloroform, collecting the multiple-dose containers, preferably of Type I glass.
chloroform extracts to a 50-mL beaker. Evaporate the • USP REFERENCE STANDARDS (11)
chloroform extracts with the aid of gentle heat and a USP Benztropine Mesylate RS
current of air to dryness, and dissolve the residue in 1 mL
of chloroform.
Chromatographic system
Adsorbent: 0.25-mm layer of chromatographic silica gel
Application volume: 1 IJL Benztropine Mesylate Tablets
Developing solvent system: Chloroform, methanol, and
a 1-in-4 solution of ammonium hydroxide (40:10:1) DEFINITION
Analysis Benztropine Mesylate Tablets contain NLT90.0% and NMT
Samples: Standardsolution and Sample solution 110.0% of the labeled amount of benztropine mesylate
Allow the applications to dry, and develop the (C2l H2S NO . CH403S).
chromatogram in the Developing solventsystem until the
solvent front has moved about three-fourths of the length IDENTIFICATION
of the plate. Remove the plate from the developing • A.
chamber, mark the solvent front, and allow the solvent to Standard stock solution: 0.2 mg/mL of USP Benztropine
evaporate. Locate the spots on the plate by lightly Mesylate RS
spraying with potassium iodoplatinate TS. Standard solution: In a separator containing the Standard
Acceptance criteria: The R F value of the principal spot of stock solution add 2 mL of 1 N sodium hydroxide. Extract
the Sample solution corresponds to that of the Standard with three 1O-mL portions of chloroform, collecting the
solution. chloroform extracts to a 50-mL beaker. Evaporate the
chloroform extracts with the aid of gentle heat and a
ASSAY current of air to dryness, and dissolve the residue in 1 mL
• PROCEDURE of chloroform.
Buffer: Transfer 0.83 mL of octylamine to a l-L volumetric Sample stock solution: Dissolve a portion of finely
flask, dilute with water to volume, and adjust with powdered Tablets, equivalent to 10 mg of benztropine
phosphoric acid to a pH of 3.0. mesylate, in 50 mL of water, shake by mechanical means
Mobile phase: Acetonitrile and Buffer (65: 35) for 30 min, and filter into a separator (0.2 mg/mL).
Standard solution: 1 mg/mL of USP Benztropine Mesylate Sample solution: In a separator containing the Sample stock
RS solution add 2 mL of 1 N sodium hydroxide. Extract with
Sample solution: Nominally 1 mg/mL of benztropine three 1O-mL portions of chloroform, collecting the
mesylate from the volume of Injection chloroform extracts to a 50-mL beaker. Evaporate the
Chromatographic system chloroform extracts with the aid of gentle heat and a
(See Chromatography (621), System Suitability.) current of air to dryness, and dissolve the residue in 1 mL
Mode: LC of chloroform.
Detector: UV 259 nm Chromatographic system
Column: 4.6-mm x 25-cm; packing L7 Adsorbent: 0.25-mm layer of chromatographic silica gel
Flow rate: 1.3 mL/min adjusted, as needed, to obtain a Application volume: 1 IJL
retention time of 7 min for benztropine mesylate Developing solvent system: Chloroform, methanol, and
Injection volume: 25 IJL a 1-in-4 solution of ammonium hydroxide (40:10:1)
System suitability Analysis
Sample: Standardsolution Samples: Standardsolution and Sample solution
Suitability requirements Allow the applications to dry, and develop the
Relative standard deviation: NMT 2.0% chromatogram in the Developing solventsystem until the
Analysis solvent front has moved about three-fourths of the length
Samples: Standardsolution and Sample solution of the plate. Remove the plate from the developing
Calculate the percentage of labeled amount of benztropine chamber, mark the solvent front, and allow the solvent to
mesylate (C2l H2sNO . CH4 0 3S) in each mLof the Injection: evaporate. Locate the spots on the plate by lightly
spraying with potassium iodoplatinate TS.
Result = (r vir s) x (C siC v) x 100 Acceptance criteria: The RF value of the principal spot of the
Sample solution corresponds to that of the Standard
ru = peak response from the Sample solution solution.

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534 Benztropine / OfficialMonographs USP 43

ASSAY Injection volume: 300 ~L

• PROCEDURE System suitability
Buffer: Transfer 0.83 mL of octylamine to a 1-L volumetric Sample: Standard solution
flask, dilute with water to volume, and adjust with Suitability requirements
phosphoric acid to a pH of 3.0. Tailing factor: NMT 2.0
Diluent: Isopropyl alcohol, water, and phosphoric acid (40: Relative standard deviation: NMT 3.0%
60: 0.1) Analysis
Mobile phase: Acetonitrile and Buffer(45:55) c Samples: Standardsolution and Sample solution
Standard solution: 0.04 mg/mL of USP Benztropine Calculate the percentage of the labeled amount of
Mesylate RS in Diluent benztropine mesylate (C21H2SNO . CH40 3S) dissolved:
Sample solution: Nominally 0.04 mg/mL of benztropine
mesylate from a suitable amount of powdered Tablets in Result = (rvlrs) x Cs x V x (lIL) x 100
Diluent prepared as follows. Add a suitable amount of fine
powder from NLT 20 Tablets to a portion of Diluent tu = peak response from the Sample solution
corresponding to 60% of the final volume. Mix by rs = peak response from the Standardsolution
mechanical means for NLT 60 min, and dilute with Diluent Cs = concentration of USP Benztropine Mesylate RS in
to volume. Centrifuge a portion of this mixture, and filter the Standardsolution (mg/mL)
the supernatant layer. V = volume of the Medium, 900 mL
Chromatographic system L = label claim (mg/Tablet)
(See Chromatography (621), System Suitability.)
Mode: LC Acceptance criteria: NLT 80% (Q) of the labeled amount of
Detector: UV 259 nm benztropine mesylate (C21H2SNO . CH40 3S) is dissolved.
Column: 4.6-mm x 25-cm; packing L7 • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Flow rate: 0.7 mL/min requirements
Injection volume: 50 ~L
System suitability ADDITIONAL REQUIREMENTS
Sample: Standardsolution • PACKAGING AND STORAGE: Preserve in well-closed
Suitability requirements containers.
Tailing factor: NMT 4.0 • USP REFERENCE STANDARDS (11)
Relative standard deviation: NMT 2.0% USP Benztropine Mesylate RS
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of
benztropine mesylate (C21H2SNO . CH40 3S) in the portion
of Tablets taken:
Benzyl Benzoate
Result = (rufrs) x (CsICv) x 100

tu = peak responsefrom the Sample solution

ts = peak response from the Standardsolution
Cs =concentration of USP Benztropine Mesylate RS in C14H1202 212.24
the Standardsolution (mg/mL) . Benzoic acid, phenyl methyl ester;
Cu = nominal concentration of benztropine mesylate Benzyl benzoate [120~51-4].
in the Sample solution (mg/mL) DEFINITION
Benzyl Benzoate contains NLT 99.0% and NMT 100.5% of
Acceptance criteria: 90.0%-110.0% C14H1202'
PERFORMANCE TESTS IDENTIFICATION
• DISSOLUTION (711)
Medium: 0.1 N hydrochloric acid; 900 mL
Apparatus 2: 50 rpm
Time: 30 min
Determine the amount of benztropine mesylate (C21H2SNO .
CH40 3S) dissolved by using the following method.
Buffer: Transfer 0.83 mL of octylamine to a 1-L volumetric ASSAY
flask, dilute to volume, and adjust with phosphoric acid to • PROCEDURE
a pH of 3.0. Sample: 2 g of Benzyl Benzoate
Mobile phase: Acetonitrile and Buffer (65:35) Analysis: Transfer the Sample to a conical flask fitted with a
Standard solution: USP Benztropine Mesylate RS in reflux condenser. Add 50.0 mL of 0.5 N alcoholic potassium
Medium. Dilute to obtain a solution having a known hydroxide VS, and boil gently for 1 h. Cool, add
concentration similar to that of the Sample solution. phenolphthalein TS, and titrate with 0.5 N hydrochloric
Sample solution: Useafiltered portion of the solution under acid VS. Perform a blank determination (see Titrimetry
test from the dissolution vessel. (541». Each mL of 0.5 N alcoholic potassium hydroxide is
Chromatographic system equivalent to 106.1 mg of Benzyl Benzoate (C14H1202)'
(See Chromatography (621), System Suitability.) Acceptance criteria: 99.0%-100.5%
Mode: LC IMPURITIES
Detector: UV 220 nm
• LIMIT OF ALDEHYDES
Column: 4.6-mm x 25-cm; packing L7 Sample solution: Transfer 10.0 g to a 125-mL conical flask
Flow rate: 2 mL/min containing 50 mL of alcohol and 5 mL of hydroxylamine

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 USP 43 Official Monographs / Benzylpenicilloyl 535

hydrochloride solution (3.5 in 100), mix, and allow to stand Back-titrant. Perform a blank determination. Each mL of 0.5
for 10 min. N alcoholic potassium hydroxide is equivalent to 106.1 mg
Analysis: Add 1 mL of bromophenol blue TS, and titrate of benzyl benzoate (C14H1202).
with 0.1 N sodium hydroxide VS to a light green endpoint. Acceptance criteria: 26.0%-30.0% (w/w)
Perform a blank determination, and match the color of the
endpoint with that of the titrated Sample solution. SPECIFICTESTS
Acceptance criteria: The net volume of 0.1 N sodium • pH (791): 8.5-9.2
hydroxide consumed does not exceed 0.50 mL (0.05% as ADDITIONAL REQUIREMENTS
benzaldehyde). • PACKAGING AND STORAGE: Package in tight containers.
SPECIFICTESTS
• SPECIFIC GRAVITY (841): 1.116-1.120
• CONGEALING TEMPERATURE (651): Congelation may be
brought about by the addition of a fragment of previously
congealed Benzyl Benzoate when the temperature has Benzylpenicilloyl Polylysine
reached the expected congealing temperature. Concentrate
Acceptance criteria: NLT 18.0°
• REFRACTIVE INDEX (831): 1.568-1.570 at 20°
• ACIDITY: Add 2 drops of phenolphthalein TS to 25 mL of » Benzylpenicilloyl Polylysine Concentrate has a
alcohol, and add 0.020 N sodium hydroxide until a pink molar concentration of benzylpenicilloyl moiety
color is produced. Add 5.0 g of Benzyl Benzoate, and titrate (C16H19N20SS) of not less than 0.0125 M and not
with 0.020 N sodium hydroxide.
Acceptance criteria: NMT 1.5 mL of 0.020 N sodium more than 0.020 M. It contains one or more
hydroxide is required to restore the pink color. suitable buffers.
ADDITIONAL REQUIREMENTS Packaging and storage-Preserve in tight containers.
• PACKAGING AND STORAGE: Preserve in tight, well-filled, Labeling-The label states that this article is not intended for
light-resistant containers, and avoid exposure to excessive direct administration to humans or animals.
heat. VSP Reference standards (11 )-
• USP REFERENCE STANDARDS (11) USP L-Lysine Hydrochloride RS
USP Benzyl Benzoate RS pH (791): between 6.5 and 8.5, the undiluted Concentrate
being used.
Limit of penicillenate and penamaldate-Transfer 1 mL
of Concentrate to a 50-mL volumetric flask, dilute with Saline
phosphate buffer, prepared as directed in the Assay, to volume,
Benzyl Benzoate Lotion and mix. Using a suitable spectrophotometer and using Saline
phosphate buffer as a blank, determine the absorbances at the
DEFINITION
wavelengths of maximum absorption at about 322 nm and
Benzyl Benzoate Lotion contains NLT 26.0% and NMT 30.0%
282 nm. Calculate the molar concentration of penicillenate
(w/w) of benzyl benzoate (C14H1202)' taken by the formula:

Benzyl Benzoate 250 mL 50A 322/26,600b

Triethanolamine 5g
in which A322 is the absorbance at 322 nm, 26,600 is the molar
Oleic Acid 20g absorptivity of the penicillenate moiety at pH 7.6, and b is the
Purified Water 750 mL length of the cell, in cm: not more than 0.00020 M is found.
Calculate the molar concentration of penamaldate taken by
To make about 1000 mL the formula:

Mix the Triethanolamine with the OleicAcid, add the Benzyl 50A 282/22,325b
Benzoate, and mix. Transfer the mixture to a suitable
container of 2000-mL capacity, add 250 mL of Purified in which A282 is the absorbance at 282 nm, 22,325 is the molar
Water, and shakethe mixture thoroughly. Add the remaining absorptivity of the penamaldate moiety at pH 7.6, and b isthe
Purified Water, and again shake thoroughly. length of the cell, in cm: not more than 0.00060 M is found.
ASSAY Benzylpenicilloyl substitution-
• PROCEDURE Citratebuffer-Dissolve 19.69 g of sodium citrate dihydrate,
Sample: 5 g of Lotion, accurately weighed, in a conical flask 0.1 mL of pentachlorophenol, and 5 mL of 2,2'-thiodiethanol
Titrimetric system in 900 mL of 0.2 N hydrochloric acid, adjust with hydrochloric
(See Titrimetry (541 ).) acid to a pH of 2.2, dilute with water to 1000 mL, and mix.
Mode: Residual titration Ninhydrin reagent-Dissolve 18 g of ninhydrin and 0.7 g of
Titrant: 0.1 N sodium hydroxide VS hydrindantin in 675 mL of dimethyl sulfoxide, add 225 mL of
Back-titrant: 0.5 N hydrochloric acid VS 4 M lithium acetate solution previously adjusted with glacial
Endpoint detection: Visual acetic acid to a pH of 5.2, and mix.
Analysis: To the Sample add 25 mL of alcohol and 2 drops Standard preparation-Dissolve an accurately weighed
of phenolphthalein TS. Cool the solution to 15°, and titrate quantity of USP L-Lysine Hydrochloride RS in Citratebufferto
quickly with Titrant to a slight pink color. Add 50.0 mL of obtain a solution having a known concentration of about 91
0.5 N alcoholic potassium hydroxide VS, connect the flask I-Ig per mL (5 x 10- 4 M).
to a reflux condenser, and boil gently for 1 h. Cool. Test preparation-Transfer 1.0 mL of Concentrate to a
Promptly add phenolphthalein TS and titrate with 1O-mL volumetric flask, dilute with water to volume, and mix.

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 536 Benzylpenicilloyl / Official Monographs USP 43

Transfer 1.0 mL of this solution to an ampul, add 1.5 mL of 6 in which Am is the highest absorbance observed; AI is the initial
N hydrochloric acid, and seal the ampul under nitrogen. Heat absorbance, n is the number of 0.02-mL portions of Mercuric
the ampul at 110° for 22 hours. Transfer the contents of the chloride solution added to the Assay preparation to obtain the
ampul to a round-bottom, 50-mL flask, and dry by vacuum maximum absorbance; 22,325 is the molar absorptivity of the
rotary evaporation. Dissolve the residue three times, using penamaldate formed by the reaction of benzylpenicilloyl with
5-mL portions of water, evaporating to dryness after each mercuric chloride at pH 7.6; and b is the length of the cell, in
dissolution. Dissolve the residue in 10 mL of Citratebuffer. cm: between 0.0125 M and 0.020 M is found.
Chromatographic system (see Chromatography (621 »- The
liquid chromatograph is equipped with a 1.75-mm x 50-cm
column that contains a packing of 8-lJm 8% cross-linked
sulfonated divinylbenzene polystyrene cation-exchange resin.
The column effluent is mixed continuously with flowing Benzylpenicilloyl Polylysine Injection
Ninhydrin reagent, and the flOWing mixture is heated at 130°
for 1.5 minutes in a reaction coil. The absorbance of the
reaction mixture is measured continuously by a 570-nm » Benzylpenicilloyl Polylysine Injection has a molar
detector. Chromatograph the Standardpreparation, and concentration of benzylpenicilloyl moiety
record the peak responses as directed for Procedure: the (C16N2H1905S) of not less than 5.4 x 10- 5 M and not
column efficiency determined from the analyte peak is not less
than 1800 theoretical plates, and the relative standard
more than 7.0 x 10-5 M. It contains one or more
deviation for replicate injections is not more than 4.0%. suitable buffers.
Procedure-Separately inject equal volumes (about 20 IJL)
of the Standard preparation and the Test preparation into the Packaging and storage-Preserve in single-dose or in
chromatograph, record the chromatograms, and measure the multiple-dose containers, preferably of Type I glass, in a
responses for the major peaks. The retention time is about 57 refrigerator.
minutes for L-Iysine. Calculate the molar concentration of Bacterial Endotoxins Test (85) -It contains not more than
lysine in the Concentrate taken by the formula: 5833.0 USP Endotoxin Units per mL.
Sterility Tests (71) -It meets the requirements when tested
(0.1C/182.65)(ru/rs) -as directed for Membrane Filtration under Test for Sterilityof the
Product to be Examined.
in which C is the concentration, in IJg per mL, of USP L-Lysine pH (791): between 6.5 and 8.5.
Hydrochloride RS in the Standard preparation: 182.65 is the Assay-
molecular weight of anhydrous lysine hydrochloride; and ru Saline phosphatebuffer and Mercuric chloride solution-
and rs are the peak responses obtained from the Test Prepare as directed in the Assay- under Benzylpenicilloyl
preparation and the Standardpreparation, respectively. Polylysine Concentrate.
Calculate the percentage of benzylpenicilloyl substitution Assay preparation-Combine the contents of a sufficient
taken by the formula: number of containers to obtain not less than 3 mL of Injection.
Transfer 3.0 mL of Injection to a 1O-mL volumetric flask,
100(B/L) dilute with Saline phosphate buffer to volume, and mix.
Procedure-Proceed as-directed for Procedure in the Assay
in which B is the molar concentration of benzylpenicilloyl - under Benzylpenicilloyl Polylysine Concentrate. Calculate the
moiety in the Concentrate, as determined in the AssaYi and L molar concentration of benzylpenicilloyl moiety in the
is the molar concentration of lysine in the Concentrate: not Injection taken by the formula:
less than 50% and not more than 70% is found.
Assay- (10/3){[A m (3 + 0.02n)/3] - A I }/22,325b
Saline phosphate buffer-Dissolve 9 g of sodium chloride
and 1.38 g of monobasic sodium phosphate in 900 mL of in which the terms are as defined therein.
water, adjust with 5 N sodium hydroxide or phosphoric acid
to a pH of 7.6, dilute with water to 1000 mL, and mix.
Mercuric chloride solution-Dissolve 35 mg of mercuric
chloride in 500 mL of water, and mix.
Assay preparation-Transfer 1.0 mL of Concentrate to a Beta Carotene
500-mL volumetric flask, dilute with Saline phosphate buffer to H,C
H, CH,
volume, and mix. H,C CH,

Procedure-Transfer 3.0 mL of Assay preparation to a

spectrophotometric cell. Using a suitable spectrophotometer CH3 CH, H,C CH,
CH,
and using Saline phosphate buffer as the blank, determine the
initial absorbance at the wavelength of maximum absorbance C4oH s6 536.87
at about 282 nm. Add 0.02 mL of Mercuric chloride solution p,p-Carotene;
to the Assay preparation in the spectrophotometric cell, mix, all-trans-p-Carotene;
and determine the absorbance at the same wavelength after (all-E)-l,l'-(3,7, 12, 16-Tetramethyl-l,3,5,7,9,ll,13, 15,17-
1 and 3 minutes. Repeat the addition of 0.02-mL portions of octadecanonaene-l,18-diyl)bis[2,6,6-
Mercuric chloride solution until a maximum absorbance reading trimethylcyclohexene] [7235-40-7].
is obtained. Calculate the molar concentration of
benzylpenicilloyl moiety in the Concentrate taken by the DEFINITION
formula: Beta Carotene contains NLT 96.0% and NMT 101.0% of total
carotenoids calculated as beta carotene (C4oHs6) . It contains
500{[Am(3 + 0.02n)/3] - A;}/22,325b NLT 95% of all-trans-beta carotene in the total carotenoids
content.

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USP 43 Official Monographs / Beta Carotene 537

IDENTIfICATION Column: 4.6-mm x 25-cm; 5-lJm packing L68

• A. Column temperature: 30°
Sample solution: Prepare as directed in the Sample solution Flow rate: 0.6 mL/min
in the test for Contentof Total Carotenoids. Injection volume: 20 IJL
Analysis: Record the UV-Vis spectrum from 300-600 nrn. System suitability
Acceptance criteria: The Sample solution shows a shoulder Samples: System suitability solution and Standardsolution
at about 427 nm, an absorption maximum at about 455 The approximate relative retention times of the
nm, and another maximum at about 483 nm. The components in the System suitability solution are listed in
absorbance ratio A4ss1A483 is between 1.14 and 1.18. Table 7.
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as Table 1
obtained in the test for Content of Beta Carotene. Relative Relative
Retention Response
COMPOSITION Analyte Time Factor
• CONTENT OF TOTAL CAROTENOIDS
[NOTE-Use low-actinic glassware.] all-trans-Alpha carotene 0.93 1.0
Sample stock solution: 0.1 mg/mL of Beta Carotene in all-trans-Beta carotene 1.00 1.0
tetrahyd rofuran
Sample solution: Transfer 3.0 mLof Sample stocksolution to 9-cis-Beta carotene 1.07 1.0
a 1OO-mL volumetric flask, and dilute with cyclohexane to 1 3-cis-Beta carotene 1.17 1.2
volume.
15-cis-Beta carotene 1.21 1.4
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).)
Analytical wavelength: 456 nm Suitability requirements
Cell path: 1 cm Chromatogram similarity: The chromatogram from the
Blank: Cyclohexane System suitability solution is similar to the reference
Analysis chromatogram provided with the lot of USP Beta
Sample: Sample solution _ Carotene System Suitability RS being used.
Calculate the percentage of total carotenoids (T) as beta Resolution: NLT 1.5 between all-trans-beta carotene and
carotene (C4oHs6) : all-trans-alpha carotene; NLT 1.2 between all-trans-beta
carotene and 9-cis-beta carotene, System suitability
T= AI(Fx C) solution
Tailing factor: NMT 2.0 for the all-trans-beta carotene
A = absorbance of the Sample solution peak, Standardsolution
F = 2505, coefficient of extinction (E 1%) of pure Relative standard deviation: NMT 2.0% for the
all-trans-beta carotene in cyclohexane (100 mL . all-trans-beta carotene peak from replicate injections,
g-l . crrr') Standardsolution
C = concentration of the Sample solution (g/mL) Analysis
Sample: Sample solution
Acceptance criteria: 96.0%-101.0% of total carotenoids as Record the chromatograms, and identify the peaks of the
beta carotene (C4oH s6) relevant analytes of the Sample solution by comparing
• CONTENT OF BETA CAROTENE with those of the System suitability solution. Measure the
[NOTE-Use low-actinic glassware.] peak area responses.
Mobile phase: Transfer 50 mg of butylated hydroxytoluene Calculate the percentage of all-trans-beta carotene relative
to a l-L volumetric flask, and dissolve with 20 mL of to total carotenoids in the sample taken:
2-propanol. Add 0.2 mLof N-ethyldiisopropylamine, 25 mL
of 0.2% ammonium acetate solution, 455 mL of Result = (r vir T) x 100
acetonitrile, and about 450 mL of methanol. Allow the
solution to reach room temperature, and dilute with ru = peak area of all-trans-beta carotene from the
methanol to volume. Sample solution
Diluent: 50 IJg/mL of butylated hydroxy toluene in alcohol rT = [(peak area of all-trans-alpha carotene x 1.0) +
System suitability solution: Transfer 20 mg of USP Beta (peak area of all-trans-beta carotene) + (peak area
Carotene System Suitability RS to a 50-mL volumetric flask. of 9-cis-beta carotene) + (peak area of 13-cis-beta
Add 1 mL of water and 4 mL of tetrahydrofuran, and carotene x 1.2) + (peak area of 15-cis-beta
sonicate for 5 min. Dilute with Diluent to volume, and carotene x 1.4) + (sum of peak areas of other
sonicate for 5 min. Cool to room temperature, pass the cis-isomers of beta carotene)] from the Sample
suspension through a membrane filter of 0.45-lJm pore solution
size, and use the clear filtrate.
Standard solution: 10 IJg/mL of USP Beta Carotene RS in Acceptance criteria: NLT 95% of aJl-trans-beta carotene in
tetrahydrofuran and Diluent (1:9). Dissolve an appropriate the total carotenoids content
amount of USP Beta Carotene RS in a volumetric flask first • ALPHA CAROTENE AND OTHER RELATED COMPOUNDS
with tetrahydrofuran, using 10% of the volume of the flask, Mobile phase, System suitability solution, Standard
then dilute with Diluent to volume. solution, Sample solution, and Chromatographic
Sample solution: Dilute the freshly prepared Sample stock system: Proceed as directed in the test for Content of Beta
solution as prepared in the test for Content of Total Carotene.
Carotenoids (1 inl 0) with Diluent. Analysis
Chromatographic system Sample: Sample solution
(See Chromatography(621), System Suitability.) Calculate the percentage of alpha carotene and other
Mode: LC individual related compounds relative to total carotenoids
Detector: UV 448 nm in the portion of the Sample taken:

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538 Beta Carotene / Official Monographs USP43

Result = (r vir r) x 100 ASSAY

ru = (peak area of all-trans-alpha carotene x 1.0) or
(peak area response of other individual related
compounds x appropriate relative response • CONTENT OF TOTAL BETA CAROTENE
factor, Table 7) in the Sample solution [NOTE-Use low-actinic glassware.]
rT = [(peak area of all-trans-alpha carotene x 1.0) + Mobile phase: Transfer 50 mg of butylated hydroxy toluene
(peak area of all-trans-beta carotene) + (peak area into a I-L volumetric flask, and dissolve with 20 mL of
of 9-cis-beta carotene) + (peak area of 1 3-cis-beta 2-propanol. Add 0.2 mL of N-ethyldiisopropylamine, 25
carotene x 1.2) + (peak area of 15-cis-beta mL of 0.2% ammonium acetate solution, 455 mL of
carotene x 1.4) + (sum of peak areas of other acetonitrile, and about 450 mL of methanol. Allow the
cis-isomers of beta carotene)] from the Sample solution to reach room temperature, and dilute with
solution methanol to volume.
Diluent: 50 mg/L of butylated hydroxy toluene in alcohol
Acceptance criteria System suitability solution: Transfer 20 mg of USP Beta
Alpha carotene: NMT 1.0% Carotene System Suitability RS to a 50-mL volumetric flask.
Total related compounds (including alpha carotene): Add 1 mL of water and 4 mL of tetrahydrofuran, and
NMT5% sonicate for 5 min. Dilute with Diluent to volume, and
sonicate for 5 min. Cool to room temperature, pass through
IMPURITIES a membrane filter of 0.45-J,lm pore size, and use the clear
• RESIDUE ON IGNITION (281): NMT 0.2%, 2 g of specimen filtrate.
being used Standard stock solution: 60 J,lg/mL of USP Beta Carotene
SPECIFIC TESTS RS in tetrahydrofuran. [NOTE-The USP Beta Carotene RS is
• Loss ON DRYING (731) subjected to the spectrophotometric purity test at the time
Analysis: Dry under vacuum over phosphorus pentoxide at of analysis; see the determination of the concentration of
40° for 4 h. Standard solution A below.]
Acceptance criteria: NMT 0.2% Standard solution A: Transfer 5.0 mL of the Standard stock
solution into a 1OO-mL volumetric flask, add 5.0 mL of
ADDITIONAL REQUIREMENTS tetrahydrofuran, and dilute with Diluent to volume.
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Determine the concentration of Standard solution A
containers. according to the Analysis of Standard solution B.
• USP REFERENCE STANDARDS (11) [NOTE-The concentration of Standard solution B equals
USP Beta Carotene RS the concentration of Standard solution A]
(all-E)-l,l '-(3',7,12, 16-Tetramethyl- Standard solution B: Transfer 5.0 mL of the Standard stock
1,3,5,7,9,11,13,15,17-octadecarionaene-l,18- diyl) solution into a 1OO-mL volumetric flask, and dilute with
bis[2,6,6-trimethylcyclohexene]. cyclohexane to volume. Prepare in triplicate.
USP Beta Carotene System Suitability RS Instrumental conditions
(See Ultraviolet- Visible Spect~o~?~eX"~.~~.~~'?><.Y.7
Analytical wavelength:~.~-?Q>nm"(I.JSP;1SMaYlz()f~)
Cell: 1 cm
Blank: Cyclohexane
Beta Carotene Capsules Analysis
DEFINITION Sample: Standard solution B
Calculate the concentration of total beta carotene (J,lg/mL)
as all-trans-beta carotene (C4oHs6) in Standard solution B:

Beta Carotene Capsules contain NLT Result =(Au!a) x F

and NMT 125.0% of the labeled
carotene Au = average absorbance of the three preparations of
Standard solution B
a = absorptivity ?~;;~~.~~<~"t~;f~~~~-beta carotene in
IDENTIFICATION cyciohexane~"'IlJ~P1H0~Y;2()j"~), 250
• A. F =conversion factor, 1000 J,lg/mg
Sample solution: Dilute the Sample stock solution from the
test for Content of Total Beta Carotene with cyclohexane to Sample stock solution: Randomly select a number of
a final concentration of 1-5 J,lg/mL of beta carotene. Pass Capsules, equivalent to 10-50 mg of beta carotene, with a
through a membrane filter of 0.45-J,lm pore size. total weight not exceeding 5 g. For powder-containing
Analysis: Record the UV-Vis spectrum from 300 to 600 nm. Capsules, empty the shell, and transfer shell and contents
Acceptance criteria: The Sample solution shows a shoulder into a 250-mL volumetric flask. For Capsules containing
at about 427 nm, an absorption maximum at about 455 liquid formulations, place the Capsules directly into a
nm, and another maximum at about 483 nm. The 250-mL volumetric flask. Add 250 rnq of butylated
absorbance ratio A4ss/~83 is between 1.14 and 1.18. hydroxy toluene, 0.5 mL of alkaline protease R, and 15 mL
• B. The retention time of the major peak of the Sample of water. Swirl the flask gently to wet the entire contents.
solution corresponds to that of the Standard solution, as Sonicate the flask at 50° for 30 min, and swirl the flask every
obtained in the test for Content of Total Beta Carotene. 10 min. Add 100 mL of alcohol to the warm suspension,
and shake Vigorously. Add 110 mL of methylene chloride,
and shake vigorously again. Disperse any clumps with a
homogenizer, and rinse the homogenizer probe with 15
mL of methylene chloride into the flask. Allow the solution

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USP 43 OfficialMonographs / Beta Carotene 539

to stand in the dark until it reaches room temperature Calculatethe percentage of all-trans-beta carotene in the
(about 2 h), dilute with methylene chloride to volume, portion of Capsules taken:
shake vigorously, and allow the solidsto settle.
Sample solution: Dilute a volume of the Sample stock Result = (rall.trans/Zru) x 100
solution with a Diluent-methylenechloride mixture (1 :1) so
that the final concentration of beta carotene is 1-5 ~g/mL. rall.trans = peak area of all-trans-beta carotene from the
Pass through a membrane filter of 0.45-~m pore size. Sample solution
Chromatographic system ru = (peak area of all-trans-beta carotene) + (peak area
(See Chromatography (621), System Suitability.) of 9-cis-betacarotene) + (peak area of 13-cis-beta
Mode: LC carotene x 1.2) + (peak area of 15-cis-beta
Detector: UV 448 nm carotene x 1.4) from the Sample solution
Column: 4.6-mm x 25-cmi 5-~m packing L68
Column temperature: 30° Acceptance criteria: .~Q,9.()o/O~;i<U~~lE~~Yj~~1~j-125.0% of the
Flow rate: 0.6 mL/min labeled amount of total beta carotene (C4oHs6)
Injection volume: 20 ~L
System suitability
Samples: System suitabilitysolution and StandardsolutionA SPECIFIC TESTS
[NoTE-The approximate relativeretention times of
the components in the System suitabilitysolution are
listed in Table 1.]
• ALPHA CAROTENE AND OTHER RELATED COMPOUNDS
Table 1 Mobile phase, System suitability solution, Sample
solution, and Chromatographic system: Proceed as
Relative Relative directed in the test for Content of Total Beta Carotene.
Retention Response
Name Time Factor Analysis
'i)
Sample: Sample solution
all-trans-Alpha carotene 0.93 ',.~\.I~r .• '!\'I~y,"'II'.~) Calculatethe percentage of alpha carotene and other
all-trans-Beta carotene 1.00 1 individual related compounds relative to total beta
'- carotene in the portion of Capsules taken:
9-cis-Beta carotene 1.07 1
1 3-cis-Beta carotene 1.17 1.2 Result = (ru/rr) x 100
15-cis-Beta carotene 1.21 1.4 ru = peak area of alpha carotene or other individual
related compounds
Suitability requirements rr = sum of the areas of all the peaks
Chromatogram similarity: The chromatogram from the
System suitability solution is similarto the reference Acceptance criteria
chromatogram provided with the lot of USP Beta Alpha carotene: NMT 1.0%
Carotene System"S,~i~~~Ili~~,~,~.,~~!~g used. Total related compounds (including alpha
Resolution: NLT~,1"-?(US(>1'Mi'lYl2019) between carotene): NMT'+'S9(o-'.(USPllMilYl2019)
all-trans-beta carotene and all-trans-alpha carotene and PERFORMANCE TESTS
between all-trans-beta carotene and 9-cis-:beta carotene, • UNIFORMITY OF DOSAGE UNITS (90S): Meet the
System suitability solution requirements
Tailing factor: NMT 2.0 for the all-trans-beta carotene
peak, Standardsolution A . ADDITIONAL REQUIREMENTS
Relative standard deviation: NMT 2.0% for the • PACKAGING AND STORAGE: Preserve in tight, light-resistant
all-trans-beta carotene peak from replicate injections, containers.
Standardsolution A
Analysis
Samples: Standardsolution A and Sample solution
Identifythe peaks of the relevant analytes of the Sample • LABELING: The label states the name and content of
solution by comparing with those of the System suitability carriers and antioxidants added to the fnrlY\l '11::Itinn
solution. Measure the peak area responses. of total as
Calculate the percentage of the labeled amount of total
beta carotene in the portion of Capsulestaken:
Result = (Zrulrs) x (Cs/Cu) x 100 • RIEI=II:RIEN,l"'1I= )l'AN[)AIRI~S (11)
USP BetaCarotene RS
= (peak area of all-trans-beta carotene) + (peak area (all-£)-l /1'-(3/7/12/16-Tetramethyl-
of 9-cis-betacarotene) + (peak area of 13-cis-beta 1/3/5/7/9/11/13,15/17-octadecanonaene-l /18- diyl)
carotene x 1.2) + (peak area of 15-cis-beta bis[2,6, 6-trimethylcyclohexene].
carotene x 1.4) from the Sample solution C4oHs6 536.87
= peak area of all-trans-beta carotene from USP Beta Carotene System Suitability RS
StandardsolutionA
=concentration of all-trans-beta carotene in
StandardsolutionA as determined above
(uq/rnl)
= nominal concentration of total beta carotene in
the Sample solution (uq/rnt)

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540 Betahistine / OfficialMonographs USP 43

Betahistine Hydrochloride IMPURITIES

• RESIDUE ON IGNITION (281): NMT 0.1 %
• ORGANIC IMPURITIES
• 2HCI Solution A, Mobile phase, Sample solution, and
Chromatographic system: Proceed as directed in the
Assay.
CsH 12N 2·2HCI 209.12 Analysis
2-Pyridineethanamine, N-methyl-, dihydrochloride; Sample: Sample solution
2-[2-(Methylamino)ethyl]pyridine, dihydrochloride [5579- Calculate the percentage of each impurity in the portion of
84-0]. Betahistine Hydrochloride taken:
DEFINITION Result = (rVlrT) x 1IF x 100
Betahistine Hydrochloride contains NLT99.0% and NMT
101.0% of betahistine hydrochloride (CSH 12N2 ·2HCI), to = peak response of each impurity from the Sample
calculated on the dried basis. solution
IDENTIfiCATION rr = sum of all the peak responses from the Sample
solution
F = relative response factor of each impurity (see
Table 1); 1.0 for all other peaks

Acceptance criteria: See Table 1.

requirements.
Table 1
• B. The retention time of the major peak of the Sample
solution corresponds to that of the Standardsolution, as Relative Relative Acceptance
obtained in the Assay. Retention Response Criteria,
Name Time Factor NMT (%)
• C. IDENTIFICATION TESTS-GENERAL (191), Chemical
Identification Tests, Chloride: Meets the requirements 2-(2-Hydroxyethyl)
pyridine" 0.3 1.5 0.2
ASSAY
2-Vinylpyridinea 0.4 2.0 0.2
• PROCEDURE
Solution A: 0.69 mg/mL of ammonium acetate in water. N-Meth-
Adjust with glacial acetic acid to a pH of 4.7. yl-N,N-bis(2-pyridin-
Mobile phase: Acetonitrile and Solution A containing 2.88 2-yl-ethyl) arnlne" 2.4 1.1 0.2
mg/mL of sodium lauryl sulfate (7:13) Anyunspecified
Standard solution: 0.38 mg/mL of USP Betahistine impurity - - 0.10
Hydrochloride RS in Mobilephase Totalimpurities - - 0.5
Sample solution: 0.38 mg/mL of Betahistine
Hydrochloride in Mobilephase a Thisimpurityis a hydrochloride salt.
Chromatographic system b Thisimpurityexistsas a trihydrochloride.
(See Chromatography (621), System Suitablltty.)
Mode: LC SPECifiC TESTS
Detector: UV 254 nm • pH (791)
Column: 3.0-mm x 15-cm; 5-lJm packing L1 Sample: 100 mg/mL of Betahistine Hydrochloride in water
Column temperature: 40° Acceptance criteria: 2.0-3.0
Flow rate: 0.5 mL/min • Loss ON DRVING (731)
Injection volume: 10 IJL Analysis: Dry between 100° and 105° to constant weight.
System suitability Acceptance criteria: NMT 1.0%
Sample: Standardsolution
Suitability requirements ADDITIONAL REQUIREMENTS
Tailing factor: NMT 2.0 • USP REFERENCE STANDARDS (11)
Relative standard deviation: NMT 0.73% USP Betahistine Hydrochloride RS
Analysis
Samples: Standardsolution and Sample solution
Calculate the percentage of betahistine hydrochloride
(CSH12N 2 • 2HCI) in the portion of Betahistine
Hydrochloride taken: Betaine Hydrochloride
Result =(rulrs) x (CslCv ) x 100
ru = peak response of betahistine from the Sample
solution
CSH llN02 • HCI 153.61
rs = peak response of betahistine from the Standard
solution Methanaminium, 1-carboxy-N, N, N-trimethyl-, chloride.
Cs = concentration of USP Betahistine Hydrochloride Betaine hydrochloride.
RS in the Standardsolution (mg/mL) (Carboxymethyl)trimethylammonium chloride [590-46-5].
Cu = concentration of Betahistine Hydrochloride in the
Sample solution (mg/mL) » Betaine Hydrochloride contains not less than 98.0
percent and not more than 100.5 percent of
Acceptance criteria: 99.00/0-1-01.0% on the dried basis CSH ll NOz . HCI, calculated on the anhydrous basis.

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 USP 43 Official Monographs / Betamethasone 541

Packaging and storage-Preserve in well-closed Loss on drying (731 )-Dry it at 105° for 3 hours: it loses not
containers. more than 1.0% of its weight.
USP Reference standards (11)- Residue on ignition (281): not more than 0.2%, a platinum
USP Betaine Hydrochloride RS crucible being used.
Identification- Ordinary impurities (466)-
Test solution: methanol.
Standard solution: methanol.
Application volume: 10 IJL.
Eluant: a mixture of toluene, acetone, methyl ethyl ketone,
and formic acid (55:20:20:5), in a nonequilibrated chamber.
.. "'con .... '''',... to the tests for Chloride Visualization: 5.
(191 ).
pH (791): between 0.8 and 1.2, in a solution (1 in 4). Assay-
Water Determination, Method I (921): not more than 0.5%. Mobile phase-Prepare a filtered and degassed mixture of
Residue on ignition (281): not more than 0.1 %. water and acetonitrile (63:37). Make adjustments if necessary
Assay-Transfer about 400 mg of Betaine Hydrochloride, (see System Suitability under Chromatography (621».
accurately weighed, to a conical flask, add 50 mL of glacial Internal standard solution-Prepare a solution of
acetic acid, and heat gently with swirling until solution is propylparaben in alcohol having a known concentration of
complete. Add 25 mLof mercuric acetate TS,cool, add 2 drops about 0.25 mg per mL.
of crystal violet TS, and titrate with 0.1 N perchloric acid VS to Standardpreparation-Dissolve an accurately weighed
a green endpoint. Perform a blank determination, and make quantity of USP Betamethasone RS in alcohol to obtain a
any necessary correction. Each mL of 0.1 N perchloric acid is solution having a known concentration of about 0.2 mg per
equivalent to 15.36 mg of CSH 11N02· HCI. mL.Transfer 10.0 mL of this solution to a suitable vial, and add
10.0 mL of Internal standardsolution, to obtain a Standard
preparation having known concentrations of about 0.1 mg of
betamethasone and about 0.125 mg of propylparaben per
mL.
Betamethasone 'Assay preparation-Using about 80 mg of Betameth asone,
accurately weighed, prepare as directed for Standard
preparation.
o Chromatographic system (see Chromatography (621»-The
flquld chromatograph is equipped with a 240-nm detector
and a 4.6-mm x 25-cm column that contains packing L1. The
flow rate is about 1.0 mL per minute. Chromatograph the
o Standardpreparation, and record the peak responses as
directed for Procedure: the relative retention times are about
C22H29FOs 392.46 1.0 for betamethasone and 1.4 for propylparaben; the
Pregna-1,4-diene-3,20-dione, 9-fluoro-11, 17,21-trihydroxy- resolution, R, between betamethasone and propylparaben is
1 e-rnethyl-; (11~, 1 6~)-. not less than 3.0; and the relative standard deviation for
9-Fluoro-11~, 17,21-trihydroxy-16~-methylpregna-l ,4-diene- replicate injections is not more than 2.0%.
3,20-dione [378-44-9]. . Procedure-Separately inject equal volumes (about 10 IJL)
of the Standard preparationand the Assay preparation into the
» Betamethasone contains not less than 97.0 chromatograph, record the chromatograms, and measure the
percent and not more than 103.0 percent of responses for the major peaks. Calculate the quantity, in mg,
of C22H29FOs in the portion of Betamethasone taken by the
C22H29FOSf calculated on the dried basis. formula:
Packaging and storage-Preserve in tight containers. Store
between 2° and 30°.
USP Reference standards (11 )- in which C is the concentration, in mg per mL, of USP
USP Betamethasone RS Betamethasone RS in the Standard preparation; and Ru and Rs
Identification- are the peak height ratios of the betamethasone peak and the
internal standard peak obtained from the Assay preparation
and the Standard preparation, respectively.

hromatoaraohk: Identification Test (201)-

Test solution-Prepare a of Betamethasone in
Betamethasone Cream
dehydrated alcohol containing 0.5 mg per mL. DEFINITION
Developing solvent system: a mixture of chloroform and Betamethasone Cream contains NLT 90.0% and NMT 115.0%
diethylamine (2:1). of the labeled amount of betamethasone (C22H29FOs) in a
Procedure-Proceed as directed in the chapter, except to
suitable cream base.
locate the spots by lightly spraying with dilute sulfuric acid (1
in 2) and heating on a hot plate or under a lamp until spots IDENTIFICATION
appear. • A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST
Specific rotation (781 S): between +118° and +126°, (201) .
calculated on the dried basis. Standard solution: 1 mg/mL of USP Betamethasone RS in
Test solution: 5 mg per mL, in methanol. dehydrated alcohol

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542 Betamethasone / OfficialMonographs USP43

Sample solution: Nominally 1 mg/mL of betamethasone SPECIFIC TESTS

prepared by concentrating 10 mL of the Sample solution • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
from the Assay on a steam bath to 1 mL SPECIFIED MICROORGANISMS (62): It meets the
Chromatographic system requirements of the tests for the absence of Staphylococcus
Developing solvent system: Chloroform and aureus and Pseudomonas aeruginosa.
diethylamine (2:1)
Spray reagent: Methanol, sulfuric acid, and nitric acid ADDITIONAL REQUIREMENTS
(10:10:1 ) • PACKAGING AND STORAGE: Preserve in collapsible tubes or
Analysis in tight containers. .
Samples: Standard solution and Sample solution • USP REFERENCE STANDARDS (11)
Proceed as directed in the chapter, except spray the plate USP Betamethasone RS
with the Spray reagent, and heat at 105° for 10 min.
Acceptance criteria: Meets the requirements
ASSAY
• PROCEDURE Betamethasone Oral Solution
Mobile phase: Acetonitrile and water (37:63)
Internal standard solution: 0.25 mg/mL of propylparaben DEFINITION
in alcohol Betamethasone Oral Solution contains NLT 90.0% and NMT
Standard stock solution: 0.2 mg/mL of USP 115.0% of the labeled amount of betamethasone
Betamethasone RS in alcohol (C22H29FOs)'
Standard solution: 0.1 mg/mL of USP Betamethasone RS IDENTIFICATION
prepared by combining 10.0 mL of the Internal ~tandard • A. THIN-LAVER CHROMATOGRAPHIC IDENTIFICATION TEST
solution and 10.0 mL of the Standardstock solution (201)
Sample solution: Nominally 0.1 mg/mL of betamethasone Diluent: Chloroform and methanol (1:1)
prepared as follows. To a portion of Cream, nominally Standard solution:' 1 mg/mL of USP Betamethasone RS in
equivalent to 2 mg of betamethasone, add 10.0 mL of alcohol
Internal standardsolution and 10.0 mL of alcohol. Mix by Sample solution: Place a volume of Oral Sol~tion, .
rotation for 20 min. Centrifuge at 2500 rpm for 10 min. equivalent to about 1 mg of betamethasone, In a centrifuge
Transfer a portion of the supernatant to a suitable vial. tube. Add 15 mL of 0.1 N hydrochloric acid and 20 mL of
Chromatographic system ethyl acetate. Shake the tube for about 1 min. Centrifuge
(See Chromatography (621), System SUitability.) to separate the phases. Transfer the upper phase (ethyl
Mode: LC acetate) to a suitable container. Evaporate to dryness on a
Detector: UV'240 nm steam bath under a gentle stream of nitrogen. . .
Column: 4.6-mm x 25-cm; packing L1 Allow to cool to room temperature. Dissolve the residue In
Flow rate: 1 mL/min about 0.5 mL of Diluent by using a vortex mixer. Transfer
Injection volume: 10 fJL the solution to a 2-mL volumetric flask with small
System suitability portions of Diluent. Dilute with Diluent to volume, and
Sample: Standardsolution mix.
[NoTE-The relative retention tlmes for betamethasone Evaporate 1 mL of the resulting solution on a steam bath
and propylparaben are 1.0 and 1.4, respectively.] just to dryness, and dissolve the residue in 0.5 mL of
Suitability requirements . alcohol.
Resolution: NLT 3.0 between betamethasone and Chromatographic system
propylparaben Application volume: 10 fJL
Relative standard deviation: NMT 2.0% Developing solvent system: Chloroform and
Analysis diethylamine (2:1)
Samples: Standard solution and Sample solution Spray reagent: Dilute sulfuric acid (1 in 2)
Calculate the percentage of the labeled amount of Analysis: Proceed as directed in the chapter. Locate the
betamethasone (C22H29FOs) in the portion of Cream spots by lightly spraying with Spray reagent, and heat on a
taken: hot plate or under a lamp until spots appear.
Acceptance criteria: Meets the requirements
Result = (RulR s) x (CslCu) x 100 • B. The retention time of the major peak of the Sample
solution corresponds to that of the Standardsolution, as
= peak height ratio of the betamethasone peak to obtained in the Assay.
the internal standard peak from the Sample
solution ASSAY
= peak height ratio of the betamethasone peak to • PROCEDURE
the internal standard peak from the Standard Protect all standard and sample solutions from light.
solution Buffer: 6.8 giL of monobasic potassium phosphate in water.
=concentration of USP Betamethasone RS in the Adjust with phosphoric acid to a pH of 2.9.
Standardsolution (mg/mL) Solution A: Acetonitrile and Buffer (25:75)
= nominal concentration of betamethasone in the Solution B: Acetonitrile and Buffer (45:55)
Sample solution (mg/mL) Diluent: Dehydrated alcohol and water (2:3)
Mobile phase: See Table 1.
Acceptance criteria: 90.00/0-115.0%
Table 1
PERFORMANCE TESTS
• MINIMUM FILL (755): Meets the requirements Time Solution A Solution B
(min) (0/0) (0/0)
0 100 0

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USP 43 OffiCial Monographs / Betamethasone 543

Table 1 (continued) IMPURITIES

Time Solution A Solution B • ORGANIC IMPURITIES
(min) (%) (%) Protect all sample and standard solutions from light.
25.0 0 100
Buffer, Solution A, Solution B, Diluent, Mobile phase,
Standard stock solution, System suitability solution,
25.1 100 0 Sample solution, and Chromatographic system:
35.0 100 0 Proceed as directed in the Assay.
Standard solution: 0.48 IJg/mL of USP Betamethasone RS
in Diluent from the Standard stock solution
Standard stock solution: 0.12 mg/mL of USP Sensitivity solution: 0.024 IJg/mL of USP Betamethasone
Betamethasone RS prepared as follows. Transfer a RS in Diluent from the Standard solution
quantity of USP Betamethasone RS to a suitable container, System suitability
and dilute, using sonication, with dehydrated alcohol to Samples: System suitability solution and Sensitivity solution
obtain a solution containing 0.3 mg/mL. Quantitatively [NOTE-The relative retention times for betamethasone
dilute an aliquot of this solution with water to obtain a and beclomethasone are 1.0 and 1.2, respectively.]
0.12-mg/mL solution of betamethasone. Suitability requirements
Standard solution: 0.048 mg/mL of USP Betamethasone RS Resolution: NLT 4.0 between betamethasone and
in Diluent from Standard stocksolution beclomethasone, System suitability solution
Beclomethasone solution: 0.12 mg/mL of Relative standard deviation: NMT 10% for
beclomethasone prepared as follows. Transfer a quantity of betamethasone, Sensitivity solution
beclomethasone to a suitable container, and dilute, using Analysis
sonication, with dehydrated alcohol to obtain a solution Samples: Standard solution and Sample solution
containing 0.3 mg/mL. Quantitatively dilute an aliquot of Calculate the percentage of each related compound in the
this solution with water to obtain a 0.12 mg/mL solution of portion of Oral Solution taken:
beclomethasone.
System suitability solution: 0.048 mg/mL each of USP Result = (rufrs) x (Cs/Cu) x 100
Betamethasone RS and beclomethasone in Diluent,
prepared from the Standard stock solution and = peak response for individual related compounds
Beclomethasone solution from the Sample solution
Sample solution: Nominally 0.048 mg/mL of = peak response for betamethasone from the
betamethasone prepared as follows. Transfer a measured Standard solution
volume of Oral Solution, containing a known amount of = concentration of USP Betamethasone RS in the
betamethasone, to a suitable volumetric flask, and dilute Standard solution (lJg/mL)
with Diluent to volume. = nominal concentration of betamethasone in the
Chromatographic system Sample solution (lJg/mL)
(See Chromatography (621), System Suitability.)
Mode: LC Acceptance criteria: See Table 2.
Detector: UV 254 nm
Column: 4.6-mm x 15-cm; 4-~m packing L1 Table 2
Flow rate: 1.5 mL/min Relative Acceptance
Injection volume: 50 IJL Retention Criteria,
System suitability . Name Time NMT (%)
Samples: Standard solution and System suitability solution Betamethasone 1.0 -
[NOTE-The relative retention times for betarnethasone
and beclomethasone are 1.0 and 1.2, respectively.] 9,11-Expoxy-17u,21.dihydroxy-
16~-methylpregna.l,4 dlene-
Suitability requirements 3,20-dione 1.25 1.3
Resolution: NLT4.0 between betamethasone and
beclomethasone, System suitability solution 17u,21.Dihydroxy.16~·methylpre·
Tailing factor: NMT 1.5 for betamethasone, System gna-l,4,ll·triene-3,20-dione 1.33 0.7
suitability solution
Relative standard deviation: NMT 2.0%, Standard SPECIFIC TESTS
solution • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
Analysis SPECIFIED MICROORGANISMS (62): It meets the
Samples: Standard solution and Sample solution requirements of the test for the absence of Escherichia coli.
Calculate the percentage of the labeled amount of The total aerobic microbial count is NMT 10 2 cfu/mL, and
betamethasone (C22H29FOs) in the portion of Oral the total combined molds and yeasts count is NMT 10 1
Solution taken: cfu/mL.
• pH (791): 2.8-3.6
Result = (rulrs) x (Cs/Cu) x 100 • DELIVERABLE VOLUME (698): Meets the requirements for
oral solution packaged in multiple-unit containers
= peak response from the Sample solution
ADDITIONAL REQUIREMENTS
= peak response from the Standard solution
• PACKAGING AND STORAGE: Store at controlled room
= concentration of USP Betamethasone RS in the
temperature, protected from light. Preserve in tight
Standard solution (mg/mL) containers.
= nominal concentration of betamethasone in the
• USP REFERENCE STANDARDS (11)
Sample solution (rnq/ml.) USP Betamethasone RS
Acceptance criteria: 90.0%-11.5.0%

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 544 Betamethasone / OfficialMonographs USP 43

and a 4-mm x 30-cm column that contains packing L1. The

Betamethasone Acetate flow rate is about 1 mL per minute. Chromatograph the
Standardpreparation and record the peak responses as
C24H31F06 434.50 directed for Procedure: the relative retention times are about 3
Pregna-l,4-diene-3,20-dione, 9-fluoro-ll, 17-dihydroxy-16- for progesterone and 1.0 for betamethasone acetate; the
methyl-21-(acetyloxy)-, (11 P,16P)-. resolution, R, between the analyte and internal standard peaks
9-Fluoro.-l1 P,17,21-trihydroxy-16p-methylpregna-l ,4-diene- is not less than 2; and the relative standard deviation for
3,20-dlone 21-acetate [987-24-6]. replicate injections is not more than 2.0%.
Procedure-Separately inject equal volumes (about 25 J.lL)
» Betamethasone Acetate contains not less than of the Standardpreparation and the Assay preparation into the
chromatograph, record the chromatograms, and measure the
97.0 percent and not more than 103.0 percent of responses for the major peaks. Calculate the quantity, in mg,
C24H 31 F0 6f calculated on the anhydrous basis. of C24H31F0 6 in the portion of Betamethasone Acetate taken
by the formula:
Packaging and storage-Preserve in tight containers. Store
between 2° and 30°.
YiP Reference standards (11)-
USP Betamethasone Acetate RS in which C is the concentration, in mg per mL, of USP
Betamethasone Acetate RS in the Standardpreparation;and Ru
Identification-
and R, are the peak response ratios obtained from the Assay
preparation and the Standard preparation, respectively.

~: !~~f3~gtt~§goPl9Cl~~~~
Sp~qtrgst:ppy:l:gZ;ryrl..(GN1._j.
B: Thin-Layer Chromatographic Identification Test (201)- Betamethasone Benzoate
Test solution: 0.5 mg per mL in dehydrated alcohol.
Developing solvent system: a mixture of chloroform and
diethylamine (2:1).
Procedure--Proceed as directed in the chapter. Locate the
spots on the plate by lightly spraying with 10% sulfuric acid
in alcohol and heating on a hot plate or under a lamp until
spots appear.
Specific rotation (781 S): between +120° and +128°.
Test solution: 10 mg per mL, in dioxane.
Water Determination, MethodI (921): not more than 4.0%.
Residue on ignition (281): not more than 0.2%, a platinum C29H3l06 496.57
crucible being used. . Pregna-l,4-diene-3,20-dione, 17-(benzoyloxy)-9-fluoro-
Ordinary impurities (466)- 11,21-dihydroxy-16-methyl-, (11 P,16P)-;
9-Fluoro-ll p, 17,21-trihydroxy-16p-methylpregna-l ,4-diene-
Test solution: methanol.
Standardsolution: methanol. 3,20-dione 17-benzoate [22298-29-9].
Application volume: 10 J.lL. DEFINITION
Eluant: a mixture of toluene and isopropyl alcohol (90:10), Betamethasone Benzoate contains NLT 98.0% and NMT
in a nonequilibrated chamber. . 102.0% of betamethasone benzoate (C29H33F06), calculated
Visualization: 5. on the dried basis.
Assay- IDENTIFICATION
Mobilephase-Prepare a filtered and degassed mixture of
water, acetonitrile, and glacial acetic acid (800: 700: 1.5).
Make adjustments if necessary (see System Suitability under
Chromatography (621 »,
Internal standard solution-Transfer about 35 mg of
progesterone to a 50-mL volumetric flask, add Mobilephaseto
volume, and mix. ASSAY
Standardpreparation-Dissolve an accurately weighed • PROCEDURE
quantity of USP Betamethasone Acetate RS in Mobile phase, Mobile phase: Acetonitrile and water (60:40)
and quantitatively dilute with Mobilephase to obtain a solution Internal standard solution: 0.6 mg/mL of betamethasone
containing about 0.5 mg per mL. Transfer 10.0 mL of the dipropionate in methanol
resulting solution to a 50-mL volumetric flask, add 10.0 mL of Standard stock solution: 0.6 mg/mL of USP
Internal standard solution, dilute with Mobilephase to volume, Betamethasone Benzoate RS in methanol
and mix to obtain a solution having a known concentration of Standard solution: 0.2 mg/mL of USP Betamethasone
about 0.1 mg of USP Betamethasone Acetate RS per mL. Benzoate RS prepared by mixing 5.0 mL of Standardstock
Assay preparation-Transfer about 50 mg of Betameth solution and 10.0 mL of Internal standard solution
asone Acetate, accurately weighed, to a 1OO-mL volumetric Sample stock solution: 0.6 mg/mL of Betamethasone
flask, add Mobilephaseto volume, and mix. Transfer 10.0 mL Benzoate in methanol
of the resulting solution to a 50-mL volumetric flask, add 10.0 Sample solution: 0.2 mg/mL of Betamethasone Benzoate
mL of Internal standardsolution, dilute with Mobilephase to prepared by mixing 5.0 mL of Sample stock solution and
volume, and mix. 10.0 mL of Internal standardsolution
Chromatographic system (see Chromatography (621 »- The Chromatographic system
(See Chromatography (621), System Suitability.)
liquid chromatograph is equipped with a 254-nm detector

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 USP 43 OfficialMonographs / Betamethasone 545

Mode: LC ADDITIONAL REQUIREMENTS

Detector: UV 254 nm • PACKAGING AND STORAGE: Preserve in tight containers.
Column: 4-mm x 30-cm; 5-lJm packing L1 Store between 2° and 30°.
Flow rate: 1 mL/min • USP REFERENCE STANDARDS (11)
Injection volume: 15 IJL USP Betamethasone Benzoate RS
System suitability
Sample: Standardsolution
[NOTE-The relative retention times for betamethasone
benzoate and betamethasone dipropionate are 1.0
and 1.4, respectively.] Betamethasone Benzoate Gel
Suitability requirements
Resolution: NLT 3 between betamethasone benzoate DEFINITION
and the internal standard Betamethasone Benzoate Gel contains an amount of
Relative standard deviation: NMT 2.0% from three betamethasone benzoate (Cz9H33F06) equivalent to NLT
replicate injections 90.0% and NMT 110.0% of the labeled amount of
Analysis betamethasone benzoate (Cz9H33F06)'
Samples: Standardsolution and Sample solution IDENTIFICATION
Calculate the percentage of betamethasone benzoate
(Cz9H3l06) in the portion of Betamethasone Benzoate • A. The retention time of the major peak of the Sample
solution corresponds to that of the Standardsolution, both
taken: relative to the internal standard, as obtained in the Assay.
Result = (Ru/R s) x (Cs/Cu) x 100 ASSAY
• PROCEDURE
Ru = peak response ratio of the betamethasone Mobile phase: Methanol, acetonitrile, and water (23:9:18)
benzoate peak to the internal standard peak Internal standard solution: 250 IJg/mL of USP
from the Sample solution Methyltestosterone RS in methanol
Rs = peak response ratio of the betamethasone Standard stock solution: 0.5 mg/mL of USP
benzoate peak to the internal standard peak Betamethasone Benzoate RS in methanol
from the Standardsolution Standard solution: 0.05 mg/mL of USP Betamethasone
Cs = concentration of USP Betamethasone Benzoate Benzoate RS prepared as follows. Transfer 5.0 mL of
RS in the Standardsolution (mg/mL) Standardstock solution to a 50-mL volumetric flask, add
Cu = concentration of Betamethasone Benzoate in the 10.0 mL of Internal standard solution, and dilute with
Sample ~olution (mg/mL) methanol to volume.
Sample solution: 0.05 mg/mL of betarnethasone benzoate
Acceptance criteria: 98.00/0-102.0% on the dried basis in methanol prepared as follows. Transfer a portion of Gel,
IMPURITIES nominally equivalent to 0.5 mg of betamethasone
• RELATED STEROIDS
benzoate, into a 125-mL separatory funnel. Add 20 mL of
Standard solution A: 5 mg/mL of USP Betamethasone water and 2 mL of saturated sodium acetate solution, shake
Benzoate RS in methanol to disperse the Gel, and add 2.0 mL of Internal standard
Standard solution B: 100 IJg/mL of USP Betamethasone solution. Extract this solution with one 50-mL portion of
Benzoate RS from StandardsolutionA in methanol chloroform, followed by three 40-mL portions of
Sample solution: 20.0 mg/mL of Betarnethasone Benzoate chloroform. Discard the aqueous layer. Wash the
in methanol chloroform extract with 10 mL of water, allow to stand for
Chromatographic system 10 min, then pass through chloroform-wetted glass fiber
(See Chromatography (621), Thin-Layer Chromatography.) filter paper and anhydrous sodium sulfate into a suitable
Mode: TLC container. Evaporate to dryness under a vacuum at 30°.
Adsorbent: 0.25-mm layer of chromatographic silica gel Dissolve the residue in 10 mL of methanol.
mixture Chromatographic system
Application volume: 10 IJL (See Chromatography (621), System Suitability.)
Developing solvent system: Toluene, acetone, and Mode: LC
methanol (75:25:4) Detector: UV 236 nm
Analysis Column: 3.9-mm x 30-cm; packing L1
Samples: Standardsolutions and Sample solution Column temperature: 30°
Proceed as directed in the chapter. Examine the plate under Flow rate: 1.5 mL/min
short-wavelength UVlight. Injection volume: 10 IJL
Acceptance criteria: The principal spot from the Sample System suitability
solution corresponds in RF value to that of Standardsolution Sample: Standardsolution
[NoTE-The relative retention times for
A; and the Sample solution shows NMT 3 additional spots,
the intensity and size of which do not exceed those of the methyltestosterone and betarnethasone benzoate
are about 1.0 and 1.33, respectively.]
spot from Standardsolution B.
Suitability requirements
SPECIFIC TESTS Resolution: NLT 3.0 between methyltestosterone and
• OPTICAL ROTATION, Specific Rotation (781 S) betamethasone benzoate
Sample solution: 40 mg/mL, in dioxane Relative standard deviation: NMT 1.0%
Acceptance criteria: +60° to +66° Analysis
• Loss ON DRYING (731) Samples: Standardsolution and Sample solution
Sample: 200 mg Calculate the percentage of the labeled amount of
Analysis: Dry the Sample at 105°for 3 h. betamethasone benzoate (Cz9H33F06) in the portion of
Acceptance criteria: NMT 0.5% Gel taken:

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546 Betamethasone / OfficialMonographs USP 43

Result = (Ru/R s) x (Cs/Cu) x 100 ASSAY

• PROCEDURE
Ru = peak response ratio of the betamethasone Mobile phase: Acetonitrile and water (1 in 2) such that the
benzoate peak to the internal standard peak retention times for betamethasone dipropionate and
from the Sample solution beclomethasone dipropionate are 14 and 18 min,
Rs = peak response ratio of the betamethasone respectively. Degas by sonicating for 5-10 min. Do not
benzoate peak to the internal standard peak leave Mobile phase in the column overnight, but flush the
from the Standard solution system after use with water for 15 min, followed by
Cs = concentration of USP Betamethasone Benzoate methanol for 15 min.
RS in the Standard solution (mg/mL) Diluent: Acetic acid and methanol (1 in 1000)
Cu = nominal concentration of betamethasone Internal standard solution: 0.9 mg/mL of USP
benzoate in the Sample solution (mg/mL) Beclomethasone Dipropionate RS in Diluent
Standard stock solution: 0.6 mg/mL of USP
Acceptance criteria: 90.00/0-110.0% Betamethasone Dipropionate RS in Diluent
PERfORMANCE TESTS Standard solution: 0.3 mg/mL of USP Betamethasone
• MINIMUM fiLL (755): Meets the requirements Dipropionate RS and 0.45 mg/mL of USP Beclomethasone
Dipropionate RS, prepared by combining 5.0 mL each of
SPECifiC TESTS the Internal standard solution and the Standard stock solution
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR Sample stock solution: 0.6 mg/mL of Betarnethasone
SPECIFIED MICROORGANISMS (62): It meets the Dipropionate in Diluent
requirements of the tests for absence of Staphylococcus Sample solution: 0.3 mg/mL of Betamethasone
aureus and Pseudomonas aeruginosa. Dipropionate, prepared by cornbininq 5.0 mL each of the
Internal standard solution and the Sample stock solution
ADDITIONAL REQUIREMENTS
Chromatographic system
• PACKAGING AND STORAGE: Preserve in tight containers. (See Chromatography (621), System Suitability.)
Store at 25°, excursions permitted between 15° and 30°. Mode: LC
Protect from freezing. Detector: UV 254 or 240 nm
• USP REFERENCE STANDARDS (11) Column: 4-mm x 30-cm; packing L1
USP Betamethasone Benzoate RS Injection volume: 5-25 ~L
USP Methyltestosterone RS System suitability
Sample: Standard solution
Suitability requirements
Peak area ratios: The lowest and highest peak area ratios
Betamethasone Dipropionate of three successive injections agree within 2.0%.
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of betamethasone dipropionate
(C2sH37F07) in the portion of Betamethasone
Dipropionate taken:
Result =(Ru/R s) x (Cs/Cu) x 100

C2sH37F07 504.59 = peak height ratio of betamethasone dipropionate

Pregna-1,4-diene-3,20-dione, 9-fluoro-11-hydroxy-16- to the internal standard from the Sample solution
methyl-1 7,21-bis(1-oxopropoxy)-, (11 P,16P); = peak height ratio of betamethasone dipropionate
9-Fluoro-11 p,17,21-trihydroxy-16p-methylpregna-1 ,4-diene- to the internal standard from the Standard
. 3,20-dione 17,21-dipropionate [5593-20-4]. solution
= concentration of USP Betamethasone
DEfiNITION Dipropionate RS in the Standard solution
Betamethasone Dipropionate contains NLT 97.0% and NMT (mg/mL)
103.0% of betamethasone dipropionate (C2sH3l07), = concentration of Betamethasone Dipropionate
calculated on the dried basis. in the Sample solution (mg/mL)
IDENTifiCATION Acceptance criteria: 97.00/0-103.0% on the dried basis
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.2%, using a platinum
crucible
• ORGANIC IMPURITIES
• B. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Mobile phase: Acetonitrile and water (65:35)
(201 ) System suitability solution: 0.05 mg/mL of USP
Sample solution: 1 mg/mL, in chloroform Betamethasone Dipropionate RS and 0.05 mg/mL of USP
Chromatographic system Betamethasone Valerate RS in Mobile phase
Developing solvent system: Chloroform and acetone Sample solution: 0.3 mg/mL of Betamethasone
(7:1 ) Dipropionate in Mobile phase. Shake until dissolved.
Acceptance criteria: Meets the requirements Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 nm
Column: 4.6-mm x 15-cm; packing L1

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USP43 Official Monographs / Betamethasone 547

Flow rate: 1 mL/min Developing solvent system: Chloroform and acetone

Injection volume: 10 IJL (7:1 )
System suitability Analysis
Sample: System suitability solution Samples: Standard solution and Sample solution
Suitability requirements Proceed as directed in the chapter.
Resolution: NLT 4.0 between betamethasone valerate Acceptance criteria: Meets the requirements
and betamethasone dipropionate
Column efficiency: NLT 8000 theoretical plates ASSAY
Analysis • PROCEDURE
Sample: Sample solution Mobile phase: Acetonitrile and water (1 in 2) such that the
Calculate the percentage of each 'impurity in the portion of retention times for betamethasone dipropionate and
Betamethasone Dipropionate taken: beclomethasone dipropionate are 14 and 18 min,
respectively. Degas by sonicating for 5-10 min. Do not
Result = (ru/rr) x 100 leave the Mobilephase in the column overnight, but flush
the system after use with water for 15 min, followed by
ru = peak response for each impurity methanol for 15 min.
rr = sum of the responses for all the peaks Diluent: Acetic acid in methanol (1 in 1000)
Internal standard solution: 0.45 mg/mL of USP
Acceptance criteria Beclomethasone Dipropionate RS in Diluent
Individual impurities: NMT 1.0% Standard stock solution: 0.2 mg/mL of USP
Total impurities: NMT 2.0% Betamethasone Dipropionate RS in Diluent
Standard solution: 0.133 mg/mL of USP Betamethasone
SPECIFIC TESTS Dipropionate RS and 0.15 mg/mL of USP Beclomethasone
• OPTICAL ROTATION, Specific Rotation (781S) Dipropionate RS prepared by combining 10.0 mL of the
Sample solution: 10 mg/mL, in dioxane Standard stock solution and 5.0 mL of the Internal standard
Acceptance criteria: +63° to +70° solution
• Loss ON DRYING (731) Sample solution: Nominally equivalent to 0.1 mg/mL of
Analysis: Dry at 105° for 3 h. -betarnethasone, prepared as follows. Transfer a portion of
Acceptance criteria: NMT 1.0% Cream, equivalentto 2 mg of betamethasone dipropionate
(1 .6 mg of betamethasone), into a capped 50-mL
ADDITIONAL REQUIREMENTS centrifuge tube. Add 5.0 mL of Internal standardsolution
• PACKAGING AND STORAGE: Preserve in tight containers. and 10.0 mL of Diluent. Heatin a water bath at 60°,shaking
Storeat 25°, excursions permitted between 15° and 30°. intermittently, until the Cream melts. Remove from the
• USP REFERENCE STANDARDS (11) bath, and shake vigorously untilthe Cream has resolidified.
USP Beclomethasone Dipropionate RS Repeatthe heating and shaking. Freeze in an ice-methanol
USP Betamethasone Dipropionate RS bath for 15 min, and centrifugeat 2500 rpm for 5 min. Use
USP BetamethasoneValerate RS the supernatant.
Chromatographic system
(See Chromatography (621), System SUitability.)
Mode: LC
Betamethasone Dipropionate Cream Detector: UV 254 or 240 nm
Column: 4-mm x 30-cm; packing L1
DEFINITION Injection volume: 5-25 IJL
Betamethasone Dipropionate Cream contains an amount of System suitability
betamethasone dipropionate (C2sH 3l 0 7 ) equivalentto NLT Sample: Standard solution
90.0% and NMT 110.0% of the labeled amount of Suitability requirements
betamethasone (C22H29FOs), in a suitable cream base. Peak area ratios: The lowestand highest peakarea ratios
of three successive injections agree within 2.0%.
IDENTIFICATION Analysis
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Samples: Standard solution and Sample solution
(201) Calculate the percentage of the labeled amount of
Standard solution: 150 IJg/mL of USP Betamethasone betamethasone (C22H29FOs) in the portion of Cream
Dipropionate RS in chloroform taken:
Sample solution: Nominally equivalent to 150 IJg/mL of
betamethasone dipropionate, prepared asfollows. Transfer Result = (Ru/R s) x (Cs/C u) x (MrdMr2 ) x 100
1.5 g of Creamto a glass-stoppered,50-mL centrifugetube.
Add 15 mL of a methanol-hydrochloric acid solution =peak height ratio of betamethasone dipropionate
prepared by mixing 1 volume of dilute hydrochloric acid (1 to the internal standard from the Sample solution
in 120) with 4 volumesof methanol. Shaketo obtain a =peak height ratioof betamethasone dipropionate
homogeneous mixture.Add 30 mL of solvent hexane, mix to the internal standard from the Standard
for 10 min, and centrifuge. Using a syringe, transfer the solution
loweraqueous phase to a second centrifugetube, and add = concentration of USP Betamethasone
20 mL of water. Extract this aqueous mixture with Dipropionate RS in the Standard solution
chloroform by shaking, centrifuging, and removing the (mg/mL)
lower, chloroform phase with a syringe. Evaporate the = nominal concentration of betamethasone in the
chloroform on a steam bath with the aid of a stream of Sample solution (mg/mL)
nitrogen to dryness, cool, and dissolve the residue in = molecular weight of betamethasone, 392.46
chloroform. = molecular weight of betamethasone
Chromatographic system dipropionate, 504.59
Application volume: 40 IJL

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548 Betamethasone / OfficialMonographs USP 43

Acceptance criteria: 90.00/0-110.0% temperature to dryness. Cool the vial to room temperature,
add 4.0 mL of methanol, and swirl to dissolve the residue.
PERfORMANCE TESTS Sample solution: Nominally 0.23 mg/mL of
• MINIMUM fILL (755): Meets the requirements betamethasone, prepared as follows. Transfer a portion of
ADDITIONAL REQUIREMENTS Lotion, equivalent to 1.2 mg of betamethasone .
• PACKAGING AND STORAGJE: Preserve in collapsible tubes or dipropionate (0.93 mg of betamethasone), into a capped
tight containers. Store at 25°, excursions permitted . 50-mL centrifuge tube. Add 10.0 mL of 0.1 N hydrochloric
between 15° and 30°. Protect from freezing. acid, shake to disperse, then add 2.0 mL of Internal standard
• USP REFERENCE STANDARDS (11) solution and 2.0 mL of chloroform. Cap, and shake
USP Beclomethasone Dipropionate RS vigorously for about 2 min, or disperse on avortex mixer
USP Betamethasone Dipropionate RS for about 1 min. Centrifuge at 2500 rpm for about 3 min.
Transfer the chloroform phase to a suitable vial. Evaporate
the chloroform under a stream of nitrogen at a slightly
elevated temperature to dryness. Cool the vial to room
temperature, add 4.0 mL of methanol, and swirl to dissolve
Betamethasone Dipropionate Lotion the residue.
Chromatographic system
DEfiNITION (See Chromatography (621), System Suitability.)
Betamethasone Dipropionate Lotion contains an amount of Mode: LC
betamethasone dipropionate (C2sH3l07) equivalent to NLT Detector: UV 254 or 240 nm
90.0% and NMT 110.0% of the labeled amount of Column: 4-mm x 30-cm; packing L1
betamethasone (C22H29FOs), in a suitable lotion base. Injection volume: 5-25 IJL
System suitability
IDENTifICATION Sample: Standard solution
• A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST Suitability requirements
(201) . Peak area ratios: The lowest and highest peak area ratios
Standard solution: 150 IJg/mL of USP Betamethasone of three successive injections agree within 2.0%.
Dipropionate RS in chloroform Analysis
Sample solution: Nominally 150 IJg/mL of betamethasone Samples: Standard solution and Sample solution
dipropionate, prepared as follows. Transfer a portion of Calculate the percentage of the labeled amount of
Lotion, equivalent to 0.6 mg of betamethasone betamethasone (C22H29FOs) in the portion of Lotion taken:
dipropionate, to a 50-mL vial; add 10 mL of 0.1 N
hydrochloric acid; then add 4 mL of chloroform. Disperse Result = (Ru/R s) x (Cs/Cu) x (MrdMr2 ) x 100
on a vortex mixer for about 1 min, shake vigorously for 10
min,' and centrifuge at 2000 rpm for about 5 min. Transfer Ru =peak height ratio of betamethasone dipropionate
the chloroform layer to a suitable vial. to the internal standard from the Sample solution
Chromatographic system Rs = peak height ratio of betamethasone dipropionate
Application volume: 40 IJL to the internal standard from the Standard
Developing solvent system: Chloroform and acetone solution
(7:1 ) Cs = concentration of USP Betamethasone
Analysis Dipropionate RS in the Standard solution
Samples: Standard solution and Sample solution (mg/mL)
Proceed as directed in the chapter. Cu = nominal concentration of betamethasone in the
Acceptance criteria: Meets the requirements Sample solution (mg/mL)
ASSAY Mr1 = molecular weight of betamethasone, 392.46
• PROCEDURE Mr2 = molecular weight of betamethasone
Mobile phase: Acetonitrile and water (1 in 2) such that the dipropionate, 504.59
retention times for betamethasone dipropionate and
beclomethasone dipropionate are 14 and 18 min, Acceptance criteria: 90.00/0-110.0%
respectively. Degas by sonicating for 5-10 min. Do not SPECIFIC TESTS
leave the Mobile phase in the column overnight, but flush • MINIMUM FILL (755): Meets the requirements
the system after use with water for 15 min, followed by
methanol for 15 min. ADDITIONAL REQUIREMENTS
Internal standard solution: 0.9 mg/mL of USP • PACKAGING AND STORAGE: Preserve in tight containers.
Beclomethasone Dipropionate RS in chloroform Store at 25°, excursions permitted between 15° and 30°.
Standard stock solution A: 0.6 mg/mL of USP Protect from light and freezing.
Betamethasone Dipropionate RS in chloroform • USP REFERENCE STANDARDS (11)
Standard stock solution B: 0.3 mg/mL of betamethasone USP Beclomethasone Dipropionate RS
dipropionate and 0.45 mg/mL of beclomethasone USP Betamethasone Dipropionate RS
dipropionate, prepared by combining 5.0 mL each of
Internal standard solution and Standard stock solution A
Standard solution: To 10.0 mL of 0.1 N hydrochloric acid
in a capped 5-mL centrifuge tube, add 4.0 mL of Standard
stock solution B. Cap the tube, and shake vigorously for Betamethasone Dipropionate Ointment
about 2 min, or disperse on a vortex mixer for about 1 min.
Centrifuge at 2500 rpm for about 3 min. Transfer the DEfiNITION
chloroform phase to a suitable vial. Evaporate the Betamethasone Dipropionate Ointment contains an amount
chloroform under a stream of nitrogen at a slightly elevated of betamethasone dipropionate (C2sH37F07) equivalent to

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USP 43 OffiCial Monographs / Betamethasone 549

NLT 90.0% and NMT 110.0% of the labeled amount of Suitability requirements
betamethasone (CnHz9FOs), in a suitable ointment base. Peak area ratios: The lowest and highest peak area ratios
of three successive injections agree within 2.0%.
IDENTlflCAnON Analysis
• A. THIN-LAVER CHROMATOGRAPHIC IDENTIFICATION TEST Samples: Standardsolution and Sample solution
(201 ) Calculate the percentage of the labeled amount of
Standard solution: 150 IJg/mL of USP Betamethasone betamethasone (CzzHz9FOs) in the portion of Ointment
Dipropionate RS in chloroform taken:
Sample solution: Nominally 150 IJg/mL of betamethasone
dipropionate, prepared as follows. Transfer 1.5 g of Result = (Ru/R s) x (Cs/Cu) x (M rtlMr2 ) x 100
Ointment to a glass-stoppered, 50-mL centrifuge tube. Add
15 mL of methanol-hydrochloric acid solution prepared by =peak height ratio of betamethasone dipropionate
mixing 1 volume of dilute hydrochloric acid (1 in 120) with to the internal standard from the Sample solution
4 volumes of methanol. Shake to obtain a homogeneous = peak height ratio of betamethasone dipropionate
mixture. Add 30 mLof solvent hexane, mix for 10 min, and to the internal standard from the Standard
centrifuge. Using a syringe, transfer the lower aqueous solution
phase to a second centrifuge tube, and add 20 mLof water. = concentration of USP Betamethasone
Extract this aqueous mixture with chloroform by shaking, Dipropionate RS in the Standardsolution
centrifuging, and removing the lower, chloroform phase (mg/mL)
with a syringe. Evaporate the chloroform on a steam bath = nominal concentration of betamethasone from
with the aid of a stream of nitrogen to dryness, cool, and the Sample solution (mg/mL)
dissolve the residue in chloroform. = molecular weight of betamethasone, 392.46
Chromatographic system = molecular weight of betamethasone
Application volume: 40 IJL dipropionate, 504.59
Developing solvent system: Chloroform and acetone
(7:1 ) Acceptance criteria: 90.0%-110.0%
Analysis
Samples: Standard solution and Sample solution PERFORMANCETESTS
Proceed as directed in the chapter. • MINIMUM FILL (755): Meets the requirements
Acceptance criteria: Meets the requirements ADDITIONAL REQUIREMENTS
ASSAY • PACKAGING AND STORAGE: Preserve in collapsible tubes or
• PROCEDURE tight containers. Store at 25°, with excursions permitted
Mobile phase: Acetonitrile and water (1 in 2) such that the between 15° and 30°. Protect from freezing.
retention times for betamethasone dipropionate and • USP REFERENCE STANDARDS (11)
beclomethasone dipropionate are 14 and 18 min, USP Beclomethasone Dipropionate RS
respectively. Degas by sonicating for 5-10 min. Do not USP Betamethasone Dipropionate RS .
leave the Mobilephase in the column overnight, but flush
the system after use with water for 15 min, followed by
methanol for 15 min.
Diluent: Acetic acid and alcohol (1 in 1000)
Internal standard solution: 0.45 mg/mL of USP Betamethasone Sodium Phosphate
Beclomethasone Dipropionate RS in Diluent
Standard stock solution: 0.2 mg/mL of USP
Betamethasone Dipropionate RS in Diluent .
Standard solution: 0.133 mg/mL of USP Betamethasone
Dipropionate RS and 0.15 mg/mL of USP Beclomethasone
Dipropionate RS prepared by combining 10.0 mL of the
Standardstock solution and 5.0 mL of the Internal standard
solution C2zHzsFNazOsP 516.40
Sample solution: Nominally equivalent to 0.1 mg/mL of Pregna-l,4-diene-3,20-dione, 9-fluoro-l1, 17-dihydroxy-16-
betamethasone, prepared as follows. Transfer a portion of methyl-21-(phosphonooxy)-, disodium salt, (11 ~,16~)-;
Ointment, equivalent to 2 mg of betamethasone 9-Fluoro-l1~, 1 7,21-trihydroxy-16~-methylpregna-l ,4-diene-
dipropionate (1.6 mg of betamethasone), into a capped 3,20-dione 21-(disodium phosphate) [151-73-5].
50-mL centrifuge tube. Add 5.0 mL of Internal standard DEFINITION
solution and 10.0 mL of Diluent. Heat in a water bath at 70°, Betamethasone Sodium Phosphate contains NLT 97.0% and
shaking intermittently, until the sample melts. Remove NMT 103.0% of betamethasone sodium phosphate
from the bath, and shake vigorously until the Ointment has (C2zHzsFNazOsP), calculated on the anhydrous basis.
solidified. Repeat the heating and shaking. Freeze in an ice-
methanol bath for 15 min, and centrifuge at 2500 rpm for IDENTIFICATION
5 min. Use the supernatant.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 254 or 240 nm
Column: 4-mm x 30-cm; packing L1
Injection volume: 5-25 IJL (201 )
System suitability Standard solution: 1 mg/mL of USP Betamethasone
Sample: Standard solution Sodium Phosphate RS in methanol

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 550 Betamethasone / Official Monographs USP 43

Sample solution: 1 mg/mL of Betamethasone Sodium Standard solution: Dilute 5.0 mL of Standard phosphate
Phosphate in methanol . solution in a mixture of 10 mL of water and 5 mLof 2 N
Chromatographic system sulfuric acid contained in a 25-mLvolumetricflask, by
Application volume: 10 IJL warming if necessary. Add 1 mLeach of Phosphate reagent
Developing solvent system: 500 mL of butyl alcohol and A and Phosphate reagent B, dilute with water to 25.0 mL,
200 mLof dilute hydrochloric acid (1 in 12). Place in a mix, and allowto stand at room temperature for 30 min.
separatory funnel, and mix. Usethe organic layeras the Sample solution: Dissolve 50 mg of BetamethasoneSodium
developing solvent. Phosphate in a mixture of 10 mLof water and 5 mL of 2 N
Spray reagent: Sulfuric acid, methanol, and nitric acid sulfuric acid contained in a 25-mLvolumetricflask, by
(10:10:1 ) warming if necessary. Add 1 mL each of Phosphate reagent
Analysis A and Phosphate reagent B, dilute with water to 25.0 rnl,
Samples: Standard solution and Sample solution mix, and allowto stand at room temperature for 30 min.
Proceed as directed in the chapter, except to spray the Instrumental conditions
plate with Spray reagent, and heat at 105 for 10 min.
0
Mode: Vis
Acceptance criteria: Meets the requirements Analytical wavelength: Maximum absorbance at about
• C. IDENTIFICATION TESTS-GENERAL, Sodium (191) and 730 nm
Phosphate (191) Cell: 1 cm
Analysis: Ignite it at 800 (see Residue on Ignition (281 ».
0
Blank: Water
Acceptance criteria: The residue meets the requirements Analysis
for sodium and phosphate. . Samples: Standard solution and Sample solution
Acceptance criteria: The absorbance of the Sample solution
ASSAY is NMT that of the Standard solution. The limitis 1.0% of
• PROCEDURE phosphate (P0 4) .
~ Mobile phase: Methanol and 0.07 Manhydrous monobasic • LIMIT OF FREE BETAMETHASONE
potassium phosphate (3:2) Sample stock solution: 1.0 mg/mL of Betamethasone
Diluent: Methanol and water (3:2) Sodium Phosphate in water, prepared as follows. Dissolve
Standard solution: 0.1 7 mg/mL of USP Betamethasone 25.0 mg of Betamethasone Sodium Phosphate in water to
Sodium Phosphate RS in Diluent make 25.0 mL.
Sample solution: 0.17 mg/mL of Betamethasone Sodium Sample solution: Transfer 5.0 mLof the Sample stock
Phosphate in Diluent solution to a separator, and extract with three 25-mL
Chromatographic system portions of chloroform. Filter each extract through a
(See Chromatography (621), System Suitability.) chloroform-saturated cotton pledget, combining the
Mode: LC filtrates in a conicalflask. Evaporate the chloroform on a
Detector: UV· 254 nm steam bath to dryness with the aid of a current of air, and
Column: 3.9-mm x 30-cm; packing L1 dissolve the residue in methanol to make 25.0 mL.
Flow rate: 1.5 mL/min Blank solution: Transfer5.0 mL of water to a separator.
Injection volume: 20 IJL Proceed as directed in Sample solution, beginning with
System suitability "extract with three 25-mLportions of chloroform".
Sample: Standard solution Instrumental conditions
Suitability requirements Mode: UV
Tailing factor: NMT 2 Analytical wavelength: Maximum absorbance at about
Relative standard deviation: NMT 3.0% 239 nm
Analysis Cell: 1 cm
Samples: Standard solution and Sample solution Blank: Blank solution
Calculatethe percentage of betamethasone sodium Analysis
phosphate (C22H2sFNa20sP) in the portion of Sample: Sample solution
Betamethasone Sodium Phosphate taken: Calculatethe quantity, in mg, of free betamethasone in
the portion of Betamethasone Sodium Phosphate taken:
Result =(ru/rs) x (CslCu) x 100
Result = A x 3.125
:= peak response from the Sample solution
= peak response from the Standard solution A = absorbance of the Sample solution
=concentration of USP Betamethasone Sodium
Phosphate RS in the Standard solution (mg/mL) Acceptance criteria: NMT 0.25 mg (1.0%)
= concentration of Betamethasone Sodium .
SPECIFIC TESTS
Phosphate in the Sample solution (mg/mL)
• OPTICAL ROTATION, Specific Rotation (781S)
Acceptance criteria: 97.0%-103.0% on the anhydrous Sample solution: 10 mg/mL
0
basis Acceptance criteria: +99 to +105
0

• WATER DETERMINATION, Method I (921): NMT 10.0%

IMPURITIES
• LIMIT OF PHOSPHATE IONS ADDITIONAL REQUIREMENTS
Standard phosphate solution and Phosphate reagent A: • PACKAGING AND STORAGE: Preserve in tight containers.
Prepare as directed in Phosphate in Reagents (see Reagents, • USP REFERENCE STANDARDS (11)
Indicators, and Solutions-General Tests for Reagents). USP Betamethasone Sodium Phosphate RS
Phosphate reagent B: Dissolve 350 mg of
p-methylaminophenol sulfate in 50 mL of water. Add 20 9
of sodium metabisulfite, mix to dissolve, and dilute with
water to 100 mL.

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 USP 43 Official Monographs / Betamethasone 551

and internal standard peaksis not less than 5; and the relative
Betamethasone Sodium Phosphate standard deviation for replicate injections is not more than
Injection 2.0%.
Procedure-Separately injectequal volumes(about 20 IJL)
of the Standardpreparation and the Assay preparation into the
» Betamethasone Sodium Phosphate Injection is a chromatograph, record the chromatograms, and measurethe
sterile solution of Betamethasone Sodium responses for the major peaks. The relative retention times are
Phosphate in Water for Injection. It contains an about 2.4 for butylparabenand 1.0 for betamethasone sodium
amount of betamethasone sodium phosphate phosphate. Calculate the quantity, in mg, of C22H29FOs in each
mL of the Injection taken by the formula:
(CZZHZ8FNaz08P) equivalent to not less than 90.0
percent and not more than 110.0 percent of the (392.47/516.41)(25C/\I)(R u/R 5)
labeled amount of betamethasone (CZZHZ9FOs).
in which 392.47 and 516.41 are the molecular weights of
Packaging and storage-Preserve in slnqle-dose or in betamethasone and betamethasone sodium phosphate,
multiple-dose containers, preferably of Type I glass. respectively; C is the concentration, in mg per mL, of USP
USP Reference standards (11 )- BetamethasoneSodium Phosphate RS in the Standard
USP Betamethasone Sodium Phosphate RS preparation; V isthe volume, in mL, of Injection taken; and R u
Identification-Dilute the Injection with methanol, if and R 5 are the peak response ratios obtained from the Assay
necessary, to obtain a solution containing about 2 mg of preparation and the Standardpreparation, respectively.
betamethasone sodium phosphate per mL. Separately apply
10 IJL of this test solution and 10 IJL of a solution of USP
BetamethasoneSodium Phosphate RS in methanol containing
2 mg per mL to a thin-layer chromatographic plate (see
Chromatography (621» coated with chromatographic silica Betamethasone Sodium Phosphate and
gel mixture. Develop the chromatogram in an equilibrated Betamethasone Acetate Injectable
chamber containing n-butyl alcohol previously shaken with 1
N hydrochloric acid, until the solventfront has moved about Suspension
three-fourthsof the length ofthe plate. Remove the plate from
the developing chamber, air-dry, then spraywith a mixtureof Betamethasone Sodium Phosphate and
»
sulfuric acid, methanol, and nitric acid (10:10:1). Heat the
Betamethasone Acetate Injectable Suspension is a
plate at 105°for 10 minutes:the R .vatue of the principal spot
sterile preparation of Betamethasone Sodium
from the test solution corresponds to that obtained from the
Standard solution. Phosphate in solution and Betamethasone Acetate
Bacterial Endotoxins Test (85) -It contains not more than in suspension in Water for Injection. It contains an
29.2 USP Endotoxin Units per mg of betamethasone. amount of betamethasone sodium phosphate
pH (791): between 8.0 and 9.0.
Particulate Matter in Injections (788): meets the (CzzHzaFNaz08P) equivalent to not less than 90.0
requirementsfor small-volume injections. percent and not more than 115.0 percent of the
Other requirements-It meets the requirements under labeled amount of betamethasone (CZZHZ9FOs),
Injections and Implanted Drug Products (1).
and not less than 90.0 percent and not more than
Assay- 115.0 percent of the labeled amount of
Mobile phase-Prepare a filtered and degassed mixtureof betamethasone acetate (Cz4H 31 F0 6) .
methanol and 0.05 M monobasic potassium phosphate (1:1).
Make adjustments if necessary (see System Suitability under Packaging and storage-Preserve in multiple-dose
Chromatography (621 ». containers, preferablyof Type I glass.
Internal standard solution-Transfer about 100 mg of USPReference standards (11 )-
butylparaben to a 1OO-mL volumetric flask, add methanol to USP BetamethasoneAcetate RS
volume, and mix. USP BetamethasoneSodium Phosphate RS
Standardpreparation-Using an accurately weighed
quantity of USP Betamethasone Sodium Phosphate RS, Identification-
prepare a solution in water containing 4 mg per mL. Transfer A: Thin-layer chromatographic identification test (201)-
3.0 mL of this solution to a 25-mL volumetric flask, add 5.0 Test solution-Dilute 2 mL with 2 mL of methanol.
mL of Internal standard solution, dilute with water to volume, Standardsolution-Prepare a solution of USP Betamethasone
and mix to obtain a solution havinga known concentration of Sodium Phosphate RS in a mixture of methanol and water
about 0.5 mg of USP BetamethasoneSodium Phosphate RS (1:1) having a concentration of 2 mg per mL.
per mL. Developing solventsystem, Spray reagent, and Procedure -
Assay preparation-Transfer an accurately measured Proceedas directed for Identification test B under
volume of Injection, equivalentto about 9 mg of Betamethasone Sodium Phosphate.
betamethasone, to a 25-mL volumetric flask. Add 5.0 mL of B: Test solution-Use the Test solution prepared for
the Internal standardsolution, dilutewith water to volume,and Identification test A.
mix. Standardsolution-Prepare a solution of USP Betamethasone
Chromatographic system (see Chromatography (621 »- Acetate RS in a mixture of methanol and water (1:1) having a
The liquid chromatograph isequipped with a 254-nm concentration of 1.5 mg per mL.
detector and a 3.9-mm x 30-cmcolumnthat contains packing Developing solventsystem and Procedure-Proceed as directed
L1. The flow rate is about 2 mL per minute. Chromatograph for Identification test B under Betamethasone
the Standardpreparation, and record the peak responses as Bacterial Endotoxins Test (85) -It contains not more than
directed for Procedure: the resolution, R, between the analyte 29.2 USP Endotoxin Units per mg of betamethasone.

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552 Betamethasone / Official Monographs USP 43

pH (791): between 6.8 and 7.2. Betamethasone Sodium phosphate RS in the Standard
Other requirements-It meets the requirements under preparation,' Vis the Volume, in mL, of Injectable Suspension
Injections and Implanted Drug Products (1). taken; and R u and R s are the peak response ratios obtained
Assay- for betamethasone phosphate and methyltestosterone from
Mobilephase-Prepare a filtered and degassed mixture of the Assay preparation and the Standard preparation,
methanol and 0.075 M monobasic potassium phosphate respectively.
(7:5). Make adjustments if necessary (see System Suitability
under Chromatography (621 ».
Internal standard solution-Transfer about 50 mg of
methyltestosterone to a 50-mL volumetric flask, add methanol Betamethasone Valerate
to volume, and mix.
Standardpreparation-Transfer about 63 mg of USP
Betamethasone Sodium Phosphate RS, accurately weighed, to
a 25-mL volumetric flask, add Mobile phase to volume, and
mix (Solution 7). Transfer about 45 mg of USP Betamethasone OH

Acetate RS, accurately weighed, to a 25-mL volumetric flask,

add methanol to volume, and mix (Solution 2). Pipet 5 mL
each of Solution 7 and Solution 2 into a 1OO-mL volumetric o
flask. Add 10.0 mL of Internal standard solution, dilute with
Mobilephase to volume, and mix to obtain a Standard
C27H37F06 476.58
preparation havinq known concentrations of about 126 ~g of
USP Betamethasone Sodium Phosphate RS per mL and 90 ~g Pregna-1,4-diene-3,20-dione, 9-fluoro-11,21-dihydroxy-16-
of USP Betamethasone Acetate RS per mL. methyl-1 7-[(l-oxopentyl)oxy]-, (11 ~,16~)-.
Assay preparation-Using a "To contain" pipet transfer an 9-Fluoro-11 ~,17,21-trihydroxy-16~-methylpregna-1 ,4-diene-
accurately measured volume of the well-mixed Injectable 3,20-dione 17-valerate [2152-44-5].
Suspension, equivalent to about 9 mg of betamethasone
acetate, to a 1OO-mL volumetric flask. Rinse the pipet with » Betamethasone Valerate contains not less than
about 10 mL of Mobile phase, collecting the rinse in the .97.0 percent and not more than 103.0 percent of
volumetric flask. Add 10.0 mL of Internal standard solution, C27H3l061 calculated on the dried basis.
dilute with Mobile phase to volume, and mix.
Chromatographic system (see Chromatography (621»- Packaging and storage-Preserve in tight containers.
The liquid chromatograph is equipped with a 254-nm USP Reference standards (11)-
detector and a 3.9-mm x 30-cm column that contains packing USP Beclomethasone Dipropionate RS
L1. The flow rate is about 1.2 mL per minute. USP Betamethasone Valerate RS
Chromatograph the Standardpreparation, and record the
peak responses as directed for Procedure: the resolution, R, Identification-
between the betamethasone phosphate and betamethasone
acetate peaks is not less than 5.0, and the resolution, R,
between the betarnethasone acetate and internal standard
peaks is not less than 3.0, and the relative standard deviation
for replicate injections is not more than 2.0~.
" " ,··'-uv· e-, Chrrvmrttrutrrtrmir Identification Test (201)-
Procedure-Separately inject equal volumes (about 20 ~L)
of the Standardpreparation and the Assay preparation into the Test solution: mg per in alcohol.
chromatograph, record the chromatograms, and measure the Developing solvent system: a mixture of toluene and ethyl
responses for the major peaks. The relative retention times are acetate (1:1).
about 0.5 for betamethasone phosphate, 1.7 for Procedure-Proceed as directed in the chapter. Spray the plate
methyltestosterone, and 1.0 for betamethasone acetate. with a mixture of sulfuric acid, methanol, and nitric acid
Calculate the quantity, in mg, of betamethasone acetate (10:10:1), and heat at 105° for 15 minutes.
(C24H 3,F06) in each mL of the Injectable Suspension taken by Specific rotation (781 S): between +75° and +82°.
the formula: Test solution: 10 mg per mL, in dioxane.
Loss on drying (731 )-Dry it at 105° for 3 hours: it loses not
0.1C/V(R v/R s) more than 0.5% of its weight.
Residue on ignition (281): not more than 0.2%, a platinum
in which C is the concentration, in ~g per mL, of USP crucible being used.
Betamethasone Acetate RS in the Standardpreparation,' Visthe Chromatographic purity-
volume, in mL, of Injectable Suspension taken; and R u and R s Mobilephase-Prepare a filtered and degassed mixture of
are the peak response ratios obtained for betamethasone acetonitrile, water, and glacial acetic acid (550:450:1). Make
acetate and methyltestosterone from the Assay preparation adjustments if necessary (see System Suitability under
and the Standardpreparation, respectively. Calculate the Chromatography (621».
quantity, in mg, of betamethasone (C22H29FOs) equivalent to Test solution-Transfer about 4 mg of Betamethasone
the quantity of betamethasone sodium phosphate Valerate, accuratelyweighed, to a suitable flask. Add 10 mL of
(C22H2aFNa20aP), in each mL of the Injectable Suspension Mobilephase, and shake until dissolved.
taken by the formula: Chromatographic system (see Chromatography (621»-The
llquld chromatograph is equipped with a 254-nm detector
(392.46/516.41)(0.1 C/V)(R v/R s) and a 4.6-mm x 15-cm column that contains packing L1. The
flow rate is about 1 mL per minute. Chromatograph the Test
in which 392.46 and 516.41 are the molecular weights of solution, and record the peak responses as directed for
betamethasone and betamethasone sodium phosphate, Procedure: the resolution, R, between betamethasone valerate
respectively; C is the concentration, in ~g per mL, of USP

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 USP 43 Official Monographs / Betamethasone 553

and any impurity is not less than 1.5; and the column efficiency 90.0% and NMT 110.0% of the labeled amount of
is not less than 9000 theoretical plates. betamethasone (C22H29FOs), in a suitable cream base.
Procedure-Inject a volume (about 10 I-IL) of the Test
solution into the chromatograph, record the chromatogram, IDENTDFICATION
and measure all of the peak responses. Calculate the • A. The retention time of the major peak of the Sample
percentage of each impurity in the portion of Betamethasone solution corresponds to that of the Standardsolution, as
Valerate taken by the formula: obtained in the Assay.
• B. The UV spectrum of the major peak of the Sample
100(r/rs) solution corresponds to that of the Standardsolution, as
obtained in the Assay.
in which r; is the peak response for each impurity; and rs is the ASSAY
sum of all the peak responses: not more than 1.0% of any • PROCEDURE
individual impurity is found; and not more than 2.0% of total Solution A: Water
impurities is found. Solution B: Acetonitrile
Assay- Mobile phase: See Table 1.
Mobilephase-Prepare a filtered and degassed mixture of
acetonitrile and water (3:2). Make adjustments if necessary Table 1
(see System Suitability under Chromatography (621». Time Solution A Solution B
Internal standard solution-Transfer about 40 mg of (min) (%) (%)
beclomethasone dipropionate to a 1OO-mL volumetric flask, 0.0 63 37
add a solution of glacial acetic acid in methanol (1 in 1000) to
volume, and mix. 7.0 63 37
Standardpreparation-Transfer about 30 mg of USP 15.0 30 70
Betamethasone Valerate RS, accurately weighed, to a 50-mL
volumetric flask, add a solution of glacial acetic acid in 19.0 30 70
methanol (1 in 1000) to volume, and mix. Transfer 5.0 mL of 19.1 10 90
this solution to a suitable stoppered vial, add 10.0 mL of
Internal standard solution, and mix to obtain a solution having 21.0 10 90
a known concentration of about 0.2 mg of USP 21.1 63 37
Betamethasone Valerate RS per mL.
Assay preparation-Transfer about 60 mg of Betameth 25.0 63 37
asone Valerate, accurately weighed, to a 1OO-mL volumetric
flask, add a solution' of glacial acetic acid in methanol (1 in Diluent A: Tetrahydrofuran and water (50:50)
1000) to volume, and mix. Transfer 5.0 mL of this solution to Diluent B: Acetonitrile and water (40:60)
a suitable stoppered vial, add 10.0 mL of Internal standard System suitability solution: 25 I-Ig/mL of USP
solution, and mix. Betamethasone Valerate RS and 10 I-Ig/mL of USP
Chromatographic system (see Chromatography (621 )-The Betamethasone Valerate Related Compound A RS in Diluent
liquid chromatograph is equipped with a 254-nm detector B. Sonicate to dissolve if necessary.
and a 4-mm x 30-cm column that contains packing L1. The Standard solution: 25 I-Ig/mL of USP Betamethasone
flow rate is about 1.2 mL per minute. Chromatograph the Valerate RS in Diluent B. Sonicate to dissolve if necessary.
Standardpreparation, and record the peak responses as Sample solution: Nominally 20 I-Ig/mL of betamethasone,
directed for Procedure: the relative retention times are about prepared as follows. Transfer 1.0 mg of betamethasone
1.7 for beclomethasone dipropionate and 1.0 for from a portion of Cream to a suitable glass centrifuge tube.
betamethasone valerate; the resolution, R, between Add 15.0 mL of Diluent A and mix with a vortex mixer to
betamethasone valerate and beclomethasone dipropionate is disperse the sample thoroughly. Add 35.0 mL of Diluent B
not less than 4.5; and the relative standard deviation for and sonicate for 10 min with intermittent shaking.
replicate injections is not more than 2.0%. Centrifuge to obtain a clear supernatant. Pass through a
Procedure-Separately inject equal volumes (about 10 I-IL) suitable filter of 0.2-l-Im pore size using a glass syringe.
of the Standardpreparation and the Assay preparation into the Discard the first 1 mL.
chromatograph, record the chromatograms, and measure the Chromatographic system
responses for the major peaks. Calculate the quantity, in mg, (See Chromatography (621), System SUitability.)
of C27H37F06 in the portion of Betamethasone Valerate taken Mode: LC
by the formula: Detector: UV240 nm. For Identification B,use a diode array
detector in the range of 200-400 nm.
Column: 4.6-mm x 15-cm; 3.5-l.Jm packing L1
Temperatures
in which C is the concentration, in mg per mL, of USP Autosampler: 4 0
Betamethasone Valerate RS in the Standardpreparation; and Column: Ambient
Ru and Rs are the peak response ratios obtained from the Assay Flow rate: 1 mL/min
preparation and the Standardpreparation, respectively. Injection volume: 100 I-IL
System suitability
Samples: System suitability solution and Standardsolution
[NoTE-See Table 2 for relative retention times.]
Suitability requirements
Betamethasone Valerate Cream Resolution: NLT 2.0 between betamethasone valerate
and betamethasone valerate related compound A,
DEFINITION System suitabilitysolution
Betamethasone Valerate Cream contains an amount of Tailing factor: NMT 2.0, Standardsolution
betamethasone valerate (C 27H3l06) equivalent to NLT

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554 Betamethasone / OfficialMonographs USP 43

Relative standard deviation: NMT 1.0%, Standard Cv = nominal concentration of betamethasone in the
solution Sample solution (~g/mL)
Analysis Mrt = molecular weight of betamethasone, 392.46
Samples: Standardsolution and Sample solution Mr2 = molecular weight of betamethasone valerate,
Calculatethe percentage of the labeled amount of 476.58
betamethasone (C22H29FOs) in the portion of Cream
taken: Acceptance criteria: See Table 2. Disregardany impurity
peak lessthan 0.1%.
Result = (rufrs) x (CslCu) x (M r1IMr2 ) x 100
Table 2
to = peak response from the Sample solution Relative Acceptance
ts = peak response from the Standardsolution Retention Criteria,
C, = concentration of USP Betamethasone ValerateRS Name Time NMT(%)
in the Standard solution (~g/mL) Betamethasone 0.30 1.0
Cv = nominal concentration of betamethasone in the
Sample solution (~g/mL) Betamethasone valerate 1.00 -
Mrt = molecular weight of betamethasone, 392.46 Betamethasonevalerate related corn-
Mr2 = molecular weight of betamethasone valerate, pound A 1.04 1.0
476.58 Anyindividual unspecified
degradation product - 1.0
Acceptance criteria: 90.0%-110.0%
Totaldegradation products - 2.0
IMPURITIES
• ORGANIC IMPURITIES SPECIFIC TESTS
Solution A, Solution B, Mobile phase, Diluent A, Diluent • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
B, System suitability solution, Sample solution, and SPECIFIED MICROORGANISMS (62): It meets the
Chromatographic system: Proceed as directed in the requirements of the tests for absence of Staphylococcus
Assay. aureus and Pseudomonas aeruginosa.
Standard solution: 0.25 ~g/mL each of USP • MINIMUM FILL (755): Meets the requirements
Betamethasone RS, USP Betamethasone Valerate RS, and
USP Betamethasone Valerate Related Compound A RS in ADDITIONAL REQUIREMENTS
Diluent B. Sonicate to dissolveif necessary. • PACKAGING AND STORAGE: Preserve in collapsible tubes or
System suitability in tight containers.
Samples: System sUitability solution and Standardsolution • USP REFERENCE STANDARDS (11)
[NoTE-See Table 2 for relative retention times.] USP Betamethasone RS
Suitability requirements USP Betamethasone Valerate RS
Resolution: NLT 2.0 between betamethasone valerate USP Betamethasone Valerate Related Compound A RS
and betamethasone valerate related compound A, 9-Fluoro-11~, 17-dihydroxy-16~-methyl-3,20­
System suitability solution dioxopregna-1,4-dien-21-yl valerate.
Relative standard deviation: NMT 5%, Standard C27H37F06 476:58
solution
Analysis
Samples: Sample solution and Standard solution
Calculate the percentage of each specified deqradation
product in the portion of Cream taken: Betamethasone Valerate Lotion
Result = (rvlrs) x (CslCu) x 100 DEFINITION
Betamethasone Valerate Lotion contains an amount of
tu = peak response of each specifieddegradation betamethasone valerate (C27H3l06) equivalent to NLT
product from the Sample solution 95.0% and NMT 115.0% of the labeled amount of
rs = peak response of the corresponding USP betamethasone (C22H29FOs).
Reference Standard from the Standardsolution
Cs = concentration of the corresponding USP IDENTIFICATION
Reference Standard in the Standardsolution (~gl • A. The retention time of the major peak of the Sample
mL) solution corresponds to that of the Standard solution, as
Cv = nominal concentration of betamethasone in the obtained in the Assay.
Sample solution (~g/mL) • B. The UV spectrum of the major peak of the Sample
solution corresponds to that of the Standardsolution, as
Calculate the percentage of each unspecified degradation obtained in the Assay.
product in the portion of Cream taken: ASSAY
Result = (rufrs) x (Cs/Cu) x (M rtlMr2 ) x 100 • PROCEDURE
Solution A: Water
rv = peak response of each unspecified degradation Solution B: Acetonitrile
product from the Sample solution Mobile phase: See Table 7.
rs = peak response of betamethasone valerate from
Table 1
the Standardsolution
Cs = concentration of USP Betamethasone ValerateRS Time Solution A Solution B
(min) (%) (%)
in the Standard solution (~g/mL)
0.0 63 37

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USP 43 Official Monographs / Betamethasone 555

Table 1 (continued) IMPURITIES

Time Solution A Solution B • ORGANIC IMPURITIES
(min) (%) (%) Solution A, Solution B, Mobile phase, Diluent, System
7.0 63 37 suitability solution, Sample solution, and
Chromatographic system: Proceed as directed in the
15.0r 30 70 Assay. .
19.0 30 70 Standard solution: 0.001 mg/mL each of USP
Betamethasone RS, USP Betamethasone Valerate RS, and
19.1 10 90 USP Betamethasone Valerate Related Compound A RS in
21.0 10 90 Diluent. Sonicate to dissolve if necessary.
System suitability
21.1 63 37 Samples: System SUitability solution and Standardsolution
25.0 63 37 [NOTE-See Table 2 for relative retention times.]
Suitability requirements
Resolution: NLT 2.0 between betamethasone valerate
Diluent: Acetonitrile and water (40:60) and betamethasone valerate related compound A,
System suitability solution: 0.05 mg/mL of USP System SUitability solution
Betamethasone Valerate RS and 0.01 mg/mL of USP Relative standard deviation: NMT 5.0%, Standard
Betamethasone Valerate Related Compound A RS in solution
Diluent. Sonicate to dissolve if necessary. Analysis
Standard solution: 0.05 mg/mL of USP Betamethasone Samples: Sample solution and Standardsolution
Valerate RS in Diluent. Sonicate to dissolve if necessary. Calculate the percentage of each specified degradation
Sample solution: Nominally 0.04 mg/mL of product in the portion of Lotion taken:
betamethasone in Diluent, prepared as follows. Accurately
weigh and transfer a portion of Lotion to a suitable Result = (rulr s) x (CsICu) x 100
volumetric flask. Add about 80% of the final flask volume
of Diluent. Sonicate for about 5 min. Dilute with Diluent to t» = peak response of each specified degradation
volume. Pass through a suitable filter of 0.2-lJm pore size. product from the Sample solution
Chromatographic system ts = peak response of the corresponding USP
(See Chromatography (621), System Suitability.) Reference Standard from the Standardsolution
Mode: LC Cs = concentration of the corresponding USP
Detector: UV 240 nm. For Identification B, use a diode array Reference Standard in the Standardsolution(mgl
detector in the range of 200-400 nm. mL)
Column: 4.6-m'm x 15-cm; 3.5-lJm packing L1 Cu = nominal concentration of betamethasone in the
Temperatures Sample solution (mg/mL)
Column: Ambient
Autosampler: 4 0 Calculate the percentage of each unspecified degradation
Flow rate: 1 mL/min product in the portion of Lotion taken:
Injection volume: 50 IJL
System suitability Result = (rulr s) x (CslCu) x (M,dM'2) X 100
Samples: System suitability solution and Standardsolution
[NoTE-See Table 2 for relative retention times.] to = peak response of each unspecified deqradation
Suitability requirements . product from the Sample solution
Resolution: NLT 2.0 between betamethasone valerate rs = peak response of betamethasone valerate from
and betamethasone valerate related compound A, the Standardsolution
System sUitabilitysolution Cs =concentration of USP Betamethasone Valerate RS
Tailing factor: NMT 2.0, Standardsolution in the Standardsolution (mg/mL)
Relative standard deviation: NMT 1.0%, Standard Cu = nominal concentration of betamethasone in the
solution Sample solution (mg/mL)
Analysis M'l = molecular weight of betamethasone, 392.46
Samples: Standardsolution and Sample solution M'2 = molecular weight of betamethasone valerate,
Calculate the percentage of the labeled amount of 476.58
betamethasone (C22H29FOs) in the portion of Lotion taken:
Acceptance criteria: See Table 2. Disregard any impurity
Result = (rulrs) x (CslCu) x (M,dM'2) X 100 peak less than 0.1 %.
= peak response from the Sample solution Table 2
= peak response from the Standardsolution Relative Acceptance
= concentration of USP Betamethasone Valerate RS Retention Criteria,
in the Standardsolution (mg/mL) Name Time NMT (%)
= nominal concentration of betamethasone in the Betamethasone 0.30 1.0
Sample solution (mg/mL) .
= molecular weight of betamethasone, 392.46 Betamethasone
valerate 1.00 -
= molecular weight of betamethasone valerate,
476.58 Betamethasone
valerate related
compound A 1.04 10.0
Acceptance criteria: 95.0%-11 5.0%

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556 Betamethasone / Official Monographs USP 43

Table 2 (continued) Diluent A: Tetrahydrofuran and water (50:50)

Relative Acceptance Diluent B: Acetonitrile and water (40:60)
Retention Criteria, System suitability solution: 25 uq/m], of USP
Name Time NMT(%) Betamethasone Valerate RS and 10 J.lg/mL of USP
Any individual Betamethasone Valerate Related Compound ARS in Diluent
unspecified B. Sonicate to dissolve if necessary.
degradation -
Standard solution: 25 J.lg/mL of USP Betamethasone
product 1.0
Valerate RS in Diluent B. Sonicateto dissolve if necessary.
Total degradation prod-
- Sample solution: Nominally 20 J.lg/mL of betamethasone,
ucts 12.0 prepared as follows. Transfer 1.0 mg of betamethasone
from a portion of Ointment to a suitable glass centrifuge
SPECIFIC TESTS tube. Add 15.0 mL of Diluent A and mixwith a vortex mixer
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR to disperse the sample thoroughly. Add 35.0 mL of Diluent
SPECIFIED MICROORGANISMS (62): Meets the requirements B and sonicate for 10 min with intermittent shaking.
of the tests for absence of Staphylococcus aureus and Centrifuge to obtain a clear supernatant and use the clear
Pseudomonas aeruginosa supernatant.
• MINIMUM FILL (755): Meets the requirements Chromatographic system
• pH (791): 4.0-6.0 (See Chromatography (621), System SUitability.)
Mode: LC
ADDITIONAL REQUIREMENTS Detector: UV 240 nm. For Identification B, use a diode array
• PACKAGING AND STORAGE: Preserve in tight, light-resistant detector in the range of 200-400 nm.
containers,and store at controlled room temperature. Column: 4.6-mm x 15-cm; 3.5-J.lm packing L1
• USP REFERENCE STANDARDS (11) Flow rate: 1 mL/min
USP Betamethasone RS Injection volume: 100 J.lL
USP Betamethasone Valerate RS Autosampler temperature: 4°
USP Betamethasone Valerate Related Compound A RS System suitability
9-Fluoro-11~, 17-dihydroxy-16~-methyl-3,20­ Samples: System suitability solution and Standard solution
dloxopreqna-La-dien-Z'l-yl valerate, [NoTE-See Table 2 for relative retention times.]
C27H3l06 476.58 Suitability requirements
Resolution: NLT 2.0 between betamethasone valerate
and betamethasone valerate related compound A,
System suitability solution
Tailing factor: NMT 2.0, Standard solution
Betamethasone Valerate Ointment Relative standard deviation: NMT 1.0%, Standard
solution
DEFINITION Analysis
Betamethasone Valerate Ointment contains an amount of Samples: Standard solution and Sample solution
betamethasone valerate (C27H37F06) equivalent to NLT Calculatethe percentage of the labeled amount of
90.0% and NMT 110.0% of the labeled amount of betamethasone (C22H29FOs) in the portion of Ointment
betamethasone (C22H29FOs), in a suitable ointment base. taken:
IDENTIFICAnON
• A. The retention time of the major peak of the Sample Result = (rulrs) x (CsICu) x (Mr dMr2 ) x 100
solution corresponds to that of the Standard solution, as
obtained in the Assay. = peak response from the Sample solution
• B. The UV spectrum of the major peak of the Sample = peak response from the Standard solution
solution corresponds to that of the Standard solution, as = concentration of USP BetamethasoneValerateRS
obtained in the Assay. in the Standard solution (J.lg/mL)
= nominal concentration of betamethasone in the
ASSAY Sample solution (J.lg/mL)
• PROCEDURE = molecular weight of betamethasone, 392.46
Solution A: Water = molecular weight of betamethasone valerate,
Solution B: Acetonitrile 476.58
Mobile phase: See Table 1.
Acceptance criteria: 90.0%-110.0%
Table 1
IMPURITIES
Time Solution A Solution B
(min) (%) (%) • ORGANIC IMPURITIES
Solution A, Solution B, Mobile phase, Diluent A, Diluent
0.0 63 37 B, System suitability solution, Sample solution, and
7.0 63 37 Chromatographic system: Proceed as directed in the
Assay.
15.0 30 70 Standard solution: 0.25 J.lg/mL each of USP
19.0 30 70 Betamethasone RS, USP Betamethasone Valerate RS, and
USP Betamethasone Valerate Related Compound A RS in
19.1 10 90 Diluent B. Sonicate to dissolve ifnecessary.
21.0 10 90 System suitability
Samples: System suitability solution and Standard solution
21.1 63 37
[NOTE-See Table 2 for relative retention tlrnes.]
25.0 .63 37

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 USP 43 Official Mon~graphs / Betaxolol 557

Suitability requirements USP Betamethasone Valerate RS

Resolution: NLT 2.0 between betamethasone valerate USP Betamethasone Valerate Related Compound A RS
and betamethasone valerate related compound A, 9-Fluoro-l1 p,17-dihydroxy-16p-methyl-3,20-
System suitability solution dioxopregna-l ,4-dien-21-yl valerate.
Tailing factor: NMT 2.0, Standardsolution C27H37F06 476.58
Relative standard deviation: NMT 5.0%, Standard
solution
Analysis
Samples: Sample solution and Standardsolution
Calculate the percentage of each specified degradation Betaxolol Hydrochloride
product in the portion of Ointment taken:
Result = (ru/rs) x (Cs/Cu) x 100
• Hel
ru = peak response of each specified degradation
product from the Sample solution
rs = peak response of the corresponding USP ClsH29N03' HCI 343.89
Reference Standard from the Standardsolution 2-Propanol, 1-[4-[2-(cyclopropylmethoxy)ethyl]phenoxy]-3-
Cs = concentration of the corresponding USP [(l-methylethyl)amino]-, hydrochloride, (±)-;
Reference Standard in the Standard solution (~g/ (±)-1-[p-[2-(Cyclopropylmethoxy)ethyl]phenoxy]-3-
mL) (isopropylamino)-2-propanol hydrochloride [63659-19-8].
Cu = nominal concentration of betarnethasone in the
Sample solution (~g/mL) DEfiNITION
Betaxolol Hydrochloride contains NLT98.0% and NMT
Calculate the percentage of each unspecified degradation 102.0% of betaxolol hydrochloride (ClsH29N03 . HCI),
product in the portion of Ointment taken: calculated on the dried basis.
Result = (ru/r s) x (Cs/Cu) x (M rdMr2 ) x 100 IDENTIFICATION

ru = peak response of each unspecified degradation

product from the Sample solution
ts = peak response of betamethasone valerate from
the Standardsolution
Cs = concentration of USP Betamethasone Valerate RS
in the Standardsolution (pq/rnt)
Cu = nominal concentration of betamethasone in the
Sample solution (pq/rnt)
Mr 1 = molecular weight of betamethasone, -392.46
M r2 = molecular weight of betamethasone valerate,
476.58

Acceptance criteria: See Table 2. Disregard any impurity

peak less than 0.1 %. ·~~~I(Q&~1"MgY;i~~O)IDENTIFICATION TESTS-GENERAL (191),
Chemical Identification Tests, Chloride
Table 2 Analysis: Proceed as directed for test B.
Acceptance criteria: Meets the requirements
Relative Acceptance
Retention Criteria, ASSAY
Name Time NMT (%)
Betamethasone 0.30 1.0
Betamethasone valerate 1.00 -
Betamethasone valerate
related compound A 1.04 1.0
Any individual unspecified
degradation product - 1.0
Total degradation products - 2.0

SPECIfiC TESTS
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
SPECIFIED MICROORGANISMS (62): Meets the requirements
of the tests for absence of Staphylococcus aureus and
Pseudomonas aeruginosa
• MINIMUM flu (755): Meets the requirements
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in collapsible tubes or
in tight containers, and avoid exposure to excessive heat.
• USP REFERENCE STANDARDS (11)
USP Betamethasone RS

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558 Betaxolol / Official Monographs USP 43

SLlitabili Table 1
"17~lililig llelatiy~ Relative ~cc~ptiinc~
Relative tipn,:';NfV1'r(J~~~o/i~ici~ril~~~i~2()2() Reten~iori Response Criteria,
Analysis -- Name Time .Factol' NfI.1](%)
Samples: Standard solution and Sample solution Betcixoiol
Calculate the percentage of betaxolol hydrochloride hy~roxyethyl
(ClsH29N03' HCI) in the portion of Betaxolol arialog~ 0;3 1:3 OS
Hydrochloride taken: Bet~){olol
related
cOrnPoundAb q:9 1.2 tr5
Result =(rvlrs) x (CslCv) x 100
Betaxolol 1.0 1.0 ...,..
t» =peak response from the Sample solution Betaxololphel1ol
rs = peak response from the Standard solution analogC 1.0 1-.3 0.5
Cs = concentration of USP Betaxolol Hydrochloride RS
in the Standard solution (mg/mL) Betclxolotopen
chain'a!1alog~ 1'.7 0.95 0.5
Cv = concentration of Betaxolol Hydrochloride in the
Sample solution (mg/mL) Beta'~~~16Xil"ane
anal e 3.7 L3 0.5
Acceptance criteria: 98.0%-102.0% on the dried basis A.ny. unspecified ~

IMPURITIES impuritY LO 0,'0

• RESIDUE ON IGNITION (281): NMT 0.1 % Total impurities - - 1;0

a • _ :: lamino)pr6pilO.2~OI.
b pan-2-ol;
c4-[2~( yc opr .. . .... ..
d 1-[4-(2~Butoxyethyl)pheno _. s ropylamino)propan.2-ol.
.e 2-({[4-(2-:Cyclopropylmethoxy)ethyl]phenoxyjrnethyl)oxirane. & (USP 1.M~Y.2020)

SPECIFIC TESTS
• pH (791)
Sample solution: 20 mg/mL
Acceptance criteria: 4.5-6.5
• Loss ON DRVING (731)
Analysis: Dry under vacuum at 65° for 2 h.
-'Qn Acceptance criteria: NMT 1.0%
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers.
Store at room temperature.

Change .to read:

so utidn • USP REFERENCE STANDARDS (11)
Relative standard deviatiori:NMT 5~0%,:Standard USP Betaxolol Hydrochloride RS
solution .. . ·USP BetaxololRelated C ound A RS
Analysis 1-(4 Iphenoxy)-3-(i :ylamino)propan~2-01.
Samples: C14H 2 237.34.6. (USP ay-2020)
Id . ~:~asisof

Betaxolol Ophthalmic Solution

Resulf':; (r ~rrsrx. (C~X<::'vrxOjFi}(fP(j
DEFINITION
!r~ nseOfeacb·irripuritYfrorTftri~-$dmjJte Betaxolol Ophthalmic Solution is a sterile, aqueous, isotonic
solution of Betaxolol Hydrochloride. It contains a suitable
.ns<=-ofQe"taxofQ1fromthe:§[andattJ antimicrobial preservative. It contains the equivalent of NLT
90.0% and NMT 110.0% of the labeled amount of betaxolol
(ClsH29N03)'
"9~QJ9.ricle. RS
IDENTIFICATION
• A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
F obtained in the Assay.
• B. The UV spectrum of the major peak of the Sample
solution corresponds to that of the Standard solution, as
obtained in. the Assay.

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USP 43 Official Monographs / Betaxolol 559

ASSAY Calculate the percentage of each impurity in the portion of

• PROCEDURE Ophthalmic Solution taken:
Buffer: Dissolve 7.1 g of anhydrous dibasic sodium
phosphate in 800 mL of water, adjust with phosphoric acid Result = (rulrs) x (CslCu) x (M,dMr2) x 100
to a pH of 3.0, and dilute with water to 1000 mL.
Mobile phase: Acetonitrile and Buffer (1:1) to = peak response of each impurity from the Sample
Standard solution: 0.11 mg/mL of USP Betaxolol solution
Hydrochloride RS in Buffer ts = peak response of betaxolol from the Standard
Sample solution: Nominally 0.1 mg/mL of betaxolol in solution
Buffer from Ophthalmic Solution Cs = concentration of USP Betaxolol Hydrochloride RS
Chromatographic system in the Standard solution(mg/mL)
(See Chromatography (621), System Suitability.) Cv =nominal concentration of betaxolol in the Sample
Mode: LC solution(mg/mL)
Detector: UV or diode array 280 nm. [NoTE-Use the diode Mr1 = molecular weight of betaxolol, 307.43
array detector to perform Identification test 8.] Mr2 = molecular weight of betaxolol hydrochloride,
Column: 4-mm x 25-cm; packing L1 343.89
Flow rate: 1.1 mL/min
Injection volume: 10 J-1L Acceptance criteria
System suitability Single largest individual impurity: NMT 1%
Sample: Standard solution Any other individual impurity: NMT 0.3%
Suitability requirements
SPECIFIC TESTS
Tailing factor: 0.8-2.0
• STERILITY TESTS (71): It meets the requirements when
Relative standard deviation: NMT 2.0%
tested as directed for Test for Sterility of the Product to Be
Analysis
Examined, Membrane Filtration.
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
• pH (791): 4.0-8.0
betaxolol (ClsH29N03) in the portion of Ophthalmic A~DITIONAL REQUIREMENTS
Solution taken: • PACKAGING AND STORAGE: Preserve in tight containers.
Store at room temperature.
Result =(rulr s) x (CsICu) x (MrdMr2) x 100 • USP REFERENCE STANDARDS (11)
USP Betaxolol Hydrochloride RS
ru =peak responsefrom the Sample solution
rs = peak responsefrom the Standard solution
Cs = concentration of USP Betaxolol Hydrochloride RS
in the Standard solution (mg/mL)
Cv =nominal concentration of betaxolol in the Sample Betaxolol Tablets
solution (mg/mL) .
M r1 = molecular weight of betaxolol, 307.43 DEFINITION
Mr2 =molecular weight of betaxolol hydrochloride, Betaxolol Tablets contain an amount of Betaxolol
343.89 Hydrochloride equivalent to NLT 90.0% and NMT 110.0%
of the labeled amount of betaxolol hydrochloride
Acceptance criteria: 90.0%-110.0% (C1SH29N03' HCI).

IMPURITIES IDENTIFICATION
• ORGANIC IMPURITIES • A. The retention time of the major peak of the Sample
Mobile phase: Add 5 mL of phosphoric acid to 990 mL of solution corresponds to that of the Standard solution, as
water. Adjust with 2 M ammonium hydroxide to a pH of obtained in the Assay.
3.0, and dilute with water to 1000 mL. Prepare a mixture
of this solution and acetonitrile (45:55). Dissolve 3 g of
sodium dodecyl sulfate in 450 mL of the mixture.
Standard solution: 2.2 IJg/mL of USP Betaxolol
Hydrochloride RS in Mobilephase
Sample solution: Nominally equivalent to 0.2 mg/mL of
betaxolol in Mobile phase from Ophthalmic Solution ASSAY
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: 220 nm
Column: 4.6-mm x 25-cm; 1O-J-1m packing L1
Flow rate: 1.5 mL/min
Injection volume: 20 J-1L
System suitability
Sample: Standard solution
Suitability requirements
Relative standard deviation: NMT 5%
Tailing factor: NMT 2.5
Analysis
Samples: Standard solution and Sample solution

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560 Betaxolol / OfficialMonographs USP 43

and add 15 niL ofMobilephas~. Sonicate for 10 min, and • UNIFORMITY OF DOSAGE UNITS (90S)
centrifuge for 30 min. Use the clear supernataht " Procedure for content uniformity
Chromatographic system .. . Standard solution: 0.1 mg/mL of USP Betaxolol
(See.Chro rnatography'(621); System SUitability.) Hydrochloride RS in 0.1 N hydrochloric acid
Mode:LC . .. . ... Sample solution: Place 1 Tablet in a suitable volumetric
Detector: .UV 222 nm.For IdeHtificatlor1B; use-adiode array flaskto obtain a concentration of betaxolol hydrochloride,
detector' in the range of 210-400 nm~ based on the label claim, of 0.1 mg/mL. Add an amount
Column: 4.6-mmx 25-cm;5-lJmpackfrig' Ll of 0.1 N hydrochloric acid equal to 70% of the volume of
How rate: .1 ;5 mL/miri " . the flask. Shake by mechanical means until dissolved,
Inject'ion' volume:' JO'~L dilute with 0.1 N hydrochloric acid to volume, and mix.
System sUitability Filter, and discard the first 20 mL of the filtrate.
Sample: $tandardsolutIon Instrumental conditions
Suitability requirem s Mode: UV
Tailing factor: NM .0 Analytical wavelength: 274 nm
Relative standard deviation:NMT 1.Q%... (USP 1~May.2020) Cell path length: 1 cm
Analysis .. - Blank: 0.1 N hydrochloric acid
Samples: Standard solutionand Sample solution Analysis
Calculate the percentage of the labeled amount of Samples: Standard solution and Sample solution
betaxolol hydrochloride (ClsH29N03 . HCI) in the portion Calculate the percentage of the labeled amount of
of Tablets taken: betaxolol hydrochloride (C1SH29N03 . HC!) in the
Tablet taken:
Result = (rulrs) x (CsiCu) x 100
Result = (AulAs) x (CsICu) x 100
= peak response from the Sample solution
= absorbance of the Sample solution
= peak response from the Standard solution
= absorbance of the Standard solution
= concentration of USP Betaxolol Hydrochloride RS
in the Standard solution(mg/mL) = concentration of USP Betaxolol Hydrochloride RS
= nominal concentration of betaxolol in the Standard solution (mg/mL)
hydrochloride in the Sample solution(mg/mL) = nominal concentration of betaxolol
hydrochloride in the Sample solution(mg/mL)
Acceptance criteria: 90.0%-110.0%
Acceptance criteria: Meet the requirements
PERFORMANCE ,TESTS
IMPURITIES
Change to read:
Add. the followin
• DISSOLUTION (711)
...··ORGANIC
Medium: 0.01 N hydrochloric acid; 500 mL
Apparatus 2: 50 rpm Buffer, Mo rid
Time: 30 min . Chromat irected in the
Standard solution: USP Betaxolol Hydrochloride RS in Assay.
Memum ' System suitability solution: 1 mg/mL of USPBetaxolol
Sample solution: Sample per Dissolution (711). Dilute with Hydrochloride RS and 30 IJg!mL of USP Betaxolol Related
Mediumto a concentration that is similar to that of the Compound A in Mobile phase .
Standard solution. St ImL of USP Betaxolol
Instrumental conditions ilephase
Mode: UV S
Analytical wavelength: 274 nm
Cell path length: A 5-cm path length cell may be used for
lower dosage levels.
"'Analysis
Samples: .Standard solution ents
Determine the percentage .0 betw
betaxolol hydrochlorid~ (C betaxol
Relative standard deviatio
(AulAs) xCs xD x(VI L) x 100 Solution ,.
Analysis
Au == absorbance ofthe Samplesolution Samples:. Sample solutionand Stcmdifrd'solution
As =absorbance'of the5taodard"solution Calculate the percentage of eelch.degradation" prod!J(:fin
Cs = concen the portion of Tablets taken: '
D ~ dilution
V = volume Result :;;(rulrs)x ~CslCu) x (1/ f)x 10Q
'-. ='Iabel clar
= peak' response ofeac:n' impurity from the Sample
Tolerances: NLT 80% (Q) of the labeled amount of solution r.rt : . . '. • ••.••

betaxolol hydrochloride (C,sH29N0 3· HCI) is dissolved. = peak response fromfne Standard'soluti0n.

== concentration of USP Betaxolol Hydro'cnloriHe'R'S
in the Standard solution(mg/mL)' ' , ..

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 USP 43 Official Monographs / Bethanechol 561

=nominal concentration of betaxolol DEFINITION

hydrochloride in the Samplesolution (mg!mL) Bethanechol Chloride contains NLT 98.0% and NMT 101 .5%
F == relative respdnsefactor for eachinclividual of bethanechol chloride (C7H17CIN 202), calculated on the
impurity (see. Table 7) . dried basis.

Acceptance criteria: See Table 7. IDENTIFICATION

Table 1
Relative Relative J\cc~pt:a(lc~ IDEN'f.FlcATION'TESTS i(197),.lnfratea
Retention Response Criteria,
Name Tjm~ . Factor NfV11l{O/O) ~ .... . . ....... A (CNI-May-2020) ..
It B. The retention time of the major peak of the Sample
Betaxolol solution corresponds to that of the Standard solution, as
hydroxyethyl obtained in the Assay.
analog" 0.3 L3 0;5
• C. IDENTIFICATION TESTS-GENERAL, Chloride (191): Meets
Betaxolol related the requirements
compound Ab.~
~

0.9 L2
ASSAY
Betaxolol 1.0 1.0 ...... • PROCEDURE
8etaxolol phenol Buffer: 29 mg/L of edetic acid in a solution prepared as
artalog d 1.6 13 0.5 follows. Transfer a portion of edetic acid to a suitable
volumetric flask. Dissolve in water, using 50% of the final
Betaxolol open flask volume. Add 0.3 mL of nitric acid per L, and dilute with
chelin analoge 1.7 '0.95 0:5
water to volume.
aetaxolol oxirane ...... Mobile phase: Acetonitrile and Buffer (5:95)
amilogC,t 3.7 L3 System suitability solution: 0.1 mg/mL of bethanechol
Anyunspecified chloride in a solution prepared asfollows. Transfera portion
degradation ...... of bethanechol chloride to a suitable volumetric flask. Add
prodljct 1.0 0.2 4% of the final flask volume of 0.1 N sodium hydroxide,
and allow to stand for 15 min. Add 4% of the final flask
Total
degradation - ...... volume of 0.1 N hydrochloric acid. Dissolve in and dilute
products 1.0 with Mobilephase to volume.
Standard solution: 0.1 mg/mL of USP Bethanechol
a 1-[4-(2-Hydroxyethyl)phenoxy]-3-(iSopropylamino)propan-2-01; Chloride RS in Mobilephase
b 1-(4-Ehenoxy)-3-(is /n6)pr,opan-2-01. Sample solution: 0.1 mg/mL of Bethanechol Chloride in
C It isa ss impurityan for identification only.It is . Mobilephase
drug su ce, isnot reportedforthe drug product, and shoul _ Chromatographic system
in the t egradation products.
(See Chromatography (621), System Suitability.)
d 4-[2-Cycopropylmethoxy)ethyl]phenol. .
Mode: LC
e 1-[4-(2~Butoxyethyl)phenoxy]~3-(isopropylamino)propal1-2-01.
Detector: Conductivity
f 2-({[4:{2-Cydopropylmethoxy)ethyl]phenoxy}methyl)oxirane;... ·(~SP;-M~y:io20)
Column: 3.9-mm x 15.0-cm; packing L55
Temperatures
ADDITIONAL REQUIREMENTS Detector: 35°
• PACKAGING AND STORAGE: Preserve in tight containers.
Column: 30°
• LABELING: Label the Tablets to state both the content of the
Flow rate: 1 mL/min
betaxolol active moiety and the content of betaxolol Injection volume: 25 IJL
hydrochloride used in formulating them. System suitability
Samples: System suitability solution and Standard solution
Change. to read: [NoTE-See Table 7 for the relative retention times.]
• USP REFERENCE STANDARDS (11) Suitability requirements
USP Betaxolol Hydrochloride RS Resolution: NLT 0.8 between desacetyl methacholine
AUSP Betaxolol Related Compound A RS and bethanechol chloride, System suitability solution
Tailing factor: NMT 3.5, Standard solution
1-(4-Ethylphenoxy)-3-(isopropylamino)propan-2-oL
Relative standard deviation: NMT 3.0%, Standard
C14H23N02 237.34 A (USP l-May-2020) solution
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of bethanechol chloride
Bethanechol Chloride (C 7H 17CIN 202) in the portion of Bethanechol Chloride
taken:

Result = (r vir s) x (C siC v) x 100

CI

=peak response from the Sample solution

= peak response from the Standard solution
C7H 17CIN 202 196.68
1-Propanaminium, 2-[(aminocarbonyl)oxy]-N,N,N-trimethyl-
=concentration of USP Bethanechol Chloride RS in
the Standard solution(mg/mL)
, chloride, (±)-; = concentration of Bethanechol Chloride in the
(±)-(2-Hydroxypropyl)trimethylammonium chloride Sample solution (rnq/rnt)
carbamate [590-63-6].

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 562 Bethanechol / OfficialMonographs USP43

Acceptance criteria: 98.0%-101.5% on the dried basis SPECIFIC TESTS

IMPURITIES • pH (791)
Sample solution: 10 mg/mL of Bethanechol Chloride in
• RESIDUE ON IGNITION (281): NMT 0.1 % water
• ORGANIC IMPURITIES Acceptance criteria: 5.5-6.5
Buffer: 0.48 giL of methanesulfonic acid in water
• Loss ON DRYING (731 )
Mobile phase: Acetonitrile and Buffer (5:95) Analysis: Dry at 105 0 for 2 h.
System suitability solution: 0.1 mg/mL of bethanechol Acceptance criteria: NMT 1.0%
chloride in a solution prepared as follows. Transfer a portion
of bethanechol chloride to a suitable volumetric flask. Add ADDITIONAL REQUIREMENTS
4% of the final flask volume of 0.1 N sodium hydroxide, • PACKAGING AND STORAGE: Preserve in tight containers.
and allow to stand for 15 min. Add 4% of the final flask • USP REFERENCE STANDARDS (11)
volume of 0.1 N hydrochloric acid. Dissolve in and dilute USP Bethanechol Chloride RS
with Mobilephase to volume.
Standard solution: 1 J.lg/mL of USP Bethanechol Chloride
RS in Mobilephase
Sample solution: 0.1 mg/mL of Bethanechol Chloride in
Mobilephase Bethanechol Chloride Injection
. Chromatographic system
(See Chromatography (621), System Suitability.) DEFINITION
Mode: LC Bethanechol Chloride Injection is a sterile solution of
Detector: Conductivity Bethanechol Chloride in Water for Injection. It contains NLT
Column: 3.9-mm x 15.0-cm; packing L55 95.0% and NMT 105.0% of the labeled amount of
Temperatures bethanechol chloride (C7H17CIN20 2) .
Detector: 35 0 IDENTIFICATION
Column: 30 0 • A. The retention time of the major peak of the Sample
Flow rate: 1 mL/min solution corresponds to that of the Standardsolution, as
Injection volume: 50 J.lL obtained in the Assay.
System suitability • B. IDENTIFICATION. TESTS-GENERAL, Chloride (191): Meets
Samples: System suitability solution and Standardsolution the requirements
[NoTE-See Tablel for the relative retention times.]
Suitability requirements ASSAY
Resolution: NLT 0.8 between desacetyl methacholine • PROCEDURE
and bethanechol chloride, System SUitability solution Mobile phase: 20 mM methanesulfonic acid
Relativestandard deviation: NMT 10.0% for Diluent: 0.1 mg/mL of calcium chloride and 0.1 mg/mL of
bethanechol chloride, Standardsolution magnesium chloride in water
Analysis System suitability solution: 1 mg/mL of USP Bethanechol
Samples: Standardsolution and Sample solution Chloride RS in solution prepared as follows. Transfer a
Calculate the percentage of each impurity in the portion of portion of USP Bethanechol Chloride RS to: a suitable
Bethanechol Chloride taken: volumetric flask. Add 60% of the final volume of water, 8%
of the final volume of Diluent, and 2% of the final volume
Result = (r vir s) x (C siC v) x (1IF) x 100 of 0.1 N of sodium hydroxide. Dilute with water to volume.
Standard solution: 1.0 mg/mL of USP Bethanechol
ru = peak response of any impurity from the Sample Chloride RS in water
solution Sample solution: Nominally 1.0 mg/mL of bethanechol
rs = peak response of bethanechol chloride from the chloride from a volume of Injection in water
Standardsolution Chromatographic system
Cs = concentration of USP Bethanechol Chloride RS in (See Chromatography (621), System Suitability.)
the Standardsolution (mg/mL) Mode: LC
Cu = concentration of Bethanechol Chloride in the Detector: Conductivity
. Sample solution (mg/mL) Column: 4-mm x 25-cm; packing L53
F = relative response factor (see Table 1) Flow rate: 1 mL/min
Injection volume: 50 J.lL
Acceptance criteria: See Table 7. System suitability
Sample: System suitability solution
Table 1 [NoTE-See Table 1 for the relative retention times.]
Relative Relative Acceptance Suitability requirements
Retention Response Criteria, Resolution: NLT 2.0 between the calcium ion and
Name Time Factor NMT (%) desacetyl methacholine
Desacetyl metha- Tailing factor: NMT 4.5 for bethanechol chloride
choline- 0.9 1.2 1.0 Relative standard deviation: NMT 2.0% for
Bethanechol bethanechol chloride
chloride 1.0
-'- - Analysis
Samples: Standard solution and Sample solution
Anyindividual un- Calculate the percentage of the labeled amount of
specified impurity - 1.0 0.1
bethanechol chloride (C7H 17CIN2 0 2) in each mL of the
Total impurities - - 1.5 Injection taken:
a 2-Hydroxypropyltrimethyl ammonium chloride. Result = (r vir s) x (C siC v) x 100

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USP 43 OfficialMonographs / Bethanechol 563

ru = peak responsefrom the Sample solution

rs = peak responsefrom the Standard solution
Bethanechol Chloride Compounded
Cs = concentration of USP Bethanechol Chloride RS in Oral Solution
the Standard solution (mg/mL)
Cu = nominal concentration of the Sample solution DEFINITION
(mg/mL) Bethanechol Chloride Compounded Oral Solution contains
NLT 90.0% and NMT 110.0% of the labeled amount of
Acceptance criteria: 95.0%-105.0% bethanechol chloride (C7H17CIN202)'
Prepare Bethanechol Chloride Compounded Oral Solution 5
IMPURITIES mg/mL as follows (see Pharmaceutical Compounding-
• ORGANIC IMPURITIES Nonsterile Preparations (795).
Mobile phase, Diluent, System suitability solution,
Standard solution, Sample solution, Chromatographic
Bethanechol Chloride 500mg
system, and System suitability: Proceed as directed in the
Assay. Vehicle for Oral Solution (regular or suqar-free), NF, a
Analysis sufficient quantity to make 100 mL
Samples: Standard solution and Sample solution
Calculate the percentage of desacetyl methacholine in each Add Bethanechol Chloride powder and about 20 mL of Vehicle
mL of Injection taken: for Oral Solutionto a mortar, and mix. Add the Vehicle for Oral
Solution in small portions almost to volume, and mix
Result =(r vir s) x (C siC v) x 100 thoroughly after each addition. Transfer the contents of the
mortar, stepwise and quantitatively, to a calibrated bottle.
ru = peak response of desacetyl methacholine from Add enough Vehicle for OralSolution to bring to final volume,
the Sample solution and mix wel/.
rs = peak response of bethanechol chloride from the
Standard solution ASSAY
Cs = concentration of USP Bethanechol Chloride RS in • PROCEDURE
the Standard solution (mg/mL) Mobile phase: Acetonitrile and water (33:67)
Cu = nominal concentration of bethanechol chloride Standard solution: 500 IJg/mL of USP Bethanechol
in the Sample solution (mg/mL) Chloride RS in Mobilephase
Sample solution: Agitate the container of Oral Solution for
Acceptance criteria: See Table 7. 30 min on a rotating mixer, remove a 1O-mL sample, and
store in a clear glass vial at -700 until analyzed. At the time
Table 1 of analysis, remove the sample from the freezer, allow it to
Relative Acceptance reach room temperature, and mix with a vortex mixer for
Retention Criteria, 30 s. Dilute a suitable volume of Oral Solution with Mobile
Name Time NMT (%) phase to obtain a nominal concentration of 500 IJg/mL of
Sodium" 1.0 - bethanechol chloride.
Chromatographic system
Magnesium a 1.4 - (See Chromatography (621), System SUitability.)
Calcium- 1.6 - Mode: LC
Detector: UV 200 nm
Desacetyl methacholine" 2.0 4.0 Column: 4.6-mm x 25-cm; 5-lJm packing L11
Bethanechol chloride 2.8 - Flow rate: 0.7 mL/min
Injection volume: 20 IJL
a Included for identification purposes only. System suitability
b 2-Hydroxypropyltrimethyl ammonium chloride. Sample: Standardsolution
[NOTE-The retention time of bethanechol chloride is
SPECIFIC TESTS about 3 min.]
• BACTERIAL ENDOTOXINS TEST (85): NMT 25.0 USP Suitability requirements
Endotoxin Units/mg of bethanechol chloride Relative standard deviation: NMT 3.1 % for replicate
• pH (791): 5.5-7.5 injections
• OTHER REQ.UIREMENTS: It meets the requirements in Analysis
Injections and ImplantedDrug Products (1). Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
ADDITIONAL REQUIREMENTS bethanechol chloride (C7H 17CIN 20 2) in the portion of Oral
• PACKAGING AND STORAGE: Preserve in single-dose Solution taken:
containers, preferably of Type I glass.
• USP REFERENCE STANDARDS (11) Result = (rvlr s) x (CslCv) x 100
USP Bethanechol Chloride RS
= peak responsefrom the Sample solution
= peak response from the Standard solution
= concentration of USP Bethanechol Chloride RS in
the Standard solution (lJg/mL)
Cv = nominal concentration of bethanechol chloride
in the Sample solution (lJg/mL)

Acceptance criteria: 90.0%-110.0%

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564 Bethanechol / Official Monographs USP 43

SPECIFIC TESTS Analysis

II pH (791): 3.9-4.9 Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of
ADDITIONAL REQUIREMENTS bethanechol chloride (C7H 17CIN 202) in the volume of Oral
• PACKA.GING AND STORAGE: Packagein tight, light-resistant
containers. Store at room temperature or in a refrigerator. Suspension taken:
• BEYOND-Use DATE: NMT 60 days after the day on which it Result = (rvlrs) x (CslCv) x 100
was compounded when stored at room temperature or in
a refrigerator = peak responsefrom the Sample solution
• LABELING: Label it to state the Beyond-Use Date.
= peak responsefrom the Standardsolution
• USP REFERENCE STANDARDS (11)
USP Bethanechol Chloride RS
=concentration of USP Bethanechol Chloride RS in
the Standardsolution (lJg/mL)
Cv =nominal concentration of bethanechol chloride
in the Sample solution (lJg/mL)

Bethanechol Chloride Compounded Acceptance criteria: 90.0%-110.0%

Oral Suspension SPECIFIC TESTS

• pH (791): 3.9-4.9
DEFINITION ADDITIONAL REQUIREMENTS
Bethanechol Chloride Compounded Oral Suspensioncontains
• PACKA,GING AND STORAGE: Package in tight, light-resistant
NLT 90.0% and NMT 110.0% of the labeled amount of containers. Store at room temperature, or in a refrigerator.
bethanechol chloride (C7H,7CIN202)' • BEYOND-USE DATE: NMT 60 days after the day on which it
Prepare Bethanechol Chloride Compounded Oral Suspension was compounded when stored at room temperature or in
5 mg/mL as follows (see Pharmaceutical Compounding- a refrigerator '
Nonsterile Preparations (795». • LABELING: Label it to state that it is to be well shaken, and
. to state the Beyond-Use Date.
Bethanechol Chloride 500mg • USP REFERENCE STANDARDS (11)
Vehicle: a 1:1 mixture of Vehicle for Oral
USP Bethanechol Chloride RS
Sol~tion (regular or sugar-free), NF, and
Vehicle for Oral Suspension, NF, a sufficient quantity
to make 100 mL

If using Bethanechol Chloride tablets, add to a suitable mortar Bethanechol Chloride Tablets
and comminute to a fine powder, or add the Bethanechol
Chloride powder to the mortar. Add about 20 mL of the DEFINITION
Vehi~/e, and mix to a uniform paste. Add the' Vehicle in small
Bethanechol Chloride Tablets contain NLT 90.0% and NMT
pon;l<;>ns almost to volume, and mix thoroughly after each 110.0% of the labeled amount of bethanechol chloride
addltl~>n ..Transfer the contents of the mortar, stepwise and (C7H,7CIN202)'
quantitatively, to a calibrated bottle. Add sufficient Vehicle to IDENTIFICATION
final volume, and mix well. .
ASSAY
• PROCEDURE .
Mobile phase: Acetonitrile and water (33:67) • A•. . •. ~~~~~~~~~()~I~I~~~~I.~;I~~~.()~.rEsTs.(·1;9'J;);/1 Hfrrltfea
Standard solution: 500 IJg/mL of USP Bethanechol Spe.st,.q~J;qPY:J ?7M.. <l::N J~lv1iW"ZOZO)
Chloride RS in Mobilephase Sample: Nominally 100 mg of bethanechol chloride from a
Sample solution: Agitate the container of Oral Suspension suitable portion of pulverized Tablets prepared as follows.
for 30 mi~ on a rotating n:ixer, remove a 1O-mL sample, Pulverize a portion of Tablets equivalent to 100 mg of
and store m a clear glass Vial at -70 0 until analyzed. At the bethanechol chloride. Add 15 mL of ether and allow to
~ime of analysis, remove the sample from the freezer, allow digest for 15 min. Decant the ether, again' extract the
It to reach room temperature, and mix with a vortex mixer residue with 10 mL of ether, and discard the ether extracts.
for 3~ s. Dilute a suitable volume of Oral Suspension with Add 30 mL of alcohol to the residue. Shakefor 10 min and
MobIlephaseto obtain a nominal concentration of 500 IJgI allow to stand for 1 h with frequent agitation. Filter ~ith
mL of bethanechol chloride. suction, and evaporate the filtrate on a steam bath to
Chromatographic system dryness: the bethanechol chloride so obtained is
(See Chromatography (621), System Suitability.) recrystallized from alcohol and dried at 105 0 for 2 h.
Mode: LC • B. Th.e retention time of the major peak of the Sample
Detector: UV 200 nm solution corresponds to that of the Standardsolution as
Column: 4.6-mm x 25-cm; 5-lJm packing L11 obtained in the Assay. '
Flow rate: 0.7 mL/min ASSAY
Injection volume: 20 IJL • PROCEDURE
System suitability Buffer: 29 mg/L of edetic acid in solution prepared as
Sample: Standardsolution follows. Transfer a portion of edetic acid to a suitable
[NoTE-The retention time for bethanechol chloride is volumetric flask. Dissolvewith water, using 50% of the final
about 3 min.] . volume. Add 0.3 mL of nitric acid per L, and dilute with
Suitability requirements water to volume.
Relative standard deviation: NMT 3.1% for replicate Mobile phase: Acetonitrile and Buffer (5:95)
injections

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 USP 43 Official Monographs / Bethanechol 565

System suitability solution: 0.1 mg/mL of bethanechol Injection volumes

chloride in solution prepared as follows. Transfer a portion For the System suitability solution: 50 ~L
of bethanechol chloride to a suitable volumetric flask. Add For the Standard solution and Sample solution: 100 ~L
4% of the final volume of 0.1 N sodium hydroxide, and Analysis
allow to stand for 15 min. Add 4% of the final volume of Samples: Standardsolution and Sample solution
0.1 N hydrochloric acid. Dissolve in and dilute with Calculate the percentage of the labeled amount (Q) of
Mobile phase to volume. bethanechol chloride (C7H,7CINzOz) dissolved:
Standard solution: 0.1 mg/mL of USP Bethanechol
Chloride RS in Mobilephase Result =(rvlrs) x Cs x V x (1 I L) x 100
Sample solution: Nominally 0.1 mg/mL of bethanechol
chloride from a suitable amount of powdered Tablets in to = peak response from the Sample solution
solution prepared as follows. Add a portion of fine powder, rs = peak response from the Standardsolution
equivalent to 1 Tablet, from NLT 20 Tablets to a suitable Cs = concentration of USP Bethanechol Chloride RS in
volumetric flask. Dissolve in Mobilephase, using 60%-70% the Standardsolution (mg/mL)
of the final volume. Sonicate for 20 min. Shake by V = volume of the Medium, 900 mL
mechanical means for 15 min. Dilute with Mobilephase to L = label claim (mg/Tablet)
volume, and mix. Allow to stand for 10 min, and pass
through a I-urn glass filter, discarding the first 3 mL of the Tolerances: NLT 80% (Q) of the labeled amount of
filtrate. bethanechol chloride (C7H,7CINzOz) is dissolved.
Chromatographic system • UNIFORMITY OF DOSAGE UNITS (90S): Meet the
(See Chromatography (621), System Suitability.) requirements
Mode: LC
Detector: Conductivity IMPURITIES
Column: 3.9-mm x 15.0-cm; packing L55 • ORGANIC IMPURITIES
Temperatures Buffer: 0,48 giL of methanesulfonic acid in water
Detector: 35° Mobile phase: Acetonitrile and Buffer(5:95)
Column: 30° System suitability solution: 0.1 mg/mL of bethanechol
Flow rate: 1 mL/min chloride in solution prepared as follows. Transfer the
Injection volume: 50 ~L bethanechol chloride to a suitable volumetric flask. Add 4%
System suitability of the final volume of 0.1 N sodium hydroxide, and allow
Samples: System suitability solution and Standardsolution to stand for 15 min. Add 4% of the final volume of 0.1 N
[NoTE-See Table 1 for the relative retention times.] hydrochloric acid. Dissolve in and dilute with Mobile phase
Suitability requirements to volume.
Resolution: NLT 0.8 between desacetyl methacholine Standard solution: 1 ~g/mL of USP Bethanechol Chloride
and bethanechol chloride, System suitability solution RS in Mobilephase
Tailing factor: NMT 3.5, Standardsolution Sample solution: Nominally 0.1 mg/mL of bethanechol
Relative standard deviation: NMT 3.0%, Standard chloride from a suitable amount of powdered Tablets in
solution solution prepared as follows. Add a portion of fine powder,
Analysis equivalent to 1 Tablet, from NLT 20 Tablets to a suitable
Samples: Standardsolution and Sample solution volumetric flask. Dissolve in Mobilephase, using 600/0-70%
Calculate the percentage of the labeled amount of of the final volume. Sonicate for 20 min. Shake by
bethanechol chloride (C7H,7CINzOz) in the portion of mechanical means for 15 min. Dilute with Mobilephase to
Tablets taken: volume, and mix. Allow to stand for 10 min, and pass
through a T-urn glass filter, discarding the first 3 mL of the
Result = (rv/rs) x (CslCv) x 100 filtrate.
Chromatographic system
to = peak response from the Sample solution (See Chromatography (621), System Suitability.)
rs = peak response from the Standardsolution Mode: LC
Cs = concentration of USP Bethanechol Chloride RS in Detector: Conductivity
the Standardsolution (mg/mL) Column: 3.9-mm x 15.0-cm; packing L55
Cv = nominal concentration of the Sample solution Temperatures
(mg/mL) Detector: 35°
Column: 30°
Acceptance criteria: 90.0%-110.0% Flow rate: 1 mL/min
Injection volume: 50 ~L
PERFORMANCE TESTS System suitability
• DISSOLUTION (711) Samples: System suitability solution and Standard solution
Medium: 0.1 N hydrochloric acid; 900 mL [NOTE-See Table 1 for the relative retention times.]
Apparatus 2: 50 rpm Suitability requirements
Time: 30 min Resolution: NLT0.8 between desacetyl methacholine
Buffer, Mobile phase, and System suitability solution: and bethanechol chloride, System suitabilitysolution
Proceed as directed in the Assay. Relative standard deviation: NMT 10.0% for
Standard solution: (LI900) mg/mL of USP Bethanechol bethanechol chloride, Standardsolution
Chloride RS in Medium, where L is the label claim in mgl Analysis
Tablet Samples: Standardsolution and Sample solution
Sample solution: A portion of solution under test Calculate the percentage of each impurity in the portion of
Chromatographic system and System suitability: Proceed Tablets taken:
as directed in the Assay, except for the following
parameters: Result =(rvlrs) x (CsICv) x (liP) x 100

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566 Bethanechol / OfficialMonographs USP 43

tu = peak response for any impurity in the Sample Standard solution: 0.05 mg/mL of USP Bicalutamide RS in
solution Diluent
ts = peak response of bethanechol chloride from the Sample solution: 0.05 mg/mL of Bicalutamide in Diluent
Standard solution Chromatographic system
Cs = concentration of USP Bethanechol Chloride RS in (See Chromatography(621), System Suitability.)
the Standard solution (mg/mL) Mode: LC
Cv = nominal concentration of bethanechol chloride Detector: UV 270 nm
in the Sample solution (mg/mL) Column: 4.0-mm x 10-cm; 3-lJm packing L1
F = relative response factor (see Table 7) Flow rate: 1 mL/min
Injection size: 10 IJL
Acceptance criteria: See Table 1. System suitability
Samples: System suitability solution and Standard solution
Table 1 [NOTE-The relative retention times for bicalutamide
Relative Relative Acceptance related compound A isomer A and bicalutamide
Retention Response Criteria, related compound A isomer Bare 0.75 and 0.78,
Name Time Factor NMT(%) respectively.]
Desacetyl metha- Suitability requirements
choline" 0.9 1.2 1.0 Resolution: NLT2.0 between bicalutamide related
compound A isomer Band bicalutamide, System
Bethanechol
chloride 1.0 - - suitability solution
Relative standard deviation: NMT 2%, Standard
Any unspecified degra- solution
dation product - 1.0 0.2
Analysis
Total impurities - - 1.5 Samples: Standard solution and Sample solution
Calculate the percentage of C18Hll4N204S in the portion
a 2-Hydroxypropyltrimethyl ammonium chloride. of Bicalutamide taken:
ADDITIONAL REQUIREMENTS Result = (ru/r s) x (Cs/Cu) x 100
• PACKAGING AND $TORAGE: Preserve in tight containers.
• USP REFERENCE STANDARDS (11) = peak response from the Sample solution
USP Bethanechol Chloride RS = peak response from the Standard solution
= concentration of USP Bicalutamide RS in the
Standardsolution (mg/mL)
= concentration of the Sample solution (mg/mL)
Bicalutamide Acceptance criteria: 98.0%-102.0% on the anhydrous and
solvent-free basis
IMPURITIES
INORGANIC IMPURITIES
• Residue on Ignition (281): NMT 0.1%
C18H14F4N204S 430.37 ORGANIC IMPURITIES
Propanamide, N-[ 4-cyano-3-(trifluoromethyl)phenyl]-3-[(4- ~ Procedure
fluorophenyl)sulfonyl]-2-hydroxy-2-methyl-, (±)-; Solution A, Solution B, Diluent, System suitability solution,
(±)-4'-Cyano-u,u,u-trifluoro-3-[(p-fluorophenyl)sulfonyl]-2- and Chromatographic system: Proceed as directed in the
methyl-m-Iactotoluidide [90357-06-5]. Assay.
Mobile phase: See the gradient table below.
DEFINITION
Bicalutamide contains NLT 98.0% and NMT 102.0% of
C18H14F4N204S, calculated on the anhydrous and solvent-free Time Solution A Solution B
(min) (%) (%)
basis. "
0 67 33
IDENTIFICATION
16.5 67 33
26.5 40 60
32.5 5 95
32.6 67 33
• B. The of the major peak of the Sample
35 33
solution corresponds to that of the Standard solution, as 67
obtained in the Assay.
Standard solution: 1 IJg/mL of USP Bicalutamide RS in
ASSAY Diluent
• PROCEDURE Sample solution: 1 mg/mL of Bicalutamide in Diluent
Solution A: 0.01% (v/v) of trifluoroacetic acid in water System suitability
Solution B: 0.01% (v/v) of trifluoroacetic acid in acetonitrile Sample: System sUitability solution
Mobile phase: Solution A and Solution B(52:48) Suitability requirements
Diluent: Solution A and Solution B (1:2) Resohrtlon 1: NLT 0.8 between bicalutamide related
System suitability solution: 5 IJg/mL of USP Bicalutamide compound A isomer A and bicalutamide related
RelatedCompound A RS and 50 IJg/mL of USP Bicalutamide compound A isomer B
RS in Diluent

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 USP 43 Official Monographs / Bicalutamide 567

Resolution 2: NLT 8.5 between bicalutamide related

compound A isomer Band bicalutamide Bicalutamide Tablets
Analysis DEFINITION
Samples: Standard solution and Sample solution Bicalutamide Tabletscontain NLT 90.0% and NMT 110.0% of
Calculatethe percentage of each impurity in the portion of the labeled amount of bicalutamide (ClsH14F4N204S),
Bicalutamide taken:
IDENTIFICATION
Result = (ru/rs) x (Cs/Cu) x (1 IF) x 100 • A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as
= peak area of each impurity from the Sample obtained in the Assay.
solution
= peak area of bicalutamide from the Standard
solution
=concentration of bicalutamide in the Standard
solution(mg/mL)
=concentration of Bicalutamide in the Sample
solution(mg/mL)
F = relative response factor (see Impurity Table 7) ASSAY

Acceptance criteria
Individual impurities: See Impurity Table 7.
Total impurities: NMT 0.5% • PROCEDURE
Mobile phase: Tetrahydrofuran, acetonitrile, and water
Impurity Table 1 (20:15:65)
Relative Relative Acceptance
System suitability stock solution: 0.8 mg/mL of USP
Retention Response Criteria, Bicalutamide RS and 0.4 mg/mL of USP Bicalutamide
Name Time Factor NMT (%) Related Compound B RS in tetrahydrofuran
System suitability solution: 0.04 mg/mL of USP
Bicalutamide aminobenzo-
nitrile" 0.30 1.4 0.1 Bicalutamide RS and 0.02 mg/mL of USP Bicalutamide
Related Compound B RS in Mobilephasefrom the System
Bicalutamide related com- suitabilitystock solution
pound A isomer Ab 0.64 1.0 0.1
Standard stock solution: 0.8 mg/mL of USP Bicalutamide
Bicalutamide related com- RS in tetrahydrofuran
pound A isomer Bb I 0.67 1.0 0.1 Standard solution: 0.04 mg/mL of USP Bicalutamide RS in
Desfluoro blcalutamlde- 0.83 1.1 0.2
Mobilephasefrom the Standard stock solution .
Sample stock solution: 0.5 mg/mL of bicalutamide in
2-Fluoro blcalutarnide" 0.94 1.0 0.2 tetrahydrofuran prepared as follows. Transfer an equivalent
Bicalutamide 1.00 - - to 50 mg of bicalutamide from finely powdered Tablets
(NLT 20) into a 1OO-mL volumetricflask. Add 50 mLof
Deoxyblcalutarnldes 1.33 1.0 0.2 tetrahydrofuran, and sonicate for NLT 10 min to cornplete
Bicalutamide sulfide! 1.56 1.0 0.1
dissolution. Allow to cool to room temperature, and
dilute with tetrahydrofuran to volume. Pass through a
Any unspecified impurity - 1.0 0.1 suitable filterof 0.45-lJm pore size.
Sample solution: 0.04 mg/mL of bicalutamide in Mobile
a 4-Amino-2-(trifluoromethyl)benzonitrile. phasefrom the Sample stock solution
b N-[4-Cyano-3-(trifluoromethyl)phenyl]-3-[(4-fluorophenyl)sulfinyl]-2- Chromatographic system
hydroxy-2-methylpropanamide.
c N-[4-Cyano-3-(trifluoromethyl)phenyl]-2-hydroxy-2-methyl-3-
(See Chromatography (621), System Spitability.)
(phenylsulfonyl)propanamide. Mode: LC
d N-[4-Cyano-3-(trifluoromethyl)phenyl]-3-(2-fluorophenylsulfonyl)-2-
hydroxy-2-methylpropanamide.
e N-[ 4-Cyano-3-(trifluoromethyl)phenyl]-3-(4-fluorophenylsulfonyl)-2- x
methylpropanamide.
f N-[ 4-Cyano- 3-(trifluoromethyl)phenyl]-3-(4-fluorophenylthio)-2-hydroxy-2-
Column temperature:
methylpropanamide. Flow rate: 1.5 mL/min
Injection volume: 10 IJL
SPECIFIC TESTS System suitability
• WATER DETERMINATION, Method I (921): NMT 0.2% Sample: System suitability solution
[NoTE-The relative retention times for bicalutamide
ADDITIONAL REQUIREMENTS and bicalutamide related compound Bare 1.0 and
• PACKAGING AND STORAGE: Preserve in tight containers, and 1.1, respectively.]
store at room temperature. Suitability requirements
• USP REFERENCE STANDARDS (11) Resolution: Greater than 1.9 between bicalutamide and
USP Bicalutamide RS bicalutamide related compound B
USP Bicalutamide Related Compound A RS Tailing factor: Less than 1.3 for orcaiutamroe
[N-[ 4-cyano-3-(trifluoromethyl)phenyl]-3-[(4- Relative standard deviation: NMT for
fluorophenyl)sulfinyl]-2-hydroxy-2- bicalutamide
methylpropanamide] Analysis
(ClsH14F4N203S 414.37) Samples: Standard solution and Sample solution

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568 Bicalutamide / Official Monographs USP 43

Calculate the percentage of the labeled amount of

bicalutamide (ClsH14F4N204S) in the portion of Tablets
taken:
Result = (rulrs) x (CsICu) x 100
= peak area from the Sample solution
= peak area from the Standardsolution al' cQnditions
= concentration of USP BicalutamideRS in the rolet-Visible Spectroscopy (857).)
Standardsolution (mg/mL)
= nominal concentration of bicalutamide in the v~lerigtfl:,t(O hn}
Sample solution (mg/mL) m
A
Acceptance criteria: 90.0%-110.0% Samples: Standard sol .-lion
PERFORMANCE TESTS Calculatethe perce"ta ount of
bicalutamige (C1sH14
Change to read:
Resulf; {Au!As)?'. C;x.DfV~(V LfxJOO
• DISSOLUTION (711)
Test 1 Au . =absorbance of th~ SarripJe:sol
Medium: 1.0% w/v sodium lauryl sulfate in water; 1000 As ='absorbance of tne5tanaard·s
mL Cs =c '. .. amideRS. ih the
Apparatus 2: 50 rpm 5
Time: 45 min D =dil
Standard solution: 0.05 mg/mL of USP Bicalutamide RS in V 7' vo ..
Medium prepared as follows. Transfer USP Bicalutamide L:. == label claim (l11g
RS to a suitable volumetric flask, dissolve in
tetrahydrofuran equivalent to 1% of the final volume, and Tolerances: 80% (. . (the I .
dilute with Medium to volume. bkaJutami (C1sH14F4 S) is d ~i018)
Sample solution: Pass a portion of the solution under test • UNIFORMITY OF DOSAGE UNITS (905)
through a suitable filter of 0.45-l.lm pore size. Procedure for content uniformity
Instrumental conditions Diluent: 10 mg/mL of sodium lauryl sulf~te in wa!er .
(See Ultraviolet-Visible Spectroscopy (857).) Standard solution: 0.05 mg/mL of USP Blcalutamlde RS In
Mode: UV Diluent. [NOTE-Dissolve USP Bicalutamide RS in a
Analytical wavelength: 270 nm minimum volume of tetrahydrofuran before dilution with
Blank: Medium Diluent.]
Analysis Sample stock solution: Transfer 1 Tablet to a 1.00-mL
Samples: Standardsolution and Sample solution volumetric flask. Add 10 mLof water, and sonicate for
Calculate the percentage of the labeled amount of approximately 30 min. Add80 mLof tetrahydrofuran, and
bicalutamide (ClsH14F4N204S) dissolved: sonicate for 30 min to complete dissolution of
bicalutamide. Allow to cool to room temperature, and
Result = (AulAs) x Cs x V x (1IL) x 100 dilute withtetrahydrofuran to volume. Pass a portion of
=absorbance of the Sample solution the solution through a suitable filterof 0.45-J.Jm pore size.
= absorbance of the Standardsolution
Sample solution: Transfer 10.0 ~L of the Sample sto~k
solution into a 1OO-mL volumetrrc flask, and dilute With
= concentration of USP Bicalutamide RS in the Diluent to volume.
Standardsolution (mg/mL) Instrumental conditions
V = volume of Medium (mL) (See Ultraviolet- Visible Spectroscopy (857).)
L =label claim (mg/Tablet) Mode: UV
Analytical wavelength: 270 nm
Tolerances: NLT 80% (Q) of the labeled amount of Blank: Diluent
bicalutamide (ClsH14F4N204S) is dissolved. Analysis
Test 2: Ifthe product complies with this test, the labeling Samples: Standardsolution and Sample solution
indicates that the product meets USP Dissolution Test 2. Calculate the percentage of the labeled amount of
Medium, Apparatus 2, Time, Standard solution, Sample bicalutamide (ClsH14F4N204S) in the Tablet taken:
solution, Instrumental conditions, and Analysis:
Proceed as directed for Test 1. Result = (AulAs) x (CsiCu) x 100
Tolerances: NLT 75% (Q) of the labeled amount of
bicalutamide (ClsH14F4N204S) is dissolved. ..' .. ' =absorbance of the Sample solution
·Test 3:. ifthe productcompli test, t abeling = absorbance of the Standardsolution
indicates that the product mee .. olutio 3: = concentration of USP Bicalutamide RS in the
in: 1.0% (w/v) sodiumlauryl sulfate inw r; Standardsolution (mg/mL)
L = nominal concentration of bicalutamide in the
us 2: .75 rpm Sample solution (mg/mL)
60mii,
S .. ard solution: O~Olmg/mLofU$P Bkalutarniae RSin Acceptance criteria: Meet the requirements
Medium, sonicateto aid dis.solution. Passa portion of the

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 USP 43 OfficialMonographs / Biotin 569

IMPURITIES
• LIMIT OF 4-AMINO-2-(TRIFLUOROMETHYL)BENZONITRILE
Biotin
Mobile phase and System suitability solution: Prepare as
directed in the Assay.
Standard stock solution: 0.2 mg/mL of USP Bicalutamide
rHNKi····~
o
NH H 0
OH
RS in tetrahydrofuran H S
Standard solution: 0.02 mg/mL of USP Bicalutamide RS in
Mobile phasefrom the Standardstock solution ClOH,6NZ03S 244.31
Sample solution: Transferthe equivalent to 50 mg of 1H-Thieno[3,4-cI]imidazole-4-pentanoic acid, hexahydro-2-
bicalutamide from powdered Tablets (NLT 20) to a 25-mL oxo-, [3aS-(3aa,4~,6aa)]-;
volumetric flask. Add 2 mL of tetrahydrofuran, and allow to (3aS,4S,6aR)-Hexahydro-2-oxo-l H-thieno[3,4-cI]imidazole-
st~nd for 5 min. Add 20 mL of Mobilephase, sonicate for 10
4-valeric acid [58-85-5]. .
min, .and allow to cool to room temperature. Dilute with DEFINITION
MobIlephase to volume, and pass through a suitable filter Biotin contains NLT 97.5% and NMT 102.0% of biotin
of 0.2-lJm pore size. (C'OH'6 NZ03S),
Chromatographic system
(See Chromatography (621), System Suitability.) IDENTIFICATION
Mode: LC
Detector: UV 220 nm
Column: 5-mm x 12.5-cm; 3-lJm packing II
Column temperature: 50°
Flow rate: 1.5 mL/min )
Injection volume: 10 IJl Specific Tests for Optical
System suitability .. Rotation (781S), Rotation.
Sample: System suitability solution • C. The retention of the major peak of the Sample
[NoTE-The relative retention times of 4-amino- solution corresponds to that of the Standardsolution as
2-(trifluoromethyl)benzonitrile, bicalutamide, and obtained in the Assay. '
bicalutamide related compound B are about 004, 1.0, ASSAY
and about 1.1, respectively.]
• PROCEDURE
Suitability requirements Buffer solution: Dissolve 1 9 of sodium perchlorate
Resolution: Greater than 1.9 between bicalutamide and monohydrate in 500 mL of water, add 1 ml of phosphoric
bicalutamide related compound B acid, and dilute with water to 1000 ml.
Tailing factor: Less than 1.3 for bicalutamide Mobile phase: Acetonitrile and Buffer solution (8.5: 91.5)
Relative standard deviation: NMT 2.0% for Diluent: Acetonitrile and water (1:4)
bicalutamide Standard solution: 0.1 mg/ml of USP Biotin RS in Diluent.
Analysis Sonicate if necessary to dissolve.
Samples: Standard solution and Sample solution Sample solution: 0.1 mg/mL of Biotin in Diluent. Sonicate
Calculate the percentage of 4-amino-2-(trifluoromethyl) if necessary to dissolve.
benzonitrile in the portion of Tablets taken: Chromatographic system
(See Chromatography (621), System Suitability.)
Result = (ru/rs) x (Cs/Cu) x (1/ F) x 100
Mode: lC
= peak area of 4-amino-2-(trifluoromethyl) Detector: UV 200 nm
benzonitrile from the Sample solution . Column: 4.6-mm x 15-cm; 3-lJm packing l7
= peak area of bicalutamide from the Standard Flow rate: 1.2 mL/min
solution Injection volume: 50 IJL
=concentration of USP Bicalutamide RS in the System suitability
Standard solution (mg/ml) Sample: Standardsolution
= nominal concentration of bicalutamide in the Suitability requirements
Sample solution (mg/mL) Tailing factor: NMT 1.5
F = relative response factor for 4-amino- Relative standard deviation: NMT 2.0% for replicate
2-(trifluoromethyl)benzonitrile, 1 A injections
Analysis
Acceptance criteria: NMT 0.1% Samples: Standardsolution and Sample solution
Calculate the percentage of biotin (ClOH16Nz03S) in the
ADDITIONAL REQUIREMENTS portion of Biotin taken:
• PACKAGING AND STORAGE: Preserve in tight containers, and
store at controlled room temperature. Result =(ru/rs) x (Cs/Cu) x 100
• LABELING: When more than one Dissolution test is given, the
labeling states the test used only if Test 1 is not used. = peak responsefrom the Sample solution
• USP REFERENCE STANDARDS (11) = peak responsefrom the Standardsolution
USP Bicalutamide RS· =concentration of USP Biotin RS in the Standard
USP Bicalutamide Related Compound B RS solution (mg/mL)
(RS)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(3- =concentration of Biotin in the Sample solution
fluorophenylsulfonyl)-2-hydroxy-2- (mg/ml)
methylpropanamide.
C'SH'4F4NZ04S 430.37 Acceptance criteria: 97.50/0--102.0%

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570 Biotin / OfficialMonographs USP 43

IMPURITIES Chromatographic system

• RElATED COMPOUNDS (See Chromatography (621), System Suitability.)
Buffer solution, Mobile phase, Diluent, Standard Mode: LC
solution, Sample solution, Chromatographic system, Detector: UV 200 nm
and System suitability: Proceed as directed in the Assay. Column: 4.6-mm x 15-cm; 3-l..Jm packing L7
Analysis Flow rate: 1.2 mL/min
Sample: Sample solution Injection volume: 50 I..JL
Measure the peak responses of the Sample solution. System suitability
Calculatethe percentage of each impurity in the portion of Sample: Standardsolution
Biotin taken: Suitability requirements
Tailing factor: NMT 1.5
Result =(rVlrT) x 100 Relative standard deviation: NMT 2.0% for replicate
injections
tu = peak response of each impurity from the Sample Analysis
solution Samples: Standardsolution and Sample solution
rr = sum of the peak responses of all the peaks from Calculate the percentage of biotin (ClOH16Nz03S) in the
the Sample solution portion of Capsules taken:
Acceptance criteria Result = (rufrs) x (CsICv) x 100
Individual impurity: NMT1.0%
Total impurities: NMT 2.0% to = peak response from the Sample solution
SPECIFIC TESTS rs = peak response from the Standardsolution
• OPTICAL ROTATION, Specific Rotation (781S) Cs =concentration of USP Biotin RS in the Standard
Sample solution: 20 mg/mL in 0.1 N sodium hydroxide solution (mg/mL)
Acceptance criteria: +890 to +930 Cu = nominal concentration of biotin in the Sample
solution (mg/mL)
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Store in tight containers. Acceptance criteria: 90.0%-110.0%
• USP REFERENCE STANDARDS (11)
USP Biotin RS PERFORMANCE TESTS
• DISSOLUTION (711)
Buffer solution: 0.02 N anhydrous disodium hydrogen
phosphate adjusted with phosphoric acid to a pH of 7.4
Medium: Buffer solution; 500 mL
Biotin Capsules Apparatus 1: 100 rpm
Time: 1 h
DEFINITION Standard solution: Dissolve a suitable amount of USP Biotin
Biotin Capsules contain NLT 90.0% and NMT110.0% of the RS in Buffer solution to obtain a concentration similarto that
labeled amount of biotin (CloH16Nz03S), expected in the Sample solution.
Sample solution: Withdraw a portion of the solution under
IDENTIFICATION test, pass through a suitable filter, and use the pooled
• A. The retention time of the major peak of the Sample sample as the test specimen.
solution corresponds to that of the Standardsolution as Analysis: Proceed as directed in the Assay, making any
obtained in the Assay. necessary adjustments.
ASSAY Calculate the percentage of the labeled amount of biotin
• PROCEDURE (CloH16Nz03S) dissolved:
Buffer solution: Dissolve 1 g of sodium perchlorate
monohydrate in 500 mLof water, add 1 mLof phosphoric Result = (rvlrs) x (C, x VIL) x 100
acid, and dilute with water to 1000 mL.
Mobile phase: Acetonitrile and Buffer solution (8.5: 91.5) tu = peak area from the Sample solution
Dlluentr Acetonitrile and water (1:4) rs = peak area from the Standard solution
Standard solution: 0.05 mg/mL of USP Biotin RS in Diluent. Cs = concentration of USP Biotin RS in the Standard
Sonicate, if necessary, to dissolve. solution (l..Jg/mL)
Sample solution: Weigh NlT 20 Capsules in a tared V =volume of Medium, 500 mL
weighing bottle. Open the Capsules, without the loss of L = labeled amount of biotin (l..Jg/Capsule)
shell material, and transfer the contents to a 100-mL
beaker. Remove any contents adhering to the empty shells Tolerances: NLT 75% (Q) of the labeled amount of biotin
by washing, if necessary, with several portions of ether. (CloH16Nz03S) is dissolved.
Discard the washings, and dry the Capsule shellswith the • UNIFORMITY OF DOSAGE UNITS (905): Meet the
aid of a current of dry air until the odor of ether is no longer requirements
perceptible. Weigh the empty Capsule shells in the tared ADDITIONAL REQUIREMENTS
weighing bottle, and calculate the averaqe net weight per • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Capsule. Transfera portion of the Capsule contents, containers.
equivalent to a nominal amount of 5 mg of biotin to a • USP REFER.ENCE STANDARDS (11)
1OO-mL volumetricflask, add 60 mLof water, and shake in USP Biotin RS
a water bath at 65 for 20 min. Sonicate for 5 min, shake
0

by mechanical means for 15 min, and cool to room

temperature. Add 20 mLof acetonitrile, dilute with water
to volume, and filter. .

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USP 43 Official Monographs / Biotin 571

Add the fol8owing: SPECIFIC TESTS

-pH (l91):'3.l-4.7
A.Biotin Compounded '.0..,11 Sus, ADDITIONAL 'REQUIREMENTS
• PACKAGING AND STORAGE: . Package. infight, light~resist~nt
DEFINITION ,,' ,,',', " ".~' , " " ' '" con·~ainers. Storeatcohtrolled roomtemperature oi'ina
Biotin Compounded Oral Suspension contains NLT90'.0%,and refrige.rator.
NMT 110.0% of the labeled amount of biotin •
(CloH16N203S), ,' , " .."
PrepClre BjotinCompounaed 'dral'Suspension JOn1glinL',a~
follows (see Pharmaceutical CQmpounding~N6nsterile
Pr:eparations (795».

Biotin powder
Vehicle: 1:1. rnlxtute of Ora-plusaand
Ora-Sweet;a a sufficient quantity to make 100 mL

a Perrigo Pharmaceuticals, Allegan, MI.

Biotin Tablets
Place the Biotin powder in a suitable container DEFINITION
a fine powder. Add a small amount of Vehic Biotin Tablets contain NLT 90.0% and NMT 110.0% of the
to form a smooth· paste. Ad ufficient amoun , 0 labeled amount of biotin (Cl0H16N203S),
to make the mortar conten
contents stepwis~and qua IDENTIFICATION
container using the remainderof the Vehicle. A d ,sufficient • A. The retention time of the major peak of the Sample
Vehicle to bring to final volume. Mix well. solution corresponds to that of the Standard solution, as
obtained in the Assay.
ASSAY
• PROCEDURE ASSAY
Solution A:' Add 1 9 of sodiumperchlorat '. onohydrate to • PROCEDURE
500 mL of water. Add 1 mL of phosph , c:id anqthen an Buffer solution: Dissolve 1 g of sodium perchlorate
additional 500 mL ater. monohydrate in 500 mL of water, add 1 mL of phosphoric
Mobile phase: A~et ile and SolutlonA (8.S:'9L.5) acid, and dilute with water to 1000 mL.
Diluent: Acetonitrile and water (20:80) . . .. ' .. Mobile phase: Acetonitrile and Buffer solution (8.5: 91.5)
Standard solution: 0.1 mg/mL of USPBiotin RSin Diluent Diluent: Acetonitrile and water (1:4)
Sample solution: Transfer 1.0 mL of Oral Suspension into a Standard solution: 0.05 mg/mL of USP Biotin RS in Diluent.
1OO-mL volumetric flask, and add Diluent to volume. Pass Sonicate, if necessary, to dissolve.
Sample solution: Transfer a portion equivalent to 5 mg of
through a filter qfO.22-lJm pore size~
biotin from NLT 30 finely powdered Tablets to a 100-mL
Chromatographic system ,, .
volumetric flask, add 60 mL of water, and shake in a water
(See Chromatography (621), System Suitability.)
bath at 65 0 for 20 min. Sonicate for 5 min, shake by
Mode: LC mechanical means for 15 min, and cool to room
Detector: UV 200 nm temperature. Add 20 mL of acetonitrile, dilute with water
Column: 4.6-mm x 15-cm; 3.5-lJm packing L7 to volume, and filter.
Flow rate: 1.2 mL/min Chromatographic system
Injection volume: 50 IJL (See Chromatography (621), System SUitability.)
System suitability Mode: LC
Sample: .Standardsolution Detector: UV 200 nm
[NOTE-The retention time for biotiF1 isabout 11.8 Column: 4.6-mm x 15-cm; 3-lJm packing L7
min.] Flow rate: 1.2 mL/min
Suitability requirements Injection volume: 50 IJL
Tailing factor: , NMT2.0 System suitability
Relative standard. deviation: NMT 2.0% for replicate Sample: Standard solution
injections Suitability requirements
Analysis Tailing factor: NMT 1.5
Samples: ' Standardsolution and Sample solution. ,c Relative standard deviation: NMT 2.0% for replicate
Calculate the, percentage of the,labeled amount of.biotin injections
(ClOH16N203S) in the portion of Oral Suspension taken; Analysis
Samples: Standard solution and Sample solution
Result ='(rvlrs) x (CslC{J) X'100 Calculate the percentage of biotin (ClOH16N203S) in the
portion of Tablets taken:
= peak response of biotin fr()m the Sample solution
= peak response'ofbiotin'fromthe Sti:mdard~olution Result = (rv/rs) x (CslCu) x 100
= concentration of USP Biotin RS in the Standard
solution(mglmL) . . ' ' ','- tu = peak response from the Sample solution
Cv = nominal concentration of bi.otinin theSa:JIlple rs = peak response from the Standard solution
solution (mgJ.rnL) Cs = concentration of USP Biotin RS in the Standard
solution (mg/mL)
Acceptance criteria: 90.0%~110.0%

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 572 Biotin / Official Monographs USP 43

= nominal concentration of biotin in the Sample IDENTIFICATION

solution (mg/mL)

Acceptance criteria: 90.00/0-110.0%

PERFORMANCE TESTS
Ic!;);:
• DISSOLUTION (711)
requirements."
Buffer solution: 0.02 N anhydrous disodium hydrogen
phosphate adjusted with phosphoric acid to a pH of 7.4 • B. The retention time of the major peak of the Sample
Medium: Buffer solution; 500 mL solution corresponds to that of the Standard solution, as
Apparatus 2: 75 rpm obtained in the Assay.
Time: 1 h ASSAY
Standard solution: Dissolve a suitable amount of USP Biotin • PROCEDURE
RS in Buffer solution to obtain a concentration similar to that Buffer: 1.58 giL of ammonium formate in water; adjusted
expected in the Sample solution. with formic acid to a pH of 5.0
Sample solution: Withdraw a portion of the solution under Mobile phase: Acetonitrile and Buffer (45:55)
test, pass through a suitable filter, and use the pooled Diluent: Acetonitrile, acetic acid, and water (30:4:66)
sample as the test specimen. Standard solution: 0.5 mg/mL of USP Bisacodyl RS in
Analysis: Proceed as directed in the Assay, making any Diluent
necessary adjustments. . Sample solution: 0.5 mg/mL of Bisacodyl in Diluent
Calculate the percentage of the labeled amount of biotin Chromatographic system
(ClOH16Nz03S) dissolved: (See Chromatography (621), System Suitability.)
Mode: LC
Result = (ru/rs) x (Cs x VIL) x 100 Detector: UV 265 nm
Column: 4.6-mm x 25-cm; 5-~m packing L1
ru =peak area from the Sample solution Flow rate: 1.5 mL/min
rs =peak area from the Standardsolution Injection volume: 10 ~L
Cs = concentration of USP Biotin RS in the Standard Run time: NLT 2 times the retention time of bisacodyl
solution (~g/mL) System suitability
V = volume of Medium, 500 mL Sample: Standardsolution
L =labeled amount of biotin (~g/Tablet) Suitability requirements
Tailing factor: NMT 1.5
Tolerances: NLT 75% (Q) of the labeled amount of biotin Relative standard deviation: NMT 0.73%
(CloH16Nz03S) is dissolved. Analysis
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Samples: Standardsolution and Sample solution
requirements Calculate the percentage of bisacodyl (CZZH19N04) in the
portion of Bisacodyl taken:
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Result = (rulrs) x (CslCu) x 100
containers. '
• USP REFERENCE STANDARDS (11) ru = peak response of bisacodyl from the Sample
USP Biotin RS solution
ts = peak response of bisacodyl from the Standard
solution
Cs =concentration of USP BisacodylRS in the Standard
Bisacodyl solution (mg/mL)
Cu = concentration of Bisacodyl in the Sample solution
(mg/mL)

Acceptance criteria: 98.00/0-102.0% on the dried basis

IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1%
• ORGANIC IMPURITIES
CZZH19N04 361.39 Buffer: 1.58 giL of ammonium formate in water; adjusted
Phenol, 4,4'-(2-pyridinylmethylene)bis-, diacetate (ester); with formic acid to a pH of 5.0
4,4'-(2-Pyridylmethylene)diphenol diacetate (ester); Mobile phase: Acetonitrile and Buffer (45:55)
4,4'-(Pyridin-2-ylmethylene)diphenyl diacetate [603-50-9]. Diluent: Acetonitrile and water (35:5)
System suitability solution: 0.8 mg/mL of USP Bisacodyl
DEFINITION RS; 2 ~g/mL each of USP Bisacodyl Related Compound A
Bisacodyl contains NLT 98.0% and NMT 102.0% of bisacodyl RS, USP Bisacodyl Related Compound C RS, and USP
(CZZH19N04), calculated on the dried basis. Bisacodyl Related Compound E RS; and 4 ~g/mL of USP
[CAUTION-Avoid inhalation and contact with the eyes, skin, Bisacodyl Related Compound B RS in Diluent
and mucous membranes.] Sensitivity solution: 0.0003 mg/mL of USP Bisacodyl RS in
Diluent
Standard stock solution: 1.0 mg/mL of USP Bisacodyl RS in
Diluent
Standard solution: 1:0 ~g/mL of USP Bisacodyl RS in Diluent
Sample solution: 1.0 mg/mL of Blsacodyl in Diluent

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 USP 43 Official Monographs / Bisacodyl 573

Chromatographic system ADDITIONAL REQUIREMENTS

(See Chromatography (621), System Suitability.) CD PACKAGING AND STORAGE: Preserve in well-closed

Mode: LC containers, protected from light. Store at room

Detector: UV 265 nm temperature.
Column: 4.6-mm. x 25-cm; 4- or 5-!Jm packing L1 CD USP REFERENCE STANDARDS (11)

Flow rate: 1.5 mL/min USP Bisacodyl RS

Injection volume: 20 IJL USP Bisacodyl Related Compound A RS
Run time: NLT 3.5 times the retention time of bisacodyl 4,4'-(Pyridin-2-ylmethylene)diphenol.
System suitability ClsH1SNOz 277.32
Samples: System suitabilitysolution, Sensitivity solution, and USP Bisacodyl Related Compound B RS
Standard solution 2,4'-(Pyridin-2-ylmethylene)diphenol.
[NoTE-See Table 7 for the relative retention times.] ClsH1SNOz 277.32
Suitability requirements USP Bisacodyl Related Compound C RS
Resolution: NLT 1.5 between the bisacodyl related 4-[(4-Hydroxyphenyl)(pyridi n-2-yl)methyl]phenyl
compound E and bisacodyl peaks, System suitability acetate.
solution CZOH 17N03 319.35
Tailing factor: NMT 2.0 for the bisacodyl peak, System USP Bisacodyl Related Compound E RS
sUitability solution 2-[(4-Acetoxyphenyl)(pyridin-2-yl)methyl]phenyl
Relative standard deviation: NMT 5.0% for the acetate.
bisacodyl peak, Standard solution CZZH19N04 361.39
Signal-to-noise ratio: NLT 10, Sensitivity solution
Analysis
Samples: Standardsolution and Sample solution
Calculatethe percentage of each individual impurity in the
portion of Bisacodyl taken: Bisacodyl Suppositories
Result = (rulrs) x (CsICu) x (1IF) x 100
» Bisacodyl Suppositories contain not less than
to = peak response of each individual impurity from 90.0 percent and not more than 110.0 percent of
the Sample solution
rs = peak response of bisacodyl from the Standard the labeled amount of C22H19N04'
solution
Cs = concentration of the Standard solution (mg/mL) Packaging and storage-Preserve in well-closed containers
Cu = concentration of the Sample solution (mg/mL) at a temperature not exceeding 30°.
USPReference standards (11)-
F = relative response factor (see Table 7)
USP Bisacodyl RS
Acceptance criteria: See Table 7.The reporting threshold is Identification-
0.05%. A: Transfer a quantity of Suppositories, equivalentto about
150 mg of bisacodyl, to a 500-mL conicalflask, add 75 mL of
Table 1 solvent hexane, and heat on a steam bath until they are
Relative Relative Acceptance melted. Filter the solution, with the aid of vacuum, through a
Retention Response Criteria, medium-porosity, sintered-glassfunnel, and wash the residue
Name Time Factor NMT(%) with about 100 mL of warm solvent hexane until it isfree from
Bisacodyl related fat. Continue the vacuum until the residue appears dry.
compound A 0.20 1.7 0.15 Dissolve the residue by rinsing the filter with about 50 mLof
Bisacodyl related
warm acetone, collecting the filtrate in a 150-mLbeaker, and
compound B 0.40 1.5 0.15 evaporate the filtrate on a steam bath to a volume of about 5
mL. To the residual liquid add about 75 mL of water, heat on
Bisacodyl related a steam bath for 15 minutes, and cool. Scratch the sides of the
compound C 0.45 1.3 0.50
beaker to induce crystallization, filter the crystals, and dry at
Specified unidentified 100° for about 15 minutes: the bisacodyl so obtained melts
impurity 1 0.85 1.0 0.20 between 129° and 135°, and responds to Identification test A
Bisacodyl related under Bisacodyl
compound E 0.90 1.0 0.50 B: The chromatogram of the Assay preparation obtained as
directed in the Assay exhibits a major peak for bisacodyl, the
Bisacodyl 1.0 - - retention time of which corresponds to that exhibited in the
Specified unidentified chromatogram of the Standard preparation.
impurity 2 2.6 1.0 0.30
Assay- .
Any individual
Mobilephase-Prepare a filtered and degassed mixture of
unspecified - 0.074 M sodium acetate in water [adjusted with 2.5% (v/v)
impurity 1.0 0.10
acetic acid to a pH of 7.4] and acetonitrile (55:45). Make
Total impurities - - 1.0 adjustments if necessary (see System Suitability under
Chromatography (621 »
SPECIFIC TESTS Standard preparation-Dissolve an accurately weighed
• Loss ON DRYING (731) quantity of USP Bisacodyl RS in acetonitrileto obtain a
Analysis: Dry at 105° for 2 h. Standard preparation having a known concentration of about
Acceptance criteria: NMT 0.5% 0.5 mg per mL.
Assay preparation-Transfer a number of Suppositories,
equivalent to about 100 mg of bisacodyl, to a 500-mL

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 574 Bisacodyl / Official Monographs USP 43

separator, add 150 mL of n-hexane, and shake until all the Chromatographic system (see Chromatography (621»-
suppositories are dissolved. Add 50 mL of acetonitrile, shake The llquid chromatograph is equipped with-a 254-nm
for 1 minute, and allow the layers to separate. Drain the lower detector and a 3.9-mm x 30-cm column containing packing
layer into a 200-mL volumetric flask, and extract the n-hexane L1. The flow rate is about 2 mL per minute. Chromatograph
layer remaining in the separator with two 50-mL portions of the Standardpreparation, and record the peak responses as
acetonitrile, combining the lower layers in the volumetric flask. directed for Procedure: the relative retention times are about
Dilute the combined extracts in the volumetric flask with 2.0 for bisacodyl and 1.0 for ethylparaben; the resolution, R,
acetonitrile to volume, mix, and filter. between bisacodyl and the internal standard is not less than
Chromatographic system (see Chromatography (621 »- 7.0; the column efficiency, determined for the analyte peak, is
The liquid chromatograph is equipped with a 265-nm not less than 2000 theoretical plates; the tailing factor is not
detector, a 3.9-mm x 30-cm column that contains packing L1, more than 1.2; and the relative standard deviation for replicate
and a guard column that contains packing L2. The flow rate is injections is not more than 2.0%.
about 2 mL per minute. Chromatograph the Standard Procedure-Separately inject equal volumes (about 10 I.IL)
preparation, and record the peak responses as directed for of the Standardpreparation and the Assay preparation into the
Procedure: the tailing factor is not more than 2.0; and the chromatograph, record the chromatograms, and measure the
relative standard deviation for replicate injections is not more responses for the major peaks. Calculate the quantity, in mg,
than 2.0%. of C22H,9N04 in the portion of Rectal Suspension taken by the
Procedure-Separately inject equal volumes (about 10 I.IL) formula:
of the Standardpreparation and the Assay preparation into the
chromatograph, record the chromatograms, and measure the
responses for the major peaks. Calculate the quantity, in mg,
of C22H,9N04 in the Suppositories taken by the formula: in which Cis the concentration, in mg per mL,of USP Bisacodyl
RS in the Standardpreparation; and Ru and Rs are the peak
200C(r u/rs) response ratios of the bisacodyl peak to the internal standard
peak obtained from the Assay preparation and the Standard
in which C isthe concentration, in mg per mL, of USPBisacodyl preparation, respectively.
RS in the Standard preparation; and ru and rs are the peak
responses obtained from the Assay preparation and the
Standardpreparation, respectively.
Bisacodyl Delayed-Release Tablets
DEFINITION
Bisacodyl Rectal Suspension Bisacodyl Delayed-Release Tablets contain. NLT 90.0% and
NMT 110.0% of the labeled amount of bisacodyl
(Cn H,9N 0 4)'
» Bisacodyl Rectal Suspension is a suspension of
Bisacodyl in a suitable aqueous medium. It contains IDENTIFICATION
not less than 90.0 percent and not more than
115.0 percent of the labeled amount of C22H19N04'
Packaging and storage-Preserve in unit-dose containers
at a temperature not exceeding 30°. mm
USPReference standards (11)- Sample solution: Macerate a portion of powdered Tablets,
USP Bisacodyl RS equivalent to 300 mg of bisacodyl, with 100 mLof acetone.
Identification-The retention time of the major peak in the Heat on a steam bath to boiling, filter, and evaporate to '
chromatogram of the Assay preparation corresponds to that of about 20 mL. Add 200 mL of water, and warm the mixture
the Standardpreparation as obtained in the Assay. on the steam bath, passing a stream of nitrogen over the
pH (791): between 5.0 and 6.8. surface to evaporate the acetone. After 30 min, cool the
mixture, and filter through a sintered-glass funnel. Discard
Assay- - the filtrate, and dissolve the crystals in 50 mL of acetone.
Mobile phase-Prepare a filtered and degassed mixture of Evaporate the solution to about 15 mL, add about 75 mL
methanol and 0.01 M monobasic potassium phosphate of water, heat on a steam bath for 15 min, and then cool.
(60:40). Make adjustments if necessary (see System Suitability Scratch the sides of the beaker to induce crystallization,
under Chromatography(621 ». filter the crystals, and dry at 100° for about 15 min. Using
Internal standard solution-Dissolve a suitable quantity of the crystals, prepare a solution (1 in 200) in chloroform.
ethylparaben in methanol, and dilute with an equal volume of Acceptance criteria: Meet the requirements
water to obtain a solution containing about 5.0 mg per mL • B. The retention time of the major peak of the Sample
Standardpreparation-Dissolve an accurately weighed solution corresponds to that of the Standardsolution, as
quantity of USP Bisacodyl RS in methanol, add an accurately obtained in the Assay.
measured volume of Internal standard solution, and dilute
quantitatively, and stepwise if necessary, with methanol to ASSAY
obtain a solution having known concentrations of about 671.1g • PROCEDURE
per mL and 250 I.Ig per mL for bisacodyl and ethylparaben, Buffer: 0.074 M sodium acetate in water, adjusted with
respectively. 2.5% (v/v) acetic acid to a pH of 7.4
Assay preparation-Transfer an accurately measured Mobile phase: Acetonitrile and Buffer(45:55)
volume of Rectal Suspension, equivalent to 6.7 mg of Standard solution: 0.5 mg/mL of USP Bisacodyl RS in
bisacodyl, to a 1OO-mL volumetric flask. Add 5.0 mL of Internal acetonitrile -
standard solution, dilute with m-ethanol to volume, and mix.

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 USP 43 Official Monographs / Bismuth 575

Sample solution: Transfer a portion of finely powdered

Tablets equivalent to 100 mg of bisacodyl, to a 200-mL
Strong Ammonia Solution,
volumetric flask, add 25 mL of water, and shake by' Purified Water, each, a sufficient quan-
mechanical means for 15 min followed by sonication for 15
min. Add 100 mL of acetonitrile, and shake by mechanical tity, to make....................................... 1000 mL
means for 15 min followed by sonication for 15 min. Dilute
with acetonitrile to volume, mix, and filter. Mix the Bismuth Subnitrate with 60 mL of Purified
Chromatographic system Water and 60 mL of the Nitric Acid in a suitable
(See Chromatography (621), System Suitability.)
Mode: LC container, and agitate, warming gently until
Detector: UV 265 nm solution is effected. Pour this solution, with
Columns constant stirring, into 5000 mL of Purified Water
Guard: Packing L2 containing 60 mL of the Nitric Acid. Dilute 160 mL
Analytical: 3.9-mm x 30-cm; packing L1 of Strong Ammonia Solution with 4300 mL of
Flow rate: 2 mL/min
Injection volume: 10 I-IL Purified Water in a glazed or glass vessel of at least
System suitability 12,000-mL capacity. Dissolve the Ammonium
Sample: Standard solution Carbonate in this solution, and then pour the
Suitability requirements bismuth solution quickly into it with constant
Tailing factor: NMT 2.0 stirring. Add sufficient 6 N ammonium hydroxide,
Relative standard deviation: NMT 2.0%
Analysis if necessary, to render the mixture distinctly
Samples: Standard solution and Sample solution alkaline, allow to stand until the precipitate has
Calculate the percentage of the labeled amount of settled, then pour or siphon off the supernatant,
bisacodyl (C22H19N04) in the portion of Tablets taken: and wash the precipitate twice with Purified Water,
Result =(rulrs) x (CslCu) x 100 by decantation. Transfer the magma to a strainer
of close texture, so as to provide continuous
ru = peak response from the Sample solution washing with Purified Water, the outlet tube being
rs = peak response from the Standard solution elevated to preventthe surface of the magma from
Cs =concentration of USP Bisacodyl RS in the Standard becoming dry. When the washings no longer yield
solution (mg/mL) a pink color with phenolphthalein TS, drain the
Cu =nominal concentration of bisacodyl in the Sample moist preparation, transfer to a graduated vessel,
solution (mg/ml)
add sufficient Purified Water to make 1000 mL, and
Acceptance criteria: 90.0%-110.0% mix.
PERFORMANCE TESTS [NOTE-This method of preparation may be varied,
• DISINTEGRATION (701): Proceed as directed for provided the product meets the following
Delayed-Release (Enteric-Coated) Tablets. The Tablets do not requirements.]
disintegrate after 1 h of agitation in simulated gastric fluid
Packaging and storage-Preserve in tight containers, and
TS, but then disintegrate within 45 min in simulated
protect from freezing.
intestinal fluid TS.
• UNIFORMITV OF DOSAGE UNITS (905): Meet the Identification-
requirements A: It responds to the tests for Bismuth (191) and for
Carbonate (191).
ADDITIONAL REQUIREMENTS B: Add 1 mL of 3 N hydrochloric acid to 1 mL of Milk of
• PACKAGING AND STORAGE: Preserve in well-closed Bismuth: a clear solution is produced. Pour the clear solution
containers at a temperature not exceeding 30°. into 10 volumes of water: a white precipitate is formed.
• USP REFERENCE STANDARDS (11) Microbial enumeration tests (61) and Tests for specified
USP Blsacodyl RS microorganisms (62)-The total bacterial count does not
exceed 100 du per mL and the test for Escherichia coli is
negative.
Water-soluble substances-Boil 10 mL with 90 mL of
water for 10 minutes, cool, add water to make the total
Milk of Bismuth volume 100 mL, mix, and filter. Evaporate 50 mL of the filtrate
to dryness, and ignite it gently: the weight of the residue does
» Milk of Bismuth contains bismuth hydroxide and not exceed 5 mg (0.1 %).
Bismuth Subcarbonate in suspension in water, and Arsenic, Method I (211)-Evaporate 3.75 mL on a steam bath
to dryness, add 2 mL of sulfuric acid, and heat until copious
yields not less than 5.2 percent and not more than fumes of sulfur trioxide are evolved. The limit is 0.8 ppm.
5.8 percent (w/w) of bismuth trioxide (Biz0 3) . Lead-To 5 mL add warm nitric acid, dropwise, until it is just
dissolved, and pour the solution into 50 mL of water: a white
precipitate may form. Filter, if necessary, evaporate the filtrate
Bismuth Subnitrate . 80 g on a steam bath to 15 mL, again filter, and to 10 mL of the
Nitric Acid . 120 mL filtrate add an equal volume of 2 N sulfuric acid: no precipitate
is formed.
Ammonium Carbonate .. 10 g Limit of alkalies and alkaline earths-Dissolve 2.0 mL in
5 mL of hydrochloric acid, dilute with water to 100 mL, add
hydrogen sulfide to precipitate the bismuth completely, and

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576 Bismuth / OfficialMonographs USP 43

filter. To 50 mL of the clear filtrate add 5 drops of sulfuric acid, Test solution-Ignite about 3 g of Bismuth Citrate,
evaporate to dryness, and ignite: the weight of the residue accurately weighed, in a porcelain crucible, cool, and
does not exceed 3 mg (0.3%). cautiously add 6 N nitric acid to dissolve the residue. Add 100
Assay-Evaporate an accurately weighed quantity of Milk of mL of water, and mix. A white precipitate forms. Filter this
Bismuth to dryness, and ignite the residue to constant weight. mixture, evaporate on a steam bath to obtain about 15 mL of
From the weight of the Bi20 3 so obtained determine the solution, and filter again. Dilute the filtrate with water to 20.0
percentage in the assay specimen. mL.
Procedure--Concomitantly determine the absorbances of
the Standard solution and the Test solution at the emission lines
of 324.7 nm, 217 nrn, and 328.1 nm for copper, lead, and
silver, respectively, with an atomic absorption
Bismuth Citrate spectrophotometer (see Atomic Absorption Spectroscopy (852»
equipped with copper, lead, and silver hollow-cathode lamps
and an oxidizing flame. The absorbances of the Test solution

~
OHO 0- _
do not exceed those of the Standard solution for each element
(10 ~g per g).
813+ -
_ 0
o Limit of soluble bismuth-
o Standard solution-Transfer 242.0 mg of bismuth nitrate
BiC6H s0 7 398.08 pentahydrate to a 1OO-mL volumetric flask. Add 3 mL of 1.5
N nitric acid, swirl to dissolve, dilute with water to volume, and
[813-93-4].
mix. Transfer 1.0 mL of this solution to a 500-mL volumetric
flask, add 250 mL of 1.5 N nitric acid, dilute with water to
» Bismuth Citrate contains not less than 49 percent volume, and mix. This solution contains 2.0 ~g of bismuth (Bi)
and not more than 54 percent of bismuth (Bi). per mL. [NoTE-The concentration of bismuth in this solution
may be modified by using a different quantity or by further
Packaging and storage-Preserve in tight, light-resistant dilution to bring the absorption responses within the working
containers, store at controlled room temperature, and prevent . range of the atomic absorption spectrophotometer.]
exposure to excessive heat. Test solution-Prepare a mixture of 5.0 g of Bismuth Citrate
USPReference standards (11) - and 100 mL of water, and stir by mechanical means the
USP Bismuth Citrate RS suspension thus obtained for 2 hours. Pass through filter
Identification- paper. Pass the filtrate thus obtained through a filter having a
o.l-prn orfiner porosity. To 10.0 mL of the filtrate add 0.1 mL
of nitric acid.
Procedure--Concomitantly determine the absorbances of
the Standard solution and the Test solution at the emission line
on specimen. of 223.06 nm for bismuth with an atomic absorption
ctrr\nnh, heated, salt chars, and on ignition spectrophotometer (see Atomic Absorption Spectroscopy (852»
leaves blackened residue having a yellow equipped with a bismuth hollow-cathode lamp and an
surface. The residue is soluble in warm nitric acid, and this oxidizing flame. The absorbances of the Test solution do not
solution, when dropped into a large excessof water, produces exceed those of the Standard solution (40 ~g per g).
a white turbidity. . Assay-Transfer about 300 mg of Bismuth Citrate, accurately
C: Dissolve 1 g in ammonia TS. When treated with weighed, to a porcelain crucible, and ignite. Allow to cool, add
hydrogen sulfide in excess, a black precipitate is obtained. 2 mL of nitric acid to the residue, dropwise, and warm until
Filter this mixture, drive off the excess hydrogen sulfide by complete solution has been effected. Add about 60 mL of
heating, and allow to cool. To a portion of this cooled solution water and 0.3 mL of xylenol orange TS, and titrate with 0.05
add an excess of calcium hydroxide TS, and boil: a white N edetate disodium VS to a yellow endpoint. Each mL of 0.05
precipitate is formed. Reserve a second portion of the cooled N edetate disodium is equivalent to 10.45 mg of bismuth (Bi).
solution for the test for Limit of nitrate.
Arsenic, Method I (211 )-Prepare the Test Preparation as
follows. Triturate 300 mg with an equal weight of calcium
hydroxide, and ignite. Dissolve the residue in 5 mL of 3 N Bismuth Subcarbonate
hydrochloric acid: the limit is 10 ~g per g.
Limit of nitrate-To the second portion of cooled solution DEFINITION
reserved from Identification test C, add an equal volume of Bismuth Subcarbonate contains NLT 97.6% and NMT 100.7%
sulfuric acid, mix, and allow to cool. Into the liquid, drop a of bismuth subcarbonate [(BiO)2C03], calculated on the
crystal of ferrous sulfate, and allow to stand for 30 minutes: no dried basis.
brown or brownish black color appears around the crystal.
IDENTIFICATION
Limit of copper, lead, and silver- • A. IDENTIFICATION TESTS-GENERAL, Bismuth and Carbonate
Standard solution-Prepare a solution containing 1000 ~g (191 )
of copper per mL, a solution containing 1000 ~g of lead per
mL, and a solution containing 1000 ~g of silver per mL. ASSAY
Transfer 3.0 mL of each solution to a 2000-mL volumetric flask, • PROCEDURE
dilute with 1 N nitric acid to volume, and mix. [NoTE-The Sample solution: 500 mg of Bismuth Subcarbonate in 3 mL
concentrations of copper, lead, and silver in this solution may of nitric acid. Dilute with water to 250 mL, and add 0.3 mL
be modified by using a different quantity or by further dilution of xylenol orange TS.
to bring the absorption responses within the working range of Titrimetric system
the atomic absorption spectrophotometer.] Mode: Direct titration
Titrant: 0.05 M edetate disodium VS

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 USP 43 OfficialMonographs / Bismuth 577

Endpoint detection: Visual . Standard stock solution 2: 10 IJg/mL of copper in 2 N nitric

Analysis: Titrate with Titrant to a yellow endpoint. Each mL acid from Standardstock solution 1
of 0.05 M edetate disodium is equivalent to 12.75 mg of Standard solution: Mix 0.25 mL of Standardstocksolution
bismuth subcarbonate [(BiO)2C03]' 2 and 9.75 mL of water.
Acceptance criteria: 97.6%-100.7% on the dried basis Sample solution: To 5 mL of th~ Sample stock soluti?n
retained from the test for Chloride and Sulfate, Chloride add
IMPURITIES 2 rnl, of 6 N ammonium hydroxide, dilute with water to 50
• CHLORIDE AND SULFATE, Chloride (221) mL, mix, and filter.
Sample stock solution: 5.0 gin 10 mL of water. Add 20 mL Analysis: To 10 mL each of the Standardsolution and the
of nitric acid, warm to achieve dissolution, and allow to Sample solution add 1 mL of a solution of sodium
cool. Dilute with water to obtain 100 mL of solution. diethyldithiocarbamate (1 in 1000).
Sample solution: To 6.6 mL of the Sample stock solution add Acceptance criteria: No more color is obtained from the
4 mL of nitric acid, and dilute with water to 50 mL. . Sample solution than is obtained from the Standardsolution
Acceptance criteria: A 15.0-mL portion of the Sample (0.005%).
solution shows no more chloride than corresponds to 70 IJL • LIMIT OF LEAD
of 0.020 N hydrochloric acid (0.05%). Diluent: 6 N nitric acid, lead-free
• LIMIT OF ALKALIES AND ALKALINE EARTHS Standard stock solution: 0.1598 mg/mL of lead nitrate in
Sample solution: Boil 1.0 9 with.20 mL of a mi~ture of acetic Diluent. This solution contains 100 IJg/mL of lead.
acid and water (1:1). After 2 min, cool, and.filter. Standard solutions: 1.0, 2.0, and 3.0 f.Jg/mL of lead from
Analysis: Collect the filtrate, wash the residue with 20 mL of the Standardstocksolution in Diluent
water, and add the washing to the filtrate. To this solution Sample solution: 12.5 9 of Bismuth Subcarbonate in 75
add 2 mL of 2 N hydrochloric acid and 20 mL of water. Heat mL of Diluent. Heat to boiling for 1 min, cool, and dilute
to boiling, and precipitate the bismuth by adding hydrogen with water to 100 mL.
sulfide. Cool the mixture, and filter. Collect the filtrate, Analysis: Concomitantly determine the absorbances of the
wash the residue with water, and add the washing to the Standardsolutions and the Sample solution at the lead
filtrate. Evaporate this solution to dryness on a water bath. emission line of 283.3 nm with an atomic absorption
To the residue add 0.5 mL of sulfuric acid, dry slowly, and spectrophotometer (see AtomicAbsorption Spectroscopy
cool. (852» equipped with a lead hollow-cathode lamp and an
Acceptance criteria: The weight of the residue does not air-acetylene flame, using a 1:5 dilution of the Diluent as
exceed 10 mg (1.0%). the blank. Plot the absorbances of the Standardsolutions
• LIMIT OF NITRATE versus concentration,in IJg/mL, of lead, and draw the
Indigo carmine titrant: To 4 g of indigo carmine in 900 mL straight line best fitting the three plotted points. From the
of water add 2 mL of sulfuric acid, and dilute with water to graph, determine the concentration, C, in IJg/mL, of lead
1000 mL. I
in the Sample solution.
Standard solution: 0.0815 mg/mL of potassium nitrate Calculate the percentage of lead (Pb) in the portion of
(equivalent to 0.05 mg/mL of nitrate) in water. Place 20.0 Bismuth Subcarbonate taken:
mL in a 125-mL conical flask.
Sample solution: To 250 mg of Bismuth Subcarb~nate in a Result= C/1250
125-mL conical flask add 20 mL of water, and swirl to
suspend.- C = concentration of lead in the Sample solution (lJg/
Analysis: To the Standardsolution and the Sample solution mL)
add 0.05 mL of Indigo carmine titrant. Carefully add 30 mL
of sulfuric acid, and immediately titrate with Indigo carmine Acceptance criteria: NMT 0.002%
titrant to a stable blue endpoint. .
SPECIFIC TESTS
Acceptance criteria: The volume of Indigo carmine titrant
consumed by the Sample solution does not exceed that • Loss ON DRYING (731)
consumed by the Standard solution (0.4%). Analysis: Dry at 105° to constant weight.
• LIMIT OF SilVER
Acceptance criteria: NMT 1.0% of its weight
Standard solution: 7.87 IJg/mL of silver nitrate ADDITIONAL REQUIREMENTS
Sample solution: To 2.0 9 of Bismuth Subcarbonate add 1 • PACKAGING AND STORAGE: Preserve in well-closed
mL of water and 4 mL of nitric acid. containers, protected from light.
Analysis: Heat the Sample solution gently to ach~eve
dissolution, add water to obtain 11 mL of solution, and
cool. Add 2 mL of 1 N hydrochloric acid, and allow.to stand
in a dark place for 5 min. Treat the Standardsolution
concomitantly with 1 mL of nitric acid and 2 mL of 1 N Bismuth Subgallate
hydrochloric acid.
O
Acceptance criteria: The turbidity produced from the
Sample solution is NMT that produced from the Standard
solution (0.0025%).
IOV
HO-Sf
'0
I
~
OH

• LIMIT OF ARSENIC, Method 1(211)

OH
Test preparation: 600 mg in 35 mL of 3 N hydrochloric acid
Acceptance criteria: NMT 5 ppm C7H s Bi0 6 394.09
• LIMIT OF COPPER Gallic acid bismuth basic salt [99-26-3].
Standard stock solution 1: 5 mg/mL of copper prepared as
follows. To a 1OO-mL volumetric flask add 1.34 9 of cupric DEFINITION
chloride, 109 of ammonium chloride, and.3 mL o.fsodium Bismuth Subgallate is a basic salt that, when dried at 105° for
metabisulfite solution (275 mg/mL), and dilute With water 3 h, contains the equivalent of NLT 52.0% and NMT 57.0%
to volume. . of bismuth trioxide (Bi203).

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578 Bismuth / Official Monographs USP 43

IDENTIFICATION • LIMIT OF FREE GALLIC ACID

• A. IDENTIFICATION TESTS-GENERAL, Bismuth (191) Sample: 1.0 g
Sample: When heated to redness, it at first chars, leaving Analysis: Shakethe Sample with 20 mL of alcohol for 1 min,
finally a yellow residue. Use the residue for analysis. filter, and evaporate thefiltr~te to dryne~s on a stea~ bath,
Acceptance criteria: Meets the requirements then dry the residue at 105 for 1 h. Weigh the residue.
• B. Acceptance criteria: NMT 5 mg (0.5%)
Sample: 100 mg
Analysis: Agitate the Sample thoroughly with an excess of SPECIFIC TESTS
hydrogen sulfide TS, filter, and boil the filtrate to expel the • Loss ON DRYING (731)
dissolved gas. Cool, and add 1 drop of ferric chloride TS. Analysis: Dry 'a sample at 105° for 3 h.
Acceptance criteria: A purplish blue mixture is produced. Acceptance criteria: NMT 7.0%

ASSAY ADDITIONAL REQUIREMENTS

• PROCEDURE • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Sample solution: Dry 1 g of Bismuth Subgallate at 105° for containers.
3 h, then weigh and ignite in a porcelain crucible. Allow it
to cool, and add nitric acid to the residue, dropwise,
warming until complete solution has been effected.
Analysis: Evaporate the Sample solution to dryness, and
carefully ignite the residue to constant weight. From the Bismuth Subnitrate
weight of the residue, determine the percentage of Bi203 in
the portion of Bismuth Subgallate taken. BisO(OH)9(N0 3)4 1461.99
Acceptance criteria: 52.0%-57.0% on the dried basis Bismuth hydroxide nitrate oxide BisO(OH)9(N0 3) 4'
IMPURITIES Bismuth hydroxide nitrate oxide Bis O(OH)9(N0 3)4 [1304-85-
• ARSENIC (211) 4].
Test preparation: 400 mg
Analysis: Triturate the Test preparation with 400 mg of »Bismuth Subnitrate isa basicsalt that contains the
calcium hydroxide, and ignite. Dissolve the residue in 5 mL . equivalent.ofnot less than 79.0 pe~c~nt of b~smuth
of 3 N hydrochloric acid. trioxide (BI 2 0 3) , calculated on the dried basis.
Acceptance criteria: NMT 7.5 ppm; the solution, without
further treatment, meets the requirements. Packaging and storage-Preserve in well-closed
• LIMIT OF NITRATE containers.
Sample: 100 mg Identification-It responds to the tests for Bismuth (191 ) and
Analysis: Mix the Sample with 5 mL of 2 N sulfuric acid and for Nitrate (191).
5 mL of ferrous sulfate TS, filter the mixture, and carefully Loss on drying (731)-Dry it at 105° for 2 hours: it loses not
superimpose the filtrate, without mixing, on 5 mL of sulfuric more than 3.0% of its weight.
acid, in a test tube. °
Carbonate-Add 3 g to 3 mL of warm nitric acid: no
Acceptance criteria: No reddish brown color appears at the effervescence occurs. Pour the solution into 100 mL of water:
zone of contact of the two liquids. a white precipitate forms. Filter, evaporate the filtrate on a
• LIMITS OF COPPER, LEAD, AND SILVER steam bath to 30 mL, again filter the liquid, divide the latter
Sample: 3 g °
filtrate into portions of 5 mL each, and use these several
Analysis: Ignite the Sample in a porcelain crucible, cool, and portions in the tests for Chloride, Sulfate, Copper, Lead, and
cautiously add, dropwise, just sufficient nitric acid to Silver.
dissolve the residue upon warming. Evaporate the solution Chloride (221)-A 1O-mL portion of the test liquid retained in
to dryness, again ignite, and cool. Cautiously dissolve the the test for Carbonate shows no more chloride than
residue in just sufficient nitric acid with the aid of gentle corresponds to 0.50 mL of 0.020 N hydrochloric acid
heat, concentrate the solution to about 4 mL, and pour it (0.035%).
into 100 mL of water. Filter, evaporate the filtrate on a Sulfate (221)-To a 5-mL portion of the test liquid retained in
steam bath to 20 mL, again filter, and divide this filtrate into the test for Carbonate add 5 drops of barium nitrate TS: no
portions of 5 mL each. turbidity is produced immediately.
Acceptance criteria Limit of ammonium salts-Boil about 100 mg with 5 mL
Copper: To 5 mL of the filtrate add a slight excess of 6 N of 1 N sodium hydroxide: the vapor does not turn moistened
ammonium hydroxide: the liquid does not exhibit a bluish red litmus paper blue.
color. Arsenic, Method I (211 )-Mix 375 mg with 5 mL of water,
Lead: To 5 mL of the filtrate add 5 mL of 2 N sulfuric acid: cautiously add 2 mL of sulfuric acid, and heat the mixture until
the liquid does not become cloudy. fumes of sulfur trioxide are copiously evolved. Cool, cautiously
Silver: To 5 mL of the filtrate add hydrochloric acid, add 10 mL of water, and again evaporate to strong fuming,
dropwise: no precipitate is formed that is insoluble in a repeating, if necessary, to remove any trace of nitric acid. The
slight excess of hydrochloric acid, but that is soluble in 6 limit is 8 ppm.
N ammonium hydroxide. Copper-To a 5-mL portion of the test liquid retained in the
• LIMIT OF ALKALIES AND ALKALINE EARTHS test for Carbonate add a slight excess of 6 N ammonium
Sample: 1.0 g hydroxide: the llquld does not exhibit a bluish color.
Analysis: Boil the Sample with 20 mL of a mixture of equal Lead-Mix a 5-mL portion of the test liquid retained in the
volumes of 6 N acetic acid and water, cool, and filter. test for Carbonate with an equal volume of 2 N sulfuric acid:
Precipitate the bismuth from the filtrate by the addition of the liquid does not become cloudy.
hydrogen sulfide, boil the mixture, and filter. Add 5 drops Silver-To a 5-mL portion of the test liquid retained in the test
of sulfuric acid to the filtrate, evaporate to dryness, and for Carbonate add hydrochloric acid, dropwise: no precipitate
ignite to constant weight. Weigh the residue. is formed that is insoluble in a slight excess of hydrochloric
Acceptance criteria: NMT 5 mg (0.5%) acid, but that is soluble in 6 N ammonium hydroxide.

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USP 43 Official Monographs / Bismuth 579

Limit of alkalies and alkaline earths-Boil 1.0 g with 20 Sample solution: Transfer 52 mg of Bismuth Subsalicylate,
mL of a mixture of equal volumes of 6 N acetic acid and water, previously dried at 105° for 3 h, into a 200-mL volumetric
cool, and filter. Add 2 mL of 3 N hydrochloric acid, precipitate flask. Add 10 mL of 0.5 N sodium hydroxide, heat on a
the bismuth by the addition of hydrogen sulfide, boil the steam bath for 15 min, allow to cool, and dilute with water
mixture, and filter it. Add 5 drops of sulfuric acid to the filtrate, to volume. Centrifuge 70 mL, and then transfer 50.0 mL of
evaporate to dryness, and ignite to constant weight: the the clear supernatant to a beaker. Add about 40 mL of
weight of the residue does not exceed 5 mg (0.5%). water, and adjust with 0.5 N sodium hydroxide or 1 N
Assay-Transfer about 400 mg of Bismuth Subnitrate, hydrochloric acid to a pH of 4.5. Transfer this solution to a
accurately weighed, to a 250-mL beaker. Add 5 mL of water, 1OO-mL volumetric flask with the aid of water, and dilute
then add 2 mL of nitric acid, and warm, if necessary, to effect with water to volume.
solution. Dilute with water to 100 mL, add 0.3 mL of xylenol Reacted sample solution: To 25.0 mL of Sample solution
orange TS, and titrate with 0.05 M edetate disodium VS to a add 1.0 mL of Solution A.
yellow endpoint. Each mL of 0.05 M edetate disodium is Unreacted sample solution: To 25.0 mL of the Sample
equivalent to 11.65 mg of Bi203 • solution add 1.0 mL of 0.05 N hydrochloric acid.
Blank: Water, adjusted with 0.5 N sodium hydroxide or 1 N
hydrochloric acid to a pH of 4.5
Reacted blank solution: To 25.0 mL of Blank add 1.0 mL of
Solution A.
Bismuth Subsalicylate Unreacted blank: To 25.0 mL of Blank add 1.0 mL of 0.05
N hydrochloric acid.
C7H sBi0 4 362.09 Instrumental conditions
(2-Hydroxybenzoato-0')-oxobismuth; Mode: UV
2-Hydroxybenzoic acid bismuth (3+) salt, basic [14882-18- Analytical wavelength: 525 nm
9]. Analysis
Samples: Reacted standard solution, Unreacted standard
DEFINITION
Bismuth Subsalicylate is a basic salt that contains NLT 56.0%
solution, Reacted sample solution, Unreacted sample
and NMT 59.4% of bismuth (Bi) and NLT 36.5% and NMT
solution, Reacted blank solution, and Unreacted blank
Concomitantly determine the absorbances of the Samples.
39.3% of total salicylates on the dried basis.
Calculate the percentage of total salicylates in the portion
IDENTIFICATION of dried Bismuth Subsalicylatetaken:

Result = [(AUR - Auu - B)/(AsR- Asu - B)] x (CslCu) x 100

• A.' AUR = absorbance of the Reacted sample solution

S Auu = absorbance of the Unreacted sample solution
• B. IDENTiFICATION TESTS-GENERAL, Bismuth (191): Meets B = difference in the absorption of the Reacted blank
the requirements solution and the absorption of the Unreacted
blank
ASSAY ASR = absorbance of the Reacted standard solution
• BISMUTH Asu = absorbance of the Unreacted standard solution
Sample solution: Transfer an equivalent to 300 mg of
Bismuth Subsalicylate, previously dried at 105° for 3 h, to a Cs = concentration of USP Salicylic Acid RS in the
porcelain crucible, and ignite. Allow it to cool, and add Standard solution (mg/mL)
about 2 mL of nitric acid to the residue, dropwise, warrninq Cu = concentration of Bismuth Subsalicylate in the
until dissolved. Add about 60 mL of water and 0.3 mL of Sample solution (mg/mL)
xylenol orange TS.
Acceptance criteria: 36.50/0-39.3% of total salicylates on
Titrimetric system
the dried basis
Mode: Direct titration
Titrant: 0.05 M edetate disodium VS IMPURITIES
Endpoint detection: Visual • ARSENIC, Method I (211)
Analysis: Titrate the Sample solution with Titrant to a yellow Sample: 300 mg of Bismuth Subsalicylate with 300 mg of
endpoint. Each mL of Titrant is equivalent to 10.45 mg of calcium hydroxide
bismuth (Bi). Analysis: Triturate the Sample, and ignite. Dissolvethe
Acceptance criteria: 56.0%-59.4% of bismuth (Bi) on the residue in 5 mL of 3 N hydrochloric acid.
dried basis Acceptance criteria: 10 ppm
• TOTAL SALICYLATES • LIMIT OF COPPER, LEAD, AND SILVER
Solution A: Ferric ammonium sulfate TS, 1 N hydrochloric Standard stock solution: Add 3.0 mL each of 1000-lJg/mL
acid, and water (4:1 :15) solutions of copper, lead, and silver, respectively, to a
Standard stock solution: 0.2 mg/mL of USP Salicylic Acid 2000-mL flask, and dilute with 1 M nitric acid to volume.
RS in water Standard solution: 1.5 IJg/mL of copper, 1.5 IJg/mL of lead,
Standard solution: 0.05 mg/mL of USP Salicylic Acid RS in and 1.5 IJg/mL of silver, in 1 M nitric acid from the Standard
water, prepared by adding 25.0 mL of Standard stock stock solution. The concentrations of copper, lead, and silver
solution and 70 mL of water to a 1OO-mL volumetric flask. may be modified by using different volumes or
Adjust with 0.5 N sodium hydroxide or 1 N hydrochloric concentrations to bring the absorption response within the
acid to a pH of 4.5, before dilution with water to volume. working range of the atomic absorption
Reacted standard solution: To 25.0 mL of Standard spectrophotometer.
solution add 1.0 mL of Solution A. Sample solution: Ignite 3 g of sample in a porcelain
Unreacted standard solution: To 25.0 mL of the Standard crucible, cool, and cautiously add 6 M nitric acid to dissolve
solution add 1.0 mL of 0.05 N hydrochloric acid. the residue, and evaporate on a steam bath. Ignite the
residue, cool, transfer the residue to a tared conical flask,

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580 Bismuth / OfficialMonographs USP 43

and wash the flask with about 5 mLof 6 M nitric acid, the acetonitrile addition, shaking, centrifuging, and
adding the wash to the conical flask. Dissolve the residue decanting. Combine the decanted liquid with the first
with the aid of heat, and add water to obtain a solution decantate. Pass the combined liquid through a filterof
weighing 20.0 g. The concentrate of Bismuth 0.5-~m or finer pore size,and collectthe filtrate in a 50-mL
Subsalicyclate may be modified by using the same volumetric flask. Wash the container with 5 mL of
proportions used for modifying the Standard solution, by acetonitrile, and filterthe wash, collectingthe filtrate in the
using a different quantity, or by further dilution. volumetric flask. Dilute with water to volume.
Instrumental conditions Chromatographic system
(See Atomic Absorption Spectroscopy (852).) (See Chromatography(621):, System Suitability.)
Mode: Atomic absorption spectrophotometry Mode: LC
Analytical wavelength: 324.7 nm for copper; 217 nm for Detector: UV 300 nm
lead; 328.1 nm for silver Columns
Lamps: Copper, lead, and silverhollow-cathode, and Guard: 3.2-mm x 1.5-cm; 5-~m packing L1
oxidizing flames Analytical: 4.6-mm x 30-cm; s-um packing L1
Analysis Flow rate: 1 mL/min
Samples: Standard solution and Sample solution Injection volume: 20 ~L
Acceptance criteria: 10 ppm; the absorbances of the System suitability
Sample solutions do not exceed those of the Standard Sample: Standard solution
solutions for each element. Suitability requirements
• LIMIT OF SOLUBLE BISMUTH Tailing factor: NMT 2.0
Standard solution: 2 ~g/mL of bismuth (Bi), prepared as Relative standard deviation: NMT 2.0%
follows. Add 242.0 mg of bismuth nitrate pentahydrate to Analysis
a 1OO-mL volumetricflask, add 3 mLof 1.5 M nitric acid, Samples: Standard solution and Sample solution
swirl to dissolve, and dilute with water to volume. Add 1.0 Calculatethe percentage of free salicylic acid in the portion
mL of this solution to a 500-mLvolumetricflask, add 250 of Bismuth Subsalicylate taken:
mLof 1.5 M nitricacid, and dilute with water to volume.
The concentration of bismuth in this solution may be Result = (rulrs) x (CslCu) x 100
modified by using a lesserdilution or by further dilution to .
bring the absorption response within the working range of ru = peak area of salicylic acidfrom the Sample solution
the atomic absorption spectrophotometer. ts = peak area of salicylic acid from the Standard
Sample solution: 5.0 g of Bismuth Subsalicylate in 100 mL solution
of water, and stir the suspension thus obtained for 2 h at Cs =concentration of USP Salicylic Acid RS in the
20°-23°. Passthrough filter paper. Pass the filtrate thus Standard solution (mg/mL)
obtained through a filter of O.l-~m or less pore size. Add Cu =concentration of the Bismuth Subsalicylate in the
0.1 mLof nitric acid to 10.0 mLof the filtrate. The Sample solution (mg/mL)
concentrate of Bismuth Subsalicyclate may be modified by
using the same proportions used for modifying the Acceptance criteria: NMT 0.2%
Standard solution, by using a different quantity, or by
SPECIFIC TESTS
further dilution.
Instrumental conditions • pH (791)
(See Atomic Absorption Spectroscopy (852).) Sample solution: 109 of Bismuth Subsalicylate in 90 mLof
Mode: Atomic absorption spectrophotometry water
Analytical wavelength: 223.06 nm for bismuth Analysis: Shake by mechanical means for 10 min, and filter.
Lamp: Bismuth hollow-cathode and an oxidlzinq flame Acceptance criteria: 2.7-5.0
• Loss ON DRYING (731)
Analysis Analysis: Dryat 105 for 3 h.
0

Samples: Standard solution and Sample solution Acceptance criteria: NMT 1.0%
Concomitantly determine the absorbances of the
Standard solution and the Sample solution. ADDITIONAL REQUIREMENTS
Acceptance criteria: 40 ppm; the absorbances of the • PACKAGING AND STORAGE: Preserve intight, light-resistant
Samplesolution do not exceed those of the Standard containers.
solution. • USP REFERENCE STANDARDS (11)
• LIMIT OF NITRATE USP BismuthSubsalicylate RS
Standard solution: To 0.1 g of salicylic acid add 6 mLof USP Salicylic Acid RS
water, 4.0 mLof a solution containing 100 ~g of nitrate per
mL, and 20 mLof sulfuric acid. Prepare concomitantly
with the Sample solution
Sample solution: Add 10 mLof water to 0.1 g of Bismuth
Subsalicylate. Carefully add 20 mLof sulfuric acid. Bismuth Subsalicylate Magma
Acceptance criteria: 0.4%; the Sample solution should not
be more yellow than the Standard solution. DEFINITION
• LIMIT OF FREE SALICYLIC ACID Bismuth Subsalicylate Magma is a suspension of Bismuth
Mobile phase: Methanol and 0.06 M acetic acid (55:45) Subsalicylate in water that contains NLT 90.0% and NMT
Diluent: Acetonitrile and water (1:1) 110.0% of the labeled amount of bismuth subsalicylate
Standard solution: 0.02 mg/mL of USP Salicylic Acid RS in (C7H sBi04) . Bismuth subsalicylate is a basic salt that when
Diluent dried at 105 for 3 h contains NLT 56.0% and NMT 59.4%
0

Sample solution: Add 260 mg of Bismuth Subsalicylate to bismuth (Bi) and NLT 36.5% and NMT 39.3% of total
a glass centrifuge tube, add about 12 mLof acetonitrile, salicylates.
shake by mechanical means for 20 min, and centrifuge.
Decant the supernatant into a suitable container. Repeat

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USP 43 OfficialMonographs / Bismuth 581

Dry at 105° for 3 h to determine the solids content and, after Analysis
determining the solids content, perform all tests on a portion Samples: Reacted standardsolution, Unreacted standard
of the dried Magma. solution, Reaaed sample solution, Unreacted sample
solution, Reacted blank solution, and Unreacted blank
IDENTIFICAnON
Concomitantly determine the absorbances of the Samples.
Calculate the percentage of total salicylates in the portion
of-dried Magma taken:
• A.~~~ Result = [(AUR - Auu - B)/(AsR- Asu - B)] x (Cs/Cu) x 100
$pgC{r
• B. IDENTIFICATION TESTs-GENERAL, Bismuth (191): Meets AUR = absorbance of the Reacted sample solution
the requirements Auu = absorbance of the Unreacted sample solution
ASSAY B =difference in the absorption of the Reacted blank
• BISMUTH solution and the absorption of the Unreacted
Sa~ple solutio~: Transfer a.n equivalent to 300 mg of blank
bismuth subsallcylate, previously dried at 105° for 3 h, to a = absorbance of the Reacted standard solution
porcelain crucible, and ignite. Allow it to cool, and add =absorbance of the Unreacted standard solution
about 2 mL of nitric acid to the residue, dropwise, warming = concentration of USP Salicylic Acid RS in the
until dissolved. Add about 60 mL of water and 0.3 mL of Standardsolution (mg/mL)
xylenol orange TS. Cu = concentration of bismuth subsalicylate in the
Titrimetric system Sample solution (mg/mL)
Mode: Direct titration
Titrant: 0.05 Medetate disodium VS Acceptance criteria: 36.5%-39.3% of total salicylates on
Endpoint detection: Visual the previously dried basis
Analysis: Titrate the Sample solution with Titrant to a yellow
endpoint. Each mL of Titrant is equivalent to 10.45 mg of IMPURITIES
bismuth (Bi). • LIMIT OF COPPER, LEAD, AND SILVER
Acceptance criteria: 56.0%-59.4% of bismuth on the Standard stock solution: Add 3.0 mL each of 1OOO-~g/mL
previously dried basis solutions of copper, lead, and silver, respectively, to a
• TOTAL SALICYLATES
2000-mL flask, and dilute with 1 M nitric acid to volume.
Solution A: Ferric ammonium sulfate TS, 1 N hydrochloric Standard solution: 1.5 ~g/mL of copper, 1.5 ~g/mL of lead
acid, and water (4:1 :15) and 1.5 ~g/mL of silver, in 1 M nitric acid from the Standard
Standard stock solution: 0.2 mg/mL of USP Salicylic Acid stocksolution. The concentrations of copper, lead, and silver
RS in water may be modified by using different volumes or
Standard solution: 0.05 mg/mL of USP Salicylic Acid RS in concentrations to bring the absorption response within the
water, prepared by adding 25.0 mL of Standardstock working range of the atomic absorption
solution and 70 mL of water to a 1OO-mL volumetric flask. spectrophotometer.
Adjust with 0.5 N sodium hydroxide or 1 N hydrochloric Sample solution: Ignite 3 g of sample in a porcelain
acid to a pH of 4.5, before dilution with water to volume. crucible, cool, cautiously add 6 M nitric acid to dissolve the
Reacted standard solution: To 25.0 mL of Standard residue, and evaporate on a steam bath. Ignite the residue
solution add 1.0 mL of Solution A. cool, transfer the residue to a tared conical flask, and wash
Unreacted standard solution: To 25.0 mL of the Standard the flask with about 5 mL of 6 M nitric acid, adding the
solution add 1.0 mL of 0.05 N hydrochloric acid. wash to the conical flask. Dissolve the residue with the aid
Sa~ple solution: Transfer an equivalent to 52 mg of
of heat, and add water to obtain a solution weighing 20.0
bismuth subsalicylate from previously dried Magma at 105 ° g. The concentrate of bismuth subsalicyclate may be
for 3 h to a 200-mL volumetric flask. Add 10 mL of 0.5 N modified by using the same proportions used for
sodium hydroxide, heat on a steam bath for 15 min, allow modifying the Standard solution, by using a different
to cool, and dilute with water to volume. Centrifuge 70 mL, quantity, or by further dilution.
and then transfer 50.0 mL of the clear supernatant to a Instrumental conditions
bea~er. Add about 40 mL of water, and adjust with 0.5 N
(See Atomic Absorption Spectroscopy (852).)
sodium hydroxide or 1 N hydrochloric acid to a pH of 4.5. Mode: Atomic absorption spectrophotometry
Transfer this solution to a 1OO-mL volumetric flask with the Analytical waveleng~h: 324.7 nm for copper; 217 nm for
aid of water, and dilute with water to volume. lead; 328.1 nm for silver
Reacted sample solution: To 25.0 mL of Sample solution Lamps: Copper, lead and silver hollow-cathode and
add 1.0 mL of Solution A. oxidizing flames ' .
Unreacted sample solution: To 25.0 mL of the Sample Analysis
solution add 1.0 mL of 0.05 N hydrochloric acid. Samples: Standardsolution and Sample solution
Blank: Water, adjusted with 0.5 N sodium hydroxide or 1 N Concomitantly determine the absorbances of the
hydrochloric acid to a pH of 4.5 Standardsolution and the Sample solution
Reacted blank solution: To 25.0 mL of Blankadd 1.0 mL of Acceptance criteria: 10 ppm; the absorbances of the
Solution A. Sample solutions do not exceed those of the Standard
Unreacted blank: To 25.0 mL of Blank add 1.0 mL of 0.05 solutions for each element.
• LIMIT OF SOLUBLE BISMUTH
N hydrochloric acid.
Instrumental conditions Standard solution: 2 ~g/mL of bismuth (Bi), prepared as
Mode: UV follows. Add 242.0 mg of bismuth nitrate pentahydrate to
Analytical wavelength: 525 nm a 1OO-mL volumetric flask, add 3 mL of 1.5 M nitric acid
swirl to dissolve, and dilute with water to volume. Add 1'.0
mL of this solution to a 500-mL volumetric flask, add 250
mL of 1.5 M nitric acid, and dilute with water to volume.
The concentration of bismuth in this solution may be

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582 Bismuth / OfficialMonographs USP 43

m?dified by using a lesserdilution or by further dilution to Result = (rufrs) x (CslC u) x 100

bring the absorption response within the working range of
the atomic absorption spectrophotometer. = peakarea of salicylic acid from the Sample solution
Sample sc;>lution: 5.0 g of bismuth subsalicylate from dried = peak area of salicylic acid from of the Standard
Mag!l1a In 100 mLof water, and stir the suspension thus solution
obta!ned for 2 h at 20°-23°. Pass through filter paper. Pass =concentration of USP Salicylic Acid RS in the
the filtrate thus obtained through a filter of O.l-JJm or less Standard solution (mgfmL)
pore size.Add0.1 mL of nitricacid to 10.0 mLof the filtrate. =concentration of the bismuth subsalicylate in the
The concentrate of bismuth subsalicyclate may be modified Sample solution (rnq/rnl.)
by using the same proportions used for modifying the
Standard solution, by using a differentquantity or by Acceptance criteria: NMT 0.2%
further dilution. '
Instrumental conditions ADDITIONAL REQUIREMENTS
(See Atomic Absorption Spectroscopy (852).) • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Mode: Atomic absorption spectrophotometry containers.
Analytical wavelength: 223.06 nm for bismuth • LABELING: The label states that this article is not intended
lamp: Bismuth hollow-cathode and an oxidizing flame for direct administration to humans or animals.
Analysis • USP REFERENCE STANDARDS (11)
Samples: Standard solution and Sample solution USP Bismuth Subsalicylate RS
Concomitantly determine the absorbances of the USP Salicylic Acid RS
Standard solution and the Sample solution.
Acceptance criteria: 40 ppm; the absorbances of the
Sample solution do not exceed those of the Standard
solution. Bismuth Subsalicylate Oral Suspension
• LIMIT OF NITRATE
Standard solution: To 0.1 g of salicylic acid add 6 mL of DEFINITION
water, 4.0 mL of a solution containing 100 JJg of nitrate per . Bismuth Subsalicylate Oral Suspension contains NLT 90.0%
mL, and 20 mL of sulfuric acid. Prepare concomitantly and NMT 110.0% of the labeled amount of bismuth
with the Sample solution. subsalicylate (C7H sBi04) . It may contain one or more suitable
Sample solution: Add 10 mLof water to 0.1 g of Magma. buffers, coloring agents, flavors, preservatives, stabilizers
Carefully add 20 mLof sulfuric acid, and mix. sweeteners, and suspending agents. '
Acceptance criteria: 0.4%; the Sample solution should not
be more yellow than the Standard solution. IDENTIFICAnON
• LIMIT OF FREE SALICYLIC ACID • A. IDENTIFICATION TESTS-GENERAL (191), Bismuth: Meets
Mobile phase: Methanol and 0.06 M acetic acid the requirements
(550:450) • B. IDENTI~ICATION TESTs-GENERAL (191), Salicylate: Meets
Diluent: Acetonitrile and water (1 :1) the requirements of test A after acidifying with nitric acid
Standard solution: 0.02 mgfmL of USP Salicylic Acid RS in
ASSAY
Diluent
Sample solution: Add 260 mg of bismuth subsalicylate • PROCEDURE
from dried Magma to a glass centrifugetube, add about 12 Standard stock solution: 2.5 rnq/rnl, of bismuth in nitric
mLof ace~onitrile, shake by mechanical means for 20 min, acid. Prepare by dissolving in 6% of the flask volume of
and c~ntrlfuge. Decant the supernatant into a suitable nitric acid and diluting with 0.01 N nitric acid to volume.
cont~lne~. Repeat the ac~tonitrile addition, shaking,
Standard solution: 0.05 mgfmL of bismuth in 1 N nitric
centrifuging, and decanting, combining the decanted acid from the Standard stocksolution
liquid with .the first decantate. Pass the combined liquid Sample solution: Transfer109 of Oral Suspension,
through a filterof 0.5-JJm pore size,collecting the filtrate in previously well shaken in its original container to ensure
a 50-mLvolumetric flask. Wash the container with 5 mL of homogeneity, to a 200-mLvolumetricflask. Addabout 100
acetonitrile, and filterthe wash, collecting the filtrate in the mL of 1 N nitric acid, and dilute with 1 N nitric acid to
volumetric flask. Dilute with water to volume. volume. Mix well without shaking, transfer 10.0 mLof this
Chromatographic system mixture to a 1OO-mL volumetric flask, and dilute with 1 N
(See Chromatography (621), System Suitability.) nitricacid to volume. Centrifuge about 20 mL at 4500 rpm
Mode: LC for at least 10 min. .
Detector: UV 300 nm Instrumental conditions
Columns (See Ultraviolet-Visible Spectroscopy (857).)
Guard: 3.2-mm x 1.5-cm; 5-JJm packing L1 Mode: UV-Vis
Analytical: 4.6-mm x 30-cm; 5-JJm packing L1 Analytical wavelength: 463 nm
Flow rate: 1 mt/rnln Cell: 1 cm
Injection volume: 20 JJL Blank: 1 N nitric acid
System suitability Analysis
Sample: Standard solution Samples: Standard solution, Sample solution, and Blank
Suitability requirements Transfera measured volume of the Sample solution that
Tailing factor: NMT 2.0 contains 0.9 mg of bismuth subsalicylate and 10 mLofthe
Relative standard deviation: NMT 2.0% Standard solution to separate 50-mLvolumetricflasks. Add
Analysis 10.0 mLof 10% ascorbic acid solution and 25.0 mLof
Samples: Standard solution and Sample solution . 20% potassium iodide solution to each volumetricflask
Calculatethe percentage of free salicylic acid in the portion and dil~te with water to volume. Concomitantly ,
of Magma taken: determine the absorbances of both solutions, using the
Blank to set the spectrophotometer.

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 USP 43 OfficialMonographs / Bisoctrizole 583

Calculate the percentage of the labeled amount of bismuth Transfer 10.0 mL of the Standard solution and the Sample
subsalicylate (C 7HsBi0 4 ) in the portion of Oral Suspension solution to separate 50.0-mL volumetric flasks, and dilute
taken: with the Blank to volume. Concomitantly determine the
absorbance of the solutions at the wavelength of
Result =(A viA s) x (C siC v) x (M ,JiM (2) x 100 maximum absorbance at 463 nm with a suitable
spectrophotometer, using the combined reagent
Au = absorbance of the Sample solution solutions as the blank.
As = absorbance of the Standard solution Calculate the percentage of the labeled amount of bismuth
Cs = concentration of bismuth in the Standardsolution subsalicylate (C 7HsBi04) in the portion of Tablets taken:
(mg/mL)
Cu = nominal concentration of bismuth subsalicylate Result = (A viA s) x (C siC v) x (M ,JiM (2) x 100
in the Sample solution (mg/mL)
Mt = molecular weight of bismuth subsalicylate, Au =absorbance of the Sample solution
362.09 As =absorbance of the Standardsolution
M ,2 = molecular weight of bismuth, 208.98 Cs =concentration of bismuth in the Standardsolution
(mg/mL)
Acceptance criteria: 90.0%-110.0% Cu =nominal concentration of the Sample solution
(mg/ml)
SPECIFIC TESTS M ,1 =molecular weight of bismuth subsalicylate,
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR 362.09
SPECIFIED MICROORGANISMS (62): The total aerobic M ,2 =molecular weight of bismuth, 208.98
microbial count is NMT 102 cfu/g, and the total combined
molds and yeasts count is NMT 5 x 10 1 du/g. It meets the Acceptance criteria: 90.0%-110.0%
requirements of the test for the absence of Escherichia coli.
• pH (791): 3.0-5.5 PERFORMANCE TESTS
• DISINTEGRATION (701)
ADDITIONAL REQUIREMENTS This test does not apply to Tablets labeled as chewable.
• PACKAGING AND STORAGE: Preserve in tight containers. Time: 10 min
Protect from freezing. Avoid excessive heat (over 40°). Acceptance criteria: Meet the requirements
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight containers.
Avoid excessive heat (over 40°).
Bismuth Sub$alicylate Tablets • LABELING: Label chewable Tablets to indicate that they are
to be chewed before swallowing.
DEFINITION
Bismuth Subsalicylate Tablets contain NLT 90.0% and NMT
110.0% of the labeled amount of bismuth subsalkylate
(C 7H sBi04 ) ·
IDENTIFICATION
Bisoctrizole
• A. IDENTIFICATION TESTS-GENERAL, Bismuth (191): Meet
the requirements .
• B. IDENTIFICATION TESTS-GENERAL (191), Salicylate: After
acidifying with nitric acid, it meets the requirement? of test
A.
ASSAY
, • PROCEDURE
Standard stock solution: 2.5 mg/mL of bismuth in nitric
acid. Prepare by dissolving in 6% of the flask volume of C41 HsoN 602 658.87
nitric acid, and diluting with 0.01 N nitric acid to volume. Phenol, 2,2'-methylenebis[6-(2H-benzotriazol-2-yl)-4-
Standard solution: 0.05 mg/mL of bismuth in 1 N nitric (l,l,3,3-tetramethylbutyl)]-;
acid from the Standardstock solution 2,2'-Methylenebis[6-(2H-benzotriazol-2-yl)-4-(l,l,3,3-
Sample stock solution: Equivalent to 90 mg of bismuth tetramethylbutyl)phenol] [103597-45-1].
subsalicylate from finely powdered Tablets in a 200-mL
volumetric flask. Add 150 mL of 1 N nitric acid, and sonicate DEFINITION
for 2 min. Dilute with 1 N nitric acid to volume. Bisoctrizole contains NlT 96.0% and NMT 102.0% of
Sample solution: Transfer 20.0 mL of the Sample stock bisoctrizole (C41 HsoN 6 0 2) , calculated on the as-is basis.
solution to a 1OO-mL volumetric flask, and dilute with 1 N
IDENTIFICAnON
nitric acid to volume. Centrifuge a portion at 4500 rpm for
at least 10 min.
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).) • A.ASPE¢TROSCOPIC IDENTIFICATION TESTS (197), Infrared
Mode: UV-Vis
Spectr9scopy: ~97Kj. (eN l-May:2020)
Analytical wavelength: 463 nm
• B. The retention time of the major peak of the Sample
Cell: 1 cm solution corresponds to that of the Standardsolution, as
Blank: 10% ascorbic acid solution, 20% potassium iodide
obtained in the Assay.
solution, and 1 N nitric acid (2:5:1)
Analysis
Samples: Standardsolution and Sample solution

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584 Bisoctrizole / OfficialMonographs USP 43

ASSAY IMPURITIES
• PROCEDURE • LIMIT OF BISOCTRIZOLE RELATED COMPOUND A AND
Diluent: Tetrahydrofuran and 0.2% (wlv) aqueous solution BISOCTRIZOLE ISOMER
of 1-pentane sulfonic acid sodium salt (60:40) Diluent, Solution A, Solution B, Mobile phase, System
Solution A: 0.4 9 of 1-pentane sulfonic acid sodium salt, 800 suitability solution, Sample solution, and
mL of methanol, 200 mLof water, and 0.5 mL of Chromatographic system: Proceed as directed in the
phosphoric acid Assay.
Solution B: 0.4 9 of 1-pentane sulfonic acid sodium salt, Standard stock solution A: 0.65 mg/mL of USP Bisoctrizole
1000 mLof methanol, and 0.5 mL of phosphoric acid RS in tetrahydrofuran
Mobile phase: See Table 1. Return to original conditions Standard stock solution B: 0.40 mg/mL of USP Bisoctrizole
and re-equilibrate the system. Related Compound A RS in tetrahydrofuran
Standard solution: Transfer 5 mLof Standardstocksolution
Table 1 A and 1.0 mL of Standard stock solution B to a 100-mL
Time Solution A Solution B volumetric flask. Add 60 mL of tetrahydrofuran, and dilute
(min) (%) (%) with Diluent to volume.
0 70 30
System suitability
Sample: System suitabilitysolution
1 70 30 [NOTE-See Table 2 for the relative retention times for
11 3 97
bisoctrizole related compound A and the bisoctrizole
isomer.]
40 3 97 Suitability requirements
Resolution: NLT 1.5 between bisoctrizole and the
System suitability solution: 0.8 mg/mL of bisoctrizole from bisoctrizole isomer
USP Bisoctrizole Resolution Mixture RS prepared as follows. Analysis
TransferUSP Bisoctrizole Resolution MixtureRS to a suitable Samples: Standard solution and Sample solution
volumetric flask, dissolve intetrahydrofuran, and dilutewith Calculatethe percentage of bisoctrizole related compound
Diluent to volume. A in the portion of Bisoctrizole taken:
Standard solution: 0.8 mg/mL of USP Bisoctrizole RS
prepared as follows. Transfer USP Bisoctrizole RS to a Result = (r vir s) x (C siC v) x 100
suitable volumetric flask, dissolve in tetrahydrofuran
equivalent to 60% of the final volume, and dilute with ru =peak response of bisoctrizole related compound
Diluent to volume. A from the Sample solution
Sample solution: Transfer 80 mg of Bisoctrizole to a 100-mL rs = peak response of bisoctrizole related compound
volumetric flask. Dissolve in 60 mLof tetrahydrofuran, and Afrom the Standard solution
dilute with Diluent to volume. Cs =concentration of USP Bisoctrizole Related
Chromatographic system Compound A RS in the Standardsolution
(See Chromatography (621), System Suitability.) (mg/mL)
Mode: LC Cu =concentration of Bisoctrizole in the Sample
Detector: UV 346 nm solution(mg/mL)
Column: 3.0-mm x 25-cm; 5-J.lm packing L1 Calculatethe percentage of bisoctrizole isomer in the
Column temperature: 40° portion of Bisoctrizole taken:
Flow rate: 0.8 mL/min
Injection volume: 10 J.lL Result = (r vir s) x (C siC v) x 100
System suitability .
Samples: System suitabilltv solution and Standardsolution ru = peak response of bisoctrizole isomer from the
[NoTE-See Table 2 for the relative retention times for Sample solution
bisoctrizole and the bisoctrizole isomer.] rs =peak response of bisoctrizole from the Standard
Suitability requirements . solution
Resolution: NLT 1.5 between bisoctrizole and the Cs = concentration of USP Bisoctrizole RS in the
bisoctrizofe isomer, System suitability solution Standard solution(mg/mL)
Relative standard deviation: NMT 2.0%, Standard
solution
Cu =
concentration of Bisoctrizole in the Sample
solution(mg/mL)
Analysis
Samples: Standardsolution and Sample solution Acceptance criteria: See Table 2.
Calculatethe percentage of bisoctrizole (C41 HsoN 60 2) in the • ORGANIC IMPURITIES
portion of Bisoctrizole taken: Diluent, Solution A, Solution B, Mobile phase, Standard
solution, Sample solution, Chromatographic system,
Result = (r vir s) x (C siC v) x 100 and System suitability: Proceed as directed in the Assay.
Analysis
ru = peak response from the Sample solution Sample: Sample solution
rs = peak response from the Standard solution Calculatethe percentage of each individual unspecified
Cs = concentration of USP Bisoctrizole RS in the impurity in the portion of Bisoctrizole taken:
Standardsolution (mg/mL)
Cu =concentration of Bisoctrizole in the Sample Result = (r vir r) x 100
solution (mg/mL)
ru = peak response of each individual impurity
Acceptance criteria: 96.00/0-102.0% on the as-is basis rT = sum of the responses of all the peaks

Acceptance criteria: See Table 2.

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 USP 43 Official Monographs / Bisoprolol 585

Table .2 Specific rotation (781): between _2° and +2°.

Relative Acceptance Test solution: 10 mg per mL, in methanol.
Retention Criteria, Water Determination, Method I (921): not more than 0.5%.
Name Time NMT(%) Residue on ignition (281): not more than 0.1%.
Bisoctrizole related Chromatographic purity-
compound A" 0.42 0.5
Diluent, Mobile phase, System suitability solution, and
Bisoctrizole 1.0 - Chromatographic system-Proceed as directed in the Assay.
Bisoctrizole isomer!' 1.1 4.0 Standard solution-Prepare as directed for Standard
preparation in the Assay.
Anyindividual
- Test solution-Prepare as directed for Assay preparation in
unspecified impurity 0.10 the Assay.
Total impurities - 4.0 Procedure-Inject a volume (about 10 J.JL) of the Test
solution into the chromatograph, record the chromatogram,
a 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol. and measure the peak areas. Calculate the percentage of total
b Phenol, 2,2'-methylenebis[6-(2H-benzotriazol-2-yl)-4-(1,1,3,3- impurities in the portion of Bisoprolol Fumarate taken by the
tetramethylbutyl)] formula:
ADDITIONAL REQUIREMENTS 1OO(rir s)
• PACKAGING AND STORAGE: Preserve in well-closed
containers, and store at controlled room temperature. in which r i is the sum of areasfor all the peaks, excluding the
• USP REFERENCE STANDARDS (11) fumaric acid and bisoprolol peaks; and r s is the sum of the
USP Bisoctrizole RS areas of all the peaks in the chromatogram: not more than
USP Bisoctrizole Related Compound A RS 0.5% of total impurities is found.
2-(2H-Benzotriazol-2-yl)-4-(l,l,3,3-tetramethylbutyl) Content of fumaric acid-Transfer about 500 mg of
phenol. Bisoprolol Fumarate, accurately weighed, to a beaker, and
C2oH2SN3 323.43 dissolve in 70 mL of dehydrated alcohol. Add 8.0 mL of 0.1 N
USP Bisoctrizole Resolution Mixture RS tetrabutylammonium hydroxide VS, and stir for 2 minutes.
A mixture of approximately 1.5% of bisoctrizole isomer Titrate with 0.1 N tetrabutylammonium hydroxide VS,
[phenol, 2,2'-methylenebis[6-(2H-benzotriazol-2-yl)-4- determining the endpoint potentiometrically, using a
(l,l,3,3-tetramethylbutyl)]] in a matrix of bisoctrizole. glass-calomel electrode system. Perform a blank
determination, and make any necessary correction (see
».
Titrimetry (541 Each mL of 0.1 N tetrabutylammonium
hydroxide is equivalent to 5.804 mg of fumaric acid: not less
than 14.8% and not more than 15.4% offumaric acid isfound,
Bisoprolol Fumarate calculated on' the anhydrous basis.
Assay-
o Diluent-Prepare a mixture of water and acetonitrile
HO)'~OH (65:35).
o Mobile phase-To a 1-L portion of Diluent add 5 mL of
heptafluorobutyric acid, 5 mL of diethylamine, and 2.5 mL of
(ClsH31N04)2'C4H404 766.96 formic acid. Mix, filter, and degas. Make adjustments if
2-Propanol, 1-[4-[[2-(1-methylethoxy)ethoxy]methyl]phen necessary (see System SUitability under Chromatography
oxy]-3-[(1-methylethyl)amino]-, (±)-, (t)-2-butenedioate (621»,
(2:1) (salt). System suitability solution-Prepare a solution in Diluent
(±)-1-[[a-(2-lsoproproxyethoxy)-p-tolyl]oxy]-3-(isopropyI containing about 0.5 mg of propranolol hydrochloride and 1
amino)-2-propanol fumarate (2:1) (salt) [104344-23-2]. mg of Bisoprolol Fumarate per mL.
Standard preparation-Quantitatively dissolvean accurately
» Bisoprolol Fumarate contains not less than 97.5 weighed quantity of USP Bisoprolol Fumarate RS in Diluent to
obtain a solution having a known concentration of about 1 mg
percent and not more than 102.0 percent of (C 18 per mL.
H 31 NO 4) 2 • C 4 H 4 0 4/ calculated on the Assay preparation-Transfer about 50 mg of Bisoprolol
anhydrous basis. Fumarate, accurately weighed, to a 50-mL volumetric flask.
Dissolve in and dilute with Diluent to volume, and mix.
Packaging and storage-Preserve in tight, light-resistant Chromatographic system (see Chromatography (621 »- The
containers. Store at controlled room temperature. liquid chromatograph is equipped with a 273-nm detector
VSP Reference standards (11)- and a 4.6-mm x 12.5-cm column that contains packing L7.
USP Bisoprolol Fumarate RS The flow rate is about 1 mL per minute. Chromatograph the
System suitability solution, and record the peak areas as
Identificatlon- directed for Procedure: the resolution, R, between bisoprolol
and propranolol is not less than 7.0. Chromatograph the
Standard preparation, and record the peak areasasdirected for
Procedure: the tailing factor is not more than 2.0; and the
relative standard deviation for replicate injections is not more
than 2.0%.
retention time of the-major peak in the Procedure-Separately inject equal volumes (about 10 J.JL)
chromatogram of the Assay preparation corresponds to that in of the Standard preparation and the Assay preparation into the
the chromatogram of the Standard preparation, as obtained chromatograph, record the chromatograms, and measure the
in the Assay. ' areas for the major peaks. Calculate the quantity, in mg, of (C

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586 Bisoprolol / Official Monographs USP 43

18 H 31 NO 4)Z . C 4H 4
taken by the formula:
° 4in the portion of Bisoprolol Fumarate Calculate the percentage of (C18H31N04)Z' C4H404 in the
portion of Tablets taken:

50C(r ulr s) Result = (rulrs) x (CslCu) x 100

in which C is the concentration, in mg per mL, of USP =peak response from the Sample solution
Bisoprolol Fumarate RS in the Standard preparation; and r u and = peak response from the Standard solution
r s are the peak areasobtained from the Assay preparation and = concentration of USP Bisoprolol Fumarate RS in
the Standard preparation, respectively. the Standard solution (mg/mL)
Cu = nominal concentration of bisoprolol fumarate in
the Sample solution (mg/mL)

Acceptance criteria: 90.00/0-105.0%

Bisoprolol Fumarate Tablets
PERFORMANCE TESTS
DEFINITION • DISSOLUTION (711)
Bisoprolol Fumarate Tablets contain NLT 90.0% and NMT Test 1
105.0% of the labeled amount of bisoprolol fumarate Medium: Water; 900 mL
[(C18H31N04)Z' C4H404]· Apparatus 2: 75 rpm
Time: 20 min
IDENTIFICATION Determine the amount of (C 18H31N04)z . C4H404dissolved
• THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST (201) by using the following method.
Sample solution: Equivalent to 40 mg of bisoprolol Diluent: Methanol, triethylamine, phosphoric acid, and
fumarate, from powdered Tablets (NLT 5), in a 50-mL flask. water (160: 5: 2.5: 35)
Add about 20 mL of a mixture of dichloromethane and Mobile phase: Methanol, triethylamine, and water
methanol (7:3), shake for 30 min, centrifuge, and use the (34:1 :50). Adjust with phosphoric acid to a pH of 4.0
clear solution. ± 0.1.
Application volume: 20 ~L Standard stock solution: USP Bisoprolol Fumarate RS in
Developing solvent system: Dichloromethane, methanol, water to obtain a solution having a known concentration
and ammonia TS, stronger (70: 10: 0.8) of about twice the concentration of bisoprolol fumarate
Analysis in the Sample solution
Sample: Sample solution Standard solution: Standard stock solution and Diluent
Proceed as directed in the chapter, except to develop the (1:1)
chromatogram until the solvent front has moved about Sample solution: Sample per Dissolution (711). Withdraw
two-thirds of the length of the plate and to dry the plate a portion of the solution under test, filter, and dilute with
in a current of cold air. an equal volume of Diluent:
ASSAY Chromatographic system
(See Chromatography (621), System Suitability.)
• PROCEDURE
Diluent: Acetonitrile and water (7:13) Mode: LC
Mobile phase: A 1-L portion of Diluent. Add 5 mL of Detector: UV 227 nm
heptafluorobutyric acid,S mL of diethylamine, and 2.5 mL Column: 4.6-mm x 33-mm; packing L7
of formic acid. Flow rate: 1 mL/min
System suitability solution: 0.5 mg/ml of propranolol Injection size: 50 ~L
hydrochloride and 1 mg/mL of bisoprolol fumarate in System suitability
Diluent Sample: Standard solution
Standard solution: 1 mg/mL of USP Bisoprolol Fumarate RS Suitability requirements
in Diluent Relative standard deviation: NMT 2.0%
Sample solution: Transfer an equivalent of 25 mg of Analysis
bisoprolol fumarate, from powdered Tablets (NLT 20), to a Samples: Standard solution and Sample solution
25-mL volumetric flask. Add 10 mL of Diluent, and sonicate Tolerances: NlT 80% (Q) of the labeled amount of
for 10 min. Cool, dilute with Diluent to volume,and mix. (C18H31N04)Z' C4H404 is dissolved.
Centrifuge for 20 min, and use the clear supernatant. Test 2: If the product complies with this test, the labeling
Chromatographic system indicates that it meets USP Dissolution Test 2.
(See Chromatography (621), System Suitability.) Medium: 0.5 M sodium chloride; 900 mL
Mode: LC Apparatus 2: 75 rpm
Detector: UV 273 nm Time: 20 min
Column: 4.6-mm x 12.5-cm; packing L7 Analysis: Proceed as directed for Test 1 with the following
Flow rate: 1 mL/min modifications.
Injection size: 10 ~L Diluent: Prepare a mixture of methanol, 0.1 N
System suitability hydrochloric acid, triethylamine, and phosphoric acid
Samples: System suitability solution and Standard solution (160: 35: 5: 2.5). The dimensions of the column are 4.6
Suitability requirements mm x 25 em.
Resolution: NLT 7.0 between bisoprolol and Tolerances: NLT 80% (Q) of the labeled amount of
propranolol, System suitability solution (C18H31N04)Z' C4H40 4 is dissolved.
Tailing factor: NMT 2.0, Standard solution • UNIFORMITY OF DOSAGE UNITS (905): Meet the,
Relative standard deviation: NMT 2.0%, Standard requirements
solution ADDITIONAL REQUIREMENTS ., ,,', . '
Analysis • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Samples: Standard solution and Sample solution containers, and store at controlled room temperature.

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 USP43 OfficialMonographs / Bisoprolol 587

• LABELING: When more than one Dissolution test is given, the Standard stock solution l-Quantitatively dissolve an
labeling states the Dissolution test used only if Test 1 is not accurately weighed quantity of USP Bisoprolol Fumarate RS in
used. Medium to obtain a solution having a known concentration of
• USP REFERENCE STANDARDS (11) about 0.5 mg per mL.
USP Bisoprolol Fumarate RS Standard stock solution 2-Transfer about 30 mg of USP
2-Propanol, 1-[4-[[2-(1-methylethoxy)ethoxy]methyl] Hydrochlorothiazide RS, accurately weighed, to a 50-mL
phenoxy]-3-[(1-methylethyl)amino]-, (±)-, (E)- volumetric flask, dissolve in 5 mL of methanol, dilute with
2-butenedioate (2:1) (salt). Medium to volume, and mix.
(ClsH31N04)Z' C4H404 766.96 Standard solution-Dilute accurately measured volumes of
Standard stock solution 1 and Standard stock solution 2 with
Medium to obtain a solution having known concentrations of
bisoprolol fumarate and hydrochlorothiazide corresponding
to those of the solution under test.
Bisoprolol Fumarate and Chromatographic system (see Chromatography (621»)-The
liquid chromatograph is equipped with a UV detector capable
Hydrochlorothiazide Tablets of measuring peak responses at 227 nm and 272 nm,
simultaneously, and a 3.9-mm x 15-cm column that contains
» Bisoprolol Fumarate and Hydrochlorothiazide packing L11. The flow rate is about 1.5 mL per minute.
Chromatograph the Standard solution, and record the peak
Tablets contain not less than 90.0 percent and not areas as directed for Procedure: the relative standard deviation
more than 110.0 percent of the labeled amounts for replicate injections is not more than 2.0%.
of bisoprolol fumarate (C1sH31N04)z . C4H404 and Procedure-Separately inject equal volumes (about 20 ~L)
hydrochlorothiazide (C7HsCIN304Sz). of the Standard solution and the filtered portions of the
solution under test into the chromatograph, record the
Packaging and storage-Preserve in tight, light-resistant chromatograms, and measure the peak areasfor bisoprolol at
containers. Store at controlled room temperature. 227 nm and for hydrochlorothiazide at 272 nm. Calculate the
USP Reference standards (11)- quantities, in mg, of bisoprolol fumarate (C18H31N04)Z'
USP Bisoprolol Fumarate RS C4H404 and hydrochlorothiazide (C7HsClN304Sz) dissolved.
USP Chlorothiazide RS Tolerances-Not less than 80% (Q) of the labeled amount
USP Hydrochlorothiazide RS of (ClsH31N04)Z'C4H404 is dissolved in 20 minutes and not
less than 80% (Q) of the labeled amount of C7HsCIN304Sz is
Identification- dissolved in 30 minutes.
A: Thin-Layer Chr;omatographic Identification Test (201)- Uniformity of dosage units (905): Meet the requirements
Test solution-Finely powder 1 Tablet, and transfer the powder with respect to bisoprolol fumarate and to
to a 5-mL volumetric flask. Dilute with methanol to volume, hyd roch lorothiazide.
sonicate for 5 minutes, centrifuge, and use the supernatant.
Standard solution l-Dissolve a suitable quantity .of USP Chromatographic purity-
Bisoprolol Fumarate RS in methanol to obtain a solution Diluent, Solution A, Solution B, Mobile phase, and System
containing 1 mg per mL. suitability solution-Proceed as directed in the Assay.
Standard solution 2-Dissolve a suitable quantity of USP Standard solution-Dissolve an accurately weighed quantity
Hydrochlorothiazide RS in methanol to obtain a solution of USP Hydrochlorothiazide RS in Diluent, and quantitatively
containing 1 mg per mL. dilute with Diluent, if necessary, to obtain a solution having a
Application volume: 25 ~L. known concentration of about 2 ~g per mL.
Developing solvent system: a mixture of methylene chloride, Test stock solution-Proceed as directed for Assay stock
methanol, and 14.5 M ammonium hydroxide solution preparation in the Assay.
(43:20:8). Test solution-Quantitatively dilute an accurately measured
Procedure-Locate the spots on the plate under volume of the Test stock solution with Diluent to obtain a
short-wavelength UV light and by exposure to iodine vapors: solution having a concentration of about 100 ~g of bisoprolol
the RF values of the principal spots in the chromatogram fumarate per mL.
obtained from the Test solution correspond to those of the Chromatographic system (see Chromatography (621»)-
principal spots in the chromatograms obtained from Standard Prepare as directed in the Assay, but use a 260-nm detector.
solution 1 and Standard solution 2. Chromatograph the System suitability solution, and record the
B: The retention times of the major peaks in the peak responses as directed for Procedure: the resolution, R,
chromatograms of the Bisoprolol fumarate assay preparation between chlorothiazide and hydrochlorothiazide is not less
and the Hydrochlorothiazide assay preparation correspond to than 1.5. Chromatograph the Standard solution and record
those in the chromatogram of the Standard preparation, as the peak responses as directed for Procedure: the tailing factor
obtained in the Assay. is not more than 1.3; and the relative standard deviation for
replicate injections is not more than 2.0%.
Dissolution (711)- Procedure-Separately inject equal volumes (about 10 ~L)
Medium: 0.1 N hydrochloric acid; 900 mL. of the Standard solution and the Test solution into the
Apparatus 2: 75 rpm. chromatograph, record the chromatograms, and measure the
Times: 20 minutes for bisoprolol fumarate; 30 minutes for responses for all the peaks. Calculate the percentage of each
hydrochlorothiazide. impurity in the portion of Tablets taken by the formula:
Triethylamine solution-Mix 2 mL of triethylamine with
1000 mL of water, and adjust with phosphoric acid to a pH of (1001F)(W81 WH ) ( CsiC8)(r;/ rs)
3.0.
Mobile phase-Prepare a filtered and degassed mixture of in which Fis the responsefactor, equal to 1.2 for the peak with
acetonitrile and Triethylamine solution (1 :4). Make adjustments a relative retention time of 0.69 and 1.4 for the peak with a
if necessary (see System Suitability under Chromatography relative retention time of 1.2, both retention times relative to
(621»

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588 Bisoprolol / OfficialMonographs USP 43

that of the hydrochlorothiazide peak; WB and WH are the relative standard deviation for replicate injections is not more
labeled quantities, in mg, of bisoprolol fumarate and than 2.0%.
hydrochlorothiazide, respectively, in each Tablet; Cs is the Procedure-Separately inject equal volumes (about 10 IJL)
concentration, in mg per mL, of USP Hydrochlorothiazide RS of the Standard preparation, Bisoprolol fumarate assay
in the Standard solution; CB is the concentration, in mg per mL, preparation, and Hydrochlorothiazide assay preparationinto the
of bisoprolol fumarate in the Test solution; r,is the peak chromatograph, record the chromatograms, and measure the
response of each of the two impurities obtained from the Test areas for the major peaks. Calculate the quantities, in mg, of
solution; and rs is the response for the hydrochlorothiazide bisoprolol fumarate (C18H31N04)Z' C4H404 and
peak obtained from the Standard solution: not more than 1.0% hydrochlorothiazide (C7H8C1N304Sz) in the portion of Tablets
for the impurity with a relative retention time of 0.69 isfound; taken by the formula:
and not more than 2.0% for the impurity with a relative
retention time of 1.2 is found. 5000( C/I/)(rvirs)

Assay- in which C is the concentration, in mg per mL, of USP

Diluent-Mix 10 mL of 1 M dibutylammonium phosphate Bisoprolol Fumarate RS or USP Hydrochlorothiazide RS in the
with 1000 mL of a mixture of water and acetronitrile (1:1). Standard preparation, as appropriate; V is the volume of the
Solution A-Mix 10 mL of 1 M dibutylammonium Assay stock preparation used to prepare the Bisoprolol fumarate
phosphate with 1000 mL of water. assay preparation or the Hydrochlorothiazide assay
Solution B-Prepare a mixture of acetonitrile and water preparation; rv is the peak area obtained from the Bisoprolol
(3:2). Add 10 mL of 1 M dibutylammonium phosphate per fumarateassay preparation or the Hydrochlorothiazide assay
liter, stir vigorously for 2 minutes, filter, and degas. preparation, as appropriate; and ts is the corresponding peak
Mobilephase-Use variable mixtures of Solution A and area obtained from the Standard preparation.
Solution B as directed for Chromatographic system. Make
adjustments if necessary (see System Suitability under
Chromatography (621 »,
System suitability solution-Prepare a solution of USP
Chlorothiazide RS and USP Hydrochlorothiazide RS in Diluent . Bleomycin for Injection
containing 40 IJg of each per mL.
Standard preparation-Dissolve suitable quantities of USP
Bisoprolol Fumarate RS and USP Hydrochlorothiazide RS in » Bleomycin for Injection contains an amount of
Diluent to obtain a solution having known concentrations of Bleomycin Sulfate equivalent to not less than 90.0
about 100 IJg of each per mL. Stir by mechanical means for 1 percent and not more than 120.0 percent of the
hour. •
Assay stock preparation-Weigh 10 Tablets, and transfer to
labeled amount of bleomycin.
a 1OO-mL volumetric flask. Add about 50 mL of Diluent,
Packaging and storage-Preserve as described in
sonicate for 10 minutes, and cool. Dilute with Diluent to
Packaging and Storage Requirements (659), Injection Packaging,
volume, stir by mechanical means for 1 hour.and centrifuge.
Packaging for constitution.
Bisoprolol fumarate assay preparation-Quantitatively USP R.eference standards (11)-
transfer a portion of the Assay stock preparation to a 50"rnL
USP Bleomycin Sulfate RS
volumetric flask, and dilute with Diluent to volume to obtain a
Constituted solution-At the time of use, it meets the
solution having a concentration of about 100 IJg of bisoprolol
requirements for Injections and Implanted Drug Products (1),
fumarate per mL. .
Specific Tests, Completeness and clarity of solutions.
Hydrochlorothiazide assay preparation-Quantitatively
transfer a portion of the Assay stock preparation toa 50-mL Identification-
volumetric flask, and dilute with Diluent to volume to obtain a
solution having a concentration of about 62.5 IJg of
hydrochlorothiazide per mL.
Chromatographic system (see Chromatography (621 »-The )\:.. . ~~p~£tt9Af9PJSlg~g~jti~~ti~nTest$(lQ7);;If1fraied
liquid chromatograph is equipped with 225-nm detector and Spgc:tr()§C:(JpY:ilQ'4t<.~·(C:N.l~MclY-2040).
an 8-mm x 1O-cm column that contains packing L11. The flow B: It responds to the tests for Sulfate (191 ).
rate is about 3 mL per minute. The chromatograph is Bacterial Endotoxins Test (85) -It contains not more than
programmed as follows. 10.0 USP Endotoxin Units per Bleomycin Unit.
Sterility Tests (71) -It meets the requirements when tested
Time Solution A Solution B as directed for Membrane Filtration under Test for Sterility of the
(minutes) (%) (%) Elution Product to be Examined, the entire contents of each container
being used.
0 100 0 equilibration
Water Determination, Method Ie(921) : not more than
0-9.0 100--+40 0--+60 linear gradient 6.0%. Prepare the specimen for test as follows. Use a dry
9.0-9.1 40--+100 60-0 linear gradient
syringe to inject 4 mL of anhydrous methanol through the
stoppers of two tared containers, respectively, and shake to
9.1-12.0 100 0 re-equilibration dissolve. Using the same syringe, aspirate the contents of the
two containers, transfer to the titration vessel, and titrate.
Chromatograph the System suitability solution, and record the Perform a blank determination on 8 mL of the anhydrous
peak areasasdirected for Procedure: the resolution, R, between methanol. Determine the weights of the empty containers,
chlorothiazide and hydrochlorothiazide is not less than 1.5. and calculate the percentage of water.
Chromatograph the Standard preparation, and record the peak Other requirements-It meets the requirements for pH,
areas as directed for Procedure: the tailing factor for the Copper, and Content of bleomycins under Bleomycin Sulfate. It
hydrochlorothiazide peak is not more than 1.3; and the meets also the requirements for Uniformity of Dosage Units

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 USP 43 Official Monographs / Bleomycin 589

(905) and for Labeling (7), Labels and Labeling for Injectable Procedure-Concomitantly determine the absorbances of
Products. the Standardpreparations and the Test preparation at the
copper emission line at 324.8 nm, with a suitable atomic
Assay- absorption spectrophotometer (see AtomicAbsorption
Assay preparation-Constitute Bleomycin for Injection as Spectroscopy (852») equipped with a copper hollow-cathode
directed in the labeling. Withdraw all of the withdrawable lamp .and an air-acetylene flame, using Dilutenitric acid asthe
contents, using a suitable hypodermic needle and syringe, and blank. Plot the absorbancesof the Standardpreparations versus
quantitatively dilute with Buffer B. 16 to obtain a solution concentration, in IJg per mL, of copper, and draw the straight
having a convenient concentration. line best fitting the three plotted points. From the graph so
Procedure-Proceed as directed under Antibiotics- obtained, determine the concentration, C, in IJg per mL, of
MicrobialAssays (81), using an accurately measured volume of copper in the Test preparation. Calculate the percentage of
Assay preparation diluted quantitatively and stepwise with copper in the portion of Bleomycin Sulfate taken by the
Buffer B.16 to yield a Test Dilution having a concentration formula:
assumed to be equal to the median dose level of the Standard.
C/W
in which W is the weight, in mg, of Bleomycin Sulfate taken
to prepare the Test preparation: not more than 0.1% is found.
Bleomycin Sulfate
Content of bleomycins-
Mobile phase-Dissolve 960 mg of sodium 1-pentane
Bleomycin sulfate (salt). sulfonate in 1000 mL of deaerated 0.08 N acetic acid, adjust
Bleomycin sulfate (salt) [9041-93-4]. with ammonium hydroxide to a pH of 4.3, filter,and degas.
[NOTE-l .86 g of edetate disodium may be included if needed
» Bleomycin Sulfate is the sulfate salt of bleomycin, to obtain satisfactory chromatography.] Usea linear gradient
a mixture of basic cytotoxic glycopeptides of 10% to 40% methanol mixed with this solution, with a
produced by the growth of Streptomyces verticil/us, gradient mixing time of 60 minutes, and allow
chromatography to proceed with the final gradient mixture
or produced by other means. It has a potency of for a further 20 minutes or until demethylbleomycin A2 has
not less than 1.5 Bleomycin Units and not more been eluted.
than 2.0 Bleomycin Units per mg. Test preparation-Dissolve Bleomycin Sulfate in deaerated
water to obtain a solution having a concentration of about 2.5
Packaging and storage-Preserve in tight containers. Bleomycin Units per mL. Store this solution in a refrigerator
Labeling-Where it is intended for usein preparing injectable until just prior to use.
dosage forms, the label states that it is sterile or must be Chromatographic system (see Chromatography (621»)-
subjected to further processing during the preparation of The liquid chromatograph is equipped with a 254-nm
injectable dosage forms. detector and a 4.6-mm x 250-mm stainless steel column
USPReference standards (11) - containing packing L1. The flow rate is about 1.2 mL per
USP Bleomycin Sulfate RS minute.
Identification- Procedure-Inject about 10 IJLof the Test preparation into
the chromatograph by means of a suitable microsyringe or
sampling valve, record the chromatogram, and measure the
peak responses for all peaks. The elution order is bleomycinic
acid, bleomycin A2 (major peak), bleomycin As, bleomycin B2
~:~t~~ (major peak), bleomycin B4, and demethylbleomycin A2 •
'Spggt[g~~Q .~l§l.
B: It responds to the tests for (191). Calculate the percentage contents of bleomycin A2,
pH (791): between 4.5 and 6.0, in a solution containing 10 bleomycin B2 , and bleomycin B4 taken by the formula:
Bleomycin Units per mL.
Loss on drying (731)-Dry it in vacuum at a pressure not 1OOr,lr t
exceeding 5 mm of mercury at 60° for 3 hours: it loses not
more than 6.0% of its weight. in which r f is the peak response corresponding tothe
particular bleomycin and r t is the total of the responses of all
Copper- peaks:the content of bleomycin A2 is between 55% and 70%;
Dilute nitric acid-Dilute 20 mL of nitric acid to 2000 mL
with water. the content of bleomycin B2 is between 25% and 32%; the
Copper stock solution-Transfer 1.000 g of copper to a content of bleomycin B4 is not more than 1%; and the
1OOO-mL volumetric flask, dissolve in 20 mL of nitric acid, combined percentage of bleomycin A2 and bleomycin B2 is not
dilute with Dilute nitric acid to volume, and mix. Store in a less than 90%.
polyethylene bottle. This solution contains 1000 IJg of copper Other requirements-Where the label states that
per mL. Bleomycin Sulfate is sterile, it meets the requirements for
Standardpreparations-Transfer 5.0 mL of Copper stock Sterility and Bacterial endotoxins under Bleomycin for Injection.
solution to a 1OO-mL volumetric flask, dilute with Dilute nitric Where the label states that Bleomycin Sulfate must be
acid to volume, and mix. Transfer 3.0, 9.0, and 15.0 mL, subjected to further processing during the preparation of
respectively, of this solution to separate 1OO-mL volumetric injectable dosage forms, it meets the requirements for Bacterial
flasks, dilute the contents of each flask with Dilutenitric acid to endotoxins under Bleomycin for Injection.
volume, and mix. These Standardpreparations contain,
Assay-
respectively, 1.5,4.5, and 7.5 IJg of copper per mL.
Assay preparation-Dissolve a suitable quantity of
Test preparation-Dissolve about 75 mg of Bleomycin Bleomycin Sulfate, accurately weighed, in Buffer B. 16, and
Sulfate, accurately weighed, in 10.0 mL of Dilute nitric acid.

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 590 Bleomycin / Official Monographs USP43

quantitatively dilute with Buffer B.16 to obtain a solution chromatograph, record the chromatograms, and measurethe
having a convenient concentration. peak responses. The relative retention times are about 0.25,
Procedure-Proceed as directed under Antibiotics- 0.74, 1.0, 1.27, 1.40 for tosylate ion,
Microbial Assays(81), using an accurately measured volume of o-bromobenzyldimethylamine, bretylium,
Assaypreparation diluted quantitatively and stepwise with m-bromobenzyldimethylamine, and
Buffer B.16 to yield a Test Dilution having a concentration p-bromobenzyldimethylamine, respectively. The sum of the
assumedto be equal to the median dose level of the Standard. responses for all the peaks, excluding those of the bretylium
and tosylate peaks,from the Testsolution is not more than two
times the bretylium response from the Standard solution (2%);
and no individual peak response is greater than that of the
bretylium peak from the Standard solution (1 %).
Bretylium Tosylate Assay-Dissolve about 300 mg of Bretylium Tosylate,
accurately weighed, in 50 mL of dioxane in a conical flask. Add
2 drops of crystal violet TS, and titrate with 0.025 N perchloric
acid in dioxane to a blue-green endpoint. Perform a blank
determination (see Titrimetry (541», and make any necessary
correction. Each mL of 0.025 N perchloric acid is equivalent
to 10.36 mg of C18H24BrN03S,
C18H24BrN03S 414.36
Benzenemethanaminium, 2-bromo-N-ethyl-N N-dimethyl-
salt with 4-methylbenzenesulfonic acid (1:1)~ ,
(o-Bromobenzyl)ethyldimethylammonium p-
toluenesulfonate [61-75-6]. Bretylium Tosylate Injection
» Bretylium Tosylate contains not less than 98.0
» Bretylium Tosylate Injection is a sterilesolution of
percent and not more than 101.0 percent of
C,8H24BrN03S, calculated on the dried basis. .Bretylium Tosylate in Water for Injection. It
contains not less than 90.0 percent and not more
Packaging and storage-Preserve in tight containers. Store than 110.0 percent of the labeled amount of
at 25°, excursions permitted between 15° and 30°. C'8H24BrN03S,
USP Reference standards (11)-
USP Bretylium T~sylate RS Packaging and storage-Preserve in single-dose
Identification- containers, preferably of Type I glass.
USP Reference standards (11)-
USP Bretylium Tosylate RS
Identification-The retention time of the major peak in the
chromatogram of the Assaypreparation corresponds to that of
the Standard preparation, both relative to the internal
o standard, as obtained in the Assay.
chromatogram of Testsolution rnr'rocnnr,\r1C' Bacterial Endotoxins Test (85) -It contains not more than
chromatogram of the Standard solution, as nh11-",ir'orl 0.20 USP Endotoxin Unit per mg of bretylium tosylate.
for Related compounds. pH (791): between 3.5 and 7.0.
Loss on drying (731)-Dry it in vacuum at 75° for 2 hours: it Particulate Matter in Injections (788): meets the
loses not more than 3.0% of its weight. requirements for small-volume injections.
Residue on ignition (281): not more than 0.1 %. Other requirements-It meets the requirements under
Injections and Implanted Drug Products (1).
Related compounds-
0.01 M Sodium 7-octanesulfonate solution-Dissolve 1.0814 Assay-
g of l-sodium octanesulfonate in 500 mL of water. pH 3.1 Tetramethylammonium phosphate buffer-Dissolve
Mobile phase-Prepare a mixture of 0.01 M Sodium 1.38 g of monobasic sodium phosphate and 2.0 mL of 25%
7:octanes~/fonate solution, acetonitrile, glacial acetic acid, and tetra-methylammonium hydroxide solution in methanol in
triethylamine (81: 19: 2: 0.5). Make adjustments if necessary 800 mL of water, adjust with phosphoric acid to a pH of 3.1
(see System Suitability under Chromatography (621». ± 0.1, dilute with water to 1000 mL, and mix.
Standard solution-Dissolve an accurately weighed quantity Mobile phase-Transfer 15 mL of tetrahydrofuran and 75
of USP Bretylium Tosylate RS in Mobile phase, and dilute mL of acetonitrile to a 1OOO-mL volumetric flask, and dilute
quantitatively, and stepwise if necessary, to obtain a solution with pH 3.7 Tetramethylammonium phosphate buffer to
having a known concentration of about 20 ~g per mL. volume.
Testsolutio,!-Transfer about 200 mg of ~retylium Tosylate, Standard preparation--Dissolve an accurately weighed
accurately weighed, to a 1OO-mL volumetric flask, dissolve in quantity of USP Bretylium Tosylate RS in water, and dilute
and dilute with Mobile phase to volume, and mix. quantitatively, and stepwise if necessary, with water to obtain
. ~hromatographic system (see Chromatography(621»-The a solution having a known concentration of about 0.2 mg per
liquid chromatograph is equipped with a 265-nm detector mL.
and a4.6-mm x 25-cm column that contains packing L11. The Assay preparation-Transfer an accurately measured
flow rate is about 1.9 mL per minute. Chromatograph the volume of Injection, equivalent to about 10 mg of bretylium
Standard solution, record the chromatograms, and record the tosylate, to a 50-mL volumetric flask, dilute with water to
volume, and mix.
peak responses as directed for Procedure: the relative standard
deviation for replicate injections is not more than 3.0%. Chromatographic system (see Chromatography (621 »-
Procedure-Separately inject equal volumes (about 30 ~L) The liquid chromatograph is equipped with a 220-nm
of the Testsolution and the Standard solution into the detector and a 3.9-mm x 30-cm column that contains packing

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 USP 43 Official Monographs / Brimonidine 591

L1. The flow rate is about 2 mL per minute. Chromatograph of bretylium tosylate (C'SH24BrN0 3S) in each mL of the
the Standardpreparation, and record the peak responses as Injection taken by the formula:
directed for Procedure: the relative retention times are about
0.7 for tosylate and 1.0 for bretylium; the resolution, R, 50(C/V)(r vir s)
between the bretylium and tosylate peaks is not less than 3.0;
and the relative standard deviation for replicate injections is in which C is the concentration, in mg per mL, of USP
not more than 1.4%. Bretylium Tosylate RS in the Standardpreparation; Vis the
Procedure-Separately inject equal volumes (about 20 IJL) volume, in mL, of Injection taken; and r u and r sare the
of the Standardpreparation and the Assay preparation into the bretylium peak responses from the Assay preparation and the
chromatograph, record the chromatograms, and measure the Standardpreparation, respectively.
responses for the major peaks. Calculate the quantity, in mg, Assay for dextrose-Transfer an accurately measured
of C'SH 24BrN0 3S in each mL of the Injection taken by the volume of Injection, containing 2 to 5 g of dextrose, to a
formula: 1OO-mL volumetric flask. Add 0.2 mL of 6 N ammonium
hydroxide, dilute with water to volume, and mix. Determine
50(C/V)(r vir s) the angular rotation in a suitable polarimeter tube (see Optical
Rotation (781). Calculate the percentage (g per 100 mL) of
in which C is the concentration, in mg per mL, of USP dextrose (C6H,206 . H20) in the portion of Injection taken by
Bretylium Tosylate RS in the Standardpreparation; Vis the the formula:
volume, in mL, of Injection taken; and r v and r sare the
bretylium peak responses obtained from the Assay preparation (100/52.9)(198.17/180.16)AR
and the Standardpreparation, respectively.
in which 100 is the percentage; 52.9 is the midpoint of the
specific rotation range for anhydrous dextrose, in degrees;
198.17 and 180.16 are the molecular weights for dextrose
monohydrate and anhydrous dextrose, respectively; A is 100
Bretylium Tosylate in Dextrose Injection mm divided by the length of the polarimeter tube, in mm; and
R is the observed rotation, in degrees.
» Bretylium Tosylate in Dextrose Injection is a sterile
solution of Bretylium Tosylate and Dextrose in
Water for Injection. It contains not less than 95.0
percent and not more than 105.0 percent of the
Brimonidine Tartrate
labeled amounts of bretylium tosylate
(C'SH24BrN0 3 S) and dextrose (C6H,206 . H20). It
contains no antimicrobial agents.
(NO: ~y\ . Jlrr
~;,;
N
h HN-.I
HO
OH
H

0
OH

Packaging and storage-Preserve in single-dose glass or C"H,oBrN s ' C4H606 442.22

plastic containers. Glass containers are preferably of Type I or 6-Quinoxalinamine, 5-bromo-N-(4,5-dihydro-1 H-imidazol-2-
Type II glass. yl)-, [R-(R*,R*)]-2,3-dihydroxybutanedioate (1:1);
VSP Reference standards (11)- 5-Bromo-6-(2-imidazolin-2-ylam ino)quinoxaline L-tartrate
USP Bretylium Tosylate RS (1:1). [70359-46-5].
Identification- . DEFINITION
A: The retention time of the major peak in the Brimonidine Tartrate contains NLT 98.0% and NMT 102.0%
chromatogram of the Assay preparation corresponds to that in of brimonidine tartrate (C'l HlOBrN s . C4H606), calculated on
the chromatogram of the Standardpreparation, as obtained in the dried basis.
the Assay for bretylium tosylate.
IDENTIFICATION
B: Add a few drops of a solution (1 in 20) to 5 mL of hot
alkaline cupric tartrate TS.A copious red precipitate of cuprous
oxide is formed.
Bacterial Endotoxins Test (85) -It contains not more than
0.20 USP Endotoxin Unit per mg of bretylium tosylate. ~~~~i~\I~~~1"IFlc~~i~~(!~~~~l(~;0~5;l/~f[g[ecl
pH (791): between 3.0 and 6.5.
Sp~Cl(gy:\·1·Q.7.1ftjl.:97Kj.·Qrl·Qz.tYh",.(cN1,MaY·2Q2(l)
• B. The retention time of the major peak of the Sample
Other requirements-It meets the requirements under
solution corresponds to that of the Standardsolution, as
Injections and Implanted Drug Products (1). obtained in the Assay.
Assay for bretylium tosylate- ASSAY
pH 3.1 Tetramethylammonium phosphate buffer, Mobile
• PROCEDURE
phase, Standardpreparation, and Chromatographic system- Protect the solutions containing brimonidine tartrate from
Proceed as directed in the Assay under Bretylium Tosylate
light.
Injection. Mobile phase: In a 1-L volumetric flask, dissolve 2.6 g of
Assay preparation-Transfer an accurately measured sodium 1-heptanesulfonate in 310 mL of methanol, add 2.5
volume of Injection, equivalent to about 10 mg of bretylium mL of triethylamine and 7.5 mL of glacial acetic acid, and
tosylate, to a 50-mL volumetric flask, dilute with water to dilute with water to volume.
volume, and mix. Standard solution: 1.3 mg/mL of USP Brimonidine Tartrate
Procedure-Separately inject equal volumes (about 20 IJL) RS in water
of the Standardpreparation and the Assay preparation into the
Sample solution: 1.3 mg/mL of Brimonidine Tartrate in
chromatograph, record the chromatograms, and measure the
water
responses for the major peaks. Calculate the quantity, in mg,

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592 Brimonidine / Official Monographs USP 43

Chromatographic system Cu = concentration of Brimonidine Tartrate in the

(See Chromatography (621), System Suitability.) Sample solution (mg/mL)
Mode: LC
Detector: UV 264 nm Acceptance criteria: See Table 1. Disregard the tartrate
Column: 4.6-mm x 25-cm; 5-J.Jm packing L1 peak at a relative retention time of 0.09. The reporting
Column temperature: 30° threshold is 0.05%.
Flow rate: 1 mL/min
Injection volume: 20 J.JL Table 1
System suitability Relative Acceptance
Sample: Standard solution Retention Criteria,
Suitability requirements Name Time NMT(%)
Tailing factor: NMT 1.5 Desbrornobrlmonldine- 0.86 0.1
Relative standard deviation: NMT 0.73%
Analysis Brimonidine 1.00 -
Samples: Standard solution and Sample solution Any individual
Calculate the percentage of brimonidine tartrate unspecified -
(Cll H10BrN s . C4H 606) in the portion of Brimonidine impurity 0.1
Tartrate taken: Total impurities - 0.3

Result = (rulrs) x (CsICu) x 100 a N-(Quinoxafin-6-yl)imidazofidin-2-imine.

tu = peak response from the Sample solution SPECIFIC TESTS

ts = peak response from the Standard solution • OPTICAL ROTATION (781 S), Procedures, Specific Rotation
Cs =concentration of USP Brimonidine Tartrate RS in Sample solution: 10 mg/mL in water
the Standard solution (mg/mL) Acceptance criteria: +9.0° to +10.5°
Cu = concentration of Brimonidine Tartrate in the • Loss ON DRVING (731)
Sample solution (mg/mL) Analysis: Dry 1 9 at 105° for 3 h.
Acceptance criteria: NMT 0.5%
Acceptance criteria: 98.0%-102.0% on the dried basis
ADDITIONAL REQUIREMENTS
IMPURITIES • PACKAGING AND STORAGE: Preserve in well-closed,
• RESIDUE ON IGNITION (281): NMT 0.3% light-resistant containers, and store at room temperature.
• ORGANIC IMPURITIES • USP REFERENCE STANDARDS (11)
Protect the solutions containing brimonidine tartrate and USP Brimonidine Tartrate RS
related substances from light. USP Brimonidine Related Compound E RS
Mobile phase: Proceed as directed in the Assay. 2-(5-Bromoquinoxalin-6-yl)guanidine.
System suitability solution: 1.3 mg/mL of USP Brimonidine C9H sBrNs 266.10
Tartrate RS and 1.3 J.Jg/mL of USP Brimonidine Related
Compound E RS in water
Sensitivity solution: 0.65 J.Jg/mL of USP Brimonidine
Tartrate RS in water
Standard solution: 1.3 J.Jg/mL of USP Brimonidine Tartrate Brinzolamide
RS in water
Sample solution: 1.3 mg/mL of Brimonidine Tartrate in
water
Chromatographic system: Proceed as directed in the
Assay, except for the Run time.
Run time: NLT 3 times the retention time of brimonidine
System suitability
Samples: System suitability solution and Sensitivity solution
[NOTE-The relative retention times for brimonidine C12H21N30SS3 383.51
related compound Eand brimonidine are about 0.93 2H-Thieno[3,2-e]-1,2-thiazine-6-sulfonamide, 4-
and 1.00, respectively.] (ethylamino)-3,4-dihydro-2-(3-methoxypropyl)-, 1,1-
Suitability requirements dioxide, (R)-;
Resolution: NLT 1.5 between brimonidine related (R)-4-(Ethylamino)-3,4-dihydro-2-(3-methoxypropyl)-2H-
compound E and brimonidine, System suitability solution thieno[3,2-e]-1,2-thiazine-6-sulfonamide 1,1-dioxide
[138890-62-7].
Signal-to-noise ratio: NLT 10, Sensitivity solution
Analysis DEFINITION
Samples: Standard solution and Sample solution Brinzolamide contains NLT98.0% and NMT 102.0% of
Calculate the percentage of each impurity in the portion of brinzolamide (C12H21N30SS3) , calculated on the dried basis.
Brimonidine Tartrate taken:
IDENTIFICATION
Result = (rulrs) x (Cslt u) x 100

= peak response of each individual impurity from

the Sample solution
= peak response of brimonidine from the Standard
solution peak of the Sample
= concentration of USP Brimonidine Tartrate RS in System suitability
the Standard solution (mg/mL)

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USP43 OfficialMonographs / Brinzolamide 593

solution, as obtained in Limit of Brinzolamide Related Analysis

Compound A. Sample: Sample solution
Calculate the percentage of brinzolamide related
ASSAY compound A in the portion of Brinzolamide taken:
• PROCEDURE
Buffer: Add 4.0 mL of triethylamine to 1000 mL of water, Result = (r vir r) x 100
and adjust with phosphoric acid to a pH of 3.0.
Mobile phase: Acetonitrile and Buffer (25:75) = peak response for brinzolamide related
Standard solution: 0.1 mg/mL of USP Brinzolamide RS in compound A
Mobilephase = sum of the peak responses for brinzolamide and
Sample solution: 0.1 mg/mL of Brinzolamide in Mobile brinzolamide related compound A
phase
Chromatographic system Acceptance criteria: NMT 0.5%
(See Chromatography (621), System Suitability.) • ORGANIC IMPURITIES
Mode: LC Buffer: Prepare as directed in the Assay.
Detector: UV 254 nm Mobile phase A: Prepare asdirected for Mobilephase in the
Column: 4.6-mm x 25-cm; 5-~m packing L1 Assay.
Flow rate: 1.0 mL/min Mobile phase B: Acetonitrile and Buffer(35:65)
Injection volume: 20 ~L System suitability solution: 0.1 mg/mL each of USP
System suitability Brinzolamide RS and USP Brinzolamide Related Compound
Sample: Standardsolution B RS in Mobilephase A
Suitability requirements Sample solution: 1 mg/mL of Brinzolamide in Mobile
Column efficiency: NLT 1200 theoretical plates phaseA
Tailing factor: NMT 2;0 Chromatographic system
Relative standard deviation: NMT 2.0% (See Chromatography (621), System Suitability.)
Analysis Mode: LC
Samples: Standardsolution and Sample solution Detector: UV 230 nm
Calculate the percentage of brinzolamide (C12H21 N 30 SS 3) in . Column: 4.6-mm x 25-cm; 5-~m packing L1
the portion of Brinzolamide taken: Flow rate: 1.0 mL/min
Injection volume: 10 ~L
Result = (r vir s) x (C siC v) x 100 System suitability
Sample: System suitability solution
= peak responsefrom the Sample solution Use Mobilephase A.
=peak responsefrom the Standardsolution [NOTE-The relative retention times for brinzolamide
=concentration of USP Brinzolamide RS in the related compound Band brinzolamide are 0.8 and
Standardsolution (mg/mL) 1.0, respectively.]
Cu = concentration of Brinzolamide in the Sample Suitability requirements
solution (mg/mL) . Resolution: NLT 2.0 between the brinzolamide and
brinzolamide related compound B peaks
Acceptance criteria: 98.0%-102.0% on the dried basis Column efficiency: NLT 1200 theoretical plates for the
brinzolamide peak
IMPURITIES
Tailing factor: NMT 2.0 for the brinzolamide peak
• RESIDUE ON IGNITION (281): NMT 0.1%
Analysis 1
• LIMIT OF BRINZOLAMIDE RELATED COMPOUND A
Use Mobile phaseA.
Mobile phase: Dehydrated alcohol, chromatographic
Sample: Sample solution
hexane, methanol, and diethylamine (55: 40: 5: 0.2)
Allow the elution to continue for 20 min, and measurethe
System suitability solution: 0.4 mg/mL of ~SP . areasfor all the peaks, excluding the peaks of Mobilephase
Brinzolamide RS and 0.02 mg/mL of USP Brinzolarnlde
Related Compound A RS in dehydrated alcohol
A. .
Calculate the percentage of each impurity in the portion of
Sample solution: 0.5 mg/mL of Brinzolamide in dehydrated
Brinzolamide taken:
alcohol
Chromatographic system Result =(r vir r) x 100
(See Chromatography (621), System Suitability.)
Mode: LC ru = peak response for each impurity
Detector: UV 254 nm rT = sum of all the peak responses
Column: 4.6-mm x 25-cm; packing L51
Flow rate: 0.75 mL/min Acceptance criteria 1: NMT 0.3% for any individual
Injection volume: 5 ~L impurity
System suitability Analysis 2
Sample: System suitability solution Use Mobile phase B.
[NOTE-The relative retention times for brinzolamide Sample: Sample solution
and brinzolamide related compound A are 1.0 and Allow the elution to continue for 20 min, and measure the
1.2, respectively.] areas for brinzolamide and all the peaks having a relative
Suitability requirements retention greater than 6.
Resolution: NLT 1.8 between brinzolamide and Calculate the percentage of each impurity in the portion of
brinzolamide related compound A peaks Brinzolamide taken:
Column efficiency: NLT 2000 theoretical plates for the
brinzolamide peak Result= (r vir r) x 100
Tailing factor: NMT 1.8 for the brinzolamide peak
ru = peak responsefor each impurity

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594 Brinzolamide / OfficialMonographs USP 43

rr = sum of all the peak responses Suitability requirements

Resolution: NLT 4.5 between the brinzolamide and
Acceptance criteria 2: NMT0.3% for any individual brinzolamide related compound B peaks, System
impurity; NMT1.0% for total impuritiesfrom Analysis 7 and suitabilitysolution
Analysis 2 Tailing factor: NMT2.0, System SUitability solution
Relative standard deviation: NMT2.0%, Standard
SPECIFICTESTS . solution A
• Loss ON DRYING (731) Analysis
Analysis: Dry under vacuum at 100°-105° for 3 h. Samples: Standardsolution A and Sample solution
Acceptance criteria: NMT 0.5% Calculate the percentage of the labeled amount of
ADDITIONAL REQUIREMENTS brinzolamide(C12H21N30SS3) inthe portion of Ophthalmic
• PACKAGING AND STORAGE: Preserve in well-closed Suspension taken:
containers.
• USP REFERENCE STANDARDS (11) Result = (rulrs) x (CslCu) x 100
USP Brinzolamide RS
USP Brinzolamide Related Compound A RS to = peak response from the Sample solution
Brinzolamide (S)-isomer. ts = peak response from StandardsolutionA
C12H21N30SS3 383.52 Cs = concentration of USP Brinzolamide RS in Standard
USP Brinzolamide Related Compound B RS solutionA (mg/mL)
(R)-4-Amino-2-(3-methoxypropyl)-3,4-dihydro-2H- Cu = nominal concentration of brinzolamide in the
thieno[3,2-e][1,2]thiazine-6-sulfonamide 1,I-dioxide Sample solution (mg/mL)
oxalate.
ClOH17N30SS3 . C2H20 4 445.49 Acceptance criteria: 90.0%-110.0%
IMPURITIES
• LIMIT OF BRINZOLAMIDE RELATED COMPOUND A
Mobile phase: Dehydrated alcohol, chromatographic
Brinzolamide Ophthalmic Suspension hexane, methanol, and diethylamine (55: 40: 5: 0.2)
System suitability solution: 0.4 mg/mL of USP
DEFINITION Brinzolamide RS and 0.02 mg/mL of USP Brinzolamide
Brinzolamide Ophthalmic Suspension is a sterile, aqueous Related Compound A RS in dehydrated alcohol
suspension of Brinzolamide containing a suitable Sample solution: Transfer a volume of Ophthalmic
antimicrobial preservative. It contains NLT 90.0% and NMT Suspension, equivalent to 10 mg of brinzolamide, to a
110.0% of the labeled amount of brinzolamide 25-mL volumetric flask. Dilute'with alcohol to volume.
(C12H21N30SS 3). Chromatographic system
(See Chromatography (621), System SUitability.)
IDENTIFICATION Mode: LC
• A. The retention time of the major peak of the Sample Detector: UV 254 nm
solution corresponds to that of Standard solutionA, as Column: 4.6-mm x 25-cm; packing L51
obtained in the Assay. Flow rate: 0.75 mL/min
Injection volume: 5 ~L
ASSAY System suitability
• PROCEDURE Sample: System SUitability solution
Buffer: 11.75 giL of ammonium acetate in water. Adjust [NoTE-The relative retention times for brinzolamide
with acetic acid to a pH of 5.2. . and brinzolamide related compound A are 1.0 and
Mobile phase: Methanol and Buffer (35:65) 1.2, respectively.]
Standard solution A: 0.2 mg/mL of USP Brinzolamide RS in Suitability requirements
Mobile phase Resolution: NLT 1.8 between the brinzolamide and
System suitability solution: 0.06 mg/mL of USP brinzolamide related compound A peaks
Brinzolamide Related Compound B RS in Standardsolution Column efficiency: NLT 2000 theoretical plates for the
A , brinzolamide peak
Sample solution: Nominally 0.2 mg/mL of brinzolamide in Tailing factor: NMT 1.8 for the brinzolamide peak
Mobile phase prepared as follows. Transfera volume of Analysis
Ophthalmic Suspension, equivalent to 10 mg of Sample: Sample solution
brinzolamide, into a 50-mLvolumetric flask, and dilute with Calculate the percentage of brinzolamide related
Mobile phase to volume. compound A in the portion of Ophthalmic Suspension
Chromatographic system taken:
(See Chromatography (621), System SUitability.)
Mode: LC Result =(rulrr) x 100
Detector: UV 254 nm
Column: 4.6-mm x 15-cm; 5-~m packing L1 tu = peak response for brinzolamide related
Flow rate: 1.0 mL/min compound A
Injection volume: 20 ~L rr = sum of the peak responses for brinzolamide and
System suitability brinzolamide related compound A
Samples: StandardsolutionA and System suitabilitysolution
[NoTE-The relative retention times for brinzolamide Acceptance criteria: NMT 1.5%
related compound Bare between 0.48 and 0.61, • ORGANIC IMPURITIES
and the relative retention time for brinzolamide is Buffer, Mobile phase, Standard solution A, System
1.0.] suitability solution, Sample solution, Chromatographic

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 USP 43 OfficialMonographs / Bromocriptine 595

system, and System suitability: Proceed asdirected in the DEFINITION

Assay. Bromocriptine Mesylate contains NLT 98.0% and NMT
Standard solution B: 2.5 ~g/mL of USP Brinzolamide 102.0% ofC32H4oBrNsOs . CH4S03, calculated on the dried
Related Compound B RS in Mobilephase basis. '
Analysis
Samples: Sample solution and Standardsolution B .IDENTIFICATION
Calculate the percentage of each impurity in the portion of
Ophthalmic Suspension taken:

Result = (rufrs) x (Cs/Cu) x (MrdMr2) x 100

=peak response for each impurity from the

Sample solution
rs = peak response for brinzolamide related
compound B from Standard solution B
= concentration of USP Brinzolamide Related
Compound B RS in Standard solution B (mg/mL)
= nominal concentration of brinzolamide in the
Sample solution (mg/mL)
= molecular weight of des-ethyl brinzolamide, ASSAY
356.46
• PROCEDURE
= molecular weight of des-ethyl brinzolamide Sample solution: 600 mg of Bromocriptine Mesylate
oxalate, 445.49 Analysis: Dissolve with 80 mL of a mixture of acetic
anhydride and glacial acetic acid (7:1). Titrate with 0.1 N
Acceptance criteria
perchloric acid VS. Perform a blank determination, and
Any individual impurity: NMT 0.5% make any necessary correction (see Titrimetry(541». Each
Total impurities: NMT 2.0% . mL of 0.1 N perchloric acid is equivalent to 75.07 mg of
SPECIFICTESTS C32H40BrNsOs . CH4S03·
• STERILITY TESTS (71): It meets the requirements when Acceptance criteria: 98.0%-102.0% on the dried basis
tested as directed for Test for Sterility of the Product to Be
Examined, Membrane Filtration. IMPURITIES
INORGANIC IMPURITIES
• pH (791): 6.5-8.5
• Residue on Ignition (281): NMT 0.1%
ADDITIONAL REQUIREMENTS ORGANIC IMPURITIES
• PACKAGING AND STORAGE: Preserve in tight containers. • Procedure 1: Limit of Methanesulfonic Acid Content
Store at a temperature between 4° and 30°. Sample solution: 400 mg of Bromocriptine Mesylate
• USP REfERENCE STANDARDS (11) Analysis: Dissolve with 70 mL of methanol. Titrate under
USP Brinzolamide RS nitrogen with 0.1 N methanolic potassium hydroxide VS.
USP Brinzolamide Related Compound A RS Perform a blank determination, and make any necessary
Brinzolamide (S)-isomer. correction (see Titrimetry(541». Each mL of 0.1 N
C12H21N30SS3 383.52 methanolic potassium hydroxide is equivalent to 9.61 mg
USP Brinzolamide Related Compound B RS . of CH3S03H.
(R)-4-Am ino-2-(3-methoxypropyl)- 3,4-dihydro-2 H- Acceptance criteria: NLT 12.5% and NMT 13.4% of
thieno[3,2-e][1,2]thiazine-6-sulfonamide 1,1-dioxide CH3S03H on the dried basis
oxalate. • Procedure 2
Cl0H17N30SS3 . C2H204 445.49 Solution A: 0.1 N citric acid solution. Adjust with
hydrochloric acid to a pH of 2.0.
Diluent: Methanol and Solution A (1:1)
Solution B: Acetonitrile and 0.01 M phosphate buffer, pH 7.0
(2:3)
Bromocriptine Mesylate Solution C: Acetonitrile and 0.01 M phosphate buffer, pH 7.0
(3:2)

': ~ 0
Nyo
HO~.... ..,••,.A
H )..,.", CH3
N
Mobile phase: See the gradient table below.

Time Solution B Solution C

?Cd:
CH3
(%) (%)
HN
h r N';W.
H~_.~O
(min)

- H3C CH3 0 100 0

N
Br H I 100
CH3 18 0

C32H40BrNsOs' CH4S03 750.70 30 a 100

Ergotaman-3',6', 18-trione, 2-bromo-12'-hydroxy-2'-(1- 40 a 100
methylethyl)-5'-(2-methylpropyl)-, monomethanesulfonate
(salt), (5'a)-; 41 100 a
2-Bromoergocryptine monomethanesulfonate (salt)
[22260-51-1 ]. System suitability solution: 2.0 mg/mL each of
a-ergocryptine and Bromocriptine Mesylate in Diluent
Standard stock solution: 46 ~g/mL of USP Bromocriptine
Mesylate RS in methanol and Solution A (1:1).

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596 Bromocriptine / OfficialMonographs USP 43

[NOTE-Dissolvein 50% of the flask volume of methanol and Acceptance criteria: +95° to +105°
dilute with Solution A to volume.] • loss ON DRYING
Standard solution: 4.6 J..Ig/mL of USP Bromocriptine (See Thermal Analysis (891 ).)
Mesylate RS in Diluent from the Standardstock solution Analysis: Determine the percentage of volatile substances
Sample solution: 4.6 mg/mL of Bromocriptine Mesylate in by thermogravimetric analysis using 10 mg of
methanol and Solution A (1:1). [NOTE-Dissolve in 50% of the Bromocriptine Mesylate. Heat the specimen under test at
flask volume of methanol and dilute with Solution A to the rate of 10°Imin in an atmosphere of nitrogen at a flow
volume.] rate of 45 mL/min. Record the thermogram from ambient
Chromatographic system temperature to 160°.
(See Chromatography (621), System Suitability.) Acceptance criteria: It loses NMT 4.0% of its weight.
Mode: LC
Detector: UV 300 nm ADDITIONAL REQUIREMENTS
Column: 4.6-mm x 15-cm; 3-J..Im packing L1 • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Flow rate: 2 mL/min containers, in a cold place.
Injection size: 20 JJL • USP REFERENCE STANDARDS (11)
System suitability USP Bromocriptine Mesylate RS
Samples: System SUitability solution and Standardsolution
[NoTE-The relative retention times for a-ergocryptine
and bromocriptine mesylate are 0.46 and 1.0,
respectively.] Bromocriptine Mesylate Capsules
Suitability requirements
Resolution: NLT 15 between a-ergocryptine and DEFINITION
bromocriptine mesylate, System suitability solution Bromocriptine Mesylate Capsules contain bromocriptine
Tailing factor: NMT 1.5, System SUitability solution mesylate (C32H40BrNsOs . CH4S03) equivalent to NLT 90.0%
Relative standard deviation: NMT 10.0%, Standard and NMT 110.0% of the labeled amount of bromocriptine
solution (C32H40BrNsOs).
Analysis
Samples: Standardsolution and Sample solution IDENTIFICATION
[NoTE-The relative retention times for bromocriptine • A~ The principal spot of the Sample solution corresponds, in
and bromocriptinine are 1.0 and 1.7, respectively.] R F value and color, to that of the Standardsolution, as
Calculate the percentage of each impurity in the portion of obtained in the test for OrganicImpurities.
Bromocriptine Mesylate taken:
ASSAY
Result =(ru/rs) x (Cs/Cu) x (1 IF) x 100 • PROCEDURE
Conduct this procedure without exposure to daylight and
ru = peak response of each impurity from the Sample with minimum exposure to artificial light.
solution Buffer: 0.125 giL of ammonium carbonate in water
rs = peak response of bromocriptine from the Mobile phase: Acetonitrile and Buffer(3:2)
Standardsolution Standard solution: 1.0 mg/mL of bromocriptine from USP
Cs = concentration of USP Bromocriptine Mesylate RS Bromocriptine Mesylate RS in dehydrated alcohol. Sonicate
in the Standardsolution (mg/mL) as needed.
Cu = concentration of Bromocriptine Mesylate in the Sample solution: 1.0 mg/mL of bromocriptine in methanol,
Sample solution (mg/mL) prepared as follows. Remove, as completely as possible, the
F = relative response factor equal to 1.4 for.any peak contents of NLT 10 Capsules. Weigh and determine the
eluting at a relative retention time of about 0.9 average weight per Capsule. Mix the combined contents,
or less, and equal to 1.0 for all other peaks and transfer a weighed quantity of the powder, nominally
equivalent to 50 mg of bromocriptine, to a 50-mL
Acceptance criteria volumetric flask. Add 30 mL of dehydrated alcohol, and
Individual impurities: NMT 0.4% of bromocriptinine is shake for 15 min. Dilute with dehydrated alcohol to
found; NMT 0.1 % of any individual impurity is found. volume, mix, and filter. Use this solution without delay.
Total impurities: NMT 1.0% Chromatographic system
(See Chromatography (621), System Suitability.)
SPECIFIC TESTS Mode: LC
• COLOR OF SOLUTION (631) Detector: UV 300 nm
Matching solutions: Prepare three solutions, A, B, and C, Column: 4-mm x 25-cm; packing L7
containing, respectively, the following parts of cobaltous Flow rate: 2 mL/min
chloride CS, ferric chloride CS, cupric sulfate CS, and dilute Injection volume: 20 J..IL
hydrochloric acid (1 in 40). System suitability
A: 3.0:3.0:2.4:31.6 Sample: Standard solution
B: 1.0:2.4:0.4:36.2 Suitability requirements
C: 0.6:2.4:0:37.0 Column efficiency: NLT 1000 theoretical plates
Sample solution: 10 mg/mL of Bromocriptine Mesylate in Tailing factor: NMT 2.0
methanol Relative standard deviation: NMT 2.0%
Analysis: Compare the Sample solution with 1O-mL portions Analysis
of the Matching solutions in suitable matched tubes. Samples: Standard solution and Sample solution
Acceptance criteria: The solution is clear and not darker in Calculate the percentage of the labeled amount
color than Matching solutions A, B, and C. bromocriptine (C32H40BrNsOs) in the portion of Capsules
• OPTICAL ROTATION, Specific Rotation (781 S)
taken:
Sample solution: 10 mg/mL, in a mixture of methylene
chloride and methanol (1:1)

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USP43 Official Monographs / Bromocriptine 597

Result = (r vIr s) x (C sIC v) x 100 Cu = nominal concentration of bromocriptine in the

Sample solution (mg/mL)
= peak response from the Sample solution M r7 = molecular weight of bromocriptine, 654.59
= peak response from the Standardsolution M r2 = molecular weight of bromocriptine mesylate,
= concentration of bromocriptine, from USP 750.70
Bromocriptine Mesylate RS, in the Standard
solution(mg/mL) Acceptance criteria: Meet the requirements
=nominal concentration of bromocriptine in the IMPURITIES
Sample solution (mg/mL)
• ORGANIC IMPURITIES
Acceptance criteria: 90.0%-110.0% Conduct this test without exposure to daylight and with
minimum exposure to artificial light. Perform the test
PERFORMANCE TESTS rapidly, preparing and spotting the Sample solution last.
• DISSOLUTION (711) Standard stock solution: 2.3 mg/mL of USP
Medium: 0.1 N hydrochloric acid; 500 mL BromocrlptlneMesylate RS in methanol, equivalent to 2
Apparatus 2: 50 rpm mg/mL of bromocriptine
Time: 60 min Standard solution 1: 0.06 mg/mL (3.0%) of bromocriptine
Standard solution: USP Bromocriptine Mesylate RS in in methanol, from Standardstock solution
Medium, at a concentration similar to the Sample solution. Standard solution 2: 0.04 mg/mL (2.0%) of bromocriptine
[NOTE-A volume of alcohol not to exceed 5% of the total in methanol, from Standardstock solution
volume of the Standardsolution may be used to bring the Standard solution 3: 0.02 mg/mL (1.0%) of bromocriptine
Standard into solution before dilution with Medium.] in methanol, from Standardstock solution
Sample solution: Sample per Dissolution (711), passed Standard solution 4: 0.01 mg/mL (0.50%) of
through a glass-fiber filter. bromocriptine in methanol, from Standard stocksolution
Instrumental conditions Sample solution: 2.0 mg/mL of bromocriptine in methanol,
(See Fluorescence Spectroscopy (853).) prepared as follows. Transfer a quantity of the Capsule
Mode: Fluorometry contents, equivalent to 20 mg of bromocriptine, to a
Excitation wavelength: 315 nm . conical flask. Add 10 mL of methanol, and stir by
Emission wavelength: 445 nm mechanical means for 20 min. Centrifuge the suspension
Blank: Medium for 10 min at about 3500 rpm. Use the clear supernatant.
Analysis Chromatographic system
Samples: Standard solution and Sample solution (See Chromatography (621), Thin-Layer Chromatography.)
Calculate the percentage of the labeled amount of Mode: TLC
bromocriptine rnesylate (C32H40BrNsOs . CH4S03) Adsorbent: 0.25-mm layer of chromatographic silica gel
dissolved. mixture
Tolerances: NLT 75% (Q) of the labeled amount of Application volume: 50 ~L as 1.5-cm bands
bromocriptine mesylate (C32H40BrNsOs . CH4S03) is Developing solvent: Methylene chloride, dioxane,
dissolved. alcohol, and ammonium hydroxide (180:15:5:1)
e UNIFORMITY OF DOSAGE UNITS (905) Spray reagent: 0.2% o-phthalaldehyde in sulfuric acid
Procedure for content uniformity Analysis
Protect all solutions from light. Samples: Standardstock solution, Standardsolutions, and
Diluent: Dissolve 1.0 g of tartaric acid in 500 mL of water, Sample solution
add 500 mL of methanol, and mix. . Develop under the exclusion of light in a tank lined with
Standard solution: 0.04 mg/mL of USP Bromocriptine filter paper, previously equilibrated for 30 min, using
Mesylate RS in Diluent Developing solvent until the solvent front has moved a
Sample solution: Transfer the contents of 1 Capsule into distance of 15 cm onthe plate. Dry the plate briefly in a
a 25-mL volumetric flask. Add 15 mL of Diluent, and shake current of cold air. Spray evenly with the Spray reagent,
by mechanical means for 20 min. Dilute with Diluent to and view the plate under long-wavelength UV light.
volume, and mix. Filter, and dilute 10.0 mL of the clear Acceptance criteria: Any major secondary spot, other than
filtrate with Diluent to 50.0 mL. the principal spot, obtained from the Sample solution is not
Instrumental conditions greater in size and intensity than the spot obtained from
(See Ultraviolet-Visible Spectroscopy (857).) Standardsolution 1 (3.0%). Any remaining spots are not
Mode: UV greater in size and intensity than the spot obtained from
Analytical wavelength: Maximum absorbance (about Standardsolution 3 (1.0%). The sum of the organic
306 nm) impurities is NMT 5.0%.
Cell: 1 cm ADDITIONAL REQUIREMENTS
Blank: Diluent • PACKAGING AND STORAGE: Preserve in tight, light-resistant
Analysis containers.
Samples: Standard solution, Sample solution, and Blank • USP REFERENCE STANDARDS (11)
Calculate the percentage of the labeled amount of USP Bromocriptine Mesylate RS
bromocriptine (C32H40BrNsOs) in the Capsule taken:

Result = (A vIA s) x (C sIC v) x (M rdM r2) X 100

= absorbance of the Sample solution Bromocriptine Mesylate Tablets

= absorbance of the Standardsolution
= concentration of USP Bromocriptine Mesylate RS DEFINITION
in the Standardsolution (mg/mL) Bromocriptine Mesylate Tablets contain bromocriptine
mesylate (C32H40BrNsOs . CH4S03) equivalent to NLT 90.0%

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598 Bromocriptine / OfficialMonographs USP 43

and NMT 110.0% of the labeled amount of bromocriptine Mode: Fluorometry

(C32H40BrNsOs). Excitation wavelength: 315 nm
Emission wavelength: 445 nm
IDENTIFICATION Analysis
• A. The principal spot of the Sample solution corresponds, in Samples: Standardsolution, Sample solution, and Blank
RF value and color, to that of the Standardstock solution, as Calculate the percentage of the labeled amount of
obtained in the test for OrganicImpurities. bromocriptine (C32H40BrNsOs) dissolved.
ASSAY Tolerances: NLT 80% (Q) of the labeled amount of
• PROCEDURE bromocriptine (C32H40BrNsOs) is dissolved.
Buffer: 0.01 M ammonium carbonate in water Test 2: Ifthe product complies with this test, the labeling
Mobile phase: Acetonitrile and Buffer(65:35) indicates that it meets USP Dissolution Test 2.
Standard solution: 0.22 mg/mL of USP Bromocriptine Medium: 0.1 N hydrochloric acid; 500 mL
Mesylate RS in methanol Apparatus 2: 50 rpm
Sample solution: Transfer a quantity of powdered Tablets Time: 30 min
(NLT 20), equivalent to 10 mg of bromocriptine, to an Buffer: 0.01 M ammonium carbonate in water
appropriate container. Add 40 mLof methanol, and stirfor Mobile phase: Acetonitrile and Buffer(65:35)
20 min, protected from light. Quantitativelyfilter through Standard solution: Dissolve USP Bromocriptine Mesylate
a fine glass filtering funnel into a 50-mLvolumetric flask. RS in methanol, and quantitativelydilute with Medium to
Rinse the filterwith methanol, adding the rinsing to the obtain a solution havinga known concentration similar to
filtrate, and dilute with methanol to volume. the expected concentration of the Sample solution.
Chromatographic system Sample solution: Sample per Dissolution (711).
(See Chromatography (621), System Suitability.) Chromatographic system
Mode: LC (See Chromatography (621), System Suitability.)
Detector: UV 300 nm Mode: LC
Column: 4-mm x 25-cm; packing L1 Detector: UV 300 nm
Flow rate: 2 mL/min Column: 3.9-mm x 30-cm; packing L1
Injection volume: 50 IJL Flow rate: 1.5 mL/min
System suitability Injection volume: 100 IJL
Sample: Standardsolution System suitability
Suitability requirements Sample: Standardsolution
Coefficient of variation: NMT 3.0% for 3 replicate Suitability requirements
injections Relative standard deviation: NMT 2.0%
Analysis Analysis
Samples: Standardsolution and Sample solution Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of Calculate the percentage of the labeled amount of
bromocriptine (C32H40BrNsOs) in the portion of Tablets bromocriptine (C32H40BrNsOs) dissolved.
taken: Tolerances: NLT 80% (Q) of the labeled amount of
bromocriptine (C32H40BrNsOs) is dissolved.
Result = (rulrs) x (CsICu) x (M,t/M'2) x 100 • UNIFORMITY OF DOSAGE UNITS (905)
Procedure for content uniformity
= peak response from the Sample solution [NOTE-Protect all solutionsfrom light.]
= peak response from the Standardsolution Diluent: Dissolve 1.0 g of tartaric acid in 500 mL of water,
= concentration of USP Bromocriptine Mesylate RS add 500 mL of methanol, and mix.
in the Standardsolution (mg/mL) Standard solution: 0.04 mg/mL of USP Bromocriptine
= nominal concentration of bromocriptine in the Mesylate RS in Diluent
Sample solution (mg/mL) Sample solution: Transfer 1 Tablet into a 25-mL
= molecularweight of bromocriptine, 654.59 volumetric flask. Add 15 mLof Diluent, and shake by
= molecular weight of bromocriptine mesylate, mechanical means for 30 min. Dilutewith Diluent to
750.70 volume, and mix. Filter, and dilute 10.0 mLof the clear
filtrate with Diluent to 50.0 mL.
Acceptance criteria: 90.0%-110.0% Instrumental conditions
(See Ultraviolet- Visible Spectroscopy (857).)
PERFORMANCE TESTS Mode: UV
• DISSOLUTION (711> Analytical wavelength: 306 nm
Test 1: Ifthe product complieswith this test, the labeling Cell: 1 em
indicates that it meets USP Dissolution Test 1. Blank: Diluent
Medium: 0.1 N hydrochloric acid; 500 mL Analysis
Apparatus 1: 120 rpm Samples: Standardsolution, Sample solution, and Blank
Time: 60 min Calculatethe percentage of the labeled amount of
Standard solution: USP Bromocriptine Mesylate RS at a bromocriptine (C32H40BrNsOs) in the Tablet taken:
known concentration in Medium
[NOTE-A volume of alcohol not to exceed 5% of the Result =(Au/As) x (CslCu) x (M,r/M'2) X 100
total volume of the Standardsolution may be used to
dissolve the Standard before dilution with Medium.] Au =absorbance of the Samplesolution
Sample solution: Sample per Dissolution (711), passed As = absorbance of the Standardsolution
through a glass-fiber filter. Cs =concentration of USP Bromocriptine Mesylate RS
Blank: Medium in the Standard solution (mg/mL)
Instrumental conditions Cu = nominal concentration of bromocriptine in the
(See Fluorescence Spectroscopy (853).) Sample solution(mg/mL)

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 USP 43 Official Monographs / Bromodiphenhydramine 599

= molecular weight of bromocriptine, 654.59

= molecular weight of bromocriptine mesylate, Bromodiphenhydramine Hydrochloride
750.70

Acceptance criteria: Meet the requirements

IMPURITIES
• ORGANIC IMPURITIES
[NOTE-Conduct this test without exposure to daylight
and with minimum exposure to artificial light.
Perform the test rapidly, preparing and spotting the C17H 2oBrNO· HCI 370.71
Sample solution last.] Ethanamine,2-(4-bromophenyl)phenylmethoxy-N,N-
Standard stock solution: 1.2 mg/mL of USP dimethyl-, hydrochloride.
Bromocriptine Mesylate RS in methanol, equivalent to 1 2-(p-Bromo-a.-phenylbenzyl)oxy-N,N-dimethylethylamine
mg/mL of bromocriptine hydrochloride [1808-12-4].
Standard solution 1: 0.50 mg/mL (5%) of bromocriptine in
methanol, from Standard stock solution » Bromodiphenhydramine Hydrochloride contains
Standard solution 2: 0.30 mg/mL (3%) of bromocriptine in not less than 98.0 percent and not more than
methanol, from Standard stock solution
Standard solution 3: 0.10 mg/mL (1%) of bromocriptine in 101.0 percent of C17H20BrNO . HCI, calculated on
methanol, from Standard stock solution the dried basis.
Sample solution: Transfer an equivalent to 20 mg of
bromocriptine, from powdered Tablets, to a conical flask. Packaging and storage-Preserve in tight containers.
Add 10 mL of methanol, and mix for 20 min. Centrifuge USP Reference standards (11)-
the suspension for 10 min at 4000 rpm. Use the clear USP Bromodiphenhydramine Hydrochloride RS
supernatant. Identification-
Chromatographic system
(See Chromatography (621), Thin-Layer Chromatography.)
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica gel
mixture
Application volumes
Standard solutions: 10-J.1L, as 1.5-cm bands
Sample solution: 50-J.1L, as 1.5-cm bands
Developing solvent system: Methylene chloride,
dioxane, alcohol, and ammonium hydroxide (180: 15: 5:
0.1) J.1g per
Spray reagent: 0.2% o-phthalaldehyde in sulfuric acid Medium: 0.1 N sulfuric acid.
Analysis Absorptivities at 228 nrn, calculated on the dried basis, do
Samples: Standard stocksolution, Standard solutions, and not differ by more than 3.0%.
Sample solution Melting range (741): between 148° and 152°.
Proceed as directed in Chromatography (621)i Thin-Layer Loss on drying (731 )-Dry it at 1OSO for 3 hours: it loses not
Chromatography. Dry the plate for 5 min in a current of more than 0.5% of its weight.
cold air. Develop in a tank lined with filter paper, . Assay-Dissolve about 700 mg of Bromodiphenhydramine
previously equilibrated for 20 min, using Developing Hydrochloride, accurately weighed, in 50 mL of glacial acetic
solvent system until the solvent front has moved a distance acid, and add 10 mL of benzene and 15 mL of mercuric acetate
of 10 cm on the plate. Dry the plate under vacuum at TS. Add 2 drops of crystal violet TS, and titrate with 0.1 N
room temperature for 15 min. Spray evenly with the perchloric acid VS to a green endpoint. Perform a blank
Spray reagent,and view the plate under long-wavelength determination, and make any necessary correction. Each mL
UV light. of 0.1 N perchloric acid is equivalent to 37.07 mg of
Acceptance criteria: Any spot, other than the principal C17H20BrNO . HCI.
spot, from the Sample solution is not greater in size and
intensity than the spot from Standard solution 2 (3.0%). Any
remaining spots are not greater in size and intensity than
the spot obtained from Standard solution 3 (1.0%). The sum
of the organic impurities is NMT 5.0%. Bromodiphenhydramine Hydrochloride
ADDITIONAL REQUIREMENTS Oral Solution
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
containers.
• LABELING: The labeling indicates the Dissolution test with »Bromodiphenhydramine Hydrochloride Oral
which the product complies. Solution contains not less than 93.0 percent and
• USP REFERENCE STANDARDS (11) not more than 107.0 percent of the labeled
USP Bromocriptine Mesylate RS
amount of bromodiphenhydramine hydrochloride
(C 17H 20BrNO . HCI).
Packaging and storage-Preserve in tight, light-resistant
containers.

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600 Bromodiphenhydramine / Official Monographs USP 43

US!' Reference standards (11)- Test solution-Transfer a volume of Oral Solution, equivalent
USP Bromodiphenhydramine Hydrochloride RS to about 10 mg of codeine phosphate, to a separator, and add
5 mL of water, 5 mL of methylene chloride, and 1 mL of
ammonium hydroxide. Shake for 1 minute, allow the layers to
separate, and use the clear, lower layer.
Id~.~.!i!~c~!i().~t . ;~~~~Cj!~~~~il~t~ Standardsolution-Prepare a solution of USP
Infr.qr~CfSpe9tT()$C()PX:;1.~~~~(Gt~fJ! ~., 2Q) - Bromodiphenhydramine Hydrochloride RS and USP Codeine
Test specimen-Transfer the final solution obtained from the Phosphate RS in methanol containing 10 mg of each per mL.
titration in the Assay to a separator, add about 1 mL of 0.1 N Developing solvent system: a mixture of alcohol and
sulfuric acid, and shake with 25 mL of ether. (Methyl red enters ammonium hydroxide (49:1).
the ether phase.) Drain the aqueous layer into another B: The retention times of the major peaks in the
separator, add 5 mL of 1 N sodium hydroxide, and shake with chromatogram of the Assay preparation correspond to those
10 mL of chloroform. Drain the chloroform layer into a small in the chromatogram of the Standard preparation, asobtained
flask containing 2 g of anhydrous sodium sulfate, and swirl. in the Assay.
Pour the chloroform solution through a small cotton pledget, Microbial enumeration tests (61) and Tests for specified
pre-rinsed with chloroform, into a beaker, and evaporate to microorganisms (62)-lt meets the requirements of the tests
about 5 mL. Apply a few drops of the solution directly to a for absence of Salmonella species, Escherichia coli,
potassium bromide plate, and completely remove the Staphylococcus aureus, and Pseudomonas aeruginosa. The total
chloroform by warming for 2 to 3 minutes under an IR lamp. aerobic microbial count does not exceed 100 cfu per mL, and
Alcohol Determination, Method I (611): between 12.0% the total combined molds and yeasts count does not exceed
and 15.0% of C2H sOH. 50 cfu per mL.
Assay-Evaporate an accurately measured volume of Oral pH (791): between 4.5 and 6.5.
Solution, equivalent to about 250 mg of Alcohol Determination, Method /I (611): between 4.0% and
bromodiphenhydramine hydrochloride, to about half the 6.0% is found.
original volume, using a suitable vacuum evaporator. Transfer
the concentrated solution to a 250-mL separator, with the aid Assay-
of sufficient warm water to bring the volume to the original Diluent-Prepare a mixture of methanol and water (80:20).
volume. Add 20 g of sodium chloride, and shake until Mobilephase-Prepare a filtered and degassed mixture of
dissolved. Add 5 mL of 1 N sodium hydroxide, shakewith 100 methanol, water, 0.1 N ammonium hydroxide solution, and
mL of ether, and drain the aqueous layer into a second 0.1 N ammonium nitrate solution (27:3:2:1). Make
adjustments if necessary (see System SUitability under
separator containing 50 mL of ether. Shake, and discard the
aqueous layer. Wash the ether solutions with two 20-mL Chromatography (621 ».
portions of water, shaking each aqueous portion successively Standardpreparation-Dissolve accurately weighed
quantities of USP Bromodiphenhydramine Hydrochloride RS
in the two separators, and then discard the aqueous solutions.
Extract the ether solutions successively with 10.0 mL of 0.1 N and USP Codeine Phosphate RS in Diluent, and dilute
sulfuric acid VS, followed by two 5-mL portions of water, and quantitatively, and stepwise if necessary, with Diluentto obtain
collect the aqueous extracts in a conical flask.Add methyl red a solution having known concentrations of about 100 IJg per
TS to the solution in the flask, and titrate the excess acid with mL and 80 IJg per mL, respectively.
0.02 N sodium hydroxide VS. Perform a blank determination Assay preparation--Using a pipet calibrated "to contain",
(see Residual Titrations under Titrimetry(541»). Each mL of 0.1 transfer an accurately measured volume of Oral Solution,
N sulfuric acid is equivalent to 37.07 mg of equivalent to about 10 mg of bromodiphenhydramine
bromodiphenhydramine hydrochloride (C17H20BrNO. HCI). hydrochloride and 8 mg of codeine phosphate, to a 100-mL
volumetric flask, dissolve in and dilute with Diluent to volume,
and mix.
Chromatographic system (see Chromatography (621»)-The
liquid chromatograph is equipped with a 254-nm detector
and a 3.9-mm x 30.0-cm column that contains packing L3.
Bromodiphenhydramine Hydrochloride The flow rate is about 1.0 mL per minute. Chromatograph the
and Codeine Phosphate Oral Solution Standardpreparation, and record the peak responses as
directed for Procedure: the relative retention times are about
1.0 for bromodiphenhydramine and 1.4 for codeine; the
» Bromodiphenhydramine Hydrochloride and resolution, R, between bromodiphenhydramine and codeine
Codeine Phosphate Oral Solution contains not less is not less than 2.0; and the relative standard deviation for
than 90.0 percent and not more than 110.0 replicate injections is not more than 2.0%.
percent of the labeled amounts of bromodiphen Procedure-Separately inject equal volumes (about 20 IJL)
of the Standard preparation and the Assay preparation into the
hydramine hydrochloride (C17H2o BrNO . HCI) and chromatograph, record the chromatograms, and measure the
codeine phosphate hemihydrate (C18H21 N0 3 • peak responses for bromodiphenhydramine and codeine.
H3P04 • V2H 2 0 ). Calculate the quantity, in mg, of bromodiphen hydramine
hydrochloride (C17H 20BrNO . HCI) in each mL of the Oral
Packaging and storage-Preserve in tight, light-resistant Solution taken by the formula: .
containers.
Labeling-Label it to indicate the alcohol content. 1OO( CI V)( rvirs)
USPReference standards (11)-
USP Bromodiphenhydramine Hydrochloride RS in which C is the concentration, in mg per mL, of USP
USP Codeine Phosphate RS Bromodiphenhydramine Hydrochloride RS in the Standard
preparation; V is the volume, in mL, of Oral Solution taken to
Identification- prepare the Assay preparation; and tu and r s are the
A: Thin-Layer Chromatographic Identification Test (201)- bromodiphenhydramine peak responses obtained from the
Assay preparation and the Standardpreparation, respectively.

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 USP 43 OfficialMonographs / Brompheniramine 601

Calculate the quantity, in mg, of codeine phosphate RS, and USP Chlorpheniramine Related Compound B RS in
hemihydrate (C18Hz1N0 3 • H3P04 • Y2H 20) in each mL of the Diluent. Sonicate for 1 min.
Oral Solution taken by the formula: System suitability solution: 0.5 mg/mL of USP
Brompheniramine Maleate RS and 2 ~g/mL each of USP
(406.37/397.36)(100CIV)(rvlrs) Pheniramine Maleate RS, USP Chlorpheniramine Maleate
RS, and USP Chlorpheniramine Related Compound B RS in
in which 406.37 and 397.36 are the molecular weights of Diluent, prepared as follows. Transfer 5.0 mg of USP
codeine phosphate hemihydrate and anhydrous codeine Brompheniramine Maleate RS to a 1O-mL volumetric flask,
phosphate, respectively; C is the concentration, in mg per mL, add 5.0 mL of Diluent, and 1.0 mL of the System suitability
of USP Codeine Phosphate RS in the Standardpreparation; V is stocksolution, and dilute with Diluent to volume.
the volume, in mL, of Oral Solution taken to prepare the Assay Standard solution: 0.5 mg/mL of USP Brompheniramine
preparation; and rvand rs are the codeine peak responses Maleate RS in Diluent. Sonicate for 1 min.
obtained from the Assay preparation and the Standard Sample solution: 0.5 mg/mL of Brompheniramine
preparation, respectively. Maleate in Diluent. Sonicate for 1 min.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 225 nm
Brompheniramine Maleate Column: 4.6-mm x 25-cm; 5-~m packing L1
Column temperature: 30°

~
": Flow rate: 1 mL/min

Br
I
":
h
,.-:N

.
N
/CH,

6H,
¢
o

o
OH

OH
Injection volume: 10 ~L
System suitability
[NOTE-The relative retention times for maleic acid and
brompheniramine are 0.18 and 1.0, respectively.]
Samples: System suitability solution and Standardsolution
C16H19BrNz· C4H 404 435.31 Suitability requirements
2-Pyridinepropanamine, y-(4-bromophenyl)-N, N-dimethyl-, Resolution: NLT 1.5 between chlorpheniramine and
(±)-, (Z)-2-butenedioate (1:1); brompheniramine; and NLT 2.0 between
(±)- 2-p-Bromo-a- 2-(dimethylamino)ethylbenzylpyridine chlorpheniramine related compound Band
maleate (1:1) [980-71-2]. phenlrarnlne, System suitability solution
DEFINITION Tailing factor: NMT 2.0 for brompheniramine, Standard
Brompheniramine Maleate, dried at 105° for 3 h, contains NLT solution
98.0% and NMT 102.0% of brompheniramine maleate Relative standard deviation: NMT 0.73%, Standard
(C16H19BrNz· C4H404) · solution
Analysis
IDENTIFICATION Samples: Standardsolution and Sample solution
Calculate the percentage of brompheniramine maleate
(C16H19BrNz . C4H 404) in the portion of Brompheniramine
Maleate taken:

. . ) Result = (rvlrs) x (CsICv) x 100

of the maleic acid and
brompheniramine peaks of the Sample solution correspond = peak response of brompheniramine from the
to those of the Standardsolution, as obtained in the Assay. Sample solution
= peak response of brompheniramine from the
ASSAY Standardsolution
• PROCEDURE = concentration of USP Brompheniramine Maleate
Solution A: 5.44 giL of monobasic potassium phosphate. RS in the Standardsolution (mg/mL)
Adjust with phosphoric acid to a pH of 3.0 ± 0.1. = concentration of Brompheniramine Maleate in
Solution B:' Acetonitrile the Sample solution (mg/mL)
Mobile phase: See Table 1.
Acceptance criteria: NLT 98.0o/o-NMT 102.0% on the
Table 1 previously dried basis
Time Solution.A Solution B
(min) (%) (%) IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.2%
0 95 5 • ORGANIC IMPURITIES
1 95 5 Solution A, Solution B, Diluent, Mobile phase, System
suitability solution, and Chromatographic system:
20 70 30
Proceed as directed in the Assay.
30 70 30 Standard solution: 2.7 ~g/mL of USP Brompheniramine
Maleate RS in Diluent, equivalent to 2.0 ~g/mL of
31 95 5
brompheniramine. Sonicate 1 min.
40 95 5 Sensitivity solution: 0.74 ~g/mL of USP Pheniramine
Maleate RS in Diluent
Diluent: Acetonitrile and Solution A (5:95) Sample solution: 0.5 mg/mL of Brompheniramine
System suitability stock solution: 0.02 mg/mL each ofUSP Maleate in Diluent. Sonicate for 1 min.
Pheniramine Maleate RS, USP Chlorpheniramine Maleate

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 602 Brompheniramine / Official Monographs USP 43

System suitability USP Chlorpheniramine Related Compound B RS

Samples: System suitability solution, Standard solution, and Di(pyridin-2-yl)amine.
Sensitivity solution C,oHgN3 171.20
Suitability requirements USP Pheniramine Maleate RS
Resolution: NLT 1.5 between chlorpheniramine and
brompheniramine; and NLT 2.0 between
chlorpheniramine related compound Band
pheniramine, System suitability solution
Signal-to-noise ratio: NLT 10 for pheniramine, Bromphentramlne Maleate Injection
Sensitivity solution
Relative standard deviation: NMT 5.0% for
brompheniramine, Standard solution » Brompheniramine Maleate Injection is a sterile
Analysis solution of Brompheniramine Maleate in Water for
Samples: Standard solution and Sample solution Injection. It contains not lessthan 90.0 percent and
Calculate the percentage of each impurity in the portion of not more than 110.0 percent of the labeled
Brompheniramine Maleate taken:
amount of C16H19BrNz . C4H404 •
Result = (rvlrs) x (Cs/Cu) x (1 IF) x 100
Packaging and storage-Preserve in single-dose or in
= peak response of each impurity from the Sample multiple-dose containers, preferably of Type I glass, protected
solution from light.
= peak response of brompheniramine from the USPReference standards (11) -
Standard solution USP Brompheniramine Maleate RS
= concentration of brornphenlrarnlne in the Identification-Dilute a volume of Injection, equivalent to
Standard solution (mg/mL) about 50 mg of brompheniramine maleate, with dilute
= concentration of Brompheniramine Maleate in hydrochloric acid (1 in 1200) to 25 mL, and proceed as
the Sample solution (mg/mL) .directed under Identification-Organic Nitrogenous Bases
F = relative response factor (see Table 2) (181), beginning with "Transfer the liquid to a separator:" the
Injection meets the requirements of the test.
Acceptance criteria: See Table 2. Disregard any peak Bacterial Endotoxins Test (85) -It contains not more than
having areas less than 0.05% of brompheniramine. 35.7 USP Endotoxin Units per mg of brompheniramine
maleate.
Table 2 pH (791): between 6.3 and 7.3.
Relative Relative Acceptance Other requirements-It meets the requirements under
Retention Response Criteria, Injections and Implanted Drug Products (1).
Name Time Factor NMT (%) Assay-Proceed with Injection as directed under Salts of
Maleic acid" 0.18 - - Organic Nitrogenous Bases (501), to prepare the solution
employed for the determination of the absorbance, A u, at 262
Chlbrphel1irarnil1e relat- nm. For the determination of A s, dissolve about 25 mg of USP
ed compound Bb 0.46 - -
Brompheniramine Maleate RS, accurately weighed, in 20 mL
Pheniramine 0.53 0.45 0.4 of dilute sulfuric acid (1 in 350), and treat this solution the
Chlorpheniramine 0.94 1.1 0.4
same as the portion of Injection being assayed. Calculate the
quantity, in mg, of C'6H,gBrNz . C4H4 0 4 in each mL of the
Brompheniramine 1.0 - - Injection taken by the formula:
Any other unspecifiedirn-
purity - 1.0 0.10 (WIV)(A ulA s)
Total impurities - - 1 in which W is the weight, in mg, of USP Brompheniramine
aSaltcounter ion is included in the table for identification purposes only.
Maleate RS in the Standard Preparation and V is the volume,
b Di(pyridin-2-yl)amine. Usedonly to establish the system suitability,
in mL, of Injection taken. .

SPECIFICTESTS
• OPTICAL ROTATION (781)
Sample: 100 mg/mL i'n water at 20°
Acceptance criteria: -0.2° to +0.2°, measured in a 20-cm
Brompheniramine Maleate Oral
tube Solution
• pH (791)
Sample: 10 mg/mL
Acceptance criteria: 4.0-5.0 » Brompheniramine Maleate Oral Solution
• Loss ON DRYING (731) contains not less than 95.0 percent and not more
Analysis: Dry at 105° for 3 h. than 105.0 percent of the labeled amount of
Acceptance criteria: NMT 0.5% brompheniramine maleate (C16H19BrNz . C4H404 ) .
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Packaging and storage-Preserve in well-closed,
containers. light-resistant containers.
• USP REFERENCE STANDARDS (11) USP Reference standards (11) -
USP Brompheniramine Maleate RS USP Brompheniramine Maleate RS
USP Chlorpheniramine Maleate RS Identification-Transfer a volume of Oral Solution,
equivalent to about 50 mg of brompheniramine maleate, to a

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 USP 43 Official Monographs / Brompheniramine 603

separator, render distinctly alkaline with 1 N sodium mL of this solution to a separator containing 25 mL of water,
hydroxide, and extract with two 50-mL portions of mix, and proceed as directed under Assay preparation,
chloroform, shaking gently to avoid emulsification. Wash the beginning with "adjust with sodium hydroxide solution (1 in
combined chloroform extracts with 10 mL of water, and 10) to a pH of 11." The concentration of USP
discard the aqueous phase. Filter the combined chloroform Brompheniramine Maleate RS in the Standardpreparation is
extracts into a conical flask, and evaporate the solvent on a about 20 I-Ig per mL.
steam bath, with the aid of a current of air. To the residue add Assay preparation-Weigh and finely powder not fewer
25 mL of dilute hydrochloric acid (1 in 1200), and proceed as than 20 Tablets. Weigh accurately a portion of the powder,
directed under Identification-Organic Nitrogenous Bases equivalent to about 4 mg of brompheniramine maleate, mix
(181), beginning with "Transfer the liquid to a separator." The with 50 mL of water for 10 minutes, adjust with sodium
Oral Solution meets the requirements of the test. hydroxide solution (1 in 10) to a pH of 11, and cool to room
pH (791): between 2.5 and 3.5. temperature. Extract the mixture with two 75-mL portions of
Alcohol Determination, Method I (611): between 2.7% and solvent hexane, and combine the extracts in a second
3.3% of C2H sOH. separator. Extract the solvent hexane solution with three
Assay-Transfer an accurately measured volume of Oral 50-mL portions of dilute hydrochloric acid (1 in 120),
Solution, equivalent to about 20 mg of brompheniramine combining the acid extracts in a 200-mL volumetric flask. Add
maleate, to a separator, render distinctly alkaline with 1 N dilute hydrochloric acid (1 in 120) to volume, and mix.
sodium hydroxide, and extract with ten 1O-mL portions of Procedure-Concomitantly determine the absorbances of
chloroform, shaking gently to avoid emulsification. Wash the the Assay preparation and the Standard preparation, in 1-cm
combined chloroform extracts with 10 mL of water, wash the cells at the wavelength of maximum absorbance at about 264
latter with 20 mL of chloroform, and discard the aqueous nm, with a suitable spectrophotometer, using dilute
phase. Quantitatively filter the combined chloroform extracts hydrochloric acid (1 in 120) as the blank. Calculate the
and washings into a conical flask, and evaporate the solvent quantity, in mg, of C16H19Br N 2 . C4H 4 0 4 in the portion of
on a steam bath, with the aid of a current of air. To the residue Tablets taken by the formula:
add 25 mL of glacial acetic acid and 5 mL of acetic anhydride,
agitate, and allow to stand for about 15 minutes. Add 1 drop 0.2C(Au/As)
of crystal violet TS, and titrate with 0.01 N perchloric acid VS
to a blue-green endpoint. Perform a blank determination; and in which C is the concentration, in I-Ig per mL, of USP
make any necessary correction. Each mL of 0.01 N perchloric Brompheniramine Maleate RS in the Standardpreparation;and
acid is equivalent to 2.1 77 mg of brompheniramine maleate Auand As are the absorbances of the Assay preparation and the
(C16H19BrN2 . C4 H4 0 4 ) · Standardpreparation, respectively.

Brompheniramine Maleate Tablets Brompheniramine Maleate and

Pseudoephedrine Sulfate Oral Solution
» Brompheniramine Maleate Tablets contain not
less than 95.0 percent and not more than 105.0 » Brompheniramine Maleate and Pseudoephedrine
percent of the labeled amount of C'6H19BrN2 . Sulfate Oral Solution contains not less than 90.0
C4H 404 • percent and not more than 110.0 percent of the
labeled amounts of brompheniramine maleate
Packaging and storage-Preserve in tight containers. (C'6H19BrN2' C4H 404 ) and pseudoephedrine
USP Reference standards (11)-
USP Brompheniramine Maleate RS sulfate [(C,oH15NOh . H2S04 ] ,
Identification-Tablets meet the requirements under
Identification-Organic Nitrogenous Bases (181). USP Reference standards (11)-
USP Brompheniramine Maleate RS
Dissolution (711)- USP Pseudoephedrine Sulfate RS
Medium: water; 500 mL.
Apparatus 7: 100 rpm. Identification-
Time: 45 minutes. A: The retention times of the major peaks in the
Procedure-Determine the amount of C16H19BrN2 . C4H4 0 4 chromatogram of the Assay preparation correspond to those
dissolved from UV absorbances at the wavelength of in the chromatogram of the Standard preparation, as
maximum absorbance at about 264 nm on filtered portions of obtained in the Assay.
the solution under test, suitably diluted with 3 N hydrochloric B: A solution of it meets the requirements of the test for
acid, using 5-cm cuvettes, in comparison with a Standard Sulfate (191).
solution having a known concentration of USP C: Transfer a volume of Oral Solution, equivalent to about
Brompheniramine Maleate RS in the same Medium. 6 mg of brompheniramine maleate, to a separator, add 0.5
Tolerances-Not lessthan 75% (Q) of the labeled amount mL of ammonium hydroxide and 5 mL of methylene chloride,
of C16H19BrN2' C4H 4 0 4 is dissolved in 45 minutes. shake for 1 minute, and allow the layers to separate. Use the
Uniformity of dosage units (905): meet the requirements. clear, lower layer as the test solution. Prepare separate
Standard solutions in methanol containing, respectively, 1.2
Assay- mg of USP Brompheniramine Maleate RS and 9 mg of USP
Standardpreparation-Dissolve an accurately weighed Pseudoephedrine Sulfate RS per mL. Separately apply 5 IJLof
quantity of USP Brompheniramine Maleate RS in water, and each solution to a suitable thin-layer chromatographic plate
dilute quantitatively with water to obtain a solution having a (see Chromatography (621» coated with a 0.25-mm layer of
known concentration of about 160 I-Ig per mL. Transfer 25.0 chromatographic silica gel mixture. Allow the spots to dry, and

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 604 Brompheniramine / Official Monographs USP 43

develop the chromatogram in a solvent system consisting of a responsesfor the major peaks. Calculate the quantity, in mg,
mixture of ethyl ether, methanol, and ammonium hydroxide of brompheniramine maleate (C16H19BrN2' C4H404) in each
(16:3:1) until the solvent front has moved about three-fourths mL of the Oral Solution taken by the formula:
of the length of the plate. Remove the plate from the
developing chamber, mark the solvent front, and allow the 25CV(R u/Rs)
solvent to evaporate. Locate the spots on the plate by
examination under short-wavelength UV light: the RFvalues of in which C is the concentration, in mg per mL, of USP
the two principal spots obtained from the test solution Brompheniramine Maleate RS in the Standardpreparation; V
correspond to those obtained from the Standard solutions. is the volume, in mL, of Oral Solution taken; and Ru and Rs are
the peak response ratios obtained for brompheniramine
Uniformity of dosage units (905)- maleate and naphazoline hydrochloride from the Assay
FOR ORAL SOLUTION PACKAGED IN SINGLE-UNIT CONTAINERS:
preparation and the Standard preparation, respectively.
meets the requirements. Calculate the quantity, in mg, of pseudoephedrine sulfate
Deliverable volume (698)- (C10H15NO)2' H2S04 in each mL of the Oral Solution taken by
FOR ORAL SOLUTION PACKAGED IN MULTIPLE-UNIT CONTAINERS: the same formula, changing the terms to refer to
meets the requirements. pseudoephedrine sulfate.
Assay-
Mobile phase-Prepare a mixture of water, acetonitrile,
methanol, and tetrahydrofuran (550:320:80:50). Transfer 1.0
mL of phosphoric acid, followed by 4.33 g of dodecyl sulfate Budesonide
sodium to this mixture, and mix. Adjust with ammonium
hydroxide to a pH of 3.50 ± 0.05, filter, and degas. Make OH

adjustments if necessary (see System Suitability under

Chromatography (621). [NoTE-The pH of the Mobilephase is
critical and may cause 1 to 4 minutes of differences in the
retention times of internal standard and brompheniramine
maleate.]
Internal standard solution-Transfer about 50 mg of
naphazoline hydrochloride to a 1OO-mL volumetric flask, add C25H3406 430.53
Mobilephase to volume, and mix. Pregna-l,4-diene-3,20-dione, 16, 17-butylidenebis(oxy)-
Standardpreparation-Dissolve an accurately weighed 11,21-dihydroxy-,[11 b, 16a(R)], and 16a,17-[(S)-
quantity of USP Brompheniramine Maleate RS in Mobilephase, Butylidenebis(oxy)]-11 b,21-dihydroxypregna-l ,4-diene-
a
and quantitatively dilute with Mobilephaseto obtain solution 3,20-dione;
having a known concentration of about 6000j IJg per mL, j (RS)-ll b, 16a,17,21-Tetrahydroxypregna-l ,4-diene-3,20-
being the ratio of the labeled amount, in mg, of dione cyclic 16, 17-acetal with butyraldehyde [51333-22-
brompheniramine maleate to the labeled amount, in mg, of 3].
pseudoephedrine sulfate per mL (Solution P). Transfer about S epimer [51372-28-2].
30 mg of USP Pseudoephedrine Sulfate RS, accurately R epimer [51372-29~3].
weighed, to a 25-mL volumetric flask, add 5.0 mL each of
Solution P and Internal standard solution, dilute with Mobile DEFINITION
phase to volume, and mix to obtain a Standard preparation Budesonide is a mixture of two epimeric forms, epimer A (C-
having known concentrations of about 1200j IJg of USP 22S) and epimer B (C-22R). It contains NLT 40.0% and NMT
Brompheniramine Maleate RS per mL and about 1.2 mg of 51.0% of epimer A, and the sum of both epimers is NLT
USP Pseudoephedrine Sulfate RS per mL. 98.0% and NMT 102.0% of budesonide (C25H3406),
Assay preparation-Using a "To contain" pipet transfer an calculated on the dried basis.
accurately measured volume of Oral Solution, equivalent to [NOTE-Protect all solutions containing Budesonide from
about 30 mg of pseudoephedrine sulfate, to a 25-mL light.]
volumetric flask. Rinse the pipet with about 5 mL of Mobile IDENTIFICATION
phase, collecting the rinse in the volumetric flask. Add 5.0 mL
of lntematstandard solution, dilute with Mobilephase to
volume, and mix.
Chromatographic system (see Chromatography (621) -The
liquid chromatograph is equipped with a 254-nm detector
and a 4-mm x 30-cm column that contains packing L11. The
flow rate is about 1.5 mL per minute. Chromatograph the
Standardpreparation, and record the peak responses as
directed for Procedure: the relative retention times are about
1.0 for pseudoephedrine sulfate, 1.5 for naphazoline
hydrochloride, and 2.5 for brompheniramine maleate; the
resolution, R, between the pseudoephedrine sulfate and
naphazoline hydrochloride peaks is not less than 3, and Meets the requirements
between the brompheniramine maleate and naphazoline
hydrochloride peaks is not less than 3; and the relative ASSAY
standard deviation for replicate injections is not more than • PROCEDURE
2.0%. Buffer: 0.5 mL of glacial acetic acid in 1 L of water. Adjust
Procedure-Separately inject equal volumes (about 10 IJL) with potassium hydroxide to a pH of 3.9.
of the Standardpreparation and the Assay preparation into the Mobile phase: Acetonitrile and Buffer (45:55)
chromatograph, record the chromatograms, and measure the Standard solution: 0.06 mg/mL of USP Budesonide RS
prepared as follows. Transfer USP Budesonide RS to a

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 USP 43 Official Monographs / Budesonide 605

suitable volumetric flask, dissolve in acetonitrile equivalent Table 1 (continued)

to 30% of the flask volume, and dilute with water to Time Solution A Solution B
volume. (min) (%) (%)
Sample solution: 0.06 mg/mL of Budesonide prepared as
60 75 25
follows. Transfer Budesonide to a suitable volumetric flask,
dissolve in acetonitrile equivalent to 30% of the flask 70 75 25
volume, and dilute with water to volume.
Chromatographic system Diluent: Acetonitrile and water (3:7)
(See Chromatography (621), System Suitability.) System suitability stock solution A: 0.3 mg/mL of USP
Mode: LC Budesonide Related Compound E RS in acetonitrile
Detector: UV 240 nm System suitability stock solution B: 0.3 mg/mL of USP
Column: 4.6-mm x 25-cm; 3-lJm packing L1 Budesonide Related Compound G RS in acetonitrile
Column temperature: 50° System suitability stock solution C: 0.3 mg/mL of USP
Flow rate: 1 mL/min Budesonide Related Compound L RS in acetonitrile
Injection volume: 20 IJL System suitability solution: 0.6 mg/mL of USP
System suitability Budesonide RS and 3 IJg/mL each of USP Budesonide
Sample: Standardsolution Related Compound E RS, USP Budesonide Related
[NOTE-The relative retention time for epimer B is 0.96 Compound G RS, and USP Budesonide Related Compound
with respect to epimer A.] L RS in Diluent prepared as follows. Transfer USP
Suitability requirements . Budesonide RS to a suitable volumetric flask and add
Resolution: NLT 1.2 between the two budesonide suitable quantities of System suitability stock solution A,
epimer peaks System suitabilitystock solution B, and System suitabilitystock
Relative standard deviation NMT 1.0%, for the sum of solution C. Dilute with acetonitrile equivalent to 30% of the
the peak areas of the two budesonide epimers final volume and dilute with water to volume.
Analysis Standard stock solution: 0.6 mg/mL of USP Budesonide RS
Samples: Standardsolution and Sample solution prepared asfollows. Transfer USP Budesonide RS to a
Calculate the percentage of budesonide epimer A . suitable volumetric flask, dissolve in acetonitrile equivalent
(C2sH3406) in the portion of Budesonide taken: to 30% of the flask volume, and dilute with water to
volume.
Result = [rUA/(rUA + rUB)] x 100 . Sensitivity solution: 0.3 Ilg/mL of USP Budesonide RS in
Diluent from the Standard stock solution
rUA = peak area of epimer A from the Sample solution Standard solution: 6 IJg/mL of USP Budesonide RS in
rUB = peak area of epimer B from the Sample solution Diluent from the Standardstock solution
Sample solution: 0.6 mg/mL of Budesonide prepared as
Calculate the percentage of budesonide (C2sH3406) in the follows. Transfer Budesonide to a suitable volumetric flask,
portion of Budesonide taken: dissolve in acetonitrile equivalent to 30% of the flask
volume, and dilute with water to volume.
Result = [(rUA + rUB)/(rSA + rSB)] x (Cs/Cu) x 100 Chromatographic system
(See Chromatography (621), System SUitability.)
= peak area of epimer A from the Sample solution Mode: LC
= peak area of epimer B from the Sample solution Detector: UV l40 nm
= peak area of epimer A from the Standardsolution Column: 4.6-mm x 25-cm; 3-lJm packing L1
= peak area of epimer B from the Standardsolution Temperatures
= concentration of USP Budesonide RS in the Autosampler: 4°
Standardsolution (mg/ml) Column: 50°. [NOTE-The resolution between
= concentration of Budesonide in the Sample budesonide related compound Eand budesonide
solution (mg/mL) related compound L may be improved by lowering the
temperature, but to NLT 40°.]
Acceptance criteria Flow rate: 1.5 mL/min
Epimer A: 40.0%-51 .0% .. Injection volume: 50 IJL
Both epimers: 98.0%-102.0% on the dried baSIS System suitability
Samples: System suitability solution,Sensitivity solution, and
IMPURITIES Standardsolution
• ORGANIC IMPURITIES [NOTE-The relative retention times of the two epimers
Solution A: 0.5 mL of glacial acetic acid in 1 L of water. of budesonide are 0.96 (epimer B) and 1.00 (epimer
Adjust with potassium hydroxide to a pH of 3.9. A), respectively.]
Solution B: Acetonitrile Suitability requirements
Mobile phase: See Table 7. Resolution: NLT 1.2 between budesonide related
compound E and budesonide related compound Land
Table 1 NlT 3.0 between budesonide epimer A and the first
Time Solution A Solution B epimer of budesonide related compound G, System
(min) (%) (%) suitability solution
0 75 25 Tailing factor: NMT 1.5 for the budesonide epimer B
peak, Standardsolution
5 75 25 Relative standard deviation: NMT 5.0%, for the sum of
35 68 32 the peak areasof the two budesonide epimers, Standard
solution
42 59 41
Signal-to-noise ratio: NLT 10, Sensitivity solution
59 25 75

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 606 Budesonide / OfficialMonographs USP 43

Analysis microbial count is NMT 10 3 cfu/g, and the total combined

Samples: Standard solution and Sample solution molds and yeast count is NMT 102 cfu/g.
Calculate the percentage of each impurity in the portion of • loss ON DRYING (731)
Budesonide taken: Analysis: Dry at 105 0 to constant weight.
Acceptance criteria: NMT 0.3%
Result = (ru/rs) x (Cs/Cu) x 100
ADDITIONAL REQUIREMENTS
= peak response of each individual impurity from • PACKAGING AND STORAGE: Preserve in tight, light-resistant
the Sample solution containers. Store at controlled room temperature.
= peak response of the sum of budesonide epimers • USP REFERENCE STANDARDS (11)
from the Standard solution USP Budesonide RS
= concentration of USP Budesonide RS in the USP Budesonide Related Compound E RS
Standard solution (mg/mL) 16a, 17-[Butylidenebis(oxy)]-11 ~,21-dihydroxypregna­
= concentration of Budesonide in the Sample 1,4,14-triene-3,20-dione.
solution (mg/mL) C2sH320 6 428.52
USP Budesonide Related Compound G RS
Acceptance criteria: See Table 2. Disregard peaks that are 16a, 17-[Butylidenebis(oxy)]-11 ~,21-dihydroxypregna-4­
less than 0.05% of the total peak areas of the budesonide ene-3,20-dione.
epimers. Disregard peaks eluting after 60 min, which, if C2sH3606 432.55
present, are due to the change in the gradient. USP Budesonide Related Compound L RS
16a,17-[Butylidenebis(oxy)]-21-hydroxypregna-1,4-
Table 2 diene-3, 11,20-trione.
Relative Acceptance C2sH320 6 428.52
Retention Criteria,
Name Time NMT (0/0)
16a-Hydroxyprednisolonea 0.12 0.2
Budesonide acetaldehyde Bumetanide
acetal (eplmers)" 0.39,0.40 0.10'
Budesonide o-homo analog d• e 0.47 0.10
Desonldev f 0.51 0.10
Budesonide glyoxal (epirners)s 0.76,0.78 0.07c
Budesonide related compound Eh 0.86 0.10
Budesonide related compound L' 0.88 0.2
Budesonide epimer B 0.96 - C17H20N20SS 364.42
Budesonide epimer A 1.00 - Benzoic acid, 3-(aminosulfonyl)-5-(butylamino)-4-phenoxy-;
3-(Butylamino)-4-phenoxy-5-sulfamoylbenzoic acid
Budesonide related compound G
(epimers)i 1.07, 1.08 0.10c [28395-03-1 ].

Budesonide 21-acetate (eplmers)" 1.39, 1.40 0.10c DEFINITION

Bumetanide contains NLT 98.0% and NMT 102.0% of
Budesonide 21-butyratel 1.48 0.10 bumetanide (C17H20N20SS), calculated on the dried basis.
Any other individual impurity - 0.10
IDENTIFICATION
Total specified impurities - 0.4
Total unspecified impurities - 0.4

a 11~, 16a, 17,21-Tetrahydroxypregna-l ,4-diene-3,20-dione.

b 16a, 17 -[Ethylidenebis(oxy)]-ll ~,21-dihydroxypregna-l ,4-diene-3,20-dione.
C Limit includes both epimers.
d 16u, 17-[Butylidenebis(oxy)]-ll ~-hydroxy-17-(hydroxymethyl)-o-
homoandrosta-l ,4-diene-3, 17a-dione; also known as o-homobudesonide.
e This impurity is to be reported under total unspecified impurities. Do not report • B. ASPECTROSCOPICIDENTIF'ICATIONTESTS(197);
it under total specified impurities. Ultraviolet-Visible Spectroscopy: 19.7U.l (eN 1~MaY-2020)
f 16a, 17{'l-Methylethylkienebistoxyjl-l l S, 21-dihydroxypregna-l ,4-diene- Sample solution: 50 ~g/mL in isopropyl alcohol
3,20-dione.
9 16u, 17-[Butylidenebis(oxy)]-ll ~-hydroxy-3,20-dioxopregna-l,4-dien-21-al;
Acceptance criteria: Meets the requirements
also known as 21-dehydrobudesonide. • C. The principal spot of the Sample solution exhibits an RF
h Also known as 14, 15-dehydrobudesonide or budesonide 14-ene. value corresponding to that of Standard solution A, as
i Also known as ll-ketobudesonide. obtained in the test for Organic Impurities.
J Also known as 1,2-dihydrobudesonide. . '
k16a, 17-[Butylidenebis(oxy)]-ll ~,21-dihydroxypregna-l,4-diene-3,20-dione- ASSAY·
21-acetate. • PROCEDURE
116a,17-[Butylidenebis(oxy)]-11 ~,21-dihydroxypregna-l ,4-diene-3,20-dione- Sample solution: Dissolve 1 g of Bumetanide in 150 mL of
21-butyrate. . alcohol, and add phenol red TS.
Titrimetric system
SPECIFIC TESTS Mode: Direct titration
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR Titrant: 0.1 N sodium hydroxide VS
SPECIFIED MICROORGANISMS (62): The total aerobic

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 USP 43 Official Monographs / Burnetanide 607

Analysis: Perform a blank determination, and make any USP Bumetanide Related Compound A RS
».
necessary correction (see Titrimetry (541 Each mL of 0.1 3-Amino-4-phenoxy-5-sulfamoylbenzoic acid.
N sodium hydroxide is equivalent to 36.44 mg of C13H12N 20SS 308.31
bumetanide (C17H20N20SS.) USP Bumetanide Related Compound B RS
Acceptance criteria: 98.0%-102.0% on the dried basis 3-Nitro-4-phenoxy-5-sulfamoylbenzoic acid.
C13HlON207S 338.29
IMPURITIES
USP Butyl 3-(butylamino)-4-phenoxy-
• RESIDUE ON IGNITION (281): NMT 0.1%, on a 1-g specimen
5-sulfamoylbenzoate RS
• ORGANIC IMPURITIES
Standard solution A: 25 mg/mL of USP Bumetanide RS in C2l H28N20SS 420.53
methanol
Standard solutionB: 50 ~g/mL of USP Bumetanide RS from
Standardsolution A in methanol
Standard solution C: 50 ~g/mL of USP Bumetanide Related
Compound B RS in methanol Bumetanide Injection.
Standard solution D: 25 ~g/mL of USP Bumetanide Related
Compound A RS in methanol » Bumetanide Injection is a sterile solution of
Standard solution E: 25 ~g/mL of USP Butyl Bumetanide in Water for Injection, prepared with
3-(butylamino)-4-phenoxy-5-sulfamoylbenzoate RS in
methanol the aid of Sodium Hydroxide. It contains not less
Sample solution: 25 mg/mL of Bumetanide in methanol than 90.0 percent and not more than 110.0
Chromatographic system percent of the labeled amount of bumetanide
(See Chromatography (621), Thin-Layer Chromatography.) (C17HzoNzOsS).
Mode: TLC
Adsorbent: 0.25-mm layer of chromatographic silica gel Packaging and storage-Preserve in single-dose or
mixture multiple-dose containers, preferably of Type I glass, protected
Developing solvent system: Chloroform, cyclohexane, from light.
glacial acetic acid, and methanol (160:20:20:5) USP Reference standards (11)-
Application volume: 20 ~L USP Bumetanide RS
Analysis USP Bumetanide Related Compound A RS
Samples: Standardsolution A, Standardsolution 8, Standard 3-Amino-4-phenoxy-5-sulfamoylbenzoic acid.
solution C, Standard solution D, Standard solution E, and C13H12N 20SS 308.31
Sample solution
Proceedasdirected in Chromatography(621). After drying Identification-
the application spots, place the plate in an unlined and A: The relative retention time of the major peak in the
unsaturated chromatographic chamber. Examine the chromatogram of the Assay preparation corresponds to that in
plate under short-wavelength UV light. the chromatogram of the Standardpreparation, both relative
Acceptance criteria: See Table 1. Any secondary spots from to the internal standard, as obtained in the Assay.
the Sample solution are not larger or more intense than the 8: The principal spot obtained from the chromatogram of
corresponding principal spots from the corresponding the Test solution exhibits an R F value corresponding to that of
standard solution identified in Table 1. the Identification solution, as obtained in the test for Related
compounds.
Table 1 Bacterial Endotoxins Test (85) -It contains not more than
Corresponding Acceptance 350 USP Endotoxin Units per mg of bumetanide.
Standard Criteria, pH (791): between 6.8 and7.8.
Name Solution NMT (%)
Related compounds-
Bumetaniderelated compound
Aa Standard solution D 0.1 Adsorbent: O.25-mm layer of chromatographic silica gel
mixture.
Bumetanlderelated compound Test solution-Pipet a volume of Injection, equivalent to 5
Ba Standard solutionC 0.2
mg of bumetanide, into a 125-mL separator, and adjust with
Butyl 3-(butylamino)-4-phenoxy- 0.1 N sodium hydroxide to a pH of 12. Extract with two 20-mL
5-sulfamoylbenzoate Standard solutionE 0.1 portions of ethyl ether, discard the ethyl ether extracts, and
Other individual impurities Standard solution B 0.2 adjust the aqueous layer with 1 N acetic acid to a pH of 4.
Extract with two 20-mL portions of ethyl ether, passing the
Sum of other individual impuri- extracts through anhydrous sodium sulfate. Wash the sodium
ties' - 0.4 sulfate with about 5 mL of ethyl ether. Evaporate the
combined ethyl ether extracts with the aid of a stream of
a Excluding bumetanide related compound A, bumetanide related compound
B,and butyl 3-(butylamino)-4-phenoxy-5-sulfamoylbenzoate. nitrogen to dryness, and dissolve the residue in 0.5 mL of
methanol.
SPECIFIC TESTS Identification solution-Dissolve USP Bumetanide RS in
• Loss ON DRYING (731) methanol to obtain a solution having a concentration of about
Sample: Dry a sample at 105° for 4 h. 10 mg per mL.
Acceptance criteria: NMT 0.5% Standard solutions-Dilute a volume of the Identification
solution quantitatively, and stepwise if necessary, with
ADDITIONAL REQUIREMENTS methanol to obtain a solution having a known concentration
• PACKAGING AND STORAGE: Preserve in tight, light-resistant of about 0.08 mg of USP Bumetanide RS per mL.
containers. Store at controlled room temperature. Quantitatively dilute with methanol to obtain Standard
• USP REFERENCE STANDARDS (11) solutions having the following compositions.
USP Bumetanide RS

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 608 Bumetanide / OfficialMonographs USP 43

Percentage (%, directed for Procedure: the relative retention times are about
for comparison 0.7 for 4-ethylbenzaldehyde and 1.0 for bumetanide; the
Standard Concentration with test resolution R between the analyte and internal standard peaks
solution Dilution (Ilg of RS per ml) specimen) is not less th~n 1.5 the tailing factor for the analyte peak is not
undiluted 80 0.8 more than 1.4, and the relative standard deviation for replicate
injections is not more than 2.0%.
2 3 in 4 60 0.6
Procedure-Separately inject equal volumes (about 20 IJL)
3 1 in 2 40 0.4 of the Standard preparation and the Assay preparation into the
4 1 in 4 20 0.2
chromatograph, record the chromatograms, and measure the
responsesfor the major peaks. Calculate the quantity, in mg,
5 1 in 8 10 0.1 of C17HzoNzOsS in each mL of the Injection taken by the
formula:
Standardsolution 6-Dissolve an accurately weighed
quantity of USP Bumetanide Related Compound A RS in (2C/V)(R u/R s)
methanol, and dilute quantitatively, and stepwise if necessary,
with methanol to obtain a solution having a known in which C is the concentration, in mg per mL, of USP
concentration of about 0.02 mg per mL. Bumetanide RS in the Standard preparation;V is the volume, in
Application volume: 50 IJL. mL, of Injection taken; and R u and R s are the peak response
Developing solvent system: a mixture of chloroform, ratios obtained from the Assay preparation and the Standard
cyclohexane, glacial acetic acid, and methanol (80: 10: 10: preparation, respectively.
2.5).
Procedure-Proceed as directed for Thin-Layer
Chromatography under Chromatography (621). Examine the
plate under short-wavelength UV light. Any secondary spot
obtained from the chromatogram of the Test solution having Bumetanide Tablets
an R Fvalue corresponding to the R Fvalueof the principal spot
obtained from the chromatogram of Standardsolution 6 is not
DEFINITION
Bumetanide Tablets contain NLT 90.0% and NMT 110.0% of
larger or more intense than the principal spot obtained from
the chromatogram of Standardsolution 6: not more than 0.2% the labeled amount of bumetanide (C17HzoNzOsS).
of bumetanide related compound A is found. For all other IDENTIFICATION
secondary spots obtained from the chromatogram of the Test • A. The relative retention time of the major peak of the
solution, compare the intensity of each spot with the principal Sample solution corresponds to that of the Standard
spots obtained from the chromatograms of Standardsolutions solution, as obtained in the Assay.
7 through 5: not more than 0.2% of any individual other • B. The principal spot of the Sample solution exhibits an RF
impurity is found; and not more than 0.8% of the sum of all value corresponding to that of the Identification solution, as
other impurities is found (excluding bumetanide related obtained in the test for OrganicImpurities.
compound A). '
Other requirements-It meets the requirements under ASSAY
Injections and Implanted Drug Products (1).
Assay- .
Mobilephase-Prepare a filtered and degassed mixture of • PROCEDURE
methanol, water, tetrahydrofuran, and glacial acetic acid Mobile phase: Methanol, tetrahydrofuran, glacial acetic
(50:45:5:2). Make adjustments if necessary(see System acid, and water (50: 5:2: 45)
SUitabilityunder Chromatography (621 »). Internal standard stock solution: 0.5 mg/mL of
Internal standard solution-Transfer about 50 mg of 4-ethylbenzaldehyde in methanol
4-ethylbenzaldehyde to a 1OO-mL volumetric flask. Dissolve in Internal standard solution: Add 10.0 mL of Internal
and dilute with methanol to volume, and mix. Transfer 10.0 standardstock solution, 10.0 mL of tetra hyd rofu ran, and 4.0
mL of the resulting solution to a 1OO-mL volumetric flask, add mL of glacial acetic acid to a 1OO-mL volumetric flask, and
10.0 mL of tetrahydrofuran and 4.0 mL of glacial acetic acid, dilute with methanol to volume.
dilute with methanol to volume, and mix. Standard stock solution: 250 IJg/mL of USP Bumetanide RS
Standardpreparation-Dissolve an accurately weighed in Internal standardsolution
quantity of USP Bumetanide RS in Internal standard solution, Standard solution: 125 IJg/mL from Standardstock solution
and quantitatively dilute with Internal standardsolution to in water
obtain a solution having a known concentration of about 250 Sample solution: of
IJg per mL. Transfer 5.0 mL ofthe resulting solution to a 10-mL bumetanide prepared as a
volumetric flask, dilute with water to volume, and mix to equivalent to 0.5 mg of bumetanide, from finely powdered
obtain a solution having a known concentration of about 125 Tablets (NLT 20), to a 1O-mL volumetric flask. Add 2.0 mL
IJg of USP Bumetanide RS per mL. of Internal standardsolution and sonicate for 5 min. Add 2.0
Assay preparation-Transfer an accurately measured mL of water. Cool and filter, discarding the first 1 mL of the
volume of Injection, equivalent to about 0.25 mg of filtrate.
bumetanide, to a flask. Add an equal volume of Internal Chromatographic system
standardsolution, accurately measured, insert the stopper, and (See Chromatography (621), System Suitability.)
mix. Mode: LC
Chromatographic system (see Chromatography (621 »)-The Detector: UV 254 nm
liquid chromatograph is equlpped with a 254-nm detector Column: 3.9-mm x 30-cm; packing L1
and a 3.9-mm x 30-cm column that contains packing L1. The Flow rate: 1 mL/min
flow rate is about 1 mL per minute. Chromatograph the Injection volume: 20 IJL
Standardpreparation, and record the peak responses as

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USP 43 Official Monographs / Bumetanide 609

System suitability Chromatographic system

Sample: Standardsolution (See Chromatography (621), System Suitability.)
[NoTE-The relative retention times for Mode: LC
4-ethylbenzaldehyde and bumetanide are 0.7 and Detector: UV 222 nm
1.0, respectively.] Column: 4.6-mm x 15-cm; 5-~m packing L1
Suitability requirements Column temperature: 35 0

Resolution: NLT 1.5 between 4-ethylbenzaldehyde and Flow rate: 1.5 mL/min
bumetanide Injection volume: 100 ~L
Tailing factor: NMT 1.4 Run time: NLTl .7 times retention time of bumetanide
Relative standard deviation: NMT 2.0% System suitability
Analysis Sample: Standardsolution
Samples: Standardsolution and Sample solution Suitability requirements
Calculatethe percentage of bumetanide (C17H20N20SS) in Tailing factor: NMT 2.0
the portion of Tablets taken: Relative standard deviation: NMT 2.0%
Analysis
Result = (RuIR s) x (CslCu) x 100 Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of
Ru = peak response ratio of bumetanide to the internal bumetanide (C17H20N20SS) dissolved:
standard from the Sample solution
Rs = peak response ratio of bumetanide to the internal Result = (rulrs) x Cs x V x (lIL) x 100
standard from the Standardsolution
Cs = concentration of USP Bumetanide RS in the ru = peak response of bumetanide from the Sample
Standardsolution (mg/mL) solution
Cu = nominal concentration of the bumetanide in the ts = peak response of bumetanide from the Standard
Sample solution (mg/mL) solution
Cs =concentration of USP Bumetanide RS in the
Acceptance criteria: 90.00/0-110.0% Standardsolution (mg/mL)
PERFORMANCE TESTS
V =volume of Medium, 900 mL
• DISSOLUTION (711)
L = label claim (mg/Tablet)
Test 1 Tolerances: NLT 80% (Q) of the labeled amount of
Medium: Water; 900 mL bumetanide (C17H20N20SS) is dissolved.
Apparatus 2: 50 rpm • UNIFORMITY OF DOSAGE UNITS (905): Meet the
Time: 30 min ' requirements
Solution A: 7.505 gIL of glycine and 5.85 gIL of sodium
chloride in water IMPURITIES
Solution B: Solution A, 0.1 N hydrochloric acid, and water ORGANIC IMPURITIES
(4:1:45). Adjust, ifnecessary,with 0.1 N hydrochloric acid Identification solution: 20 mg/mL of USP Bumetanide RS
or 0.1 N sodium hydroxide to a pH of 2.9. in methanol
Standard solution: USP Bumetanide RS at a known Standard solution 1: 160 ~g/mL of USP Bumetanide RS
concentration in Medium from Identification solution in methanol
Sample solution: Dilutewith Solution B as needed. Standard solution 2: 120 ~g/mL of USP Bumetanide RS
Instrumental conditions from Standardsolution 1 in methanol
Mode: Fluorescence Standard solution 3: 80 uq/rnl, of USP Bumetanide RS
Detectors from Standardsolution 1 in methanol
Excitation wavelength: 350 nm Standard solution 4: 40 ~g/mL of USP Bumetanide RS
Emission wavelength: 450 nm from Standardsolution 1 in methanol
Analysis Standard solution 5: 20 ~g/mL of USP Bumetanide RS
Samples: Standardsolution and Sample solution from Standardsolution 1 in methanol
Determine the percentage of the labeled amount of Standard solution 6: 40 ~g/mL of USP Bumetanide
bLimetanide (C17H20N20SS) dissolved. Related Compound A RS in methanol
Tolerances: NLT 85% (Q) of the labeled amount of Sample solution: Nominally 20 mg/mL of bumetanide
bumetanide (C17H20N20SS) is dissolved. prepared as follows. Equivalent to 10 mg of bumetanide
Test 2: Ifthe product complies with this test, the labeling from powdered Tablets in a 50-mL centrifuge tube. Add
indicates that it meets USP Dissolution Test 2. 20 mLof acetone (spectrophotometric or HPLC quality),
Medium, Apparatus 2, and Time: Proceed as directed in and shake by mechanical means for 10 min. Centrifuge
Test 1. for 10 min, decant the supernatant into a glass-stoppered,
Buffer: 2.72 gIL of potassium phosphate, monobasic in 25-mLconicalflask, and evaporate withthe aid of a stream
water. Adjust with 1.8 N potassium hydroxide to a pH of of nitrogen to dryness. Dissolve the residue in 0.5 mLof
7.0. methanol.
Mobile phase: Acetonitrile and Buffer (30:70) Chromatographic system
Diluent: Acetonitrile and water (50:50) (See Chromatography (621), General Procedures,
Standard stock solution: 55.5 ~g/mL of USP Bumetanide Thin-Layer Chromatography.)
RS in Diluent Mode: TLC
Standard solution: (L/l 000) ~g/mL of USP Bumetanide Adsorbent: 0.25-mm layerof chromatographic silica gel
RS in Medium, from Standardstock solution, where L isthe mixture
label claim in mg/Tablet Application volume: 25 ~L
Sample solution: Passa portion of the solution under test Visualization: Short-wavelength UV light
through a suitable filter of 0.45-~m pore size. Developing solvent system: Methanol, cyclohexane,
glacial acetic acid, and chloroform (2.5: 10: 10: 80)

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 610 Bumetanide / OfficialMonographs USP 43

Analysis nitrogen, and dry the residue in vacuum. Add 2 mL of

Samples: Standard solutions 7-6 and Sample solution chloroform to the residue, and dissolve.
Acceptance criteria
Bumetanide related compound A: Any secondary spot
from the Sample solution with an RF value corresponding
to the RF value of the principal spot from Standard
solution 6 is not larger or more intense than the principal
spot from Standard solution 6; NMT 0.2%. ~g per
Any individual other impurity: For all other secondary Medium: 0.1 N hydrochloric acid.
spots from the Sample solution, compare the intensity of Absorptivities at 271 nm, calculated on the anhydrous basis,
each spot with the principal spots from Standard do not differ by more than 3.0%.
solutions 7-5; NMT 0.2% of any individual other C: Dissolve about 50 mg in 10 mL of water in a small
impurity is found. separator, render alkaline with 6 N ammonium hydroxide, and
Sum of all other impurities: NMT 0.8% of the sum of all extract with 10 mL of ether: the aqueous layer meets the
other impurities is found (excluding bumetanide related requirements of the tests for Chloride (191).
compound A). pH (791): between 4.5 and 6.0, in a solution (1 in 100).
Water Determination, Method I (921): between 4.0% and
ADDITIONAL REQUIREMENTS 6.0%.
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Residue on ignition (281): not more than 0.1%.
containers.
• LABELING: When more than one Dissolution test is given, the limit of residual solvents-
labeling states the Dissolution test used only if Test 7 is not Alcohol standard solution-Pipet 2 mL of dehydrated alcohol
used. into a 1OO-mL volumetric flask, dilute with water to volume,
• USP REFERENCE STANDARDS (11) and mix. Transfer 2.0 mL of this solution to a 50-mL volumetric
USP Bumetanide RS flask, dilute with water to volume, and mix. The resulting
USP Bumetanide Related Compound A R5 solution contains 0.08% of alcohol.
3-Amino-4-phenoxy-5-sulfamoylbenzoic acid. Isopropyl alcohol standard solution-Pipet 2 mL of isopropyl
C13H12N 20s5 308.31 alcohol into a 1OOO-mL volumetric flask, dilute with water to
volume, and mix. Transfer 2.0 mL of this solution to a 100-mL
volumetric flask, dilute with water to volume, and mix. The
resulting solution contains 0.004% of isopropyl alcohol.
Test solution-Transfer 1.0 g of Bupivacaine Hydrochloride,
Bupivacaine Hydrochloride accurately weighed, to a 25-mL volumetric flask, dilute with
water to volume, and mix.
Chromatographic system-Under typical conditions, the
instrument is equipped with a flame-ionization detector and a
• Hel
• . H20 4-mm x 2-m column that contains packing 53. The carrier gas
is nitrogen, flowing at a rate of about 40 mL per minute. The
column temperature is maintained at about 175°, the injection
port temperature is maintained at about 200°, and the
C18H28N20. HCI . H20 342.90 detector temperature is maintained at about 280°.
2-Piperidinecarboxamide, l-butyl-N-(2,6-dimethylphenyl)-, Procedure-Inject equal volumes (about 5 ~L) of the Test
monohydrochloride, monohydrate, (±)-. solution, the Alcohol standard solution, and the Isopropyl alcohol
(±)-1-Butyl-2',6'-pipecoloxylidide monohydrochloride, standard solution successively into the gas chromatograph.
monohydrate [73360-54-0]. Measure the responses of the alcohol peak and the isopropyl
Anhydrous 324.90 alcohol peak in each chromatogram. Determine the
[18010-40-7]. percentage of alcohol taken by the formula:

»Bupivacaine Hydrochloride contains not less than 2(r vir 5)

98.5 percent and not more than 101.5 percent of and determine the percentage of isopropyl alcohol taken by
C'8H28N20· HCI, calculated on the anhydrous the formula:
basis.
0.1 (r vir 5)
Packaging and storage-Preserve in well-closed
containers. in which r u and r 5 are the responses of the respective analytes
USP Reference standards (11)- in the Test solution and of the corresponding analytes in the
U5P Bupivacaine Hydrochloride R5 Alcohol standard solution and the Isopropyl alcohol standard
solution, respectively. The sum of the content of alcohol and
Identification- the content of isopropyl alcohol does not exceed 2%.
Chromatographic purity-
Adsorbent: 0.25-mm layer of chromatographic silica gel
mixture.
Solvent: a mixture of chloroform and isopropylamine
mg in 15 mL of water in a (99:1 ).
separator, add 1 mL of 6 N ammonium hydroxide, and extract Test solution-Dissolve a suitable quantity of Bupivacaine
with three 30-mL portions of chloroform. Evaporate the Hydrochloride in Solvent to obtain a solution containing 20.0
chloroform at room temperature with the aid of a stream of mg per mL.

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 USP 43 OfficialMonographs / Bupivacaine 611

Standard solution-Dissolve a suitable quantity of USP Internal standard solution: 1.3 mg/mL of dibutyl phthalate
Bupivacaine Hydrochloride RS, accurately weighed, in Solvent in methanol '
to obtain a solution containing 20.0 mg per mL. Standard solution: 0.5 mg/mL of USP Bupivacaine
Dilutedstandard solution-Quantitatively dilute a'portion Hydrochloride RS, prepared as follows. In a 100-mL
of the Standardsolution in Solvent to obtain a solution having volumetric flask, dissolve 50 mg of USP Bupivacaine
a concentration of 100 IJg per mL. Hydrochloride RS in 10.0 mL of water, using sonication if
Developing solvent system: a mixture of hexanes and necessary. Add 10 mL of Internal standard solution, and
isopropylamine (97:3). dilute with methanol to volume.
Procedure-Apply separate 1O-IJL portions of the Test Sample solution: Nominally 0.5 mg/mL of bupivacaine
Solution, the Standardsolution, and the Dilutedstandard hydrochloride, prepared asfollows. In a 1OO-mL volumetric
solution on the starting line of suitable thin-layer flask, transfer an amount of Injection equivalent to 50 mg
chromatographic plate as directed for Thin-Layer of bupivacaine hydrochloride, add 10.0 mL of Internal
Chromatography under Chromatography (621). Develop the standard solution, and dilute with methanol to volume.
chromatogram in a suitable chamber until the solvent has Chromatographic system
moved about three-fourths of the length of the plate. Remove (See Chromatography (621), System Suitability.)
the plate from the chamber, mark the solvent front, and dry it Mode: LC
in warm air. Place the plate in a closed chamber with a dish Detector: UV 263 nm
containing 1 g of iodine in a shallow layer, and allow to remain Column: 4-mm x 30-cm; packing L1
for about 5 minutes. Remove the plate from the chamber, Flow rate: 2 mL/min
spray it with 7 N sulfuric acid, and examine the Injection volume: 20 IJL
chromatogram: the R F value of the principal spot from the System suitability
Test solution corresponds to that of the Standard solution, and Sample: Standardsolution
the estimated size and intensity of any other spot obtained [NoTE-The relative retention times for bupivacaine
from the Test solution does not exceed that of the principal hydrochloride and dibutyl phthalate are about 1.0
spot obtained from the Diluted standardsolution (0.5%); and and 1.2, respectively.]
the total of the estimated sizesand intensities of all of the other Suitability requirements
spots obtained from the Test solution does not exceed four Resolution: NLT 2.0 between bupivacaine hydrochloride
times that of the principal spot obtained from the Diluted and dibutyl phthalate
standardsolution (2.0%). Relative standard deviation: NMT 1.0% for the ratio of
Assay-Transfer about 600 mg of Bupivacaine Hydrochloride, bupivacaine to the internal standard from three
accurately weighed, to a 250-mL conical flask, and dissolve in replicate injections
20 mL of glacial acetic acid. Add 10 mL of mercuric acetate TS Analysis
and 3 drops of crystal violet TS, and titrate with 0.1 N Samples: Standardsolution and Sample solution
perchloric acid VS to a green endpoint. Perform a blank Calculate the percentage of the labeled amount of
determination, and make any necessary correction. Each mL bupivacaine hydrochloride (ClsH2SN20 . HCI) in the
of 0.1 N perchloric acid is equivalent to 32.49 mg of portion of Injection taken:
ClsH2SN20· HCI.
Result = (R viR s) x (C siC v) x 100

Ru = peak response ratio of bupivacaine to the internal

standard from the Sample solution
Bupivacaine Hydrochloride Injection Rs = peak response ratio of bupivacaine to the internal
standard from the Standardsolution
DEFINITION Cs = concentration of USP Bupivacaine Hydrochloride
Bupivacaine Hydrochloride Injection is a sterile solution of RS, calculated on the anhydrous basis, in the
Bupivacaine Hydrochloride in Water for Injection. It contains Standardsolution (mg/mL)
NLT 93.0% and NMT 107.0% of the labeled amount of Cu = nominal concentration of bupivacaine
bupivacaine hydrochloride (C1SH2SN20 . HCI). hydrochloride in the Sample solution (mg/mL)
IDENTIFICATION Acceptance criteria: 93.0%-107.0%
• A. IDENTIFICATION-ORGANIC NITROGENOUS BASES (181)
Sample solution: 2 mg/mL of bupivacaine hydrochloride in SPECIFICTESTS
0.01 N hydrochloric acid, from Injection • BACTERIAL ENDOTOXINS TEST (85): NMT 2.5 USP Endotoxin
Analysis: Proceed as directed in the chapter beginning with Units/mg of bupivacaine hydrochloride
"Transfer the liquid to a separator". • pH (791): 4.0-6.5
Acceptance criteria: Meets the requirements • OTHER REQ.UIREMENTS: It meets the requirements in
• B. The retention time of the bupivacaine peak in the Injections and Implanted Drug Products (1).
Sample solution corresponds to that of the Standard
ADDITIONAL REQUIREMENTS
solution, as obtained in the Assay.
• PACKAGING AND STORAGE: Preserve in single-dose or in
ASSAY multiple-dose containers, preferably of Type I glass.
• PROCEDURE Injection labeled to contain 0.5% or less of bupivacaine
Buffer: 1.94 giL of monobasic potassium phosphate and hydrochloride may be packaged in 50-mL, multiple-dose
2.48 giL of dibasic potassium phosphate in water. Adjust, containers.
if necessary, with 1 N potassium hydroxide or 1 M • USP REFERENCE STANDARDS (11)
phosphoric acid to a pH of 6.8. USP Bupivacaine Hydrochloride RS
Mobile phase: Acetonitrile and Buffer (65: 35). Adjust, if
necessary, with 1 M phosphoric acid to a pH of 7.7 ± 0.2.
Filter the solution through a membrane filter of t-urn or
finer pore size, and degas.

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 612 Bupivacaine / OfficialMonographs USP43

Assay for buplvacalne hydrochloride-

Bupivacaine Hydrochloride in Dextrose pH 6.8 Phosphate buffer, Mobilephase, Internal standard
Injection solution, Standard preparation, Chromatographic system, and
Procedure-Proceed asdirected in the Assay under Bupivacaine
Hydrochloride Injection.
» Bupivacaine Hydrochloride in Dextrose Injection Assay preparation-Transfer an accurately measured
is a sterile solution of Bupivacaine Hydrochloride volume of Injection, equivalent to about 50 mg of bupivacaine
and Dextrose in Water for Injection. It contains not hydrochloride, to a 1OO-mL volumetric flask, add 10.0 mL of
less than 93.0 percent and not more than 107.0 Internalstandardsolution, dilute with methanol to volume, and
mix.
percent of the labeled amounts of bupivacaine Assay for dextrose-Determine the angular rotation of
hydrochloride (ClsH2SN20 . HC!) and dextrose Injection in a suitable polarimeter tube (see OpticalRotation
(C6H 120 6) . It contains no preservative. ».
(781 Calculate the percentage (g per 100 mL) of dextrose
(C6H1206) in the portion of Injection taken by the formula:
Packaging and storage-Preserve in single-dose
containers, preferably of Type I glass. (100152.9)AR
USP Reference standards (11) -
USP Bupivacaine Hydrochloride RS in which 100 is the percentage; 52.9 is the midpoint of the
USP Dextrose RS specific rotation range for anhydrous dextrose, in degrees; A
is 100 mm divided by the length of the polarimeter tube, in
Identification- mm; and R is the observed rotation, in degrees.
A: Thin-Layer Chromatographic Identification Test (201)-
Adsorbent: chromatographic silica gel mixture; 0.25 mm.
Developing solvent: mixture of butyl alcohol, water,
dehydrated alcohol, and glacial acetic acid (6:2: 1:1).
Test preparation: Bupivacaine Hydrochloride in Dextrose Bupivacaine Hydrochloride and
Injection. .Epinephrine Injection
Standardpreparations A, B, and C-Separately prepare (A) a
solution of USP Bupivacaine Hydrochloride RS in water, (B) a DEFINITION
solution of USP Dextrose RS in water, and (C) a solution of USP Bupivacaine Hydrochloride and Epinephrine Injection is a
Bupivacaine Hydrochloride RS in (B) to obtain solutions having sterile solution of Bupivacaine Hydrochloride and
concentrations corresponding to the labeled concentrations of Epinephrine or Epinephrine Bitartrate in Waterfor Injection.
bupivacaine hydsochlorlde and dextrose in the Injection. It contains NLT 93.0% and NMT 107.0% of the labeled
Naphthalenediol reagent-Dissolve 20 mg of amount of bupivacaine hydrochloride (ClsH2sN20 . HC!).
1,3-naphthalenediol in 10 mL of dehydrated alcohol The content of epinephrine (C9H13N03) does not exceed
containing 0.2 mL of sulfuric acid. 0.001 % (1 in 100,000). It contains the equivalent of NLT
lodoplatinate reagent-Mix equal volumes of platinic chloride 90.0% and NMT 115.0% of the labeled amount of
solution (3 in 1000) and potassium iodide solution (6 in 100). epinephrine (C9H13N03) .
Procedure-Separately apply 10 I.JL each of the Test
preparation and Standardpreparations A and C to a portion of IDENTIFICATION
the chromatographic plate, and separately apply 1 I.JL each • A.
of the Test preparation and Standard preparation B to the Procedure 1
remaining portion of the plate. Dry the applications in a Sample solution: Nominally 2 mg/mL of bupivacaine
current of warm air, develop the chromatograms, remove the hydrochloride in 0.01 N hydrochloric acid from Injection
plate from the developing chamber, and mark the solvent Analysis: Proceed as directed in Identification-Organic
front. Dry the plate in warm circulating air, and examine the Nitrogenous Bases (181), beginning with "Transfer the
plate under short-wavelength UV light: the R F value of the liquid to a separator".
principal spot obtained from the Test preparation corresponds Acceptance criteria:' Meets the requirements
to the spots obtained from the adjacent chromatograms of Procedure 2
Standardpreparations A and C. Spray the plate with Sample solution: Use the' Sample solution from Procedure
Naphthalenediolreagent, heat at 90° for 5 minutes, and 1: Bupivacaine Hydrochloride in the Assay.
examine the plate: the R F value of the principal blue-purple Acceptance criteria: The retention time of the
spot obtained from the Test preparation corresponds to that bupivacaine peak of the Sample solution corresponds to
obtained in the adjacent chromatogram of Standard that of the Standardsolution, as obtained in Procedure 1:
preparation B. Cool the plate, spray it with lodoplatinate Bupivacaine Hydrochloride in the Assay.
reagent, and examine the plate: bupivacaine appears as a • B.
blue-purple spot on a salmon-colored background, and the Sample: Nominally equivalent to 50 I.Jg of epinephrine from
dextrose spots fade slightly: the R F value of the bupivacaine Injection
spot obtained from the Test preparation corresponds to those Analysis: Pipet the Sample into a suitable container, add 0.1
obtained from the adjacent chromatograms of Standard mL of Ferro-citrate solution and 2.0 mL of Buffer solution
preparations A and C. »,
(prepared as directed in Epinephrine Assay (391 mix, and
B: It responds to Identification test B under Bupivacaine allow the solution to stand for 10 min. Filter the solution.
Hydrochloride Injection. Acceptance criteria: The filtrate is violet in color and may
Bacterial Endotoxins Test (85) -It contains not more than turn brownish.
1.8 USP Endotoxin Units per mg of bupivacaine hydrochloride. ASSAY
pH (791): between 4.0 and 6.5. • PROCEDURE·1: BUPIVACAINE HYDROCHLORIDE
Other requirements-It meets the requirements under Buffer: 1.94 giL of monobasic potassium phosphate and
Injections and Implanted Drug Products (1). 2.48 giL of dibasic potassium phosphate in water. Adjust,

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 USP 43 OfficialMonographs / Bupivacaine 613

if necessary, with 1 N potassium hydroxide or 1 M amount of Injection equivalent to 25 IJg of epinephrine,

phosphoric acid to a pH of 6.8. and dilute with Mobilephase to volume.
Mobile phase: Acetonitrile and Buffer(65:35). Adjust, if Chromatographic system
necessary, with 1 M phosphoric acid to a pH of 7.7 ± 0.2. (See Chromatography (621), System Suitability.)
Pass the solution through a membrane filter of t-urn or finer Mode: LC
pore size, and degas. Detector: Electrochemical held at a potential of +0.75 V
Internal standard solution: 1.3 mg/mL of dibutyl phthalate Column: 4.6-mm x 25-cm; packing L1
in methanol Flow rate: 1.2 mL/min
Standard solution: 0.5 mg/mL of USP Bupivacaine Injection volume: 20 IJL
Hydrochloride RS, prepared as follows. In a 100-mL System suitability
volumetric flask, dissolve 50 mg of USP Bupivacaine Samples: System suitability solution and Standardsolution
Hydrochloride RS in 10.0 mL of water, using sonication if [NOTE-The relative retention times for epinephrine
necessary. Add 10 mL of Internal standard solution, and and dopamine are about 1.0 and 2, respectively.]
dilute with methanol to volume. Suitability requirements
Sample solution: Nominally 0.5 mg/mL of bupivacaine Resolution: NLT 6.0 between the epinephrine and
hydrochloride, prepared asfollows. In a 1OO-mL volumetric dopamine peaks, System suitability solution
flask, transfer an amount of Injection equivalent to 50 mg Relative standard deviation: NMT 2.0%, Standard
of bupivacaine hydrochloride, add 10.0 mL of Internal solution
standard solution, and dilute with methanol to volume. Analysis
Chromatographic system Samples: Standardsolution and Sample solution
(See Chromatography (621), System Suitability.) Calculate the percentage of the labeled amount of
Mode: LC epinephrine (C9H13N03) in the portion of Injection taken:
Detector: UV 263 nm
Column: 4-mm x 30-cm; packing L1 Result = (r ulr s) x (C sIC u) x (M rdM r2) X 100
Flow rate: 2 mL/min
Injection volume: 20 IJL ru = peak response of epinephrine from the Sample
System suitability solution
Sample: Standardsolution rs =peak response of epinephrine from the Standard
[NOTE-The relative retention times for bupivacaine solution
and dibutyl phthalate are about 1.0 and 1.2, Cs =concentration of USP Epinephrine Bitartrate RS in
respectively.] the Standardsolution (lJg/mL)
Suitability requirements Cu = nominal concentration of epinephrine in the
Resolution: NLT 2.0 between bupivacaine and dibutyl Sample solution (lJg/mL)
phthalate M rl =molecular weight of epinephrine, 183.21
Relative standard deviation: NMT 1.0% for the ratio of M r2 =molecular weight of epinephrine bitartrate,
bupivacaine to the internal standard from three 333.30
replicate Injections
Analysis Acceptance criteria: 90.00/0-115.0%
Samples: Standardsolution and Sample solution
SPECIFIC TESTS
Calculate the percentage of the labeled amount of
• COLOR AND CLARITY
bupivacaine hydrochloride (ClsH2sN20 . H<;:I) in the
Standard solution:' Transfer 2.0 mL of 0.100 N iodine VSto
portion of Injection taken: a 500-mL volumetric flask and dilute with water to volume.
Result = (R ulR s) x (C sIC u) x 100 Sample solution: Injection
Analysis 1: Visually examine a portion of the Sample solution
Ru = peak response ratio of bupivacaine to the internal in a suitable clear glass test tube against a white
standard from the Sample solution background.
Rs = peak response ratio of bupivacaine to the internal Acceptance criteria 1: The Sample solution is not pinkish,
standard from the Standardsolution and it contains no precipitate.
Cs = concentration of USP Bupivacaine Hydrochloride Analysis 2: Perform Analysis 2 if any yellow color is observed
RS, calculated on the anhydrous basis, in the in the Sample solution. Concomitantly determine the
Standardsolution (mg/mL) absorbances of the Sample solution and the Standard
Cu = nominal concentration of bupivacaine solution in 1-cm cells with a suitable spectrophotometer set
hydrochloride in the Sample solution(mg/mL) at 460 nm.
Acceptance criteria 2: The absorbance of the Sample
Acceptance criteria: 93.0%-107.0% solution does not exceed that of the Standardsolution.
• PROCEDURE 2: EPINEPHRINE • BACTERIAL ENDOTOXINS TEST (85): NMT 1.6 USP Endotoxin
Mobile phase: Prepare a mixture of water, methanol, and 2 Units/mg of bupivacaine hydrochloride
M monobasic sodium phosphate (900:50:50), containing • pH (791): 3.3-5.5
40 mg/L of edetate disodium, 0.4 mL/L of phosphoric acid, • OTHER REQUIREMENTS: It meets the requirements in
and 0.4 gIL of sodium l-octanesulfonate. Make Injections and Implanted Drug Products (1).
adjustments, if necessary, to obtain a retention time of NLT ADDITIONAL REQUIREMENTS
11 min for the epinephrine peak. • PACKAGING AND STORAGE: Preserve in single-dose or
System suitability solution: 2 IJg/mL each of epinephrine multiple-dose containers, preferably of Type I glass,
bitartrate and dopamine hydrochloride in Mobilephase protected from light. Injection labeled to contain 0.5% or
Standard solution: 2 IJg/mL of USP Epinephrine Bitartrate less of bupivacaine hydrochloride may be packaged in
RS in Mobile phase 50-mL multiple-dose containers.
Sample solution: Nominally 1 IJg/mL of epinephrine,
prepared asfollows. In a 25-mL volumetric flask, transfer an

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614 Bupivacaine / Official Monographs USP 43

• LABELING: The label indicates that Injection is not to be used Table 1 (continued)
if its color is pinkish or darker than slightly yellow, or if it Time Solution A Solution B
. contains a precipitate. (min) (%) (%)
• USP REFERENCE STANDARDS (11)
20 39 61
USP Bupivacaine Hydrochloride RS
USP Epinephrine Bitartrate RS
Diluent: Methanol and water (80:20)
Standard solution: 2.0 mg/mL of USP Buprenorphine
Hydrochloride RS in Diluent
Sample solution: 2.0 mg/mL of Buprenorphine
Buprenorphine Hydrochloride Hydrochloride in Diluent
Chrornatoqraphlc system
(See Chromatography (621)/ System Suitability.)
Mode: LC
CH3 • HCI Detector: UV 240 nm
Column: 4.6-mm x 10-cm; 3.5-l..Im packing l1
Column temperature: 30°
Flow rate: 1.3 ml/min
C29H41N04' HCI 504.11 Injection volume: 5 I..IL
6,14-Ethenomorphinan-7-methanol, 17-(cyclopropylmethyl)- System suitability
0.-(1,1 -dimethylethyl)-4,5-epoxy-18,19-dihydro-3-hydroxy- Sample: Standard solution
6-methoxy-a-methyl-, hydrochloride, [50.,70.(5)]-; Suitability requirements
21-Cyclopropyl-7a-[(S)-l-hydroxy-1,2,2-trimethylpropyl]- Tailing factor: NMT 2.0
6,14-endo-ethano-6,7/8, 14-tetrahydrooripavine Relative standard deviation: NMT 0.73%
hydrochloride [53152-21 -9]. Analysis
DEFINITION Samples: Standard solution and Sample solution
Buprenorphine Hydrochloride contains NLT 98.0% and NMT Calculate the percentage of buprenorphine hydrochloride
102.0% of buprenorphine hydrochloride (C29H41NO,l . HCI), (C29H41N04. HCI) in the portion of Buprenorphine
calculated on the anhydrous basis. Hydrochloride taken:

IDENTIFICATION Result = (r ulr s) x (C siC u) x 100

ru = peak response of buprenorphine from the

Sample solution
·A rs =peak response of buprenorphine from the
i~fJ .. J _ Standard solution
• B. The retention time of the buprenorphine peak of the Cs = concentration of USP Buprenorphine
Sample solution corresponds to that of the Standard Hydrochloride RS in the Standard solution
solution, as obtained in the Assay. (mg/mL)
• C. IDENTIFICATION TESTS-GENERAL (191), Chemical Cu =concentration of Buprenorphine Hydrochloride
Identification Tests, Chloride in the Sample solution(mg/mL)
Sample stock solution: 50 mg/mL of Buprenorphine
Hydrochloride in methanol Acceptance criteria: 98.00/0-102.0% on the anhydrous
Sample solution: 10 mg/ml of Buprenorphine . basis
Hydrochloride in carbon dioxide-free water from the
IMPURITIES
Sample stocksolution • RESIDUE ON IGNITION (281): NMT 0.1%
Analysis: Use 3 mL of the Sample solution.
• ORGANIC IMPURITIES
Acceptance criteria: Meetsthe requirements Buffer, Solution A, Solution B, and Mobile phase: Prepare
ASSAY as directed in the Assay.
• PROCEDURE System suitability solution: 5.0 mg/mL of USP
Buffer: 5.44 gil of monobasic potassium phosphate Buprenorphine System Suitability Mixture RS in methanol
prepared as follows. Initially dissolve in 90% volume of Standard solution: 0.005 mg/mL of USP Buprenorphine
water, and adjust with 5% (v/v) of phosphoric acid to a pH Hydrochloride RS and 0.01 mg/mL of USP Buprenorphine
of 4.5. Dilute with water to volume. Related Compound A RS in methanol
Solution A: Acetonitrile and Buffer (10:90) Sample solution: 5.0 mg/ml of Buprenorphine
Solution B: Acetonitrile Hydrochloride in methanol _
Mobile phase: See Table 1. Returnto the original conditions Chromatographic system: Proceedasdirected in the Assay
and re-equilibrate the system. except for the Column.
Column: 4.6-mm x 5.0-cm; 3.5-l..Im packing L1
Table 1 System suitability
Time Solution A Solution B
Samples: System suitability solution and Standardsolution
(min) (%) (%) Suitability requirements
Resolution: NLT 1.5 between buprenorphine and
0 89 11 ethenobuprenorphine, System suitability solution
2 89 11 Relative standard deviation: NMT 5% for
buprenorphine and buprenorphine related compound
12 64 36
A, Standard solution
15 41 59 Analysis
Samples: Standard solution and Sample solution

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 USP 43 Official Monographs / Buprenorphine 615

Calculate the percentage of buprenorphine related Acceptance criteria: NMT 0.2 mL of 0.02 N sodium
compound A in the portion of Buprenorphine hydroxide or 0.02 N hydrochloric acid is required to change
Hydrochloride taken: the color of the indicator.
• WATER DETERMINATION (921), Method I: NMT 1.0%
Result = (r vir s) x (C siC v) x 100
ADDITIONAL REQUIREMENTS
ru = peak response of buprenorphine related • PACKAGING AND STORAGE: Preserve in tight, light-resistant
compound A from the Sample solution containers.
rs =peak response of buprenorphine related • USP REFERENCE STANDARDS (11)
compound A from the Standardsolution USP Buprenorphine Hydrochloride RS
Cs =concentration of USP Buprenorphine Related USP Buprenorphine Related Compound A RS
Compound A RS in the Standardsolution (S)-2-[17-(But-3-en-1-yl)-4,5a-epoxy-3-hydroxy-6-
(mg/mL) methoxy-6a,14-ethanomorphinan-7a-yl]-3,3-
Cu = concentration of Buprenorphine Hydrochloride dimethylbutan-2-ol.
in the Sample solution (mg/mL) Cz9H 4,N04 467.65
USP Buprenorphine System Suitability Mixture RS
Calculate the percentage of each other individual impurity It contains buprenorphine and about 0.5% of
in the portion of Buprenorphine Hydrochloride taken: ethenobuprenorphine. The chemical information for
ethenobuprenorphine is asfollows:
Result = (r vir s) x (C siC v) x (1 IF) x 100 (5)- 2-[1 7-(Cyclopropylmethyl)-4,5a-epoxy- 3-hydroxy-6-
methoxy-6a,14-ethenomorphinan-7a-yl]-3,3-
rv = peak response of each other individual impurity dimethylbutan-2-ol.
from the Sample solution CZ9H39N04 465.63
rs = peak response of buprenorphine from the
Standard solution
Cs = concentration of USP Buprenorphine
Hydrochloride RS in the Standard solution
(mg/mL) Buprenorphine Compounded Buccal
Cu = concentration of Buprenorphine Hydrochloride
in the Sample solution(mg/mL)
Solution, Veterinary
F = relative response factor (see Table 2) DEFINITION
Buprenorphine Compounded Buccal Solution, Veterinary,
Acceptance criteria: See Table 2. The reporting threshold is
0.05%. I
contains NLT 90.0% and NMT 110.0% of the labeled
amount of buprenorphine (Cz9H 4,N04) .
Table 2 Prepare Buprenorphine Compounded Buccal Solution,
Veterinary 3 mg/mL as follows (see Pharmaceutical
Relative Relative Acceptance Compounding-Nonsterile Preparations (795».
Retention Response Criteria,
Name Time Factor NMT (0/0)
Buprenorphine (as hydrochloride) 30 mg (32.4 mg)
Buprenorphine 1.0 - -
Dextrose 500 mg
Ethenobuprenorphtne- 1.1 1.0 0.10
Sodium Citrate (anhydrous) 20mg
Buprenorphine related com-
pound A 1.4
- 0.20 Citric Acid Monohydrate 25 mg
Buprenorphine 2,2'-dimerb 1.8 3.3 0.10 Purified Water, a sufficient quantity to make 10ml
Any other individual unspeci-
fied impurity - 1.0 0.10 Dissolvethe Dextrose, Sodium CitrateAnhydrous, and CitricAcid
Total impurities - - 0.65 Monohydrate in 5 mL of Purified Waterin a suitable calibrated
container. Add the Buprenorphine hydrochloride powder into
a(5)-2-[17-(Cyclo'propylmethyl)-4,5a-epoxy-3-hydroxy-6-methoxy-6a,14- the mixture and add sufficient Purified Waterto bring to final
ethenomorphinan-7a-yl]- 3,3-dimethylbutan-2 -01. volume, and mix well.
b 2,2'-Bi{17-(cyclopropylmethyl)-4,5a-epoxy-3-hydroxy-7a-[(S)-2-hydroxy-3,3-
dimethylbutan-2-yl]-6-methoxy-6n,14-ethanomorphinan). ASSAY
• PROCEDURE
SPECIFIC TESTS Mobile phase: Acetonitrile and 10 mM ammonium acetate
• OPTICAL ROTATION (781 S), Procedures, Specific Rotation (80:20)
Sample solution: 20 mg/mL in methanol Standard solution: 0.3 mg/mL of buprenorphine
Acceptance criteria: -92 0 to -98 0 prepared from USP Buprenorphine Hydrochloride RS in
• ACIDITY OR ALKALINITY methanol
Sample stock solution: 50 mg/mL of Buprenorphine Sample solution: Transfer 1 mL of Buccal Solution,
Hydrochloride in methanol Veterinary into a 1O-mL volumetric flask, dilute with
Sample solution: 10 mg/mL of Buprenorphlne methanol to volume, and mix well.
Hydrochloride in carbon dioxide-free water from Sample Chromatographic system
stock solution (See Chromatography (621), System Suitability.)
Analysis: Add 0.05 mL of methyl red TS2 to 10 mL of Sample Mode: LC
solution and titrate with 0.02 N sodium hydroxide or 0.02 Detector: UV 280 nm
N hydrochloric acid. Column: 2.1-mrn x 5-cm; 5-~m packing L7
Column temperature: 40 0
Flow rate: 0.25 mL/min

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 616 Buprenorphine / OfficialMonographs USP 43

Injection volume: 10 J,lL Buffer: 9 mM of dibasic ammonium phosphate in water.

System suitability Adjust with a solution of phosphoric acid and water (1:1)
Sample: Standardsolution to a pH of 6.2.
[NoTE-The retention time for buprenorphine is about Solution A: Acetonitrile, methanol, and Buffer (7:3:90)
5.8 min.] . Solution B: Acetonitrile, methanol, and Buffer (56:24:20)
Suitability requirements Mobile phase: See Table 1.
Tailing factor: NMT 2.0
Relative standard deviation: NMT 2.0% for replicate Table 1
injections Time Solution A Solution B
Analysis (min) (0/0) (0/0)
Samples: Standardsolution and Sample solution
0 99 1
Calculate the percentage of the labeled amount of
buprenorphine (C29H41N04) in the portion of Buccal 30 1 99
Solution, Veterinary taken: 45 1 99
Result = (rulrs) x (CsICu) x 100 45.1 99 1
55 99 1
= peak response of buprenorphine from the
Sample solution .
= peak response of buprenorphine from the Solution C: Phosphoric acid and water (1:1000)
Standardsolution Diluent: Acetonitrile, methanol, and Solution C (7:3:90)
= concentration of buprenorphine in the Standard Standard solution: 0.57 mg/mL of USP Buprenorphine
solution (mg/mL) Hydrochloride RS and 0.13 mg/mL of USP Naloxone RS in
Cu = nominal concentration of buprenorphine in the Diluent
Sample solution (mg/mL) Sample solution: Nominally 0.52 mg/mL of buprenorphine
and 0.13 mg/mL of naloxone prepared as follows. Transfer
Acceptance criteria: 90.0%-110.0% NLT 13 Tablets to a suitable volumetric flask, and add about
70% of the final volume of Diluent. Sonicatefor 15 min with
SPECIFIC TESTS occasional swirling and shake for 15 min. Dilute with
• pH (791): 3.5-4.5 Diluent to volume. Pass a portion through a suitable filter of
ADDITIONAL REQUIREMENTS 0.45-J,lm pore size. Discard the first 5 mL of filtrate.
• PACKAGING AND STORAGE: Packagein tight, light-resistant Chromatographic system
containers. Store at 2°_8°. . (See Chromatography (621), System Suitability.)
• LABELING: Label it to indicate that it is for veterinary use Mode: LC
only. Label to indicate that it is for buccal administration, Detector: UV 280 nm. For Identification B, usea diode array
and to state the Beyond-Use Date. detector in the range of 210-400 nm.
• BEYOND-USE DATE: NMT 90 days after the date on which it Column: 4.6-mm x 25-cm; 5-J,lm packing L11
was compounded when stored at 2°_8° Column temperature: 60°
• USP REFERENCE STANDARDS (11) Flow rate: 0.8 ITlL/min
USP Buprenorphine Hydrochloride RS Injection volume: 100 J,lL
System suitability
Sample: Standardsolution
Suitability requirements
Tailing factor: NMT 2.0 for both buprenorphine and
Buprenorphine and Naloxone naloxone
Relative standard deviation: NMT 2.0% for both
Sublingual Tablets buprenorphine and naloxone
DEFINITION Analysis
Buprenorphine and Naloxone Sublingual Tablets contain Samples: Standardsolution and Sample solution
amounts.of buprenorphine hydrochloride and naloxone Calculate the percentage of the labeled amount of
hydrochloride equivalent to NLT 94.0% and NMT 106.0% buprenorphine (C29H41N04) in the portion of Tablets
of the labeled amount of buprenorphine (C29H41N04) and taken:
naloxone (C19H21N04).
Result = (r ulr s) x (C siC u) x (M,dM '2) x 100
IDENTIFICATION
• A. The retention times of the buprenorphine and naloxone =peak response of buprenorphine from the
peaks of the Sample solution correspond to those of the Sample solution
Standardsolution, as obtained in the Assay. = peak response of buprenorphine from the
• B. The UV absorption spectra of the buprenorphine and Standardsolution
naloxone peaks of the Sample solution and those of the =concentration of USP Buprenorphine
Standardsolution exhibit maxima and minima at the same Hydrochloride RS in the Standardsolution
wavelengths, as obtained in the Assay. (mg/mL) .
= nominal concentration of buprenorphine in the
ASSAY Sample solution (mg/mL)
• PROCEDURE M'I =molecular weight of buprenorphine, 467.65
[NOTE-It is suggested to protect all solutions M ,2 =molecular weight of buprenorphine
containing buprenorphine and naloxone from hydrochloride, 504.11
light.] .

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USP 43 OfficialMonographs / Buprenorphine 617

Calculate the percentage of the labeled amount of Calculate the percentage of the labeled amount of
naloxone (C19H21N04) in the portion of Tablets taken: buprenorphine (C29H41N0 4) dissolved:

Result = (r vir s) x (C siC v) x 100 Result = (r vir s) x C s x V x (M r11 M (2) x (1IL) x 100
ru = peak response of naloxone from the Sample ru =peak response of buprenorphine from the
solution Sample solution
rs =peak response of naloxone from the Standard rs = peak response of buprenorphine from the
solution Standard solution
Cs = concentration of USP Naloxone RS in the Cs = concentration of USP Buprenorphine
Standard solution(mg/mL) Hydrochloride RS in the Standard solution
Cu =nominal concentration of naloxone in the Sample (mg/mL)
solution (mg/mL) V = volume of Medium, 500 mL
M'I = molecular weight of buprenorphine, 467.65
Acceptance criteria: 94.0%-106.0% of the labeled amount M r2 = molecular weight of buprenorphine
of buprenorphine (C29H41N04) and naloxone (C19H21N04) hydrochloride, 504.11
PERFORMANCE TESTS L = label claim of buprenorphine (mg/Tablet)
• DISSOLUTION (711)
Calculate the percentage of the labeled amount of
Medium: Water (deaerated for 5 min); 500 mL
naloxone (C19H21N04) dissolved:
Apparatus 1: 100 rpm
Time: 10 min
Result =(r vir s) xes x V x (1I L) x 100
Buffer: 0.018 M monobasic potassium phosphate in water
prepared asfollows. Dissolve2,4 g of monobasic potassium ru = peak response of naloxone from the Sample
phosphate and 0.5 g of sodium hydroxide in each liter of solution
water. Adjust with phosphoric acid to a pH of 6.8. rs =peak response of naloxone from the Standard
Solution A: Acetonitrile, methanol, and Buffer (40:20:40) solution
Solution B: Acetonitrile and Buffer (78:22) .C s = concentration of USP Naloxone RS in the
Mobile phase: See Table 2. Standard solution(mg/mL)
Table 2
V =volume of Medium, 500 mL
L =label claim of naloxone (mg/Tablet)
Time Solution A Solution B
(min) (%) (%) Tolerances: NLT 80% (Q) of the labeled amount of
0 100 0 buprenorphine (C29H41N04) and naloxone (C19H21N04) is
dissolved.
2.0 100 0
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
3.0 0 . 100 requirements
6.0 0 100 IMPURITIES
• ORGANIC IMPURITIES
6.1 100 0
[NOTE-It is suggested to protect all solutions
8.0 100 0 containing buprenorphine and naloxone from light.]
Buffer, Solution A, Solution B, Mobile phase, Solution C,
Diluent: Methanol and water (50:50) Diluent, Sample solution, and Chromatographic system:
Standard solution: 0.01 mg/mL of USP Buprenorphine Proceed as directed in the Assay.
Hydrochloride RS and 0.0025 mg/mL of USP Naloxone RS
Standard solution: 0.0015 mg/mL of USP Buprenorphine
in Diluent. Sonicate if necessary. Pass a portion through a
suitable filter of 0,45-lJm pore size. Discard the first 4 mL of Hydrochloride RS and 0.0004 mg/mL of USP Naloxone RS
filtrate. in Diluent
Sample solution: Pass a portion of the solution under test System suitability
through a suitable filter of 0,45-lJm pore size. Sample: Standard solution
Chromatographic system Suitability requirements
(See Chromatography (621), System Suitability.) Relative standard deviation: NMT 5% for
Mode: LC buprenorphine and naloxone
Detector: UV 230 nm Analysis
Column: 4.6-mm x 5-cm; 5-lJm packing L7 Samples: Sample solution and Standard solution
Column temperature: 25° Identify the buprenorphine degradation products using the
Flow rate: 1.0 mL/min relative retention times given in Table 3.
Injection volume: 40 IJL Calculate the percentage of each buprenorphine related
System suitability degradation product in the portion of Tablets taken:
Sample: Standard solution
Suitability requirements . Result = (r vir s) x (C siC v) x (M ,dM (2) x 100
Tailing factor: NMT 2.0 for both buprenorphine and
naloxone ru =peak response of each individual buprenorphine
Relative standard deviation: NMT 2.0% for both related degradation product from the Sample
buprenorphine and naloxone solution
Analysis rs =peak response of buprenorphine from the
Samples: Standard solution and Sample solution Standard solution

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618 Buprenorphine / Official Monographs USP 43

Cs = concentration of USP Buprenorphine Table 3 (continued)

Hydrochloride RS in the Standard solution Relative Acceptance
(mg/mL) Retention Criteria,
Cu =nominal concentration of buprenorphine in the Name Time NMT (%)
Sample solution(mg/mL) Total degradation products - 3.0
M rl = molecular weight of buprenorphine, 467.65
M r2 = molecular weight of buprenorphine a Quantified relative to naloxone.
hydrochloride, 504.11 b (S)-2-(4,5a-Epoxy-3-hydroxy-6-methoxy-6a, 14-ethanomorphinan-7a-yl)-3, 3-
dimethylbutan-2-ol.
C These are process impurities and are excluded from the total degradation
Identify the naloxone degradation products using the
products.
relative retention times given in Table 3.
d 4,5a-Epoxy-7 a-[(S)-2-hydroxy-3,3-dimethylbutan-2-yl]-3,6-dimethoxy-6a,
Calculate the percentage of each naloxone related 14-ethanomorphinan-17-carbonitrile.
degradation product and any other degradation product e (S)-2-[17-(Cyclopropylmethyl)-4,5a-epoxy-3,6-dihydroxy-6a, 14-
in the portion of Tablets taken: ethanomorphinan-7a-yl]-3,3-dimethylbutan-2-ol.
f Quantified relative to buprenorphine.
Result = (r vir s) x (C siC v) x 100 9 (S)-2-[17-(Cyclopropylmethyl)-4,5a-epoxy-3-hydroxy-6-methoxy-6a,14-
ethanomorphinan-7p-yl]-3,3-dimethylbutan-2-ol.
ru =peak response of each naloxone related h (S)-2-[17-(But-3-en-1-yl)-4,5a-epoxy-3-hydroxy-6-methoxy-6a,14-
ethanomorphinan-7a-yl]-3,3-dimethylbutan-2-ol.
degradation product or any other degradation
i (5)-2-[17 -(Cyclopropylmethyl)-4,5a-epoxy-3,6-dimethoxy-6a,14-
product from the Sample solution ethanomorphinan-7a-yl]-3,3-dimethylbutan-2-ol.
rs =peak response of naloxone from. the Standard
solution ADDITIONAL REQUIREMENTS
Cs = concentration of USP Naloxone RS in the • PACKAGING AND STORAGE: Preserve in tight containers, and
Standard solution(mg/mL) store at controlled room temperature.
Cu = nominal concentration of naloxone in the Sample • USP REFERENCE STANDARDS (11)
solution (mg/mL) USP Buprenorphine Hydrochloride RS
USP Naloxone RS
Acceptance criteria: See Table 3. Disregard any peaks
below 0.05%.

Table 3
Relative Acceptance Bupropion Hydrochloride
Retention Criteria,
Na~e Time NMT (0/0)
Naloxone degradation product 1a 0.30 0.5
Naloxone degradation product 2a 0.54 0.5
Dealkyl buprenorphinev c 0.55 - C13H1SCINO· HCI 276.20
1-Propanone, 1-(3-chlorophenyl)-2-[(1,1-dimethylethyl)
Naloxone 0.61 - amino]-, hydrochloride, (±)-;
Naloxone degradation product 3a 0.67 0.5 (±)-2-(tert-Butylamino)-3' -chloropropiophenone
hydrochloride [31677-93-7].
Buprenorphine nltrllevd 0.90 -
DEFINITION
6-0-Desmethylbuprenorphinec, e 0.91 - Bupropion Hydrochloride contains NLT 98.0% and NMT
Buprenorphine degradation 102.0% of bupropion hydrochloride (C13H 1SCINO . HCI),
product 1f 0.95 0.3 calculated on the anhydrous basis.
Buprenorphine 7-(5)-epimer<- 9 0.99 - IDENTIFICATION
Buprenorphine 1.00 -
Buprenorphine butenyl analog e, h 1.03 -
3-0-Methylbuprenorphinec, I 1.16 -
Any unspecified degradation product" - 0.3
peak of the Sample
solution corresponds to that of Standard solution, as
obtained in the Assay.

IDENTIFICATION TESTS-GENERAL (191), Chloride

• C.
Sample solution: 1 mg/mL of Bupropion Hydr~;hloride
A~~~e~~~~~Z(~~i~~~i~.; ...~i~~~.<~.~~\)~~quirements~Qf
tgs~..: ·«CN<1-MaY-2oj8)~.;l\J.(ERR1"Ati9-201'8)
ASSAY
• PROCEDURE
Diluent: Methanol and water (50:50)
Buffer: 3.4 giL of monobasic potassium phosphate in water.
Adjust with 1 N sodium hydroxide to a pH of 7.0.

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USP 43 OfficialMonographs / Bupropion 619

Mobile phase: Methanol, tetrahydrofuran, and Buffer mg/mL of USP Bupropion Hydrochloride Related
(39:11 :50) Compound FRS, and 0.012 mg/mL of USP
Standard solution: 1 mg/mL of USP Bupropion 3-Chlorobenzoic Acid RS in methanol
Hydrochloride RS and 2 IJg/mL each of USP Bupropion System suitability solution: 0.002 mg/mL of bupropion
Hydrochloride Related Compound A RS and USP Bupropion hydrochloride related compound C, 0.002 mg/mL of
Hydrochloride Related Compound B RS in Diluent bupropion hydrochloride related compound F, and 0.0012
Sample solution: 1 mg/mL of Bupropion Hydrochloride in mg/mL of 3-chlorobenzoic acid from System SUitability stock
Diluent solution in Diluent
Chromatographic system Standard stock solution: 0.06 mg/mL of USP
(See Chromatography (621), System Suitability.) 3-Chlorobenzoic Acid RSin methanol
Mode: LC Standard solution: 1.2 IJg/mL of USP 3-Chlorobenzoic Acid
Detector: UV 250 nm RS from Standard stock solution in Diluent
Column: 3.9-mm x 15-cm; 5-lJm packing L7 Sample solution: 600 IJg/mL of Bupropion Hydrochloride
Flow rate: 1.1 mL/min in Diluent
Injection volume: 20 IJL Chromatographic system
System suitability (See Chromatography (621), System Suitability.)
Sample: Standard solution Mode: LC
[NoTE-See Table 3 for the relative retention times.] Detector: UV 226 nm
Suitability requirements Column: 4.6-mm x 10-cm; 3.5-lJm packing L1
Resolution: NLT 1.3 between bupropion hydrochloride Column temperature: 40°
related compound A and bupropion; NLT 1.3 between Flow rate: 1.5 mL/min
bupropion and bupropion hydrochloride related Injection volume: 5 IJL
compound B System suitability
Relative standard deviation: NMT 2.0% for bupropion Samples: System suitability solution and Standard solution
Analysis [NOTE-See Table 2 for the relative retention times.]
Samples: Standard solution and Sample solution Suitability requirements
Calculate the percentage of bupropion hydrochloride Resolution: NLT 1.3 between bupropion hydrochloride
(C13H1sCINO· HCI) in the portion of Bupropion related compound F and bupropion hydrochloride
Hydrochloride taken: related compound C, System suitabilitysolution; NLT 1.5
between bupropion hydrochloride related compound C
Result =(rulrs) x (CsICu) x 100 and 3-chlorobenzoic acid, System suitability solution
Relative standard deviation: NMT 5.0%, Standard
ru = peak response from the Sample solution solution
rs =peak response from the Standard solution Analysis
Cs = concentration of USP Bupropion Hydrochloride Samples: Standard solution and Sample solution
RS in the Standard solution(mg/mL) Calculate the percentage of 3-chlorobenzoic acid in the
Cu =concentration of Bupropion Hydrochloride in the portion of Bupropion Hydrochloride taken:
Sample solution (mg/mL)
Result = (rulrs) x (CsICu) x 100
Acceptance criteria: 98.00/0-102.0% on the anhydrous
basis tu = peak response of 3-chlorobenzoic acid from the
Sample solution
IMPURITIES ts = peak response of 3-chlorobenzoic acid from the
• LIMIT OF 3-(HLOROBENZOIC ACID . Standard solution
Protect all analytical solutions from light and use within one Cs = concentration of USP 3-Chlorobenzoic Acid RS in
day. the Standard solution (lJg/mL)
Diluent: Methanol and 0.001 N hydrochloric acid (20:80) Cu = concentration of Bupropion Hydrochloride in the
Solution A: Acetonitrile and water (10:90). Add 0.4 mL of Sample solution (lJg/mL)
trifluoroacetic acid per L of the mixture.
Solution B: Acetonitrile and water (95:5). Add 0.3 mL of Acceptance criteria: See Table 2.
trifluoroacetic acid per L of the mixture.
Mobile phase: See Table 1. Table 2
Relative Acceptance
Table 1 Retention Criteria,
Time Solution A Solution B Name Time NMT(%)
(min) (%) (%)
Bupropion 1.0 -
0 90 10
Bupropion hydrochloride
related compound Fa 1.71 -
3.4 87 13
Bupropion hydrochloride
10.0 15 85
related compound C· 1.75 -
10.1 0 100
3-Chlorobenzoic acid 1.80 0.2
13.0 0 100
a Included for system suitability purposes only.
13.2 90 10
19.0 90 10 • ORGANIC IMPURITIES
Diluent, Buffer, Mobile phase, Standard solution, Sample
System suitability stock solution: 0.02 mg/mL of USP solution, and Chromatographic system: Proceed as
Bupropion Hydrochloride Related Compound C RS, 0.02 directed in the Assay.

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620 Bupropion / Official Monographs USP 43

System suitability Table 3 (continued)

Sample: Standard solution Relative Relative Acceptance.
[NoTE-See Table 3 for the relative retention times.] Retention Response Criteria,
Suitability requirements Name Time Factor NMT (%)
Resolution: NLT 1.3 between bupropion hydrochloride Total lrnpurlttes" - - 1.0
related compound A and bupropion; NLT 1.3 between
bupropion and bupropion hydrochloride related a 2-(tert-Butylamino)-1-phenylpropan-l-one; also known as 2-(tert-butylamino)
compound B ~ropiophenone.
Relative standard deviation: NMT 2.0% for bupropion; 1.(3-Chlorophenyl)propane.l,2-dione; alsoknownas 1-(3-chlorophenyl)-1 ,2-
NMT 5.0% for bupropion hydrochloride related propanedione.
compound B c2-(tert-Butylamino)-1-(2-chlorophenyl)propan-l-one; also known as 2-(tert-
butylamino)-2'-chloropropiophenone.
Analysis d 1-(3-Chlorophenyl)propan-l-one; also knownas 3'.chloropropiophenone.
Samples: Standard solution and Sample solution e 2-Bromo.l-(3-chlorophenyl)propan.1-one; also knownas 2-bromo·3'-
Calculate the percentage of each impurity in the portion of chloropropiophenone.
Bupropion Hydrochloride taken: f 2-(tert-Butylamino)-1-(3,4-dichlorophenyl)propan-l-one; also known as 2-
(tert-butylamino)- 3',4'-dichloropropiophenone.
Result = (rulrs) x (CsICu) x (liP) x 100 9 2-(tert-Butylamino)-1-(3,5-dichlorophenyl)propan-l-one; also knownas 2-
(tert-butylamino)- 3',5'.dichloropropiophenone.
t» = peak response for each impurity from the hSum of allimpurities found in the tests for Limit of 3-Chlorobenzoic Acid and
Organic Impurities.
Sample solution
ts = peak response for bupropion from the Standard SPECIFIC TESTS
solution
• WATER DETERMINATION, Method I (921): NMT 0.5%
C, = concentration of USP Bupropion Hydrochloride
RS in the Standard solution (mg/mL) ADDITIONAL REQUIREMENTS
Cu = concentration of Bupropion Hydrochloride in the • PACKAGING AND STORAGE: Preserve in well-closed,
Sample solution (mg/mL) light-resistant containers. Store at room temperature.
F = relative response factor for each impurity relative • USP REFERENCE STANDARDS (11)
to bupropion (see Table 3) USP Bupropion Hydrochloride RS
USP Bupropion Hydrochloride Related Compound A RS
Acceptance criteria: See Table 3. 2-( tert-Butylamino)-4'-ch loropropiophenone
hydrochloride.
Table 3 C13H1SCINO . HCI 276.20
Relative Relative Acceptance USP Bupropion Hydrochloride Related Compound B RS
Retention Response Criteria, 2-(tert-Butylamino)-3'-bromopropiophenone
Name Time Fador NMT(%) hydrochloride.
Deschloro buproplon- 0.38 1.5 0.5 C13H1SBrNO· HCI 320.66
USPBupropion Hydrochloride Related Compound C RS
Bupropion dione
derivative" 0.58 1.0 0.2 1-(3-Chlorophenyl)-2-hydroxypropan-1-one.
CgHgOzCI 184.62
o-Buproplon" 0.71 0.45 0.1 'USP Bupropion Hydrochloride Related Compound F RS
Chloropropiophenone'' 0.78 1.2- 0.1 1-(3-Chlorophenyl)-1-hydroxypropan-2-one.
CgHgOzCI 184.62
Bupropion USP 3-Chlorobenzoic Acid RS
hydrochloride related com-
pound A 0.92 1.4 0.2 3-Chlorobenzoic acid.
C7H sCIOz 156.57
Bupropion 1.0 - -
Bupropion
hydrochloride related com-
pound B 1.14 0.81 0.2
Brornochlorcproplo- Bupropion Hydrochloride Tablets
phenones 1.63 0.88 0.1
DEFINITION
4-Chlorobupropion f 2.30 1.1 0.2 Bupropion Hydrochloride Tablets contain NLT 90.0% and
5-Chlorobupropion9 2.74 0.69 0.2 NMT 110.0% of the labeled amount of bupropion
hydrochloride (C13H1sCINO . HCI).
Any individual impurity - 1.0 0.1
IDENTIFICATION

Sample: Crush 1 Tablet using a mortar and pestle. Prepare

an approximate 1% (w/w) dispersion of the sample in
potassium bromide.
Acceptance criteria: The Sample shows strong bands at
about 1690, 1560, and 1240 crrr" and a weaker band at
about 740 crrr', similar to the reference preparation.

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 USP 43 Official Monographs / Bupropion 621

• B. The retention time of the major peak of the Sample ADDITIONAL REQUIREMENTS
solution corresponds to that of the Standardsolution, as • PACKAGING AND STORAGE: Preserve in tight containers.
obtained in the Assay. • USP REFERENCE STANDARDS (11)
USP Bupropion Hydrochloride RS
ASSAY
• PROCEDURE
Buffer: 6.8 giL of monobasic potassium phosphate and
1.164 giL of sodium hydroxide in water
Mobile phase: Methanol and Buffer(65:35) Bupropion Hydrochloride
Diluent: Methanol and water (65:35)
Standard solution: 0.6 mg/mL of USP Bupropion Extended-Release Tablets
Hydrochloride RS in Diluent
Sample stock solution: Nominally 3.0 mg/ml of bupropion DEFINITION
hydrochloride in Diluent prepared as follows. Transfer an Bupropion Hydrochloride Extended-Release Tablets contain
appropriate number of Tablets to a suitable volumetric NLT 90.0% and NMT 110.0% of the labeled amount of
flask. Add 50% of the flask volume of Diluent, and shake by bupropion hydrochloride (C13H18CINO· HCI).
mechanical means until the Tablets have disintegrated (30- IDENTIFICATION
60 min). Sonicate for 5 min, dilute with Diluent to volume,
and mix. Allow to stand for at least 30 min. Use the
supernatant.
Sample solution: Nominally 0.6 mg/mL of bupropion • A.
hydrochloride from the Sample stock solution in Diluent ~1ig
Chromatographic system Sample: Crush 1 Tablet using a mortar and pestle. Prepare
(See Chromatography (621), System Suitability.) an approximate 1% (w/w) dispersion of the sample in
Mode: LC potassium bromide.
Detector: UV 224 nm Acceptance criteria: The Sample shows strong bands at
Column: 4.6-mm x 15-cm; s-um base-deactivated about 1690, 1560, and 1240 cm- 1 and a weaker band at
packing Ll about 740 crrr', similar to the reference preparation.
Flow rate: 1.2 mL/min • B. The retention time of the major peak of Sample solution
Injection volume: 10 I.JL A or Sample solution B corresponds to that of the Standard
System suitability solution, as obtained in the Assay.
Sample: Standardsolution
Suitability requirements ASSAY
Tailing factor: 'NMT 2.5
Relative standard deviation: NMT 2.0%
Analysis
Calculate the percentage of the labeled amount of • PROCEDURE
bupropion hydrochloride (C13H18CINO . HCI) in the Diluent 1: Methanol and 0.001 N hydrochloric acid
portion of Tablets taken: (20:80)
Solution A: Acetonitrile, trifluoroacetic acid, and water (10:
Result = (rulrs) x (CsICu) x 100 0.04: 90)
Solution B: Acetonitrile, trifluoroacetic acid, and water (95:
= peak area from the Sample solution 0.03: 5)
= peak area from the Standardsolution . Mobile phase: See Table 1.
= concentration of USP Bupropion Hydrochloride
Table 1
RS in the Standardsolution (mg/mL)
= nominal concentration of bupropion Time Solution A Solution B
(min) (%) (%)
hydrochloride in the Sample solution (mg/mL)
0 90 10
Acceptance criteria: 90.0%-110.0%
3.4 87 13
PERFORMANCE TESTS 10.0 15 85
• DISSOLUTION (711)
Medium: Water; 900 mL 10.1 0 100
Apparatus 2: 50 rpm 13.0 0 100
Time: 45 min
Standard solution: USP Bupropion Hydrochloride RS at a 13.2 90 10
known concentration in 0.1 N hydrochloric acid 19.0 90 10
Sample solution: Pass a portion of the solution under test
through a suitable filter, and dilute with 0.1 N hydrochloric
acid, if necessary. System suitability stock solution: 0.02 mg/ml of USP
Instrumental conditions Bupropion Hydrochloride Related Compound C RS and 0.2
Mode: UV mg/mL of USP Bupropion Hydrochloride Related
Analytical wavelength: 252 nm Compound F RS in methanol
Analysis System suitability solution: 0.002 mg/mL of bupropion
Samples: Standardsolution and Sample solution hydrochloride related compound C and 0.02 mg/mL of
Tolerances: NlT 80% (Q) of the labeled amount of bupropion hydrochloride related compound F from the
bupropion hydrochloride (C13H18CINO . HCI) is dissolved. System suitability stock solution in Diluent 1
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Standard solution: 0.6 mg/mL of USP Bupropion
requirements Hydrochloride RS in Diluent 1

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622 Bupropion / Official Monographs USP43

Sample stock solution A: Transfer a number of Tablets, PERFORMANCE TESTS

intact or crushed, to a suitable homogenizer vessel
containing sufficient methanol to obtain a concentration of
3.0 mg/mL of bupropion hydrochloride. Immediately
homogenize the sample for 30 s at 20,000 rpm. Allow • DISSOLUTION (711)
extraction for 3 min, and follow by two additional 1O-s For products labeled for dosing every 12 h
pulses, each at 20,000 rpm, pausing 3 min between these Test 1
pulses to ensure complete extraction. Pass a portion of the Medium: Water; 900 mL
solution through a nylon filter of 0.45-lJm pore size, Apparatus 2: 50 rpm
discarding the first 2-4 mL of the filtrate. Times: 1, 4, and 8 h
Sample solution A: Nominally 0.6 mg/mL of bupropion Standard solution: (LI900) mg/mL of USP Bupropion
hydrochloride from Sample stocksolution A in 0.001 N Hydrochloride RS in Medium, where L is the label claim,
hydrochloric acid in mg/Tablet. Dilute with Medium, if necessary.
Alternatively, the Sample solution can be prepared asfollows. Sample solution: Pass a portion of the solution under test
Buffer: Dissolve 100 g of anhydrous dibasic sodium through asuitable filter, and dilute with Medium, if
phosphate in 1 L of water. Add 50 mL of phosphoric acid, necessary.
stir or sonicate until dissolved, and mix. Adjust with Instrumental conditions
phosphoric acid to a pH of 3.0. (See Ultraviolet-Visible Spectroscopy (857).)
Diluent 2: Methanol and Buffer(20:80) Mode: UV-Vis
Sample stock solution B: Weigh and grind NLT 20 Tablets Analytical wavelength: 298 nm
to prepare a solution having a nominal concentration of 3 Blank: Medium
mg/mL. Initially add Diluent 2 (75% of the volume of the Analysis
flask), stir for 30 min, and sonicate for 15 min. Dilute with Samples: Standardsolution and Sample solution
Diluent 2 to volume. Centrifuge a portion of the resulting Determine the percentages of the labeled amount of
solution, and use the supernatant. bupropion hydrochloride (C13H 1sCINO . HCI)
Sample solution B: Nominally 0.6 mg/mL of bupropion dissolved.
hydrochloride from Sample stock solution 8 in Diluent 2 Tolerances: See Table 2.
Chromatographic system
(See Chromatography (621), System Suitability.) Table 2
Mode: LC Time Amount
Detector: UV 226 nm (h) Dissolved (0/0)
Column: 4.6-mm x 10-cm; 3.5-lJm packing L1 25-45
1
Column temperature: 40°
Flow rate: 1.5 mL/min 4 60-85
Injection volume: 5 lJL 8 NLT80
System suitability
Samples: Syst~I11..~~itC1bi,lif~.~~/~~i~~. and Standardsolution
[NoTE-See "T.C1l;'.!e.ggj.(Ra.J~Ff!tJ:..].019) for the relative The percentages of the labeled amount of bupropion
retention times.] . hydrochloride (C 13H 1SCINO . HCI) dissolved at the
Suitability requirements times specified conform to Dissolution (711),
Resolution: NLT 1.3 between bupropion hydrochloride Acceptance Table 2.
related compound F and bupropion hydrochloride Test 2: If the product complies with this test, the labeling
related compound C, System suitability solution indicates that it meets USP Dissolution Test 2.
Tailing factor: NMT 1.9, Standardsolution Medium: 0.1 N hydrochloric acid, pH 1.5 (prepared by
Relative standard deviation: NMT 1.5%, Standard transferring 50 mL of hydrochloric acid to 6000 mL of
solution water, adding 18 g of sodium hydroxide, mixing, and
Analysis adjusting with either diluted sodium hydroxide or
Samples: Standard solution and Sample solution A or hydrochloric acid to a pH of 1.5); 900 mL, deaerated
Sample solutionB Apparatus 1: 50 rpm
Calculate the percentage of the labeled amount of Times: 1, 2, 4, and 6 h
bupropion hydrochloride (C 13H1SCINO . HCI) in the Buffer: 3.45 g of monobasic sodium phosphate in 996
portion of Tablets taken: mL of water. Add 4.0 mL of triethylamine, and adjust
with phosphoric acid to a pH of 2.80.
Result = (rulrs) x (Cs/Cu) x 100 Mobile phase: Methanol and Buffer (35:65)
Standard solution: (LI900) mg/mL of USP Bupropion
=peak response of bupropion hydrochloride from Hydrochloride RS in Medium, where L is the label claim,
Sample solutionA or Sample solution B in mg/Tablet
= peak response of bupropion hydrochloride from Sample solution: Use portions of the solution under test,
the Standardsolution and pass through a nylon filter of 0.45-lJm pore size.
= concentration of USP Bupropion Hydrochloride Chromatographic system
RS in the Standard solution (mg/mL) (See Chromatography (621), System Suitability.)
=nominal concentration of bupropion Mode: LC
hydrochloride in Sample solutionA or Sample Detector: UV 298 nm
solution8 (mg/mL) Column: 4.6-mm x 15-cm; packing L1
Flow rate: 1 mL/min
Acceptance criteria: 90.0%-110.0% Injection volume: 20 lJL
System suitability
Sample: Standardsolution

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USP 43 OfficialMonographs / Bupropion 623

Suitability requirements Apparatus 2: 50 rpm

Column efficiency: NLT 2000 theoretical plates Times: 1, 3, and 6 h
Tailing factor: NMT 2.0 Standard solution: (L/900) mg/mL of USP Bupropion
Relative standard deviation: NMT 2.0% Hydrochloride RS in Medium, where L is the label claim,
Analysis in mg/Tablet. Dilute with Medium, if necessary.
Samples: Standard solution and Sample solution Sample solution: Pass a portion of the solution under test
Determine the percentages of the labeled amount of through a suitable filter, and dilute with Medium, if
bupropion hydrochloride (C13H 1SCINO . HCI) necessary.
dissolved. Instrumental conditions
Tolerances: See Table 3. (See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis
Table 3 Analytical wavelength: 298 nm
Time Amount Cell: 0.5 cm
(h) Dissolved (%) Blank: Medium
Analysis
1 25-50
Samples: Standard solution and Sample solution
2 40-65 Determine the percentages of the labeled amount of
bupropion hydrochloride (C13H1SCINO . HCI)
4 65-90
dissolved.
6 NLT 80 Tolerances: See Table 5.

The percentages of the labeled amount of bupropion Table 5

hydrochloride (C13H 1SCINO· HCI) dissolved at the Time Amount
times specified conform to Dissolution (711), (h) Dissolved (%)
Acceptance Table 2. 1 35-55
Test 3: If the product complies with this test, the labeling
indicates that it meets USP Dissolution Test 3. 3 65-85
Medium: Water; 900 mL 6 NLT 80
Apparatus 2: 50 rpm. Use wire coil sinkers, if necessary.
Times: 1,2,4, and 6 h The percentages of the labeled amount of bupropion
Standard solution: (L/900) mg/mL of USP Bupropion hydrochloride (C13H1SCINO . HCI) dissolved at the
Hydrochloride RS in Medium, where L is the label claim,
in mg/Tablet. Dilute with Medium, if necessary. times specified conform to Dissolution (711),
Sample solution: Pass a portion of the solution. under test Acceptance Table 2.
through a suitable filter, and dilute with Medium, if Test 7: If the product complies with this test, the labeling
indicates that it meets USP Dissolution Test 7.
necessary.
Medium: 0.1 N hydrochloric acid, pH 1.5 (prepared by
Instrumental conditions
transferring 50 mL of hydrochloric acid to 6000 mL of
(See Ultraviolet- Visible Spectroscopy (857).)
water, adding 18 g of sodium hydroxide, mixing, and
Mode: UV-Vis
adjusting with either diluted sodium hydroxide or
Analytical wavelength: 250 nm
hydrochloric acid to a pH of 1.5); 900 mL, deaerated
Blank: Medium
Analysis Apparatus 1: 50 rpm
Times: 1,2,4, and 6 h
Samples: Standard solution and Sample solution .
Determine the percentages of the labeled amount of Buffer: 3.45 g of monobasic sodium phosphate in 996
mL of water. Add 4.0 mL of triethylamine, and adjust
bupropion hydrochloride (C13H 1SCINO . HCI)
with phosphoric acid to a pH of 2.80.
dissolved. Mobile phase: Methanol and Buffer (45:55)
Tolerances: See Table 4. Standard solution: (L/900) mg/mL of USP Bupropion
Hydrochloride RS in Medium, where L is the label claim,
Table 4
in mg/Tablet
Amount Dissolved Sample solution: Use portions of the solution under test,
Amount Dissolved (for Tablets that
(for Tablets that contain all other and pass through a nylon filter of 0.45-J.Im pore size.
contain 200 mg of strengths of Chromatographic system
Time bupropion bupropion (See Chromatography (621), System Suitability.)
(h) hydrochloride) (%) hydrochloride) (%) Mode: LC
1 30-50 30-55 Detector: UV 298 nm
Column: 4.6-mm x 15-cm; packing L1
2 45-65 50-75 Flow rate: 1 mL/min
4 65-85 70-90 Injection volume: 20 J.IL
System suitability
6 NLT 78 NLT80 Sample: Standard solution
Suitability requirements
The percentages of the labeled amount of bupropion Column efficiency: NLT 2000 theoretical plates
hydrochloride (C13H,sCINO . HCI) dissolved at the Tailing factor: NMT 2.0
times specified conform to Dissolution (711), Relative standard deviation: NMT 2.0%
Acceptance Table 2. Analysis
Test 5: If the product complies with this test, the labeling Samples: Standard solution and Sample solution
indicates that it meets USP Dissolution Test 5.
Medium: Water; 900 mL

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624 Bupropion / Official Monographs USP 43

Determine the percentages of the labeled amount of Sample solution: Pass a portion of the solution under test
bupropion hydrochloride (C13H 18CINO . HCI) through a suitable filter.
dissolved. Instrumental conditions
Tolerances: See Table 6. (See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis
Table 6 Analytical wavelength: 298 nm
Time Amount Cell: 0.5 cm
(h) Dissolved (%) Blank: Medium
System suitability
1 25-50
Sample: Standardsolution
2 45-70 Suitability requirements
4 NLT70
Relative standard deviation: NMT 2.0%
Analysis .
6 NLT80 Samples: Standardsolution and Sample solution
Calculate the percentage of the labeled amount of
The percentages of the labeled amount of bupropion bupropion hydrochloride (C13H 18CINO· HCI) dissolved
hydrochloride (C13H18CINO . HCI) dissolved at the at each time point (I):
times specified conform to Dissolution (711),
Acceptance Table 2. Result, = (A/As) x Cs x V x (11L) x 100
Test 9: If the product complies with this test, the labeling
indicates that it meets USP Dissolution Test 9. A, =absorbance of bupropion hydrochloride from the
Medium: 0.1 N hydrochloric acid, pH 1.5 (prepared by Sample solution at time point i
transferring 50 mL of hydrochloric acid to 6000 mL of As =absorbance of bupropion hydrochloride from the
water, adding 18 g of sodium hydroxide, mixing, and
Standard solution
adjusting with either diluted sodium hydroxide or
Cs =concentration of US!' Bupropion Hydrochloride
hydrochloric acid to a pH of 1.5); 900 mL RS in the Standard solution(mg/mL)
Apparatus 1: 50 rpm V = volume of Medium, 900 mL
Times: 1, 2,4, and 8 h L = label claim (mg/Tablet)
Standard solution: (Lll000) mg/mL of USP Bupropion Tolerances: See Table 8.
Hydrochloride RS in Medium, where L is the label claim,
in mg/Tablet TableS
Sample solution: Pass a portion of the solution under test
through a suitable filter. Time Point Time Amount
(I) (h) Dissolved (%)
Instrumental conditions
(See Ultraviolet-Visible Spectroscopy (857).) 1 1 20-40
Mode: UV-Vis
2 2 35-60
Analytical wavelength: 298 nm
Blank: Medium 3 4 55-85
Analysis 4 8 NLT80
Samples: Standard solution and Sample solution
Determine the percentages of the labeled amount of
bupropion hydrochloride (C13H 18CINO . HC/) The percentages of the labeled amount of bupropion
dissolved. hydrochloride (C13H 18CINO . HCI) dissolved at the
Tolerances: See Table 7. times specified conform to Dissolution (711),
Acceptance Table 2. .
Table 7 Test 17: If the product complies with this test, the labeling
indicates that it meets USP Dissolution Test 17.
Time Amount ,
(h) Dissolved (%) Medium: 0.1 N hydrochloric acid, pH 1.5 (prepared by
transferring 50 mL of hydrochloric acid to 6 L of water.
1 20-45 containing 18 g of sodium hydroxide, mixing, and
2 35-55 adjusting with either diluted sodium hydroxide or
diluted hydrochloric acid to a pH of 1.5); 900 mL,
4 55-85 deaerated
8 NLT80 Apparatus 1: 50 rpm
Times: 1, 2, 4, and 8 h
Buffer: To each liter of water add 6.8 g of monobasic
The percentages of the labeled amount of bupropion
potassium phosphate. Adjust with phosphoric acid to a
hydrochloride (C13H18CINO . HCI) dissolved at the pH of 3.0.
times specified conform to Dissolution (711), Mobile phase: Methanol and Buffer (60:40)
Acceptance Table 2. Standard solution: (LI900) mg/mL of USP Bupropion
Test 10: If the product complies with this test, the labeling Hydrochloride R.S in Medium, where L is the label claim,
indicates that it meets USP Dissolution Test 10. in mg/Tablet. Sonication may be used to promote
Medium: Water; 900 mL dissolution.
Apparatus 2: 50 rpm Sample solution: Pass a portion of the solution under test
Times: 1, 2, 4, and 8 h through a suitable filter. [NOTE-A 0.45-lJm nylon
Standard solution: (LI900) mg/mL of USP Bupropion membrane filter may be suitable.]
Hydrochloride RS in Medium, where L is the label claim, Chromatographic system
in mg/Tablet (See Chromatography (621), System Suitability.)
Mode: LC

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USP 43 Official Monographs / Bupropion 625

Detector: UV 298 nm Apparatus 1: 50 rpm

Column: 4.6-mm x 15-cm; 5-l.Im packing L7 Times: 1, 2, 4, and 8 h
Flow rate: 1 mL/min Standard stock solution: 0.56 mg/mL of USP Bupropion
Injection volume: 25 I.IL Hydrochloride RS in Medium
Run time: NLT 1.5 times the retention time of Standard solution: (LI900) mg/mL of USP Bupropion
bupropion Hydrochloride RS in Medium, where L is the label claim,
System suitability in mg/Tablet
Sample: Standard solution Sample solution: Pass a portion of the solution under test
Suitability requirements through a suitable filter of 10-l.Im pore size.
Tailing factor: NMT 2.0 Instrumental conditions
Relative standard deviation: NMT 2.0% (See Ultraviolet-Visible Spectroscopy (857).)
Analysis Mode: UV-Vis
Samples: Standardsolution and Sample solution Analytical wavelength: 298 nm
Calculate the concentration (C;) of bupropion Cell: 1 cm
hydrochloride (C13H1SCINO . HCI) in the sample Blank: Medium
withdrawn from the vessel at time point i: System suitability
Sample: Standardsolution
Result; = (r;/rs) x Cs Suitability requirements
Relative standard deviation: NMT 2.0%
r; = peak response of bupropion from the Sample Analysis
solution at time point i Samples: Standardsolution and Sample solution
rs =peak response of bupropion from the Standard Calculate the percentage of the labeled amount of
solution bupropion hydrochloride (C13H1SCINO . HCI) dissolved
Cs = concentration of USP Bupropion Hydrochloride at each time point (I):
RS in the Standard solution (mg/mL)
Result; = (A;/As) x Cs x V x (1I L) x 100
Calculate the percentage of the labeled amount of
bupropion hydrochloride (C13H1SCINO . HCI) dissolved =absorbance of bupropion from the Sample
at each time point (I): solution at time point i
=absorbance of bupropion from the Standard
Result, = C1 x Vx (lIL) x 100 solution
Result, = {[C2 x (V - Vs)] + (C1 x Vs)} x (lIL) x 100 = concentration of USP Bupropion Hydrochloride
Result, = ({C3 X. [V - (2 x Vs)]} + [(C2 + C1) x VsD x (lIL) RS in the Standardsolution (mg/mL)
x 100 V = volume of Medium, 900 mL
Result, = ({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C1) x Vs]) x L =label claim (mg/Tablet)
(lIL) x 100
Tolerances: See Table 7O.
C; = concentration of bupropion hydrochloride in the
portion of the sample withdrawn at time point i Table 10
(mg/mL) Amount
V =volume of Medium, 900 mL . Amount Dissolved (for
Dissolved (for Tablets that con-
L = label claim (mg/Tablet) Tablets that con- tain 1SOor 200
v, = volume of Sample solution withdrawn at each tain 100 mg of mgof
time point (mL) bupropion buprokion
Time Point Time hydrochloride) hydroch oride)
(I) (h) (%) (%)
Tolerances: See Table 9.
1 1 32-52 25-45
Table 9
2 2 50-70 45-65
Amount Amount
Dissolved (for Dissolved (for 3 4 NLT 75 65-85
Tablets that Tablets that
contain 100 mg contain 1SOmg 4 8 NLT 85 NLT85
of or 200 mg of
buproplon bupropion
TIme Point Time hydrochloride) hydrochloride) The percentages of the labeled amount of bupropion
(I) (h) (0/0) (0/0) hydrochloride (C13H 1SCINO . HCI) dissolved at the
1 20-40 15-35 times specified conform to Dissolution (711),
1
Acceptance Table 2.
2 2 40-60 35-55 For products labeled for dosing every 24 h
3 4 60-85 55-80 Test 4: If the product complies with this test, the labeling
indicates that it meets USP Dissolution Test 4.
4 8 NLT 85 NLT80 Medium: 0.1 N hydrochloric acid; 900 mL, deaerated
Apparatus 1: 75 rpm
The percentages of the labeled amount of bupropion Times: 2, 4, 8, and 16 h
hydrochloride (C13H1SCINO. HCI) dissolved at the Standard solution: (L/900) mg/mL of USP Bupropion
times specified conform to Dissolution (711), Hydrochloride RS in Medium, where L is the label claim,
Acceptance Table 2. . in mg/Tablet. Dilute with Medium, if necessary.
Test 19: If the product complies with this test, the labeling Sample solution: Pass a portion of the solution under test
indicates that it meets USP Dissolution Test 79. through a suitable filter, and dilute with Medium, if
Medium: Water, degassed; 900 mL necessary.

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626 Bupropion / OfficialMonographs USP 43

Instrumental conditions Apparatus 1: 75 rpm

(See Ultraviolet-Visible Spectroscopy (857).) Times: 2 h in Acidstagemedium; 3, 8, and 16 h in Buffer
Mode: UV-Vis stagemedium. The time in the Buffer stage medium
Analytical wavelength: 252 nm includes the time in the Acidstage medium.
Blank: Medium Standard solution: (LI900) mg/mL of USP Bupropion
Analysis Hydrochloride RS in Acidstagemedium, where L is the
Samples: Standardsolution and Sample solution label claim, in mg/Tablet
Determine the percentages of the labeled amount of Sample solution: Passa portion of the solution under test
bupropion hydrochloride (C13H1SCINO . HCI) through a suitable filter of 0.45-lJm pore size.
dissolved. Instrumental conditions
Tolerances: See Table 11. (See Ultraviolet-Visible Spectroscopy (857).)
Mode: UV-Vis
Table 11 Analytical wavelength: 298 nm
Time Amount Cell: 0.5 cm
(h) Dissolved (%) Blank: Medium
Analysis
2 NMT20
Samples: Standardsolution and Sample solution
4 20-45 Determine the percentages of the labeled amount of
bupropion hydrochloride (C13H1SCINO· HCI)
8 65-90
dissolved.
16 NLT 80 Tolerances: See Table 13.

The percentages of the labeled amount of bupropion Table 13

hydrochloride (C13H1SCINO . HCI) dissolved at the Time Amount
times specified conform to Dissolution (711), (h) Dissolved (%)
Acceptance Table 2. 2 NMT10
Test 6: If the product complies with this test, the labeling
3 10-30
indicates that it meets USP Dissolution Test 6.
Medium: 0.1 N hydrochloric acid; 900 mL, deaerated 8 60-90
Apparatus 1: 75 rpm
16 NLT 80
Times: 1,2,4,8, and 12 h
Standard solution: (LI900) mg/mL of USP Bupropion
Hydrochlo'ride RS in Medium, where L is the label claim, The percentages of the labeled amount of bupropion
in mg/Tablet. Dilute with Medium, if necessary. hydrochloride (C13H1SCINO· HCI) dissolved at the
Sample solution: Passa portion of the solution under test times specified conform to Dissolution (711),
through a suitable filter, and dilute with Medium, if Acceptance Table 2.
necessary. . Test 11: Ifthe product complies with this test, the labeling
Instrumental conditions indicates that it meets USP Dissolution Test 11.
(See Ultraviolet- Visible Spectroscopy (857).) Acid stage medium: 0.1 N hydrochloric acid; 750 mL
Mode: UV-Vis Buffer stage medium: pH 6.8 phosphate buffer (add 250
Analytical wavelength: 298 nm mL of 76 giL tribasic sodium phosphate to the Acidstage
Blank: Medium medium, adjust with 2 N hydrochloric acid or 2 N sodium
Analysis . hydroxide to a pH of 6.8, if necessary); 1000 mL
Samples: Standardsolution and Sample solution Apparatus 2: 50 rpm
Determine the percentages of the labeled amount of Times: 2 h in Acidstagemedium; 3, 8, and 16 h in Buffer
bupropion hydrochloride (C13H 1SCINO . HCI) stagemedium. The time in the Buffer stage medium
dissolved. includes the time in the Acidstagemedium.
Tolerances: See Table 12. Acid stage standard solution: 0.06 mg/mL of USP
Bupropion Hydrochloride RS in Acidstagemedium.
Table 12 Sonication may be used to aid in dissolution.
Time Amount Buffer stage standard solution: 0.15 mg/mL of USP
(h) Dissolved (%) Bupropion Hydrochloride RS in Buffer stage medium.
Sonication may be used to aid in dissolution.
1 15-35
Sample solution: Passa portion of the solution under test
2 25-50 through a suitable filter of 0.45-lJm pore size.
Instrumental conditions
4 40-65
(See Ultraviolet- Visible Spectroscopy (857).)
8 65-90 Mode: UV-Vis
NLT 80
Analytical wavelength: 298 nm
12
Cell: 0.5 cm
Blank: Acid stagemedium or Buffer stagemedium
The percentages of the labeled amount of bupropion Analysis
hydrochloride (C13H 1sCINO . HCI) dissolved at the Samples: Acidstagestandardsolution, Buffer stage
times specified conform to Dissolution (711), standardsolution, and Sample solution .
Acceptance Table 2. Calculate the concentration (Cj) of bupropion
Test 8: If the product complies with this test, the labeling hydrochloride (C13H1SCINO . HCI) in the sample
indicates that it meets USP Dissolution Test 8. withdrawn from the vessel at time point i:
Acid stage medium: 0.1 N hydrochloric acid; 900 mL
Buffer stage medium: pH 6.8 phosphate buffer; 900 mL Result, = (A/As) x Cs

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USP 43 OfficialMonographs / Bupropion 627

A; =absorbance of bupropion hydrochloridefrom the Calculate the concentration (C;) of bupropion

Sample solution at time point i hydrochloride (C13H,sCINO . HCI) in the sample
=absorbance of bupropion hydrochloridefrom the withdrawn from the vessel at time point i:
Acidstagestandard solution or Buffer stage
standardsolution Result; = (A;/As) x Cs
= concentration of USP Bupropion Hydrochloride
RS in the Acidstagestandard solution or Buffer A; =absorbance of bupropion hydrochloridefrom the
stage standard solution (mg/mL) Sample solution at time point i
=absorbance of bupropion hydrochloridefrom the
Calculatethe percentage of the labeled amount of Standardsolution
bupropion hydrochloride (C13H,sCINO. HCI) dissolved =concentration of USP Bupropion Hydrochloride
at each time point (I): RS in the Standardsolution(mg/mL)
Result, = C, x VA x (1I L) x 100 Calculate the percentage of the labeled amount of
Result, = {[C2 x (VB - Vs)] + (C, x Vs)} x (lIL) x 100 bupropion hydrochloride(C13H,sCINO . HCI) dissolved
Result, =({C3 x (VB - (2 x Vs)]} + [(C2 + C,) x VsD x (1I L) at each time point (I):
x 100
Result, =({C4 x (VB - (3 x Vs)]} + [(C3 + C2 + C,) x VsD x Result, = C, x V x (1I L) x 100
(lIL) x 100 Result, = HC2 x (V- Vs)] + (C, x Vs)} x (lIL) x 100
Result 3 = ({C3 x [V - (2 x Vs)]} + [(C2 + C,) x VsD x (1 I L)
c; = concentration of bupropion hydrochloride in the x 100
portion of the sample withdrawn at time point i Result, = ({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C1) x VsD x
(mg/mL) (lIL) x 100
=volume of Acidstage medium, 750 mL
= label claim (mg/Tablet) C; =concentration of bupropion hydrochloride in the
= volume of Buffer stagemedium, 1000 mL portion of the sample withdrawn at time point i
= volume of Sample solution withdrawn from the (mg/mL)
Acidstage medium or Buffer stage medium (mL) V = volume of Medium, 900 mL
L = label claim (mg/Tablet)
Tolerances: See Table 14. Vs = volume of Sample solution withdrawn from the
Medium (mL)
Table 14
Time Point
I
Time Amount
Tolerances: See Table 15.
(I) (h) Dissolved (%)
Table 15
1 2 NMT10
Time Point Time Amount
2 3 10:..30 (I) (h) Dissolved (%)

3 8 55-85 1 2 NMT25

4 16 NLT 75 2 4 25-50

3 8 60-85
The percentages of the labeled amount of bupropion 4 12 NLT 80
hydrochloride (C13H,sCINO· HCI) dissolved at the
times specified conform to Dissolution (711),
Acceptance Table 2. The percentages of the labeled amount of bupropion
Test 12: Ifthe product complies with this test, the labeling hydrochloride (C13H,sCINO· HCI) dissolved at the
indicates that it meets USP Dissolution Test 12. times specified conform to Dissolution (711),.
Medium: 0.1 N hydrochloric acid; 900 mL Acceptance Table 2.
Apparatus 1: 75 rpm Test 13: Ifthe product complieswith this test, the labeling
Times: 2, -4, 8, and 12 h indicates that it meets USP Dissolution Test 13.
Standard solution: (LI900) mg/mL of USP Bupropion Medium: 0.1 N hydrochloric acid; 900 mL, deaerated
Hydrochloride RS in Medium, where L is the label claim, Apparatus 1: 75 rpm
in mg/Tablet Times: 2, 4, 8, and 12 h
Sample solution: Withdrawat least 10 mLof the solution Standard solution: (L/900) mg/mL of USP Bupropion
under test and pass through a suitable filter. Hydrochloride RS in Medium, where L is the label claim,
Instrumental conditions in mg/Tablet .
(See Ultraviolet-Visible Spectroscopy (857).) Sample solution: Withdrawat least 10 mL of the solution
Mode: UV-Vis under test and centrifuge. Use the supernatant. .
Analytical wavelength: 252 nm Instrumental conditions
Cell (See Ultraviolet-Visible Spectroscopy (857).)
For Tablets labeled to contain 150 mg: 0.1 cm Mode: UV-Vis
For Tablets labeled to contain 300 mg: 0.05 cm Analytical wavelength: 252 nm
Blank: Medium Cell: 0.1 cm
System suitability Blank: Medium
Sample: Standardsolution System suitability
Suitability requirements Sample: Standardsolution
Relative standard deviation: NMT 3.0% Suitability requirements
Analysis . Relative standard deviation: NMT 2.0%
Samples: Standardsolution and Sample solution

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628 Bupropion / Official Monographs USP 43

Analysis Blank: Medium

Samples: Standard solution and Sample solution Analysis
Calculate the concentration (C;) of bupropion Samples: Standard solution and Sample solution
hydrochloride (C13H,8CINO . HCI) in the sample Calculate the concentration (Cj) of bupropion
withdrawn from the vessel at time point i: hydrochloride (C13H,8C1NO . HCI) in the sample
withdrawn from the vessel at time point i:
Result, = (AiAs) x Cs
Result, = (AiAs) x Cs x D
Aj =absorbance of bupropion hydrochloride from the
Samplesolution at time point i Aj = absorbance from the Sample solution at time
As =absorbance of bupropion hydrochloride from the point i
Standard solution = absorbance from the Standard solution
Cs =concentration of USP Bupropion Hydrochloride = concentration of USP Bupropion Hydrochloride
RS in the Standard solution (mg/mL) RS in the Standard solution (mg/mL)
D = dilution factor for the Sample solution, if needed
Calculate the percentage of the labeled amount of
bupropion hydrochloride (C13H,8CINO . HCI) dissolved Calculate the percentage of the labeled amount of
at each time point (I): bupropion hydrochloride (C13H,8CINO· HCI) dissolved
at each time point (I):
Result, = C1 x V x (1I L) x 100
Result, = {[C2 x (V- Vs) ] + (C1 x Vs)} x (lIL) x 100 Result, = C1 x Vx (lIL) x 100
Result, = ({C3 x [V - (2 x Vs)]} + [(C2 + C1) x Vs]) x (lIL) Result, = [(C2 x V) + (C1 x Vs)] x (lIL) x 100
x 100 Result, = {(C3 x V) + [(C2 + C1) x Vs]} x (lIL) x 100
Result, = ({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C1) x Vs]) x Result, ={(C4 x V) + [(C3 + C2 + C1) x Vs]} x (lIL) x 100
(1IL) x 100
c, =concentration of bupropion hydrochloride in the
Cj = concentration of bupropion hydrochloride in the portion of the sample withdrawn at time point i
portion of the sample withdrawn at time point i (mg/mL)
(mg/mL) V =volume of Medium, 900 mL
V = volume of Medium, 900 mL L = label claim (mg/Tablet)
L = label claim (mg/Tablet) Vs =volume of Sample solution withdrawn at each
Vs = volume of Sample solution withdrawn from the time point and replaced with Medium (mL)
Medium (mL)
Tolerances: See Table 17.
Tolerances: See Table 16.
Table 17
Table 16 Time Point Time Amount
Amount Amount (I) (h) Dissolved (%)
Dissolved Dissolved
Time Point Time (150 mg/Tablet) (300 mg/ 1 2 NMT20
(I) (h) (%) Tablet) (%)
2 4 20-45
1 2 NMT25 NMT25
3 8 55-85
2 4 30-55 25-45
4 16 NLT80
3 8 65-90 60-80
4 12 NLT80 NLT80 The percentages of the labeled amount of bupropion
hydrochloride (C13H,8CINO . HCI) dissolved at the
The percentages of the labeled amount of bupropion times specified conform to Dissolution (711),
hydrochloride (C13H,8CINO. Hel) dissolved at the Acceptance Table 2.
times specified conform to Dissolution (711), Test 15: If the product complies with this test, the labeling
Acceptance Table 2. indicates that it meets USP Dissolution Test 15.
Test 14: If the product complies with this test, the labeling Acid stage
indicates that it meets USP Dissolution Test 14. Acid stage medium: 0.1 N hydrochloric acid,
Medium: 0.1 N hydrochloric acid; 900 mL degassed; 900 mL
Apparatus 1: 75 rpm Apparatus 1: 100 rpm
Times: 2,4, 8, and 16 h Time: 2 h in Acid stagemedium
Standard solution: (LI900) mg/mL of USP Bupropion Buffer: 3.5 giL of monobasic sodium phosphate
Hydrochloride RS in Medium, where L is the label claim, prepared as follows. Dissolve 3.45 g of monobasic
in mg/Tablet. If necessary, dilute the solution with sodium phosphate in 996 mL of water, add 4.0 mL of
Medium. triethylamine, and adjust with phosphoric acid to a pH
of 2.8.
Sample solution: Pass a portion of the solution under test
Mobile phase: Methanol and Buffer (45:55)
through a suitable filter. Replace the portion removed
with the same volume of Medium. If necessary, dilute the Acid stage standard solution: 0.033 mg/mL of USP
filtrate with Medium. Bupropion Hydrochloride RS in Acid stage medium.
Instrumental conditions Sonication may be used to promote dissolution.
(See Ultraviolet-Visible Spectroscopy (857).) Acid stage sample solution: Pass a portion of the
Mode: UV-Vis solution under test through a suitable filter, discard the
Analytical wavelength: 252 nm first 5 mL, and use the filtrate. Then discard the Tablets

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USP 43 OfficialMonographs / Bupropion 629

and remaining solution. [NOTE-A 0.45-l..Jm nylon Analysis

membrane filter may be suitable.] Samples: Bufferstage standard solution and Buffer
Chromatographic system stagesample solution
(See Chromatography(621), System Suitability.) Calculate the concentration (C;) of bupropion
Mode: LC hydrochloride (C13H 1SCINO . HCI) in the sample
Detector: UV 298 nm withdrawn from the vessel at time point i:
Column: 4.6-mm x 15-cm; 5-l..Jm packing L1
Flow rate: 1 mL/min Result; = (r;/r s) x Cs
Injection volume: 10 I..JL
Run time: NLT 1.5 times the retention time of r; = peak response of bupropion from the Buffer stage
bupropion sample solution at time point i
System suitability rs =peak response of bupropion from the Buffer stage
Sample: Acid stage standard solution standard solution
Suitability.requirements Cs = concentration of USP Bupropion Hydrochloride
Tailing factor: NMT 2.0 RS in the Bufferstagestandard solution (mg/mL)
Relative standard deviation: NMT 2.0%
Analysis Calculate the percentage of the labeled amount of
Samples: Acid stage standard solution and Acid stage bupropion hydrochloride (C13H,sCINO . HCI)
sample solution dissolved at each time point (I):
Calculate the percentage of the labeled amount of
bupropion hydrochloride (C13H,sCINO . HCI) Result, = C7 x V x (1I L) x 100
dissolved: Result, = {[C2 x (V - Vs)] + (C7 x Vs)} x (lIL) x 100
Result, = ({C3 x [V - (2 x Vs)]} + [(C2 + C7) x VsD x
Result = (rufrs) x Cs x Vx (lIL) x 100 (lIL) x 100
Result, = ({C4 x [V - (3 x Vs)]} + [(C3 + C2 + C7) x Vs]) x
tu = peak response of bupropion from the Acid stage (lIL) x 100
samplesolution
ts =peak response of bupropion from the Acid stage C; = concentration of bupropion hydrochloride in the
standard solution portion of the sample withdrawn at time point i
Cs = concentration of USP Bupropion Hydrochloride (mg/mL)
RS in the Acid stagestandard solution (mg/mL) V =volume of Bufferstage medium, 900 mL
V = volume of Acid stage medium, 900 mL L = label claim (mg/Tablet)
L = label claim (mg/Tablet) Vs = volume of Bufferstagesamplesolution withdrawn
at each time point (mL)
Buffer stage: Usefresh Tablets.
Buffer stage medium: pH 6.8 tribasic sodium Tolerances
phosphate buffer and 0.5% sodium laurylsulfate Acid stage: NMT 10%; the percentage of the labeled
(Dissolve 19 g of tribasic sodium phosphate in 1 L of amount of bupropion hydrochloride (C13H 1SClNO·
water, add 7 mL of hydrochloric acid, and adjust with HCI) dissolved at the time specified conforms to
0.2 N sodium hydroxide or dilute hydrochloric acid to Dissolution (711), Acceptance Table 3.
a pH of 6.8. Add 5 g of sodium dodecyl sulfate. To Buffer stage: See Table 78.
promote dissolution, the resulting solution can be
continuously stirred and heated to 41°. Allow the Table 18
solution to cool to 37° before use. Do not allow the
Time Point Time Amount
temperature to fall below 36.5° before beginning the (I) (h) Dissolved (%)
test.); 900 mL
Apparatus 1: 100 rpm 1 1 5-25
Times: 1, 2, 4, and 8 h 2 2 25-45
Buffer: 1.4 giL of dibasic ammonium phosphate and
0.5 giL of sodium l-hexanesulfonate prepared as 3 4 60-85
follows. Dissolve 1.4 g of dibasic ammonium 4 8 NLT 85
phosphate and 0.5 g of sodium l-hexanesulfonate in
1 L of water. To each 1 L of this solution, add 2.0 mL
of triethylamine, and adjust with phosphoric acid to a The percentages of the labeled amount of bupropion
hydrochloride (C13H,sCINO . HCI) dissolved at the
pH of 7.0.
Mobile phase: Acetonitrile and Buffer(60:40) times specified conform to Dissolution (711),
Buffer stage standard solution: 0.33 mg/mL of USP Acceptance Table 2.
Bupropion Hydrochloride RS in Buffer stagemedium Test 16: If the product complies with this test, the labeling
Buffer stage sample solution: Pass a portion of the indicates that it meets USP Dissolution Test 76.
solution under test through a suitable filter, discard the Medium: 0.1 N hydrochloric acid; 900 mL, deaerated
first 5 mL, and use the filtrate. Apparatus 1: 75 rpm
Chromatographic system: Proceed as directed under Times: 2, 5, 8, and 16 h
the Acid stage. Buffer: 3.5 giL of monobasic sodium phosphate
System suitability prepared asfollows. Dissolve 3.45 g of monobasic
Sample: Bufferstage standard solution sodium phosphate in 996 mL of water, add 4.0 mL of
Suitability requirements triethylamine, and adjust with phosphoric acid to a pH
Tailing factor: NMT 2.0 of 2.8.
Relative standard deviation: NMT 2.0% Mobile phase: Methanol and Buffer(35:65)

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630 Bupropion / Official Monographs USP 43

Standard solution: 0.17 mg/mL of USP Bupropion The percentages of the labeled amount of bupropion
Hydrochloride RS in Medium. Sonication may be used to hydrochloride (C13H,8C1NO . HCI) dissolved at the
promote dissolution times specified conform to Dissolution (711),
Sample solution: Pass a portion of the solution under test Acceptance Table 2.
through a suitable filter, and discard NLT 1 mL. Dilute Test 18: If the product complies with this test, the labeling
the filtrate with Medium if necessary. Replace the portion indicates that it meets USP Dissolution Test 18.
removed with the same volume of Medium. [NOTE-A Medium: 0.1 N hydrochloric acid; 900 mL, deaerated
0.45-l..Im nylon membrane filter may be suitable.] Apparatus 1: 75 rpm
Chromatographic system Times: 2, 4, 8, and 16 h
(See Chromatography (621), System Suitability.) Buffer: 6.8 giL of monobasic potassium phosphate in
Mode: LC water adjusted with phosphoric acid to a pH of 3.0
Detector: UV 298 nm Mobile phase: Methanol and Buffer(60:40)
Column: 4.6-mm x 15-cm; 5-l..Im packing L1 Standard solution: (LI900) mg/mL of USP Bupropion
Column temperature: 30° Hydrochloride RS in Medium, where L is the label claim,
Flow rate: 1 mL/min in mg/Tablet. Sonication may be used to promote
Injection volume: 20 I..IL dissolution.
Run time: NLT 1.5 times the retention time of Sample solution: Centrifuge a portion of the solution
bupropion under test for 15 min.
System suitability Chromatographic system
Sample: Standardsolution (See Chromatography (621), System Suitability.)
Suitability requirements Mode: LC
Tailing factor: NMT 2.0' Detector: UV 298 nm
Relative standard deviation: NMT 2.0% Column: 4.6-mm x 15-cm; 5-l..Im packing L7
Analysis Flow rate: 1 mL/min
Samples: Standardsolution and Sample solution Injection volume: 25 I..IL
Calculate the concentration (C;) of bupropion Run time: NLT 1.5 times the retention time of
hydrochloride (C13H,8CINO . HCI) in the sample bupropion
withdrawn from the vessel at time point i: System suitability
Sample: Standardsolution
Result, = (r/rs) x Cs x D Suitability requirements
Tailing factor: NMT 2.0
r, = peak response of bupropion from the Sample Relative standard deviation: NMT 2.0%
solution at time point i Analysis
= peak response of bupropion from the Standard Samples: Standardsolution and Sample solution
solution Calculate the concentration (C;) of bupropion
= concentration of USP Bupropion Hydrochloride hydrochloride (C13H 18CINO . HCI) in the sample
RS in the Standardsolution (mg/mL) withdrawn from the vessel at time point i:
D = dilution factor for the Sample solution, if needed
Result, = (r/rs) x Cs
Calculate the percentage of the labeled amount of
bupropion hydrochloride (C13H18CIN9 . HCI) dissolved rj = peak response of bupropion from the Sample
at each time point (I): solution at time point i
= peak response of bupropion from the Standard
Result, = C1 X V x (1I L) x 100 . solution
= [(C2 x V) + (C, x Vs)] x (lIL) x 100
Result, = concentration of USP Bupropion Hydrochloride
Result, = {(C3 x V) + [(C2 + C,) x Vs]} x (1I L) x 100 RS in the Standardsolution (mg/mL)
Result, = {(C4 x V) + [(C3 + C2 + C,) x Vs]} x (1I L) x 100
Calculate the percentage of the labeled amount of
c, = concentration of bupropion hydrochloride in the bupropion hydrochloride (C13H,8C1NO . HC!) dissolved
portion of the sample withdrawn at time point i at each time point (J):
(mg/mL)
V = volume of Medium, 900 mL Result, = C1 x V x (1I L) x 100
L = label claim (mg/Tablet) Result, = {[C2 x (V - Vs)] + (C, x Vs)} x (lIL) x 100
v, = volume of Sample solution withdrawn at each Result, = ({C3 x [V - (2 x Vs)]} + [(C2 + C,) x VsD x (1I L)
time point and replaced with Medium (mL) x 100
Result, = ({C4 x [V- (3 x Vs)]} + [(C3 + C2 + C,) x VsD x
Tolerances: See Table 19. (lIL) x 100
Table 19 c, = concentration of bupropion hydrochloride in the
Time Point Time Amount portion of the sample withdrawn at time point i
(I) (h) Dissolved (%) (mg/mL)
1 2 NMT10 V =volume of Medium, 900 mL
L = label claim (mg/Tablet)
2 5 30-60 Vs = volume of Sample solution withdrawn at each
3 8 65-88 time point (mL)
4 16 NLT85 Tolerances: See Table 20.

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USP 43 Official Monographs / Bupropion 631

Table 20 Table 21
Amount Amount Amount
Dissolved (for Dissolved (for Amount Dissolved
Tablets that Tablets that :DlssQlved (f9 rTab!eisthat
contain 150 mg contain 300 mg (for Tablets~l1<lt contain 300 mg
of of contilin) 50J;TIg , of
bupropion bupropion ofbuproRion bupropion
Time Point Time hydrochloride) hydrochloride) Time Point Time hydrochloride) hydrochloride)
(i) (h) (%) (%)
W (h) (%) (%)
1 2 NMT20 NMT20 :1 2 NMT15 NMT1S
2 4 25-50 25-50 2 4 10-35 10+35
3 8 65-95 60-85
:3 8 :S5-80 50-::75'
4 16 NLT80 NLT80
tl '16 NLT80 NLT80

The percentages of the labeled amount of bupropion ntages'ofthe labeled amount of bupropion
hydrochloride (C13H 18CINO· HCI) dissolved at the . l8CINO.~ HCI) dissolved at the
times specified conform to Dissolution (711), ormto Dissolution (711),
Acceptance Table 2. . ' . '.'
4Test 2 . f the producfcomplie' . th,is t~stthe, .
labeli dicatesthat it meets lssolu~J st 20~ .. omplies with this test, tlielabeling
Me chloric, acid ; 900 m eareated USPDissolution Test21.
A chloricacidVS; 900 ml,deareated
h
S '. . '. ..1' mg/mL of USP .Bupropion
Hydrochloride RS in Medium ,
Sam' . Pass a portion of the soluti der test
e filter/and dilute with M , it
n he portion removed wi same
v
Ins
(S
M - is
An . I wavelength: 298 nm
Bla' edium
AnalysIs .' . ,
Samples: Standard solutio
Cal~ulatethe con .
hydrochloride ( 13
withdrawn from the
Result;;:: (A/As) x Cs x 0
;:: absorbance from the Sample solution at time
point l . . ' '.. _.
;:: absorbance from the 'standard solution
;:: concentration of USP' Supro' . rochlorfde
RS in t . andard solution
D ;:: d,ilutio tor for the Sampl i If needed

centage of the labele

ochloride(C13H18CIN
int (i): Result; ;::(AiAs)xCsx· D
;; absorballce from the Samples61utiQnattlme
;:: f~:~~~ance from theSfa"dard ~o(ytion
6nof bupropion h D
the sample withdr
ge of the labeled. amount of
ride (C13H1sCINq . HCI) dissolved
;::
(I)::

(1 X .vx (lIL) x'1'00

x. V) + (C7 x Vs)] x (1/L) ><100
Tolerances: See!' Table 21.

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632 Bupropion / OfficialMonographs USP 43

V
t
y~

D
.Tolerances:,-SeeTable .22~
Tilble22

Time Point TIme

(i) (h)

4 NMI20
2 8
16 Nli-80

Table 23
Time Point 'rime Amount.DisSolved
'(1) (h) (%)
1 2 ~f\II'1Il5,
2 4, if08>_q
3 12 l'lU;r8()

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USP 43 OfficialMonographs / Bupropion 633

L/9(0) rn9!mL.qfLJSP
r stgge/nedium, . TirneP~il')t Tim,e. AmolJnt'Dissolved
blet (i) (h) .. , (0/0)
i / ution a '. er sfage sa-mp!e
1 2 NMT15
ion.:oftbe, solution ul)dertest
er~ 2 '6 5()4.75.
ns .3 16 N~"'80
.$pecjrosCopy:(?57)·)
ou bupropicm
)di datthe
B ILJtion(711),
Sy . ~j
Sa • UNIFORMITY OF DOSAGE UNITS (905): Meet the
standa1i s . n requirements
SuitabilU:yrequirements IMPURITIES
Relative standardd e
standard·solution an
Analysis
Samples: Acid stagestandard • ORGANIC IMPURITIES
standqrdsolutiOf7~ Acid stage. Diluent 1, Solution A, Solution B, Mobile phase, and
stage. sample solution Sample solution A or Sample solution B: Proceed as
Calculate the concentra . directed in the Assay.
hydrochloride (C13H 18 System suitability stock solution A: 0.02 mg/mt of USP
withdrawn from.thev ._. _~ .~ .. Bupropion Hydrochloride Related Compound C RS, 0.02
mg/mt of USP Bupropion Hydrochloride Related
Compound FRS, and 0.012 mg/mt of USP
3-Chlorobenzoic Acid RS in methanol
System suitability solution A: 0.002 mg/mt of bupropion
hydrochloride related compound C, 0.002 mg/mL of
bupropion hydrochloride related compound F, and 0.0012
mg/mt of 3-chlorobenzoic acid from System SUitability stock
Cs solution A in Diluent 1
System suitability stock solution B: 0.012 mg/mt of USP
3-Chlorobenzoic Acid RS in methanol
System suitability solution B: 0.0012 mg/mt of
Calculate the percentage of thel da of 3-chlorobenzoic acid from System SUitability stock solution 8
in Diluent 1
bupropi6n hydrochlo' .(C13 H1 o· ssolveq
Standard solution: 0.0012 mg/mL of USP Bupropion
in Acid stagemedium A • Hydrochloride RS in Diluent 1
Chromatographic system: Proceed as directed in the Assay
Result1 = C, x VA X (1/L) x,1 00 except use a Detector as follows.
Detector: UV 226 nrn, adjusted ±2 nm so that the relative
=. concentration ofbupropion hydrochloride in the response factor requirement is met. [NoTE-The peak
portion of the .sample withdrawn at time point 1 responses of the compounds of interest are very sensitive
= volume of Acid stagelJ1edium, 900 mt to changes in the detection wavelength.]
=label claim (mg!Tabl,et) System suitability
Samples: System suitability solution A, System suitability
Calculate the perceotag'e'of thel:lbe 'of solution 8, and Standard solution
bupropion hydrochloride (C13H1sCI issolved [NOTE-See ~ral:JleZ5~(RB·1.Feb'2019) for the relative
at each time point (I): . . . retention times.]
Suitability requirements
Result2 =[C2 x \Is x (1/Lrx1 b Resolution: NtT 1.3 between bupropion hydrochloride
ReslJlt3 = [C3 ,><. \Is x (ilL) x· related compound F and bupropion hydrochloride
related compound C, System suitability solution A; NLT
== concentration.of bupropi(m~ya 'iilthe 1.3 between bupropion hydrochloride C and
portionofthe sample withdra~ ointi 3-chlorobenzoic acid, System suitability solution A
(mg/ml) Relative standard deviation: NMT 10%, Standard
== volume'0(8L{(fer stage mediuf?1;~OO.rnt solution
= I.a ',' Relative response factor: 3.8-4.5 for the peak response
=p of 3-chlorobenzoic acid in System suitability solution 8
hy r divided by the peak response from bupropion in the
Standard solution
Tolerances: See Table z4., Analysis
Samples: System suitability solution 8, Standard solution,
and Sample solution A or Sample solution 8
Calculate the percentage of 3-chlorobenzoic acid in the
portion of Tablets taken:

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634 Bupropion / Official Monographs USP 43

Result =(rulr s) x (Cs/Cu) x 100

.....
1;~P.~i;~~f~f
>7<\:< (continued)
Acceptance
=peak response of 3-chlorobenzoic acid from Criteria,
Sample solution A or Sample solution B NMT (%)
= peak response of 3-chlorobenzoic acid from 100 150
System suitability solution B Relative Relative mg mg
= concentration of USP 3-Chlorobenzoic Acid RS in Retention Response or or
System suitability solution 8 (mg/mL) Name Time Factor less greater
Cu = nominal concentration of bupropion Total impurities - - 3.2 3.3
hydrochloride in Sample solution A or Sample
solution 8 (mg/mL) a 2-Amino-l-(3-chlorophenyl)-1-propanone.
b (3S,5S,6S)-6-(3-Chlorophenyl)-6-hydroxy-5-methyl-3-thiomorpholine
Calculate the percentage of each other degradation carboxylicacid.
product in the portion of Tablets taken: . c (3S,5R,6R)-6-(3-Chlorophenyl)-6-hydroxy-5-methyl-3-thiomorpholine
carboxylicacid. ..
Result = (rufr s) x (Cs/C u) x (1IF) x 100 d 1-(3-Chlorophenyl)propane-l,2-dione.

= peak response of each other degradation ADDITIONAL REQUIREMENTS

product from Sample solution A or Sample • PACKACING AND STORAGE: Preserve in well-closed
solution B containers. Store at controlled room temperature. Protect
= peak response of bupropion hydrochloride from from light.
the Standardsolution • LABELING: When more than one Dissolution test is given, the
=concentration of USP Bupropion Hydrochloride labeling states the Dissolution test used only if Test 1 is not
RS in the Standardsolution (mg/mL) used.
= nominal concentration of bupropion • USP REFERENCE STANDARDS (11)
hydrochloride in Sample solution A or Sample USP Bupropion Hydrochloride RS
solution 8 (mg/mL) USP Bupropion Hydrochloride Related Compound C RS
F =relative response factor for .;~~b,~ther 1-(3-Chlorophenyl)-2-hydroxypropan-1-one.
(see ~!1£q]:jie; C9H902C1 184.62
USP Bupropion Hydrochloride Related Compound F RS
1-(3-Chlorophenyl)-1-hydroxypropan-2-one.
Acceptance criteria: See C9H90 2C1 184.62
USP 3-Chlorobenzoic Acid RS
'tiJ.l.'i2~'i;;;;';","";;:;.'" 3-Chlorobenzoic acid.
C7H sCI02 156.57
Acceptance
Criteria,
N.MT (%)
100 150
Relative Relative mg mg
Retention Response or or Buspirol1e Hydrochloride
Name Time Factor less greater
Bupropionamine" 0.38 1.2 0.3 0.3
5,5,5- • HCl
Thiomorpholine
derivatives 0.56 1.1 1.0 1.5
5,R,R-Thiomor-
pholine deriva-
tive' 0.78 1.1 0.5 0.4 C21H31Ns02' HCI 421.96
8-Azaspiro[4,5]decane-7,9-dione, 8-[4-[4-(2-pyrimidinyl)-1-
Bupropion 1.0 - - - piperazinyl]butyl]-, monohydrochloride;
Bupropion N-[4-[ 4-(2-Pyrimidinyl)-1-piperazinyl]butyl]-1, 1-
related cyclopentanediacetamide monohydrochloride [33386-08-
compound F 1.71 1.8 1.2 2.3 2].
Bupropion DEfiNITION
related
compound C 1.75 1.7 0.3 0.3 Buspirone Hydrochloride contains NLT 97.5% and NMT
102.5% of buspirone hydrochloride (C21H31NsOz . HCI),
3-Chlorobenzoic
acid 1.80 - 0.3 0.3
calculated on the as-is basis.

Bupropiondione IDENTifiCATION
derivative" 2.25 1.00 0.4 0.4
Any unspecified
degradation -
product 1.00 0.2 0.2 • A.
Sj::/if....... . ... l
• B. The relative retention time of the major peak of the
Sample solution corresponds to that of the Standard
solution, as obtained in the Assay.
• C. IDENTIFICATION TESTS-GENERAL, Chloride (191)
Sample solution: 10 mg/mL in water

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USP 43 OfficialMonographs / Buspirone 635

Acceptance criteria: Meets the requirements Result = (r ulr s) x (C siC u) x 100

ASSAY = peak response from the Sample solution
• PROCEDURE
= peak response from the Standard solution
Buffer A: 6.8 giL of monobasic potassium phosphate and
0.93 giL of sodium 1-hexanesulfonate monohydrate. =concentration of USP Buspirone Hydrochloride RS
Adjust with phosphoric acid to a pH of 3.4. in the Standardsolution(mg/mL)
Buffer B: 3.4 giL of monobasic potassium phosphate and =concentration of Buspirone Hydrochloride in the
3.52 giL of sodium 1-hexanesulfonate monohydrate. Sample solution(mg/mL)
Adjust with phosphoric acid to a pH of 2.2.
Acceptance criteria: 97.50/0-102.5% on the as-is basis
Solution A: Acetonitrile and Buffer A (5:95)
Solution B: Acetonitrile and Buffer B (75:25) IMPURITIES
Mobile phase: See Table 7. • RESIDUE ON IGNITION (281): NMT 0.5%
• ORGANIC IMPURITIES
Table 1 Buffer A, Buffer B, Solution A, Solution B, Mobile phase,
Time Solution A Solution B Diluent, Impurities stock solution, and System suitability
(min) (%) (%) solution: Proceed as directed in the Assay.
0 90 10 Standard solution: 0.001 mg/mL each of USP Buspirone
Hydrochloride RS, USP Buspirone Related Compound A
6 90 10 RS, USP Buspirone RelatedCompound G RS, USP Buspirone
34 42 58 Related Compound K RS, USP Buspirone Related
Compound L RS, and USP Buspirone Related Compound N
45 42 58 RS in Diluent
55 0 100 Sample solution: 1.0 mg/mL of Buspirone Hydrochloride in
Diluent
56 100 0 Chromatographic system
60 100 0 (See Chromatography (621), System SUitability.)
'Mode: LC
61 90 10 Detector: UV 210 and 240 nm
Column: 4.6-mm x 15-cm; 5-j.lm·packing L1
Diluent: Solution A Column temperature: 40°
Impurities stock solution: 0.25 mg/mL each of USP Flow rate: 1 mL/min
Buspirone RelatedCompound A RS, USP Buspirone Related Injection volume: 20 ul,
Compound G RS', USP Buspirone Related Compound K RS, System suitability
USP Buspirone Related Compound L RS, and USP Buspirone Samples: System suitability solution and Standard solution
Related Compound N RS in acetonitrile [NOTE-See Table 2 and Table 3 for relative retention
System suitability solution: 1.0 mg/mL of USP Buspirone times.]
Hydrochloride RS and 0.001 mg/mL each of USP Buspirone Suitability requirements
Related Compound A RS, USP Buspirone Related Resolution at 240 nm: NLT 2.0 between buspirone and
Compound G RS, USP Buspirone Related Compound K RS, buspirone related compound G peaks, System suitability
USP Buspirone Related Compound L RS, and USP Buspirone solution
Related Compound N RS in Diluent, from Impurities stock Resolution at 210 nm: NLT 4.0 between buspirone
solution related compound Land buspirone related compound
Standard solution: 0.1 mg/mL of USP Buspirone . N peaks, System suitability solution
Hydrochloride RS in Diluent Relative standard deviation: NMT 2.0% for each peak,
Sample solution: 0.1 mg/mL of Buspirone Hydrochloride in Standardsolution
Diluent . Analysis
Chromatographic system Samples: Standard solution and Sample solution
(See Chromatography (621), System Suitability.) For impurities detected at UV 240 nm
Mode: LC Calculate the percentage of buspirone related compound
Detector:· UV 240 nm A or buspirone related compound G in the portion of
Column: 4.6-mm x 15-cm; 5-j.lm packing L1 Buspirone Hydrochloride taken:
Column temperature: 40°
Flow rate: 1 mL/min Result = (r ulr s) x (C siC u) x 100
Injection volume: 20 ut,
System suitability ru = peak response of buspirone related compound A
Samples: System suitabilitysolution and Standard solution or buspirone related compound G from the
Suitability requirements Sample solution
Resolution: NLT 2.0 between buspirone and buspirone rs = peak response of buspirone related compound A
related compound G peaks, System suitability solution or buspirone related compound G from the
Tailing factor: NMT 1.5, Standard solution Standard solution
Relative standard deviation: NMT 0.92%, Standard Cs =concentration of USP Buspirone Related
solution Compound A RS or USP Buspirone Related
Analysis Compound G RS in the Standard solution
Samples: Standard solution and Sample solution (mg/mL)
Calculate the percentage of buspirone hydrochloride Cu =concentration of Buspirone Hydrochloride in the
(C 2l H31 N S0 2 • HCI) in the portion of Buspirone Sample solution (mg/mL)
Hydrochloride taken:

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 636 Buspirone / Official Monographs USP 43

Calculate the percentage of specified impurities and any Result = (r vir s) x (C siC v) x 100
other individual impurity in the portion of Buspirone
Hydrochloride taken: = peak response of buspirone related compound K,
buspirone related compound L, or buspirone
Result = (r vir s) x (C siC v) x (1 IF) x 100 related compound N from the Sample solution
=peak response of buspirone related compound K,
ru = peak response of specified impurities and any buspirone related compound L, or buspirone
other individual impurity from the Sample related compound N from the Standard solution
solution = concentration of USP Buspirone Related
rs = peak response of buspirone from the Standard Compound K RS, USP Buspirone Related
solution Compound L RS, or USP Buspirone Related
Cs =concentration of USP Buspirone Hydrochloride RS Compound N RS in the Standard solution
in the Standardsolution (mg/mL) (mg/mL)
Cu =concentration of Buspirone Hydrochloride in the Cv = concentration of Buspirone Hydrochloride in the
Sample solution (mg/mL) Sample solution (mg/mL)
F =relative response factor (see Table 2)
Calculate the percentage of buspirone bromobutyl analog
Acceptance criteria and any other individual impurity in the portion of
For impurities detected at UV 240 nm: See Table 2. Buspirone Hydrochloride taken:
Disregard any peak below 0.05%.
Result = (r vir s) x (C siC v) x 100
Table 2
Relative Relative Acceptance = peak response of buspirone bromobutyl analog
Retention Response Criteria, and any other individual impurity from the
Name Time Factor NMT(%) Sample solution
Buspirone related = peak response of buspirone from the Standard
compound A" 0.2 - 0.10 solution
Spiroammonium saltb 0.3 1.0 0.10
=concentration of USP Buspirone Hydrochloride RS
in the Standard solution (mg/mL)
Bispyrimidinylpiperazinyl bu- = concentration of Buspirone Hydrochloride in the
tane- 0.6 1.0 0.10 Sample solution(mg/mL)
Bispyrimidinylpiperazinylbutyl
ether" . 0.7 1.0 0.10 Acceptance criteria
I
For impurities detected at UV 210 nm: See Table 3.
Buspirone open ring" 0.8 1.0 0.3 Disregard any peak below 0.05%.
Buspirone open ring dimer' 0.9 1.0 0.10
Table 3
Buspirone 1.0 - - Relative Acceptance
Buspirone related Retention Criteria,
compound G9 1.05 - 0.10 Name Time NMT(%)

Buspirone diester dimer" 1.1 1.0 0.10 Buspirone related compound Ka 0.6 0.1

Chlorobuspirone' 1.2 1.0 0.10 Buspirone 1.0 -

Buspirone open ring spirodim- Buspirone related compound Lb 1.7 0.10
erl 1.5 0.5 0.2
Buspirone bromobutyl analog' 1.8 0.10
Anyother individual
impurity - 1.0 0.10 Buspirone related compound Nd 1.9 0.10

Total impurities - - 0.4 Anyother individual impurity - 0.10

a 2-(Piperazin-l-yl)pyrimidine.
Total impurities - 0.2
b 8-(Pyrimidin-2-yl)-8-aza-5-azoniaspiro[4.5]decane. a 8-Azaspiro[4,5]decane-7,9-dione.
c 1,4-Bis[4-(pyrimidin-2-yl)piperazin-l-yl]butane. b8-(4-Chlorobutyl)-8-azaspiro[4.5]decane-7,9-dione.
d Bis{4-[1-(pyrimidine-2-yl)piperazine-4-yl]butane-l-yl} ether. c8-(4-Bromobutyl)-8-azaspiro[4.5]decane-7,9-dione.
e 2-{1-[2-0xo-2-({4-[4-(pyrimidin-2-yl)piperazin-l-yl]butyl}amino)ethyl] d 8,8'-(Butane-l,4-diyl)bis(8-azaspiro[4.5]decane-7,9-dione).
cyclopentyl}acetic acid.
f 4-[4-(Pyrimidin-2-yl)piperazin-l-yl]butyl 2-{1-[2-oxo-2-((4-[4-(pyrimidin-2-yl)
piperazin-l-yl]butyl}amino)ethyl]cyclopentyl}acetate. SPECIFIC TESTS
9 l,4-Di(pyrimidin-2-yl)piperazine. • WATER DETERMINATION, MethodI (921): NMT 0.5%
h Bis{4-[4-(pyrimidin-2-yl)piperazin-l-yl]butyl} 2,2'-(cyclopentane-l, 1-diyl)
diacetate. ADDITIONAL REQUIREMENTS
i 8-{4-[4-(5-Chloropyrimidin-2-yl)piperazin-l-yl]butyl}-8-azaspiro[4.5]decane- • PACKAGING AND STORAGE: Preserve in tight, light-resistant
7,9-dione. containers, at controlled room temperature.
j 4-(7,9-Dioxo-8-azaspiro[4.5]decan-8-yl)butyl 2-{1-[2-oxo-2-({4-[4-(pyrimidin- • USP REFERENCE STANDARDS (11)
2-yl)piperazin-l-yl]butyl}amino)ethyl]cyclopentyl}acetate. USP Buspirone Hydrochloride RS
USP Buspirone Related Compound A RS
For impurities detected at UV 210 nm 2-(Piperazin-l-yl)pyrimidine.
Calculate the percentage of buspirone related compound CSH 12N 4 164.21
K, buspirone related compound L, or buspirone related USP Buspirone Related Compound G RS
compound N in the portion of Buspirone Hydrochloride 1,4-Di(pyrimidin-2-yl)piperazine.
taken: C12H14N6 242.28

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USP 43 Official Monographs / Buspirone 637

USP Buspirone Related Compound K RS System suitability solution: 1.0 mg/mL of USP Buspirone
8-Azaspiro[4.5]decane-7, 9-dione. Hydrochloride RS and 0.001 mg/mL each of USP Buspirone
C9H nNO z 167.21 Related Compound A RS, USP Buspirone Related
USP Buspirone Related Compound L RS Compound G RS, USP Buspirone Related Compound K RS,
8-(4-Chlorobutyl)-8-azaspiro[4.5]decane-7, 9-dione. USP Buspirone RelatedCompound L RS, and USP Buspirone
C13H zoCINOz 257.76 Related Compound N RS in Diluentfrom the Impurities stock
USP Buspirone Related Compound N RS solution
8,8'-(Butane-l ,4-diyl)bis(8-azaspiro[4.5]decane-7, 9- Standard solution: 0.1 mg/mL of USP Buspirone
dione). Hydrochloride RS in Diluent
CzzH32Nz04 388.50 Sample solution: Nominally 0.1 mg/mL of buspirone
hydrochloride from NLT 20 finely powdered Tablets in
Diluent, prepared as follows. Transfer a suitable amount of
the powder to a suitable volumetric flask. Add 60% of the
flask volume of Diluent, and sonicate for 30 min. Allow the
Buspirone Hydrochloride Tablets solution to cool to room temperature, and then dilute with
Diluent to volume. Centrifuge the solution and filter the
DEFINITION supernatant. Further dilute the filtrate with Diluent as
Buspirone Hydrochloride Tablets contain NLT 90.0% and needed.
NMT 110.0% of the labeled amount of buspirone Chromatographic system
hydrochloride (CZ1H31 NsOz . HC!). (See Chromatography (621), System Suitability.)
Mode: LC
IDENTIFICATION Detector: UV 240 nm
Column: 4.6-mm x 15-cm; 5-l..Im packing L1
Column temperature: 40°
Flow rate: 1 mL/min
• A. Injection volume: 20 I..IL
~p> .. . 1 System suitability
Sample: Grind 20 Tablets to a fine powder, add 50 mL of Samples: System suitabilitysolution and Standard solution
chloroform, stir for 3-5 min, and filter into a 250-mL Suitability requirements
evaporating flask. Evaporate the solution with the aid of a Resolution: NLT 2.0 between the buspirone and
rotary evaporator to dryness at low heat. Use the residue. buspirone related compound G peaks, System suitability
Acceptance criteria: Meet the requirements . solution
• B.' The relative retention time of the major peak of the Tailing factor: NMT 1.5, Standard solution
Sample solution corresponds to that of the Standard Relative standard deviation: NMT 1.0%, Standard
solution, as obtained in the Assay. solution
ASSAY Analysis
• PROCEDURE Samples: Standard solution and Sample solution
Buffer A: 6.8 giL of monobasic potassium phosphate and Calculate the percentage of the labeled amount of
0.93 giL of sodium 1-hexanesulfonate monohydrate, buspirone hydrochloride (CZ1H31 NsOz . HCI) in the portion
adjusted with phosphoric acid to a pH of 3.4 of Tablets taken:
Buffer B: 3.4 giL of monobasic potassium phosphate and
3.52 giL of sodium 1-hexanesulfonate monohydrate, Result =(rulrs) x (CslCu) x 100
adjusted with phosphoric acid to a pH of 2.2
Solution A: Acetonitrile and Buffer A (5:95) t» = peak response from the Sample solution
Solution B: Acetonitrile and Buffer B (75:25) ts = peak response from the Standard solution
Mobile phase: See Table 1. Cs =concentration of USP Buspirone Hydrochloride RS
in the Standard solution(mg/mL)
Table 1 Cu = nominal concentration of buspirone
Time Solution A Solution B hydrochloride in the Sample solution(mg/mL)
(min). (%) (%)
Acceptance criteria: 90.0%-110.0%
0 90 10
PERFORMANCE TESTS
6 90 10
• DISSOLUTION (711)
34 42 58 Medium: 0.01 N hydrochloric acid; 500 mL
58
Apparatus 2: 50 rpm
45 42
Time: 30 min
55 0 100 Sample solution: Filter a portion of the solution under test,
0 and dilute with Medium as needed.
56 100
Standard solution: USP Buspirone Hydrochloride RS in
60 100 0 Medium having a concentration similar to that expected in
61 90 10 the Sample solution
Instrumental conditions
Mode: UV
Diluent: Solution A Analytical wavelength: Maximum at about 235 nm
Impurities stock solution: 0.25 mg/mL each of USP Analysis
Buspirone Related Compound A RS, USP Buspirone Related Samples: Sample solution and Standard solution
Compound G RS, USP Buspirone Related Compound K RS, Calculate the percentage of the labeled amount of
USP Buspirone Related Compound L RS, and USP Buspirone buspirone hydrochloride (CZ1H31 NsOz . HCI) dissolved:
Related Compound N RS in acetonitrile

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638 Buspirone / Official Monographs USP 43

Result = (AulAs) x Cs x V x (lIL) x 100 Calculate the percentage of any individual unspecified
degradation product in the portion of Tablets taken:
=absorbance of the Sample solution
= absorbance of buspirone hydrochloride from the Result = (rulrs) x (CslCu) x 100
Standardsolution
= concentration of USP Buspirone Hydrochloride RS ru = peak response of any individual unspecified
in the Standardsolution (mg/mL) degradation product from the Sample solution
V = volume of Medium, 500 mL rs = peak response of buspirone from the Standard
L = label claim (mg/Tablet) solution
Cs = concentration of USP Buspirone Hydrochloride RS
Tolerances: NLT 80% (Q) of the labeled amount of in the Standardsolution (mg/mL)
buspirone hydrochloride (C2l H3l NS0 2 • HCI) is dissolved. Cu = nominal concentration of buspirone
• UNIFORMITV OF DOSAGE UNITS (905): Meet the hydrochloride in the Sample solution(mg/mL)
requirements
Acceptance criteria
IMPURITIES For impurities detected at UV 240 nm: See Table 2.
• ORGANIC IMPURITIES Disregard any peak below 0.05%.
Buffer A, Buffer B, Solution A, Solution B, Mobile phase,
Diluent, Impurities stock solution, and System suitability Table 2
solution: Proceed as directed in the Assay. Relative Acceptance
Standard solution: 0.001 mg/mL each of USP Buspirone Retention Criteria,
Hydrochloride RS, USP Buspirone Related Compound A Name Time NMT(%)
RS, USP Buspirone RelatedCompound G RS, USP Buspirone
Buspirone related compound A" 0.2 0.20
Related Compound K RS, USP Buspirone Related
Compound L RS, and USP Buspirone RelatedCompound N Spiroammonium saltb• C 0.3 -
RS in Diluent
Sample solution: Nominally 1.0 mg/mL of buspirone
Bispyrimidinylpiperazinyl butane- d 0.6 -
hydrochloride from NLT 20 finely powdered Tablets in Bispyrimidinylpiperazinylbutyl ether- e 0.7 -
Diluent, prepared as follows. Transfer a suitable amount of
the powder to a suitable volumetric flask. Add 6()% of the
Buspirone open ring c• f 0.8 -
flask volume of Diluent, and sonicate for 30 min. Allow the Buspirone open ring dlrner- 9 0.9 -
solution to cool to room temperature, and then dilute with
Diluent to volume. Centrifuge the solution and filter the
Buspirone 1.0 -
supernatant. Use the filtrate. Buspirone related compound GC. h 1.05 -
Chromatographic system
(See Chromatography (621), System Suitability.)
Buspirone diester dlrner-! 1.1 -
Mode: LC Chlorobusplrone- j 1.2 -
Detector: UV 210 and 240 nm
Column: 4.6-mm x .15-cm; 5-l-Im packing L1
Buspirone open ring splrodlmer- k 1.5 -
Column temperature: 40° Any individual unspecified
-
Flow rate: 1 mL/min degradation product 0.2
Injection volume: 20 I-IL Total impurities - See Table 3
System suitability
Samples: System suitability solution and Standardsolution a 2-(Piperazin-l-yl)pyrimidine.
Suitability requirements . b 8-(Pyrimidin-2-yl)-8-aza-5-azoniaspiro[4.5]decane.
Resolution at 240 nm: NLT 2.0 between the buspirone C Process impurity included for identification only and not included in the
calculation of total degradation products.
and buspirone related compound G peaks, System
d 1,4-Bis[4-(pyrimidin-2-yl)piperazin-l-yl]butane.
SUitability solution
e Bis{4-[1-(pyrimidine-2-yl)piperazine-4-yl]butane-l-yl} ether.
Resolution at 210 nm: NLT 4.0 between the buspirone
f 2-{1-[2-0xo-2-«(4-[4-(pyrimidin-2-yl)piperazin-l-yl]butyl}amino)ethyl]
related compound Land buspirone related compound cyclopentyl}acetic acid.
N peaks, System SUitability solution 9 4-[4-(Pyrimidin-2-yl)piperazin-l-yl]butyl 2-{1-[2-oxo-2-({4-[4-(pyrimidin-2-yl)
Relative standard deviation: NMT 2.0% for each peak, hiperazin-l-yl]butyl}amino)ethyl]cyclopentyl}acetate.
Standardsolution 1,4-Di(pyrimidin-2-yl)piperazine.
Analysis i Bis{4-[4-(pyrimidin-2-yl)piperazin-l-yl]butyl} 2,2'-(cyclopentane-l, l-diyl)
Samples: Standardsolution and Sample solution diacetate.
j 8-{4-[4-(5-Chloropyrimidin-2-yl)piperazin-l-yl]butyl}-8-azaspiro[4.5]decane-
For impurities detected at UV 240 nm 7,9-dione.
Calculate the percentage of buspirone related compound k 4-(7,9-Dioxo-8-azaspiro[4.5]decan-8-yl)butyl 2-{l-[2-oxo-2-({4-[4-(pyrimidin-
A in the portion of Tablets taken: 2-yl)piperazin-l-yl]butyl}amino)ethyl]cyclopentyl}acetate.

Result =(rulr s) x (CslCu) x 100 For impurities detected at UV 210 nm

Calculate the percentage of any individual unspecified
= peak response of buspirone related compound A degradation product in the portion of Tablets taken:
from the Sample solution
= peak response of buspirone related compound A Result = (ru/rs) x (CslCu) x 100
from the Standardsolution
= concentration of USP Buspirone Related tu = peak response of any individual unspecified
Compound A RS in the Standard solution degradation product from the Sample solution
(mg/mL) rs = peak response of buspirone from the Standard
= nominal concentration of buspirone solution
hydrochloride in the Sample solution (mg/mL)

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 USP 43 OfficialMonographs / Busulfan 639

= concentration of USPBuspirone Hydrochloride RS IDENTIFICATION

in the Standardsolution (mg/mL) • A.
= nominal concentration of buspirone Sample: 100 mg
hydrochloride in the Sample solution (mg/mL) Analysis: Fusethe Sample with 100 mg of potassium nitrate
and a pellet of potassium hydroxide weighing 250 mg.
Acceptance criteria Cool, dissolve the residue in water, acidify with 3 N
For impurities detected at UV 210 nm: See Table 3. hydrochloric acid, and add a few drops of barium chloride
Disregard any peak below 0.05%. TS.
Acceptance criteria: A white precipitate is formed.
Table 3 • B.
Relative Acceptance Sample: 100 mg
Retention Criteria, Analysis: Add 10 mL of water and 5 mL of 1 N sodium
Name Time NMT (%) hydroxide to the Sample. Heat until a clear solution is
Buspirone related compound Ka, b 0.6 - obtained. .
Acceptance criteria: An odor characteristic of
Buspirone 1.0 - methanesulfonic acid is perceptible.
Buspirone related compound Lb,c 1.7 - • c.
Sample solution: Use the solution from the Analysis in
Buspirone bromobutyl analogb, d 1.8 - Identification test B.
Buspirone related compound Nb, e 1.9 - Analysis: Cool the Sample solution, and divide it into two
equal portions. To the first portion add 1 drop of potassium
Any individual unspecified permanganate TS. Acidify the second portion of the
degradation product - 0.2
solution with 2 N sulfuric acid, and add 1 drop of potassium
Total impurities - 2.0 f permanganate TS.
Acceptance criteria
a 8-Azaspiro[4.5]decane-7,9-dione, For first portion: The purple color changes to violet, then
b Process impurityincludedfor identification only and not includedin the to blue, and finally to emerald-green.
calculationof total degradation products.
c8-(4-Chlorobutyl)-8-azaspiro[4.5]decane-7,9-dione.
For second portion: The color of the permanganate is not
d 8-(4-Bromobutyl)-8-azaspiro[4.5]decane-7,9-dione. discharged.
e 8,8'-(Butane-l,4-diyl)bis(8-azaspiro[4.5]decane-7,9-dione). ASSAY
fTotal impurities include impuritiesdetected at UV 240 nrn. • PROCEDURE
Sample solution: Transfer 80 mg of Busulfan into a 250-mL
ADDITIONAL REQUIREMENTS conical flask. Add 30 mL of water, swirl, add
• PACKAGING AND STORAGE: Preserve in tight, light-resistant phenolphthalein TS, and neutralize with 0.05 N sodium
containers at controlled room temperature. hydroxide. Connect the flask to a reflux air condenser, and
• USP REFERENCE STANDARDS (11) boil the mixture gently for NLT 30 min, adding water
USP Buspirone Hydrochloride RS occasionally to maintain the volume. Cool to room
USP Buspirone Related Compound A RS temperature.
2-(Piperazin-1-yl)pyrimidine. Titrimetric system
CSH 12N4 164.21 Mode: Direct titration
USP Buspirone Related Compound G RS Titrant: 0.05 N sodium hydroxide VS
1,4-Di(pyrimidin-2-yl)piperazine. Endpoint detection: Visual
C12H 14N6 242.28 Analysis: Add phenolphthalein TS to the Sample solution,
USP Buspirone Related Compound K RS and titrate with Titrant. Each mL of Titrant is equivalent to
8-Azaspiro[4.5]decane-7,9-dione. 6.158 mg of busulfan (C6H1406SZ)'
C9H13N02 167.21 Acceptance criteria: 98.0%-100.5% on the dried basis
USP Buspirone Related Compound L RS
8-(4-Chlorobutyl)-8-azaspiro[ 4.5]decane-7,9-dione. IMPURITIES
C13H2oCIN0 2 257.76 • RESIDUE ON IGNITION (281): NMT 0.1 %
USP Buspirone Related Compound N RS SPECIFIC TESTS
8,8'-(Butane-1,4-diyl)bis(8-azaspiro[4.5]decane-7,9- • MELTING RANGE OR TEMPERATURE (741): 115°-118°
dione). • Loss ON DRYING (731)
C22H3ZNz04 388.50 Analysis: Dry a sample under vacuum at 60° to constant
weight.
Acceptance criteria: NMT 2.0%
ADDITIONAL REQUIREMENTS
Busulfan • PACKAGING AND STORAGE: Preserve in tight containers.
• LABELING: The label bears a warning that great care should
be taken to prevent inhaling particles of Busulfan and
exposing the skin to it.

C6H1406SZ 246.30
1,4-Butanediol, dimethanesulfonate;
1,4-Butanediol dimethanesulfonate [55-98-1].
DEFINITION
Busulfan contains NLT 98.0% and NMT 100.5% of busulfan
(C6H1406SZ), calculated on the dried basis.

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640 Busulfan / OfficialMonographs USP 43

Busulfan Tablets PERFORMANCE TESTS

• DISINTEGRATION (701)
DEFINITION Time: 30 min the use of disks being omitted
Busulfan Tablets contain NLT 93.0% and NMT 107.0% of the Acceptance c;iteria: Meet the requirements
• UNIFORMITY OF DOSAGE UNITS (90S): Meet the
labeled amount of busulfan (C6H,406S2)'
requirements
IDENTIFICATION
ADDITIONAL REQUIREMENTS
• A. • PACKAGING AND STORAGE: Preserve in well-closed
Sample: Asuitable number of Tablets . containers.
Analysis: Pulverize the Sample and extract the powder with
several portions of acetone. Evaporate the combined
acetone extracts, with the aid of a current of air, on a steam
bath.
Acceptance criteria: The dry residue melts at about 115°. Butabarbital
• B.
Sample: 100 mg of the powder obtained in Identification 0 ~~o
test A
Analysis: Fuse the Sample with 100 mg of potassium nitrate
and a pellet of potassium hydroxide weighing 250 mg.
Cool, dissolve the residue in water, acidify with 3 N
H3C
$
H3C

CH 0
3
I
NH

hydrochloric acid, and add a few drops of barium chloride ClOH16N203 212.25
TS. 2,4,6(1H,3H,5H)-Pyrimidinetrione, 5-ethyl-5-(1-
Acceptance criteria: Awhite-precipitate isformed. methylpropyl)-;
• C. 5-sec-Butyl-5-ethylbarbituric acid [125-40-6].
Sample: 100 mg of the powder obtained in Identification
test A DEFINITION
Analysis: Add 10 mL of water and 5 mL of 1 N sodium Butabarbital contains NLT 98.5% and NMT 101.0% of
hydroxide to the Sample. Heat until a clear solution is butabarbital (C'OH 16N203), calculated on the dried basis.
obtained. IDENTIFICATION
Acceptance criteria: An odor characteristicof
methanesulfonic acid is perceptible.
• D.
Sample solution: Use the solution from Identification test C.
Analysis: Cool the Sample solution, and divide it into two
equal portions. To the first portion add 1 drop of potassium
permanganate TS. Acidify the second portion of the . ASSAY
solutionwith 2 N sulfuric acid, and add 1 drop of potassium • PROCEDURE
permanganate TS. Internal standard solution: 2 mg/mL of tetracosane in
Acceptance criteria chloroform
For first portion: The purple color changes to violet, then Standard stock solution: 2 mg/mL of USP Butabarbital RS
to blue, and finally to emerald-green. in chloroform
For second portion: The color of the permanganate is not Standard solution: 1 mg/mL of USP Butabarbital RS from
discharged. Standardstock solution in Internal standardsolution
prepared as follows. Combine 10.0 mL of Stan.dard stock
ASSAY solution and 10.0 mLof Internal standardsolution.
• PROCEDURE Sample stock solution: 2 mg/mL of Butabarbital in
Guard against accidental inhalation of the fine powder. chloroform .
Sample solution: Transferan equivalent to 80 mg of Sample solution: 1 mg/mL of Butabarbital from Sample
busulfan, from finely powdered Tablets (NLT 40), to a stock solution in Internal standard solution prepared as
1OO-mL beaker. Extractwith four 20-mL portions of follows. Combine 10.0 mL of Sample stock solutionand 10.0
acetone, each time stirring the mixture well. Allow the mLof Internal standard solution.
insoluble matter to settle, and decant the supernatant Chromatographic system
through a sintered-glassfilter into a 250-mLconicalflask. (See Chromatography (621), System Suitability.)
Evaporate the combined acetone extracts to about 10 mL, Mode: GC
add phenolphthalein TS, and neutralize with 0.05 N Detector: Flame ionization
sodium hydroxide. Evaporate to dryness, and add about 30 Column: 4-mm x 1.8-m; 10% phase G37 on support Sl AB
mL of water. Connect the flask to a reflux air condenser, Temperatures
and boil the mixture gently for NLT 30 min, adding water Injection port: 260 0

occasionally to maintain the volume. Cool to room Detector: 300 0

temperature. Column: 260°

Titrimetric system Carrier gas: Drynitrogen
Mode: Directtitration Flow rate: 50 mL/min
Titrant: 0.05 N sodium hydroxide VS Injection volume: 2 J.IL
Endpoint detection: Visual System suitability
Analysis: Add phenolphthalein TS to the Sample solution, Sample: Standardsolution .
and titrate with Titrant. Each mLof Titrant is equivalent to [NoTE-The relative retention times for butabarbital
6.158 mg of the labeled amount of busulfan (C6H1406S2)' and tetracosane are 0.6 and 1.0, respectively.]
Acceptance criteria: 93.0%-107.0%

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 USP 43 OfficialMonographs / Butabarbital 641

Suitability requirements • USP REFERENCE STANDARDS (11)

Resolution: NLT 3.0 between butabarbital and USP Butabarbital RS
tetracosane
Tailing factor: NMT 1.3 for butabarbital and NMT 1.2
for tetracosane
Relative standard deviation: NMT 1.0%
Analysis Butabarbital Sodium
Samples: Standardsolution and Sample solution
Calculate the percentage of butabarbital (ClOH,6N203) in 0 ~'V0Na
the portion of Butabarbital taken:
Result =(Ru/R s) x (Cs/Cu) x 100
$
H,C

H,C CH,O
II
N

= peak response ratio of butabarbital to the internal ClOH,sN2Na0 3 234.23

standard from the Sample solution 2,4,6(1 H,3H,5H)-Pyrimidinetrione, 5-ethyl-5-(1-
= peak response ratio of butabarbital to the internal methylpropyl)-, monosodium salt;
standard from the Standardsolution Sodium 5-sec-butyl-5-ethylbarbiturate [143-81-7].
= concentration of USP Butabarbital RS in the DEFINITION
Standardsolution (mg/mL) Butabarbital Sodium contains NLT 98.2% and NMT 100.5%
= concentration of Butabarbital in the Sample of butabarbital sodium (C,oH,sN2Na03), calculated on the
solution (mg/mL) dried basis.
Acceptance criteria: 98.5%-101.0% on the dried basis IDENTIFICATION
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1 %
• ORGANIC IMPURITIES
Diluent: Chloroform and methanol (50:50) • A·.;~~~~~~~~~9~1~;'~~~~!~~!~~t~9Ni~~$]+$i(1M;)iZ)~;i;rtirrare(J
Standard solution A: 4.0 mg/mL of USP Butabarbital RS in §pg.9tq:)S.~()RY;i)1.·~;7,~ ••(CN'1"'MaY+2020)
Sample: 150 mg of Butabarbital Sodium
Diluent
Analysis: Transfer the Sample to a suitable separator,
Standard solution B: 0.4 mg/mL of USP Butabarbital RS dissolve in 10 mL of water, and add 15 mL of 3 N
from Standardsolution A in Diluent hydrochloric acid. Extract with three 20-mL portions ?f
Sample solution: 40 mg/mL of Butabarbital in Diluent chloroform, filter the extracts through anhydrous sodium
Chromatographic system sulfate, and collect the extracts in a suitable beaker.
(See Chromatography (621), Thin-Layer Chromatography.) Evaporate the combined extracts on a steam bath with the
Mode: TLC aid of a current of air to dryness, and dry the residue at 105°
Adsorbent: 0.25-mm layer of chromatographic silica gel for 2 h.
mixture Acceptance criteria: Meets the requirements
Application volume: 10 f..JL
Developing solvent system: Acetone, meth):'lene
chloride methanol, and ammonium hydroxide (5:3:1 :1)
Spray re~gent: A solution of mercurous nitrate dihydrate • B.: ;~
in 0.15 N nitric acid (1 in 100)
Analysis
.(j£i~
Analytical wavelength: 240 nm
Samples: Standardsolution A, Standardsolution B, and Buffer: pH 9.6 alkaline borate buffer (see Reagents,
Sample solution Indicators, and Solutions-Buffer Solutions)
Proceed as directed in the chapter. Develop the . Standard solution: 10 uq/rn], of USP Butabarbital RS in
chromatogram in the Developing solventsystem until the Buffer
solvent front has moved about three-fourths of the length Sample solution: 10 uq/m], of Butabarbital Sodium in Bu~er
of the plate. Remove the plate from the developing Acceptance criteria: Absorptivities, calculated on the dried
chamber, and dry under a current of air. Spray the plate basis, do not differ by more than 3.0%.
with Spray reagent, and immediately estim.ate the • C. IDENTIFICATION TESTS-GENERAL, Sodium (191)
intensities of any spots of the Sample solution, other .than Sample: 100 mg of Butabarbital Sodium . .
the principal spot, in comparison with S~am!ard soiutionB. Analysis: Ignite the Sample, and proceed as directed In the
Acceptance criteria: The RF value of the principal spot ?f the chapter using the residue.
Sample solution corresponds to that of Standardsolution A; Acceptance criteria: Meets the requirements
and the sum of the intensities of any secondary spots of the
Sample solution is NMT the intensity of the principal spot ASSAY
produced by Standardsolution B, corresponding to NMT a • PROCEDURE
total of 1% of impurities. Buffer: pH 9.6 alkaline borate buffer (see Reagents,
Indicators, and Solutions-Buffer Solutions)
SPECIFIC TESTS Standard stock solution: 0.125 mg/mL of USP Butabarbital
• MELTING RANGE OR TEMPERATURE, Class la (741): 164°- RS in Buffer
167° Standard solution: 0.0125 mg/mL of USP Butabarbital RS
• Loss ON DRYING (731) from Standardstock solution in Buffer
Analysis: Dry at 1 OSO for 2 h. Sample stock solution: 0.140 mg/mL of Butabarbital
Acceptance criteria: NMT 1.0% Sodium in Buffer
ADDITIONAL REQUIREMENTS Sample solutlon: 0.0140 mg/mL from Sample stock solution
• PACKAGING AND STORAGE: Preserve in tight containers. in Buffer

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642 Butabarbital / OfficialMonographs USP 43

Instrumental conditions • PH (791)

Mode: UV Sample solution: Use the Sample solution from the test for
Analytical wavelength: Maximum absorbance at about Completeness of Solution.
240 nm Acceptance criteria: 10.0-11.2
Blank: Buffer • Loss ON DRYING (731)
Analysis Analysis: Dry at 150 to constant weight.
0

Samples: Standardsolution, Sample solution, and Blank Acceptance criteria: NMT 5.0%
Concomitantly determine the absorbances of the solutions.
Calculate the percentage of butabarbital sodium APDITIONAL REQUIREMENTS
(ClOH1SN2Na03) in the portion of Butabarbital Sodium • PACKAGING AND STORAGE: Preserve in tight containers.
taken: • USP REFERENCE STANDARDS (11)
USP Butabarbital RS
Result = (A ulA s) x (C siC u) x (M rtiM r2) X 100
=absorbance of the Sample solution
=absorbance of the Standardsolution Butabarbital Sodium Oral Solution
=concentration of USP Butabarbital RS in the
Standardsolution (~g/ml) DEFINITION
= concentration of Butabarbital Sodium in the Butabarbital Sodium Oral Solution contains NlT 90.0% and
Sample solution (~g/ml) NMT 110.0% of the labeled amount of butabarbital sodium
M r7 =molecularweight of butabarbital sodium, 234.23 (CloH1SN2Na03)'
M r2 = molecular weight of butabarbital, 212.25
IDENTIFICATION
Acceptance criteria: 98.2%-100.5% on the dried basis
IMPURITIES
• ORGANIC IMPURITIES • A•.·~.~·~.~~T'~~~~~ISjil~~~~I~!~~~IC)N>Tis"l'$«lQi/)~rnrfc{Eeli
Diluent: Chloroform and methanol (50:50) SpggtrqsqqpY;«~9'1.~"'(cNJ,MaY~~92Q)
Standard solution A: 4.0 mg/ml of USP Butabarbital RS in Sample: Transferan equivalent to 150 mg of butabarbital
Diluent sodium from a volume of Oral Solution, to a separator.
Standard solution B: 0.4 mg/ml of USP Butabarbital RS Render it distinctlyalkaline by the addition of 1 N sodium
from StandardsolutionA in Diluent hydroxide, and saturate it with sodium chloride. Extract the
Sample solution: 44 mg/ml of Butabarbital Sodium in mixture with two 15-ml portions of ether, and discard the
Diluent ' ether. Acidify the solution with hydrochloricacid, and
Chromatographic system render it just alkaline to litmus by adding small portions of
(See Chromatography (621), Thin-Layer Chromatography.) sodium bicarbonate (carbonate-free). Extract the liberated
Mode: TlC acid barbiturate using five20-ml portions of chloroform.
Adsorbent: 0.25-mm layerof chromatographic silica gel Wash the combined chloroform extracts with 10 ml of
mixture water acidified with 1 drop of hydrochloric acid, then
Application volume: 10 ~l extract the water with 10 mLof chloroform, adding the
Developing solvent system: Acetone, methylene latter to the main chloroform solution. Pass the chloroform
chloride, methanol, and ammonium hydroxide (5:3:1:1) solution through a pledget of cotton or other suitable filter,
Spray reagent: Asolution of mercurous nitrate dihydrate previously washed with chloroform, into a tared beaker,
in 0.15 N nitric acid (1 in 100) and finally wash the separator and the filterwith three 5-mL
Analysis portions of chloroform. Evaporate the combined
Samples: StandardsolutionA, Standardsolution B, and chloroformsolution and washings on a steam bath with the
Sample solution aid of a current of airto dryness, and dry the residue at 105 0

Develop the chromatogram in the Developing solvent for 2 h.

system until the solvent front has moved about Acceptance criteria: Meets the requirements
three-fourths of the length of the plate. Remove the plate • B. The retention time of the butabarbital peak of the
from-the developing chamber, and dry under a current of Sample solution corresponds to that of the Standard
air. Spray the plate with Sprayreagent, and immediately solution, as obtained in the Assay.
estimate the intensitiesofany spots of the Sample solution,
other than the principalspot, in comparison with ASSAY
Standardsolution B. • PROCEDURE
Acceptance criteria: The R F value of the principal spot of Solution A: Dissolve 2.0 mL of bromine and 109 of
the Sample solution corresponds to that of Standardsolution potassium bromide in 60 mL of water.
A; and the sum of the intensities of any secondary spots of Solution B: Sodium metabisulfite in water (1 in 10)
the Sample solution is NMT the intensity of the principal Internal standard solution: 0.7 mg/mL of secobarbital in
spot produced by Standardsolution B, corresponding to chloroform
NMT a total of 1% of impurities. Standard solution: 1 mg/ml of USP ButabarbitalRS and 1.4
mg/mL of secobarbital in chloroform .
SPECIFIC TESTS Sample stock solution: Nominally 0.3 mg/mL of
• COMPLETENESS OF SOLUTION butabarbital sodium from Oral Solution prepared as
Sample solution: Dissolve 1.0 g of Butabarbital Sodium in follows. Transfera volume of Oral Solution, equivalent to
10 mL of carbon dioxide-free water. 30 mg of butabarbital sodium, to a separator. Add 1 mLof
Acceptance criteria: After 1 min, the solution is clear and Solution A, and swirl. Allow to stand for 5 min, add 1 mLof
free from undissolved solid. Solution B, and swirl. Add 300 mg of sodium bicarbonate in
small portions, with mixing, and extract with four 10-mL
portions of chloroform. Pass the extracts through about 15

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 USP43 OfficialMonographs / Butabarbital 643

g of anhydrous sodium sulfate that is supported on a funnel

by a small pledget of glass wool. Collect the combined
Butabarbital Sodium Tablets
filtrates in a 50-ml volumetric flask, wash the sodium sulfate DEFINITION
with 5 ml of chloroform, collecting the washing with the Butabarbital Sodium Tablets contain NlT 90.0% and NMT
filtrate, dilute with chloroform to volume, and mix. 110.0% of the labeled amount of butabarbital sodium
[NOTE-This solution includes a bromination step for (ClOH 15N2Na03). .
elimination of parabens and a carbonate-chloroform
extraction for elimination of benzoic acid.] IDENTIFICATION
SaJ!lple solution: Combine 2.0 ml of Sample stock solution • A. The retention time of the butabarbital peak of the
with 2.0 ml of Internal standard solution in a suitable Sample solution corresponds to that of the Standard
container, and reduce the volume to about 1 ml by solution, as obtained in the Assay.
evaporation, with the aid of a stream of dry nitrogen, at
room temperature. ASSAY
Chromatographic system • PROCEDURE
(See Chromatography (621), System Suitability.) Solution A: Ammonium hydroxide in water (1 in 25)
Mode: GC Internal standard solution: 1.2 mg/ml of secobarbital in
Detector: Flame ionization chloroform
Column: 4-mm x 0.9-m glass; packed with 3% liquid Standard solution: 0.8 mg/ml of USP Butabarbital RS and
phase G10 support on 80- to 10-mesh SlA 1 mg/ml of secobarbital in chloroform
Temperatures Sample stock solution: Finely powder NlT 20 Tablets.
Injection port: 225 0 Transfer a portion of the powder, equivalent to 50 mg of
Detector: 225 0 butabarbital sodium, to a 50-ml volumetric flask. Add 35
Column: 200 ±1 0 0 .
ml of Solution A, and dilute with water to volume. Filter, if
Carrier gas: A suitable gas such as dry nltroqen necessary, discarding the first 15 ml of the filtrate, and
Flow rate: 60-80 ml/min transfer 25.0 ml of the clear solution to a separator. Add 2
Injection volume: 5 J,Jl ml of hydrochloric acid, and extract with three 25-ml
System suitability portions of chloroform. Filterthe extracts through about 15
Sample: Standard solution 9 of anhydrous sodium sulfate that is supported on a funnel
[NoTE-The relative retention times for butabarbital and by a small pledget of glass wool. Collect the combined
secobarbital are 0.6 and 1.0, respectively.] filtrate in a 1OO-ml volumetric flask, and wash the sodium
Resolution: NlT 2.4 between butabarbital and sulfate with 15 ml of chloroform, collecting the washing
secobarbital with the filtrate. Dilute with chloroform to volume.
Tailing factor: NMT 2.0 each for butabarbital and Sample solution: Combine 4.0 ml of Sample stock solution
secobarbital with 1.0 ml of Internal standard solution in a suitable
Relative standard deviation: NMT 1.5% for the peak container. Reduce the volume to about 1 ml by
response ratio of butabarbital to the internal standard evaporation, with the aid of a stream of nitrogen, at room
Analysis . temperature.
Samples: Standard solution and Sample solution Chromatographic system
Calculate the percentage of the labeled amount of (See Chromatography (621), System Suitability.)
butabarbital sodium (CloH15N2Na03) in the portion of Mode: GC
Oral Solution taken: Detector: Flame ionization
Column: 4-mm x 0.9-m glass; packed with 3% liquid
Result =(Ru/R s) x (Cs/Cu) x (Mr1/Mr2 ) x 100 phase Gl 0 support on 80- to 10-mesh SlA
Temperatures
= peak response ratio of butabarbital to the internal Injection port: 225 0
standard from the Sample solution Detector: 225 0
=peak response ratio of butabarbltal to the internal Column: 200 ± 10 0
standard from the Standard solution Carrier gas: A suitable gas such as dry nitrogen
=concentration of USP Butabarbital RS in the Flow rate: 60-80 ml/min
Standard solution (uq/rnl) Injection volume: 5 J,Jl
= nominal concentration of butabarbital sodium System suitability
in the Sample solution (J,Jg/ml) Sample: Standard solution
=molecular weight of butabarbital sodium, 234.23 [NoTE-The relative retention times for butabarbital and
= molecular weight of butabarbital, 212.25 secobarbital are 0.6 and 1.0, respectively.]
Resolution: NlT 2.4 between butabarbital and
Acceptance criteria: 90.0%-110.0% secobarbital
Tailing factor: NMT 2.0 each for butabarbital and
OTHER COMPONENTS . secobarbital
• ALCOHOL DETERMINATION, Method II (611): Between Relative standard deviation: NMT 1.5% for the peak
95.0% and 115.0% of the labeled amount of alcohol response ratio of butabarbital to the internal standard
(C2H sOH) Analysis
Samples: Standard solution and Sample solution
ADDITIONAL REQUIREMENTS Calculate the percentage of the labeled amount of
• PACKAGING AND STORAGE: Preserve in tight containers. butabarbital sodium (CloH1SN2Na03) in the portion of
Store at controlled room temperature. Tablets taken: .
• USP REFERENCE STANDARDS (11)
USP Butabarbital RS Result = (Ru/R s) x (Cs/Cu) x (Mr dMr2) x 100
Ru =peak response ratio of butabarbital to the internal
standard from the Sample solution

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 644 Butabarbital / Official Monographs USP 43

= peak response ratio of butabarbital to the internal IDENTIFICATION

standard from the Standardsolution
= concentration of USP Butabarbital RS in the
Standardsolution (mg/ml)
= nominal concentration of butabarbital sodium
in the Sample solution (mg/mL) @~2Q)
= molecular weight of butabarbital sodium, 234.23 • time of t e major peak of the Sample
= molecular weight of butabarbital, 212.25 solution corresponds to that of the Standardsolution, as
obtained in the Assay. .
Acceptance criteria: 90.0%-110.0% ASSAY
PERFORMANCE TESTS • PROCEDURE
• DISSOLUTION (711) Buffer: 1.0 mL of phosphoric acid diluted with water to 1 L
Medium: Water; 900 mL Mobile phase: Acetonitrile and Buffer (25:75)
Apparatus 1: 100 rpm System suitability solution: 0.1·mg/ml each of USP
Time: 45 min Butalbital RS and USP Salicylic Acid RS in Mobilephase.
Standard solution: USP Butabarbital RS in Medium Sonication may be used to aid in dissolution.
Sample solution: Pass a portion of the solution under test Standard solution: 0.1 mg/mL of USP Butalbital RS in
through a suitable filter, and mix with sufficient ammonium Mobile phase. Sonication may be used to aid in dissolution.
hydroxide to provide a concentration of 0.5 N ammonium Sample solution: 0.1 mg/mL of Butalbital in Mobilephase.
hydroxide. Dilute with Medium, if necessary. Sonication may be used to aid in dissolution.
Instrumental conditions Chromatographic system
Mode: UV (See Chromatography (621), System Suitability.)
Analytical wavelength: 239 nm Mode: lC
Analysis Detector: UV 214 nm
Samples: Standardsolution and Sample solution Column: 4.6-mm x 1O-cm; 5-~m packing L1
Calculate the percentage of the labeled amount of Column temperature: 30°
butabarbital sodium (ClOH1SN2Na03) dissolved: Flow rate: 1 mL/min
Injection volume: 20 ~l
Result = (AvIAs) x (CsICu) x D x (MrdMr2 ) x 100 System suitability
Samples: System suitability solution and Standard solution
= absorbance of the Sample solution [NoTE-The relative retention times of salicylicacid and
= absorbance of the Standardsolution butalbital are 0.86 and 1.0, respectively.]
= concentration of USP Butabarbital RS in the Suitability requirements
Standardsolution (mg/ml) Resolution: NlT 3.0 between salicylic acid and
= nominal concentration of butabarbital sodium butalbital, System suitability solution
in the Sample solution (mg/mL) . Tailing factor: NMT 1.5, Standard solution
= dilution factor for the Sample solution Relative standard deviation: NMT 1.0%, Standard
= molecular weight of butabarbital sodium, 234.23 solution
= molecular weight of butabarbital, 212.25 Analysis
Samples: Standardsolution and Sample solution
Tolerances: NLT 75% (Q) of the labeled 'amount of Calculate the percentage of butalbital (Cl1H16N203) in the
butabarbital sodium (Cl0H1SN2Na03) is dissolved. portion of Butalbital taken:
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements Result = (r vir s) x (C siC v) x 100

ADDITIONAL REQUIREMENTS = peak response from the Sample solution

• PACKAGING AND STORAGE: Preserve in tight containers, and = peak response from the Standardsolution
store at controlled room temperature. = concentration of USPButalbital RS in the Standard
• USP REFERENCE STANDARDS (11) solution (mg/ml)
USP Butabarbital RS = concentration of Butalbital in the Sample solution
(mg/ml)

Acceptance criteria: 98.0%-102.0% on the dried basis

Butalbital IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1 %
• ORGANIC IMPURITIES
H'C~~YO Buffer: 4.1 giL of monobasic potassium phosphate adjusted
H3C)--IllNH with 1 N sodium hydroxide to a pH of 6.0
H3C 0
Mobile phase: Acetonitrile and Buffer (22: 78)
System suitability solution: 10 ~g/mL each of USP
CllH16N203 224.26 Butalbital RS and USP Butabarbital RS in Mobile phase.
2,4,6(1 H,3H,5H)-Pyrimidinetrione, 5-(2-methylpropyl)-5-(2- Sonication may be used to aid in dissolution. .
propenyl)-; Sample solution: 1 mg/mL of Butalbital in Mobilephase.
5-Allyl-5-isobutylbarbituric acid [77-26-9]. Sonication may be used to aid in dissolution.
Chromatographic system
DEFINITION (See Chromatography (621), System Suitability.)
Butalbital contains NlT 98.0% and NMT 102.0% of butalbital Mode: lC
(Cl1H16N203), calculated on the dried basis. Detector: UV 214 nm

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 USP 43 Official Monographs / Butalbital 645

Column: 4.6-mm x 15-cm; 5-J.Im packing L78 Apparatus 7: 100 rpm.

Column temperature: 30° Time: 60 minutes.
Flow rate: 1 mL/min Mobile phase and Chromatographic system-Prepare as
Injection volume: 20 J.IL directed in the Assay under Butalbital, Acetaminophen, and
System suitability Caffeine Tablets.
Sample: System suitability solution Standard preparation-Prepare a solution in methanol
[NoTE-The relative retention times of butabarbital having known concentrations of about 0.02A mg of USP
and butalbital are 0.83 and 1.0, respectively.] Acetaminophen RS per mL, 0.02B mg of USP Butalbital RS per
Suitability requirements mL, and 0.02C mg of USP Caffeine RS per mL, in which A, B,
Resolution: NLT 2.0 between butabarbital and butalbital and C are the labeled amounts, in mg of acetaminophen,
Tailing factor: NMT 1.5 for butalbital butalbital, and caffeine, respectively, per Capsule.Transfer5.0
Relative standard deviation: NMT 5.0% for butalbital mL of this solution to a 1OO-mL volumetricflask, dilute with
Analysis water to volume, and mix.
Sample: Sample solution Procedure-Pass a portion of the solution under test
Calculatethe percentage of each impurity in the portion of through a filter of 10-J.Im or finer porosity. Separately inject
Butalbital taken: equal volumes (about 20 J.IL) of the filtrate and the Standard
preparation into the chromatograph, record the
Result = (r vir r) x 100 chromatograms, and measure the responses for the major
peaks. Calculatethe quantities, in mg, of butalbital
ru = peak response of each impurity from the Sample (CllH16Nz03), acetaminophen (CsHgNOz), and caffeine
solution (CSH10N40Z) dissolved by the same formula:
rT = sum of the peak responses from the Sample
solution 900 C(rvirs)
Acceptance criteria in which C is the concentration, in mg per mL, of the
Any individual unspecified impurity: NMT 0.10% appropriate USP Reference Standard in the Standard
Total impurities: NMT 1% preparation; and rv and ts are the peak responses of the
SPECIFICTESTS corresponding analyte obtained from the solution under test
• Loss ON DRYING (731) and the Standard preparation, respectively.
Analysis: Dry under vacuum at room temperature to Tolerances-Not lessthan 80%(Q) of the labeled amounts
constant weight. of CllH16NzO, CsHgNO z, and CSH10N40Z is dissolved in 60
Acceptance criteria: NMT 0.2% minutes.
Uniformity of dosage units (90S): meet the requirements.
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in well-closed Assay-
containers. Mobile phase, Internal standard solution, Butalbital standard
• USP REFERENCE STANDARDS (11) stock solution, Caffeine standard stock solution, Standard
USP Butabarbital RS preparation, and Chromatographic system-Proceed as
USP Butalbital RS directed in the Assay under Butalbital, Acetaminophen, and
USP Salicylic Acid RS Caffeine Tablets
Assay preparation-Remove, as completely as possible, the
contents of not fewer than 20 Capsules. Transferan accurately
weighed portion of the powder, equivalent to about the
weight of the contents of 1 Capsule, to a 200-mLvolumetric
Butalbital, Acetaminophen, and flask, add Internal standard solution to volume, and mix.
Sonicate for 15 minutes, mix, and allowto cool and settle.
Caffeine Capsules Transfer20.0 mL of the clear supernatant to a 50-mL
volumetric flask, dilute with water to volume, and mix. Passa
» Butalbital, Acetaminophen, and Caffeine portion of this solution through a filter of 0.5 J.Im or finer
porosity, discarding the first 5 mL of the filtrate. Usethe clear
Capsules contain not less than 90.0 percent and filtrate as the Assay preparation.
not more than 110.0 percent of the labeled Procedure-Separately inject equal volumes (about 10 J.IL)
amounts of butalbital (Cl1H16Nz03), of the Standard preparation and the Assay preparation into the
acetaminophen (CSH 9N02) , and caffeine chromatograph, record the chromatograms, and measure the
peak responses for the major peaks. Calculatethe quantities,
(CSH10N40Z)' in mg, of butalbital (Cll H16Nz03), acetaminophen (CsHgNO z),
and caffeine (CSH10N40Z) in the portion of Capsules taken by
Packaging and storage-Preserve in tight containers. the formula:
USP Reference standards (1 1)-
USP Acetaminophen RS
USP Butalbital RS
USP Caffeine RS in which 0 is the concentration, in mg per mL, of the
Identification-The retention times of the butalbital, appropriate USP Reference Standard in the Standard
acetaminophen, and caffeine peaks in the chromatogram of preparation; and Rv and Rs are the peak response ratios of the
the Assay preparation correspond to those of the butalbital, corresponding analyte to phenacetin obtained from the Assay
acetaminophen, and caffeine peaks in the chromatogram of preparation and the Standard preparation, respectively.
the Standard preparation, as obtained in the Assay.
Dissolution (711)-
Medium: water; 900 mL.

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646 Butalbital / OfficialMonographs USP 43

solution to obtain a solution having a known concentration of

Butalbital, Acetaminophen, and about 0.018 mg per mL, 8 being the labeled amount, in mQ,
Caffeine Tablets of butalbital per Tablet, sonicatin~ and ~haking the solution, If
necessary, to achieve complet~ dlssol~tlon.
Caffeine standardstock solutIOn-Dissolve an accurately
» Butalbital, Acetaminophen, and Caffeine Tablets weighed quantity of USP Caffeine RS in Internal standar~
contain not less than 90.0 percent and not more solution to obtain a solution having a known concentration of
than 110.0 percent of the labeled amounts of about 0.01C mg per mL, ~ b~ing the label.ed amount, !n m~,
of caffeine per Tablet, sontcatlnq and shaking the solution, If
butalbital (Cl l H16N z0 3) , acetaminophen necessary, to achieve complete dissolution. . .
(CgH 9NO z), and caffeine (CgH lON4 0 Z) ' Standardpreparation-Transfer to a 50-mLvolumetric flask
about 0.1A mg of USP Acetaminophen RS, A being the labeled
Packaging and storage-Preserve in tight containers. amount, in mg, of acetaminophen per Tablet, 10.0 m~ of
USPReference standards (11)- Butalbitalstandard stock solution, and1 0.0 mLof Caffeme
USP Acetaminophen RS standardstock solution, sonicate for 5 minutes, dilute with
USP Butalbital RS water to volume and mix. This solution contains about
USP Caffeine RS 0.002B mg of b~talbital, 0.002A mg of ac~taminof?hen, a.nd
Identification-The retention times of the butalbital peak, 0.002C mg of caffeine per ~L. Passa portlo.n of this s?lutlon
the acetaminophen peak, and the caffeine peak in the through a suitablefilter having a 0.5-lJmor finer porosity, and
chromatogram of the Assay preparation correspond to those use the filtrate as the Standardpreparation.
of the butalbital peak, the acetaminophen peak, and the Assay preparation-Weigh and finely powder not fewer
caffeine peak in the chromatogram of the Standard than 20Tablets. Transferan accuratelyweighed portion of the
preparation, as obtained in the Assay. powder, equivalent to about 1 average Tablet weig~t, to a
200-mL volumetricflask, add Internal standardsolution to
Dissolution, Procedure for a Pooled Sample (711)- volume, and mix. Sonicate for 15 minutes, mix, and allowto
Medium: water; 900 mL. cool and settle. Transfer20.0 mL of.the clear supernatant to a
Apparatus 2: 50 rpm. 50-mL volumetric flask, dilute with water to volume, and mix.
Time: 30 minutes. . Pass a portion of this solution through a suitable filter having
Mobile phase and Chromatographic system-Prepare as a 0.5-lJm or finer porosity, discarding the first 5 mLof the
directed in the Assay. filtrate. Usethe clear filtrate as the Assay preparation.
Standardpreparation-Prepare a solution in methanol
having known concentrations of about 0.02A mg o~ USP
Chromatographic system (see Chromatography (621 »- The
liquid chromatograph is equipped with a 216-nm detector
Acetaminophen RS per mL, 0.028 mg of USP Butalbltal RS per -and a 4-mm x 25-cm column that contains packing L1. The
mL, and 0.02C mg of USP Caffeine RS per mL, in which A, 8, flow rate is about 2 mL per minute. Chromatograph the
and C are the labeled amounts, in mg, of acetaminophen, Standard preparation, and recor.d the pea.k responses as
butalbital, and caffeine, respectively, per Tablet. Transfer5.0 directed for Procedure: the relative retention times are about
mLof this solution to a 1OO-mL volumetricflask, dilute with 0.16 for acetaminophen, 0.33 for caffeine, 0.77 for
water to volume, and mix. . phenacetin, and 1.0 for butalbital; the resolution, .R~ between
Procedure-Pass a portion of the solution under test any two peaks is not lessthan 1.2; the column efficiency,
through a suitable filter having a 10-lJmor finer poros.ity. calculatedfrom the butalbital peak, is not lessthan 1000
Separately inject equal volumes (about 20 IJL) of the filtrate theoretical plates; and the relative standard deviations of the
and the Standardpreparation into the chrornatoqraph, record acetaminophen, caffeine, and butalbital responses for
the chromatograms, and measure the responsesfor the major replicate injections are not more than 2.0%. .
peaks. Calculate the quantities, in mg, of butalbital Procedure-Separately inject equal volumes (about 10 IJL)
(CllH,6N203), acetaminophen (CSH 9N0 2), and caffeine of the Standard preparation and the Assay preparation into the
(CsH,oN402) dissolved by the same formula: chromatograph, record the chromatograms, and measure the
peak responsesfor the major peaks. Calculate the quantities,
900C(ru/rs) in mg, of butalbital (C,lH,6N203), acetaminophen (CSH9N0 2),
and caffeine (CsH, oN 402) in the portion ofTabletstaken by the
in which C is the concentration, in mg per mL, of the
appropriate USP Reference Standard in the Standard same formula:
preparation; and ru and rs are the peak responses of the 500D(Ru/Rs)
corresponding analyte obtained from the solution under test
and the Standardpreparation, respectively. in which 0 is the concentration, in mg per mL, of the
Tolerances-Not lessthan 80% (Q) of the labeled appropriate USP Reference Standard in the Standar~
amounts of Cll H'6N203, CSH9N0 2, and CSH10N402 is dissolved preparation; and Ru and Rs are the peak response ratios of the
in 30 minutes. corresponding analyte to phenacetin obtained from the Assay
Uniformity of dosage units (905): meet the requirements. preparation and the Standardpreparation, respectively.
Assay- . .
Mobile phase-Transfer 800 mg of monobasic potassium
phosphate to a 2000-mLvolumetricflask. Dissolv~ in 1100 mL
of water, dilute with methanol to volume, and mix. Pass Butalbital and Aspirin Tablets
through a suitable filter having a 0.5-lJm or fi~er p.orosity.
Make adjustments if necessary(see System SUItabilIty under DEFINITION
Chromatography (621 ». Butalbital and Aspirin Tablets contain NLT 90.0% and NMT
Internal standardsolution-Prepare a solution of phenacetin 110.0% of the labeled amounts of butalbital (C"H'6N203)
in methanol containing 0.65 mg per mL.
Butalbital standard stock solution-Dissolve an accurately and aspirin (C9H s04).
weighed quantity of USP Butalbital RS in Internal standard

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USP 43 OfficialMonographs / Butalbital 647

IDENTIFICATION = peak response of aspirin from the Sample solution

18 A. The retention times of the butalbital and aspirin peaks of = peak response of aspirin from StandardsolutionB
the Sample solution correspond to that of the butalbital = concentration of USP Aspirin RS in Standard
peak of StandardsolutionA and the aspirin peak of Standard solution B (lJg/mL)
solution B, as obtained in the Assay. = nominal concentration of aspirin in the Sample
ASSAY solution (lJg/mL)
18 PROCEDURE
Mobile phase: Acetonitrile, water, and phosphoric acid Acceptance criteria: 90.00/0-110.0% of the labeled
(725:3100:4) amounts of butalbital (CllH16N203) and aspirin (C9H s04)
Diluent: Add 10 mL of formic acid to each Lof acetonitrile. PERFORMANCE TESTS
System suitability solution: 2601 IJg/mL of USP Butalbital 18 DISSOLUTION (711)
RS and 12 IJg/mL of USP SalicylicAcid RS in Diluent, where Medium: Water; 900 mL
1 is the ratio of the labeled amount of butalbital, in mgl Apparatus 1: 100 rpm
Tablet, relative to the labeled amount of aspirin, in mgl Time: 60 min
Tablet Butalbital
Standard solution A: 3251 IJg/mL of USP Butalbital RS and Mobile phase: Acetonitrile, water, and phosphoric acid
2.4 IJg/mL of USP SalicylicAcid RS in Diluent, where 1 is the (725:3100:4).
ratio of the labeled amount of butalbital, in mg/Tablet, . Standard solution: L IJg/ml of USP Butalbital RS and 30
relative to the labeled amount of aspirin, in mg/Tablet IJg/mL of salicylic acid in Mobilephase, where L is the
Standard solution B: 325 IJg/mL of USPAspirin RS in Diluent labeled amount of butalbital, in mg/Tablet
Sample solution: Nominally 320 IJg/mL of aspirin from a Sample solution: Use portions of the solution under test
suitable amount of powdered Tables in solution prepared passed through a suitable filter of 0.5-lJm pore size.
as follows. Weigh and finely powder NLT 20 Tablets. Chromatographic system
Transfer a portion of this fine powder to a suitable (See Chromatography (621 ), System Suitability.)
volumetric flask. Dilute with Diluent to volume, and Mode: LC
sonicate for 15 min. Pass a portion of the solution through Detector: UV 214 nm
a filter of 0.5-lJm pore size. , Column: 3.9-mm x 30-cm; 10-lJm packing L1
Chromatographic system Flow rate: 1.5 ml/min
(See Chromatography (621), System Suitability.) Injection volume: 10-25 IJl; equal volumes of the
Mode: LC Standardsolution and Sample solution
Detector: UV 214 nm System suitability
Column: 3.9-mm x 30-cm; 10-lJm packing Ll Sample: Standardsolution
Flow rate: 1.5 m'L/min [NOTE-The relative retention times for aspirin,
Injection volume: 10 IJl salicylic acid, and butalbital are about 0.6,0.85, and
System suitability 1.0, respectively.]
Samples: System suitability solution, StandardsolutionA, Suitability requirements
and Standardsolution B ' Resolution: NLT 3.0 between salicylic acid and
[NoTE-The relative retention times for aspirin, salicylic . butalbital
acid, and butalbital are about 0.6, 0.85, and 1.0, Relative standard deviation: NMT 3.0% for butalbital
respectively.] Analysis
Suitability requirements ' Samples: Standardsolution and Sample solution
Resolution: NLT 3.0 between salicylic acid and Calculate the percentage of the labeled amount of
butalbital, System suitability solution butalbital (CllH16N203) dissolved:
Relative standard deviation
Standard solution A: NMT 3.0% for butalbital; NMT Result = (rulr s) x Cs x V x (1I L) x 100
6.0% for salicylic acid
Standard solution B: NMT 3.0% for aspirin = peak response of butalbital from the Sample
Analysis solution
Samples: Standardsolution A, Standardsolution B, and = peak response of butalbital from the Standard
Sample solution solution
Calculate the percentage of butalbital (CllH16N203) in the = concentration of USPButalbital RS in the Standard
portion of Tablets taken: solution (lJg/mL)
V = volume of Medium, 900 mL
Result =(rulr s) x (CslCu) x 100 L = label claim of butalbital (mg/Tablet)
ru = peak response of butalbital from the Sample Aspirin
solution Buffer: Dissolve 5.98 9 of sodium acetate trihydrate in 500
rs = peak response of butalbital from Standardsolution ml of water, add 2.5 mL of glacial acetic acid, dilute with
A water to 1000 mL, and adjust with glacial acetic acid to a
C, = concentration of USP Butalbital RS in Standard pH of 4.5.
solution A (lJg/mL) Sample solution: Use a filtered portion of the solution
Cu = nominal concentration of butalbital in the Sample under test diluted with 4 volumes of Buffer.
solution (lJg/mL) Standard solution: A known concentration of USP
Aspirin RS in water diluted with 4 volumes of Buffer.
Calculate the percentage of aspirin (C9H s04 ) in the portion Prepare the Standardsolution at the time of use.
of Tablets taken: [NOTE- An amount of alcohol not to exceed 1% of the
total volume of the Standardsolution may be used to
Result =(ru/rs) x (Cs/Cu) x 100 bring the Reference Standard into solution before

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 648 Butalbital / Official Monographs USP 43

dilution first with water and then with 4 volumes of Mobile phase: Methanol and Buffer (45:55) initially
Buffer.] adjusted with phosphoric acid to a pH of 3.9. Ifthe
Instrumental conditions retention time of the salicylic acid peak differs from that of
Mode: UV-Vis the aspirin peak,adjust the pHof the Mobilephasewith 0.2
Analytical wavelength: The isosbestic point of aspirin N potassium hydroxideor 1. M phosphoric acid so that the
and salicylic acid at 265 ± 2 nm salicylic acid peak has the same retention time as that of the
Analysis aspirin peak. [NOTE-The retention time of the salicylic acid
Samples: Standardsolution and Sample solution peak decreases about 0.3 min for each 0.1 pHincrease. The
Determine the percentage of the labeled amount of retention time of the aspirin peak is essentially unaffected
aspirin (C9H S04) dissolved. by such pH adjustments.]
Tolerances: NLT 75% (Q) of the labeled amounts of Diluent: Methanol and Buffer (45:55) adjusted with
butalbital (CllH16N203) and aspirin (C9HS04 ) are phosphoric acid to a pH of 2.5 ± 0.05.
dissolved. Salicylic acid solution: 0.1 mg/mL of salicylic acid in
• UNIFORMITY OF DOSAGE UNITS (905) Diluent. Pass this solutionthrough a suitablefilter of 0.5-lJm
Analysis: Proceed as directed in the chapter. or finer pore size.
Acceptance criteria Standard stock solution: 1.6 mg/mL of USP Aspirin RS in
Butalbital: Meet the requirements for Content Uniformity Diluent. Sonication and shaking may be used to promote
Aspirin: Meet the requirements for Weight Variation dissolution. Use this solution within 24 h.
Standard solution: USP Reference Standards in Standard
IMPURITIES stock solution as listed below. Sonication and shaking the
• LIMIT OF FREE SALICYLIC ACID solution may be used to promote dissolution. Use this
Mobile phase, System suitability solution, Standard solution within 24 h.
solution A, Standard solution B, Sample solution, Butalbital: 1.6}mg/mL of USP Butalbital RS, where} is the
Chromatographic system, and System suitability: ratio of the labeled amount, in mg, of butalbital relative
Proceed as directed in the Assay. to the labeled amount of aspirin in mg/Capsule
Analysis Caffeine: 1.6j' mg/mL of USP Caffeine RS, where l' is the
Samples: Standardsolution A and Sample solution ratio of the labeled amount, in mg, of caffeine relative to
Calculatethe percentage of free salicylic acid in the Tablets the labeled amount of aspirin in mg/Capsule
taken: Sample solution: Nominally 1.6 mg/mL of aspirin from the
contents of Capsules in solution prepared as follows.
Result = (rulrs) x (CsICu) x 100 Transfera suitable portion of the contents of NLT 20
Capsulesto an appropriate volumetric flask. Dilute with
tu = peak response of salicylic acid from the Sample Diluent to volume, and sonicate for 30 min. Pass a portion
solution of this solution through a suitable filter of 0.5-lJm or finer
ts =peak response of salicylic acid from Standard pore size. Use the filtratewithin 24 h.
solutionA Chromatographic system
Cs = concentration of USP Salicylic Acid RS in Standard (See Chromatography (621), System SUitability.)
solution A (lJg/mL) Mode: LC
Cu = nominal concentration of aspirin in the Sample Detectors
solution (lJg/mL) Butalbital: UV 210 nm
Aspirin and caffeine: UV at the wavelength of the
Acceptance criteria: NMT 3.0% of free salicylic acid isosbestic point of aspirin and salicylic acid at about
ADDITIONAL REQUIREMENTS 277 nm
• PACKAGING AND STORAGE: Preserve in tight containers. Column: 3.9-mm x 30-cm; packing Ll
• USP REFERENCE STANDARDS (11) Column temperature: 35 ± 1 0

USP Aspirin RS Flow rate: 1 mL/min

USP Butalbital RS Injection volume: 10 IJL
USP Salicylic Acid RS System suitability
Samples: Salicylic acid solution and Standardsolution
[NoTE-The relative retention times for caffeine,
aspirin, salicylic acid, and butalbital are about 0.45,
0.6, 0.6, and 1.0, respectively.]
Butalbital, Aspirin, and Caffeine Suitability requirements
Capsules Resolution: NLT 2.0 between caffeineand aspirin,
Standardsolution
DEFINITION Column efficiency: NLT 2000 theoretical plates from
Butalbital, Aspirin, and Caffeine Capsules contain NLT 90.0% butalbital, Standardsolution
and NMT 110.0% of the labeled amounts of butalbital Relative standard deviation: NMT 2.0% each for
(Cl1H16N203), aspirin (CgH s0 4) , and caffeine (CSH10N402)' caffeine,aspirin, and butalbital responses, Standard
solution
IDENTIFICATION Analysis
• A. The retention times ofthe butalbital, aspirin, and caffeine Samples: Standard solution and Sample solution
peaks of the Sample solution correspond to those of the Calculate the percentage of the labeled amounts of
butalbital, aspirin, and caffeine peaks of the Standard butalbital (Cl1H16N203) and caffeine(CSH lON40 2) in the
solution, as obtained in the Assay. portion of Capsules taken:
ASSAY Result = (rulrs) x (CslCu) x 100
• PROCEDURE
Buffer: 1.36 giL of monobasic potassium phosphate in
water

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 USP 43 OfficialMonographs / Butalbital 649

t» = peak response of butalbital or caffeine from the Capsules, equivalent to about 65 mg of aspirin, to an
Sample solution appropriate container. Add 100.0 mL of Diluent, and shake
rs = peak response of butalbital or caffeine from the for 1 min. Promptlyfilter a portion of this solution,
Standardsolution discarding the first 15 mLof the filtrate, and use the clear
Cs = concentration of USP Butalbital RS or USP filtratewithin 20 min after the addition of the Diluent. Ifthe
Caffeine RS in the Standardsolution (mg/mL) intensity of the Sample solution greatly exceeds that of the
Cu = nominal concentration of butalbital or caffeine Standardsolution, the solution may be suitablydiluted with
in the Sample solution (mg/mL) Diluent.
Instrumental conditions
Determine the amount, in mg, of aspirin and salicylic acid Mode: Fluorescence
in the portion of Capsulestaken (W): Excitation wavelength: 305 nm
Emission wavelength: 444 nm
Result = (ru/rs) x Cs x V Analysis
Samples: Standardsolution and Sample solution
tu = peak response of aspirin and salicylic acid from Allow the Samples to equilibrate for 2 min in the
the Sample solution fluorimeter.
ts = peak response of aspirin and salicylic acid from Calculate the percentage of salicylic acid in the portion of
the Standard solution Capsules taken (F):
Cs = concentration of USP Aspirin RS in the Standard
solution (mg/mL) Result = (fu/ls) x (CsICu) x 100
V =volume of the Sample solution (mL)
lu = fluorescence intensity readingsfrom the Sample
Calculate the percentage of the labeled amount of aspirin solution
(C9H s04 ) in the portion of Capsules taken: Is =fluorescence intensityreadingsfrom the Standard
solution
Result = {W - [(FI1 00) x WJ}/(Cu x V) x 100 Cs = concentration of USP Salicylic Acid RS in the
Standardsolution (mg/mL)
W =amount of aspirin and salicylic acid in the portion Cu = nominal concentration of aspirin in the Sample
of Capsules taken to prepare the Sample solution solution (mg/mL)
(mg)
F = percentage of salicylic acid obtained in the Limit Acceptance criteria: NMT 2.5% of salicylic acid
of Free Salicylic Acid procedure (%)
Cu = nominal concentration of aspirin in the Sample ADDITIONAL REQUIREMENTS
solution'(mg/mL) • PACKAGING AND STORAGE: Preserve in tight containers.
V = volume of the Sample solution (mL) • USP REFERENCE STANDARDS (11)
USP Aspirin RS
Acceptance criteria: 90.0%-110.0% each of butalbital, USP Butalbital RS
aspirin, and caffeine . USP Caffeine RS
USP Salicylic Acid RS
PERFORMANCE TESTS
• DISSOLUTION (711)
Medlurn: Water; 1000 mL
Apparatus 2: 50 rpm
Time: 60 min Butalbital, Aspirin, and Caffeine Tablets
Buffer, Mobile phase, Diluent, Salicylic acid solution,
Standard solution, Chromatographic system, and DEFINITION
System suitability: Proceed as directed in the Assay. Butalbital, Aspirin, and Caffeine Tabletscontain NLT 90.0%
Sample solution: Use a portion of solution under test. and NMT 110.0% of the labeled amounts of butalbital
Analysis rc., H'6N203), asplrln (C9H s04) , and caffeine (CSH lON 402).
Samples: Standardsolution and Sample solution
IDENTIFICATION
Calculate the percentages of the labeled amounts of
butalbital (CllH,6N203), aspirin (C9H s04 ) , and caffeine • A. The retention times of the butalbital, aspirin, and caffeine
peaks of the Sample solution correspond to those of the
(CSH lON 40 2) dissolved. butalbital, aspirin, and caffeine peaks of the Standard
Tolerances: NLT 75% (Q) of the labeled amounts of solution, as obtained in the Assay.
butalbital (C"H'6N203), aspirin (C9H s04) , and caffeine
(CsH,oN 40 2) is dissolved. ASSAY
• UNIFORMITY OF DOSAGE UNITS (905): Meet the • PROCEDURE
requirements Buffer: 1.36 giL of monobasic potassium phosphate in
water
IMPURITIES Mobile phase: Methanol and Buffer (45:55) initially
• LIMIT OF FREE SALICYLIC ACID adjusted with phosphoric acid to a pH of 3.9. Ifthe
Use glassware throughout this procedure. Perform this retention time of the salicylic acid peak differs from that of
procedure on the same day the powder is removed from the aspirin peak, adjust the pHof the Mobilephasewith 0.2
the Capsules. N potassium hydroxide or 1 M phosphoric acid so that the
Diluent: Add 1 mL of phosphoric acid to each Lof methanol. salicylic acid peak has the same retention time as that of the
Standard solution: 0.0012 mg/mL of USP Salicylic Acid RS aspirin peak. [NOTE-The retention time of the salicylic acid
in Diluent. Use this solution promptly. peak decreases about 0.3 min for each 0.1 pH increase.The
Sample solution: Nominally 0.65 mg/mL of aspirin from retention time of the aspirin peak is essentially unaffected
the contents of Capsules in solution prepared as follows. by such pH adjustments.]
Transfera suitable portion of the contents of NLT 20

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650 Butalbital / Official Monographs USP 43

Diluent: Methanol and Buffer (45:55) adjusted with Determine the amount, in mg, of aspirin and salicylic acid
phosphoric acid to a pH of 2.5 ± 0.05. in the portion of Tabletstaken (W):
Salicylic acid solution: 0.1 mg/mL of salicylic acid in
Diluent. Pass this solution through a suitablefilterof 0.5-l-Im Result = (ru/rs) x Cs x V
or finer pore size.
Standard stock solution: 1.6 mg/mL of USP Aspirin RS in to = peak response of aspirin and salicylic acid from
Diluent. Sonication and shaking may be used to aid.in the Sample solution
dissolution. Usethis solution within 24 h. rs = peak response of aspirin and salicylic acid from
Standard solution: USP Reference Standards in Standard the Standardsolution
stock solution as listed below. Sonication and shaking the Cs = concentration of USP Aspirin RS in the Standard
solution may be used to promote dissolution. Usethis solution (mg/mL)
solution within 24 h. V = volume of the Sample solution (mL)
Butalbital: 1.6/ mg/mL of USP Butalbital RS, where / is the
ratio of the labeled amount, in mg, of butalbital relative Calculate the percentage of the labeled amount of aspirin
to the labeled amount of aspirin, in mg/Tablet (C9H s04) in the portion of Tabletstaken:
Caffeine: 1 .6j' mg/mL of USP Caffeine RS, where j' is the
ratio of the respective labeled amount, in mg, of caffeine Result = {W - [(F/l 00) x W]}/(Cu x V) x 100
relative to the labeled amount of aspirin in mg/Tablet
Sample solution: Nominally 1.6 mg/mL of aspirinfrom a W = amount of aspirin and salicylic acid in the portion
suitable amount of powdered Tablets in solution prepared of Tablets taken to prepare the Sample solution
as follows. Finely powder NLT 20 Tablets, and transfer a (mg)
portion of this fine powder to an appropriate volumetric F = percentage of salicylic acid obtained in the Limit
flask. Dilute with Diluent to volume, and sonicate for 30 of Free Salicylic Acid procedure (%)
min. Pass a portion of this solution through a suitablefilter Cv = nominal concentration of aspirin in the Sample
of 0.5-lJm or finer pore size, and use the filtrate within solution (mg/mL)
24h. V = volume of the Sample solution (mL)
Chromatographic system
(See Chromatography (621), System Suitability.) Acceptance criteria: 90.0%-110.0% each of butalbital,
Mode: LC . aspirin, and caffeine
Detectors PERFORMANCE TESTS
Butalbital: UV 210 nm • DISSOLUTION (711)
Aspirin and caffeine: UV at the wavelength of the Medium: Water; 900 mL
isosbestic point of aspirin and salicylic acid at about Apparatus 1: 100 rpm
277 nm Time: 60 min
Column: 3.9-mm x 30-cm; packing L1 Buffer, Mobile phase, Diluent, Salicylic acid solution,
Column temperature: 35 ± 1 0
Standard solution, Chromatographic system, and
Flow rate: 1 mL/min System suitability: Proceed as directed in the Assay.
Injection volume: 10 I-IL Sample solution: Use a portion of solution under test.
System suitability Analysis
Samples: Salicylic acid solution and Standardsolution Samples: Standardsolution and Sample solution
[NoTE-The relative retention times for caffeine, Calculate the percentages of the labeled amounts of
aspirin, salicylic acid, and butalbital are about 0.45, butalbital (CllH16N203), aspirin (C9H s04), and caffeine
0.6, 0.6, and 1.0; respectively.] (CSH lON 402) dissolved.
Suitability requirements Tolerances: NLT 80% (Q) ofthe labeled amounts of
Resolution: NLT 2.0 between caffeineand aspirin, butalbital (Cl1H16N203), aspirin (C9H s04), and caffeine
Standardsolution
Column efficiency: NLT 2000 theoretical plates from (CSH10N402) is dissolved.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
butalbital, Standardsolution
Relative standard deviation: NMT 2.0% each for requirements
caffeine, aspirin, and butalbital responses, Standard IMPURITIES
solution • LIMIT OF FREE SALICYLIC ACID
Analysis Use glassware throughout this procedure. Perform this
Samples: Standardsolution and Sample solution procedure on the same day the Tablets are powdered.
Calculate the percentage of the labeled amounts of Diluent: Add 1 mLof phosphoricacid to each Lof methanol. .
butalbital (CllH16N203) and caffeine (CSH10N402) in the Standard solution: 0.0012 mg/mL of USP Salicylic Acid RS
portion of Tablets taken: in Diluent. Use this solution promptly.
Sample solution: Nominally 0.65 mg/mL of aspirin from a
Result = (ru/rs) x (Cs/Cu) x 100 suitable amount of powdered Tablets in solution prepared
as follows. Finely powder NLT 20 Tablets, and transfer a
t» = peak response of butalbital or caffeine from the suitable portion of fine powder, equivalent to 65 mg of
Sample solution aspirin, to an appropriate container. Add 100.0 mLof
ts = peak response of butalbitalor caffeine from the Diluent, and shake by mechanical means for 15 min. Filter
Standardsolution a portion of this solution, discarding the first 15 mLof the
Cs =concentration of USP Butalbital RS or USP filtrate, and use the clearfiltrate within 20 min after the
Caffeine RS in the Standardsolution (mg/mL) addition of the Diluent. Ifthe intensityof the Sample
Cv = nominal concentration of butalbital or caffeine solution greatly exceeds that of the Standardsolution, the
in the Sample solution (mg/mL) solution may be SUitably diluted with Diluent.
Instrumental conditions
Mode: Fluorescence

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 USP 43 OfficialMonographs / Butalbital 651

Excitation wavelength: 305 nm Standard stock solution: 1.6 mg/mL of USP Aspirin RS in
Emissionwavelength: 444 nm Diluent. Sonication and shaking may be used to promote
Analysis dissolution. Usethis solution within 24 h.
Samples: Standardsolution and Sample solution Standard solution: USP Reference Standards in Standard
Allow the Samples to equilibrate for 2 min in the stock solution as listed below. Sonication and shaking the
fluorimeter. solution may be used to promote dissolution. Use this
Calculatethe percentage of salicylic acid in the portion of solution within 24 h.
Tablets taken (F): Butalbital: 1.6} mg/mL of USP Butalbital RS, where} isthe
ratio of the labeled amount of butalbital relative to the
Result = (lvlls) x (CslCv) x 100 labeled amount of aspirin in mg/Capsule
Caffeine: 1.6}' mg/mL of USP Caffeine RS, where}' is the
lo = fluorescence intensity readings from the Sample ratio of the labeled amount of caffeine relative to the
solution labeled amount of aspirin in mg/Capsule
Is =fluorescence intensity readings from the Standard Codeine phosphate: 1.6}"mg/mL of USP Codeine
solution Phosphate RS, where J" is the ratio of the labeled amount
Cs = concentration of USP Salicylic Acid RS in the of codeine phosphate relative to the labeled amount of
Standardsolution (mg/mL) aspirin in mg/Capsule
Cv = nominal concentration of aspirin in the Sample Sample solution: Nominally 1.6 mg/mL of aspirin from the
solution (mg/mL) contents of Capsules in solution prepared as follows.
Transfera suitable portion of the contents of NLT 20
Acceptance criteria: NMT 3.0% of salicylic acid Capsulesto an appropriate volumetricflask. Dilute with
ADDITIONAL REQUIREMENTS Diluent to volume, and sonicate for 30 min. Pass a portion
• PACKAGING AND STORAGE: Preserve in tight containers.
of this solution through a suitable filter of 0.5-l..Im pore size,
• USP REFERENCE STANDARDS (11)
and use the filtrate within 24 h.
USP Aspirin RS Chromatographic system
USP Butalbital RS (See Chromatography (621), System Suitability.)
USP Caffeine RS Mode: LC
USP Salicylic Acid RS Detector
Butalbital and codeine: UV 210 nm
Caffeine and aspirin: UV at the wavelength of the
isosbestic point of aspirin and salicylic acid at about
277 nm
Butalbital, Aspirin, Caffeine, and Column: 3.9-mm x 30-cm; packing Ll
Column temperature: 35 ± 1 0
Codeine Phosphate Capsules Flow rate: 1 mL/min
Injection volume: 10 I..IL
DEFINITION System suitability
Butalbital, Aspirin, Caffeine, and Codeine Phosphate Capsules Samples: Salicylic acid solution and Standard solution
contain NLT 90.0% and NMT 110.0% of the labeled [NoTE-The relative retention times for codeine,
amounts of butalbital (Cll H'6N203), aspirin (C9H s04) , caffeine, aspirin, salicylic acid, and butalbital are
caffeine (CsH,oN 402), and codeine phosphate (C,sH 2,N03· about 0.3, 0.45, 0.6, 0.6, and 1.0, respectively.]
H3P04 • V2H 20). Suitability requirements
IDENTIFICATION Resolution: NLT 2.0 between caffeine and aspirin,
Standardsolution
• A. The retention times of the butalbital, aspirin, caffeine,
and codeine peaks of the Sample solution correspond to Column efficiency: NLT 2000 theoretical plates from
those of the butalbital, aspirin, caffeine, and codeine peaks butalbital, Standardsolution
of the Standardsolution, as obtained in the Assay. Relative standard deviation: NMT 2.0% each for
codeine, caffeine, aspirin, and butalbital responses,
ASSAY Standardsolution
• PROCEDURE Analysis
Buffer: 1.36 giL of monobasic potassium phosphate in Samples: Standardsolution and Sample solution
water Calculate the percentage of the labeled amounts of
Mobile phase: Methanol and Buffer(45:55) initially butalbital (CllH'6N203) and caffeine (CsH,oN 402) in the
adjusted with phosphoric acid to a pH of 3.9. Ifthe portion of Capsules taken:
retention time of the salicylic acid peak differs from that of
the aspirin peak, adjust the pH of the Mobilephasewith 0.2 Result = (rvlrs) x (CsICv) x 100
N potassium hydroxide or 1 M phosphoric acid so that the
salicylic acid peak has the same retention time as that of the ru = peak response of butalbital or caffeine from the
aspirin peak. [NOTE-The retention time of the salicylic acid Sample solution
peak decreases about 0.3 min for each 0.1 pHincrease.The rs = peak response of butalbital or caffeinefrom the
retention time of the aspirin peak is essentially unaffected Standardsolution
by such pH adjustments.] . Cs =concentration of USP Butalbital RS or USP
Diluent: Methanol and Buffer(45:55) adjusted with Caffeine RS in the Standardsolution (mg/mL)
phosphoric acid to a pH of 2.5 ± 0.05. Cv = nominal concentration of butalbital or caffeine
Salicylicacid solution: 0.1 mg/mL of salicylic acid in in the Sample solution (mg/mL)
Diluent. Pass this solution through a suitablefilterof 0.5-l..Im
or finer pore size. Determine the amount of aspirin and salicylic acid in the
portion of Capsules taken (W):

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 652 Butalbital / Officio/Monographs USP 43

Result = (rulr s) x Cs x V Codeine phosphate: 0.16}" mg/mL of USP Codeine

Phosphate RS, where}" is the ratio of the labeled amount
ru = peak response of aspirin and salicylic acid from of codeine phosphate relative to the labeled amount of
the Sample solution aspirin in mg/Capsule
rs = peak response of aspirin and salicylic acid from Sample stock solution: ~ass 20 mL of the ~olution un.der
the Standardsolution test through a suitable filter of 0.5-~m or finer pore Size,
Cs = concentration of USP Aspirin RS in the Standard discarding the first 2 mL of the filtrate.
solution (mg/mL) Sample solution: A portion of the Sample stock solution
V . = volume of the Sample solution (mL) diluted with an equal volume Diluent
Chromatographic system and System sUi~ab.i1ity: Proceed
Calculate the percentage of the labeled amount of aspirin as directed in the Assay, except use an lniectlon volume of
(C9H s04) in the portion of Capsules taken: 100 ~L.
Analysis
Result = {W - [(F/l00) x Wj}/(C u x \I) x 100 Samples: Standardsolution and Sample solution
Calculate the percentages of the labeled amounts of
w = amount of aspirin and salicylic acid in the portion butalbital (CllH16N203), aspirin (C9H s0 4), and caffeine
of Capsules taken to prepare the Sample solution (CSH lON 402) dissolved:
F (mg)
= percentage of salicylic acid0 b l t h e L'imit.
' d In
tame Result = (rulr s) x Cs x V x 100
of Free Salicylic Acid procedure (%)
= nominal concentration of aspirin in the Sample = peak response of the butalbital, aspirin, or
solution (mg/mL) caffeine from the Sample solution
V = volume of the Sample solution (mL) = peak response of the butalbital, aspirin, or
caffeine from the Standardsolution
Calculate the percentage of the labeled amount of codeine = concentration of USP Butalbital RS, USP Aspirin
phosphate (ClsH21N03 . H3P04· Y2H 20) in the portion of RS, or USP Caffeine RS in the Standardsolution
Capsules taken: (mg/mL)
V = volume of the Medium, 1000 mL
Result = (rulrs) x (CsICu) x (M r dMr2 ) x 100
Calculate the percentage of the labeled am.ount of codeine
= peak response of codeine from the Sample phosphate (ClsH21N03' H3P04· V2H 20) dissolved:
solution
= peak response of codeine from the Standard Result = (rulr s) x Cs x V x (M rdMr2 ) x 100
solution
= concentration of USP Codeine Phosphate RS in ru = peak response of codeine from the Sample
the Standardsolution (mg/mL) solution
= nominal concentration of codeine phosphate in rs = peak response of codeine from the Standard
the Sample solution (mg/mL) . solution
Mr 1 =molecular weight of codeine phosphate Cs = concentration of USP Codeine Phosphate RS in
hemihydrate, 406.37 the Standardsolution (mg/mL)
= molecular weight of codeine phosphate V = volume of the Medium, 1000 mL
anhydrous, 397.37 Mr1 = molecular weight of codeine phosphate
hemihydrate,406.37 .
Acceptance criteria: 90.0%-110.0% each of butalbital, Mr2 = molecular weight of codeine phosphate
aspirin, caffeine, and codine phosphate . anhydrous, 397.37
PERFORMANCE TESTS
• DISSOLUTION (711) Tolerances: NLT 75% (Q) of the labeled amounts of
Medium: Water; 1000 mL butalbitalrc., H16N203), aspirin (C9H s0 4), caffeine
Apparatus 2: 50 rpm (CSH10N402), and codeine phosphate (ClsH21N03' H3P04·
Time: 60 min Y2H 20) is dissolved.
Buffer, Mobile phase, and Diluent: Prepare as directed in • UNIFORMITY OF DOSAGE UNITS (905): Meet the
the Assay. requirements
Salicylic acid solution: 0.01 mg/mL of salicylic acid in
Diluent. Pass this solution through a suitable filter of 0.5-~m IMPURITIES
or finer pore size. • LIMIT OF FREE SALICYLIC ACID .
Standard stock solution: 0.16 mg/mL of USP Aspirin RS in Use glassware throughout this procedure. Perform this
a mixture of Diluent and Medium (50:50). Use this solution procedure on the same day the powder is removed from
within 24 h. the Capsules.
Standard solution: USP Reference Standards in Standard Diluent: To each L of methanol add 1 mL of phosphoric
stock solution as listed below. Sonication and shaking the acid.
solution may be used to promote dissolution. Pass a portion Standard solution: 0.0012 mg/mL of USP Salicylic Acid RS
of the resulting solution through a suitable filter of 0.5-~m in Diluent. Use this solution promptly.
or finer pore size. Use this solution within 24 h. Sample solution: Nominally 0.65 mg/mL of aspirin from
Butalbital: 0.16} mg/mL of USP Butalbital RS, where} is the contents of Capsules in solution prepared as follows.
the ratio of the labeled amount of butalbital relative to the Transfer a suitable portion of the contents of NLT 20
labeled amount of aspirin in mg/Capsule Capsules, equivalent to about 65 mg of aspirin, to an
Caffeine: 0.1 6}' mg/mL of USP Caffeine RS, where l' is the appropriate containe~. Add 100:0 mL of piluen~, and shake
ratio of the labeled amount of caffeine relative to the for 1 min. Promptly filter a portion of this solution,
labeled amount of aspirin in mg/Capsule discarding the first 15 mL of the filtrate, and use the clear
. filtrate within 20 min after the addition of the Diluent. If the

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 USP43 Official Monographs / Butamben 653

intensity of the Sample solution greatly exceeds that of the Table 1

Standard solution, the solution may be suitably diluted with Time Solution A Solution B
Diluent. (mIn) (%) (%)
Instrumental conditions 0.0 90 10
Mode: Fluorescence
Excitation wavelength: 305 nm 1.0 90 10
Emission wavelength: 444 nm 6.0 50 50
Analysis
Samples: Standard solution and Sample solution 8.5 50 50
Allow the Samples to equilibrate for 2 min in the 8.6 90 10
fluorimeter.
Calculate the percentage of salicylic acid in the portion of 10.0 90 10
Capsules taken (F):
Standard solution: 0.2 mg/mL of USP Butamben RS
Result = (lulls) x (CslCu) x 100 prepared as follows. Transfer a suitable quantity of USP
Butamben RS to an appropriate volumetric flask. Add 10%
lu = fluorescence intensity readings from the Sample of the total flask volume of acetonitrile to dissolve, then
solution dilute with water to volume.
Is = fluorescence intensity readings from the Standard Sample solution: 0.2 mg/mL of Butamben prepared as
solution follows. Transfer a suitable quantity of Butamben to an
Cs =concentration of USP SalicylicAcid RS in the appropriate volumetric flask. Add 10% of the total flask
Standard solution (mg/mL) volume of acetonitrile to dissolve, then dilute with water to
Cu = nominal concentration of aspirin in the Sample volume.
solution (mg/mL) Chromatographic system
(See Chromatography (621), System Suitability.)
Acceptance criteria: NMT 3.0% of salicylic acid Mode: LC
ADDITIONAL REQUIREMENTS Detector: UV 285 nm
• PACKAGING AND STORAGE: Preserve in tight, light-resistant Column: 2.1-mm x 10-cm; 5-l-Im packing L1
containers. Flow rate: 0.4 mL/min
• USP REFERENCE STANDARDS (11 ) Injection volume: 1 I-IL
USPAspirin RS System suitability
USP Butalbital RS Sample: Standard solution
USP Caffeine RS I
Suitability requirements
USP Codeine Phosphate RS Tailing factor: NMT 2.0
USP SalicylicAcid RS Relative standard deviation: NMT 0.73%
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of butamben (CllH1SNOz) in the
portion of Butamben taken:
Butamben
Result =(r ulrs) x (C siCu) x 100
ru = peak response from the Sample solution
r5 = peak response from the Standard solution
C5 =concentration of USP Butamben RS in the
CllH1SNOz 193.24 Standard solution (mg/mL)
. Benzoic acid, 4-amino-, butyl ester; Cu =concentration of Butamben in the Sample
Butyl p-aminobenzoate [94-25-7]. solution (mg/mL)
DEFINITION Acceptance criteria: 98.0%-102.0% on the dried basis
Butamben contains NLT 98.0% and NMT 102.0% of
butamben (CllH1SNOz), calculated on the dried basis. IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.2%
IDENTIFICATION • ORGANIC IMPURITIES
Solution A, Solution B, Mobile phase, Sample solution,
and Chromatographic system: Proceed as directed in the
Assay.
• ~ ••< .<.>;.,;; Diluent: Acetonitrile and water (10:90)
Spg<9~g9Py;2·l;~i7/.; ;; . . . . • : Standard stock solution: 0.1 mg/mL each of USP
• B. The retention time"of the major peak of the Sample Butamben RS and USPAminobenzoic Acid RS prepared as
solution corresponds to that of the Standard solution, as follows. Transfer a suitable quantity of each Reference
obtained in the Assay. Standard to an appropriate volumetric flask. Add 10% of
the total flask volume of acetonitrile to dissolve, then dilute
ASSAY with water to volume.
• PROCEDURE Standard solution: 0.2 I-Ig/mL each of USP Butamben RS
Solution A: Add 1.0 mL of 98 percent formic acid to 1 L of
and USP Aminobenzoic Acid RS from the Standard stock
water, and mix well. solution in Diluent
Solution B: Add 1.0 mL of 98 percent formic acid to 1 L of
acetonitrile, and mix well.
Mobile phase: See Table 7.

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654 Butamben / OfficialMonographs USP 43

[NOTE-The use of blank injections of Diluent may be

suitable to reduce carryover between injections of the Butoconazole Nitrate
Sample solution and the Standardsolution, ifobserved.]
System suitability
Sample: Standardsolution
Suitability requirements
Resolution: NLT 10 between the aminobenzoic acid and
butamben peaks
Relative standard deviation: NMT 5% for each peak C19H17CI3NzS· HN0 3 474.79
corresponding to aminobenzoic acid and butamben 1H-Imidazole, 1-[4-(4-chlorophenyl)-2-[(2,6-dichlorophenyl)
Analysis thio]butyl-, mononitrate, (±)-;
Samples: Sample solution and Standardsolution (±)-1-[4-(p-Chlorophenyl)-2-[(2,6-dichlorophenyl) thio]butyl]
Calculate the percentage of aminobenzoic acid in the imidazole mononitrate [64872-77-1].
portion of Butamben taken:
DEFINITION
Result =(r vir 5) x (C siC v) x 100 Butoconazole Nitrate contains NLT 98.0% and NMT 102.0%
of butoconazole nitrate (C19H17C13NzS . HN0 3), calculated on
= peak response of aminobenzoic acid from the the dried basis.
Sample solution
= peak response of aminobenzoic acid from the IDENTIFICATION
Standardsolution
= concentration of USP Aminobenzoic Acid RS in
the Standardsolution (mg/mL)
Cv = concentration of Butamben in the Sample
solution (mg/mL)
ASSAY
Calculate the percentage of any individual unspecified • PROCEDURE
impurity in the portion of Butamben taken: Buffer: 2.18 giL of monobasic potassium phosphate and
4.18 giL of dibasic potassium phosphate in water
Result =(r vir s) x (C siC v) x 100 Mobile phase: Methanol and Buffer (3:1)
ru = peak response of any other individual impurity
Standard solution: 0.2 mg/mL of USP ButoconazoleNitrate
from the Sample solution RS in Mobilephase . .
r5 = peak-response of butamben from the Standard Sample solution: 0.2 mg/mL of Butoconazole Nitrate In
solution Mobile phase. Filter.
C5 =concentration of USP Butamben RS in the Chromatographic system
Standardsolution (mg/mL) (See Chromatography (621), System Suitability.)
Cu = concentration of Butamben in the Sample Mode: LC
solution (mg/mL) Detector: UV 229 nm
Column: 4.6"'mm x 25-cm; packing L1
Acceptance criteria: See Table 2. Disregard any impurity Column temperature: 40°
peak lessthan 0.05%. Flow rate: 2 mL/min
Injection volume: 10 IJL
Table 2 System suitability
Sample: Standardsolution
Relative Acceptance
Retention Criteria,
Suitability requirements
Name Time NMT (%) Column efficiency: NLT 2800 theoretical plates
Tailing factor: NMT 1.5
Aminobenzoic acid 0.2 0.10 Relative standard deviation: NMT 1.5%
Butamben 1.0 - Analysis ,
Samples: Standardsolution and Sample solution
Any unspecified impurity - 0.10
Calculate the percentage of butoconazole nitrate
Total impurities - 1.0 (C19H17CI3NzS· HN0 3) in the portion of Butoconazole
Nitrate taken:
SPECIFIC TESTS
• Loss ON DRYING (731) Result = (r vir 5) x (C siC v) x 100
Analysis: Dryover phosphorus pentoxide for 3 h. = peak response from the Sample solution
Acceptance criteria: NMT 1.0%
= peak response from the Standardsolution
ADDITIONAL REQUIREMENTS = concentration of USP Butoconazole Nitrate RS in
• PACKAGING AND STORAGE: Preserve in well-closed the Standardsolution (mg/mL)
containers. Cv = concentration of Butoconazole Nitrate in the
• USP REFERENCE STANDARDS (11) Sample solution (mg/mL)
USP AminobenzoicAcid RS
Benzoic acid, 4-amino. Acceptance criteria: 98.00/0-102.0% on the dried basis
C7H7NOz 137.14
USP Butamben RS IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1%

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USP 43 official Monographs / Butorphanol 655

• ORDINARY IMPURITIES (466) solution, remove them by aspiration, and discard.

Standard solutions: USP Butoconazole Nitrate RS in a Centrifuge or filter the remaining solution.
mixture of methylene chloride and methanol (2:1) Chromatographic system
Test solution: Butoconazole Nitrate in a mixture of (See Chromatography (621), System Suitability.)
methylene chloride and methanol (2:1) Mode: LC
Eluant: Chloroform, tetrahydrofuran, cyclohexane, and Detector: UV 225 nm
ammonium hydroxide (18:18:13:1) Column: 4.6-mm x 25-cm; packing L9 that has been
Visualization: 22 converted to the potassium form by the use of 0.555 M
Acceptance criteria: Meets the requirements potassium acetate solution
Flow rate: 1 mL/min
SPECIFIC TESTS Injection volume: 20 IJL
• Loss ON DRYING (731) System suitability
Analysis: Dry under vacuum at 60° for 3 h. Sample: Standardsolution
Acceptance criteria: NMT 1.0% [NoTE-The relative retention times for butoconazole
ADDITIONAL REQUIREMENTS nitrate and l-benzylimidazole are 0.6 and 1.0,
• PACKAGING AND STORAGE: Preserve in well-closed, respectively.]
light-resistant containers. Suitability requirements
• USP REFERENCE STANDARDS (11) Resolution: NLT 4.0 between the analyte and internal
USP Butoconazole Nitrate RS standard peaks
Column efficiency: NLT 1100 theoretical plates for the
analyte peak
Tailing factor: NMT 2.1 for the analyte peak
Relative standard deviation: NMT 1.5%
Butoconazole Nitrate Vaginal Cream Analysis
Samples: Standardsolution and Sample solution
DEFINITION Calculate the percentage of the labeled amount of
Butoconazole Nitrate Vaginal Cream contains Butoconazole butoconazole nitrate (C19H17CI3N2S . HN0 3) in the portion
Nitrate in a suitable cream base. It contains NLT 90.0% and of Vaginal Cream taken:
NMT 110.0% of the labeled amount of butoconazole nitrate
(C19H17CI3N2S . HN0 3)· Result = (Ru/R s) x (Cs/Cu) x 100
IDENTIFICATION Ru = peak responseratio of butoconazole nitrate to the
• A. internal standard from the Sample solution
Sample solution: Preparea mixture of the Standardsolution Rs = peak responseratio of butoconazole nitrate to the
and the Sample solution (1:1), as directed in the Assay. internal standard from the Standardsolution
Analysis: Chromatograph the Sample solution, as directed in Cs = concentration of USP Butoconazole Nitrate RS in
the Assay. the Standardsolution (mg/mL)
Acceptance criteria: The chromatogram exhibits two main Cu = nominal concentration of butoconazole nitrate
peaks that correspond to butoconazole nitrate and the in the Sample solution (mg/mL)
internal standard.
ASSAY Acceptance criteria: 90.0%-110.0% of the labeled amount
• PROCEDURE PERFORMANCE TESTS
Buffer: 1.4 g of potassium acetate in 980 mL of water. • MINIMUM FILL (755): Meets the requirements
Adjust with about 2 mL of glacial acetic acid to a pH of 4.3
± 0.1, dilute with water to 1000 mL. Adjust the buffer ADDITIONAL REQUIREMENTS
molarity (0.018-0.072 M) as necessaryto obtain suitable • PACKAGING AND STORAGE: Preserve in collapsible tubes or
chromatographic performance. Increased retention time tight containers. Avoid excessive heat, and avoid freezing.
may be achieved by a decrease in the buffer molarity. • USP REFERENCE STANDARDS (11)
Diluent: Methanol and Buffer (60:40) USP Butoconazole Nitrate RS
Mobile phase: Methanol and Buffer (65:35)
Internal standard solution: 1.6 mg/mL of
l-benzylimidazole in methanol
Standard stock solution: 0.4 mg/mL of USP Butoconazole
Nitrate RS in methanol Butorphanol Tartrate
Standard solution: Transfer 2.0 mL of the Standardstock
solution and 3.0 mL of the Internal standard solution to a
50-mL flask, and add 35.0 mL of Diluent.
Sample stock solution: Nominally 0.4 mg/mL of
butoconazole nitrate in methanol from Vaginal Cream X Jl
HO.HO
iL)\.
prepared asfollows. Add 200 mL of methanol to a 250-mL ° HO H
'OH

volumetric flask. Transfer to the flask a weighed quantity of

Vaginal Cream equivalent to about 100 mg of
butoconazole nitrate. Sonicate to dissolve, and cool to
room temperature. Dilute with methanol to volume. C21H29N02' C4H606 477.55
Sample solution: Transfer 2.0 mL of the Sample stock Morphinan-3,14-diol, 17-(cyclobutylmethyl)-, (-)-, [S-
solution and 3.0 mL of the Internal standard solution to a (R*,R*)]-2,3-dihydroxybutanedioate (1:1) (salt).
50-mL flask, and add 35.0 mL of Diluent. Allow the (-)-17-(Cyclobutylmethyl)morphinan-3,14-diol o-(-)-tartrate
precipitated excipients that form to rise to the top of the (1:1) (salt) [58786-99-5].

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 656 Butorphanol / OfficialMonographs USP 43

» Butorphanol Tartrate contains not less than 98.0 tartrate is not lessthan 15 minutes. Calculatethe percentage
percent and not more than 102.0 percent of of synthesis precursors in the test specimen by the formula:
C2l H29N02 • C4H 6061 calculated on the anhydrous 100A viA s
basis.
in which A v isthe sum of the areas of all minor peaks;and As
Packaging and storage-Preserve in tight containers.Store is the sum of the areas of the major and minor peaks.The limit
at 25°, excursions permitted between 15° and 30°. is 2.0%.
USP Reference standards (11)- Assay-Dissolve about 500 mg of Butorphanol Tartrate;
USP Butorphanol Tartrate RS accuratelyweighed, in 75 mL of glacial acetic acid. Addcrystal
Identification- violetTS, and titrate with 0.1 N perchloricacid VS. Perform a
blankdetermination, and makeany necessarycorrection. Each
mLof 0.1 N perchloric acid is equivalent to 47.76 mg of
C2,H 29N02 • C4H 60 6 •
A:-, ·~~Pgctf9~£2P/~cl~~g~l~Si~!~fJTest~'(m9~);1i)ltJXrcl[~CJ
Spect.rqscqpy: 1971\ AICNHvlaY"2020). _.. . .. -- - - ..
B: The R F value of the principalspot in the chromatogram
of the Test preparation corresponds to that in the Butorphanol Tartrate Injection
chromatogram of the Standardpreparation, as obtained for
test A in the Chromatographic purity test.
Specific rotation (781S): between -60° and -66°. » Butorphanol Tartrate Injection is a sterilesolution
Test solution: 4 mg per mL, in methanol. of Butorphanol Tartrate in Water for Injection. It
Water Determination, Method 1(921): not more than 2.0%.
Residue on ignition (281): not more than 0.1%. contains not less than 90.0 percent and not more
than 110.0 percent of the labeled amount of
Chromatographic purity- C2l H29N02 • C4H 606 • It may contain a suitable
METHOD A (Thin-Layer Chromatography)-
Standardsolution-Prepare a solution in methanol containing .preservative and a buffer.
1 mg of USP ButorphanolTartrate RS per mL.
Test solution-Transfer 100 mg of ButorphanolTartrate to a Packaging and storage-Preserve in single-dose or in
1O-mL volumetricflask. Dissolve in methanol, dilute with multiple-dose containers, preferablyof Type Iglass, protec;ted
methanol to volume, and mix. . from light.
lodoplatinate spr~1Y reagent-Prepare a 1 in 10 solution of USP Reference standards (11 )-
chloroplatinicacid in water. To 0.5 mLof this solution add 33 USPButorphanolTartrate RS
mLof water and 1 g of potassium iodide to obtain the spray Identification-Apply 1O-J.JL portions of the Injection and a
reagent. Prepare fresh daily. Standard solution of USP Butorphanol Tartrate RS having the
Procedure-Apply 50 J.JL of the Test solution, containing500 J.Jg same concentration about 2 cm apart to a line parallel to and
of butorphanol tartrate, and 5 J.JL and 10 J.JL of the Standard about 2 cm from the bottom of a thin-layerchromatographic
solution, containing 5 J.Jg and 10 J.Jg of USP Butorphanol plate (see Chromatography (621») coated with a 0.25-mm layer
Tartrate R5, respectively, about 2 cm apart to a line parallel to of chromatographic silica gel mixture. Place the plate in a
and about 2 cm from the bottom of a thin-layer developing chamber containing a mixtureof chloroform, ethyl
c~romatographic plate (see Chromatography (621») coated acetate, and methanol (40:10:9), and develop the
with a 0.25-mm layer of chromatographic silica gel mixture. chromatogram until the solventfront has moved about 10 cm
Placethe plate in a developing chamber containing, and above the line of application. Remove the plate, mark the
equilibrated with, a mixture of chloroform, methanol, solventfront, and allowthe solvent to evaporate. Examine the
benzene, and ammonium hydroxide (85:25:20:5). Develop plate under short-wavelength UV light: the R F value of the
the chromatogram untilthe solventfront has moved about 10 principal spot obtained from the solution under test
em above the line of application. Remove the plate, mark the corresponds to that obtained from the Standard solution.
solvent front, and allow the solvent to evaporate. Spraythe Benzethonium chloride, if present, isobserved as a streaked
plate ~ith lodoplatinatesprayreagent. Estimate the percentage zone near the point of application. Visualize the butorphanol
?f the Impurities present in the Test solution by comparing the spots by lightlyspraying the plate with a 1 in 250 solution of
Intensities of secondary spots, ifpresent, with the intensities of bromocresol purple in dehydrated alcohol: butorphanol
the principal spots obtained from the chromatograms of the appears as a blue spot against a light yellow background.
Standardsolution. The sum of the impurities observed is not pH (791): between 3.0 and 5.5.
greater than 2.0%. Bacterial fndotoxins Test (85) -It contains not more than
METHOD 8 (Gas Chromatography)-Dissolve a suitable 88.0 U5P Endotoxin Units per mg of butorphanol tartrate.
quantity of Butorphanol Tartrate in methanol to obtain a Other requirements-It meets the requirements under
solution containing about 10 mg per mL. Inject 1 J.JL of this Injections and Implanted Drug Products (1).
solution into a suitable gas chromatograph equipped with a Assay-
flame-ionization detector and a 1.8-m x 4-mm glass column Mobile phase-Prepare a mixture of 0.05 M ammonium
containing 3% liquid phase G3 on support 51 AB. The acetate and acetonitrile (3:1) adjusted by the addition of
temperatures of the injection port, column, and detector are glacial acetic acid to a pH of 4.1. The mixture isappropriately
maintained at about 280°, 250°, and 290°, respectively. The filtered and degassed. .
carrier gas is nitrogen. Record a 30-minute chromatogram. Internal standard solution-Dissolve about 50 mg of
Preferably using an electronic integrator, determine the areas propylparaben in 5.0 mL of methanol contained in a 250-mL
of all peaks in the chromatogram excluding the area of the volumetric flask. Add water to volume, and mix.
solvent. Ina suitablechromatogram, the retention time for the Standardpreparation-Transfer about 50 mg of USP
alpha isomer of butorphanol tartrate is 1.2 relative to 1.0 for Butorphanol Tartrate RS, accuratelyweighed, to a 25-mL
butorphanol tartrate; and the retention time of butorphanol volumetric flask containing 1.0 mLof 1 N sulfuric acid. Swirl

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 USP 43 OfficialMonographs / Butorphanol 657

the flaskto dissolve the powder completely, add water to Spray reagent: Prepare a l-in-1 0 solution of chloroplatinic
volume, and mix. Pipet 5 mL of the resulting solution into a acid inwater. To0.5 mLofthissolution, add 33 mLofwater
50-mLvolumetricflask containing 10.0 mLof Internalstandard and 1 g of potassium iodide. Preparefresh daily.
solution. Add water to volume, mix, and filter through a Analysis
microporous filter, discarding the first5 mL of the filtrate and Samples: Standardsolution and Sample solution
collecting the remainder in a suitable container. Proceed as directed in the chapter, except spray the plate
Assay preparation-Transfer an accurately measured with Spray reagent.
volume of Injection, equivalentto about 10 mg of butorphanol Acceptance criteria: The typical RF value is 0.7 for
tartrate, to a 50-mLvolumetric flask. Add 10.0 mLof Internal butorphanol tartrate.
standard solution, mix, add water to volume, and mix. Filter
through a microporous filter, discarding the first 5 mLof the ASSAY
filtrate and collecting the remainder in a suitable container. • PROCEDURE
Chromatographic system (see Chromatography (621 »- Buffer: 3.4 giL of 0.025 M monobasic potassium
phosphate. Filter.
The liquid chromatograph isequipped with a 280-nm
detector and a 4-mm x 30-cm column that contains packing Mobile phase: Acetonitrile, triethylamine, and Buffer
L11. The flow rate is about 2 mL per minute. Chromatograph (15:2:85). Mix thoroughly, and adjust with 85.0%
five replicate injectionsof the Standardpreparation, and record phosphoric acid to a pH of 3.0 ± 0.1.
the peak responses as directed for Procedure: the relative Standard solution: 0.2 mg/mL of USP Butorphanol Tartrate
standard deviation is not more than 1.5%, and the capacity RS in Mobilephase. Mix, and filter, discarding the first 2 mL
factor for butorphanol tartrate is not less than 2.0. of the filtrate. The Standardsolution isstable for at least
Procedure-Separately inject equal volumes (about 20 IJL) 108h.
of the Standardpreparation and the Assay preparation into the Sample solution: Nominally 0.2 mg/mL of butorphanol
chromatograph, adjusting the flow rate and other operating tartrate in Mobilephase prepared as follows. Prepare a
parameters, if necessary, until satisfactory chromatography composite solution by pooling a minimum of four
and peak responses are obtained. Record the chromatograms, containers of Nasal Spray into a suitable glass vessel.
and measure the responsesfor the major peaks. The relative Transferthe equivalent of 20 mg of butorphanol tartrate to
retention times are about 1.7 for propylparaben and 1.0 for a 1OO-mL volumetric flask. Dilute with Mobilephase to
butorphanol tartrate. Calculate the quantity, in mg, of volume, mix, and filter, discarding the first 2 mL of the
CZ1Hz9NOz' C4H 6 0 6 in each mLof the Injection taken by the filtrate.
formula: Chromatographic system
(See Chromatography (621), System SUitability.)
50(CIV)(R viR 5) Mode: LC
Detector: UV 280 nm
in which C is the concentration, in mg per mL, of USP Columns
Butorphanol Tartrate RS in the Standardpreparation; V is the Analytical: 4.6-mm x 15-cm; 5-lJm packing L11
volume, in mL, of Injection taken; and R vand R s are the peak Guard: 4.6-mm x 1-cm; 5-lJm packing L11
response ratios of the butorphanol tartrate peak and the Column temperature: 30°
internal standard peak obtained from the Assay preparation Flow rate: 2.0 mL/min
and the Standardpreparation, respectively. Injection volume: 20 IJL
System suitability
Sample: Standardsolution
Suitability requirements
Tailing factor: NMT 2.0
Butorphanol Tartrate Nasal Spray Relative standard deviation: NMT 2.0%
Analysis
DEFINITION Samples: Standardsolution and Sample solution
Butorphanol Tartrate Nasal Spray isan aqueous solution of Calculatethe percentage of the labeled amount of
butorphanol tartrate for administrationas a metered spray to butorphanol tartrate (CZ1HZ9NOz . C4H60 6) in the portion
the nasal mucosa. It contains the equivalent of NLT 90.0% of Nasal Spray taken:
and NMT 110.0% of the labeled amount of butorphanol
tartrate (CZ1Hz9NOz . C4H 60 6) . Result =(rvlrs) x (CsICv) x 100
IDENTIFICATION = peak response from the Sample solution
• A. The retention time of the major peak of the Sample = peak response from the Standardsolution
solution corresponds to that of the Standardsolution, as = concentration of USP ButorphanolTartrate RS in
obtained in the Assay. the Standardsolution (mg/mL)
• B. THIN-LAYER CHROMATOGRAPHIC IDENTifiCATION TEST = nominal concentration of butorphanol tartrate
(201) in the Sample solution (mg/mL)
Standard solution: 1.0 mg/mL of USP Butorphanol Tartrate
RS in methanol Acceptance criteria: 90.00/0-110.0%
Sample solution: Prepare a composite solution by pooling
the contents of three containers of Nasal Spray into a IMPURITIES
suitable vessel.Transfer1.0 mLofpooled sample to a 10-mL • ORGANIC IMPURITIES
volumetric flask, and dilute with methanol to volume. Buffer: Prepare as directed in the Assay.
Developing solvent system: Chloroform, methanol, Mobile phase: Acetonitrile, triethylamine, and Buffer(15:
benzene, and ammonium hydroxide (17:5:4:1) 5.1: 85). Mixthoroughly, and adjust with 85.0% .
[CAUTIoN-Prepare in a hood whilewearing appropriate phosphoric acid to a pH of 3.0 ± 0.1.
safety gloves, lab coat, and protective eyewear.] Standard solution: 0.005 mg/mL of USP Butorphanol
Tartrate RS

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 658 Butorphanol / Official Monographs USP43

Sensitivity solution: Transfer 2.5 mL of the Standard absence of Staphylococcus aureus and Pseudomonas
solution to a 50-mL volumetric flask, and dilute with water aeruginosa.
to volume. Do not filter. • pH (791): 4.0-6.0
Sample solution: Nominally 1 mg/mL of butorphanol • OSMOLALITY AND OSMOLARITY (785): 252-292
tartrate in water prepared as follows. Prepare a composite mOsmol/kg
solution by pooling a minimum of four containers of Nasal
Spray into a suitable glass vessel. Transfer the equivalent of ADDITIONAL REQUIREMENTS
50 mg of butorphanol tartrate to a 50-mL volumetric flask. • PACKAGING AND STORAGE: Preserve in tight containers at
Dilute with water to volume. Do not filter. controlled room temperature. Store at 25°; excursions
Chromatographic system permitted between 15° and 30°.
(See Chromatography (621), System Suitability.) • USP REFERENCE STANDARDS (11)
Mode: LC USP Butorphanol Tartrate RS
Detector: UV 280 nm
Columns
Analytical: 4.6-mm x 25-cm; 5-~m packing L11
Guard: 4.6-mm x 1-cm; 5-~m packing L11 Cabergoline
Column temperature: 40°
Flow rate: 2.0 mL/min
Injection volume: 60 ~L
Run time: 40 min
System suitability
Samples: Standardsolution and Sensitivity solution
Suitability requirements
Relative standard deviation: NMT 10.0%, Standard
solution C26H37Ns02 451.60
Sensitivity: The peak height for butorphanol tartrate is Ergoline-8p-carboxamide, N-[3-( dimethylamino)propyl]-N-
greater than or equal to three times the baseline noise, · [(ethylamino)carbonyl]-6-(2-propenyl)-;
Sensitivity solution 1-[(6-Allylergolin-8p-yl)carbonyl]-1-[3-(dimethylamino)
Analysis propyl]-3-ethylurea [81409-90-7].
Samples: Standardsolution and Sample solution
Record the chromatograms, and measure the responses DEFINITION
for the butorphanol tartrate peak in the Standard Cabergoline contains NLT98.0% and NMT 102.0% of
solution, and for all known and unknown related cabergoline (C26H37Ns02), calculated on the anhydrous basis
compounds'In the Sample solution. for the crystalline form and on the anhydrous and
Calculate the percentage of each related compound (see solvent-free basis for the amorphous form.
Table 7) and each unknown impurity in the portion of
Nasal Spray taken: . IDENTIFICATION

Result =(ru/rs) x (Cs/Cu) x 100

= peak response of each known or unknown related •
compound from the Sample solution
= peak response from the Standardsolution •
=concentration of USP Butorphanol Tartrate RS in Proceed as directed for Procedure 7 or Procedure 2. The
the Standard solution (mg/mL) criteria for Procedure 7 or Procedure 2 must be met.
= nominal concentration of butorphanol tartrate Procedure 1: Crystallinity (695)
in the Sample solution (mg/mL) Acceptance criteria
For the crystalline form: Meets the requirements
Acceptance criteria: See Table 7. For the amorphous form: Does not meet the
requirements
Table 1 Procedure 2: X-Ray Diffraction (941)
Acceptance
Acceptance criteria
Relative
Retention Criteria, For the crystalline form: A diffraction pattern is present.
Name Time NMT (%) For the amorphous form: No diffraction pattern is
present.
3,14-Dihydroxymorphinan 0.3 0.3
• C. The retention time of the major peak of the Sample
M-Butorphanol 0.7 0.5 solution corresponds to that of the Standardsolution, as
obtained in the Assay.
Butorphanol tartrate 1.0 -
ASSAY
Unknown impurity - 0.3
• PROCEDURE
Total impurities - 1.0 Prepare solutions immediately before use, and protect from
light.
SPECIFIC TESTS Buffer: Dissolve 6.8 9 of monobasic potassium phosphate in
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR 900 mL of water, adjust with phosphoric acid to a pH of
SPECIFIED MICROORGANISMS (62): The total aerobic 2.0, and dilute to 1 L. Add 0.2 mL of triethylamine to the
microbial count does not exceed 10 3 cfu/g or mL, and the resulting solution and mix.
total combined molds and yeasts count does not exceed Mobile phase: Acetonitrile and Buffer (4:21)
10 2 cfu/g or mL. It meets the requirements of the tests for Standard solution: 0.25 mg/mL of USP Cabergoline RS in
Mobile phase. Sonicate if needed.

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USP 43 OfficialMonographs / Cabergoline 659

Sample solution: 0.25 mg/mL of Cabergoline in Mobile ru = peak response of each impurity from the Sample
phase. Sonicate if needed. solution
Chromatographic system rr = sum of all the peak responses from the Sample
(See Chromatography (621), System Suitability.) solution
Mode: LC
Detector: UV 280 nm Acceptance criteria: See Table 7.
Column: 4.0-mm x 25-cm; 10-lJm packing L1
Flow rate: 1.3 mL/min Table 1
Injection volume: 100 IJL Relative Acceptance
System suitability Retention Criteria,
Sample: Standard solution Name Time NMT (%)
Suitability requirements Cabergoline related compound Da 0.3 0.1
Column efficiency: NLT 1000 theoretical plates
Relative standard deviation: NMT 2.0% for five Cabergoline related compound Bb 0.6 0.1
replicate injections Cabergoline related compound Ac 0.8 0.3
Analysis
Samples: Standard solution and Sample solution Cabergoline 1.0 -
Calculate the percentage of cabergoline (C26H37Ns02) in Cabergoline related compound Cd 2.9 0.3
the portion of Cabergoline taken:
Anyother individual unidentified impurity - 0.10
Result =(r vir s) x (C siC v) x 100 Total impurities - 0.8

= peak response from the Sample solution a (6aR,9R, 1OaR)-N-[3-(Dimethylamino)propyl]-7-(prop-2-enyl)-4,6,6a,

= peak response from the Standard solution 7,8,9,10,1Oa-octahydroindolo[4,3-fg]quinoline-9-carboxamide.
b (6aR,9R, 1OaR)-N9-[3-(Dimethylamino)propyl]-N 4- ethyl-7-(prop-2-
= concentration of USP Cabergoline RS in the enyl)6a,7,8,9,1 0,1Oa-hexahydroindolo[4,3-fg]quinoline-4,9(6H)-
Standard solution (mg/mL) dicarboxamide.
=concentration of Cabergoline in the Sample c (6aR,9R, 1OaR)-7-(Prop-2-enyl)-4,6,6a,7,8,9,10,1Oa-octahydroindolo[4,3-fg]
solution(mg/mL) quinoline-9-carboxylic acid.
d (6aR,9R,1 OaR)-N9-[3-(Dimethylamino)propyl]-N 4- ethyl-N 9_
Acceptance criteria (ethyicarbamoyl)-7-(prop-2-enyl)-6a,7,8,9,1 0,1Oa-hexahydroindolo[4,3-fg]
quinoline-4,9(6H)-dicarboxamide.
For the crystalline form: 98.0%-102.0% on the
anhydrous basis SPECIFIC TESTS
For the amorphous form: 98.00/0-102.0% on the • OPTICAL ROTATION, Specific Rotation (781 S)
anhydrous and solvent-free basis Sample solution: 1 mg/mL in alcohol
IMPURITIES Acceptance criteria
• RESIDUE ON IGNITION (281): NMT 0.1 % For the crystalline form: -77 0 to -83 0 on the anhydrous
• ORGANIC IMPURITIES basis
Prepare solutions immediately before use, and protect from For the amorphous form: -7r to -83 0 on the anhydrous
light. and solvent-tree basis
Buffer and Mobile phase: Proceed as directed in the Assay. • WATER DETERMINATION, Method 1(921)
System suitability solution: To 10 mL of 0.1 M sodium Acceptance criteria
hydroxide add 50 mg of Cabergoline and stir for about 15 For the crystalline form: NMT 0.5%
min. To 1 mL of the suspension add 1 mL of 0.1 M . For the amorphous form: NMT 1.5%
hydrochloric acid, and dilute with Mobilephase to 10.0 mL. ADDITIONAL REQUIREMENTS
Sonicate until dissolution is complete. [NOTE-The main • PACKAGING AND STORAGE: Preserve in tight containers,
degradation product obtained is cabergoline related protected from light. If it is labeled as amorphous, preserve
compound A] under nitrogen in tight containers, store cold, and protect
Sample solution: 0.25 mg/mL of Cabergoline in Mobile from light.
phase. Sonicate if needed. • LABELING: Where it is the amorphous form, the label so
Chromatographic system indicates.
(See Chromatography (621), System Suitability.) • USP REFERENCE STANDARDS (11)
Mode: LC USP Cabergoline RS
Detector: UV 280 nm
Column: 4.0-mm x 25-cm; 10-lJm packing L1
Flow rate: 1.3 mL/min
Injection volume: 100 IJL
System suitability Cabergoline Tablets
Sample: System suitability solution
[NOTE-See Table 7 for relative retention times.] DEFINITION
Suitability requirements Cabergoline Tablets contain NLT 90.0% and NMT 110.0% of
Resolution: NLT 3.0 between cabergoline and the labeled amount of cabergoline (C26H37Ns02)'
cabergoline related compound A
Analysis IDENTIFICATION
Sample: Sample solution • A. The retention time of the major peak of the Identification
Calculate the percentage of each impurity in the portion of sample solution corresponds to that of the Identification
Cabergoline taken: standard solution, as obtained in the Assay.
• B. The UV-Vis spectrum of the major peak of the
Result = (r vir r) x 100 Identification sample solution corresponds to that of the
Identification standardsolution, as obtained in the Assay.

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660 Cabergoline / OfficialMonographs USP 43

ASSAY System suitability

• PROCEDURE Sample: Standardsolution
Prepare solutions immediately before use, and protect from Suitability requirements
light. Column efficiency: NLT 3000 theoretical plates
Buffer: Transfer 6.8 9 of monobasic potassium phosphate Relative standard deviation: NMT 2%
to a 1-L volumetric flask. Dissolve the contents in 900 mL Analysis
of water. Adjust with phosphoric acid to a pH of 2.0. Dilute .Samples: Standardsolution and Sample solution
with water to volume, and add 0.2 mL of triethylamine. Calculate the percentage of the labeled amount of
Mobile phase: Acetonitrile and Buffer(16:84) cabergoline (Cz6H37NsOz) dissolved:
Standard solution: 0.25 mg/mL of USP Cabergoline RS in
Mobilephase. Sonication may be used to aid in the Result =(r ulrs) x C s x V x (1I L) x 100
dissolution of cabergoline.
Identification standard solution: 0.1 mg/mL of USP ru = peak response from the Sample solution
Cabergoline RS from the Standardsolution in Mobf/ephase. rs = peak response from the Standardsolution
[NOTE-This solution is used for Identification A and Cs =concentration of USP Cabergoline RS in the
Identification B.] Standardsolution (mg/mL)
Sample solution: Nominally 0.25 mg/mL of cabergoline V = volume of Medium, 500 mL
from finely powdered Tablets in solution prepared as L = label claim (mg/Tablet)
follows. Finely powder NLT 20 Tablets, and transfer a
suitable portion of this fine powder to an appropriate Tolerances: NLT 75% (Q) of the labeled amount of
volumetric flask. Dilute with Mobile phase to volume, and cabergoline (Cz6H37NsOz) is dissolved.
sonicate until completely dissolved. The resulting solution • UNIFORMITY OF DOSAGE UNITS (905): Meet the
may be passed through a PVDF-type filter of 0.45-l.Jm pore requirements
size before analysis.
Identification sample solution: Nominally 0.1 mg/mL of IMPURITIES
cabergoline from the Sample solution in Mobile phase. • ORGANIC IMPURITIES
[NOTE-This solution is used for Identification A and Prepare solutions immediately before use, and protect from
Identification B.] light.
Chromatographic system Buffer,' Mobile phase, and Sample solution: Prepare as
(See Chromatography (621), System Suitability.) directed in the Assay.
Mode: LC System suitability solution: To 10 mL of 0.1 N sodium
Detector: UV 280 nm. For IdentificationB, use a diode array hydroxide, add 50 mg of cabergoline. Stir for 15 min. To 1
detector in the range of 210--400 nm. mL of the suspension, add 1 mL of 0.1 N hydrochloric
Column: 4.0-mm x 25-cm; 10-l.Jm packing L1 acid, and dilute with Mobile phase to 10 mL. Sonicate until
Flow rate: 1.3 mL/min dissolution is complete. The main degradation product
Injection volume: 100 I.JL obtained is cabergoline acid.
System suitability Chromatographic system: Proceed as directed in the
Sample: Standardsolution Assay, except for the Injection volume.
Suitability requirements Injection volume
Column efficiency: NLT 1000 theoretical plates System suitability solution: 20 I.JL
Relative standard deviation: NMT 2.0% Sample solution: 100 I.JL
Analysis System suitability
Samples: Standardsolution, Identification standard Sample: System suitability solution
solution, Sample solution, and Identificationsamplesolution [NOTE-See Table 1 for relative retention times.]
Calculate the percentage of the labeled amount of Suitability requirements
cabergoline (Cz6H37NsOz) in the portion of Tablets taken: Resolution: NLT 3.0 between cabergoline and
cabergoline acid
Result = (r ulr s) x (C siC u) x 100 Analysis
Sample: Sample solution
= peak response from the Sample solution Calculate the percentage of each impurity in the portion of
.= peak response from the Standardsolution Tablets taken:
= concentration of USP Cabergoline RS in the Result = (r ulr T) x 100
Standardsolution (mg/mL)
= nominal concentration of cabergoline in the ru = peak response of each impurity from the Sample
Sample solution (mg/mL) solution
Acceptance criteria: 90.0%-110.0% rT = sum of peak responses of all impurities and
cabergoline from the Sample solution
PERFORMANCE TESTS
• DISSOLUTION (711) Calculate the percentage of total impurities in the portion
Medium: 0.1 N hydrochloric acid; 500 mL, degassed with of Tablets taken:
helium
Apparatus 2: 50 rpm Result = (r ulr T) x 100
Time: 15 min
Buffer, Mobile phase, and Chromatographic system: ru =sum of peak responses of all impurities from the
Proceed as directed in the Assay. Sample solution
Standard solution: 0.001 mg/mL of USP Cabergoline RS in rT = sum of peak responses of all impurities and
Medium cabergoline from the Sample solution
Sample solution: Pass a portion of the solution under test
through a suitable filter, discarding the first few mL. Acceptance criteria: See Table 1.

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 USP 43 Official Monographs / Caffeine 661

Table 1 Column: 4.6-mm x 15-cm; packing L1

Relative Acceptance Flow rate: 1 mL/min
Retention Criteria, Injection size: 10 IJL
Name Time NMT (%) System suitability
Cabergoline aclds 0.8 2.0 Sample: Standardsolution
[NoTE-The relative retention times for theophylline
Cabergoline 1.0 - and caffeine are 0.69 and 1.0/ respectively.]
Cabergoline N-oxide b 1.4 1.0 Suitability requirements
Resolution: NLT 6.0 between theophylline and caffeine
Any unspecified degradation product - 0.5 Tailing factor: NMT 2.0 for theophylline and caffeine
Total impurities - 2.5 peaks
Relative standard deviation: NMT 2.0%
a (6aR,9R, 1OaR)-7-Allyl-4,6,6a,7,8,9,10,1Oa-octahydroindolo[4,3-fg]quinoline- Analysis
9-carboxylic acid. Samples: Standardsolution and Sample solution
b (6aR,9 R,1OaR)-7 -Allyl.N-(3-(dimethylazinoyl)propyl)-N-(ethylcarbamoyl)- Calculate the percentage of caffeine (C8H lON 40 2) in the
4,6,6a,7,8,9,10,1Oa-octahydroindolo[4,3-fg]quinoline-9-carboxamide.
portion of Caffeine taken:
ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in Iight-resistant, tight
Result =(r vIr s) x (C sICv) x 100
containers, and store at controlled room temperature. =peak response of caffeine from the Sample solution
• USP REFERENCE STANDARDS (11)
USP Cabergoline RS = peak response of caffeine from the Standard
solution
= concentration of .USP Caffeine RS in the Standard
solution (mg/mL)
=concentration of Caffeine in the Sample solution
Caffeine (mg/mL)

Acceptance criteria: 98.50/0-101.0% on the anhydrous

basis
IMPURITIES
• RESIDUE ON IGNITION (281): NMT 0.1 %
C8H lON 40Z • H20 212.21 • ORGANIC IMPURITIES
Mobile phase, Standard solution, Sample solution,
C8H10N40Z 194.19 Chromatographic system, and System suitability:
1H-Purine-2/6-dione, 3/7-dihydro-1/3/7-trimethyl-; Proceed as directed in the Assay.
1/3/7-Trimethylxanthine [58-08-2]. Analysis
Monohydrate [5743-12-4]. Sample: Sample solution
Calculate the percentage of each impurity in the portion of
DEFINITION Caffeine taken:
Caffeine is anhydrous or contains one molecule of water of
hydration. It contains NLT 98.5% and NMT 191.0% of Result =(r vIr r) x 100
C8H lON 40Z/ calculated on the anhydrous basis.
IDENTIFICATION
= peak response for each impurity from the
Sample solution
= sum of the responses of all the peaks from the
Sample solution
• A•. ~~~~~~~.~~~~i~·;'~ Acceptance criteria
$pectr()§¢()PY:J?~M··.A:·.··(CNiin Individual impurities: NMT 0.1%
• B. The retention time of the peak of the Sample Total impurities: NMT 0.1 %
solution corresponds to that of the Standardsolution, as
obtained in the Assay. SPECIFIC TESTS
• WATER DETERMINATION, Method 11/ (921): Dry a sample at
ASSAY
80 0 for 4 h: the anhydrous form loses NMT 0.5% of its
• PROCEDURE weight, and the hydrous form loses NMT 8.5% of its
Buffer: 0.82 gIL of anhydrous sodium acetate weight.
Mobile phase: Acetonitrile, tetrahydrofuran, and Buffer
(25:20:955). Adjust with glacial acetic acid to a pH of 4.5. ADDITIONAL REQUIREMENTS
System suitability solution: 0.02 mg/mL of theophylline in • PACKAGING AND STORAGE: Preserve hydrous Caffeine in
Mobile phase. Shake, and sonicate if necessary, to dissolve. tight containers. Preserve anhydrous Caffeine in well-closed
Standard solution: Transfer 5.0 mg of USP Caffeine RS to a containers.
25-mL volumetric flask. Add 5.0 mL of the System suitability • LABELING: Label it to indicate whether it is anhydrous or
solution and 10 mL of Mobile phase. Shake, and sonicate if hydrous.
necessary. Dilute with Mobile phase to volume, and filter. • USP REFERENCE STANDARDS (11)
Sample solution: 0.2 mg/mL of Caffeine in Mobilephase. USP Caffeine RS
[NoTE-Shake, and sonicate if necessary, to dissolve.]
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 275 nm

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662 Caffeine / OfficialMonographs USP 43

Caffeine Citrate Injection rs = peak response from the Standardsolution

Cs =concentration of USP Caffeine RS in the Standard
DEfiNITION solution (mg/mL)
Caffeine Citrate Injection is a sterile solution containing M r = molecular weight of caffeine citrate, 386.31
Caffeine and citric acid in Water for Injection. It contains NLT M ,2 = molecular weight of caffeine, 194.19
90.0% and NMT 110.0% of the labeled amount of caffeine
citrate (C14H1SN409)' It contains no bacteriostat or other Calculate the percentage of the labeled amount of caffeine
preservative. citrate (C14H1SN409) in the portion of Injection taken:
IDENTifiCATION Result = (C AIC u) x 100
• A. The retention time of the major peak of the Sample
solution corresponds to that of the Standardsolution, as CA = concentration of caffeine citrate in the Sample
obtained in the Assay. solution
• B. IDENTIFICATION TESTS-GENERAL, Citrate (191): Meets Cu = nominal concentration of caffeine citrate in the
the requirements Sample solution
• C.
Solution A: Transfer 4 g of potassium iodide to a 100-mL Acceptance criteria: 90.00/0-110.0%
volumetric flask, and add 10 mL of water. Shake until the
potassium iodide is dissolved. Add 2 9 of iodine to the IMPURITIES
volumetric flask, and shake until dissolved. Dilute with • ORGANIC IMPURITIES
water to volume. Mobile phase, Theophylline stock solution, Standard
Sample solution: 5.0 mL of Injection solution, Sample solution, and Chromatographic
Analysis: Transfer the Sample solution to a 25-mL centrifuge system: Proceed as directed in the Assay.
tube, and add 5 drops of Solution A. Add 0.5 mL of 2.0 M Sensitivity solution: 5 I.Ig/mL of caffeine and 0.1 I.Ig/mL of
hydrochloric acid. theophylline from the Standardsolution in water
Acceptance criteria: A brown precipitate that dissolves on System suitability
neutralization with 0.5 mL of sodium hydroxide TS is Sample: Sensitivity solution
produced. Suitability requirements
Sensitivity: The theophylline peak is discernible.
ASSAY Analysis
• PROCEDURE Samples: Standardsolution and Sample solution
Mobile phase: Acetonitrile, tetrahydrofuran, and 0.01 M Calculate the percentage of each impurity in the portion of
sodium acetate (5:4:191) adjusted with glacial acetic acid Injection taken:
to a pH of 4.5
Theophylline stock solution: 0.02 mg/mL of theophylline Result = (r ulr s) x (C siC A) x (M ,dM (2) x (1IF) x 100
in water
Standard solution: 0.2 mg/mL of USP Caffeine RS and = peak response of each impurity from the Sample
0.004 mg/mL of theophylline from Theophylline stock solution
solution in water = peak response of caffeine from the Standard
Sample solution: Nominally 0.4 mg/mL of caffeine citrate solution
(equivalent to 0.2 mg/mL of caffeine) from Injection in = concentration of USP Caffeine RS in the Standard
water prepared as follows. Transfer a volume of Injection, solution (mg/mL)
equivalent to 50 mg of caffeine, to a 250-mL volumetric = concentration of caffeine citrate in the Sample
flask. Dilute with water to volume, pass through a solution as determined in the Assay
polyvinylidene difluoride or equivalent membrane of M" = molecular weight of caffeine citrate, 386.31
0.45-l.Im pore size, and use the filtrate. M ,2 = molecular weight of caffeine, 194.19
Chromatographic system F = relative response factor (see Table 7)
(See Chromatography (621), System Suitability.)
Mode: LC Acceptance criteria: See Table 7.
Detector: UV 275 nm
Column: 4.6-mm x 15-cm; 5-l.Im packing L1 Table 1
Flow rate: 1 mL/min Relative Relative Acceptance
Injection volume: 10 I.IL Retention Response Criteria,
System suitability Name Time Factor NMT(%)
Sample: Standardsolution Theobromine 0.4 1.13 0.10
[NOTE-See Table 7 for the relative retention times.]
Suitability requirements Paraxanthine 0.6 0.909 0.10
Resolution: NLT 6.0 between theophylline and caffeine Theophylline 0.7 1.10 0.10
Tailing factor: NMT 2.0 each for theophylline and
caffeine Caffeine 1.0 - -
Relative standard deviation: NMT 2.0% for caffeine Anyindividual
Analysis impurity - 1.0 0.10
Samples: Standardsolution and Sample solution Total impurities - - 0.1
Calculate the concentration (C A)' in mg/mL, of caffeine
citrate in the Sample solution:
SPECIFICTESTS
Result = (r ulr s) x C s x (M ,dM (2) • COLOR AND CLARITY
Analysis: Transfer a suitable portion of the Injection to a
ru = peak response from the Sample solution clear glass test tube, and visually examine the solution in a
well-lighted area.

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 USP 43 OfficialMonographs / Caffeine 663

Acceptance criteria: The solution is colorless and free of Mode: LC

haze, obvious turbidity, and precipitate. Detector: UV 275 nm
• BACTERIAL ENDOTOXINS TEST (85): NMT 0.25 USP Column: 4.6-mm x 15-cm; 5-lJm packing L1
Endotoxin Units/mg of caffeine Flow rate: 1 mL/min
• STERILITY TESTS (71): It meets the requirements of the Test Injection volume: 10 lJL
for Sterility of the Product to Be Examined, Membrane System suitability
Filtration. Sample: Standardsolution
• pH (791): 4.2-5.2 [NOTE-See Table 1 for the relative retention times.]
• PARTICULATE MATTER IN INJECTIONS (788): NMT 150 SUitability requirements
particles are equal to or greater than 10 IJm, and NMT 25 Resolution: NLT 6.0 between theophylline and caffeine
particles are equal to or greater than 25 IJm. Tailing factor: NMT 2.0 each for the theophylline and
• OTHER REQUIREMENTS: It meets the requirements under caffeine
Injections and Implanted Drug Products (1). Relative standard deviation: NMT 2.0% for caffeine
Analysis
ADDITIONAL REQUIREMENTS
Samples: Standardsolution and Sample solution
• PACKAGING AND STORAGE: Preserve in single-dose, tight
Calculate the concentration (CA) , in mg/mL, of caffeine
containers of Type I glass, and store between 15°-30°.
citrate in the Sample solution:
• USP REFERENCE STANDARDS (11)
USP Caffeine RS
Result = (r ulrs) x Cs x (Mrd Mr2)

rv =peak response from the Sample solution

rs =peak response from the Standard solution
Caffeine Citrate Oral Solution Cs = concentration of USP Caffeine RS in the Standard
solution (mg/mL)
DEFINITION Mr1 = molecular weight of caffeine citrate, 386.31
Caffeine Citrate Oral Solution is a sterile aqueous solution Mr2 = molecular weight of caffeine, 194.19
containing Caffeine and citric acid. It contains NLT 90.0%
and NMT 110.0% of the labeled amount of caffeine citrate Calculate the percentage of the labeled amount of caffeine
(C14H18N409)' It contains no bacteriostat or other citrate (C14H18N409) in the portion of Oral Solution taken:
preservative.
Result = (CAl Cu) x 100
IDENTIFICATION
• A. The retention time of the major peak of the Sample CA =concentration of caffeine citrate in the Sample
solution corresponds to that of the Standardsolution, as solution
obtained in the Assay. Cv =nominal concentration of caffeine citrate in the
• B. IDENTIFICATION TESTS-GENERAL, Citrate (191): Meets Sample solution
the requirements
.. c. Acceptance criteria: 90.00/0-110.0%
Solution A: Transfer 4 9 of potassium iodide to a 100-mL
volumetric flask, and add 10 mL of water. Shake until the IMPURITIES
potassium iodide is dissolved. Add 2 9 of iodine to the • ORGANIC IMPURITIES
volumetric flask, and shake until dissolved. Dilute with Mobile phase, Theophylline stock solution, Standard
water to volume. solution, Sample solution, and Chromatographic
Sample solution: 5.0 mL of Oral Solution system: Proceed as directed in the Assay.
Analysis: Transfer the Sample solution to a 25-mL centrifuge Sensitivity solution: 5 lJg/mL of caffeine and 0.1 IJg/mL of
tube, and add 5 drops of Solution A. Add 0.5 mL of 2.0 M theophylline from the Standardsolution in water
hydrochloric acid. System suitability
Acceptance criteria: A brown precipitate that dissolves on Sample: Sensitivity solution
neutralization with 0.5 mL of sodium hydroxide TS is SUitability requirements
produced. Sensitivity: The theophylline peak is discernible.
Analysis
ASSAY Samples: Standardsolution and Sample solution
.. PROCEDURE Calculate the percentage of each impurity in the portion of
Mobile phase: Acetonitrile, tetrahydrofuran, and 0.01 M Oral Solution taken:
sodium acetate (5:4:191) adjusted with glacial acetic acid
to a pH of 4.5 Result = (rulrs) x (CSICA) x (M r1IMr2) x (1 IF) x 100
Theophylline stock solution: 0.02 mg/mL of theophylline
in water tu =peak response of each impurity from the Sample
Standard solution: 0.2 mg/mL of USP Caffeine RS and solution
0.004 mg/mL of theophylline from Theophylline stock rs =peak response of caffeine from the Standard
solution in water solution
Sample solution: Nominally 0.4 mg/mL of caffeine citrate Cs =concentration of USP Caffeine RS in the Standard
(equivalent to 0.2 mg/mL of caffeine) from Oral Solution in solution (mg/mL)
water prepared as follows. Transfer a volume of Oral CA = concentration of caffeine citrate in theSample
Solution, equivalent to 50 mg of caffeine, to a 250-mL solution as determined in the Assay
volumetric flask. Dilute with water to volume, pass through Mr1 = molecular weight of caffeine citrate, 386.31
a polyvinylidene difluoride or equivalent membrane of Mr2 = molecular weight of caffeine, 194.19
0.45-lJm pore size, and use the filtrate. F = relative response factor, (see Table 1)
Chromatographic system .
(See Chromatography (621), System Suitability.) Acceptance criteria: See Table 1.

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 664 Caffeine / Official Monographs USP 43

Table 1 chloroform to effect complete extraction of the caffeine,

Relative Relative Acceptance passing each chloroform extract through a small filter
Retention Response Criteria, previously moistened with chloroform into a tared dish.
Name Time Factor NMT (%) Retain the water layer for the Assay for Sodium Benzoate.
Theobromine 0.4 1.13 0.10 Wash the stem of the separator, the filter, and the funnel
with 10 mL of hot chloroform, adding the washings to the
Paraxanthine 0.6 0.909 0.10 dish. Evaporate the combined chloroform solutions on a
Theophylline 0.7 1.10 0.10 steam bath, adding 2 mL of alcohol just before the lasttrace
of chloroform is expelled. Complete the evaporation of the
Caffeine 1.0 - - solvent, dry the residue, consisting of caffeine (CSH10N402),
Any individual at 80° for 4 h. Cool, and weigh.
impurity - 1.0 0.10 Acceptance criteria: 90.00/0-110.0%
Total impurities - - 0.1 e SODIUM BENZOATE
Sample solution: Use the water layer obtained in the Assay
for Caffeine.
SPECIFICTESTS Titrimetric system
e STERILITY TESTS (71): It meets the requirements of the Test
Mode: Direct titration
for Sterility of the Product to Be Examined, Membrane Titrant: 0.1 N hydrochloric acid VS
Filtration. Endpoint detection: Visual
• pH (791): 4.2-5.2 Analysis: Add 75 mL of ether and 5 drops of methyl orange
ADDITIONAL REQUIREMENTS TS to the Sample solution. Titrate with Titrant, and shake
e PACKAGING AND STORAGE: Preserve in single-dose, tight Vigorously until a permanent pink color is produced in the
containers, and store at a temperature between 15°-30°. water layer. Each mL of 0.1 N hydrochloric acid is
e USP REFERENCE STANDARDS (11) equivalent to 14.41 mg of sodium benzoate (C7H sNa0 2).
USP Caffeine RS Acceptance criteria: 90.00/0-110.0%
SPECIFIC TESTS
e BACTERIAL ENDOTOXINS TEST (85): NMT 0.7 USP Endotoxin
Unit/mg of caffeine and sodium benzoate, based on the
Caffeine and Sodium Benzoate Injection total, in mg, of the labeled amounts
e pH (791): 6.5-8.5
DEFINITION e OTHER REQUIREMENTS: It meets the requirements in
Caffeine and Sodium Benzoate Injection is a sterile solution Injections and Implanted Drug Products (1).
containing equal amounts of Caffeine and Sodium Benzoate ADDITIONAL REQUIREMENTS
in Water for Injection. It contains NLT 90.0% and NMT e PACKAGING AND STORAGE: Preserve in single-dose
110.0% of the labeled amounts of anhydrous caffeine containers, preferably of Type I glass.
(CSH10N402) and sodium benzoate (C7H sNa02). e USP REFERENCE STANDARDS (11)
IDENTIFICATION USP Caffeine RS

eA.
Sp~q[i
Calamine
Sample: Use the residue from the Assay for Caffeine. Iron oxide (Fe20 3) , mixture with zinc oxide;
Acceptance criteria: Meets the requirements
e B. Calamine (pharmaceutical preparation) [8011-96-9].
Analysis: Dip the end of a platinum wire into a portion of DEFINITION
Injection, and introduce it into a nonluminous flame. Calamine is Zinc Oxide with a small proportion of ferric oxide,
Acceptance criteria: The flame is colored intensely yellow. and contains, after ignition, NLT 98.0% and NMT 100.5%
e C. of zinc oxide (ZnO).
Analysis
Part 1: Add a few drops of ferric chloride TSto a 0.5-mL IDENTIFICATION
portion of Injection. e A. IDENTIFICATION TESTS-GENERAL, Zinc (191)
Part 2: Add 3 N hydrochloric acid to another portion of Sample: 1 g
Injection. Analysis: Treat the Sample with 10 mL of 3 N hydrochloric
Acceptance criteria: The criteria in Part 7 and Part 2 must acid, and filter.
both be met. Acceptance criteria: The filtrate meets the requirements.
Part 1: A salmon-colored precipitate is formed. e B.
Part 2: A white precipitate is formed. Sample: 1 g
Analysis: Add 10 mL of 3 N hydrochloric acid to the Sample,
ASSAY heat to boil, and filter. .
e CAFFEINE Acceptance criteria: The filtrate assumes a reddish color
Sample solution: A volume of Injection equivalent to 250 upon the addition of ammonium thiocyanate TS.
mg each of caffeine and sodium benzoate
Analysis: Transfer the Sample solution with the aid of 5 mL ASSAY
e PROCEDURE
of water to a small separator, add 1 drop of
phenolphthalein TS, and add 0.1 N sodium hydroxide, Sample solution: Digest 1.5 g of freshly ignited Calamine in
dropwise, until a permanent pink color is just produced. 50.0 mL of 1 N sulfuric acid VS, applying gentle heat until
Shakethe mixture with three or more 20-mL portions of no further solution occurs. Filter the mixture, and wash the

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 USP 43 OfficialMonographs / Calamine 665

residue on the filter with hot water until the last washing is
neutral to litmus paper. To the combined filtrate and Calamine Topical Suspension
washings add 2.5 g of ammonium chloride. Cool, and add
methyl orange TS. » Prepare Calamine Topical Suspension as follows.
Titrimetric system
Mode: Backtitration
Titrant: 1 N sodium hydroxide VS Calamine . 80 g
Endpoint detection: Visual
Analysis: Titrate the excesssulfuric acid in the Sample Zinc Oxide .. 80 g
solution with Titrant. Each mL of 1 N sulfuric acid
comsumed is equivalent to 40.69 mg of zinc oxide (ZnO). Glycerin . 20 mL
Acceptance criteria: 98.0%-100.5% on the ignited basis
Bentonite Magma .. 250 mL
IMPURITIES -----
• ARSENIC, Method J (211): NMT 8 ppm Calcium Hydroxide Topical Solution,
• CALCIUM a sufficient quantity to make .. 1000 mL
Sample: 1 g
Analysis: Digest the Sample in 25 mL of 3 N hydrochloric
acid for 30 min, filter to remove the insoluble ferric oxide,
Dilute the Bentonite Magma with an equal
and add 6 N ammonium hydroxide to the filtrate until the volume of Calcium Hydroxide Topical Solution.
precipitate first formed is redissolved, then add 5 mL more Mix the powders intimately with the Glycerin and
of 6 N ammonium hydroxide. To 10 mL of this solution add about 100 mL of the diluted magma, triturating
2 mL of ammonium oxalate TS. until a smooth, uniform paste is formed. Gradually
Acceptance criteria: NMT a slight turbidity is produced.
• CALCIUM OR MAGNESIUM
incorporate the remainder of the diluted magma.
Analysis: To another 1O-mLportion of the solution prepared Finally add enough Calcium Hydroxide Topical
in the test for Calcium, add 2 mL of dibasic sodium Solution to make 1000 mL, and shake.
phosphate TS. If a more viscous consistency in the Calamine
Acceptance criteria: NMT a slight turbidity is produced. Topical Suspension is desired, the quantity of
• LEAD .
Sample: 1 g Bentonite Magma may be increased to not more
Analysis: Add 15 mL of water to the Sample, stir, then add than 400 mL.
3 mL of glacial acetic acid, and warm on a steam bath until rNoTE-Shake the Calamine Topical Suspension
dissolved. Filter, and add 5 drops of potassium chromate before dispensing.] .
TS.
Acceptance criteria: No turbidity is produced. Packaging and storage-Preserve in tight containers.
Microbial enumeration tests (61) and Tests for specified
SPECIFIC TESTS microorganisms (62)-lt meets the requirements of the tests
• ACID-INSOLUBLE SUBSTANCES for absence of Staphylococcus aureus and Pseudomonas
Sample: 2.0 g aeruginosa.
Analysis: Dissolve the Sample in 50 mL of 3 N hydrochloric
acid. If an insoluble residue remains, collect it on a tared
filter, wash with water, and dry at 105 0 for 1'h. Cool, and
weigh. ,
Acceptance criteria: NMT 40 mg (2.0%) Phenolated Calamine Topical
• ALKALINE SUBSTANCES
Sample: 1.0 g Suspension
Analysis: Digest the Sample with 20 mL of water on a steam
bath for 15 min, filter, and add 2 drops of phenolphthalein » Prepare Phenolated Calamine Topical Suspension
TS.
Acceptance criteria: If a red color is produced, NMT 0.20 as follows:
mL of 0.10 N sulfuric acid is required to discharge it.
• Loss ON IGNITION (733) Liquefied Phenol.. .. 10 mL
Sample: 2 g
Analysis: Ignite the Sample at 500 0 to constant weight. Calamine Topical Suspension .. 990 mL
Acceptance criteria: NMT 2.0% -----
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR To make . 1000 mL
SPECIFIED MICROORGANISMS (62): It meets the
requirements of the tests for absence of Staphylococcus Mix the ingredients.
aureus and Pseudomonas aeruginosa.
rNOTE-ShaKe Phenolated Calamine Topical
ADDITIONAL REQUIREMENTS Suspension before dispensing.]
• PACKAGING AND STORAGE: Preserve in well-closed
containers. Packaging and storage-Preserve in tight containers.

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666 Calcifediol / OfficialMonographs USP 43

the ratios of the peak responses of calcifediol to testosterone

Calcifediol obtained from the Assay preparation and the Standard
preparation, respectively.
HaC OH

CHa

HO • Calcifediol Capsules
H

C27H4402 . H20 418.65 » Calcifediol Capsules contain not less than 90.0
9,1 0-Secocholesta-5,7, 10(19)-triene-3,25-diol monohydrate, percent and not more than 120.0 percent of the
(3P,5Z, 7 E)-. labeled amount of C27H4402 . H20.
25-Hydroxycholecalciferol monohydrate [63283-36-3J.
Packaging and storage-Preserve in tight, light-resistant
» Calcifediol contains not less than 97.0 percent containers.
and not more than 103.0 percent of C27H 440 2 . USP Reference standards (11 )-
H20. USP Calcifediol RS
Identification-Transfer the contents of a number of
Capsules, equivalent to about 150 IJg of calcifediol, to a
Packaging and storage-Preserve in tight, light-resistant suitable container, add 1 mL of methanol, and shake
containers at controlled room temperature. vigorously for 1 minute. Separate the layers by centrifugation,
USP Reference standards (11)- and transfer as much of the top, methanol layer as possible to
USP Calcifediol RS . a second container. Evaporate this extract to dryness, and
dissolve the residue in about 1 mL of chloroform. Proceed as
directed under Thin-layer Chromatographic Identification Test
(201), applying 20 IJL of this solution and 20 IJL of a solution
~<\<J~~j~tlJi containing about the same concentration of USP Calcifediol
<1~Z>/t!lJl,qL>'<> .....,.. RS in chloroform, and using a solvent system consisting of 60
Water Determination, Method la (921): parts of cyclohexane and 40 parts of ethyl acetate.
5.0%, determined on a 0.2-g specimen.
Dissolution (711)-
Assay- Medium: water; 500 mL.
Internal standard solution-Dissolve testosterone in ethyl Apparatus 2: 50 rpm.
acetate to obtain a solution having a concentration of about Time: 15 minutes.
0.10 mg per mL. Procedure-Place 1 Capsule in each vessel, and allow the
Mobile phase-Prepare a suitable degassed solution of Capsule to sink to the bottom of the vessel before starting
about 6 volumes of heptane, 6 volumes of water-saturated rotation of the blade. Observe the Capsules, and record the
heptane, 3 volumes of methylene chloride, and 5 volumes of time taken for each capsule shell to rupture.
ethyl acetate. Tolerances-The requirements are met if all of the Capsules
Standardpreparation-Dissolve an accurately weighed tested rupture in not more than 15 minutes. If 1 or 2 of the
quantity of USP Calcifediol RS in Internalstandardsolution, and Capsules rupture in more than 15 but not more than 30
dilute quantitatively and stepwise with Internal standard minutes, repeat the test on 12 additional Capsules. Not more
solution to obtain a solution having a known concentration of than 2 of the total of 18 Capsules tested rupture in more than
about 20 IJg per mL. . 15 but not more than 30 minutes.
Assay preparation-Transfer about 10 mg of Calcifediol, Uniformity of dosage units (905): meet the requirements.
accurately weighed, to a 50-mL volumetric flask, dissolve in
Internal standardsolution, dilute with Internalstandardsolution Assay-
to volume, and mix. Transfer 5.0 mLof this solution to a 50-mL Internal standardsolution-Dissolve testosterone in ethyl
volumetric flask, dilute with Internalstandardsolution to acetate to obtain a solution having a concentration of about
volume, and mix. 35 IJg per mL.
Chromatographic system (see Chromatography (621 »)- Mobilephase-Prepare as directed in the Assay- under
The liquid chromatograph is equipped with a 4-mm x 30-cm Calcifediol.
column that contains packing L3, a detector that monitors Standardpreparation-Dissolve an accurately weighed
absorption at 254 nm, and a pump capable of providing quantity of USPCalcifediol RS in Internalstandardsolution, and
constant flow up to a minimum of 2000 psi. dilute quantitatively and stepwise with Internal standard
System suitability-The relative standard deviation for peak solution to obtain a solution having a known concentration of
response ratios for four replicate injections of Standard about 7 IJg of USP Calcifediol RS per mL.
preparation is not more than 3.0%, and the resolution factor Assay preparation-Transfer a number of Calcifediol
is not less than 3.0. Capsules to a suitable container. Using a suitable implement,
Procedure-Introduce equal volumes of the Standard shear open a number of Capsules inside the container. Wash
preparation and the Assay preparation into the chromatograph the implement with an accurately measured volume of Internal
(see Chromatography(621»), and measure the peak responses standard solution that will yield a solution having a
obtained. Calculate the quantity, in mg, of C27H4402 . H20 in concentration of about 7 IJg of calcifediol per mL. Collect the
the portion of Calcifediol taken by the formula: rinsings in the container, and mix to obtain a homogeneous
solution of the Capsule contents.
0.5C(RulR s) Chromatographic system and System suitability-Proceed
as directed in the Assay- under Calcifediol.
in which C is the concentration, in IJg per mL, of USP Procedure-Proceed as directed for Procedure in the Assay
Calcifediol RS in the Standardpreparation; and Ru and Rs are - under Calcifediol. Calculate the quantity, in IJg, of

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USP 43 OfficialMonographs / Calcipotriene 667

C27H4402 . H20 in the portion of Capsule contents taken by the [NoTE-The relative retention times for calcipotriene
formula: related compound C and calcipotriene are 0.94 and
1.0, respectively.]
Suitability requirements
Resolution: NLT 1.5 between calcipotriene related
in which C is the concentration, in IJg per mL, of USP compound C and calcipotriene
Calcifediol RS in the Standardpreparation; Vu is the volume, in Relative standard deviation: NMT 1.0% from the
mL, of Internal standardsolution taken for the Assay calcipotriene peak
preparation; and Ru and Rs are the peak response ratios of Analysis
calcifediol to testosterone obtained from the Assay preparation Samples: Standardsolution and Sample solution
and the Standard preparation, respectively. Calculate the percentage of calcipotriene (C27H4003) in the
portion of Calcipotriene taken:
Result =(rulrs) x (CsICu) x 100

Calcipotriene = peak response from the Sample solution

= peak response from the Standardsolution
= concentration of USP Calcipotriene RS in the
Standardsolution (mg/mL) .
= concentration of Calcipotriene in the Sample
solution (mg/mL)

Acceptance criteria: 97.0%-102.0% on the dried basis

C27H4003 412.60 IMPURITIES
9,1 0-Secochola-5,7, 10(19),22-tetraene-l ,3,24-triol, 24- • ORGANIC IMPURITIES BY HPLC
cyclopropyl-, (1 a,3~,5Z,7 £,22£,245)-; Protect solutions containing calcipotriene from light and air.
(5Z, 7£,22 £,245)-24-Cyclopropyl-9, 10-secochola-5,7,10(19), . Prepare the Standardsolution and the Sample solution
22-tetraene-l a,3~,24-triol [112965-21-6]. NMT 1 h before use. Prepare the System suitability
DEFINITION solution daily.
Calcipotriene contains NLT 97.0% and NMT 102.0% of Buffer, Mobile phase, System suitability solution,
calcipotriene (C27H4003), calculated on the dried basis. Chromatographic system, and System suitability:
Proceed as directed in the Assay.
IDENTIFICATION ' Standard stock solution: Use the Standard solution in the
Assay.
Standard solution: 0.004 mg/mL of USP Calcipotriene RS
in Mobile phase from the Standardstock solution
Sample solution: 0.4 mg/mL of Calcipotriene dissolved in
10% of acetonitrile, then diluted in Mobilephase
peak of the Sample Analysis
solution corresponds to that of Standardsolution, as Samples: Standardsolution and Sample solution
obtained in the test for Assay. Calculate the percentage of any impurity in the portion of
Calcipotriene taken:
ASSAY
• PROCEDURE Result = (rulrs) x (CsICu) x 100
Protect solutions containing calcipotriene from light and air.
Prepare the Standardsolution and the Sample solution tu = peak response of any impurity from the Sample
NMT 1 h before use. Prepare the System suitability solution
solution daily. rs = peak response of calcipotriene from the Standard
Buffer: 1.0 giL of tris(hydroxymethyl)aminomethane solution
adjusted with phosphoric acid to pH 7.25 ± 0.25 Cs = concentration of USP Calcipotriene RS in the
Mobile phase: Acetonitrile and Buffer (45:55) Standardsolution (mg/mL)
System suitability solution: 0.1 mg/mL of USP Cu = concentration of Calcipotriene in the Sample
Calcipotriene RS and 0.01 mg/mL of USPCalcipotriene solution (mg/mL)
Related Compound C RS in Mobile phase
Standard solution: 0.1 mg/mL of USP Calcipotriene RS Acceptance criteria: See Table 1. Disregard any impurity
dissolved in 10% of acetonitrile, then diluted in Mobile peak less than 0.05%.
phase
Sample solution: 0.1 mg/mL of Calcipotriene dissolved in Table 1
10% of acetonitrile, then diluted in Mobile phase Relative Acceptance
Chromatographic system Retention Criteria,
(See Chromatography (621 >, System Suitability.) Name Time NMT(%)
Mode: LC Calcipotriene related corn-
Detector: UV 264 nm pound C· 0.92-0.96 1.00
Column: 4.0-mm x 25-cm; 5-lJm packing L7
Autosampler temperature: 4° Calcipotriene 1.00 -
Flow rate: 1.0 mL/min Calcipotriene impurity Db 1.13-1.17 1.00
Injection volume: 20 IJL
System suitability Any individual
unspecified impurity - 0.10
Sample: System suitability solution

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668 Calcipotriene / OfficialMonographs USP 43

Table 1 (continued) Table 2 (continued)

Relative Acceptance Relative Acceptance
Retention Criteria, Retardation Comparison Criteria,
Name Time NMT (%) Name (R,eJ Solution NMT(%)
Total impurities - 2.50 Any other
individual - Standard
a (5E,7E,22E,245)-24-Cyclopropyl-9, 10-secochola-5,7, 10(19),22-tetraene-l a, impurity solution2 0.10
3~,24-triol.
b (5Z, 7E,22E,24R)-24-Cyclopropyl-9,l 0-secochola-5,7,10(19),22-tetraene-l a, a 24,24'-Oxybis[(5Z,7E,22E,245),24-cyciopropyl-9, 10-secochola-5,7,10(19),
3~,24-triol. 22-tetraene-l a,3~-diol].
b (5Z,7 E,22E,24R)-24-Cyclopropyl-24-{[(5Z,7 E,22E,245)-24-cyclopropyl-l a,
3~-dihydroxy-9, 10-secochola-5,7, 10(19),22-tetraen-24-yl]oxy}-9, 10-
• ORGANIC IMPURITIES BY TLC secochola-5,7,1 0(19),22-tetraene-l a,3~-diol.
Prepare solutions containing calcipotriene in low actinic c (5E,6E,22E,245)-24-Cyclopropyl-9, 10-secochola-5(1 0),6,22-triene-l a,3p,24-
glassware, and keep from air. Carry out the test as rapidly triol.
as possible. d (5Z,7 Z,22E)-24-Cyclopropyl-9, 10-secochola-5,7, 10(19),22-tetraene-24-one-
Diluent: Chloroform and triethylamine (9:1) 111,3~-diol.
System suitability solution: 10 mg/mL of USP
Calcipotriene RS in Diluent. Heat in a water bath at 60° for SPECifiC TESTS
2 h to form precalcipotriene. • Loss ON DRYING
Standard solution 1: 0.025 mg/mL of USP Calcipotriene RS (See Thermal Analysis (891 ).)
in Diluent (0.25%) Sample: 5 mg
Standard solution 2: 0.01 mg/mL of USP Calcipotriene RS Analysis: Heat the Sample to 105° at a rate of 10°Imin, and
in Diluent (0.1 0%) hold at 105° for 60 min.
Sample solution: 10 mg/mL of Calcipotriene in Diluent Acceptance criteria: NMT 1.0%
Developing solvent system: Methylene chloride and
ADDITIONAL REQUIREMENTS
isobutyl alcohol (80:20)
Chromatographic system • PACKAGING AND STORAGE: Preserve in tight containers and
. store at 2°_8° or at -20° or below. Protect from light.
(See Chromatography (621), Thin-Layer Chromatography.)
Mode: TLC • USP REFERENCE STANDARDS (11)
USP Calcipotriene RS
Adsorbent: 0.25-mm layer of chromatographic plate
USP Calcipotriene Related Compound C RS,
coated with silica gel mixture
(5E,7 E,22E,24S)-24-Cyclopropyl-9, 1O-secochola-
Application volume: 10 f.JL
5,7,1 0(19),22-tetraene-1 a,3p,24-triol.
Spray reagent: Transfer 20 mL of sulfuric acid into a
1OO-mL volumetric flask, and dilute with alcohol to C27H4003 412.60
volume.
System suitability
Sample: System suitability solution
Suitability requirements Calcipotriene Ointment
Resolution: The secondary spot precalcipotriene and
principle spot calcipotriene are clearly separated. DEfiNITION
Analysis Calcipotriene Ointment contains NLT 90.0% and NMT
Samples: Standardsolution 1, Standard solution 2, and 110.0% of the labeled amount of calcipotriene (C27H4003),
Sample solution in a suitable ointment base.
Develop with Developing solventsystem until the solvent
system has moved 2/3 of the plate from the point of IDENTifiCATION
spotting. Remove the plate, and let the plate air-dry. Dry • A. The retention time of the major peak of the Sample
it again at 140° for 10 min followed by spraying the hot solution corresponds to that of the Standardsolution, as
plate with the Spray reagent. Dry the plate for NMT 1 min obtained in the Assay.
at 140°. Examine the plate under UV light at 366 nm.
Acceptance criteria: The spot of any impurity in the Sample
solution is not more intense than the spot of calcipotriene
in the appropriate Standardsolution specified in Table 2.

Table 2 Buffer, Diluent, Standard stock solution, Standard

Relative Acceptance solution, Sample stock solution, and Sample solution:
Retardation Comparison Criteria, Proceed as directed in the Assay.
Name (Rret) Solution NMT(%) Blank: Dilute 5 mLof tetrahydrofuran with Diluent to 25 mL.
Calcipotriene
Acceptance criteria: Meets the requirements
impurity Ga and cal- ASSAY
cipotriene Standard
impurity Hb 0.4 solution 1 0.25 • PROCEDURE
The solutions containing calcipotriene are stable up to 24 h
Precakipotriene- 0.9 - - at room temperature.
Calcipotriene 1.0 - - Mobile phase: Methanol and water (70:30)
Buffer: 132 giL of monobasic ammonium phosphate in
Calcipotriene Standard water
impurity Ad 1.2 solution 1 0.25
Diluent: Methanol, Buffer, and water (700:3:297)
Standard stock solution: 0.1 mg/mL of USP Calcipotriene
RS in Diluent. Sonicate if necessary.

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USP 43 Official Monographs / Calcipotriene 669

Standard solution: 2 IJg/mL of USP Calcipotriene RS Chromatographic system

prepared as follows. Transfer 5 mL of Standardstock (See Chromatography (621), System Suitability.)
solution into a 250-mL volumetric flask, add 50 mL of Mode: LC
tetrahydrofuran, and dilute with Diluent to volume. Detector: UV 264 nm
Sample stock solution: 0.01 mg/mL of calcipotriene in Column: 4.6-mm x 15-cm; 3-lJm packing L1
tetrahydrofuran prepared asfollows. Transfer Ointment Flow rate: 1.0 mL/min
equivalent to 0.25 mg of calcipotriene into a 25-mL Injection volume: 100 IJL
volumetric flask. Add 15 mL of tetrahydrofuran, and Run time: NLT 1.25 times of retention time of the
sonicate, with intermittent shaking, for 20 min in a cold calcipotriene peak
water bath. Dilute with tetrahydrofuran to volume. System suitability
Sample solution: 2 IJg/mL of calcipotriene prepared as Samples: System suitability solution and Standard solution
follows. Transfer 5 mL of the Sample stock solution into a Suitability requirements
suitable container. Add 20 mL of Diluent, mix, and sonicate Resolution: NLT 1.2 between calcipotriene related
for 10 min. Pass through a suitable filter of 0,45-lJm pore compound C and calcipotriene peaks, System suitability
size. Inject immediately after preparation. solution
Chromatographic system Relative standard deviation: NMT 5.0%, Standard
(See Chromatography (621), System Suitability.) solution
Mode: LC Analysis
Detector: UV 264 nm Samples: Standardsolution and Sample solution
Column: 4.6-mm x 15-cm; 3-lJm packing L1 Calculate the percentage of any impurity in the portion of
Flow rate: 1.0 mL/min Ointment taken:
Injection volume: 50 IJL
System suitability Result = (r vir s) x (C siC v) x 100
Sample: Standardsolution
Suitability requirements =peak response of any impurity from the Sample
Tailing factor: NMT 2.0 solution
Relative standard deviation: NMT 2.0% =peak response of calcipotriene from the Standard
Analysis solution
Samples: Standardsolution and Sample solution = concentration of USP Calcipotriene RS in the
Calculate the percentage of the labeled amount of Standardsolution (mg/mL)
calcipotriene (C27H4003) in the portion of Ointment taken: = nominal concentration of calcipotriene in the
Sample solution (mg/mL)
Result = (r vir s) x (C siC v) x 100
Acceptance criteria: See Table 1. Disregard any impurity
= peak response from the Sample solution peaks less than 0.05%.
= peak response from the Standardsolution
= concentration of USP Calcipotriene RS in the Table 1
Standardsolution (mg/mL) Relative Acceptance
= nominal concentration of calcipotriene in the Retention Criteria,
Name Time NMT (%)
Sample solution (mg/mL)
Calcipotriene impurity Ba 0.86 0.50
Acceptance criteria: 90.0%-110.0%
Calcipotriene related
PERFORMANCE TESTS compound Cb 0.92 1.00
• MINIMUM FILL (755): Meets the requirements Calcipotriene 1.0 -
IMPURITIES Calcipotriene impurity DC 1.31 1.00
• ORGANIC IMPURITIES
Specified unknown
The solutions containing calcipotriene are stable for up to 24 impurity 1.8 0.50
h at room temperature.
Mobile phase, Buffer, and Diluent: Proceed as directed in Any individual unspecified impur-
ity - 0.50
the Assay.
System suitability solution: 10.0 IJg/mL of USP Total impurities - 3.50
Calcipotriene RS and 0.1 IJg/mL of USP Calcipotriene
Related Compound C RS in Diluent a (5Z,7Z,22E,24S)-24-Cyclopropyl-9,10-secochola-5,7,10(19),22-tetraene-l c,
3~,24·triol.
Standard stock solution: 1.0 IJg/mL of USP Calcipotriene
b (5E,7E,22E,24S)-24-Cyclopropyl-9, 10-secochola-5,7,10(19),22-tetraene-l a,
RS in Diluent 3~,24-triol.
Standard solution: 0.1 IJg/mL of USP Calcipotriene RS c (5Z,7E,22E,24R)-24-Cyclopropyl-9, 10-secochola-5,7, 10(19),22-tetraene-l o;
prepared asfollows. Transfer 1.0 mL of the Standardstock 3~,24-triol.
solution into a 1O-mL volumetric flask, add 1.0 mL
tetrahydrofuran, and dilute with Diluent to volume. SPECIFIC TESTS
Sample solution: Nominally equivalent to 0.01 mg/mL of • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
calcipotriene prepared asfollows. Transfer Ointment SPECIFIED MICROORGANISMS (62): The total aerobic
equivalent to 0.1 mg of calcipotriene into a glass-stoppered microbial count is NMT 10 2 du/g. The total yeasts and
test tube, and add 1 mL of tetrahydrofuran. Sonicate for 20 molds count is NMT 5 x 10 1 du/g. It meets the
min with intermittent shaking. Add 9 mL of Diluent, and requirements of the tests for the absence of Staphylococcus
sonicate for 5 min. Shakethe test tube vigorously, and then aureus and Pseudomonas aeruginosa species.
place it in a beaker containing ice cold water for 2-3 min. • pH (791)
Pass the liquid portion through a nylon filter of 0,45-lJm Sample solution: Transfer 1 g of Ointment in a centrifuge
pore size, and discard the first few mL of the solution. test tube. Add 10 mL of water, and heat in a water bath at

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670 Calcipotriene / Official Monographs USP 43

60° for 30 min with stirring. Cool to room temperature, and Related Compound A RS and 0.2 mg/ml of USP Calcitonin
centrifuge at 2500 rpm for 10 min. Salmon RS.
Acceptance criteria: 5.5-8.5 Sample solution: 1.0 mg/ml of Calcitonin Salmon in
Solution A
ADDITIONAL REQUIREMENTS
Chromatographic system
• PACKAGING AND STORAGE: Preserve in well-closed (See Chromatography (621), System SUitability.)
containers, and store at controlled room temperature. Do Mode: lC
not freeze.
Detector: UV 220 nm
• USP REFERENCE STANDARDS (11) Column: 4.6-mm x 25-cm; packing l1
USP Calcipotriene RS Column temperature: 65°
USP Calcipotriene Related Compound C RS Flow rate: 1 mL/min
(5E, 7 E,22E,24S)-24-Cyclopropyl-9, 1O-secochola- Injection volume: 20 jJL
5,7,1 0(19),22-tetraene-1 a,3p,24-triol. System suitability
C27H4003 412.60 Sample: System suitability solution
[NOTE-The relative retention times for calcitonin
salmon and calcitonin salmon related compound A
are 1.0 and 1.15, respectively.]
Calcitonin Salmon Suitability requirements
Resolution: NLT 3 between calcitonin salmon and
r---"I calcitonin salmon related compound A
CSNLSTCVLG KLSQELHKLQ TYPRTNTGSG TP -NH.
Tailing factor: NMT 2.5 for calcitonin salmon
C14sH240N44048S2 3432 daltons Relative standard deviation: NMT 3%
[47931-85-1 ]. Analysis
Samples: Standardsolution and Sample solution
DEFINITION Calculate the percentage of calcitonin salmon
Calcitonin Salmon isa polypeptide that has the same sequence (C14sH240N44048S2) in the portion of Calcitonin Salmon
as that of the hormone that regulates calcium metabolism taken:
and is secreted by the ultimobranchial gland of salmon. It is
produced from either synthetic processes or microbial Result = (r vir s) x (C siC v) x 100
processes using recombinant DNA (rONA) technology. The
host cell-derived protein content and the host cell- or ru = peak response of calcitonin salmon from the
vector-derived DNA content of Calcitonin Salmon produced Sample solution
from an rONAprocess are determined by validated methods. rs = peak response of calcitonin salmon from the
It contains NlT'90.0% and NMT 105.0% of calcitonin Standardsolution
salmon, calculated on an acetic acid-free and anhydrous Cs =concentration of USP Calcitonin Salmon RS in the
basis. [NOTE-1 mg of acetic acid-free, anhydrous Calcitonin Standardsolution (corrected for water and acetic
Salmon is equivalent to 6000 USP Calcitonin Salmon Units. acid content) (mg/mL)
1 USP Calcitonin Salmon Unit = 1 Calcltoninlu.] Cu = concentration of the Sample solution (corrected
for water and acetic acid content) (mg/mL)
IDENTIFICATION
• A. The retention time of the major peak of the Sample Acceptance criteria: 90.0%-105.0% on an acetic acid-free
solution corresponds to that of the Standardsolution, and anhydrous basis
obtained as directed in the Assay.
OTHER COMPONENTS
ASSAY • ACETIC ACID CONTENT (503)
• PROCEDURE Sample solution: 1 mg/mL of Calcitonin Salmon in Diluent,
Solution A: Dissolve 3.26 g of tetramethylammonium prepared as directed in the chapter
hydroxide pentahydrate in 900 ml of water, and adjust Acceptance criteria: 4%-15%
with phosphoric acid to a pH of 2.5. Add 100 ml of
acetonitrile, and mix. IMPURITIES
Solution B: Dissolve 1.45 g of tetramethylammonium • PROCEDURE: RELATED PEPTIDES AND OTHER RELATED
hydroxide pentahydrate in 400 ml of water, and adjust SUBSTANCES
with phosphoric acid to a pH of 2.5. Add 600 ml of Test 1
acetonitrile, and mix. [NOTE-Thistest is performed on material produced by
Mobile phase: See Table 7. both chemical synthesis processes and rONA
processes.]
Table 1 Solution A, Solution B, Mobile phase, System suitability
Time Soiution A Solution B solution, Sample solution, Chromatographic system,
(min) (0/0) (0/0) and System suitability: Proceed as directed in the Assay.
Analysis
0 72 28
Sample: Sample solution
30 48 52 Calculate the percentage of each impurity in the portion
32 28
of Calcitonin Salmon taken:
72
55 72 28 Result = (r vir r) x 100
= peak area response of each impurity from the
Standard solution: 1.0 mg/ml of USPCalcitonin Salmon RS
in Solution A Sample solution
System suitability solution: Prepare a solution in Solution A = sum of the area responses of all the peaks from
the Sample solution
containing about 0.2 mg/ml of USP Calcitonin Salmon

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USP 43 Official Monographs / Calcitonin 671

Acceptance criteria ru = peak response for each impurity

Individual impurities: NMT 3.0% of the total area of all rr = sum of the responses of all the peaks
peaks
Total impurities: NMT 5.0% of the sum of the areasof Acceptance criteria
all the peaks including the principal peak Individual impurities: See Table 3.
[NOTE-Disregard any peaks due to the solvent and
any peaks whose area is less than 0.1% of the Table 3
principal peak.]
Relative Acceptance
Test 2 Retention Criteria
[NOTE-This test needs to be performed only on Name Time NMT (0/0)
material produced using rONA tech.nology.].
[1,7-Bis(3·sulfo-L-alanine)] calcitonin sal-
Buffer A: Dissolve 2.72 g of monobasic potassium mon-glycine 0.4 0.2
phosphate in 1000 mL of water.. .
Buffer B: Dissolve 2.72 g of monobasic potassium [l,7-Bis(3·sulfo-L-alanine)] calcitonin sal-
mon 0.6 0.2
phosphate and 29.2 g of sodium chloride in 1000 mL of
water. .. . Calcitonin salmon related compound B 0.9 0.6
Buffer C (pH 3.0 citrate buffer): Dissolve4.8 g of cltr~c acid
in 80 mL of water. Adjust with 1 M sodium hydroxide to SPECIFIC TESTS
a pH of 3.0, and dilute with water to 100.0 mL.. . • AMINO ACID PROFILE
Solution A: Acetonitrile and Buffer A (15:85). Adjust With (See Biotechnology-Derived Articles-Amino AcidAnalysis
45% (w/w) potassium hydroxide to a pH of 5.0: . (1052).)
Solution B:· Acetonitrile and Buffer B (15:85). Adjust With [NOTE-This test needs to be performed only on
45% (w/w) potassium hydroxide to a pH of 4.6. material of synthetic origin. The concen~ration of
Mobile phase: See Table 2. amino acids in the Internalstandardsolution, Standard
stock solution and Standardsolution and the amount
Table 2 of material u~ed to prepare the Sample solution can
Time Solution A Solution B be adjusted depending on the method us~d for
(min) (%) (0/0) amino acid analysis. The concentrations given are
0 100 0 based on analysis using Method 1.]
Internal standard solution: 1 mM solution of
10 0 100
y-aminobutyric acid . ..
15 0 100 Standard stock solution: Prepare a mixture contalnln~
equimolar amounts of ammonia and th~ L for~ of lysl~e,
15.1 100 0
histidine, arginine, aspartic acid, threonine, serme, proline,
22.1 100 0 valine, glutamic acid, glycine, leucine, and tyrosl~e, .
together with half the equimolar amount of L-~ystlne, In 0.1
Resolution solution: Preparea solution in wate~ . M hydrochloric acid. The final concentration IS about 2.5
containing about 0.5 mg/mL each of USP Calcltonln mM for each amino acid.
Salmon RS and USP Calcitonin Salmon Related Standard solution: Transfer 5 mL of the Internal standard
Compound B RS. To 1 mL of this solution add 100 ~L of solution and 2 mL of the Standardstock solution into a
pH 3.0 citrate buffer. . . 50-mL volumetric flask, and dilute with 0.1 M hydrochloric
Sample solution: To 1 mL of a 0.5-mg/mL solution of acid to volume.
Calcitonin Salmon add 100 mL of Buffer C. Sample solution: Place 1.5 mg of Calcitonin Salmon int<;> a
Chromatographic system heavy-wall ignition tube. Add 1.0 mL of 6 N hydrochlonc
(See Chromatography (621), System Suitability.) acid, and allow to cool. Immerse the lower half of the tube
Mode: LC in a freezing mixture until the contents are frozen~ then
Detector: UV 220 nm evacuate to approximately 1 ~ urn of ~g, purge With
Column: 4.6-mm x 20-cm; packing L9 nitrogen (repeat the evacuation and nitrogen purge three
Flow rate: 1.2 mL/min times) and seal the tube while it is under a 10 urn of Hg
Injection. volume: 50 ~L vacuu:n. Heat for 16 h at 110°-115° in an air oven. Cool,
System suitability open the tube dry in a vacuum desiccator, remove the
Sample: System suitability solution contents and ~lIow to cool to room temperature. Dissolve
[NoTE-The relative retention times for in 0.1 M'hydrochloric acid. Transfer to a 1O-mLvolumetric
[1,7-bis(3-sulfo-L-alani~e)] calcitonin .
flask, add 1 mL of Internal standardsolution, and dilute with
salmon-glycine, [1,7-bls(3-sulfo.-L-alanlne)] 0.1 M hydrochloric acid to volume.
Analysis
calcitonin salmon, and calcitonin salmon related
compound B (calcitonin salmon-glycine~ are. 0.4, Samples: Standardsolution and Sample solution
0.6, and 0.9, respectively; and the retention time for Standardize the amino acid analyzer using the Standard
calcitonin salmon is about 9 min.] solution. Inject the Sample solutio,: into the a.mino aci~
Suitability requirements analyzer, and determine the relative proportion of amino
Resolution: NLT 3.0 between calcitonin salmon and acids.
calcitonin salmon related compound B Express the content o~ eac~ amino ~cid in moles using an
Analysis internal standard calibration technique. Calculate the
Sample: Sample solution relative proportions of the amino acids by taking as
Calculate the percentage of each impurity in the portion equivalent .to one th~ su~ of the. numbe~ of mo!es of
of Calcitonin Salmon taken: . aspartic acid, qlutamlc acid, proline, qlyclne, valine,
leucine histidine, arginine, and lysine divided by 20.
Result = (r vir r) x 100 Acceptan~e criteria: The requirements are met if the values
fall within the limits in Table 4.

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672 Calcitonin / OfficialMonographs USP 43

TabRe4 Detector: UV 214 nm

Acceptance Column: 4.6-mm x 25-cm; packing II
Name Criteria Flow rate: 1.2 ml/min
Aspartic acid 1.8-2.2
Injection volume: 20 III
Analysis
Glutamic acid 2.7-3.3 Samples: Standard solution and Sample solution
Proline 1.7-2.3
[NOTE-Condition the chromatographic system by
running a blank gradient program before injecting the
Glycine 2.7-3.3 digests.]
Valine 0.9-1.1 Acceptance criteria: The chromatographic profile of the
Sample solution is similar to that of the Standard solution.
Leucine 4.5-5.3 • BIGIDENTITV
Histidine 0.9-1.1 RPMI1640 with L-glutamine: Prepare a mixture of the
ingredients in the quantities shown in Table 6 in sufficient
Arginine 0.9-1.1 water to obtain 1 l of RPMI 1640 with i-glutamine solution,
Lysine 1.8-2.2 and sterilize by filtration.
Serine 3.2-4.2 Table 6
Threonine 4.2-5.2 Calcium nitrate 100.00mg
Tyrosine 0.7-1.1 Potassium chloride 400.00 mg
Half cystine 1.4-2.1 Magnesium sulfate,"anhydrous 48.84 mg

Potassium chloride 400mg

• PEPTIDE MAPPING
(See Biotechnology-Derived Articles-Peptide Mapping Sodium chloride 6000 mg
(1055).) Sodium phosphate, dibasic, anhydrous 800 mg
[NOTE-This test needs to be performed only on
material produced using rDNA technology.] Sodium bicarbonate 2000 mg
Solution A: Water and trifluoroacetic acid (1000: 1) Glucose 2000 mg
Solution B: Acetonitrile, water, and trifluoroacetic acid
Glycine 10 mg
(800: 200: 0.85)
Mobile phase: See Table 5. L-Arginine 200 mg

L-Asparagine 50 mg
Table 5
Time Solution A Solution B L-Aspartic acid 20mg
(min) (0/0) (%)
L-Cystine dihydrochloride 65 mg
0 100 0
L-Glutamic acid 20mg
50 65 35
L-Glutamine 300mg
60 40 60
L-Histidine 15 mg
60.1 0 100
L-Hydroxyproline 20mg
65.1 0 100
L-Isoleucine 50 mg
65.2 100 0
L-Leucine 50mg
80.2 100 0
L-Lysine hydrochloride 40mg

L-Methionine 15 mg
Trypsin solution: Freshly prepare a solution containing 0.1
mg/ml of trypsin (previously treated with L-l-tosylamido- L-Phenylalanine 15 mg
2-phenylethyl chloromethyl ketone [TPCK] to remove
L-Proline 20mg
chymotrypsin activity) in water.
Tris buffer: 1 M tris(hydroxymethyl) aminomethane and 10 L-Serine 30mg
mM calcium chloride. Adjust with hydrochloric acid to a pH L-Threonine 20mg
of 8.0.
Stopping solution: Water and trifluoroacetic acid (1:1) L-Tryptophan 5 mg
Standard solution: Prepare a 1.O-mg/ml solution of USP L-Tyrosine disodium salt dihydrate 29 mg
Calcitonin Salmon RS. Transfer 1 ml of this solution to a
clean vial. Add 100 III of Tris buffer and 50 III of Trypsin L-Valine 20mg
solution. Mix, and incubate at 2°_8° for 16-20 h. Quench Biotin 0.2mg
the digestion by adding 10 III of Stopping solution.
Sample solution: 1.0 mg/ml of Calcitonin Salmon. Transfer Choline chloride 3 mg
1 ml of this solution to a clean vial. Add 100 III of Tris D-Calcium pantothenate 0.25 mg
buffer and 50 III of Trypsin solution. Mix, and incubate at
2°_8° for 16-20 h. Quench the digestion by adding 10 III Folic acid 1 mg
of Stopping solution. i-Inositol 35 mg
Chromatographic system
(See Chromatography (621 ),. System Suitability.) Niacinamide 1 mg
Mode: lC para-Aminobenzoic acid 1 mg

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USP 43 Official Monographs / Calcitonin 673

Table 6 (continued) Cell culture preparation: Prepare a cell culture of the

Pyridoxine hydrochloride 1 mg human mammary tumor cell line T-47D. Cells are
propagated using Medium A at 3r and 5% carbon dioxide.
Riboflavin 0.2mg
The medium is changed every two days, and cells are
Thiamine hydrochloride 1 mg passaged every 5-9 days using Solution C with a 1:4
subculture.
Vitamin 812 0.005 mg
Cell suspension: For the test use a cell culture that is 5-9
days old. Remove the cell culture medium from the flask by
Medium A (growth medium): Using aseptic technique aspiration, add 10 mL of Solution 0, and rock the culture
prepare the following tissue culture medium. flask to rinse the entire monolayer. Remove the liquid by
aspiration, add 2 mL of Solution C, spread over the entire
Table 7 monolayer, allow to stand for 3-5 min, and add 10 mL of
RPMI 1640 with l-glutamine 500 mL Medium A. Homogenize the cell suspension using a pipet,
transfer to a 15-mL polypropylene tube, centrifuge at
Fetal bovine serum 50 mL about 220 x g for 5 min, pour off the supernatant, and
1 M HEPES 5 mL resuspend the cell pellet in 10 mL of Medium A. Count the
cells, and adjust the cell density through dilution using
Penicillin/streptomycin solution Medium A to 2.5 x 10 4 cells/mL.
(10,000 IU/mL per 10 mg/mL) 5 mL
Procedure: Place200 IJLof the Cellsuspension into each well
Human insulin 10lU of a 96-well culture plate (the tissue culture plate), and
Hydrocortisone 0.5 mg
incubate for 18-24 h at 37° and 5% carbon dioxide. Fill
each well of an empty, round-bottomed, 96-well culture
plate (the prepared plate) with 150 IJL of one of the
Medium B (stimulation medium): Dissolve 5 9 of bovine following solutions: Positive controlsolution, Negative control
serum albumin (BSA) in 500 mL of 2 mM RPMI 1640 with solution, Standard solutions A-O, and Sample solutions A-O,
L-glutamine. so that each solution fills at least five wells on the prepared
Solution A (0.2% BSA solution): Dissolve 50 mg of BSA in plate. After incubation remove the culture medium from
25 mL of water. [NOTE-Use within one day.] . the tissue culture plate. Using an 8-channel or 12-channel
Solution B (formic acid/BSA solution): Add 25 mL of 0.1 M pipet, rapidly transfer 100 IJLof solution from each well of
formic acid and 5 mL of Solution A to a 50-mL volumetric the prepared plate to each well of the tissue culture plate.
flask, dilute with water to volume, and mix. [NOTE-Use Incubate for 15 min at ambient temperature, remove the
within two days.] solution from each well, stop stimulation by immediately
Solution C (trypsin/EDTA solution): Prepare a sterile filtered adding an appropriate cell-lysis buffer, and quantitate
solution containing 0.25% trypsin and 0.53 mM EDTA cAMP produced within the cells, using a validated kit.
(tetrasodium ethylenediaminetetraacetate). Perform the test three times, using three different 96-well
Solution D (Dulbecco's phosphate buffered saline): Dissolve culture plates. [NOTE-Some kits include a cell-lysis reagent
8 9 of sodium chloride, 1.15 9 of dibasic sodium phosphate, and a sequestering agent for the cell-lysis reagent. The
0.2 9 of monobasic potassium phosphate, 0.2 9 of range of the test kit is between 0.05 and 10 ng/mL of cAMP.
potassium chloride, 0.1 9 of calcium chloride, and 0.1 9 of The number of cells used in the assay may vary, depending
magnesium chloride in 1 L of water. on the validated kit used to quantitate cAMP.]
Standard stock solution: 20 IJg/mL of USP Calcitonin Analysis: Potency is determined by a 3-dose, 6-point
Salmon RS in Solution B parallel-line assay, using standard statistical methods. The
Positive control solution: Quantitatively dilute the calculation is carried out uslnq both the lower three
Standardstock solution in Medium B to obtain a l-ng/mL concentrations and the upper three concentrations. For the
solution of USP Calcitonin Salmon RS. assay to be valid, the requirements for regression,
Negative control solution: Medium B parallelism, and difference of quadratics must be met. If the
[NOTE-Prior analysis should be performed to identify the requirements for validity are met to the same extent in both
linear portion of the dose-response curve. For example, assessments (the lower and the higher assessments) the
the Standardsolutions and Sample solutions given final result is determined from the concentration range that
below.] shows the higher value when the common slope is divided
Standard solution A: 0.8 ng/mL of USP Calcitonin Salmon by the root mean square error.
RS from the Standardstocksolution in Medium B Acceptance criteria: Combine the three potency values by
Standard solution B: 0.4 ng/mL of USP Calcitonin Salmon using an unweighted mean on the log scale. Determine a
RS from Standardsolution A in Medium B 95% confidence interval in the log scale using standard
Standard solution C: 0.2 ng/mL of USP Calcitonin Salmon statistical methods. Convert the average and confidence
RS from Standardsolution B in Medium B interval to the potency scale using antilogs to obtain a
Standard solution D: 0.1 ng/mL of USP Calcitonin Salmon geometric mean and its confidence interval. The potency
RS from Standardsolution C in Medium B levels determined from all three performances of the test
Sample stock solution: 20 IJg/mL of Calcitonin Salmon in are valid if the data analysis indicates the three
Solution B determinations to be homogeneous, and the confidence
Sample solution A: 0.8 ng/mL of Calcitonin Salmon from interval is fully contained within 64% and 156% of the
the Sample stock solution in Medium B geometric mean. If the confidence interval requirement is
Sample solution B: 0.4 ng/mL of Calcitonin Salmon from not met, additional assays may be performed to increase
Sample solution A in Medium B the number of assays and make the confidence interval
Sample solution C: 0.2 ng/mL of Calcitonin Salmon from narrower. The determination of whether it meets the
Sample solution B in Medium B identity requirement should be done only after the
Sample solution D: 0.1 ng/mL of Calcitonin Salmon from confidence interval requirement is met. The acceptance
Sample solution C in Medium B . criterion for identity is that the geometric mean is within
80% and 125% of the Assay value.

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674 Calcitonin / Official Monographs USP 43

o MICROlBlAllENUMERATION TESTS (61) and TESTS FOR Salmon RS. Take 0.1 ml of this solution, add 0.9 ml of
SPECIFIED MICROORGANISMS (62) Solution A, and mix.
Sample: 25 mg Standard stock solution: 1.0 mg/ml of USP Calcitonin
Acceptance criteria: The total aerobic microbial count is Salmon RS in Solution A
NMT 102 cfu/g, and the total combined molds and yeasts Standard solution: 0.1 mg/ml of USP Calcitonin Salmon RS
count is NMT 102 cfu/g. from Standardstock solution diluted with Solution A
• STERILITY TESTS (71): Where the label states that Calcitonin Sample solution: Usethe solution from an undiluted
Salmon issterile, it meets the requirements when tested as Injection vial.
directed for Test for Sterilityof the Product to Be Examined, Chromatographic system
Membrane Filtration. (See Chromatography (621), System Suitability.)
• WATER DETERMINATION, Method Ie(921): NMT 10% Mode: lC
Detector: UV 220 nm
ADDITIONAL REQUIREMENTS
Column: 4.6-mm x 25-cm; packing II
• PACKAGING AND STORAGE: Preserve in tight containers. Column temperature: 65°
Store in a refrigerator or maintain in a frozen state, Flow rate: 1 ml/min
protected from light. Injection volume: 200 J,JL
• LABELING: The labeling states that the material is synthetic
System suitability
or of recombinant DNA origin. Sample: System suitability solution
• USP REFERENCE STANDARDS (11) [NoTE-The relative retention times for calcitonin
USP Calcitonin Salmon RS salmon and calcitonin salmon related compound A
C14sH24oN4404sS2 3432 daltons are 1.0 and 1.15, respectively.]
USP Calcitonin Salmon Related Compound A RS Suitability requirements
N-Acetyl-cysl-calcitonin salmon. Resolution: NLT 3 between calcitonin salmon and
USP Calcitonin Salmon Related Compound BRS (calcitonin calcitoninsalmon related compound A
salmon-glycine) Tailing factor: NMT 2.5
Calcitonin salmon-glycine. Relative standard deviation: NMT 3%
Analysis
Samples: Standardsolution and Sample solution
Calculatethe potency, in USP Calcitonin Salmon Units/mL,
Calcitonin Salmon Injection in the portion of Injection taken:

DEFINITION
Result = (r vir s) xes
Calcitonin Salmon Injection is a sterile solution of Calcitonin ru = peak area from the Sample solution
Salmon in a suitable diluent. Each ml of Calcitonin Salmon
Injection possesses an activityof NlT 80% and NMT 120% rs = peak area from the Standardsolution
of that stated on the label. Cs = concentration of USP Calcitonin SalmonRS in the
Standardsolution (USP Calcitonin Salmon
IDENTIFICATION Units/mL)
• A. The retention time of the major peak of the Sample
solution corresponds to that of the Standard solution, as Acceptance criteria: 80%-120%
obtained in the Assay.
SPECIFIC TESTS
ASSAY • BACTERIAL ENDOTOXINS TEST (85): NMT 0.625 USP
• PROCEDURE Endotoxin Units/USP Calcitonin Salmon Unit
Solution A: Dissolve 3.26 g of tetramethylammonium • STERILITY TESTS (71): Meets the requirements when tested
hydroxide pentahydrate in 900 ml of water, add 100 ml as directed in Test for Sterilityof the Product to Be Examined,
of acetonitrile, and mix. Adjustwith phosphoric acid to a Membrane Filtration
pH of 2.5, pass through a filterof 0.5-J,Jm or finer pore size, • PARTICULATE MATTER IN INJECTIONS (788): Meets the
and degas. requirements for small-volume injections
Solution B: Dissolve 1.45 g of tetramethylammonium • pH (791): 3.9-4.5
hydroxide pentahydrate in 400 ml of water, add 600 ml • INJECTIONS AND IMPLANTED DRUG PRODUCTS (1): Meets the
of acetonitrile, and mix. Adjustwith phosphoric acid to a requirements
pH of 2.5, pass through a filter of 0.5-J,Jm or finer pore size,
ADDITIONAL REQUIREMENTS
and degas.
• PACKAGING AND STORAGE: Preserve in single-dose or
Mobile phase: See Table 1.
multiple-dosecontainers, preferablyof Type I glass. Avoid
Table 1 freezing. Store in a refrigerator.
• LABELING: Label it to indicate the activityin USP Calcitonin
Time Solution A Solution B Salmon Units/mL. The labeling states that the material is
(min) (%) (%)
synthetic. Label it to state that it is to be stored in a
0 72 28 refrigerator, and that freezing is to be avoided.
30 52 • USP REFERENCE STANDARDS (11)
48
USP Calcitonin Salmon RS
32 72 28 USP Calcitonin Salmon Related Compound A RS
55 72 28 N-Acetyl-cys "-cakttonln.
C146H243N44049S2 3463
System suitability solution: Prepare a solution in Solution A
containing about 0.2 mg/ml of USP Calcitonin Salmon
Related Compound A RS and 0.2 mg/ml of USP Calcitonin

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USP 43 Official Monographs / Calcitriol 675

Calcitonin Salmon Nasal Solution Analysis

Samples: Standard solution and Sample solution
DEFINITION Calculatethe percentage of the labeled amount of
Calcitonin Salmon Nasal Solution is a solution of Calcitonin calcitonin salmon (C14SH240N4404SS2) in the portion of
Salmon in a suitable diluent. It contains suitable Nasal Solution taken:
preservatives,. and is packaged in a form suitable for nasal
administrationso that the required dosage can be controlled Result =(ru/rs) x (Cs/Cu) x 100
as required. Each mL of Calcitonin Salmon Nasal Solution
possesses an activity of NLT 80% and NMT 115% of that tu = peak area from the Sample solution
stated on the label. ts = peak area from the Standard solution
(5 = concentration of USP Calcitonin Salmon RS in the
IDENTIFICATION Standard solution (mg/mL)
• A. The retention time of the major peak of the Sample (u =nominal concentration of calcitoninsalmon in the
solution corresponds to that of the Standard solution, as Sample solution (mg/mL)
obtained in the Assay.
ASSAY
Acceptance criteria: 80%-115%
• PROCEDURE SPECIFIC TESTS
Solution A: 3.26 mg/mL of tetramethylammonium • MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
hydroxide pentahydrate in water and acetonitrile(9:1). SPECIFIED MICROORGANISMS (62): The total aerobic
Adjustwith phosphoric acid to a pH of 2.5. microbial count is NMT 102 cfu/g, and the total combined
Solution B: 1.45 mg/mL of tetramethylammonium molds and yeasts count is NMT 5 x 101 cfu/g. It meets the
hydroxide pentahydrate in acetonitrile and water (3:2). requirements for absence of Staphylococcus aureus and
Adjustwith phosphoric acid to a pH of 2.5. Pseudomonas aeruginosa.
Mobile phase: See Table 1. • pH (791): 3.5-4.5
Table 1 ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in containers suitable
Time Solution A Solution B
(min) (%) (%) for spraying the contents into the nasal cavities in a
controlled individualized dosage. Store unopened
0 72 28 containers in a refrigeratorand opened containers at room
30 48 52 temperature.
• LABELING: Label it to indicate that it isfor intranasal
32 72 28 administration only.The labelingalso states that it has been
55 72 28 prepared either with Calcitonin Salmon of synthetic origin
or Calcitonin Salmon of rDNA origin. Label it to state that
it is to be stored in a refrigerator, and that freezing is to be
Diluent: 7.5 mg/mLof sodium chloride, 2 mg/mL ofsodium avoided. Label it to indicate the activityin USP Calcitonin
acetate, and 2 mg/mL of glacial acetic acid in water Salmon Units/mL.
System suitability stock solution: Prepare a solution in • USP REFERENCE STANDARDS (11)
Solution A containing about 0.2 mg/mL of USP Calcitonin USP Calcitonin Salmon RS
Salmon Related Compound A RS and 0.2 mg/mL of USP USP Calcitonin Salmon Related Compound A RS
Calcitonin Salmon RS. . N-Acetyl-cys 1-calcitonin.
System suitability solution: System suitability stock solution C146H243N44049S2 3463
and Solution A (1 :9)
Standard stock solution: 1.0 mg/mL of USP Calcitonin
Salmon RS in Solution A
Standard solution: 0.1 mg/mL of USP Calcitonin SalmonRS
from the Standard stock solution in Solution A Calcitrio.
Sample solution: Nasal Solution in Diluent (1 :9)
Chromatographic system
(See Chromatography (621), System Suitability.) OH
Mode: LC
Detector: UV 220 nm
Column: 4.6-mm x 25-cm; packing L1
Column temperature: 65°
Flow rate: 1 mL/min C27H4403 416.64
Injection volume: 200 ~L
System suitability C27H4403 . H20 434.65
Samples: System suitability solution and Standard solution 9,10-Secocholesta-5,7,10(19)-triene-l ,3,25-triol, (1 a,3p,5Z,
[NOTE-The relative retention times for calcitonin 7£)-;
salmon and calcitoninsalmon related compound A (5Z,7 £)-9,10-Secocholesta-5,7,10(19)-triene-l a,3p,25-triol
are 1.0 and 1.15, respectively.] [32222-06-3].
Suitability requirements Monohydrate [77326-95-5].
Resolution: NLT 3 between calcitonin salmon and
calcitoninsalmon related compound A, System suitability DEFINITION
solution Calcitriol is anhydrous or contains 1 molecule of hydration.
Tailing factor: NMT 2.5, Standard solution The anhydrous form contains NLT 97.0% and NMT
Relative standard deviation: .NMT 3.0%, Standard 103.0% of calcitriol (C27H4403), calculated on the
solution solvent-free basis. The monohydrate form contains NLT

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676 Calcitriol / Official Monographs USP 43

97.0% and NMT 103.0% of calcitriol (C27H4403), calculated Acceptance criteria

on the anhydrous basis. Anhydrous form: 97.0%-103.0% on the solvent-free
[CAUTION-Care should be taken to prevent inhaling bas~ .
particlesof calcitriol, and exposing the skin to it.] Monohydrate form: 97.0%-103.0% on the anhydrous
basis
IDENTIFICATION
IMPURITIES
• ORGANIC IMPURITIES
Carry out the procedure as rapidlyas possible, and protect
all solutions containing calcitriol from light.
_ i\t,,7Qg) Buffer, Mobile phase, System suitability solution, Sample
• of the major peak of the Sample solution, Chromatographic system, and System
solution corresponds to that of the Standardsolution, as suitability: Proceed as directed in the Assay.
obtained in the Assay. Analysis
Sample: Sample solution
ASSAY Calculatethe percentage of any individual impurity in the
• PROCEDURE portion of Calcitriol taken:
Carryout the procedure as rapidly as possible, and protect
all solutions containing calcitriol from light. Result = (rvlrr) x 100
Buffer: 1.0 mg/mL of tris(hydroxymethyl)aminomethanein
water, adjusted with phosphoric acid to a pH of 7.0-7.5 ru =peak response of any individual peak other than
before final dilution the main calcitriol peak and the pre-calcitriol
Mobile phase: Acetonitrile and Buffer(55:45) peak from the Sample solution
Standard solution: 0.1 mg/mL of USP Calcitriol RS rr = sum of all the peak responsesfrom the Sample
prepared as follows. Transferan appropriate amount of USP solution
Calcitriol RS to a suitable volumetric flask, dissolve in
acetonitrile, using55% of the final volume, then dilutewith Acceptance criteria: See Table 1. The reporting threshold is
Buffer to volume. 0.05%.
System suitability solution: Heat 2.0 mLof the Standard
solution at 80° for 30 min. Table 1
Sample solution: 0.1 mg/mL of Calcitriol prepared as Relative Acceptance
follows. Transfer an appropriate amount of Calcitriol to a Retention Criteria,
suitable volumetric flask, dissolve in acetonitrile, using 55% Name Time NMT(%)
of the final volume, then dilute with Buffer to volume. Triazoline adduct of
Chromatographic system pre-calcitriol' 0.43 0.1
(See Chromatography (621), System SUitability.)
Mode: LC trans-Calcitriol b 0.96 0.25
Detector: UV 230 nm Calcitriol 1.0 -
Column: 4.6-mm x 25-cm; 5-~m packing L7 C
Column temperature: 40° 1p-Calcitriol 1.15 0.1
Flow rate: 1 mL/min Methylene caldtriol'' 1.5 0.25
Injection volume: 50 ~L
Run time: NLT 2 times the retention time of calcitriol Any unspecified
impurity - 0.1
System suitability
Samples: Standardsolution and System suitability solution Total impurities - 1.0
[NoTE-The relative retention times for pre-calcitriol
and calcitriol are 0.9 and 1.0, respectively.] a (6aR,7R,9aR)-11-[(3S,5R)-3,5-Dihydroxy-2-methylcyciohex-1-enyl ]-7-[(R)-6-
hydroxy-6-methylheptan-2-yl]-6a-methyl-2-phenyl-4a,5,6,6a,7,8,9,9a-
Suitability requirements octahydrocyclopenta[t][l ,2,4]triazolo[l ,2-a]cinnoline-l ,3(2H,11 H)-dione.
Resolution: NLT 3.5 between the pre-calcitriol and b (5E,7£)-9,1 0-Secocholesta-5,7, 10(19)-triene-l a,3p,25-triol.
calcitriol peaks, System suitability solution c (5Z,7 £)-9,1 O-Secocholesta-5, 7,1 0(19)-triene-1 p,3p,25-triol.
Relative standard deviation: NMT 1.0%, Standard d (5Z,7 £)-1 a,3p-Dihydroxy-17-[(R)-7-hydroxy-7-methyloctan-2-yl]-9,10-
solution secoandrosta-5,7,10(19)-triene.
Analysis
Samples: Standard solution and Sample solution SPECIFIC TESTS
Calculate the percentage of calcitriol (C27H4403) in the • WATER DETERMINATION (921), Method I, Method k: 3.50/0-
portion of Calcitriol taken: 5.5%, where it is labeled as a monohydrate
Result =(rvlrs) x (CsICv) x 100 ADDITIONAL REQUIREMENTS
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
to = sum of the peak responses of calcitriol and containers. Store as per labeling instructions.
pre-calcitriol from the Sample solution • LABELING: Where it is a monohydrate form, the labelso
ts = sum of the peak responses of calcitriol and indicates.
pre-calcitriol from the Standardsolution • USP REFERENCE STANDARDS (11)
Cs = concentration of USP Calcitriol RS in the Standard USP Calcitriol RS
solution(mg/mL)
Cu = concentration of CaJcitriol in the Sample solution
(mg/mL)

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 USP 43 OfficialMonographs / Calcium 677

Calcitriol Injection Calcium Acetate

» Calcitriollnjection is a sterile solution of Calcitriol.

It contains an amount of Calcitriol equivalent to not
less than 90.0 percent and not more than 115.0 C4H6Ca0 4 158.17
percent of the labeled amount of calcitriol Acetic acid, calcium salt;
Calcium acetate [62-54-4].
(Cz7H4403)' It contains no antimicrobial agents.
DEFINITION
Packaging and storage-Preserve in single-dose . Calcium Acetate contains NLT 99.0% and NMT 100.5% of
containers, preferably of Type I glass, protected from light. calcium acetate (C4H 6Ca04), calculated on the anhydrous
Store at controlled room temperature. basis.
USP Reference standards (11)-
USP Calcitriol Solution RS IDENTIFICATION
Identification-The retention time of the major peak in the • A. IDENTIFICATION TESTS-GENERAL, Calcium (191) and
chromatogram of the Assay preparation corresponds to that in Acetate (191)
the chromatogram of the Standard preparation, as obtained in Sample solution: 50 mg/mL
the Assay. . Acceptance criteria: Meets the requirements
Bacterial Endotoxins Test (85) -It contains not more than ASSAY
100 USP Endotoxin Units per J.Ig of calcitriol. • PROCEDURE
pH (791): between 5.9 and 8.0, determined on. a portio~ to Sample: 300 mg . .
which, if necessary, 0.30 mL of saturated potas~lu':l chloride Analysis: Dissolve the Sample in 150 mL of water containing
solution has been added for each 100 mL of lnjectlon. 2 mL of 3 N hydrochloric acid. While stirring, preferably
Particulate Matter in Injections (788): meets the with a magnetic stirrer, add about 30 mL of 0.05 M ede~ate
requirements for small-volume injections. disodium VS from a 50-mL buret. Add 15 mL of 1 N sodium
Other requirements-It meets the requirements under hydroxide and 300 mg of hydroxy naphthol blue, and
Injections and Implanted Drug Products (1). continue the titration to a blue endpoint. Each mL of 0.05
Assay- ... . M edetate disodium is equivalent to 7.909 mg of calcium
[NOTE-Avoid unnecessary exposure of solutions to light or acetate (C4H 6Ca04).
air.] Acceptance criteria: 99.00/0-100.5% on the anhydrous
Mobile phase-Prepare a filtered and degassed mixture of basis
methanol and water (74:26). Make adjustments if necessary IMPURITIES
(see System Suitability under Chromatography (621 ).so that • ARSENIC, Method 1(211): NMT 3 ppm
the retention time for calcitriol is not less than 20 minutes. • CHLORIDE AND SULFATE, Chloride (221)
Standardpreparation-Transfer 3.0 mL of USP Calcitri<?1 Standard: 0.70 mL of 0.020 N hydrochloric acid
Solution RS, equilibrated to room temperature, to a container; Sample: 1.0 g
add 3.0 mL of water; and mix. Acceptance criteria: 0.05%
Assay preparation-Transfer an accurately measurE;d. • CHLORIDE AND SULFATE, Sulfate (221)
volume of Injection, equivalent to about 3 J.Ig of. cakltriol, to Standard: 0.15 mL of 0.020 N sulfuric acid
a container' add a sufficient amount of water to dilute to a total Sample: 0.25 g
volume of 3.0 mL; add 3.0 mL of methanol; and mix. Acceptance criteria: 0.06%
Chromatographic system (see Chromatography (621 ))-The • LEAD (251): NMT 10 ppm
liquid chromatograph is equipped with a 2~4-nm detecto.r, a • LIMIT OFALUMINUM
4.6-mm x 4.5-cm guard column that contains 5-J.Im packing [NOTE-Use where it is labeled as intended for
, L1, and a 4.6-mm x 7.5-cm analytical column that contains parenteral use or for use in hemodialysis or peritoneal
3-J.Im packing L1. The flow rate is about 1 mL per minute. dialysis.]
Chromatograph the Standardpreparation, and record the peak Buffer: Dissolve 50 g of ammonium acetate in 150 mL of
responses as directed for Procedure: the relative standard water, adjust with glacial acetic acid to a pH of 6.0, and
deviation for replicate injections is not more than 2.0%. dilute with water to 250 mL.
Procedure-Separately inject equal volumes (ab<?ut .1 00 J.IL) Aluminum standard solution: 1.0 J.Ig/mL of aluminum.
of the Standardpreparation and the Assay preparation Into the Prepare as directed for StandardPreparations in Aluminum
chromatograph, record the chromatograms, and m.eas~re the (206).
responses for the major peaks. Calculate the quantity, In J.Ig, Standard solution: Prepare a solution containing 2.0 mL of
of calcitriol (C27H4403) in each mL of the Injection taken by the Aluminumstandard solution, 5 mL of Buffer, and 48 mL of
formula: water and extract this solution with successive portions of
10 16, and 5 mL of 0.5% 8-hydroxyquinoline in
C(r vir s) chloroform. Combine the chloroform extracts in a 50-mL
volumetric flask. Dilute the combined extracts with
in which C is the concentration, in J.Ig per mL, of calcitriol in chloroform to volume.
the USP Calcitriol Solution RS; and r u and r s are the peak Sample solution: Dissolve 1.0 g of Calcium Acetate in 50
responses obtained from the Assay preparation and the mL of water, and add 5 mL of Buffer. Extract this solution
Standardpreparation, respectively. with successive portions of 10, 10, and 5 mL of 0.5%
8-hydroxyquinoline in chloroform. Combine the
chloroform extracts in a 50-mL volumetric flask. Dilute the
combined extracts with chloroform to volume.
Blank solution: Prepare a solution containing 50 mL of
water and 5 mL of Buffer. Extract this solution with

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678 Calcium / Official Monographs USP 43

successive portions of 10, 10, and 5 mL of 0.5% the cumulative fluoride ion concentrations (0.1, 0.2, 0.5,
8-hydroxyquinoline in chloroform. Combine the and 1.0 IJg/mL) versus potential, in mY.
chloroform extracts in a 50-mL volumetric flask. Dilute the Rinse and dry the electrodes, insert them into the Sample
combined extracts with chloroform to volume. solution, stirfor 5 min, and read the potential, in mY. From
Instrumental conditions the measured potential and the standard response line
(See Fluorescence Spectroscopy (853).) determine the concentration, C, in IJg/mL, of fluoride ion
Mode: Fluorescence . in the Sample solution.
Excitation wavelength: 392 nm Calculate the amount of fluoride (ppm) in the sample taken
Emission wavelength: 518 nm by multiplying C by 50.
Analysis Acceptance criteria: 50 ppm
Samples: Standardsolution, Sample solution, and Blank • LIMIT OF MAGNESIUM
solution [NOTE-Use where it is labeled as intended for use in
Use the Blank solution to zero the instrument. hemodialysis or peritoneal dialysis. The Standard
Acceptance criteria: 2 ppm; the fluorescence of the Sample solution and the Sample solutions may be modified, if
solution is NMT that of the Standardsolution. necessary, to obtain solutions of suitable
• LIMIT OF BARIUM concentrations, adaptable to the linear or working
[NOTE-Use where it is labeled as intended for use in range of the instrument.]
hemodialysis or peritoneal dialysis.] Standard stock solution: 1000 IJg/mL of magnesium in 1
Barium chloride solution: 500 IJg/mL of barium in water N nitric acid from magnesium oxide
from anhydrous barium chloride Standard solution: 5.0 IJg/mL of magnesium from the
Buffer: Ammonium sulfate solution (1 in 10) Standardstock solution
Standard solution: To a tube add 1 g of ammonium Sample solution: 2 mg/mL of Calcium Acetate
acetate, 2 mL of 1 N hydrochloric acid, 3.0 mL of Barium Linearity solution A: Dilute 20.0 mL of the Sample solution
chloridesolution, and sufficient water to bring the volume with water to 25.0 mL (0 IJg/mL of magnesium).
to 40 mL. Linearity solution B: Dilute 2.0 mL of the Standardsolution
Sample stock solution: 250 mg/mL of Calcium Acetate and and 20.0 mL of the Sample solution with water to 25.0 mL
25 mg/mL of ammonium acetate in 1 N hydrochloric (0.4 IJg/mL of magnesium).
acid. The pH of this solution is 4.5-5.5. Filter, and cover the Linearity solution C: Dilute 4.0 mL of the Standardsolution
solution. and 20.0 mL of the Sample solution with water to 25.0 mL
Sample solutions: To four separate tubes add 1.0, 1.5, 2.0, (0.8 IJg/mL of magnesium).
and 2.5 mL of Barium chloride solution. To each tube add a Instrumental conditions
sufficient volume of the Sample stocksolution to bring the (See AtomicAbsorption Spectroscopy (852).)
volume to 40 mL. Mode: Atomic absorption spectrophotometry
Analysis: To the Sample solutions and the Standardsolution Analytical wavelength: 285.2 nm
add, with brisk stirring, 3.0 mL of Buffer, and allow to stand . Flame: Air-acetylene
for 20 min. Lamp: Magnesium hollow-cathode
Acceptance criteria: The Sample solutions containing 1.0 Blank: Water
and 1.5 mL of Barium chloride solution remain clear or are Analysis
only faintly turbid. The Sample solution containing 2.0 mL Samples: Linearitysolutions A, B, and C
of Barium chloride solution is not more turbid than the Plot the absorbances of the Linearity solutions versus their
Standardsolution. content of magnesium (0, 0.4, and 0.8 IJg/mL), draw the
• LIMIT OF FLUORIDE straight line best fitting the three points, and extrapolate
[NOTE-Prepare and store all solutions in plastic the line until it intercepts the concentration axis. From the
containers.] . intercept determine the amount, in IJg/mL, of magnesium
Buffer: 294 mg/mL of sodium citrate dihydrate in water in the Sample solution.
Standard stock solution: 1.11 mg/mL of USP Sodium Calculate the percentage of magnesium in the sample by
Fluoride RS in water multiplying this value by 0.0625.
Standard solution: Combine 20.0 mL of Standardstock Acceptance criteria: NMT 0.05%
solution with 50.0 mL of Buffer, and dilute with water to • LIMIT OF NITRATE
100.0 mL. Equivalent to 100 IJg/mL of fluoride . Sample solution: 100 mg/mL of Calcium Acetate in water
Sample solution: Transfer 2.0 g of Calcium Acetate to a Analysis: To 10 mL of the Sample solution add 5 mg of
beaker containing a plastic-coated stirring bar. Add 20.0 sodium chloride, 0.05 mL of indigo carmine TS, and, with
mL of water and 2.0 mL of hydrochloric acid, and stir until stirring, 10 mL of nitrogen-free sulfuric acid.
dissolved. Add 50.0 mL of Buffer and sufficient water to Acceptance criteria: The blue color persists for NLT 10 min.
make 100 mL. • LIMIT OF POTASSIUM
Electrode system: Usea fluoride-specific, ion-indicating [NOTE-Use where it is labeled as intended for use in
electrode and a silver-silver chloride reference electrode hemodialysis or peritoneal dialysis. The Standard
connected to a pH meter capable of measuring potentials solution and Sample solutions may be modified, if
with a minimum reproducibility of ±0.2 mY (see pH (791 ». necessary, to obtain solutions of suitable
Analysis concentrations, adaptable to the linear or working
Samples: Standardsolution and Sample solution range of the instrument.]
Transfer 50.0 mL of Buffer and 2.0 mL of hydrochloric acid Standard stock solution: 23.84 mg/mL of potassium
to a beaker, and add water to make 100 mL. Add a chloride, using potassium chloride previously dried at 105°
plastic-coated stirring bar, insert the electrodes into the for 2 h, equivalent to 12.5 mg/mL of potassium
solution, stir for 15 min, and read the potential, in mY. Standard solution: 31.25 IJg/mL of potassium from the
Continue stirring, and at 5-min intervals add 100, 100, Standardstock solution
300, and 500 IJL of the Standardsolution, reading the Sample solution: 12.5 mg/mL of Calcium Acetate
potential 5 min after each addition. Plot the logarithms of Linearity solution A: Dilute 20.0 mL of the Sample solution
with water to 25.0 mL (0 IJg/mL of potassium).

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 USP 43 OfficialMonographs / Calcium 679

Linearity solution B: Dilute 2.0 mL of the Standard solution concentrations, adaptable to the linear or working
and 20.0 mL of the Sample solution with water to 25.0 mL range of the instrument.]
(2.5 IJg/mL of potassium). Standard stock solution: 2.45 mg/mL of strontium acetate
Linearity solution C: Dilute 4.0 mL of the Standard solution in water, equivalent to 1000 IJg/mL of strontium
and 20.0 mL of the Sample solution with water to 25.0 mL Standard solution: 50.0 IJg/mL of strontium from the
(5.0 IJg/mL of potassium). Standardstock solution
Instrumental conditions Sample solution: 20 mg/mL of Calcium Acetate
(See AtomicAbsorption Spectroscopy (852).) Linearity solution A: Dilute 20.0 mL of the Sample solution
Mode: Atomic absorption spectrophotometry with water to 25.0 mL (0 IJg/mL of strontium).
Analytical wavelength: 766.7 nm Linearity solution B: Dilute 2.0 mL of the Standardsolution
Lamp: Potassium hollow-cathode and 20.0 mL of the Sample solution with water to 25.0 mL
Flame: Air-acetylene (4 IJg/mL of strontium).
Blank: Water Linearity solution C: Dilute 4.0 mL of the Standardsolution
Analysis and 20.0 mL of the Sample solution with water to 25.0 mL
Samples: Linearitysolutions A, B, and C (8 IJg/mL of strontium).
Plot the absorbances of the Linearitysolutions versus their Instrumental conditions
content of potassium (0, 2.5, and 5.0 IJg/mL), draw the (See AtomicAbsorption Spectroscopy (852).)
straight line best fitting the three points, and extrapolate Mode: Atomic absorption spectrophotometry
the line until it intercepts the concentration axis. From the Analytical wavelength: 460.7 nm
intercept determine the amount, in IJg/mL, of potassium lamp: Strontium hollow-cathode
in the Sample solution. Flame: Nitrous oxide-acetylene
Calculate the percentage of potassium in the sample by Blank: Water
multiplying this value by 0.01. Analysis
Acceptance criteria: NMT 0.05% Samples: Linearitysolutions A, B, and C
o LIMIT OF SODIUM Plot the absorbances of the Linearity solutions versustheir
[NOTE-Use where it is labeled as intended for use in content of strontium (0, 4, and 8 IJg/mL), draw the
hemodialysis or peritoneal dialysis. The Standard straight line best fitting the three points, and extrapolate
solution and the Sample solutions may be modified, if the line until it intercepts the concentration axis. From the
necessary, to obtain solutions of suitable intercept determine the amount, in IJg/mL, of strontium
concentrations, adaptable to the linear or working in the Sample solution.
range of the instrument.] Calculate the percentage of strontium in the sample by
Standard stock solution: 25.42 mg/mL of sodium chloride, multiplying this value by 0.00625.
using sodium chlpride previously dried at 105° for 2 h, Acceptance criteria: NMT 0.05%
equivalent to 10.0 mg/mL of sodium o READILY OXIDIZABLE SUBSTANCES
Standard solution: 250 IJg/mL of sodium from the Standard Sample solution: 20 mg/mL of Calcium Acetate in boiling
stocksolution water
Sample solution: 10 mg/mL of Calcium Acetate Analysis: Add a few glass beads to 100 mL of the Sample
Linearity solution A: Dilute 20.0 mL of the Sample solution solution, 6 mL of 10 N sulfuric acid, and 0.3 mL of 1 N
with water to 25.0 mL (0 IJg/mL of sodium). potassium permanganate. Mix, boil gently for 5 min, and
Linearity solution B: Dilute 2.0 mL of the Standard solution allow the precipitate to settle.
and 20.0 mL of the Sample solution with water to 25.0 mL Acceptance criteria: The pink color in the supernatant is not
(20 IJg/mL of sodium). . completely discharged.
Linearity solution C: Dilute 4.0 mL of the Standard solution
and 20.0 mL of the Sample solution with water to 25.0 mL SPECIFIC TESTS
opH(791)
(40 IJg/mL of sodium).
Instrumental conditions Sample solution: 50 mg/mL
(See Atomic Absorption Spectroscopy (852).) Acceptance criteria: 6.3-9.6
Mode: Atomic absorption spectrophotometry o WATER DETERMINATION, Method I (921)
Analytical wavelength: 589.0 nm Sample: 0.100 9
lamp: Sodium hollow-cathode Analysis: Proceed as directed in the chapter, adding 2 mL
Flame: Air-acetylene of glacial acetic acid to the titration vessel in addition to the
Blank: Water methanol.
Analysis Acceptance criteria: NMT 7.0%
Samples: Linearitysolutions A, B, and C ADDITIONAL REQUIREMENTS
Plot the absorbances of the Linearity solutions versus their o PACKAGING AND STORAGE: Preserve in tight containers.
content of sodium (0, 20, and 40 IJg/mL), draw the o LABELING: Where Calcium Acetate is intended for use in
straight line best fitting the three points, and extrapolate hemodialysis or peritoneal dialysis, it is so labeled.
the line until it intercepts the concentration axis. From the o USP REFERENCE STANDARDS (11)
intercept determine the amount, in IJg/mL, of sodium in USP Sodium Fluoride RS
the Sample solution.
Calculate the percentage of sodium in the sample by
multiplying this value by 0.0125.
Acceptance criteria: NMT 0.5%
o LIMIT OF STRONTIUM
[NOTE-Use where it is labeled as intended for use in
hemodialysis or peritoneal dialysis. The Standard
solution and Sample solutions may be modified, if
necessary, to obtain solutions of suitable

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680 Calcium / OfficialMonographs USP 43

L
es the ietelltiontlfl)eof tfie~alcium

Result= (rulr~fx~ (CslCuJ x ,10()

rv ::: peak re~p()fl~e' oJcalc h.lin fromth~Jamjj1e:solutign.
's ~ onseofcaldum·rrorll·tnt{Sta17agrg
Cs Icfurri ~Acetate -:RS~ln:the
L)
Cu caldum acetate~ ill tne

Acceptance criteria: 90.00/0:.11 O.~O%

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USP 43 Official Monographs / Calcium 681

Analysis Drift:Withint50/0, the'absorbance f5.60g1ijll

Sample: .Sample solution of USP Calcium Acetate RSfromt dard
From the linear calibration curve aet"'" inethe solutions. Se~ AtomicAbsorptiqn,S'IJ_.".'.L'v ...... 9PY ('8~L),
concentration (C), in fJg/ml.:, f~r calirLthe'Samp/~ .Procedure, Analysis;
solution. , . . '. .. . .. . ' ". Analysis ," '
Calculate the percentage of th ele8am6urifof Sample:, Sample s
calcium acetate (C4 H6Ca'01} d .. Fromthe quadratic
Correlation coeffici
Result = ex V ~ F x Dx:(fvlr1!Mi2 ) x (1 /t)x1 06 r. rc
per
,C =concentrati
determined
caldum acetate (c: H6
4

V = volume of Result =ex Vx F.x,D:J< (11L)xl ()()

F =equivalency fa r,' ~ g/~ .
o =dilution factor or the Sam le:so ~ •.ri, iflieeded C =conc~ntration()! calci ate intH~Samp"e
M;l = molecular weightqf calCiumac . ,158J7 solution determined (lJg mL)
Mr2 = molecular weight of calcium, AO.08 . .'. V = volur:ne of Med' ,900ml
L == label.c1aim (mg/Capsule) F =e . mg/~g
[) pIesolution; jfneeded
Tolerances:NlT 85% (Q) 0 [ apsule) . .
calcium acetate ( 6La
Test 3:.If the produ om hEdaheling
indicates that it me s USP
Tier l'
Medium 1: Water; 900 mL
Apparatus 2: 100 rpm, with sinkers
Time: 15 min '
Tier 2
Medium 2: ' Simulated gas T'5i900mL
Appar tus 2: 100 rpm, wi slne:rs
. in

An
and An
Samples
undert
[)issolution procedure: rmthe Result = Vs x IV! x F x (VM/VA) x OIL) x'l ()()
conditions ,under Tier. 7,.11.1 the prese == volume of Titli ed by the aliquot of
repeat the test with a new set of Capsules USI Sample stock s L)
conditions under Tier 2. ' ,
Analytical,procedure 1
M = actual Titrant concentration, hi molarity
(mmol/ml)
Blank:',O.02 N nitric acid F = equivalency factor of calcium acetate, 158.17
Standard solutions: 2.4, 3.2, 4.0~ 4!8,;and S.t? J.Ig!mlof mg/mniol
LISP CalciumAcetateRSin Blank = volume of Medium, 900mL
Sample solution: Nominally 3.7 IJg/ml6,
acetate from Sample stock solution, dilute if = volume ofthe' ati.quottaken for }\nalysis(mL)
necessary = label claim (mg/Capsule)
Ins ental conditions
( tomicAbsoli'
Mo e: Atomic ab
An . 'wavelen
lciurnholl e
Flame: Itrous oXid~acetylene
. Replicates: ' 4
System suitability
Sa!,"pl~~:Blanki Stcmdard solutions, and Samp(e ~olLJt'on
SUitabilityrequirements ., . 50
Relative staodarddeviation:NM:t3.0% 'in 4 0.2
replicate measurementS, Standard solutions arid rant
Sam lution' ..' .
Correl n coefficient:
set the instrument to zero.
the responses for each of the
Constructa quadratic calibra
the absorbance values of the ard sol ,. s
their corresponding concentrations, in micrograms'
per mil/iter. ' ,. .

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682 Calcium / OfficialMonographs USP43

\Is Apparatus 2: 50 rpm

Time: 30 min
Va Instrumental conditions
M (See AtomicAbsorption Spectroscopy (852).)
Mode: Atomic absorption spectrometry
F Analytical wavelength: 422.8 nm
Standard solution: Calcium at a known concentration in
Medium
Sample solution: Pass a portion of the solution under test
through a suitable filter. Dilute with Medium, if necessary,
to a concentration that is similar to the Standardsolution.
Acceptance criteria: NLT 80% (Q) of the labeled amount of
calcium acetate (C4H6Ca04) is dissolved.
• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
IMPURITIES
• LIMIT OF ALUMINUM
Buffer: Dissolve 50 9 of ammonium acetate in 150 mL of
water, adjust with glacial acetic acid to a pH of 6.0, and
dilute with water to 250 mL.
Aluminum standard solution 1.0 J.lg/mLof aluminum.
Prepare as directed for StandardPreparation under
Aluminum(206).
Standard solution: Preparea solution containing 2.0 mL of
Aluminum standardsolution, 5 mL of Buffer, and 48 mL of
water, and extract this solution with successive portions of
10, 10, and 5 mL of 0.5% 8-hydroxyquinoline in
chloroform. Combine the chloroform extracts in a 50-mL
volumetric flask, and dilute the combined extracts with
chloroform to volume.
Sample solution: Dissolve a portion of powdered Tablets
(NLT 10) equivalent to 1.0 9 of calcium acetate in 50 mL of
water, and add 5 mL of Buffer. Extract this solution with
successive portions of 10, 10, and 5 mL of 0.5%
8-hydroxyquinoline in chloroform. Combine the
chloroform extracts in a 50-mL volumetric flask, and dilute
the combined extracts with chloroform to volume.
Blank sclutlon: Prepare a solution containing 50 mL of
Calcium Acetate Tablets water and 5 mL of Buffer. Extract this solution with
successive portions of 10, 10, and 5 mL of 0.5%
DEFINITION 8-hydroxyquinoline in chloroform. Combine the
Calcium Acetate Tablets contain NLT 90.0% and NMT chloroform extracts in a 50-mL volumetric flask, and dilute
110.0% of the labeled amount of calcium acetate the combined extracts with chloroform to volume.
(C4H6Ca04)· Instrumental conditions
(See Fluorescence Spectroscopy (853).)
IDENTIFICATION Mode: Fluorescence
• IDENTIFICATION TESTS-GENERAL, Calcium (191 )andAcetate Excitation wavelength: 392 nm
(191 ) Emission wavelength: 51 8 nm
Sample solution: 100 mg/mL of calcium acetate from Analysis
powdered Tablets Samples: Standardsolution, Sample solution, and Blank
Acceptance criteria: Meet the requirements solution. Usethe Blank solution to zero the instrument.
Acceptance criteria: The fluorescence of the Sample
ASSAY solution does not exceed that of the Standardsolution (NMT
• PROCEDURE 2 ppm).
Sample: Amount equivalent to 300 mg of calcium acetate
from NLT 20 powdered Tablets ADDITIONAL REQUIREMENTS
Analysis: Dissolvethe Sample in 150 mL of water containing • PACKAGING AND STORAGE: Preserve in well-closed
2 mL of 3 N hydrochloric acid. While stirring, add 30 mL of containers.
0.05 M edetate disodium VS from a 50-mL buret, and add
15 mL of 1 N sodium hydroxide and 300 mg of hydroxy
naphthol blue. Continue the titration with the 0.05 M
edetate disodium VS to a blue endpoint. Each mL of 0.05
M edetate disodium is equivalent to 7.909 mg of calcium Calcium Ascorbate
acetate (C4H6Ca04)'
Acceptance criteria: 90.0%-110.0% 426.34

PERFORMANCE TESTS
• DISSOLUTION (711)
Medium: Water; 900 mL

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 USP 43 OfficialMonographs / Calcium 683

DEFINITION bar, insert the electrodes into the solution, stir for 15 min,
Calcium Ascorbate contains NlT 98.0% and NMT 101.0% of and read the potential, in mV. Continue stirring, and at
calcium ascorbate dihydrate (C12H14Ca012' 2H20), 5-min intervals add 100, 100, 300, and 500 IJl of
calculated on the as-is basis. Standardsolution, reading the potential 5 min after each
addition. Plot the logarithms of the cumulative fluoride
IDENTIFICATION ion concentrations (0.1, 0.2, 0.5, and 1.0 IJg/ml) versus
• A. IDENTIFICATION TESTs-GENERAL, Calcium (191): A 100 potential, in mV.
mg/ml solution meets the requirements. Rinse and dry the electrodes, insert them into the Sample
• B. A 100 mg/ml solution decolorizes a 100 mg/ml solution solution, stir for 5 min, and read the potential, in mV.
of dichlorophenol-indophenol. From the measured potential and the Standard
response line determine the concentration, C (in IJg/ml),
of fluoride ion in the Sample solution.
Calculate the content of fluoride in the portion of Calcium
Ascorbate taken:

ASSAY Result = (C x V)/W

• PROCEDURE C = concentration of fluoride ion in the Sample
Sample: 300 mg of Calcium Ascorbate solution (lJg/ml), obtained from the Standard
Blank: 50 ml of water response line
Titrimetric system V = volume of the Sample solution (ml)
(See Titrimetry (541).)
Mode: Direct titration
W =weight of Calcium Ascorbate taken to prepare the
Sample solution (g)
Titrant: 0.1 N iodine VS
Endpoint detection: Visual Acceptance criteria: NMT 10 ppm
Analysis: Transfer the Sample into a 250-ml conical flask,
add 50 ml of water, and mix to dissolve. Immediately SPECIFIC TE.STS
titrate with the Titrant, adding 3 ml of starch TS as the • OPTICAL ROTATION, Specific Rotation (781 S) ,_
endpoint is approached. Perform a Blank determination. Sample solution: 50 mg/ml in carbon dioxide-free water
Calculate the percentage of calcium ascorbate dihydrate [Nora-Perform measurements immediately after
(C12H14Ca012 . 2H20) in the Sample taken: preparation.]
Acceptance criteria: +95 0 to +97 0
Result = {[(V s - V 8) x N x A/w} x 100 • pH (791)
Sample solution: 100 mg/ml
Vs = Titrant volume consumed by the Sample (ml) Acceptance criteria: 6.8-7.4
V8 = Titrant volume consumed by the Blank (ml) • Loss ON DRYING (731): Dry 3 9 at 105 0 for 2 h: it loses NMT
N = Titrant normality (mEq/ml) 0.1 % of its weight.
F = equivalency factor, 106.6 mg/mEq
ADDITIONAL REQUIREMENTS
W = Sample weight (mg)
• PACKAGING AND STORAGE: Preserve in tight, light-resistant
Acceptance criteria: 98.0%-101.0% on the as-is basis containers.
• USP REFERENCE STANDARDS (11)
IMPURITIES USP Calcium Ascorbate RS
• ARSENIC, Method I (211): NMT 3 IJg/g USP Sodium Fluoride RS
• LIMIT OF FLUORIDE
[NOTE-Prepare and store all solutions in plastic
containers.]
Buffer solution: 294 mg/ml of sodium citrate dihydrate in
water Calcium Carbonate
Standard stock solution: 1.1052 mg/ml of USP Sodium
Fluoride RS in water CaC0 3 100.09
Standard solution: Transfer 20.0 ml of Standardstock Carbonic acid, calcium salt (1:1);
solution to a 1OO-mlvolumetric flask containing 50.0 ml of Calcium carbonate (1:1) [471-34-1].
Buffer solution, dilute with water to volume, and mix. Each DEFINITION
ml of Standardsolution contains 100 IJg of fluoride ion. 0
Calcium Carbonate, dried at 200 for 4 h, contains calcium
Sample solution: Transfer 2.0 9 of Calcium Ascorbate to a equivalent to NlT 98.0% and NMT 100.5% of calcium
beaker containing a plastic-coated stirring bar. Add 20 ml carbonate (CaC0 3) .
of water and 2.0 ml of hydrochloric acid, and stir until
dissolved. Add 50.0 ml of Buffer solution and sufficient IDENTIFICATION
water to make 100 ml. • A. IDENTIFICATION TESTS-GENERAL, Calcium (191): The
Electrode system: Use a fluoride-specific ion-indicating addition of acetic acid to it produces effervescence
electrode and a silver-silver chloride reference electrode (presence of carbonate), and the resulting solution, after
connected to a pH meter capable of measuring potentials boiling, meets the requirements of the tests.
with a minimum reproducibility of ± 0.2 mV (see pH
(791» ASSAY
• TITRIMETRY (541) ,
Analysis
Samples: Standardsolution and Sample solution Sample: 200 mg of Calcium Carbonate, previously dried at
0
Standard response line: Transfer 50.0 ml of Buffer 200 for 4 h
solution and 2.0 ml of hydrochloric acid to a beaker, and Blank: 100 ml of water and 15 ml of 1 N sodium hydroxide
add water to make 100 ml. Add a plastic-coated stirring Titrimetric system
(See Titrimetry (541 ).)

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 684 Calcium / Official Monographs USP 43

Mode: Direct titration • LIMIT OF FLUORIDE

Titrant: 0.05 M edetate disodium VS [NOTE-Prepare and store all solutions in plastic containers.]
Indicator: 300 mg of hydroxy naphthol blue Solution A: 294 mg/mL of sodium citrate dihydrate in water
Endpoint detection: Visual, change to distinct blue Sample: 2.0 g
Analysis: Transfer the Sample to a 250-mL beaker. Moisten Standard stock solution: 1.11 mg/mL of USP Sodium
thoroughly with a few mL of water, and add, dropwise, Fluoride RS in water
sufficient 3 N hydrochloric acid to dissolve. Add 100 mL of Standard solution: Combine 20.0 mL of the Standardstock
water, 15 mL of 1 N sodium hydroxide, and 300 mg of solution with 50.0 mL of Solution A, and dilute with water
hydroxy naphthol blue. Titrate with the Titrant. Calculate to 100.0 mL. [NOTE-Each rnl, of this solution contains 100
the percentage of calcium carbonate (CaC0 3) in the Sample IJg of fluoride ion]
taken: Electrode system: Use a fluoride-specific ion-indicating
electrode and a silver-silver chloride reference electrode
Result = [(V - B) x M x F x 100]/W connected to a pH meter capable of measuring potentials
with a minimum reproducibility of ±0.2 mV (see pH (791 ».
V = Sample titrant volume (mL) Standard response line: Transfer 50.0 mL of Solution A and,
B = Blank titrant volume (mL) 4.0 mL of hydrochloric acid to a beaker, and add water to
M =titrant molarity (mmol/mL) make 100 mL. Add a plastic-coated stirring bar, insert the
F = equivalency factor, 100.09 mg/mmol electrodes into the solution, stir for 15 min, and read the
W = weight of the Sample (mg) potential, in mY. Continue stirring, and at 5-min intervals
add 100, 100, 300, and 500 IJLof the Standardsolution,
, Acceptance criteria: 98.0%-100.5% on the dried basis reading the potential 5 min after each addition. Plot the
IMPURITIES logarithms of the cumulative fluoride ion concentrations
• ACID-INSOLUBLE SUBS'"ANCES (0.1, 0.2, 0.5, and 1.0 IJg/mL) versus potential, in mY.
Sample: 5.0 g Analysis: Transfer the Sample to a beaker containing a
Analysis: Mix the Sample with 10 mL of water, and add plastic-coated stirring bar, add 20 mL of water and 4.0 mL
hydrochloric acid, dropwise, with agitation, until it ceases of hydrochloric acid, and stir until dissolved. Add 50.0 mL
to causeeffervescence, then add water to make the mixture of Solution A and sufficient water to make 100 mL of test
measure 200 mL, and filter. Wash the insoluble residue with solution. Rinse and dry the electrodes, insert them into the
water until the last washing shows no chloride, and ignite Sample solution, stir for 5 min, and read the potential, in
and weigh the residue. mY. From the measured potential and the Standard
Acceptance criteria: NMT 0.2%; the weight of the residue response line, determine the concentration, C, in IJg/mL, of
does not exceed 10 mg. fluoride ion in the Sample solution. Calculate the content of
• ARSENIC, Method I (211 ) ,fluoride in the specimen taken:
Sample solution: Slowly dissolve 1.0 g in 15 mL of
hydrochloric acid, and dilute with water to 55 mL. Result = (V x C)/W
Analysis: Omit the addition of 20 mL of 7 N sulfuric acid V = volume of the Sample solution (mL)
specified in Arsenic (211), Method I, Procedure. C = concentration of fluoride in the
Sample solution
Acceptance criteria: NMT 3 ppm
(lJg/ m L)
• BARIUM: A platinum wire, dipped in the filtrate obtained in W =weight of Sample (g)
the test for Acid-Insoluble Substances and held in a
nonluminous flame, does not impart a green color. Acceptance criteria: NMT 50 ppm
• IRON (241) , • LIMIT OF MAGNESIUM AND ALKALI SALTS
Sample solution: 40 mg in 5 mL of 2 N hydrochloric acid. Sample solution: 1.0 g
Transfer to a beaker with the aid of water, and dilute with Analysis: Mix the Sample with 35 mL of water. Carefully add
water to 10 mL. 3 mL of hydrochloric acid, heat the solution, and boil for 1
Standard solution: Transfer 4.0 mL of the Standard Iron min. Rapidly add 40 mL of oxalic acid TS, and stir vigorously
Solution, prepared as directed in Iron (241), to a beaker, and until precipitation is well-established. Add immediately to
dilute with water to 10 mL. the warm mixture 2 drops of methyl red TS and then 6 N
Instrumental conditions ammonium hydroxide, dropwise, until the mixture is just
(See Ultraviolet-Visible Spectroscopy (857).) alkaline. Cool to room temperature, transfer to a 100-mL
Analytical wavelength: 530 nm graduated cylinder, dilute with water to 100 mL, mix, and
Blank: Water allow to stand for 4 h or overnight. Filter, and to 50 mL of
Analysis: Separately to the Sample solution and Standard the clear filtrate in a platinum dish add 0.5 mL of sulfuric
solution add 2 mL of citric acid solution (1 in 5) and 2 drops acid, and evaporate the mixture on a steam bath to a small
of thioglycolic acid, adjust with ammonia TS to a pH of 9.5 volume. Carefully heat over a free flame to dryness, and
± 0.1, dilute with water to 20 mL, and allow to stand for 5 continue heating to complete decomposition and
min. Dilute with water to 50 mL. Concomitantly determine volatilization of ammonium salts. Finally, ignite the residue
the absorbances of the solutions from the Sample solution to constant weight.
and the Standardsolution. Acceptance criteria: NMT 1.0%; the weight of the residue
Acceptance criteria: NMT 0.1%; the absorbance of the is NMT 5 mg.
solution from the Sample solution does not exceed that of • MERCURY, Method lIa (261)
the Standardsolution. Mercury stock solution and Standard mercury solution:
• LEAD (251) Proceed as directed in Mercury (261).
Sample solution: 1.0 g in 5 mL of water Standard solution: Proceed as directed in Mercury(261),
Analysis: To the Sample solution slowly add 8 mL of 3 N except use 3 mL of hydrochloric acid instead of 3 mL of
hydrochloric acid, evaporate on a steam bath to dryness, sulfuric acid.
and dissolve the residue in 5 mL of water. Sample stock solution: 4.0 g in a 1OO-mLbeaker, and
Acceptance criteria: NMT 3'ppm cautiously dissolve in 14 mL of 6 N hydrochloric acid

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 USP 43 Official Monographs / Calcium 685

Sample solution: Proceed as directed in Mercury(261) using Analytical wavelength: Calcium emission line at 422.7
the Sample stock solution, except use 3 mL of hydrochloric nm
acid instead of 3 mL of sulfuric acid. Blank: Lanthanum chloride solution
Analysis Analysis
Samples: Standard solution and Sample solution Samples: Standardsolutions, Sample solution, and Blank
Proceed as directed in Mercury (261). Plot the absorbances of the Standardsolutions versus their
Acceptance criteria: NMT 0.5 ppm concentrations of calcium carbonate, in uq/rnl, by
drawing a straight line best fitting the three plotted
SPECIFIC TESTS points. From the graph determine the concentration, C,
• Loss ON DRYING (731): Dry a sample at 200 0 for 4 h: it loses in ~g/mL, of calcium carbonate in the Sample solution.
NMT 2.0% of its weight. Calculate the percentage of label claim of calcium
ADDITIONAL REQUIREMENTS carbonate (CaC0 3) in the portion of Lozenges taken:
• PACKAGING AND STORAGE: Preserve in well-closed
containers. Result = (C/Cu) x 10'0
• USP REFERENCE STANDARDS (11)
USP Sodium Fluoride RS C = measured concentration of calcium carbonate in
the Sample solution (~g/mL), as calculated above
Cu = nominal concentration of calcium carbonate in
the Sample solution (~g/mL)

Calcium Carbonate Lozenges Acceptance criteria: 90.0%-110.0%

DEFINITION OTHER COMPONENTS

Calcium Carbonate Lozenges contain NLT90.0% and NMT • SODIUM CONTENT (if so labeled)
110.0% of the labeled amount of calcium carbonate [NoTE-The Standardsolutions and the Sample solution
(CaC0 3) · may be modified, if necessary, to obtain solutions of
suitable concentrations adaptable to the linear or
IDENTIFICATION . working range of the instrument.]
• A. IDENtiFICATION TESTS-GENERAL, Calcium (191): The Standard stock solution: Transfer 2.542 g of sodium
0
addition of 6 N hydrochloric acid to a Lozenge produces chloride, previously dried at 105 for 2 h, to a 1000-mL
effervescence, and the resulting solution, after being boiled volumetric flask. Dissolve in and dilute with water to
to expel carbon dioxide and then neutralized with 6 N volume. Transfer 10.0 mL of this solution to a 100-mL
ammonium hyd~oxide, meets the requirements of the tests. volumetric flask, and dilute with water to volume.
Standard solutions: To three separate 1OO-mL volumetric
ASSAY flasks, add 1.0, 3.0, and 5.0 mL of the Standardstock
• PROCEDURE solution, and dilute each with water to volume. These
[NoTE-The Standardsolutions and the Sample solution Standardsolutions contain 1.0, 3.0, and 5.0 ~g/mL of
may be modified, if necessary, to obtain solutions of sodium, respectively.
suitable concentrations adaptable to the linear or Sample stock solution: Prepare as directed in the Assay.
working range of the instrument.] Pass a portion of it, if necessary, through a filter of 0.5-~m
lanthanum chloride solution: .Transfer 109 of potassium or finer pore size, and use the clear solution.
chloride and 20 g of lanthanum chloride to a 2000-mL Sample solution: Transfer 10.0 mL of the Sample stock
volumetric flask. Add 1000 mL of water and 40 mL of solution to a 25-mL volumetric flask, and dilute with water
hydrochloric acid, mix, and allow to cool. Dilute with water to volume.
to volume. Instrumental conditions
Standard stock solution: Transfer 250 mg of chelornetric (See Atomic Absorption Spectroscopy (852).)
standard calcium carbonate, previously dried at 1100 for 2 Mode: Atomic absorption spectrophotometry
h and then cooled in a desiccator, to a 500-mL volumetric lamp: Sodium hollow-cathode
flask. Add 100 mL of water and 12 mL of 1 N hydrochloric Flame: Air-acetylene
acid, swirl to dissolve the calcium carbonate, and allow to Analytical wavelength: Sodium emission line at 589.6 nm
cool. Dilute with water to volume. This stock solution Blank: Water
contains about 500 ~g/mL of calcium carbonate. Analysis
Standard solutions: To three separate 1OO-mL volumetric Samples: Standardsolutions, Sample solution, and Blank
flasks add 2.0, 3.0, and 4.0 mL of the Standardstock Plot the absorbances of the Standardsolutions versus their
solution, and dilute each with Lanthanumchloride solution contents of sodium, in ~g/mL, by drawing a straight line
to volume. These Standardsolutions contain 10, 15, and 20 best fitting the three plotted points. From the graph
~g/mL of calcium carbonate, respectively. determine the quantity, C, in ~g, of sodium in each mL of
Sample stock solution: Transfer the equivalent to 3000 mg the Sample solution.
of calcium carbonate, from powdered Lozenges, to a Calculate the percentage of label claim of sodium in the
1OOO-mL volumetric flask. Add 100 mL of 1 N hydrochloric portion of Lozenges taken:
acid and 300 mL of water, and sonicate to dissolve the
powder. Dilute with water to volume. Result =(CICu) x 100
Sample solution: Transfer 5.0 mLof Sample stock solution to
a 1OOO-mL volumetric flask, and dilute with Lanthanum C = measured concentration of sodium in the Sample
chloride solution to volume. solution (~g/mL), as calculated above
Instrumental conditions Cu = nominal concentration of sodium in the Sample
(See Atomic Absorption Spectroscopy (852).) solution (~g/mL)
Mode: Atomic absorption spectrophotometry
lamp: Calcium hollow-cathode Acceptance criteria: NMT 115.0% of the labeled amount
Flame: Nitrous oxide-acetylene

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686 Calcium / OfficialMonographs USP 43

PERFORMANCE TESTS Acceptance criteria: 90.00/0-110.0%

• UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements IMPURITIES
• LIMIT OF FLUORIDE
SPECIFIC TESTS [NOTE-Prepare and store all solutions in plastic containers.]
• ACID-NEUTRALIZING CAPACITY (301) Solution A: 294 mg/mL of sodium citrate dihydrate in water
Analysis: The acid consumed by the minimum single dose Standard stock solution: 1.1 mg/mL of USP Sodium
recommended in the labeling is NLT 5 mEq of acid and NLT Fluoride RS in water
the number of mEq calculated by: Standard solution: Combine 20.0 mL of the Standardstock
solution with 50.0 mL of Solution A, and dilute with water
Result = (Fe x Q x 0.9 to 100.0 mL. [NOTE-Each mL of this solution contains 100
I-Ig of fluoride ion.]
= theoretical acid-neutralizing capacity of CaC0 3, Sample solution: Transfer a portion of Oral Suspension,
0.02 mEq equivalent to 2.0 9 of calcium carbonate, to a beaker
C = quantity of CaC0 3 in the sample tested (mg), containing a plastic-coated stirring bar. Add 20 mL of water
based on the labeled quantity and 4.0 mL of hydrochloric add. Stir until dissolved. Add
50.0 mL of Solution A and sufficient water to make 100.0
ADDITIONAL REQUIREMENTS mL.
• PACKAGING AND STORAGE: Preserve in well-closed Electrode system: Use a fluoride-specific ion-indicating
containers. electrode and a silver-silver chloride reference electrode
connected to a pH meter capable of measuring potentials
with a minimum reproducibility of to.2 mV (see pH (791 ».
Standard response line: Transfer 50.0 mL of Solution A and
4.0 mL of hydrochloric acid to a beaker. Add water to make
Calcium Carbonate Oral Suspension 100.0 mL. Add a plastic-coated stirring bar, insert the
electrodes into the solution, and stir for 15 min. Read the
DEFINITION potential, in mV. Continue stirring, and at 5-min intervals
Calcium Carbonate Oral Suspension contains NLT 90.0% and add 100, 100, 300, and 500 I-IL of the Standardsolution,
NMT 110.0% of the labeled amount of calcium carbonate reading the potential 5 min after each addition. Plot the
(CaC0 3) · logarithms of the cumulative fluoride ion concentrations
IDENTIFICATION (0.1, 0.2, 0.5, and 1.0 I-Ig/mL) versus potential, in mV.
• A. IDENTIFICATION TESTS-GENERAL, Calcium (191): The Analysis: Rinse and dry the electrodes, and insert them
addition of ace/tic acid to it produces effervescence into the Sample solution. Stir for 5 min, and read the
(presence of carbonate). The resulting solution, after potential, in mV. From the measured potential and the
boiling, meets the requirements. Standardresponse line, determine the concentration, C, in
I-Ig/mL, of fluoride ion in the Sample solution.
ASSAY Calculate the content of fluoride in the sample taken:
• PROCEDURE
Sample solution: Transfer a portion of Oral Suspension, Result = (V x Q/W
equivalent to 1 9 of calcium carbonate, previously well
shaken in its original container, to a beaker with the aid of V = volume of the Sample solution (mL)
25 mL of water. Add 20 mL of 1 N hydrochloric acid. Heat C = determined concentration of fluoride in the
on a steam bath for 30 min. Allow to cool, and transfer with Sample solution (l-Ig/mL)
the aid of water to a 1OO-mL volumetric flask. Dilute with W =nominal weight of calcium carbonate taken (g)
water to volume. Mix, and filter.
Blank: 100 mL of water, 15 mL of 1 N sodium hydroxide, Acceptance criteria: 50 I-Ig/g, with respect to the labeled
and 5 mL of triethanolamine amount of calcium carbonate
Titrimetric system • ARSENIC, Method I (211)
(See Titrimetry (541).) Test preparation: Slowly dissolve a portion of Oral
Mode: Direct titration Suspension equivalent to 1.0 9 of calcium carbonate in 15
Tltrant; 0.05 M edetate disodium VS mL of hydrochloric acid. Dilute with water to 55 mL.
Indicator: 100 mg of hydroxy naphthol blue Analysis: Proceed as directed in the chapter, except omit
Endpoint detection: Visual, change to distinct blue the addition of 20 mL of 7 N sulfuric acid specified under
Analysis: Transfer 20.0 mL of the Sample solution to a Procedure.
suitable container. Dilute with water to 100 mL. Add 15 mL Acceptance criteria: NMT 3 I-Ig/g, with respect to the
of 1 N sodium hydroxide, 5 mL of triethanolamine, and 100 labeled amount of calcium carbonate
mg of hydroxy naphthol blue. Titrate with the Titrant. • LEAD (251)
Calculate the percentage of the labeled amount of calcium Test preparation: Mix a portion of Oral Suspension
carbonate (CaC0 3) in the sample taken: equivalent to 1.0 9 of calcium carbonate in 5 mL of water.
Analysis: To the Test preparation slowly add 8 mL of 3 N
Result = [(V s- V 8) x Mx Fx 100]/W hydrochloric acid. Evaporate on a steam bath to dryness,
and dissolve the residue in 5 mL of water.
Vs =volume of the Titrant consumed by the Sample Acceptance criteria: NMT 3 I-Ig/g, with respect to the
solution (mL) labeled amount of calcium carbonate
V8 = volume of the Titrant consumed by the Blank SPECIFIC TESTS
(mL)
• MICROBIAL ENUMERATION TESTS (61) and TESTS FOR
M = Titrant molarity (mmol/mL) SPECIFIED MICROORGANISMS (62): The total aerobic
F =equivalency factor, 100.09 mg/mmol microbial count is NMT 102 cfu/mL. It meets the
W = nominal amount of calcium carbonate taken for
the Analysis (mg)

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 USP 43 Official Monographs / Calcium 687

requirements of the tests for absence of Escherichia coli and Medium: 0.1 N hydrochloric acid; 900 mL
Pseudomonas aeruginosa. Apparatus 2: 75 rpm
• pH (791): 7.5-8.7 Time: 30min
Lanthanium chloride solution: 50 mg/mL of lanthanum
ADDITIONAL REQUIREMENTS chloride in 0.1 N hydrochloric acid
• PACKAGING AND STORAGE: Preserve in tight containers, and Standard stock solution: 100 IJg/mL of calcium in 0.1 N
avoid freezing. hydrochloric acid
• USP REFERENCE STANDARDS (11) Standard solutions: Into separate 1OO-mL volumetric flasks
USP Sodium Fluoride RS containing 10.0 mL of Lanthanium chloride solution pipet 3-,
4-, 5-, and 6-mL portions of Standardstocksolution and
dilute each with 0.1 N hydrochloric acid to volume to
obtain solutions with calcium concentrations of 3, 4, 5, and
Calcium Carbonate Tablets 6 IJg/mL, respectively.
Sample solution: Filter a portion of the solution under test.
DEFINITION Pipet a volume of the filtrate, estimated to contain 1 mg of
Calcium Carbonate Tablets contain NLT 90.0% and NMT calcium, into a 250-mL volumetric flask. Add 25.0 mL of
110.0% of the labeled amount of calcium carbonate Lanthaniumchloride solution, and dilute with 0.1 N
(CaC0 3) . For Tablets labeled for any indication other than, hydrochloric acid to volume.
or in addition to antacid use,the Tablets contain NLT 90.0% Instrumental conditions
and NMT 115.0% of the labeled amount of calcium (See Atomic Absorption Spectroscopy (852).)
carbonate. Mode: Atomic absorption spectrophotometry
Analytical wavelength: 422.8 nm
IDENTIFICATION Lamp: Calcium hollow-cathode
• A. IDENTIFICATION TESTS-GENERAL, Calcium (191): The Flame: Air-acetylene
addition of 6 N acetic acid to the Tablets produces Blank: Lanthaniumchloride solution and 0.1 N hydrochloric
effervescence, and the resulting solution, after being boiled acid (1 :9)
to expel carbon dioxide and neutralized with 6 N Analysis
ammonium hydroxide, meets the requirements. Samples: Standardsolutions and Sample solution
Concomitantly determine the absorbances of the Standard
ASSAY solutions and the Sample solution against the Blank.
• PROCEDURE Construct a standard curve by plotting absorbances
Sample solution: Finely powder NLT 20 Tablets. Tra~sfer a versus calcium concentrations of the Standardsolutions,
portion of the powder, equivalent to 200 mg of calclL!m then from it obtain the concentration, C, in IJg/mL of
carbonate, to a suitable crucible. Ignite to con~tant weight. calcium, of the Sample solution.
Cool the crucible add 10 mL of water, and dissolve the Calculate the percentage of the labeled amount of calcium
residue by adding sufficient 3 N hydrochloric acid, carbonate (CaC0 3) dissolved:
dropwise, to achieve complete solution.. .
Blank: 150 mL of water and 15 mL of 1 N sodium hydroxide Result = (MjA,) x (C x 0 x V/L) x 100
Titrimetric system
(See Titrimetry(541).) M, = molecular weight of calcium carbonate, 100.09
Mode: Direct titration A, = atomic weight of calcium, 40.08
Titrant: 0.05 M edetate disodium VS
Indicator: 300 mg of hydroxy naphthol blue
C =measured concentration of calcium in the Sample
solution (mg/mL)
Endpoint detection: Visual, change to distinct blue
Analysis: Transfer the Sample solution completely to a
o = dilution factor for the Sample solution
suitable container, and dilute with water to 150 mL. Add
V = volume of Medium, 900 mL
15 mL of 1 N sodium hydroxide and 300 mg of hydroxy
L = label claim (mg/Tablet)
naphthol blue. Titrate with the Titrant. Tolerances: NLT 75% (Q) of the labeled amount of calcium
Calculate the percentage of calcium carbonate (CaC0 3) in carbonate (CaC0 3) is dissolved.
the sample taken: • UNIFORMITY OF DOSAGE UNITS (905): Meet the
requirements
Result = [(Vs - VB) x M x F x 100]/W
SPECIFIC TESTS
Vs =volume of the Titrant consumed by the Sample • ACID-NEUTRALIZING CAPACITY (301): For Tablets labeled
solution (mL) for antacid use
VB = volume of the Titrant consumed by the Blank Analysis: Proceed as directed in the chapter.
(mL) Acceptance criteria: NLT 5 mEq of aci9 is consun:ed by the
M = Titrant molarity (mmol/mL) minimum single dose recommended In the labeling, and
F =equivalency factor, 100.09 mg/mmol NLT the number of mEq calculated as follows:
W = weight of calcium carbonate taken (mg)
Result = (C x ANC) x F
Acceptance criteria: 90.0%-110.0% of the labeled amount
of CaC0 3 • ForTablets labeled for any indication other than, C = quantity of CaC0 3 in the sample tested (mg),
or in addition to, antacid use,90.00/0-115.0% of the labeled based on the labeled amount
amount of CaC0 3 ANC = theoreticalacid-neutralizing capacity of CaC0 3,
0.02 mEq/mg
PERFORMANCE TESTS
• DISSOLUTION (711)
F = acceptance factor for the lower limit of the
required acid-neutralizing capacity, 0.9
[NOTE-For Tablets labeled for any indication other
than, or in addition to, antacid use.]

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688 Calcium / Official Monographs USP43

ADDITIONAL REQUIREMENTS PERFORMANCE TESTS

• PACKAGING AND STORAGE: Preserve in well-closed • UNIFORMITY OF DOSAGE UNITS (90S): Meet the
containers. requirements for Weight Variation with respect to calcium
• LABELING: Label it to indicate whether it is for use as an carbonate and to magnesia
antacid, or as a dietary supplement, or both.
SPECIFIC TESTS
• ACID-NEUTRALIZING CAPACITY (301)
Analysis: NLT 5 mEq of acid is consumed by the minimum
single dose recommended in the labeling, and NLT the
Calcium Carbonate and Magnesia number of mEq calculated by the formula:
Tablets Result = [0.8 x (FM x M)] + [0.9 x (Fe x C)]
DEFINITION FM = theoretical acid-neutralizing capacity of
Calcium Carbonate and Magnesia Tablets contain NLT 90.0% Mg(OH)2, 0.0343 mEq
and NMT 110.0% of the labeled amount of calcium
carbonate (CaC0 3) and NLT 90.0% and NMT 115.0% of the
M =quantity of Mg(OH)2 in the sample tested (mg),
based on the labeled quantity
labeled amount of magnesium hydroxide [Mg(OH)2]' Fe = theoretical acid-neutralizing capacity of CaC0 3,
IDENTIFICATION 0.02 mEq
• A. IDENTIFICATION TESTS-GENERAL, Calcium (191): The C = quantity of CaC0 3 in the sample tested (mg),
addition of 3 N hydrochloric acid to the Tablets produces based on the labeled quantity
effervescence. The resulting solution, after being boiled to
expel carbon dioxide and neutralized with 6 N ammonium ADDITIONAL REQUIREMENTS
hydroxide, meets the requirements of the tests. • PACKAGING AND STORAGE: Preserve in well-closed
• B. IDENTIFICATION TESTS-GENERAL, Magnesium (191) containers.
Sample solution: Heat 2 Tablets in 20 mL of 1 N sulfuric
acid. Cool, add 20 mL of alcohol, mix, and allow to stand
for 30 min. Filter this solution, and add 2 mL of 1 I'J
hydrochloric acid to the filtrate.
Acceptance criteria: The solution meets the requirements.
Calcium Carbonate and Magnesia
ASSAY
Chewable Tablets
• CALCIUM CARBONATE DEFINITION
Sample solution: Finely powder NLT 20 Tablets. Transfer a Calcium Carbonate and Magnesia Chewable Tablets contain
portion of the powder, equivalent to 400 mg of calcium NLT 90.0% and NMT 110.0% of the labeled amount of
carbonate, to a beaker with 25 mL of water. Add 40 mL of calcium carbonate (CaC0 3) and NLT 90.0% and NMT
1 N hydrochloric acid. Heat on a steam bath for 30 min,
115.0% of the labeled amount of magnesium hydroxide
allow to cool, and transfer with the aid of water to a 100-mL
[Mg(OH)2]'
volumetric flask. Dilute with water to volume, mix, filter,
and use the filtrate. [NOTE-Reserve a portion of it for the IDENTIFICATION
test for Magnesium Hydroxide.] • A. IDENTIFICATION TESTS-GENERAL, Calcium (191): The
Analysis: Transfer 20.0 mL of the Sample selution to a addition of 3 N hydrochloric acid to the Chewable Tablets
suitable container, dilute with water tol 00 mL, and add 30 produces effervescence. The resulting solution, after being
mL of 1 N sodium hydroxide,S mL of trlethanolamlne, and boiled to expel carbon dioxide and neutralized with 6 N
100 mg of hydroxy naphthol blue. Titrate with 0.05 M ammonium hydroxide, meets the requirements of the tests.
edetate disodium VS until the solution is deep blue in color. • B. IDENTIFICATION TESTS-GENERAL, Magnesium (191)
Each mL of 0.05 M edetate disodium is equivalent to 5.004 Sample solution: Heat 2 Chewable Tablets in 20 mL of 1 N
mg of CaC0 3 • sulfuric acid. Cool, add 20 mL of alcohol, mix, and allow to
Acceptance criteria: 90.0%-110.0% stand for 30 min. Filter this solution, and add 2 mL of 1 N
• MAGNESIUM HYDROXIDE hydrochloric acid to the filtrate.
Sample solution: Use the Sample solution from the test for Acceptance criteria: The solution meets the requirements.
Calcium Carbonate.
Analysis: Transfer a portion of the Sample solution, ASSAY
equivalent to 120 mg of calcium carbonate and magnesium • CALCIUM CARBONATE
hydroxide combined, to a suitable container. Dilute with Sample solution: Finely powder NLT 20 Chewable Tablets.
water to 100 mL, and add 10 mL of ammonia-ammonium Transfer a portion of the powder, equivalent to 400 mg of
chloride buffer TS, 5 mL of triethanolamine, and 0.3 mL of calcium carbonate, to a beaker with 25 mL of water. Add
eriochrome black TS. Titrate with 0.05 M edetate disodium 40 mL of 1 N hydrochloric acid. Heat on a steam bath for
VS to a blue endpoint. The volume, in mL, of 0.05 M 30 min, allow to cool, and transfer with the aid of water to
edetate disodium consumed, less the volume of 0.05 M a 1OO-mL volumetric flask. Dilute with water to volume,
edetate disodium corresponding to the content of calcium mix, filter, and use the filtrate. [NOTE-Reserve a portion of
carbonate in the volume, in mL, of the Sample solution it for the test for Magnesium Hydroxide.]
taken, represents the volume, in mL, of 0.05 M edetate Analysis: Transfer 20.0 mL of the Sample solution to a
disodium equivalent to the quantity of magnesium suitable container, dilute with water to 100 mL, and add 30
hydroxide present. Each mL of 0.05 M edetate disodium is mL of 1 N sodium hydroxide,S mL of triethanolamine, and
100 mg of hydroxy naphthol blue. Titrate with 0.~5 M
equivalent to 2.916 mg of Mg(OH)2'
edetate disodium VS until the solution is deep blue In color.
Acceptance criteria: 90.00/0-115.0% Each mL of 0.05 M edetate disodium is equivalent to 5.004
mg of CaC0 3 •
Acceptance criteria: 90.0%-110.0%

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USP 43 Official Monographs / Calcium 689

• MAGNESIUM HYDROXIDE Packaging and storage-Preserve in well-closed

Sample solution: Use the Sample solution from the test for containers.
Calcium Carbonate. labeling-Label it to indicate that the Chewable Tablets are
Analysis: Transfer a portion of the Sample solution, to be chewed before swallowing. Label the Chewable Tablets
equivalent to 120 mg of calcium carbonate and magnesium to state the sodium content, in mg per Chewable Tablet, if it
hydroxide combined, to a suitable container. Dilute with is greater than 5 mg per Chewable Tablet.
water to 100 mL, and add 10 mL of ammonia-ammonium USP Reference standards (11)-
chloride buffer TS, 5 mL of triethanolamine, and 0.3 mL of USP Polydimethylsiloxane RS
eriochrome black TS. Titrate with 0.05 M edetate disodium
VS to a blue endpoint. The volume, in mL, of 0.05 M Identification-
edetate disodium consumed, less the volume of 0.05 M
edetate disodium corresponding to the content of calcium
carbonate in the volume, in mL, of the Sample solution
taken, represents the volume, in mL, of 0.05 M edetate "Cfi~',T~JJ~i<~;~~)h~il~teql{~g
fpg~t.r/d~9J:fp'2;;lgrz§Z,( a Qj- . . .
disodium equivalent to the quantity of magnesium
hydroxide present. Each mL of 0.05 M edetate disodium is Solution-Using Chewable Tablets, proceed to obtain IR
absorption spectra as directed in the Assay for
equivalent to 2.916 mg of Mg(OH)2'
polydimethylsiloxane under Alumina, Magnesia, and
Acceptance criteria: 90.0%-115.0% Simethicone Chewable Tablets
PERFORMANCE TESTS B: The addition of 1 N hydrochloric acid to a Chewable
• UNIFORMITY OF DOSAGE UNITS (905): Meet the Tablet produces effervescence, and the resulting solution, after
requirements for Weight Variation with respect to calcium having been filtered, meets the requirements of the tests for
carbonate and to magnesia Calcium (191).
C: Heat 2 Chewable Tablets in 20 mL of 1 N sulfuric acid.
SPECIFIC TESTS Cool, add 20 mL of alcohol, mix, and allow to stand for 30
• ACID-NEUTRALIZING CAPACITY (301 ) minutes. Filter this solution, and to the filtrate add 2 mL of 1
Analysis: NLT 5 mEq of acid is consumed by the minimum N hydrochloric acid: this solution meets the requirements of
single dose recommended in the labeling, and NLT the the tests for Magnesium (191).
number of mEq calculated by the formula: Uniformity of dosage units (905): meet the requirements
for Weight Variation with respect to calcium carbonate and to
Result = [0.8 x (FM x M)] + [0.9 x (Fe x C)] magnesium hydroxide.
Acid-neutralizing capacity (301 )-Not less than 5 mEq of
FM = theoretical acid-neutralizing capacity of acid is consumed by the minimum single dose recommended
Mg(OHh, 0.0343 mEq in the labeling.
M = quantity of Mg(OHh in the sample tested (mg),
based on the labeled quantity Content of sodium (if so labeled)-
Fe = theoretical acid-neutralizing capacities of Lanthanum chloride solution-Prepare as directed in the
CaC0 3, 0.02 mEq Assay for calcium carbonate and magnesium hydroxide.
C =quantity of CaC0 3 in the sample tested (mg), Dilutehydrochloric acid-Prepare as directed in the Assay for
based on the labeled quantity polydimethylsiloxane.
Standard solution-Transfer 2.542 g of sodium chloride,
ADDITIONAL REQUIREMENTS previously dried at J05 0 for 2 hours, to a 1OOO-mL volumetric
• PACKAGING AND STORAGE: Preserve in well-closed flask, dissolve in and dilute with water to volume, and mix.
containers. Transfer 5.0 mL of this solution to a 1OO-mL volumetric flask,
• LABELING: Label the Chewable Tablets to indicate that they dilute with water to volume, and mix. Transfer 4.0 mL of this
must be chewed before being swallowed. solution to a second 1OO-mL volumetric flask containing 6.0
mL of Dilutehydrochloric acid and 2.0 mL of Lanthanum chloride
solution, dilute with water to volume, and mix. This solution
contains 2.0 I-Ig of sodium (Na) per mL.
Test solution-Transfer 3.0 mL of the aqueous layer retained
Calcium Carbonate, Magnesia, and from the preparation of the Assay preparation in the Assay for
polydimethylsiloxane to a 50-mL volumetric flask containing
Simethicone Chewable Tablets 1.0 mL of Lanthanum chloride solution, dilute with water to
volume, and mix.
Former title: Calcium Carbonate, Magnesia, and Simethicone Blank solution-Transfer 15.0 mL of Dilute hydrochloric acid
Tablets and 5.0 mL of Lanthanum chloride solution to a 250:,mL
volumetric flask, dilute with water to volume, and mix.
» Calcium Carbonate, Magnesia, and Simethicone Procedure-Concomitantly determine the absorbances of
Chewable Tablets contain not less than 90.0 the Standard solution and the Test solution at the sodium
percent and not more than 110.0 percent of the emission line at 589.0 nm with a suitable atomic absorption
spectrophotometer (see AtomicAbsorption Spectroscopy (852»
labeled amounts of calcium carbonate (CaC0 3) equipped with a sodium hollow-cathode lamp and an air-
and magnesium hydroxide [Mg(OH)2], and an acetylene flame, using the Blank solution as the blank.
amount of polydimethylsiloxane [-(CH3)2SiO-]n Calculate the mg of sodium (Na) in each Chewable Tablet
that is not less than 85.0 percent and not more taken by the formula:
than 115.0 percent of the labeled amount of (5C/6)(A/W)(A u/As)
simethicone.
in which C is the concentration, in I-Ig per mL, of sodium in the
Standard solution; A is the average weight, in mg, of each

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 690 Calcium / OfficialMonographs USP 43

Chewable Tablet; W is the weight, in mg, of the portion of Calcium stock standard solution-Transfer 499.5 mg of
Chewable Tablets from the preparation of the Assay primary standard calcium carbonate to a 200-mL volumetric
preparation in the Assay for polydimethylsiloxane used to flask, and add 10 mL of water. Carefully add 5 mL of Dilute
prepare the Test solution; and Au and As are the absorbances hydrochloric acid, and swirl to dissol~e the.calciur;t carbon~te.
of the Test solution and the Standardsolution, respectively. Dilute with water to volume, and mix. This solution contains
Each Chewable Tablet contains not more than the number of 1000 I-Ig of calcium (Ca) per mL.
mg of sodium stated on the label. Magnesium stockstandard solution-Tr~nsfer 1.000 ~ ?f
magnesium metal to a 1OOO-mL volumetric flask containing
Assay for poBydimethylsiioxane- 10 mL of water, slowly add 10 mL of hydrochloric acid, and
Saccharin solution-Prepare a solution of saccharin in swirl to dissolve the metal. Dilute with water to volume, and
4-methyl-2-pentanone containing 12.5 mg per mL. mix. This solution contains 1000 I-Ig of magnesium (Mg) per
Dilute hydrochloric acid-Mix 200 mL of hydrochloric acid mL.
with sufficient water to make 1000 mL. Calcium and magnesium standardpreparation-To a
Standardpreparation-Dissolve a suitable quantity of USP 250-mL volumetric flask add 10.0 mL of Calcium stock standard
Polydimethylsiloxane RS in 4-methyl-2-pentanone to obtain a solution and 5.0 mL of Magnesium stock standard solution,
stock solution having a known concentration of about 1 mg dilute with water to volume, and mix. This solution contains
per mL. On the day of use, transfer 20.0 mL of this solution 40 I-Ig of calcium (Ca) and 20 I-Ig of magnesium (Mg) per mL.
and 5.0 mL of Saccharin solution to a 250-mL volumetric flask, On the day of use, transfer 4.0 mL of this solution to a 100-mL
dilute with 4-methyl-2-pentanone to volume, and mix. This volumetric flask containing 2.0 mL of Lanthanum chloride
solution contains about 0.08 mg of USP Polydimethylsiloxane solution, dilute with water to volume, and mix. This solution
RS per mL. contains 1.6 I-Ig of calcium (Ca) and 0.8 I-Ig of magnesium
Assay preparation-Weigh and finely powder not fewer (Mg) per mL.
than 20 Chewable Tablets. Transfer an accurately weighed Assay preparation-Transfer an accurately measured
portion of the powder, equivalent to about 20 mg of volume of the aqueous layer retained from the preparation
polydimethylsiloxane, to a 125-mL separator. Cautiously add of the Assay preparation in the Assay for polydimethylsiloxane,
50.0 mL of Dilutehydrochloric acid, and swirl until the reaction equivalent to about 28 mg of calcium carbonate, to a 200-mL
subsides. Insert the stopper, and mix. Carefully releasethe volumetric flask, dilute with water to volume, and mix.
pressure, add 50.0 mL of 4-methyl-2-pentanone, and mix for Transfer 3.0 mL of this solution to a 1OO-mL volumetric flask
10 minutes. Allow the layers to separate, and drain the containing 2.0 mL of Lanthanum chloride solution, dilute with
aqueous layer into a suitable stoppered container. water to volume, and mix.
[NOTE-Retain this aqueous layer for use in preparing the Assay Blank solution-Transfer 5.0 mL of Lanthanum chloride
preparation in the Assay for calcium carbonate and magnesium solution to a 250-mL volumetric flask, dilute with water to
hydroxide and for the preparation of the Test solutionin the test volume, and mix.
for Contentof sodium.] Filter the organic layer through a filter Procedure for calcium carbonate-eoncomitantly determine
containing 50 g of anhydrous sodium sulfate. Transfer 10.0 the absorbances of the Standardpreparation and the Assay
mL of the filtrate to a 50-mL volumetric flask, add 1.0 mL of preparation at the calcium emission line at 422.7 nm, wit~ a
Saccharin solution, dilute with methyl isobutyl ketone to suitable atomic absorption spectrophotometer (see AtomiC
volume, and mix. Absorption Spectroscopy (852») equipped with a calcium
Blank solution-Transfer 1.0 mL of Saccharin solution to a hollow-cathode lamp and a nitrous oxide-acetylene flam~,
50-mL volumetric flask, dilute with 4-methyl-2-pentanone to using the Blank solution as the blank. Calculate the quantity,
volume, and mix. in mg, of calcium carbonate (CaC0 3) in each Chewable Tablet
Procedure-eoncomitantly determine the absorbances of taken by the formula:
the Standardpreparation and the Assay preparation at the
silicon emission line at 251.6 nm, with a suitable atomic (100.09/40.08)(1 000C/3 V)(A/W)(Au/A s)
absorption spectrophotometer (see Atomic Absorption
Spectroscopy (852») equipped with a silicon hollow-cathode in which 100.09 is the molecular weight of calcium carbonate;
lamp and a nitrous OXide-acetylene flame, using the Blank 40.08 is the atomic weight of calcium; C is the concentration,
solution as the blank. Calculate the quantity, in mg, of in I-Ig per mL, of calcium in the Standard preparation; V is the
polydimethylsiloxane in each Chewable Tablet taken by the volume, in mL, of the aqueous layer retained from the
formula: preparation of the Assay preparation in the Assay for ,
polydimethylsiloxane used to prepare the Assay preparation; ,A
250QA/W)(A u/As) is the average weight, in mg, of each Chewable Tablet; W IS