1.

0 Background:

To check the safety, efficacy and quality of the drug which are not mentioned in the
pharmacopoeias recognized by Drug Act 2035, and establish the method of analysis for the
analysis of such drug products, DDA can take advice from the drug advisory committee (DAC)
as per Drug Act 2035. For this provision of Drug Act, DDA has formed a committee to assist
DDA in establishment of analytical method, product (quality control) specification for non
pharmacopoeial product. The committee has so far prepared SOP for study of documents on non
pharmacopoeial products for regulatory approval, guidelines for analytical method validation,
check lists for analytical method validation for submission of application for registration, check
list for evaluation of document submitted by the industry and protocol for the establishment of
some of methods of non pharmacopoeial products and quality control specifications. This step
will help in development of Nepal Pharmacopoeia too. The standard of drugs then will be as per
Nepal Pharmacopoeia. DDA has not been able to prepare Nepal Pharmacopoeia till now
therefore the standard of drug will be as per the quality control specifications and analytical
methods suggested by the committee after the method is approved by DDA/ DAC for non
pharmacopoeial drug products. This will be the baseline for further development of standards for
new molecules which are not come out in pharmacopoeia for future WHO activity.

2.0 Objective:

1. To provide the documented evidence that whether the analytical method submitted by
pharmaceutical industry is suitable for the analytical operation.
2. To develop the documents required during the submission of non-pharmacopoeial product.
3. To select the most appropriate method from the available different non-pharmacopoeial
methods and develop the product (quality control) specification and standard analytical
method for non pharmacopoeial drug product.

3.0 Scope:

3.1 Define the procedure, documentation, reference and acceptance criteria to be used for the
evaluation of documents of non pharmacopoeial product.
3.2 The product specification (Quality control) and analytical methods suggested by the
committee would be approved by DDA/DAC will be the standard for those non
pharmacopoeial drug products until and unless published in the monograph of any of the
pharmacopoeia recognised by Drug Standard Regulation, 2043.

1
4.0 CATEGORY OF THE NON PHARMACOPOEIAL DRUG

Based upon the study of the documents submitted by the domestic and foreign pharmaceutical
industry, seven categories of non pharmacopoeial products have been identified. The
subcategory of the products, analytical method requirement and recommendation is mentioned in
Table 1.

Category 1: The monograph of the active product ingredient (API) and dosage form is not
available in the pharmacopoeia.

Category 2: The monograph of the API is available in pharmacopoeia but dosage form is not
available in the pharmacopoeia.

Category 3: The monograph of the API and dosage form (e.g. tablet) is available but the dosage
form (e.g. capsule etc.), type of tablet dosage form (e.g. chewable tablet etc.) submitted by the
pharmaceutical industry is not available in pharmacopoeia.

Note: If the monograph of API and dosage form (e.g. tablet, liquid etc.) is available but the
salt form of API in dosage form is different from the available monograph, (e.g. diclofenac
sodium to diclofenac potassium, ferrous fumarate to ferrous ascorbate etc.) analytical
method should be based on the salt form available in the pharmacopoeial monograph and
analytical method validation is not required.

Category 4: If the monograph of the single molecule dosage (e.g. Telmisartan tablet,
Amlodipine tablet) is available but the monograph of combination dosage form (e.g. Telmisartan
and Amlodipine tablet) is not available.

Category 5: External preparations (Cream, Gel, Ointment, Liquid except eye/ear drop)

Category 6: Multivitamins, Enzymes, Mineral containing multi ingredient products.

Category 7: Biological, biosimiliar products (Vaccines, Monoclonal antibodies, Polyclonal

antibodies, rDNA product and biosimiliar products), Cytotoxic drugs and transdermal patches.

4.1 Evaluation of the document for non pharmacopoeial product

Analytical method recommendations based on the category of the non pharmacopoeial

product

Category 1:

The reference of the test method should be taken from the reliable journal or
innovator/comparator where ever possible. Complete analytical method validation should be
performed as per recognized pharmacopoeias and guidelines.

2
Category 2:

The analytical method of the dosage form should be stability indicating (HPLC preferred). If the
assay of the raw material in the pharmacopoeial monograph is based on High performance liquid
chromatography (HPLC), the analytical method of the dosage form should be based on the raw
material. The analytical method can be changed from the pharmacopoeial monograph with
suitable justification if necessary. In this category of product a complete analytical method
validation should be performed by the industry.

Category 3:

This category product is subcategorized as mentioned in Table no.1. The analytical method
should be followed as per the similar monograph of the dosage form available in pharmacopoeia
e.g. plain tablet to chewable, mouth dissolving, dispersible tablets etc. Similarly For e.g. if the
monograph of the tablet dosage form is available, the analytical method and acceptance criteria
of test parameter of tablet dosage can be applied to capsule dosage form i.e. solid dosage form to
solid dosage form wherever possible. Similarly the analytical method and acceptance criteria of
test parameter of liquid dosage form (e.g. oral solution) to liquid dosage form (suspension).
Analytical method can be changed from the available monograph with suitable justification if
necessary. Validation of the analytical method is required for this category product. The
document should be evaluated from the committee. Marketing authorization will be given after
the approval of the document from the committee.

Category 4:
For this category of product, the assay method can be developed by the pharmaceutical company
based on individual monograph of the single molecule of pharmacopoeia. However, the
dissolution test parameter in case of tablet/capsule dosage form should be as mentioned in the
individual monograph (should be narrowed but not wider e.g. if dissolution time is 45 minutes, it
can be varied to 30 minutes with justifications same is the case for RPM). Estimation of the
release of drug in case of dissolution can be done by suitable method (e.g. In the pharmacopoeial
monograph for single dose, if UV method is mentioned, it can be changed to HPLC method but
HPLC method cannot be changed to UV method) with justification unless otherwise available in
recognised guidelines (WHO, ICH, FDA and the pharmacopoeias mentioned in drug standard
regulation, 2043). Quality control specification should cover the tolerance limit of individual
monograph. Assay method and dissolution method should be validated.
Category 5:
Analysis of this category of product will be done as per the analytical method submitted by the
pharmaceutical company due to wide variation in the composition of active ingredients and their
quantity in the product. Analytical method validation should be performed and the documents

3
should be submitted to committee. The pharmaceutical company can get market authorization
from DDA after the submission of document to the committee.
Category 6:
For this product, Quality control specification should cover the tolerance limit of individual
monograph of the pharmacopoeia. The analysis of such products will be done as per the
analytical method submitted by the company. The subcategory of this category product is
mentioned in Table no.1. Analytical method validation of subcategory 6b (fat soluble vitamins)
should be submitted to the committee. Due to wide range of tolerance limit in assay and as mentioned
in Indian Pharmacopoeia, the content uniformity of such products are not required, analytical method
validation document of Category 6a and 6c should not be submitted to the committee.
Category 7: Documents of this category product should be made available to committee during
registration. The pharmaceutical company can get market authorization from DDA after the
submission of document to the committee.

4
 Table no.1: Category and sub category of non pharmacopoeial product

Category of Drug Sub category Characteristics Requirements Validation

Category 1 The monograph of API and The reference of the test method Complete analytical method
dosage form not available in should be from the reliable journal validation should be
N/A
any pharmacopoeia or innovator/comparator where ever performed.
possible.
Category 2. The monograph of the API is The analytical method of the dosage Complete analytical method
available but any dosage form form should be stability indicating validation should be
N/A
is not available in (HPLC Preferred) based on the API. the performed.
pharmacopoeia. method can be changed from the
monograph of API with justification
Category 3.a.
Tablet to Tablet
1. The analytical method should be
3 a.i. chewable, The monograph of the raw followed as per the available
dispersible, mouth material and dosage form monograph of dosage form. Complete analytical method
dissolving tablet (e.g. tablet) is available but
validation should be
Category 3. the dosage form submitted is 2. The acceptance criteria of test performed.
3 a.ii. Insert tabletnot available in parameter should be as per the available
(as per type of pharmacopoeia. monograph.
route of
administration)
Category 3.b: Tablet to capsule/vice versa
Category 3.c: liquid to liquid/suspension/powder
for oral suspension
Category 3.d: Tablet/capsule to powder e.g.
vitamin D3 powder

5
 Table no.1: Category and sub categryof non pharmacopoeial product contd.......

Category of Sub category Characteristics Requirements Validation

Drug

Category 4 The monograph of 1. Analytical method of assay

single dosage form is should be based on individual
(Fixed Dose 1. Analytical method validation should be
available but the monograph of the single molecule.
Combination) performed for assay and dissolution.
N/A monograph of 2. The dissolution test parameter
combination dosage should be as per the individual 2. Quality control specification should cover the
form is not available. monograph. tolerance limit of individual monograph.
Category 5 External preparations Quality control specification should Analytical method validation should be
(Cream, Gel, cover the tolerance limit of performed. However, the pharmaceutical
N/A Ointment, Liquid individual monograph of the company can take market authorization from
except eye/ear drop) pharmacopoeia. DDA without the approval of the document
from the committee.
Category 6 Category 6a. water soluble vitamins and
minerals
1. Analytical method validation document of
Quality control specification should Category 6 b product should be submitted to
Category 6b. fat soluble vitamins cover the tolerance limit of committee.
individual monograph of the
pharmacopoeia. 2. Analytical method validation document of
Category 6a and 6c should not be submitted to
Category 6c.Enzymes the committee due to wide range of tolerance
limit in assay and content uniformity not required
in this sub category product.

6
Table no.1: Category and sub category of non pharmacopoeial product contd.......

Category of Sub category Characteristics Requirements Validation

Drug

Category 7a. not available in

Biological, in
biosimiliar products pharmacopoeia
(Vaccines, Document Evaluation The pharmaceutical company can get market
authorization from DDA after the submission
Category 7b. not available in
of document to the committee.
Monoclonal in
antibodies, pharmacopoeia
Polyclonal
antibodies, rDNA
product and
biosimiliar products)

Category 7c not available in

in
Cytotoxic drugs and pharmacopoeia
transdermal patches

7
5.0 Procedure for the evaluation of document

Standard operating procedure for the evaluation of document of non pharmacopoeial product
(ANNEX-6) & Analytical method validation Guidelines (ANNEX-7) have been developed by
the committee. Based upon the SOP no. NPV/073/SOP-01 & Guideline no AMVP/073/01, the
evaluation of the documents of the non pharmacopoeial products submitted by the
pharmaceutical industry will be done.

All the quality control specification and analytical profile will be valid unless otherwise specified
in the individual monograph of the pharmacopoeia.

6.0 Acceptance criteria for different charecteristics of analytical method validation.

S.No. Parameters Requirement

a. Specificity
Blank values: Diluents
1 Resolution: NLT 1.5
Sample solution without
2 active Resolution: NLT 1.5
b. Linearity & Range r2 ≥ 0.98
c Repeatability RSD ≤ 2.0 %
RSD ≤ 3.0 %
Intermediate Precision For dissolution
The difference in the mean value for dissolution
results between any two conditions using the same
strength should not exceed an absolute 10 % at time
points with < 85 % dissolved nor exceed 5 % for
d. time points > 85 %.
e Accuracy 98.0 % to 102 %
Solution Stability 97.5 % to 102.5 % in comparison to the freshly
f prepared solutions
g Robustness (Optional)
h System Suitability test
Theoretical plates NLT 2000
1
Tailing factor NMT 2.0
2
RSD of five/six replicate NMT 2.0
3 injections
Resolution between two NLT 2.0
4 peaks

8
7.0. EVALUATION OF DOCUMENTS

1. All the completed documents from the domestic and foreign pharmaceutical industry will
be compiled and stored by the Analytical Method Validation Committee.

2. Committee member check the product application document and check lists for
documents required during the submission of non pharmacopoeial product (ANNEX 1-
4) using internal checklist (ANNEX 5).

3. Evaluation of the chromatogram, spectrum & calculation.

4. Compare to the acceptance criteria.

5. Prepare product specification and analytical profile of the non pharmacopoeial product.

6. Prepare deviation report if required including justification for the deviation and possible
remedies.

8.0. PRELIMINARY REQUIREMENTS

1. Analytical method reference (IP/BP/USP/JP/Any other literature)
2. Calibration of the equipments utilized in the study.
3. Grade of reagents used
4. Reference standard traceability
5. Relevant SOPs
6. Chromatogram, Spectrum & Calculation with formula should be submitted where
needed.

9
 (Annex 1): Parameters to be checked for the dosage form for the non pharmacopoeial products.
Product Specification
S.No. Parameters to be checked Dosage form
1. Description, Identification, Uniformity of weight, Disintegration test, Friability, Dissolution, Uniformity of content (if Tablet
required), Assay, Water content (if required), Related substances (if required), Leak test, Any other additional tests if required,
storage condition, pack size.
2. Description, Identification, Uniformity of weight, Disintegration test, Dissolution, Uniformity of content (if required), Assay, Capsule
Water content (if required), Related substances (if required), Leak test, Any other additional tests if required, storage
condition, pack size.
3. Description, Identification, Uniformity of volume, Uniformity of weight, Assay, Water content (if required), pH, Related Liquid, Powder for
substances (if required), Leak test, Any other additional tests if required, storage condition, pack size. oral suspension
4. Description, Identification, Filled weight variation, Assay, pH, Related substances (if required), Leak test, Any other additional Cream, Gel &
tests if required, storage condition, pack size. Ointment
5. Description, Identification, Uniformity of weight, Assay, Water content (if required), pH, Related substances (if required), Any Oral Powder
other additional tests if required, Seal test (only for sachets), storage condition, pack size.

6. Description, Identification, Uniformity of weight, Water content (if required), pH, related substances (if required), Any other Suppository
additional tests if required, leak test, storage condition, pack size.
7. Description, Identification, Uniformity of volume, Assay, Uniformity of content (if required), pH, related substances (if Sterile preparation
required), Bacterial endotoxin, sterility test, particulate matter, Any other additional tests if required, leak test, storage
condition, pack size.
8. Description, Identification, Uniformity of volume, Assay, Uniformity of content (if required), pH, related substances (if Non-sterile
required), particulate matter, Any other additional tests if required, leak test, storage condition, pack size. preparation
9. Description, Identification, Filled weight variation, Assay, pH, sterility test, isotonicity test, Related substances (if required), Sterile eye ointment
Leak test, Any other additional tests if required, storage condition, pack size.

10
 (Annex 2): Checklist of Product Specification if similar molecule is available in Pharmacopoeia.

S.No. Parameters Monograph available in pharmacopoeia Tolerance Limit

Yes (If Yes, Name of product No Pharmacopoeial Non pharmacopoeial
and Name of Pharmacopoeia) product product
1. API standard
2. Description
3. Average weight
4. Uniformity of weight
5. Disintegration test
6. Limit of water content if necessary
7. Limit of Assay
8. Method of analysis of Dissolution if
necessary
9. Limit of Dissolution if necessary
10. Method of analysis of Content
Uniformity if necessary
11. Limit of Content Uniformity if
necessary
12. Limit of Related Substance if
necessary
13. Method of analysis of Related
Substance if necessary
14. Any other tests if required

11
 (Annex 3): Analytical Method checklist.
S.No. Parameters Yes No Remarks

1. Analytical Method Reference

(IP/BP/USP/JP/Any other literature)

2. Reagents used and Grade

3. Reference standard traceability

4. Analytical Method
 Reagent Preparation
 Diluents
 Mobile phase preparation
 Standard preparation
 Sample preparation

5. Chromatogram, Spectrum & Calculation with

formula should be submitted where needed.

6. Analytical method validation

12
 (Annex 4): Analytical Method Validation checklist. (To be filled by authorized person of industry)
YES NO REMARKS
S.No Parameters Limit Requirements
1. Specificity Resolution: NLT 1.5 Should be investigated by injecting the blank (solvent)/ placebo
(matrix solution), standard solution, sample solution to demonstrate
the absence of interference with the elution of analytes.

2. Linearity r2 ≥ 0.98 Standard solutions should be prepared at minimum of 5/6

concentrations within the range of typically 80% to 120 %, of target
concentration.
3. Range Assay of drug substances (80 % to 120 % of the test concentration)
Content Uniformity (minimum 70% to 130 % of the test
concentration)
Dissolution testing (+/-20 % over the specified range)

4. Repeatability RSD ≤ 2.0 % For instrument precision determinations of five replicate of

reference standard should be made.
For the method at least nine determinations covering specified
range of 3 concentration and 3 replicates should be made.
5. Intermediate
Precision RSD ≤ 3.0 %
Test procedure Intermediate precision (within-laboratory variation)
Assay The difference in the mean value for
dissolution results between any two
should be demonstrated by at least two analysts, using at least two
conditions using the same strength HPLC/UV-vis spectrophotometer on different days and evaluating
For dissolution should not exceed an absolute 10 % at the relative percent purity data across the two systems of triplicate
time points with < 85 % dissolved nor sample of one concentration.
exceed 5 % for time points > 85 %.
6. Accuracy 98.0 % to 102 % Spiked samples should be prepared at three concentrations over the
range of 80 %, 100 % and 120 % of the target concentration. Three
individually prepared triplicates at each concentration will be
analyzed.

13
 (Annex 4): ANALYTICAL METHOD VALIDATION CHECKLIST (To be filled by authorized person of industry) contd…..

YES NO REMARKS
S.No. Parameters Limit Requirements

7. Solution Stability 97.5 % to 102.5 % Solutions of drug product should be analysed in comparison to
in comparison to the fresh prepared solutions stored at room temperature in auto
the freshly sampler and stored at 2 - 8 °C, in refrigerator at least 24 hours.
prepared solutions

8. Robustness The investigation of robustness can be done by change of flow

rate of the mobile phase, change of temperature of column,
change of composition of the mobile phase, change in the pH of
the mobile phase and use of different column.
9. System Suitability Theoretical plates
test System suitability tests should be performed on HPLC systems to
(NLT 2000) determine the accuracy and precision of the system by injecting
Tailing factor five/ six injections of a solution containing analyte (standard
solution) at 100% of test concentration. Determine relative
(NMT 2.0) standard deviation (rsd) of the replicate injections, theoretical
RSD (NMT 2.0 %) plate and tailing factor.

Note: Every page should be signed with date by the authorized person with company stamp.

Authorized Person:
Signature:
Name:
Designation:
Stamp:
Date:
14
ANNEX (5): Internal checklist for the study of document of analytical method validation

15
ANNEX (6): SOP FOR STUDY OF DOCUMENTS OF NON PHARMACOPOEIAL
PRODUCTS FOR REGULATORY APPROVAL

SOP FOR STUDY OF SOP No.: NPV/073/SOP-01

DOCUMENTS ON NON
PHARMACOPOEIAL
PRODUCTS FOR
REGULATORY APPROVAL
DEPARTMENT OF
DRUG Analytical method Validation
ADMINISTRATION Committee for Non
Pharmacopoeial product Page no.: 1 of 5
Effective Date: Review Date: Supersedes: None

Purpose:
1. To provide the documented evidence that whether the analytical method submitted by the
pharmaceutical industry is suitable for the analytical operation.
Objective:
To evaluate the available validated analytical method and give recommendation to DDA for the
approval of the Product (Quality Control) specification and standard analytical method of non
pharmacopoeial product.
Scope:
This will provide procedure for the study of documents related to analytical method validation of
non pharmacopoeial product as mentioned in the "Protocol for the Guidance and
Recommendation of documents for non pharmacopoeial product for National Regulatory
Approval."
Responsibility:
1. The entire committee member will be responsible for the guidance and recommendation
regarding the parameters for the product specification and analytical profile of the non
pharmacopoeial product.
2. Final approval of the document will be given by the Department of Drug Administration.
Prepared by: Checked by: Approved by:

…………………….. ……………………….. …………………….

16
 SOP FOR STUDY OF SOP No.: NPV/073/SOP-01
DOCUMENTS ON NON
PHARMACOPOEIAL
PRODUCTS FOR
REGULATORY APPROVAL
DEPARTMENT OF
DRUG Analytical method Validation
ADMINISTRATION Committee for Non
Pharmacopoeial product Page no.: 2 of 5
Effective Date: Review Date: Supersedes: None
Procedure:

Procedure for the incoming of documents in the committee :

1. First the pharmaceutical company registers the document of non pharmacopoeial

product along with the analytical method validation test report to Department of Drug
Administration. Domestic pharmaceutical company registers the document to
Industry section and foreign pharmaceutical company registers the document to
import section through importers.
2. The authorized person from Industrial section and Import section will check the
completeness of the document as mentioned in check list (ANNEX 1-4) prepared by
the committee.
3. From Industrial section and Import section, the authorized person prepares note &
Instruction (Tippani & Aadesh in Nepali) and submits the document file to Director
General, DDA.
4. After that the document will be send to Analytical method validation committee for
non pharmacopoeial product in order to check the document for the approval.

Prepared by: Checked by: Approved by:

…………………….. ……………………….. …………………….

17
 SOP FOR STUDY OF
DOCUMENTS ON NON SOP No.: NPV/073/SOP-01
PHARMACOPOEIAL
DEPARTMENT OF PRODUCTS FOR
DRUG REGULATORY APPROVAL
ADMINISTRATION
Analytical method Validation
Committee for Non
Pharmacopoeial product
Page no.: 3 of 5
Effective date: Review Date: Supersedes: None
5. The document of the non pharmacopoeial product will be kept by committee.
6. The document will be registered in Entry Register Book which contains all the
information regarding the entry date and remarks of the documents. The format of
the document entry book will be as follows:

S.No. Date Product API Category of Company Document Checked Date Remarks
Name Name product Name Submitted by By

Procedure for the checking of the documents

1. The received product application along with analytical method validation will be
distributed to all the member of the committee.
2. The committee member will check all the parameters of the documents and checklist
filled by the company using the internal check list (Annex 5).
3. All the documents required and acceptance criteria are available in the internal check
list.
4. If there is some deficiency and mistakes in the documents, the committee will
decide about the deficiencies and errors of the document. The committee will
correspondence the manufacturers/importers about their deficiencies.
5. The committee will recommend for the analysis of sample after obtaining the
complete documents from the manufacturers/importers.

Prepared by: Checked by: Approved by:

…………………….. ……………………….. …………………….

18
 SOP FOR STUDY OF SOP No.: NPV/073/SOP-01
DOCUMENTS ON NON
DEPARTMENT OF PHARMACOPOEIAL
DRUG PRODUCTS FOR
ADMINISTRATION REGULATORY APPROVAL

Analytical method Validation

Committee for Non
Pharmacopoeial product Page no.: 4 of 5
Effective date: Review Date: Supersedes: None

Procedure for the analysis of the finished product and approval of the report
4.2 After the completion of the document required for the AMV, the domestic pharmaceutical
company/importers will be informed to deposit the required amount of payment for the
analysis as per the NML payment rate.
4.3 The committee will request National Medicine Laboratory (NML) for analysis of the
sample.
4.4 NML will give identification number to the product for the testing purpose after the payment.
4.5 The product will be analyzed in NML using the recommended method from the committee
and report of analysis will be prepared.
4.6 AMV committee will make the discussion about the result and report of analysis of the
product.
4.7 The committee will prepare Product (Quality Control) Specification and Analytical profile.
4.8 Committee will send letter to DDA along with Product (Quality Control) Specification and
Analytical profile for the final approval.
4.9 The analytical method will be approved by the Department of Drug Administration/Drug
Advisory Committee for the official use.
4.10 Analytical report will be prepared and verification will be done by the section in charge.
4.11 Final approval of the report of analysis will be done by NML, Director.
Prepared by: Checked by: Approved by:

…………………….. ……………………….. …………………….

19
 SOP for study of documents SOP No.: NPV/073/SOP-01
on non pharmacopoeial
DEPARTMENT OF products for regulatory
DRUG approval
ADMINISTRATION
Analytical method Validation
Committee for Non
Pharmacopoeial product
Page no.: 5 of 5
Effective date: Review Date: Supersedes: None

Procedure for the numbering of the document

4.12 The name of the approved method from the DDA will be given as NML/AMV protocol.
4.13 Numbering of the product specification prepared by the committee will be done as
Product Name/Year/Number. For e.g. Numbering of Product specification of Amlodipine
& Telmisartan Tablet will be as Amlo-Telmi/073/074/001.
4.14 Similiarly, Numbering of analytical profile will be done as Product Name/Year/AP
Number. For e.g. Numbering of Analytical Profile of Amlodipine & Telmisartan Tablet will
be as Amlo-Telmi/073/074/AP001.
4.15 Numbering of the protocol of the committee will be as AMVP/Year/Number. For e.g.
AMVP/073/01
4.16 Numbering of SOP will be as NPV/Year/SOP-Number. For e.g. NPV/073/SOP-01.

Prepared by: Checked by: Approved by:

…………………….. ……………………….. …………………….

20
ANNEX (7): ANALYTICAL METHOD VALIDATION GUIDELINE FOR NON
PHARMACOPOEIAL PRODUCT

GUIDELINE NO.: AMVP/073/01

1. Requirements of Analytical method validation documents:

Identification of method type and validation approach, test method applications and validation
protocol, the intended use of each test method application, and the analytical performance
characteristics for each test method application.

Resources: This section identifies the following:

• Laboratory where the method validation is performed;

• Equipments and its calibration status used in the method validation;
• Materials: References, special instructions on handling, stability, and storage for each
material.
• Appendices: This section should contains references, a review worksheet of all personnel,
listings of all equipment, materials, test procedure(s) necessary to perform method
validation,
• Chromatogram, Spectrum & Calculation with formula should be submitted where
needed.
2. Analytical Performance Characteristics

Procedure: Before undertaking the task of methods validation, it is necessary that the analytical
system itself should be adequately designed, maintained, calibrated, and validated. All personnel
who will perform the validation testing must be properly trained. For each of the validation
characteristics in this document should defines the test procedure, documentation, and
acceptance criteria. Specific values are taken from the ICH, U.S. FDA, USP and pertinent
literature as references.

2.1. Specificity

2.1.1. Test procedure:

The specificity of the assay method should be investigated by injecting the blank (solvent)/
placebo (matrix solution), standard solution, sample solution to demonstrate the absence of
interference with the elution of analytes.

2.1.2. Documentation:
Print chromatograms.
2.1.3. Acceptance criteria:
The excipient compounds must not interfere with the analysis of the targeted analyte.
21
2.2. Linearity
2.2.1. Test procedure:
Linearity will be determined by preparing samples of at least five different concentrations within
the range of 80 % to 120 % of the target concentration. The method of standard preparation and
the number of injections should be same as used in the final procedure. Linearity curve will be
plotted for peak area response or absorbance against concentration. The linear relationship will
be evaluated by appropriate statistical methods, for example, by calculation of a regression line
by the method of least squares. Range is an expression of the lowest and highest level of analyte
that have been demonstrable to be determinable with acceptable precision, accuracy and
linearity.

2.2.2. Documentation:
Record the results on a datasheet. Calculate the mean, standard deviation, and Relative Standard
Deviation (RSD) for each concentration. Plot concentration (x-axis) versus mean response (y-
axis) for each concentration. Calculate the regression equation and coefficient of determination
(r2). Record these calculations on the datasheet.
2.2.3. Acceptance criteria:
≥0.999 for
The correlation coefficient for minimum of five/six concentration levels should be
the range of 80 to 120% of the target concentration. The y-intercept must ≤ 2 % o f th e target
concentration response. A plot of response factor versus concentration must show all values
within 2.5% of the target level response factor, for concentrations between 80 and 120% of the
target concentration.

2.3. Range
For the assay of a drug substance or a finished product: normally from 80 to 120 percent of the
test concentration; for content uniformity, covering a minimum of 70 to 130 percent of the test
concentration, unless a wider more appropriate range, based on the nature of the dosage form
(e.g., metered dose inhalers), is justified; and for dissolution testing: +/-20 % over the specified
range.
2.3.1. Test procedure:
The data obtained during the linearity and accuracy studies will be used to assess the range of the
method.
The precision data used for this assessment is the precision of the three replicate samples
analyzed at each level in the accuracy studies.
2.3.2. Documentation: Record the range on the datasheet.

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2.3.3. Acceptance criteria:
The acceptable range will be defined as the concentration interval over which linearity and
≤ 3%
accuracy are obtained per the above criteria, and in addition, that yields a precision of
RSD.
2.4. Accuracy
2.4.1. Test procedure:
Spiked samples will be prepared at three concentrations over the range of 80 %, 100 % and 120
% of the target concentration. Three individually prepared replicates at each concentration will
be analyzed. When it is (Spiked samples) difficult to prepare use a low concentration of a known
standard.
2.4.2. Documentation:
For each sample, report the theoretical value, assay value, and percent recovery. Calculate the
mean, standard deviation, RSD, and percent recovery for all samples. Record results on the
datasheet.
2.4.3. Acceptance criteria: 100 ± 2% is typical for an assay of an active ingredient in a drug
product over the range of 80 to 120% of the target concentration. The measured recovery in case
of dissolution is typically 95 % to 105 % in case of dissolution.
2.5. Precision
2.5.1 Repeatability
2.5.1.1 Test procedure:
Repeatability of system and method should be performed. For instrument precision
determinations of five replicate of reference standard should be made. For the method nine
determinations covering specified range of 3 concentration and 3 replicates should be made or
six determinations at 100 % of the test concentration. For dissolution purpose, nine
determinations covering specified range of 3 concentration and 3 replicates should be made or
six determinations at 100 % of the test concentration or 2 or 3 determinations on each of 3 days
should be performed.
2.5.1.2 Documentation
Record the retention time, peak area on the datasheet. Calculate the mean, standard deviation,
and RSD.
2.5.1.3 Acceptance criteria:

RSD should be 1% for drug substances and drug products, less than 2% for the assay and
dissolution of bulk drugs and finished products.

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2.5. 2 Intermediate Precision

2.5.2.1 Test procedure

Intermediate precision (within-laboratory variation) will be demonstrated by two analysts, using

two HPLC systems on different days and evaluating the relative percent purity data across the
two HPLC systems.

For dissolution testing purpose, if possible intermediate precision can be evaluated using a well
characterised lot of drug product with tight content uniformity. If this type of lot is not available,
premeasured placebo and active ingredients may be used to identify intermediate precision. The
dissolution procedure on the same sample may be run by at least two different analysts from the
same laboratory, with each analyst preparing the standard solutions and the medium and
following the defined quantification procedure.

2.5.2.2 Documentation: Record the relative % purity (% area) of each concentration on the
datasheet.

Calculate the mean, standard deviation, and RSD for the operators and instruments.

2.5.2.3 Acceptance criteria:

The assay results obtained by two operators using two instruments on different days should have
a statistical RSD ≤ 2%.

For dissolution, a typical acceptance criteria is the difference in mean value for dissolution
results between any two conditions, using the same strength, does not exceed an absolute 10 % at
time points with < 85 % dissolved and does not exceed 5 % for time points > 85 %.

2.6. Limit of Detection: (Not necessary for assay)

2.6.1. Test procedure:

The lowest concentration of the standard solution will be determined by sequentially diluting the
sample. Five/Six replicates should be made from this sample solution.

2.6.2. Documentation: Print the chromatogram and record the lowest detectable concentration
and RSD on the datasheet.

2.6.3. Acceptance criteria: The ICH references recommend a signal-to-noise ratio of 3:1.

2.7. Limit of Quantitation (it is not necessary for assay)

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2.7.1. Test procedure:

Limit of quantitation can be determined based on the standard deviation of the response and the
slope with the instrumental response obtained from the linearity. Establish the lowest
concentration at which an analyte in the sample matrix can be determined with the accuracy and
precision required for the method in question. This value may be the lowest concentration in the
standard curve. Make six replicates from this solution.

2.7.2. Documentation:

Print the chromatogram and record the lowest quantified concentration and RSD on the
datasheet. Provide data that demonstrates the accuracy and precision required in the acceptance
criteria.

2.7.3 Acceptance criteria:

The limit of quantitation for chromatographic methods has been described as the concentration
that gives a signal to noise ratio (a peak with height at least ten times as high as the baseline
noise level) an RSD of approximately 10% for a minimum of six replicate determinations.

2.8. System Suitability

2.8.1. Test procedure:

System suitability tests should be performed on HPLC systems to determine the accuracy and
precision of the system by injecting five/ six injections of a solution containing analyte at 100%
of test concentration. The following parameters will be determined:

• Theoretical Plate count

• Tailing factors,
• Resolution if required , and
• Reproducibility (percent RSD of retention time, peak area, and height for six injections).
2.8.2. Documentation:

Print the chromatogram and record the data on the datasheet

2.8.3. Acceptance criteria:

Retention factor (k): the peak of interest should be well resolved from other peaks and the void
volume; generally k should be ≥2.0.

Resolution (Rs): Rs should be ≥2 between the peak of interest and the closest eluted peak, which
is potentially interfering (impurity, excipient, and degradation product).

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Reproducibility: RSD for peak area, height, and retention time will be 1% for six injections.
Tailing factor (T): T should be 2.

Theoretical plates (N): ≥2000.

2.9. Robustness:

As defined by the USP, robustness measures the capacity of an analytical method to remain
unaffected by small but deliberate variations in method parameters. Robustness provides some
indication of the reliability of an analytical method during normal usage. Parameters, which will
be investigated, are percent organic content in the mobile phase or gradient ramp, pH of the
mobile phase, buffer concentration, temperature, and injection volume. These parameters may be
evaluated one factor at a time or simultaneously as part of a factorial experiment.

The effects of the following changes in chromatographic conditions will be determined:

methanol content in mobile phase adjusted by ± 2%, mobile phase pH adjusted by ±0.1 pH units,
column temperature adjusted by ±5 ˚C. If these changes are within the limits that produce
acceptable chromatography, they will be incorporated in the method procedure.

2.9.1. Stability of Standard and sample solutions

2.9.1.1 Test procedure:

Analysing solutions of drug product in comparison to the fresh prepared solutions and original
solutions stored at room temperature in auto sampler (at least 24 h) stored at 2 - 8 °C, in refrigerator (at
least 48 hour).

The stability of the standard is analyzed over the specified period of time (at least the time of the
entire dissolution procedure) using a freshly prepared standard solution at each time interval for
comparision.

2.9.1.2. Documentation

Stability should be documented by:

A table with mean values.

2.9.1.3. Acceptance criteria

The mean value of the standard solutions should be between 97.5 % and 102.5 % in comparison
to the fresh prepared standard solutions in case of the stability of the standard solution.

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