(Analytical Profiles of Drug Substances 11) Klaus Florey (Eds.) - Academic Press (1982) PDF

Analytical Profiles
of
Drug Substances
Volume 11
Edited by
Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey
Contributing Editors
Gerald S. Brenner Lee T. Grady
Glenn A. Brewer, Jr. Hans-Georg Leemann
Lester Chafetz Joseph Mollica
Nicholas DeAngelis Milton D. Yudis
Compiled under the auspices of the
Pharmaceutical Analysis and Control Section
Academy of Pharmaceutical Sciences
ACADEMIC PRESS 1982

A Subsidiary of Harcourt Brace Jovanovich,Publishers
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EDITORIAL BOARD
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Rafik Bishara Lee T. Grady
Gerald S . Brenner Boen T. Kho
Glenn A. Brewer, Jr. Hans-Georg Leemann
Lester Chafetz Joseph A. Mollica
Nicholas DeAngelis Bruce C. Rudy
John E. Fairbrother Milton D. Yudis
Klaus Florey
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AFFILIATIONS OF EDITORS,
CONTRIBUTORS, AND REVIEWERS
E . Abignente, University of Naples, Naples, Italy

H . Y. Aboul-Enein, King Saud University, Riyadh, Saudi Arabia
A. A . AZ-Badr, King Saud University, Riyadh, Saudi Arabia
I . A . Al-Meshal, King Saud University, Riyadh, Saudi Arabia
M . A . Al-Yahyu, King Saud University, Riyadh, Saudi Arabia
S. L. Ali, Zentrallaboratorium Deutscher Apotheker e. V., Eschborn, Germany
N. W. Atwater, E. R. Squibb and Sons, Princeton, New Jersey
D. M. Baaske, American Critical Care, Chicago, Illinois
R. Bishara, Eli Lilly and Company, Indianapolis, Indiana
G. S. Brenner, Merck Sharp & Dohme Research Laboratories, West Point,
Pennsylvania
G. A. Brewer, Jr., The Squibb Institute for Medical Research, New Brunswick,
New Jersey
P. de Caprariis, University of Naples, Naples, Italy
J. E . Carter, Ortho Pharmaceuticals, S o m e d e , New Jersey
L. Chajetz, Warner-Lambert Research Institute, Morris Plains, New Jersey
N. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
W. DeWitte, Ciba-Geigy, Suffern, New York
H . A . El-Obeid, King Saud University, Riyadh, Saudi Arabia
A. A. Elazzouny, National Research Center, Dokki, Cairo, Egvpt
J. Fairbroth, Stiefel Laboratories Ltd., Sligo, Ireland
K. Florey, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
S . A. Fwari, Parke-Davis, Inc., Detroit, Michigan
L. T. Grudy, The United States Pharmacopeia, Rockville, Maryland
M . M. A. Hassan, King Saud University, Riyadh, Saudi Arabia
J. G. Hoogerhde, Schering-Plough Corporation, Bloomfield, New Jersey
vii
viii AFFILIATIONS OF EDITORS, CONTRIBUTORS, A N D REVIEWERS
J. H . Johnson,American Critical Care, Chicago, Illinois

H . Kudin, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
B. T. K b , Ayerst Laboratories, Rouses Point, New York
F. Kreuzig, Biochemie GmbH, Kundl, Austria
H. G. Leemunn, Sandoz, Basel, Switzerland
E. A. Lo@, King Saud University, Riyadh, Saudi Arabia
M. E. Mohamed, King Saud University, Riyadh, Saudi Arabia
1. MoZZica, Ciba-Geigy Corporation, Summit, New Jersey
F. I. Muhtadi, King Saud University, Riyadh, Saudi Arabia
V. D. Reij, Wyeth Laboratories, Philadelphia, Pennsylvania
B. C. Rudy, Mary Kay Cosmetics, Dallas,Texas
C. M . S h r m , Wyeth Laboratories, Philadelphia, Pennsylvania
D. H . Sieh, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
H. Stober, Ciba-Geigy, Suffern, New York
K. D. Thkker, The United States Pharrnacopeia, Rockville, Maryland
T. D. Wilson, Sterling Winthrop Research Institute, Rensselaer, New York
B. E . Wyka, Schering- Plough Corporation, Bloomfield, New Jersey
M. D. Yudis, Schering- Plough Corporation, Bloomfield, New Jersey
PREFACE
It is now well over a decade that I perceived the need to supplement the official
compendial standards of drug substances with a comprehensive review of perti-
nent physical, chemical, and analytical data and methods. Ten years ago the first
volume of Analytical Projiles of Drug Substanceswas published under the auspices
of the Pharmaceutical Analysis and Control Section of the APhA Academy of Phar-
maceutical Sciences. That we were able to publish one volume per year is a tribute
to the diligence of the editors to solicit monographs and even more so to the en-
thusiastic response of our authors, an international group associated with phar-
maceutical firms,academic institutions, and compendial authorities. I would like
to express my sincere gratitude to them for making this venture possible.
I am pleased to report that five years ago a companion series entitled Phur-
macological and Biochemical Properties of Drug Substances was initiated by Mor-
ton E. Goldberg under the auspices of the section on Pharmacology and Tox-
icology, APhA Academy of Pharmaceutical Sciences. So far, three volumes have
been published.
Over the years, we have had queries concerning our publication policy. Our
goal is to cover all drug substances of medical value and, therefore, we have
welcomed any monographs of interest to an individual contributor. We also have
endeavored to solicit profiles of the most useful and used medicines, but many in
this category still need to be profiled.
Starting with this, the eleventh volume, we shall also supplement previously
published profiles with new data as we can find volunteers to write such s u p
plements. In this volume, five of the original profiles in Volume 1have been up-
dated.
The goal to cover and update all drug substances of medical value with com-
prehensive monographsis still a distant one. I estimatethat only about a quarter of
such compounds have been profiled so far. We would very much like to accelerate
ix
X PREFACE
the rate of publication and hope that even more authors can be encouraged to
write profiles. All those who have found these profiles useful are requested to con-
tribute monographs of their own. We, the editors, stand ready to receive such con-
tributions.
Klaus Florey
AMINOPHYLLINE
Kailus D.Thakker and Lee T.Grady
1. Description 2
1 . 1 Nomenclature 2
1.2 Formula, Molecular Weight, and Composition 2
1.3 Appearance, Color, and Odor 2
2. Physical Properties 3
2.1 Spectral 3
2.2 Other Properties 3
3. Methods of Preparation 9
4. Stability-Degradation 9
4.1 Stability in Solution 9
4.2 Stability in Solid State 9
5. Methods of Analysis 10
5.1 Identification Tests 10
5.2 Gravimetric Methods 11
5.3 Titrimetric Methods 11
5.4 Spectroscopic Methods 13
5.5 Chromatographic Methods 13
5.6 Immunoassays 26
6. Metabolism 31
I. Biopharmaceutics and Pharmacokinetics 31
8. Toxicity 33
9. References 34
Analytical Profilcs of Drug Substances Copyright 01982 by The American

Volume I I 1 Pharmaceutical Association
ISBN 0-12-260811-9
2 KAILAS D. THAKKER AND LEE T. GRADY
1. Description
1.1 Nomenclature
1.11 Chemical Name
Aminophylline is chemically known as 1H-

P
purine-2,6-dione, 3,7-dih dro-l,3-dimethyl-, compound wTth
1,2-ethanediamine (2:l).
1.12 Adopted Names
Aminophylline was also known as theophylline

ethylenediamine and euphylline. 1
1.13 Trade Names
Aminophylline is known as carena;

inophylline; metaphylline; theophyldine; aminocardol;
ammophylline; cardiocilina; cardophyllin; phylcardin;
tefamin; cardiomin; grifomin; minaphil; peterphylline;
stenovasan; the drox; diophylline; genophylline; phyllindon
and theolamine. P
1.2 Formula, Molecular Weight, and Composition:
11
Anhydrous: C16H24N1004 420.44
Dihydrate: C16H28N1006 456.44
Theophylline 85-87%
Ethylenediamine 12-15%
1.3 Appearance, Color and Odor
Aminophylline is available in the anhydrous form

or as the dihydrate. The dihydrate occurs as white or
slightly yellowish granules or powd r. It has a faint
ammoniacal odor and a bitter taste. f
AMINOPHYLLINE 3
2. Physical Properties
2.1 Spectral
2.11 Infrared
The infrared spectrum of aminophylline in

mineral oil mull, obtained on a B ckman 4250 spectro-
photometer, is shown in Figure l.5 It is generally
consistent wi h the reported infrared spectrum of
theophylline.5,4 The stretches for -NH2 in ethylenediamine
and -NH in theophylline appear as a broad band (combined
with the mineral oil signal) in the region of 3.0-4.0 P M .
Other signals are in the same vicinity as those for
theophylline. The fingerprint region beyond 8.5 IJM is
distinctive and can be used for identification.
2.12 Ultraviolet
Spectral characteristics of aminophylline
''
solutions in the ultraviolet region were reported by Andrade
an Inacio.'
LE1cm
Absorption max ma occurred at 243-5 nm
= 1701 and at 273-5 [E:im= 5001 in pH 9.5 borate buffer.
Figure 2 shows the ultraviolet spectrum of

aminophylline in water obtained on a Beckman 5260 recording
spectrophotometer.
2.13 Nuclear Magnetic Resonance
2.13.1 Proton NMR
An 80 MHz proton magnetic resonance

spectrum of aminoph lline in d6-dimethyl sulfoxide, obtained
on a Varian FT-80A,' containing tetramethylsilane as an
internal reference, is shown in Figure 3 . It is jimilar to
the reported (60 MHz) proton NMR of theophylline. The
assignments, based on assignments of theophylline protons,
are shown in Table I.
2.13.2 Carbon-13 NMR
The 20 MHz proton-noise decoupled

spectrum of aminophylline in d6-dimethyl sulfoxide,
obtained on a Varian FT-80A is shown in Figure 4.6 The
assignments are shown in Table 11. These are based on
assignments of dimethyluracil and 1-methylhypoxanthine.7
t
P
WAVELE W T H UM
Fig. 1. IR Spectrum of Aminophylline

0.8
0.7
a.6
y Q!5
U
z
4
m
g *1
v)
m
a
0. :
0 .i
.1
240 260 280 300 320 340 36

WAVELENGTH (nms)
Fig. 2. W Spectrum of Aminophylline
5
l ' l ~ l ' l ~ l ~~ l l ' t \ l ' l r l ~ l ~ l ' l ' l ~
10 9 a I 6 5 4 3 2 1 0
PPm
Fig. 3 . H' NMR of Aminophylline

Solvent
I I
Fig. 4. I 3 C NMR of Aminophylline

Table I
Proton Position
Proton Assignment (see structure) Chemical Shift (ppm)
-CH2- 10,ll 2.75

-N-CH3- 1 3.23
3 3.42
N-C%-
N-H 7 5.67
C-H 8 7.71
aIntensity of signal is proportional to the concentration.

This proton may be delocalized in the ring.
Table I1
Carbon Position
Carbon Assignment (see structure) Chemical Shift
N-CH3 29.7
N-CH3 27.5
c=c 109.6
c=c 148.5
N-C-N 142.7
-c=o 151.4
N-C=O-N 155.6
-CH2 on ethylenediamine is buried in the solvent signal as

shown in Figure 4.
2.2 Other Properties
2.21 Differential Scanning Calorimetry and

Melting Point
The thermogram of aminophylline2 shows two

endothermic transitions, one at 120" and another at 272°C.
The first transition reflects the melting point of
aminophylline; the second transition reflects the melting
point of theophylline. Theophylline is known to sublime on
melting. 2
2.22 Solubility
Aminophylline is soluble in water

AMINOPHYLLINE 9
(1 g/5 ml) .l It is insoluble in dehydrated alcohol and in

ether.8
3. Methods of Preparation:
Aminophylline was first prepared by Gruter' by

dissolving theophylline in aqueous solutions of ethylene-
diamine in stoichiometric proportions and evaporating in
vacuum over sodium hydroxide. Alternate methods include
treating anhydrous or h drated crystals of theophylline with
ethylenediamine vapor," and treating a 3 M solution of
theophylline in a weak organic base (pyridine, quinoline or
a-picoline) with a 2 -
M aqueous solution of ethylene-
diamine.11
4. Stabilitv-Degradation
4.1 Stability in Solution
Solutions of aminophylline become turbid on

standing due to absorption of carbon dioxide, with
subsequent precipitation of theophylline.' S 8 During the
preparation of aminophylline injection, excess
ethylenediamine is necessary to keep aminophylline from
decomposing.12
4.2 Stabilitv in Solid State
Aminophylline crystals, in the presence of

moisture, can absorb carbon dioxide fr m air and decompose
into theophylline and ethylenediamine.8 This accounts for
its characteristic odor.
Mixtures containing aminophylline and ephedrine

hydrochloride were found to be discolored13 due to an
exchange reaction between the two drugs. The ethylene-
diamine in aminophylline is presumed to liberate ephedrine
base which decomposes rapidly. The color change was
accelerated by temperature and humidity. 13.
Numerous reports are found in the literature on the
stability of aminophylline in suppository bases,14-21
especially those containing fatty acids. Dissolution of
suppositories made with cocoa butter base was markedly lower
than those ma e with macrogol base after storage at 22" for
up to a yearaP5 Other physical properties of cocoa butter
suppositories such as melting point (Tm) and melting time
have been known to increase within weeks of storage at 22"
and 30" as a result of decomposition16 . Increase in the in

vitro melting point (above 37" in some casf$) was correlated
with poor rate of release of theophylline.
Decomposition of aminophylline suppositories is

presumed to be due to the formation of insoluble amides
ethylenediamine and fatty acids of the suppository
base.
b
etwe'' our decomposition products that have been
isolated" were identified as mixturf6 of amides of oleic,
palmitic, lauric and myristic acids. Further
characterization of the decomposition products by IR
GLC, and TLC confirmed the presence of alkyl amides. 20""
Stabili rs such as hydroxylamine hydrochloride have been
useful. %f
5. Methods of Analysis
5.1 Identification Tests
Spectral identification tests include IR, W,Mass

and N M R spectroscopy. The melting point of theophylline
liberated from aminophylline is the basis of one of the
compendia1 identification tests. 22
The following color reactions are also useful

identification tests; many are used for dosage forms and for
mixtures of pharmaceuticals.
(i) Ethylenediamine present in aminophylline

reacts with sodium rhodizon85e to form a water-soluble,
violet-colored precipitate. Ethylenediamine must be
released from aminophylline and converted to acetate or
hydrochloride.
0
0
+2 a+
(ii) Ethylenediamine also foryz a yellow
precipitate with 2,4-dinitrochlorobenzene. Primary
aliphatic and aromatic amines interfere with the test.
AMINOPHYLLINE 11
(iii) Aminophylline develops an orange color

with ferric chloride and a yellow color with Ehrlich’s
reagent. Color de elopment is rapid. This test was
designed by Cooper” €or identification of pharmaceuticals
in tablets.
(iv) Reaction between dimethylglyoxime and

oxidized solutions of purines to form a colored product is
the basis of the identification test developed by Kido. 26
(V) Reaction of theophylline with potassium

chlorate in hydrochloric acid, followed by exposure to
ammonia vapors, produces a purple residue. This reaction is
the basis of on of the compendia1 identification tests for
aminophylline.2 5
(vi) Bratton-Marshall: Aminophylline in acid

solution, when treated with diazotized p-aminobenzene-sul-
fonic acid or p-aminonitrobenzene produces a yellow or a red
color, respectively.27
(vii) Addition of 1-2 drops of a 5% solution of

sodium nitroprusside to a solutig of aminophylline in
acetone produces a violet color.
(viii) Aminophylline powder, on mixing and

heatin with copper sulfate powder, also produces a violet
color.99
5.2 Gravimetric Methods
Earlier methods of analysis of aminophylline were

based on extraction of theophylline with organic solvents
such as chloroform and 2-propg~;4~ followed by gravimetric
determination of the residue.
5.3 Titrimetric Methods
5.31 Alkalimet ric
The weakly acidic nature of theophylline

lends itself to titrations with alkali. Titrations with
alkali have be59 carried out t o Jgsay aminophylline using a
potentiometric or colorimetric end-point
(thymolphthalein as indicator) in mixtures of
pha rmaceutica1s.
5.32 Acidimetric
Ethylenediamine's basic nature was used in

many early methods of analysi
strong acid were carried out.
ethylenediamin by solvent extraction31 or ion-exchange
~ h r o m a t o g r a p h 3was
~ followed by titrations with
hydrochloric acid using a colorimetric end-point (methyl
orange as indicator).
5.33 Argentometric
When theophylline is treated with solutions

of silver nitrate, it forms a silver salt that is insoluble
in water and soluble in nitric acid solutions. Basej6gtlthis
property, several assay methods have been develop
including the compendia1 assay for aminophylline.
general, silver theophyllate is precipitated, filtered,
washed, re-dissolved in nitric acid and titrated with
ammonium thio cy Aminophylline resent in mixtures of
pharmaceuticals'8:5p;39 and xanthines" can be analyzed.
Some presence of amm nia is necessary when precipitating
silver theophyllate." It has been suggested that
argentometric titrations give higher values due to
adsorption of silver ions by the very voluminous silver
theophyllate precipitate. 31
In an unusual application, silver (lloAg)

nitrate was used and radi metric titration was carried out
to analyze aminophylline. 41
5.34 Complexometric
Bosly developed a titrimetric method for

aminophylline by Precipitating Hg-(the~phylline)~with
mercuric acetate, itrating excess H g H ions with
ammonium thiocyana:zd4' Similarly, the Cu salt of
theophylline is also precipitated excess copper titrated
with ethylenediaminetetraacetate.4y94
5.35 Non-Aqueous
Non-aqueous titrations of aminophylline

using sodium methoxide as the titrant have been carried out
with dirnethylf~rmamide~~or ben~ene-methanol~~ as the
solvent for aminophylline. A differential titrimetric
method for aminophylline was dexgloped using acetous
perchloric acid as the titrant. This method allows
AMINOPHY LLINE 13
determination of ethylenediamine and theophylline content in

a single titration, and is useful for aminophylline tablets,
injections and suppositories.
5.4 Spectroscopic Methods
Aminophylline solutions obey the Beer-Lambert Law

for a zqncentration range of 0.5-1.2 mg%.5 Schack and
Waxler developed a potentiometric assay for aminophylline
in biological fluids using chloroform/2-propano1 extraction
to isolate theophylline, followed by W-absorbance
measurement at 275 nm. The method of Schack and Waxler was
used most extensively for analysis of aminophylline in early
pharmacokinetics research.a Several other
spectrophotometric methods have been developed for
aminophylline in mixtures of pharmaceuticals, each one using
an extraction t organic solvent to isolate
aminophylline. ''*kg In one case, graphical correction was
applied to the W absorbances of mixtures of aminophylline
and phen barbital, measured at two wavelengths of
maxima.58 In another application, extraction of serum
samples with a salt-solvent pair of ammonium sulfate and
ch1oroform:hexane (7:3 v/v) was carried out followed b back
extraction of theophyllinea into aqueous borate buffer ( PH
9 . 0 ) an measurement of UV absorbance at 275 nm. 51
PlavsicgZ used charcoal extraction to isolate theophyl ine
from other interfering substances. Since theophylline was
reversibly adsorbed on charcoal, elution with organic
solvent was followed by measurement of W absorbance.
Determination of aminophylline in the blood of

patients was carried out after gfidation with potassium
dichromate in an acidic medium, separation of the
oxidation product by steam distillation and measurement of
W absorbance at 257 nm.
5.5 Chromatographic Methods
5.51 Thin-Layer Chromatography
In addition to systems developed for theo-

~ h y l l i n e ,several
~ TLC systems have been developed for
"Aminophylline and theophylline are indistinguishable at

biological pH's. Therefore assays of theophylline in
biological fluids are also included here due to their
obvious applicability.
Table I11
Thin-Layer Chromatographic Systems for Aminophylline
No. Solvent Stationary Developing Solvent Detection Application Ref.

for Drug Phase Methods
1. chloroform Silica gel ch1oroform:acetic acid W,Dragen- Analysis of 54

F254 (100 :20) dorff ' s suppositories
reagent
2. water aluminum benzene:ethanol (9:l W, iodine, Mixture of 55

oxide or 9:1.5) modified pharmaceuticals
Dragendorff ' s
reagent
Silica gel (i) acetone:chloro- ferric Analysis of 56

F254 form:l-butanol:25% chloride, mixtures of
ammonium hydroxide iodine xanthines
(30:30:40:10)
(ii) ch1oroform:ethyl
ether (9O:lO)
(iii) ch1oroform:eth-
anol (9O:lO)
4. -----a Silica gel H ch1oroform:acetone: lJv Analysis of 18

methanol (8:l:l) suppositories
Table 111 (contd.)
No. Solvent St at ionary Developing Solvent Detect ion Application Ref.

for Drug Phase Methods
5. -----a Silica g e l methylene chloride: uv Analysis of tab- 57

F254 methano1:acetic acid lets ampuls,
(90:10:3) suppositories
anot mentioned.
analysis of aminophylline and its dosage forms. Table 111

lists the methods developed for aminophylline.
Zorka et alS8 separated and identified

several pharmaceuticals in a mixture including amino-
phylline using silica gel G plates and neutral, acidic, and
alkaline mobile phases. Detection was done by UV.
Riechert5' developed a "micro"-TLC method

for analysis of theophylline in biological fluids.
Kieselgel 60 F-254 DL-"Fertigplatten" thin-layer plates were
used and developed with ch1oroform:methanol (9O:lO) for a
mixture of caffeine and theophylline and with ethyl
acetate:methanol:25% ammonia (80:ZO:lO) for a mixture of
theobromine and theophylline present in saliva, plasma or
urine. Sensitivity of detection claimed was 25 ng/lO ~ 1 .
None of the dietary xanthines or other commonly co-
administered drugs appear to interfere.
5.52 PaDer ElectroDhoresis60

Whatman Paper No. 1 was used with a
potential gradient of 20 V/cm. Several xanthines were
separated. Among these, theophylline and aminophylline were
best chromatographed in Britton-Robinson buffer at pH 10.
Detection agent was 1% solution of disodium-2-hydroxy-3,6-
naphthalenedisulfonate.
5.53 Pressurized Liquid Chromatography
Numerous liquid chromatographic methods for

determinations of theophy 1 ne n biological fluids are
listed in the literature.4,f1-g' The maj ri of the
'',"
methods use reverse-phase chromatog p
li9
'l-" few are
listed that use i er normal-phase p b z ' or ion-exchange
chromatography. In many cases, chromatographic
conditions h e e n developed for a specific
application,QY,8B~'9 since adaptation of reported methods
may introduce some inttgference from co-adm nistered drugs
such as acetazo&mide, trisulfapyrimidinebl or
cephalosporins.
Table IV lists some of the recent, most

cited methods of analysis of theophylline in biological
fluids by liquid chromatography.
Table IV
No. Stationary Mobile Phase Pre-treatment of sample Comments Ref.

phase No.
1. Reverse-phase methano1:sodium Extraction of plasma with Separation of 61

C-18 acetate (pH 4 . 2 ) chloroform:2-propanol theophylline from
(ion-pair) containing 10 mM (50:50) paraxanthine.
tet rabutylammonTum
chloride (10:90)
2. Reverse-phase acetonitri1e:water Urine: pH adjusted before Analysis of theo- 62

C-18 containing 10 mM chromatography phylline in urine
(ion-pair) tet rabutylammonium Serum: ultrafiltration and serum; separation
chloride (2.5:97.5) from other xanthines
and metabolites.
3. Reverse-phase ethano1:water Assay of theophylline 6 3

C-18 (20:80) in plasma; separation
of theophylline from
sulfisoxazole and
ampic i11in.
4. Reverse-phase acetonitrile:O.Ol Molecular ultrafiltration Direct injection. 64

C-18 - sodium acetate
M to remove plasma proteins
(p-Bondapak (pH 4 . 0 ) (1:9);
C-18) 2 . 0 ml/min flow
Table IV (contd.)

phase No.
5. Reverse-phase acetonitri1e:water De-proteinization with Micromethod--only 65

C-18 ( 6 : 9 4 ) ; 3.0 ml/min 2.5 volumes of acetoni- 10-1.11sample is
flow trile required.
6. Reverse-phase acetonitri1e:ace- De-proteinization with Microscale method-- 66

C-18 tate buffer aqueous acetonitrile- only 3 0 p 1 plasma
(p-Bondapak (pH 4 . 0 ) ( 7 : 9 3 ) s olution needed for analysis;
-
Q)
7.
C-18)
Reverse-phase methanol:l% pro- Extraction with chloroform

direct injection.
50-1.1
1 sample needed. 67
C-18 pionic acid (20:80) evaporation; sample
u-Bondapak redissolved in methanol
C-18
8. Reverse-phase methano1:tetrahy- Same as Ref. 62 p-Hydroxyethyltheo-

- 68
C-18 drofuran:water con- phylline as inter-
(p-Bondapak taining 10 d/liter nal standard; sep-
C-18) sodium acetate aration from other
(40:10:50) xanthines and commonly
administered antibiotics.
Table IV (contd.)

phase NO
9. Reverse-phase methano1:sodium Deproteinization of serum B-Hydroxyethyltheo- 69

acetate (15:85) samples with two volumes phylline used as
of methanol internal standard;
no interference
from antibiotics
or metabolites.
10. Reverse-phase methano1:amonium Extraction with organic 100 u1 of serum 70

phosphate solvent before analysis sample can be
analyzed; theo-
phylline can be
analyzed in pres-
ence of anticon-
vulsants.
11. R verse-phase acet nitrile : Extraction with chloro- 8-Hydroxyethyltheo- 71

C-18 acetate buffer form:2-propanol (95:5) phylline used as
(pH 4.0) (9O:lO) internal standard;
theophylline re-
covery was found to
be between 71 and 75%.
Table IV (contd.)

phase NO
12. Reverse-phase acetonitrile: Extraction of 0.5-0.2 ml Separation of xan- 72

C-18 acetate buffer of acidified plasma thines; sensitivity
(pH 4 . 0 ) (12:88) with dichloromethane of detection 0.1 mg/
liter
13. Reverse-phase methanol:0.05 M Molecular filtration of Method applicable 73

C-18 phosphate buffer serum to separate proteins to human serum,
(pH 4 . 7 ) (12:88 v/v) urine and saliva
h)
0 samples, also sep-
aration and quan-
titation of theo-
phylline and its
metabolites.
14. Reverse-phase 2 0 ~1 of blood are 74

sufficient for
analysis; detection
by direct-curren t
pulse and differ-
ential pulse am-
perometry.
Table IV (contd.)

phase No.
15. Reverse-phase methano1:buffer Extraction with Completely automat- 75

C-8 (14:86 v/v) 2-propanol:chloroform ed analysis. De-
(25 :75) veloped on Technicon
"Fast-LC" .200-p 1
sample required. No
interference was
observed.
16. Reverse-phase sodium acetate: Adjustment of pH to 5.5 Theophylline, sul- 76

RP-8 0.02 M methanol famethoxazole,
(2:l)- ampicillin and
caffeine well
separated.
17. Reverse-phase acetonitrile: Same as Ref. 85 Authors found that 77

C-18 phosphate buffer the retention
(pH 4.8) (1:l); time of internal
3.0 ml/min flow; standard varied
50" from run t o run
when using the
conditions described
in Ref. #5; column
temp. was therefore
maintained at 50°C.
Table IV (contd.)

phase No.
18. Reverse-phase acetonitrile: Protein denaturation by No pre-column nec- 78

C-18 Whatman 10 mM phosphate acetonitrile essary; no inter-
Partisi1 buffer (10:90) ference observed.
10-ODS
19. Reverse-phase methano1:pH 2.0 Precipitation of proteins Theophylline ana- 79

C-18 hydrochloric acid by 50% v/v trichloroace- lyzed in presence
p-Bondapak solution (containing tic acid of methyl xanthines
N
N
0.02 M potassium and caffeine.
chloride)
20 * Reverse-phase acetonitri1e:ace- Plasma samples extracted Theophylline well 80

C-18 tate buffer with chloroform:2-propanol separated from
(8:92) (95:s); solvent removed paracetamol and
and samples re-dissolved and xanthines.
dissolved in mobile phase
21. Reverse-phase Solvent A: water Ion-pair extraction using Simultaneous quan- 81

C-18 5 p containing .01 - M tetrabutylammonium sul- titation of theo-
sodium acetate fate and ethyl acetate: phylline and its
and 0.005 M te- chloroform:2-propanol, major metabolites.
t rabu ty1ammonium (45:45:10 v/v) after ad-
hydrogen sulfate. justment of urine pH to
6.0-6.5
Table IV (contd.)

phase No.
21. Solvent B: ( 5 0 : 5 0 )
contd. methano1:solvent A
gradient elution
with 9% solvent B at
start, 46% solvent B
at end of run (for
program see Ref. 81).
g 22. Reverse phase methanol:10 mM Proteins precipitated 50 111 of serum are 82

p-Bondapak monobasic sodium by perchloric acid, sufficient; theophy-
C-18 phosphate (1:4); supernatant neutralized lline is well separ-
0.8 ml/min flow and injected ated from dietary
xanthines, caffeine,
theobromine and
theophylline meta-
bolites.
Table IV (contd.)

phase NO
23. Normal phase ch1orform:dioxane: Equal volume of saturated 100 u 1 of plasma 83

column formic acid ammonium sulfate is added are sufficient; theo-
(silica) (95.5:4.5:0.01 to plasma, and then phylline well sep-
v/v) extracted with chloro- arated from the
f orm:2-propanol (95 :5 metabolites.
v/v); solvent is evapor-
ated and residue
redissolved in mobile phase
*
K
3
.
24. Normal phase (i) chloroform: Extraction from plasma Mass spectrometry 84
Lichrosorb 2-propanol: with chloroform 2- used for identi-
Si-60 glacial acetic acid propanol (95:5) fication.
(92:7:1) with 40%
hexane
(ii) Ethylene chloride:
methanolic ammonium
formate (98:2)
25. Cation-exchange 0.66% acetic acid 0.1-ml sample extracted Applicable to plasma 85
Partis i1 with ethyl acetate or saliva samples;
SCx column no interference.
temp. at 5OoC
Table IV (contd.)

phase NO
26. Strongly basic acetate buffer; Sample filtered to remove General method for 86
anion ex- linear gradient particulate matter W-absorbing com-
change resin from 0-6.0 M, pounds in urine;
(35% cross- flow 0.72 a / m i n 1 30 compounds
linkage) tested.
5.54 Gas Chromatography
Earlier attempts to analyze blood levels of

theophylline resulted in evelopment of various gas
chromatographic methods. g5h_f0' Most of these methods
require extraction and derivatization before
chromatography. Table V lists some of the more recent
methods.
5.6 Immunoassays
5.61 Enzyme Immunoassay (EMIT)
The enzyme immunoassay (EMIT) developed by

Syva Corporation (Palo Alto, California) is the most widely
nSs
used method for the assay of the0 lline (in biological
fluids) in clinical laboratories.
In principle, theophylline antibodies are

preparedlo4 by injecting a solution of bovine gamma globulin
linked to theophylline ( o r a chemically similar derivative)
into sheep. Theophylline (or a derivative) is also linked
to an enzyme, in this case, glucose-6-phosphate
dehydrogenase. When a patient's serum containing free
theophylline is mixed with a solution of antibodies and
enzyme-labelled theophylline, the free drug and the enzyme-
labelled drug compete for the binding sites on antibodies.
The reduction in enzyme activity when bound to antibodies
can be monitored by using the proper substrate; in this case
NADH. This assa s rapid, specific, and requires small
sample size. s easily adaptable to ommercial
kinetic analyzers, and can be modified"' to suit the
application.
-_ Comparison of the EMIT assay with
chromat~~t;;~~#cmethods shows good agreement between the two
assays.
Vinet et all2' developed another enzyme

immunoassay. In this method, the sample is extracted with
chloroform/2-propano1, and back-extracted into aqueous
sodium hydroxide. Inhibition of beef liver phosphatase by
theophylline is determined at 25" C, using p-nitrophenyl
acetate as the substrate in a pH 9.4 2-amino-2-methyl-l-
propanol buffer system.
In another application nephelometric,

competitive immunoassay was developed. '21 A precipitate was
obtained by combining theophylline-antibody complex with a
macromolecule, and scattering of light by the precipitate
Table V
No. Stationary Conditions Pre-treatment of sample Comments Ref.

phase NO
1. 3% OV-17 on Column at 230°C; Sample extracted with 20 p 1 of plasma are 93

Gas Chrom Q nitrogen-phospho- chloroform:2-propanol sufficient; de-
rus detector ( 5 0 : 5 0 ) , evaporation and tection sensitivity
redissolution of residue 100 pmol/liter; theo-
in 0.02 M tetrabutyl- phylline well sep-
ammonium-h yd ro xid e arated from other
xanthines and co-ad-
ministered drugs.
2. Silicone sta- F.I.D. Extraction of sample with 94

tionary phase, salt-solvent pair of
2% SP 2510 DA ammonium sulfate and
methylene chloride: hex-
ane:acetic acid (80:20:0.1)
3. 5% OV-225 on Electron-capture Sample extracted with 100 p 1 of serum are 95

80/100 Gas detector; column ethyl acetate, and then sufficient; detec-
Chrom Q at 250°C treated with pentafluor- tion sensitivity
obenzyl bromide for 0.1 ug/ml.
derivatization
Table V (contd.)

phase No.
4. 3% XE-60 on Electron-capture Derivatization by penta- Sensitivity of de- 96

80/100 Gas detector; injector fluorobenzyl bromide tection 5 ng/ml.
Chrom Q and column at followed by column chrom-
220"C, detector atography prior to in-
at 225°C jection
5. 3% OV-17 on Nitrogen detector; Extraction with tetrahexyl- 25-pl sample is 97

100/120 Gas column at 240°C ammonium hydrogen sulfate sufficient.
Chrom Q in aqueous sodium hydrox-
ide
6. 3% OV-17 on Nitrogen-phospho- Off-column derivatization No interference 98

100/120 Gas rus detector; as follows: Sample is ex- from drugs or
Chrom Q column at 240°C tracted with dichloro- metabolites.
methane, dried and
reacted with --
N,N-dimethyl-
acetamide, tetramethyl-
ammonium hydroxide and 1-
iodopentane. It is then
transferred into cyclohexane:
pentane mixture (95: S), the
solvent evaporated, and it
is redissolved in methanol.
Table V

phase No.
7. 3% OV-17 on Flame-ionization Extraction of sample with 99

Gas Chrom Q detector; column ether:dichloromethane:
at 190°C 2-propanol ( 6 : 4 : 1 ) , re-
extraction of the organic
layer with 1 N sodium
hydroxide, acidification
with phosphoric acid,
re-extraction with organic
h)
solvent, evaporation and
\o redissolution of residue
in tetrapropylammonium
hydroxide
8. 3% SP 2250 on Flame-ionization Sample extracted with di- 100

100/120 detector; column chloromethane, evaporated,
Supelcoport temperature pro- and dissolved in toluene,
grammed from 160°C butylating agent is
to 240°C at 8" then added.
C/min
Table V (contd.)

phase No.
9. 3% OV-17 on Column temperature Extraction with chloro- Mass spectrometry 101

100/120 programmed from form, butylation with using probability-
Chrom W HP 180°C to 280°C; tetramethylammonium based matching.
column well detector at hydroxide and --
N,N-di-
conditioned 280"C, mass methylacetamide
spectral source
at 150°C
10. 3% SP2250 on Column temperature Samples are introduced in No interference. 102

100/120 programmed at a flash heater for
Supelcoport 190°C to 3OO0C at ethylation
type 50-50 10"/min
methylphenyl
silicone
AMINOPHYLLINE 31
was used to quantitate theophylline content.
5.62 Radioimmunoassay (RIA)
The principle of radioimmunoassay is similar

to EMIT, except that in this case decrease in radioactivity
is measured. Ra ioimmunoassays f r th ophylline have been
developed using 'H-theophylline. lY2 ,123 8-Carboxy-
theophylline was used to prepare antibodies. There is no
interference from endogenous purines or known metabolites of
theophylline at the concentrations studied. 122
6. Metabolism
UsinglJZ4C] aminophylline injection, Caldwell,

Monks and Smith determined that the metabolites of
aminophylline are the same as those of theophylline. 125,126
These are (i) 3-methylxanthine (ii) 1,3-dimethyluric acid
and (iii) 1-methyluric acid. However, the - rate and extent
of conversion to 1,3-dimethyluric acid and 3-methylxanthine
were higher or aminophylline than for theophylline.126
Therefore, "C recovery in urine (0-24 hours) was higher for
aminophylline (87%) than for theophylline ( 7 6 % ) . The
formation of 3-methylxanthine follows saturation kinetics;
therefore, the presence of circulating methylxan ines from
foods affects the elimination of aminophylline. '" The
appearance of the other wo metabolites follows first order
kinetics. Jenne et all2' determined that 1-demethylation of
theophylline to 3-methylxanthine is the dominant reaction
determining theophylline concentration in serum. Presence
of ethylenediaminelyyst affect this conversion, but how it
does is not known.
7. Biopharmaceutics and Pharmacokinetics
Pharmacokinetics of aminophylline has been studied

extensively. Since aminophylline and theophylline are
indistinguishable in biological fluids, pharmacokineticists
d o not differentiate between the two. lthough the pharma-
cokinetics of theophylline was reported' earlier, the advent
of newer analytical techniques has since led to an extensive
amount of work.
Aminophyl line is administered orally 129-133 as a
sustained-re1
lwe
dose capsulg- ordosagetablet,
fo
1329133,134,135 Or as a single-
15',130 intravenously (or by
infusion) "' ntramuscularly, or rectally as suppositories
or enema. 1389131 Among different aminophylline dosage
forms, rect 1 positories give the widest

variation.lf8,fYf
Absorption of aminophylline from tablets or capsules is

rapid, plasma levels reaching therapeutic range within 1-1 l/2
hours. Sustained-release preparations of aminophylline are
used often to maintain theophylline levels within
for about 12 hours in treatment of
as .
theraPey51'15'~f39
thma The rectal route is used often with
infants and children.
Plasma theophylline levels of 5-15 pg/ml (after

administrati of aminophylline) are considered safe and
O''
therapeutic. Although in most ca toxic symptoms do
not appear at levels above 25 ug/ml,Bz8 the patient-to-
patient variation is fglhi@ that individualization of the
therapy is necessary. In some case intra-patient
variation in doselblood levels is observedfh6 during
continuous long-term therapy. Saliva and bre ft5
milk levels
of theophylline are lower than plasma levels,
but1 &75PZ$
correlation between plasma/saliva ratio is obtained.
.'
The pharmacokinetic behavior of aminophylline once it
appears in the blood can be described by the same ode1 that
is used to describe theophylline pharmacokinetics The
volume of distri u i n at steady-state for all ages is
0.45 literlkg'158-E5P except in neonates where it is
slightly larger.152 Elimination is rapid, with a half-life
of about 6 hours in normal, healthy, non-smoking adults.
Among the factors that affect the ation of
theophylline fr sma are age,1f'i'P'9 physi lo and
disease stat 1'B-p89 co-administer 163-18 diet ,165
time of day,fb6 and smoking habits.fg7di'8s'In a case when
any of these factors is operating, the half-life of
elimination varies from 5 to 30 hours.
Very recently, Monks et have compared the

disposition and elimination of theophylline and
aminophylline. Elimination of aminophylline was faster than
theophylline in the same subjects. Although qualitatively
the disposition (and metabolism) of theophylline and
aminophylline were similar, the authors claimed there were
small but significant differences in rates of elimination
and the extent of dose eliminated within 48 hours.
AMINOPHYLLINE 33
8. Toxicitv
Toxicity of aminophylline results when bl d levels

exceed the level of 20 pg/ml of theophylline.lY8 The
severity of toxicity is directly related to plasma levels of
theophylli In mild toxicity, symptoms are nausea and
vomiting ,1981171 diarrhea, abdominal pain, nervousness,
insomnia, tachycardia and headache.
serious tachycardia, grand ma1 seizures,
cardiac arrythmias may occur In some cases, death may
result from acute toxicity. ij4
9. References
1. Merck Index, 9th ed., Merck and Co., Rahway, NJ (1976)

p. 471.
2. K. D. Thakker and M. M. Talero, unpublished results,

Drug Research and Testing Laboratory, The U.S.
Pharmacopeial Convention, Inc., Rockville, MD.
3. J. L. Cohen, "Analytical Profiles of Drug Substances ,"

Vol. IV, ed. Klaus Florey, Academic Press, NY (1975)
p. 467.
4. E. R. Blout and M. Fields, J. Am. Chem. SOC., -

72, 479
(1950).
5. M. A. Andrade and M. M. L. Inacio, Rev. Port. Farm, -

10,
141 (1960); C. A., -
55:8771d (1961).
6. L. V. Feyns, unpublished results, Drug Research

and Testing Laboratory, The U.S. Pharmacopeial
Convention, Inc., Rockville, MD.
7. E. Breitmaier, G. Haas, and W. Voelter, Atlas of C-13

NMR data, Vol. 2, Heyden & Son, Inc., NY, Spectra
82507 and 2521.
8. The British Pharmacopoeia (1980), Her Majesty's

Stationery Office, University Printing House,
Cambridge, England, p. 28.
9. U. S. Patent 919,161, (April 20, 1909).
10. U. S . Patent 2,629,717 (Feb. 24, 1953).
11. U.S. Patent 2,481,715 (Feb. 26, 1947).
12. E. E. Theimer, personal communication, The U.S.

Pharmacopeial Convention, Inc., Rockville, MD.
13. E. Kalatzis, Australas. J. Pharm., --

52 (613), S6
(1971).
14. J. Taylor, Bull. Tech./Gattefosse Rep., -

72, 6 (1979);
C. A . 93(22):210189.
15. P. Nissen and G. Juul, Arch. Pharm. Chemi., --

86 (4),
117 (1979); C. A. -
90:210027Y (1979).
AMINOPHYLLINE 35
16. C. J . de Blaey and J. J. Rutten-Kingma, Pharm. Acta.

- - 51, 186 (1977).
Helv.,
17. C. J. de B l a e y and J. J. Rutten-Kingma, Pharm. Acta.

. , 52, 11 (1978).
H e l v-
-
18. T. C i e s z y n k i , Acta. P o l . Pharm., 28 (l), 59 (1971); C.

--
A. -
74:146305c (1971).
19. T. C i e s z y n k i , Acta. P o l . Pharm., 32, 371 (1975);

- C. A.
84:79658 (1976).
-
20. J. W. Brower, E. C. J u e n g e , D. P. Page, and M. L. DOW,

69 ( 8 ) , 942 (1980).
J . Pharm. S c i . , --
21. A. A. Kassem, El Shamy, and H. A. Abdul, J. Drug Res.

4, 53 (1972).
-
22. The U.S. Pharmacopeia, 2 0 t h r e v . , Mack P u b l i s h i n g Co.,
E a s t o n , PA (1980) p. 31.
23. F. F e i g l and E. S i l v a , Anais Farm. Quim. (Sao P a u l o ) ,

6 (7), 5 (1973); C. A. -
-- 48:7099i (1954).
24. 23, 113

F. F e i g l and E. S i l v a , Drug S t a n d a r d s , -
(1955).
25. P. Cooper, Pharm. J . , 177,495 (1956).
26. 75, 103 (1957); C. A .

Y. Kido, E i s e i - S h i k e n j o Hokoku, -
52:15348 (1958).
-
27. M. P. C a s t r o and R. R. Mendoza, Inform. Quim. A n a l . ,

17 (6), 158 (1963); C. A. -
-- 61:537d (1964).
28. R. I. P e t r o v a and V. A. Z a i t s e v , Aptechn. D e l o . ,

12 (l), 77 (1963); C. A. -
-- 61:13127d (1964).
29. L. D. Ryabykh, Aptechn. Delo., 14 (l), 49 (1965);

-- C. A.
62:11628a (1965).
-
30. F. Reimers, Dansk. T i d s . Farm., 9, 11 (1935); C. A.

-
29:3841 (1935).
-
31. 20, 631
F. R e i m e r s , J. Assoc. O f f i c i a l Agr. C h e m i s t s , -
(1937).
36 KAILAS D.THAKKER A N D LEE T. GRADY
32. M. S a r s u n o v a and J. T o e l g y e s s y , Z. Anal. Chem., 196,

107 (1963).
33. A. B a r t i l u c c i and C. A. D i s c h e r , J. Am. Pharm. ASSOC.,

39, 641 (1950).
-
34. P. P. Suprun, Aptechn. Delo., --

9 (2), 49 (1960); C. A.
54:21653b (1960).
-
35. R. Foreman and M. I. B l a k e , J. Pharm. S c i . , 54 ( l ) ,

12 (1965).
36. D. D r y g i e n i e c and Z . Mlynarczyk, Farm. P o l . , 22 (6),

--
66:49269w (1967).
437 (1966); C. A. -
37. S. B h a t t a c h a r y a and S. C. B a n e r j e e , J. P r o c . I n s t .
Chemists ( I n d i a ) , 26, 33 (1954).
38. N. A. David, Drug S t a n d a r d s , 23, 54 (1955).
39. A. S i n a a n d M. A. K h a l e k , P r o c . Pharm. SOC. E g y p t ,

S c i . Ed., --35 (7), 49 (1953).
40. 21,
D. B r z e n z i n s k a and L. M a c i a s z e k , Farm. P o l s k a , -
353 (1965); C. A. - 63:11252c (1966).
41. P. W. S c h m i t t , J. Am. Pharm. Assoc., -

19, 821 (1930).
42. J. B o s l y , J. Pharm. Belg. (N. S . ) , -

4, 66 (1949); C. A .
43:6786b
- (1949).
43. M. Klera and Z . Dudzik, Farm. P o l . , --

23 (ll), 863
(1968); C. A. -68:117161 (1968).
44. M. Klera and Z. Dudzik, Farm. P o l . , -
21 (19-20), 770
(1965); C. A. -
65:5303c (1966).
45. M. A. McEniry, J. Assoc. O f f i c i a l Agr. C h e m i s t s , -
40
(3), 926 (1951).
-
46. T. Medwick and F. S c h i e s s w o h l , J. Pharm. S c i . , 52 (9),
843 (1963).
47. J. A. Schack and S. H. Waxler, J. Pharmacol. T h e r a p . ,

9, 283 (1949).
-
48. J. P. Comer and R. B. Bourne, Drug S t a n d a r d s , 28, 9
(1966)
AMINOPHYLLINE 37
37, 673
49. R. H y a t t , J. Assoc. O f f i c i a l Agr. C h e m i s t s , -
(1954).
50. M. Abdel-Hady E l s a y e d , H. Abdine, and Y. M. Elsayed,

68 (l), 9 (1979).
J. Pharm. S c i . , --
51. H. A. S c h w e r t n e r , J. E. Wallace, and K. Blum, C l i n .

24 (Z), 360 (1978).
Chem., --
-
52. F. P l a v s i c , C l i n . Chim. Acta, --
88 (3), 551 (1978).
25 (ll),
53. A. V i l l a and A. P i s t i s , Farmaco Ed. P r a t . , --
717 (1970); C. A. - 74:30565u (1971).
54. F. Schmidt, Arch. Pharm. (Weinheim), --

301 (12), 940
70:60845j (1969).
(1968); C. A. -
55. B. L. Moldaver and T. M . Sakovan, Farm. Zh. 23
(Kiev), -
(3), 28 (1968);
- 69:109839f (1968).
C. A. -
56. H. Thielemann, S c i . Pharm., --

42 (3), 179 (1974).
4, 146
57. L. R. S e l l e r and V. T o r r e , Cienc. I n d . Farm., -
(1974); C. A. - 82:160280 (1975).
58. B. Zorka, S . M i l e n a , and A. Dania, Arch. Farm., 29 (2),

--
91 (1979); C. A. -
93:13143 (1980).
59. M. R i e c h e r t , J. Chromatogr., 146, 175 (1978).
60. M. S t u c h l i k , I. C s i b a , and L. K r a s n e c , Cesk Farm.,

16 (4), 187 (1967); C A. -
-- 67:67530z (1968).
61. H. H. 26 (3),
F a r i s h and W. A. Wargin, C l i n . Chem., --
524 (1980).
62. W. V o e l t e r , K. Zech, P. Arnold, and G. Ludwig, -

J.
Chromatogr., 199, 345 (1980).
63. G. Lam, S. M. Huang, M. G. Lee, R. N a t i o n , and W. L.

25 ( l o ) , 1862 (1979).
Chiou, C l i n . Chem., --
64. L. C. F r a n c o n i , G. L. Hawk, B. J. Sandmann, and W. G.

Haney, Anal. Chem., -- 48 (2), 372 (1976).
65. G. W. Peng, M. A. F. G a d a l l a , and W. L. Chiou, C l i n .

24 (2), 357 (1978).
Chem., --
-
66. J. J. O r c u t t , P. P. Kozak, Jr., S . A . Gillman, and L.

H. Cummins, C l i n . Chem., -
23, 599 (1977).
67. R. E . H i l l , J. Chromatogr., -
1 3 5 , 419 (1977).
68. 25 ( l o ) , 1866
J. R. M i k s i c and B. Hodes, C l i n . Chem., --
(1980).
69. G. P. B u t r i m o v i t z and V . A. R a i s y s , C l i n . Chem.,

25
-- ( 8 ) , 1461 (1979).
70. P. Draper, D. S h a p c o t t , and B. Lemieux, C l i n . Biochem.,

12 ( 2 ) , 52 (1979).
--
71. S . J . S o l d i n and J. G. H i l l , C l i n . Riochem., -
1 0 , 74
(1977).
72. T. Foenander, D. J. B i r k e t t , J . D. M i n e r s , and L. M.

Wing, C l i n . Biochem., --
13 ( 3 ) , 132 (1980).
73. R. K. D e s i r a j u , E. T. S u g i t a , and R. L. Mayock, -

J.
Chromatogr. S c i . , -- 1 5 ( 1 2 ) , 563 (1977).
74. E. C. L e w i s and D. C. Johnson, C l i n . Chem., 24 ( l o ) ,

--
1711 (1978).
75. J. W. Dolan, S . van d e r Waal, S. J. B a n n i s t e r , and R.

P. Snyder, C l i n . Chem., 26 ( 7 ) , 871 (1980).
76. I. W. F r u t k o f f , G. K i d r o n i , and J. Menczel, C l i n .

Chem., --
- 26 ( 1 2 ) , 1759 (1980).
77. L. T. Mann, J r . , C l i n . Chem., 25 ( 7 ) , 1336 (1979).

--
78. J. W. Nelson, A. L. Cordry, C. G. Aron, and R . A.
B a r t e l l , C l i n . Chem., --
23 ( l ) , 124 (1977).
79. F. Nielsen-kudsk and A. K. P e d e r s e n , Acta Pharmacol.

T o x i c o l . , --
42 ( 4 ) , 298 (1978).
80. P. J. Naish, M . Cooke, and R. E. Chambers, -

J.
Chromatogr., 163,
363 (1979).
81. K. T. Muir, J. H. G. Jonkman, D. Tong, M . K u n i t o n i , and

S. Riegelman, J. Chromatogr., 221, 85 (1980).
82. M. J. Cooper, B. L. M i r k i n , and M. W. Anders, -

J.
Chromatogr., 143,
324 (1977).
AMINOPHY LLINE 39
83. P. L. von Aerde, E. Moerman, R. van S e v e r e n , and P.

Braeckman, J. Chromatogr., 222, 467 (1981).
84. K. K. Midha, S. Sved, R . D. H o s s i e , and I. J .

McGilveray, Biomedical Mass S p e c t r o m e t r y , 4 ( 3 ) , 172
(1977).
85. G. W. Peng, V. Smith, A. Peng, and W. L. Chiou, R e s .

15, 341.)6791(
Commun. Chem. P a t h o l . Pharmacol., -
86. K. S e t a , M. Washitake, T. Anmo, N. T a k a i , and T .

Okuyama, J. Chromatogr., 181, 311 (1980).
87. N. Weidner, D. N. D i e t z l e r , J. H. Ladenson, G. Kessler,

L. L a r s o n , C. H. Smith, T. James, and J. M. McDonald,
73 (l), 79 (1980).
Am. J. C l i n . P a t h o l . , --
88. G. M e n i l l e t , M. C. S a n t a i s , and F. R u f f , T o x i c o l . Eur.

2 (2), 111 (1979).
Res., --
-
89. S. A. McKenzie, A. T. Edmunds, E. B a i l l i e , and J. H.

53 (4), 322 (1978).
Meek, Arch. D i s . C h i l d , --
90. D. R. C l a r k , C l i n . Chem., --
25 (6), 1183 (1979).
91. L. W. Bond and D. L. Thornton, C l i n . Chem., --

25 (6),
1186 (1979).
92. C. A. Robinson J r . , B. M i t c h e l l , J. V a s i l i a d e s , and A.

24 ( l o ) , 1847 (1978).
L. S i e g e l , C l i n . Chem., --
93. G. M. S h i e r and I. E. T. Gan, J. Chromatogr., 182,

232 (1980).
94. H. A. S c h w e r t n e r , C l i n . Chem., --
25 (2), 212 (1979).
95. S. Sun, J. Pharm. S c i . , --

68 (4), 443 (1979).
96. A. A r b i n and P. Edlund, Acta Pharm. Suec., 12,119

(1975).
97. W. A. J o e r n , C l i n . Chem., 24 (9), 1458 (1979).
98. J. D. Lowry, L. J. Williamson, and V. A. R a i s y s , -

J.
Chromatogr., 143, 83 (1977).
99. V. P. Shah and S. Riegelman, J. Pharm. S c i . , --

63 (8),
1283 (1974).
100. G. F. J oh n so n , W. A. D e c h t i a r u k , and H. M. Solomon,

C l i n . Chem., --
2 1 ( l ) , 144 (1975).
101. M. Sheehan, R. H. Hertel, and C. T. K e l l y , C l i n .

Chem., --
- 2 3 ( l ) , 64 (1977).
102. C. V. Abraham, 0. A tk in so n , and D. Gresham, Am. J.

Med. Technol., --
4 3 ( 8 ) , 772 (1977).
103. L. Hendeles, M. Weinberger, and G. Johnson, Applied

Pharmacology, Applied T h e r a p e u t i c s , San F r a n c i s c o ,
CA (1980) p. 131.
104. K. E. R u b e n s t e i n , R. S . S c h n e i d e r , and E. F. Ullman,

Biochem. Biophys. R e s . Commun. - 47, 846 ( 1972) .
105. J . B. Gushaw, M. W. Hu, P. Singh, J. G. Miller, and R .

S. S c h n e i d e r , C l i n . Chem., -
23, 1144 ( 1977) .
106. J. R. Koup and B. Brodsky, Am. Rev. R e s p i r . D i s . ,

117, 1135 (1 9 7 8 ).
-
107. J. Y. Chang and R. J. B a s t i a n i , C l i n i c a l Study No.

41, Summary r e p o r t , S p a C o r p o r a t i o n , P a l o A l t o ,
CA (1977).
108. V. Henry, J. Deutsch, and G. Lum, C l i n . Chem., 24 ( 3 ) ,

--
514 (1978).
109. A. C a s t r o , J. I b a n e z , W. V o ig h t, T. Noto, and H.

Malkus, C l i n . Chem., -- 24 ( 6 ) , 944 ( 1978) .
110. D. N. D i e t z l e r , N. Weidner, V. L. T i e b e r , J. M.
McDonald, C. H. Smith, J. H. Ladenson, and M. P.
L a c k i e , C l i n . Chim. Acta, --
1 0 1 (2 - 3) , 163 ( 1980) .
111. N. Weidner, J. M. McDonald, V. L. T i e b e r , C. H. Smith,

G. Kessler, J. H. Ladenson, and D. N. D i e t z l e r ,
C l i n . Chim. Acta, --
97 ( l ) , 9 (1979).
112. M. O e l l e r i c h , G. W.
S y b r e c h t , and R. Haeckel, J. C l i n .
Chem. C l i n . Biochem., --17 ( 5 ) , 299 (1979).
113. N. U r quh art, W. Godolphin, and D. J . Campbell, C l i n .

Chem., --
- 25 ( 5 ) , 785 (1979).
114. H. M. He ick , A. Mohammad, and C. Golas, C l i n .

Biochem., --
12 ( 2 ) , 68 (1979).
AMINOPHYLLINE 41
115. D. A. L a c h e r , J . A. S i n n , J. S a v o r y , and M. R. Wills,

10 (4), 305 (1980).
Ann. C l i n . Lab. S c i . , --
116. H. S t o n e and B. G i l l i l a n , C l i n . Chem., --

24 (3), 520
(1978).
24 (2), 391 (1978).

117. J. P. Long, C l i n . Chem., --
118. F. D. Lasky, J. A 1 R a z i , and A. Kramen, C l i n . Chem.,

24 (8), 1381 (1978).
--
119. R. L. Boeckx, E. M. F r i t h , and F. E. Simons, "her.
1 (l), 65 (1979).
Drug Monit., _-
120. B. V i n e t and L. Z i z i a n , C l i n . Chem., --

25 (8), 1370
(1979).
121. T. Nishikawa, H. Kubo, and M. S a i t o , C l i n . Chim. Acta,

91 (l), 59 (1979).
--
122. A. L. Neese and L. F. Soyka, C l i n . Pharmacol.
T h e r a p . , 21 (5), 633 (1977).
123. C. E . Cook, M. E. Twine, M. Myers, E. Amerson, J. A.

K e p l e r , and G. F. T a y l o r , Res. Commun. Chem. P a t h .
- 13, 497 (1976).
Pharm.,
-
124. J. C a l d w e l l , T. J. Monks, and R . L. S m i t h , B r . J .
Pharmacol., - 63, 369p (1978).
125. H. H. C o r n i s h and A. A . C h r i s t m a n , J. B i o l . Chem.,

228, 315 (1957).
-
126. J. W. J e n n e , T. W. C h i c k , B. A. Miller, and R. D.

34, 408 (1977).
S t r i c k l a n d , Am. J. Hosp. Pharm., -
127. T. J . Monks, R. L. S m i t h , and J. C a l d w e l l , J. Pharm.

Pharmacol., -33, 93 (1981).
128. J. W. J e n n e , H. T. Nagasawa, and R. D. Thompson, C l i n .

Pharm. and Therap., -- 19 (3), 375 (1976).
129. L. H e n d e l e s , M. Weinberger, and L. B i g h l e y , Am. J.

Hosp. Pharm., - 34, 525 (1977).
130. M. L. S l o t f e l d t , C. E. Johnson, G. Grambau, and J. G.

Weg, Am. J. Hosp. Pharm., -- 36 (l), 66 (1979).
131. H. Lamont, E. Moermann, M. B o g a r t , M. Van Der

S t r a e t e n , and R . Pauwels, Eur. J. C l i n . Pharmacol.,
1 5 ( 6 ) , 401 (1979).
--
132. G. E. M a r l i n , M. A. B u t c h e r , J. A. Klumpp, and P. J.
Thompson, B r . J. C l i n . Pharmacol., -- 10 (3), 265
(1980).
133. P. W. Trembath and S. W. Boobis, C l i n . Pharmacol.

Ther.,
- -- 26 ( 5 ) , 654 (1979).
134. S. McKenzie and E. B a i l l i e , J. I n t . Med. Res., 7

-
Suppl. 1, 22 (1979).
135. M. C. Meyer, A. B. S t r a u g h n , and P. Lieberman, C h e s t ,

78 ( 2 ) , 300 (1980).
--
136. L. J. Lesko, A. T. Canada, G. Eastwood, D. Walker, and
D. R. Brousseau, J. Pharm. S c i . , --
6 8 ( l l ) , 1392
(1979).
137. M. W. Weinberger, R. A. Matthay, E. J. Ginchansky, C.

A. Chidsey, and T. L. P e t t y , J. Am. Med. Assoc., 235,
2110 (1978).
138. E. B. T r u i t t , J r . , V. A. McKusick, and J. C. K r a n t z ,

J r . , J. Pharmacol. Exp. Therap., 1 6 0 , 309 (1950).
139. J. Ahrens, Dtsch. Med. Wochenschr., 102 (13), 482

--
(1977).
140. M. W. Weinberger and E. A. Bronsky, J. P e d i a t r . , -

84,
421 (1974).
141. E. Ginchansky and M. Weinberger, J . P e d i a t r . , --

91 (4),
655 (1977).
142. P. R a n g s i t h i e n c h a i and R. W. Newcomb, J. P e d i a t r . ,

91 ( 2 ) , 325 (1977).
--
143. F. Nielsen-Kudsk, I. Magnussen, T. S . J e n s e n , and K.
Naeser, Acta Pharmacol. T o x i c o l . , --
46 ( 3 ) , 205
(1980).
144. F. E . Simons, K. J. Simons, G. G. S h a p i r o , W. E .

P i e r s o n , and C. W. Bierman, J. Med., 9 ( l ) , 81
(1976).
AMINOPHYLLINE 43
145. M. H. J a c o b s , R. M. S e n i o r , and G. Kessler, J. Am.

Med. Assoc., 235, 1983 (1976).
146. P. D. Watson, R. C. S t r u n k , and L. M. T a u s s i g , -

J.
91 (2), 321 (1977).
P e d i a t r . , --
147. N. N. Khanna, H. S. Bada, and S. M. Somani, -

J.
96, 494 (1980).
Pediatr., -
148. S. P. G a l a n t , S. A. Gillman, L. H. Cummins, P. P.

131 (9),
Kozak, and J. J. O r c u t t , Am. J. D i s . C h i l d . , --
970 (1977).
149. R. Koysooko, E . F. E l l i s , and G. Levy, C l i n .

15 (5), 454 (1974).
Pharmacol. Therap., --
150. P. A. Mitenko and R. I. O g i l v i e , C l i n . Pharmacol.

14, 509 (1973).
Therap., -
151. L. H e n d e l e s , M. Weinberger, and L. B i g h l e y , Am. Rev.

R e s p i r . D i s . , 118, 97 (1978).
152. 3. V. Aranda, D. S. S i t a r , W. E. P a r s o n s , P. M.
Loughnan, and A. H. N e i m s , N. Engl. J. Med., 295,
413 (1976).
153. G. G i a c o i a , W. J. J u s k o , J. Menke, and J. R. Koup, -

J.
Pediatr., - 89, 829 (1976).
154. J. P. Rosen, M. D a n i s h , M. C. Ragni.

-~ C. L. S a c c a r , S.
64, 248
J. Y a f f e , and H. I. Lecks, P e d i a t r i c s , -
(1979).
155. F. E. R. Simons and K. J. Simons, J. C l i n . Pharmacol.,

18, 472 (1978).
-
156. P. M. Loughnan, D. S. S i t a r , R. I. O g i l v i e , A. E i s e n ,
88, 874 (1976).
2. Fox, and A. H. N e i m s , J. P e d i a t r . , -
58,
157. E. F. E l l i s , R. Koysooko, and G. Levy, P e d i a t r i c s , -
542 (1976).
158. K. M. P i a f s k y , D. S. S i t a r , R. E. Rangno, and R. I.

O g i l v i e , N. Engl. J. Med., 296, 1495 (1977).
159. K. M. P i a f s k y , D. S. S i t a r , R. E. Rangno, and R. I.

O g i l v i e , C l i n . Pharmacol. Therap., -21, 310 (1977).
160. A. Mangione, T. E. I m h o f f , R. V. L e e , L. Y. Shum, and

W. J u s k o , - s t , 73, 616 (1978).
C h e-
161. N. Vicuna, J. L. McNay, T. M. Ludden, and H.

7, 33 (1979).
S c h w e r t n e r , B r . J. C l i n . Pharamcol., -
162. K. C. Chang, T. D. B e l l , B. A. L a u e r , and H. C h a i ,

n c e t , 1, 1132 (1978).
-a -
L
163. R. A. Landay, M. A. G o n z a l e z , and J. C. T a y l o r , -

J.
A l l e r g y C l i n . Immunol., 62, 27 (1978).
164. M. Weinberger, D. Hudgel, S. S p e c t o r , and C. C h i d s e y ,

J. A l l e r g y C l i n . Immunol., 59, 228 (1977).
165. A. Kappas, K. E. Anderson, A. H. Conney, and A. P.

23, 445 (1978).
Alvares, C l i n . Pharmacol. Ther., -
166. L. J. Lesko, D. B r o u s s e a u , A. T. Canada, and G .

Eastwood, J. Pharm. S c i . , -- 69 (3), 358 (1980).
167. J. J e n n e , M. Nagasawa, R. McMugh, F. McDonald, and E .

17, 195 (1975).
Wyse, L i f e S c i . , -
168. J. R. P o w e l l , J. T h i e r c e l i n , S. Vozeh, L. Sansom, and

S. Reigelman, Am. Rev. Resp. D i s . , 116, 17 (1977).
169. W. J. J u s k o , J. J. S c h e n t a g , J. H. C l a r k , M. G a r d e n e r
a n d A. M. Yurchak, C l i n . Pharmacol. T h e r a p . , 24,
406 (1978).
170. L. H e n d e l e s , L. B i g h l e y , R. H. R i c h a r d s o n , C. D.
H e p l e r , a n d J. C a r m i c h a e l , Drug I n t e l l . C l i n . Pharm.,
11, 12 (1977).
-
171. T. R. Kordash, R. G. van D e l l e n , and J. T. M c C a l l , -

J.
Am. Med. Assoc., 238, 139 (1977).
172. L. W. Z w i l l i c h , F. D. S u t t o n , J r . , T. A. N e f f , W. M.
Cohn, R. A. Matthay, and M. W. W e i n b e r g e r , Ann.
I n t e r n . Med., - 82, 784 (1975).
173. M. S. Schwartz and D. F. S c o t t , E p i l e p s i a , -

15, 501
(1974).
174. C. L. Winek, J. D. B r i c k e r , W. D. Collom, and F. W.

Fochtman, F o r e n s i c S c i . I n t . , 15 (3), 233 (1980).
ASCORBIC ACID
Ibrahim A . Al-Mesh1 and Mahmoud M . A. Hassan
1. Description 46
1.1 Nomenclature 46
1.2 Formulae 46
1.3 Molecular Weight 47
I .4 Elemental Composition 47
1.5 Appearance, Color, Odor, and Taste 47
2.1 Crystal Properties 47
2.2 Solubility 48
2.3 Specific Rotation 48
2.4 Dissociation Constant 49
2.5 Identification 49
2.6 Spectral Properties 51
3. Preparation 61
3.1 Isolation 61
3.2 Synthesis 63
4. Biosynthesis of Ascorbic Acid 63
5. Metabolism 66
6. Daily Requirement 66
7. Mode of Action 66
8. Vitamin Deficiency 67
9.2 Spectrophotometric Methods 70
9.3 Turbidimetric Method 73
9.5 Enzymatic Method 75
9.6 Polarographic Method 15
9.7 Chronometric Method 75
10. References 76
Copyrighl 0 1982 by The Amenan

Analytical Profiles of Drug Substances phrrm.ceuUcd Awduion
Volume II 45
ISBN 0-12-260811-9
46 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A. HASSAN
1, Description
1.1. Nomenclature
1.1.1 Chemical Names
a) L-Ascorbic a c i d
b) L-Xyloascorbic a c i d
c) 3-0x0-L-gluofuranolactone (enol form).
d) L-3-Ketothreohexuronic a c i d l a c t o n e
1.1.2 Generic Names
Vitamin C ; A s c o r b i c a c i d
1.1.3 Trade Names
Adenex; A l l e r c o r b ; A n t i s c o r b u t i c Vitamin;
Ascorbicap; Ascorbajen; A s c o r i l ; A sc o ri n ;
A s c o r t e a l ; A s c o r v i t ; Cantan; C a n t a x i n ; Cata-
v i n C ; Ce b i c u r e ; Cebid; Cebione; Cecon;
Cegiol an ; Cell i n ; Cenetone; Cereon; Cergona;
Ce s c o r b a t ; Cetamid; Cetan; Cetemican; Ceva-
l i n ; Ce v a t i n e ; Cevex; Cevimin; Cevi-Bid;
Ce-Vi-Sol; Cevitamin; C e v i t a n ; Cimin; C e v i t a -
mic Acid; C e v i t e x ; Ciamin; C i p c a ; C o l a s c o r ;
Concemin; C-vimin; Davitamon C ; E r i v i t C ;
Hybrin; L a r o s c o r b i n e ; Lemascorb; Megascorb;
P l a n a v i t C ; Pr o s c o r b i n C ; Redoxon; Ribena;
Sc o r b a c i d ; Scorbu-C; T e s t a s c o r b i c ; V i c e l a t ;
V i t a c e ; V i t a c i mi n ; V i t a c i n ; V i t a s c o r b o l ;
Vitix.
1.2 Formulae
1.2.1 Empirical
‘gH8’6
1.2.2 Structural CtI*OtI
I
HO
HR=o
HOCH
OH
ASCORBIC ACID 41
1.2.3 CAS No.
(50-81-7)
1.2.4 (Wiswesser Line Notation
TOSV EHJ CQ DQ EYQ IQL (1)

1.2.5 Stereochemistry
The nature o f the ring system in ascorbic

acid was determined by a study of the methy-
lated derivatives of the acid. By this means
complete confirmation was obtained of the
accuracy o f the views advanced, concerning
the stereochemical configuration of the
molecule and the nature of the reactive
enolic groups(2). The furanose structure for
ascorbic acid shown above (1.2.2) was put
forward by Lerbert el a1 ( 2 ) on the basis
o f its chemical behaviour as well as its
oxidation products.
1.3 Molecular Weight
176.12
1.4 Elemental Composition

C, 40.91%; H, 4.58%; 0 , 54.51%.
1.5 Qpearance, Color, Odor and Taste
White or slightly yellow crystals or powder. Odor-

less or almost odorless; pleasant sharp acidic
taste(3).
2.1 Crystal Properties
Usually plates, sometimes needles, monoclinic

system (4).
2.1.1 X-ray Diffraction
The available data (2) of the X-ray, reveals

that the total of 12 carbon and oxygen atoms
all but one can be accomodated in one plane

without appreciable valency strain whilst
the remaining carbon (C5) lies less than 1A"
above the plane as shown in the following
model :
2.1.2 Melting Range

Ascorbic acid melts at 190-192" with decom-
position ( 4 ) .
2.2 So lubi1ity
Ascorbic acid is soluble at 20", in 3.5 parts of

water and 25 parts of alcohol (95 per cent); 50
parts of absolute alcohol, 100 ml of glycerol, 20 ml
of propylene glycol. Solubility in hot water 40.0%
at 40°, 80% at 100". Insoluble in ether, chloroform,
benzene and light petroleum (boiling range 40-60").
2.3 Specific Rotation
[a] is+ 20.5" to 21.5" (C = 1, water)
[a] i3+ 48 (C = 1, methanol) (4).
l9 + 24 (water) (2).
['I 5780
l8 + 116 (sodium salt in neutral solution) ( 4 )
5780
l8
+ 130 (N/20 NaoH) ( 2 ) .
5780
5780 + 149 (N/l NaoH)

l8 (2).
I'[ l8
5780
+ 155 (N/2 NaoH) (2)
l8 + 161 (2N NaoH) (2)

5780
ASCORBIC ACID 49
2.4 Dissociation Constant
Ascorbic acid is a moderately strong organic acid,

two ionization constants:
pK1 4 . 1 7 and pK 11.57. pH = 3 (5mg/ml), pH = 2

2
(50 mg/ml) (4).
2.5 Identification
i) Solution of ascorbic acid decolorises, 2,6-

dicholorophenol-indophenol solution (5).
ii) Solution and ascorbic acid reduces silver

nitrate solution immediately in the cold
producing a black precipitate (5).
iii) Dissolve 0.1 g of ascorbic acid in sufficient

w a t e r to produce 100 ml and dilute 1 ml to
100 ml with 0.01M h y d r o c h l o r i c a c i d . The
light absorption of the resulting solution
exhibits a maximum only at 244 nm; A (1 per
cent, 1 cm) at 244 nm, about 560 (6).
iv) To 2 ml of a 5 per cent w/v solution add 0.5

g of s o d i u m h y d r o g e n c a r b o n a t e ; carbon
dioxide is evolved ( 6 ) .
v) To 1 ml of a 5 per cent w/v solution add

about 0.2 ml of ZM n i t r i c a c i d and 0.2 ml of
0.1M silver n i t r a t e ; a grey precipitate is
produced (6).
vi) To 5 ml of a 1 per cent w/v solution add

0.05 ml of a freshly-prepared 5 per cent w/v
solution of s o d i u m n i t r o p r u s s i d e and 2 ml of
2M s o d i u m h y d r o x i d e followed by 0.6 to 0.7
ml of h y d r o c h l o r i c a c i d , added dropwise with
stirring; the yellow color turns blue (6).
vii) Specific optical rotation, in a 10 per cent

w/v solution, +20.5' to +21.5O (6).
viii) A solution (1 in 50) reduces alkaline cupric

tartrate TS slowly at room temperature but
more readily upon heating ( 7 ) .
ix) To 2 ml of a solution (1 in 50) add 4 drops

of methylene blue TS, and warm to 40": the
deep blue color becomes appreciably lighter
or is completely discharged within 3 minutes
(7).
XI Dissolve 15 mg in 15 ml of a solution of
trichloroacetic acid (1 in 201, add about
200 mg of activated charcoal, shake the
mixture vigorously for 1 minute, and filter
through a small fluted filter, returning the
filtrate, if necessary, until clear. To 5 ml
of the filtrate add 1 drop of pyrrole, and
agitate gently until dissolved, then heat
in a bath at 50": a blue color develops ( 7 ) .
xi) For the examination of vegetable infusions

for the presence of vitamin C, the ascending
- descending paper-chromatographic method of
Block was used on the dinitrosazones. Many
combinations of solvent were used, but a
mixture of xylene and nitrobenzene (95:5)
were the most satisfactory. Of these, the
latter solvent furnished compact spots of
such definition that treatment with alcoho-
lic potash to reveal them was unnecessary
(8)
xii) Amounts of vitamin C down to 3 pg can be

detected as dark areas on the layer exposed
to short-wave UV light; brief heating to
120°C renders it fluorescent in radiation of
365 nm ( 9 ) .
xiii) The customary identification o f free ascor-

bic acid depends on its strong reducing pro-
perties and any of the reactions known from
paper chromatography may be utilized. The
limit of detection with indophenol reagent
(blue) is around 0.1 pg dipyridly-iron
(red) and molybdophosphoric acid (blue) are
almost as sensitive; after brief heating,
derivatives and decomposition products
yield the colors also. Amounts of 3-5 pg
can be visualized with iodoplatinate reagent
(yellow) and with alkaline silver nitrate
reagent (9).
ASCORBIC ACID 51
2.6 Spectral Properties
2.6.1 Ultraviolet Spectrum
The UV spectrum of ascorbic acid (0.002%) in

aqueous, acidic methanolic, ethanolic and
alkaline solution was scanned from 200 to 400
nm using Varian Carry 119 Spectrophotometer
(Fig. I ) . The UV maxima are as follows:
'max (nm)
Aqueous solution 263

Acidic solution 243
Methanol 244
Ethano 1 245
Other reported data (2) are as foJlows:
'max (nm>
Aqueous solution 260-265

Acidic solution (pH3) 245
Ethano 1 245
Methanol 263
Sodium salt in aqueous solution 265
2.6.2 Infrared Spectrum
The IR spectrum of ascorb,icacid as KBr-disc

was recorded on a Perkin-Elmer 580B FT-
spectrometer (Fig. 2 ) . The structural assign-
ments have been correlated with the following
band frequencies (Table 1).
Table - 1: IR Characteristics of Ascorbic

Acid,
Frequency Cm-I Assignment
3510
3405 OH
3306
1755 c=o
1670
1110
c-0-c
1025
52 IBRAHIM A. AL-MESHAL AND MAHMOUD M. A . HASSAN
. . .
200 210 225 230 2 4 250 260 270 284 2YO 300 310 2 0 3% 3u) 350 3m 380 390
Fig. 1. UV Spectrum of Ascorbic Acid. Ascorbic Acid

i n Water; --- Ascorbic Acid i n Ethanol; +scorbic Acid i n
-
Methanol; ....
Ascorbic Acid i n Acid Solution.
1
4w mnmlmbr(tlDm am fWe mr f4a c w I a o c o o 4 m 2 I
Fig. 2. IR Spectrum of A s c o r b i c A c i d as KBr d i s c .

Other characteristic absorption bands are:
3208, 1500, 1390, 1372, 1320, 1275, 1250,

1222, 1200, 1140, 1075, 1068, 1045, 1025,
990, 870, 820, 755, 720, 680.
2.6.3 Nuclear Magnetic Resonance Spectra
2.6.3.1 Proton Spectra
The PMR spectra of ascorbic acid in

dueterium oxide,in pyridine and in
pyridine D5 were recorded on a
Varian-T-60-A, 60 MHz spectrometer
using sodium-2,2-dimethyl-2-sila-
pentane-5-sulphonate and tetramethyl-
silane as reference standard respec-
tively (Fig.3, Fig. 4 and Fig. 5).
The following structural assignments
have been made (Table 2):
Table - 2: PMR Characteristics o f Ascorbic

Acid.
Chemical Shift (ppm)
D20 Pyridine Pyridine D5 Assignment
4.87(d) 5.43(d) 5.36 (d) 5-H

4.10(m) 4.68(m) 4.33 (m) 6-H
3.77(s)
3.68(d)
4.40(s)
4.30(s)
4.36 (s)
4.23 (d) ' 7-CH2
s=singlet, d=doublet, m=multiplet.

400
Fig. 3. PMR Spectrum of A s c o r b i c A c i d in D20.

I
L
0
z
L
1
I
0
m
0
0
0
m
;I
56
m
4
Fig. 5. PMR Spectrum of Ascorbic Acid i n Pyridine D5.

PMR data in D20 and in a mixture of

DMSO D6 and CDC13 were also reported
(10,11,12).
13
2.6.3.2 C-NMR
The 13C-NMR completely decoupled and

off-resonance spectra are shown in
Fig. 6 and Fig. 7 respectively. Both
were recorded over 5000 Hz range in
dimethylsulfoxide on Jeol FX-100, 100
MHz spectrometer. Using 10 mm sample
tube and tetramethylsilane as refe-
rence standard, at ambient tempera-
ture. The carbon chemical shifts are
assigned on the basis of the additi-
vity principles and the proton-
coupled spectrum (Table 3).
11
I
HO - c 6-
I
Table - 3: Carbon Chemical Shifts of Ascorbic

Acid.
Carbon No. Chemical Shift ppm

c-1 170.31(s)
c-2 117.93 ( s )
c-3 152.62 (s)
c-4 74.56 (d)
c-5 68.42 (d)
C -6 61.93(t)
s=singlet; d=doublet; t=triplet
13C-NMR data in Water have been also

reported (1 3) ,
Fig. 6. 13C-NMR Spectrum of Ascorbic Acid (Completely de-
coupled).
~~
Fig. 7 . 13C-NMR Spectrum of Ascorbic Acid (off-Resonance) .

61
ASCORBIC ACID
2.6.4 Mass Spectrum

The mass spectrum o f ascorbic acid obtained
by electron impact ionization, was recorded
on a Ribermag R-10-10 mas spectrometer equip-
ped with direct inlet probe. The spectrum
(Fig. 8) shows a molecular ion peak M+ at m/e
176 with a relative intensity o f 5.9%. The
most prominent fragments and their relative
intensities are shown in Table 4:
Table - 4: Prominent Mass Fragments of Ascorbic

Acid.
M/e Relative Intensity % Fragment
177 1.0 M + l
176 5.9 M +
1
116 100.0
85 36.0
71
70
24.7
23.1
HF
H
H o g
61 29.7
a'
HO-
'i'
C
PH
- 0
C
I I
H H
3. Preparation
3.1 Isolation
Many methods were reported €or the isolation o f ascor

bic acid from plants. However, the most popular is
by using freshly prepared solution of 5-6% methaphos-
phoric acid (14). This solution is a good extractant
as well as stabilizing agent f o r a limited period by
complexing metal ions and minimizing the rate of oxi-
dation. It has also been claimed that ascorbic acid
can be stablized by diluted perchloric acid solution
ASCORBIC ACID 63
o r 2,3-dimercapto-l-propanol (15). An alternative

method of extracting ascorbic acid from foods is by
forming a slurry of the frozen material with absolute
ethanol has been found to be as effective as extrac-
tion with metabphosphoric acid (16).Also a mixture
of 8% acetic acid and 0.5% oxalic acid was used (17)
3.2 Synthesis
L-ascorbic acid is conventionally synthesized (18,19)

by hydrogenating D-glucose to D-sorbitol. The latter
is made to yields L-sorbitol by oxidation with Aceto-
bacter suboxydan, this followed by introducing carbo-
xyl group at C 1 while the L-sorbose is in the form of
its diacetone derivative. The resulting diacetone-2-
keto-L-gluconic acid is then heated with hydrochloric
acid t o give ascorbic acid (Scheme 1 , p . 11).
Alternative route from sorbose by oxidation with
nitrogen peroxide.
Another method for synthesizing L-ascorbic acid was
reported ( 2 0 ) , involving a one-step oxidation of 1,2-
0-isopropyhene-a-D-glucofuranose to 1,2-0-isopropyli-
dene-a-D-xylo-hexofuranurono-6,3-Lactone-S-ulose and
acid treatment of the later followed by reduction.
4. Biosynthesis of Ascorbic Acid
In both plants and animals ascorbic acid is formed from D-

glucuronic acid. UDP-glucuronic acid is first converted to
D-glucuronic acid lactone via D-glucuronic acid-l-phos-
phate. This compound is then reduced at carbon atom 1 t o
form L-gulonic acid. (Since in the numbering of the carbon
atoms of carbohydrates the most highly oxidized carbon
atom is given the lowest possible number, the original
carbon atom 6 of glucuronic acid becomes carbon atom 1 of
gulonic acid). After the conversion of the gulonic acid
to the corresponding y-lactone, the hydroxyl group at
carbon atom 2 is oxidized to a keto group. The 2-keto-L-
gulonic acid lactone formed is subsequently converted t o
L-ascrobic acid by enolization (21). Direct conversion of
D-glucuronic acid to L-gulonic acid by isomerization at
carbon atom 5 has not yet been conclusively established
(scheme 2,~.12).
CH20H
CH20H CH20H
I I I
HO - C -
I
H IIO - C - H co
HO - C - H I I
HO- C- H HO- C--B
I H? I Acetobactv I
H -C-O€i B- C- OH H- C- OH
I cat. I suboxydans I
HO -c - B HO - C - I-; HO - c - H
I I I
CHO
Ct120H CH20€i
D-glucose
G-sorbitol L-sorbose
COOH
I
co
I
HO - c-
I
acetone + (1)*<Mn04 ~
ki - C - OH
I
H+ ( 2 ) H 2 0 (H+) I
9:CMe2 H HO - C
I
-E
CH OII
2,3,4,6-diacetone sorbose 2
2-keto-L-gulonic acid
(CH20H
C -OH
bo
HO-
II
C
11 I
eno-
lize
I lactoniz
H - C-OH
I H
HO- C-H OH O€i
HO-C-H
I I
I
CH20H
CH20H
L-ascorbic acid
(Vitamin C)
Scheme 1: Synthesis of ascorbic acid.
ASCORBIC ACID 65
-
0
II
..
6
Q"Dp-
COOH COOH C
HO Ho@o-p ,@"20H
OH OH OH
UDP-D-Glycuronic D-Glucuronic acid- D-Glucuronic
acid 1-P acid lactone
CH20H CH20H
L-Gulonic acid L-Gulonic acid
lactone
HO-C-H
I I
CH20H CH20H
2-Keto-L-gulonic L-Ascorbic
acid lactone. acid.
Scheme 2: The biosynthesis of L-ascorbic acid.

5. Metabolism
Ascorbic acid is readily absorbed and metabolised. However,
after oral administration of large quantities, only small
amounts are excreted in the urine while there is a steady
rise in the level of ascorbic acid in the plasma. If the
oral ingestion is continued for a sufficient period, the
plasma concentration rises to a maximum, after which a
rapid urinary excretion of a large part of the ingested
ascorbic acid occurs (22). Ascorbic acid (ASA) and dehydro-
ascorbic acid (DAsA) are metabolized by humans, and the
levels of AsA in blood were maximum at the 4th hr after
AsA administration but at 2nd hr after DAsA administration.
The amount of AsA and DAsA excreted in urine in 6 hr was
respectively. 60 and 70% of the amount excreted in 24 hr
after administration, About 30 and 40% of administered
dose were excreted by men and women respectively in 24 hr.
Of the vitamin excreted after administration of either
AsA or DAsA, about 90 and 10% were in the form of AsA and
DAsA, respectively (23).
The amino acids phenylalanine and tyrosine are not meta-
bolized completely in vitamin C-deficient individuals,
Under these conditions they are metabolized only partly
and are excreted in the urine as homogentisic, p-hydroxy-
phenylpyruvic and p-hydroxyphenyllactic acids. It appears
that vitamin C plays the role of a coenzyme in the meta-
bolism of tyrosine through its deaminated product, because
scorbutic liver slices cannot metabolize this amino acid
in the absence of this vitamin. Vitamin C in adequate
amounts delays the oxidation of epinephrine by the body
(24 1.
6. Daily Requirement!
Ascorbic acid is needed in daily quantities of about 70 mg
to sustain full stamina, and is an essential nutrient for
human beings. In the case of insufficiency the symptoms
of scurvy appears (21).
7. Mode o f Action
We have as yet no complete understanding o f the mode of
action of ascorbic acid. It is, however, known that this
substance is involved in certain hydroxylation reactions
which are catalysed by mixed function oxygenases in the
reduction o f folic acid to tetrahydrofolic acid as well
as well in the regulation of the redox equilibrium between
Fez+ and Fe3+ and Cu" and C U ~ +(21).
ASCORBIC ACID 61
8. Vitamin Defficiency
Patients placed on a diet deficient in vitamin C

exhibited the following: 1) 10 days, plasma fell to
a low level; 2) 30 days plasma level was zero; 3) 13
weeks, first clinical evidence of scurvy; 4) 132 days,
hyperkeratotic papules developed; 5) 141 days, wounds
failed to heal and 6) 162 days, perifollicular hemor-
rhages of scurvy developed; ascorbic acid value of
white cell platelets fell to zero. Loss of weight
occurred, accompanied by lowered blood pressure (24).
British Pharmacopoeia 1973
The B.P. 1975 has described the assay

as follows:
Weigh and powder 20 tablets. Dissolve a

quantity of the powder equivalent to 0.15 g
of ascorbic acid as completely as possible in
a mixture of 30 ml of water and 20 ml of
d i l u t e sulphuric acid and titrate with 0.lN
anunonium c e r i c sulphate, using f e r r o i n sulphate
solution as indicator. Each ml of 0 . 1 1 m o -
niwn c e ri c sulphate is equivalent to 0.008806 g
of C6H806.
British Pharmacopoeia 1980
The B.P. 1980 has described the assay as

follows :
Dissolve 0.2 g in a mixture o f 80 ml of freshly

boiled and cooled water and 10 ml of M sulfuric
acid. Titrate with 0 . 0 5 M iodine VS using 1 ml
of starch solution as indicator until a persis-
tent blue color is obtained. Each ml of 0.05M
iodine VS is equivalent to 0.00881 g of C H 0
6 8 6'
United States Pharmacopoeia 1980
a) Ascorbic Acid: Dissolve about 400 mg of

ascorbic acid, accurately weighed, in a
mixture of 100 ml of carbon dioxide-free

water and 25 ml of 2N s u l f u r i c acid. Titrate
the solution at once with 0.1 N iodine VS,
adding 3 ml o f starch TS as the end-point is
approached. Each ml of 0.1 N iodine is
equivalent to 8.806 mg of C6H806.
b) Ascorbic acid injections: Transfer to a 100-ml

volumetric flask an accurately measured volume
of ascorbic acid injection, equivalent to about
50 mg of ascorbic acid and previously diluted
with water if necessary. Add 20 ml of metaphos-
phoric-acetic acids TS, dilute with water to
volume, and mix. Accurately measure a volume
of the dilution, equivalent to about 2 mg of
ascorbic acid, into a 50-ml conical flask, add
5 ml of metaphosphoric-acetic acids TS, and
titrate with standard d i c h l o r o p h e n o l - i n d o p h e n o l
solution until a rose-pink color persists f o r
at least 5 seconds. Correct for the volume of
the dichlorophenol-indophenol solution consumed
by a mixture of 5.5 ml of metaphosphoric-
acetic acids TS and 15 ml of water. From the
ascorbic acid equivalent o f the standard
d i c h l o r o p h e n o l - i n d o p h e n o l solution calculate
the ascorbic acid content in each ml o f the
injection.
Various other titrimetric methods of assay

of vitamin C in vegetable tissues, whether as
ascorbic acid or as total vitamin C (ascorbic
plus dehydroascorbic acids), were studied and
compared. For the extraction of the vitamin,
an aqueous mixture of 8 per cent, acetic and
0.5 per cent oxalic acids was used instead of
metaphosphoric acid. Hot and cold extractions
gave practically the same results, except with
hard, dry tissues, which required hot extraction.
To effect removal of colloidal matter, which
interferes with filtration and clarification
of the extract, it is considered advisable to
add 25 to 50 per cent o f ethanol. In the
presence of ethanol causes an error o f at least
2 to 12 per cent. To establish (visually) the
titration end-point in the indophenol method,
it is recommended that a standard having the
same composition as the sample under titration
ASCORBIC ACID 69
b u t p r e v i o u s l y o x i d i s e d w i t h i o d i n e and
mercuric a c e t a t e be employed. T h i s g e n e r a l l y
e n a b l e s 93 t o 100 p e r c e n t of v i t a m i n C e x p e r i -
m e n t a l l y added t o be determined. I n t h e method
of d e t e r m i n a t i o n with methlene b l u e , t h e r a t i o
o f methylene b l u e used t o v i t a m i n C c o n t e n t
d e c r e a s e s as t h e l a t t e r v a l u e i n c r e a s e s . T h i s
d e c r e a s e of t h e r a t i o i s small; t o o b t a i n s a t i s -
f a c t o r y r e s u l t s , t h e a l i q u o t t i t r a t e d should
n o t c o n t a i n >0.04 mg o f v i t a m i n C . V i s u a l
d e t e r m i n a t i o n o f t h e end-point w i t h t h e a i d
o f a s t a n d a r d i s a g a i n recommended. The i o d i -
m e t r i c method, with potassium i o d a t e was a l s o
s t u d i e d . The c o n c l u s i o n s drawn a r e : 1) t h a t
t h e indophenol method i s s u f f i c i e n t l y a c c u r a t e ,
i s t h e most simple method and h a s t h e w i d e s t
s p h e r e of a p p l i c a t i o n ; 2) t h a t t h e methylene b l u e
method g i v e s r e s u l t s 3 t o 10 p e r c e n t lower
t h a n t h e indophenol method and 3) t h a t t h e
i o d i m e t r i c method g i v e s r ? s u l t s 20 t o 40 p e r
cmt higher (17).
Reduction i s e f f e c t e d by adding M sodium

s u l p h i d e s o l u t i o n a c i d i f i e d with HC1, and
removing t h e e x c e s s o f s u l p h i d e w i t h M
e t h a n o l i c mercuric c h l o r i d e s o l u t i o n . Reduc-
t i o n i s complete i n 10 t o 15 min. C l e a r
f i l t r a t e s a r e r e a d i l y o b t a i n a b l e €or t i t r a t i o n
w i t h dichlorophenol-indophenol ( 2 5 ) .
Simple methods o f d e t e r m i n i n g a s c o r b i c
a c i d , t h i a m i n e and n i c o t i n i c a c i d a r e
applied t o mixtures containing t h e s e
vitamins with various pharmaceutical
p r e p a r a t i o n s . Ascorbic a c i d can b e
determined i o d i m e t r i c a l l y i n t h e p r e s e n c e
o f calcium l a c t a t e , p h y t i n g l u c o s e calcium
g l y c e r o p h o s p h a t e , c a f f e i n e sodium b e n z o a t e ,
n i c o t i n i c a c i d , amidopyrine o r t h i a m i n e .
For m i x t u r e c o n t a i n i n g a s c o r b i c a c i d and
nicotinic acid, the ascorbic acid i s deter-
mined i o d i m e t r i c a l l y and t h e t o t a l a c i d i s
then titrated against 0.1 N solution of

hydrochloric acid with phenol red as indi-
cator. Thiamine i s determined in the
presence of ascorbic acid by a modifi-
cation of the U.S.S.R. Pharmacopoeia
VIII argentimetric method ( 2 6 ) .
The platinum - tungsten bimetallic elec-
trode system is applicable to the
quantitative iodimetric determination
of ascorbic acid according to the B.P.
1953. The equivalence point is given
by the very sharp break in the titration
curve. The error, = f 0.0002 on samples
of 0.05 to 0.15 g is much less than for
the usual volumetric procedure (27).
Other Sitrimetric method for the deter-
mination of ascorbic acid is by involv-
ing two stage oxidation by potasium
iodate ( 2 8 ) .
9.2 Spectrophotometric Method

9.2.1 Colorimetric
An assay method described for ascorbic acid
involves the reaction with diazotised 4-
methoxy-2-nitroaniline in acid medium, and
subsequent development of a blue color in
alkaline solution. This color, with a maximum
absorbancy at 570 nm, i s compared with stan-
dards in suitable photo-electric colorimeter.
It can be carried out directly, e.g., in the
presence of dehydro-ascorbic acid and all
other vitamins. Its sensitivity permits the
determination of quantities down to 0.5 mg
with a low limit of 10 pg per ml, when a 50-
ml sample aliquot is used (29a).
9.2.2 Ultraviolet
In aqueous solution ascorbic acid is charac-
terised by a single very intense band with
its head at 260-265.nm. The molecular extinc-
tion coefficient is approximately 7000 for
solutions containing about 2 mg/100 cc. In
stronger solutions (ca.50 mg per 100 c.c.)
wide deviations from Beer's law are encoun-
ASCORBIC ACID 71
tered. But for concentrations ranging between

0.5 and 2.5 mg per 100 C.C. Beer's law holds
with sufficient exactitude to permit of the
use of spectrophotometric measurements for
quantitative estimations of concentrations.
The intensity of the band diminishes rapidly,
falling to half value in a few hours (decom-
position of ascorbic acid by oxidation (2).
Other color reactions have been proposed as a
basis for measuring ascorbic acid: reaction
with diazotised p-aminobenzoic acid to produce
a pink color and the interaction of ascorbic
acid with dimethoxydiquinone to form a reddish
-violet color which is measured spectrophoto-
metrically at 510 nm (29b).
Ascorbic acid, cystine, thioglycollic acid,
a-mercaptopropionic acid and sodium mercapto-
butane sulphonate are determined by the fluore-
scence produce by the reduction of sodium 1 : Z -
naphthaquinone-4-sulphonate (Folin's reagent)
in U.V. The use of p-chloromercuribenzoic
acid in the determination of ascorbic acid
with 2,6-dichlorophenol-indophenol below pH3-5
is recommended because of the extent of spon-
taneous fading of indophenol that occurs. p -
Chloromercuribenzoic acid does not affect the
reaction between indophenol and ascorbic acid,
but it inhibits almost entirely the decolori-
sation of indophenol by various interfering
substances (31).
9.2.3 Spectrofluorimetric
The reaction of dehydroascorbic acid with o-

phenylenediamine to give a fluorescent quinox-
aline was used as the basis of an assay to
determine y quantities of ascorbic and dehydro-
ascorbic acids, The development of the fluore-
scent derivative of the vitamin is prevented
by forming a boric acid-dehydroascorbic acid
complex prior to addition of the diamine
solution. This provides a means of differen-
tiating between the fluorescence from the
vitamin and that from possible interfering
substances. When applied to pharmaceutical pre-
parations, beverages and special dietary foods,
the method shows a high degree of specificity.

No interfering substances were found (32).
A rapid colorimetric determination of ascorbic

acid (10 to 200 mg per 100 g) with the sodium
salt of 2,6-dichlorophenol-indophenol was
studied. An amyl acetate extract of 2,6-
dichlorophenol-indophenol has max. absorption
at 525 nm the extinction of which remains
unchanged for 4 hrs. The sample is extracted
with 2 per cent metaphosphoric acid and
diluted to produce a 0 . 4 to 1.0 mg per cent
solution of ascorbic acid. 2,6-dichlorophenol-
indophenol solution (5 ml in water) is added
to the sample solution which is rapidly ex-
tracted with amyl acetate for the extinction
to be measured. Another 5-ml portion of the
2,6-dichlorophenol-indophenol solution is
added to metaphosphoric acid and similarly
treated. The amount of ascorbic acid is
determined from the difference in the extinc-
tion of the two extracts, which is propor-.
tional to the concentration of ascorbic acid
up to 4 mg per 100 ml ( 3 3 ) .
Other method depends on the reduction of
ferric chloride by ascorbic acid and the
colorimetric determination of the ferric
chloride by means of reaction with aa-dipy-
ridly; the reaction is carried out in the
presence of phosphoric acid to eliminate in-
terference by reduction. A solution (1 ml)
containing N 0.02 mg of ascorbic acid is tre-
ted with 0.3 ml of 85 per cent. Phosphoric
acid to give a pH of 1 to 2, 5 ml of 0.5 per
cent aqueous aa-dipyridly solution and 1 ml of
1 per cent ferric chloride. The method has
been applied to orange juice, honey and urine;
it is sensitive to 5 lJg of ascorbic acid per
ml ( 3 4 ) .
A direct method for the determination of vita-
min C without removal of the reagent, 2,4-
dinitrophenylhydrazine, is applied to blood
and urine. The ascorbic acid content i s calcu-
lated from a chart (35).
Ascorbic acid can be semi-quantitatively
estimated with the comparison method after
color reaction with molybdophosphoric acid.
ASCORBIC ACID 73
The red zone of dehydroascorbic acid-DNP is

scraped off, eluted with 85% sulphuric acid,
filtered or, better, centrifuged, and the
light absorption of the solution measured at
520 to 525 nm against water as blank. The
analysis result is worked out with the help of
a standard solution which is treated identi-
cally ( 9 ) .
9.3 Turbidimetric Method

This method (36) is used €or the determination o f
ascorbic acid in foods, by the reaction of selenious
acid with ascorbic acid and stannous ions at low pH
and room temperature.

9.4.1 Paper Chromatography
Methods used €or the separation of ascorbic

acid by paper chromatography are shown in
Table 5 ( p . 21).
9.4.2 Gas-Liquid Chromatography
Gerstl and Ranf€t (42) extracted food with

metaphosphoric acid, separted the ascorbic
acid on a column of cellulose and formed the
trimethysilyl ether derivative by reaction
with N.0-bis-(trimethylsily) acetamide before
chromatographing on a column of Gas Q contain-
ing 3% SE-30.
9.4.3 High Pressure Liquid Chromatography
Packla and Kissinger ( 4 3 ) , used a strong anion

resin and elution with pH 4.75 buffer solution,
has been applied to the determination of
ascorbic acid in milk products, baby foods,
fruit juice concentrates, whole fruits and
fortified cereals. The procedure was not
directly suitable for measuring dehydroascorbic
acid. However, sood et a1 (44) used HPLC in
TABLE - 5 Methods used for separating ascorbic acid by paper chromatography
P
- -- -~ ~ ~~~ ~ ~
Chromatogram Solvent System Rf Value Reagent Application Reference
Paper Chro- 1) n-butanol saturated 2,6-dichlorophenol- Plant and 37

matography. with water + oxalic indophenol with sub- animal
sequent colorimetry. tissues.
2) Phenol saturated with
water + oxalic acid.
Paper (Sch- 1) 50% Methanol. 0.7-0.8 1) 1% silver-nitrate Lemon juice 38
leicher and solution in 10% white,red and
Schull No. Aqueous ammonia. black-currents.
602). 2) Wat er-n-butano1- 0.6-0.7 2 ) 0.005N iodine solu- tomatoes and
glacial acetic acid tion in 0,04% starch green pappers.
(50:40:14:5) so 1ut ion.
3) 0.04% aqueous solu-
tion of 2,6-dichloro-
phenol-indophenol.
4) U.V.
Paper Chro- butanol-acetic acid- 0.36 ammonium-molybdate solu- 39
matography. water (4:1 :5) tion.
Paper Chro- n-butanol-acetic acid- Alkaline tetrazolium Method f o r de- 40

matography. water (4:l:S) salts. tection of lfpg
quantity.
ASCORBIC ACID 75
the reverse phase ion-pairing mode to deter-

mine ascorbic acid in food with tridecyl ammo-
nium formate as counter-ion.
9.5 Enzymatic Method
A new enzymatic method based on the oxidation of

the ascorbic acid to dehydroascorbic acid with
ascorbic oxidase permit assaying for ascorbic acid
and dehydroascorbic acids in vegetable extracts (45).
9.6 Polarographic Method

Ascorbic acid can be analysed in a variety of mix-
tures and multivitamins preparations by cathode ray
reverse sweep polarography (46).
9.7 Chronometric Method
The reduction oxidation couple between ascorbic acid

oxidation and reduction of the semiquinoid form p-
phenylenediaimine is the basis of a new chronometric
assay of vitamin C.
Ascorbic acid interrupts formation of the colored
product by coupling its oxidation to reduction of
the semiquirioid proceeds to dye form (47).
10. References
1. G.C.G. Grasselli and N.M. Ritchey "Atlas of Spectral Data

and Physical Constants for Organic Compounds" 2nd Ed. ,
Vol. 2, CRC Press Inc., Cleveland, Ohio, 1975.
2. R.W. Herbert, E.L. Hirst, E.G.U. Percival, R.J.W. Reynolds
and Smith, J. Chem. SOC., 1270 (1933).
3. J.E. Hoover "Remington's Pharmaceutical Sciences", - 15th
Ed., Mack Publishing Company, U.S.A., (1975).
4. M. Windholz "The Merck Index" 9th Ed. Merck and Co., Inc.,
Rahaway, U.S.A. (1976).
5. "The British Pharmacopeia", Her Majesty's Stationary
Office, Cambridge, (1973).
6. "The British Pharmacopeia", Her Majesty's Stationary
Office, Cambridge, (1980).
7. "The United States Pharmacopeia", XX, Mack Publishing
Co., Easton, Pa (1980).
8. R.C.R. Barreto, Rev. Quim. Ind., Rio de Janeiro, - 24, 13,
(1955). Through, Anal. Abstract , 3, 567 (1956).
9. E. Stahl , "Thin Layer ChromatograpFy", 2nd Ed. , Springer-
Verlag., p. 304, (1966).
10. C.J. Pouchert and J.R. Campbell "The Aldrich Library of NMR
.
and Spectra" Vol XI (1974).
11. "High Resolution NMR Spectra Catalog", Varian Analytical
Instrument Division, Vol. 2, The National Press, U.S.A.
(1963) .
12. D.T. Sawyer and J.R. Brannan, Anal. Chem., 38 (2), 192,
(1966).
13. S . Berger, Tetrahedron, 33, 1587 (1977).
14. R.D. King "Developments in Food Analysis Technique-1"
Applied Science Publishers, 18 (1978).
15. C.B. Bourgeois, P.R. Mainuy, R. George and A.M. Czornomaz,
Analusis, 2, 556 (1973). Through Reference 14.
16. V.G. Randall, E.L., Pippen, A.L. Potter and R.M. McCready,
J. Fd. Sci., 40, 894. Through Reference 14.
17. G, Enachescu,%al. Inst. Cerc. Agron. Romn., 22, 463,
(1955) . Through Anal. Abstract , 3, 3474 (1956):
18. T.A. Geissman, "Principles of Organic Chemistry", 3rd. Ed.,
W.H. Freeman and Company, p. 497, (1968).
19. T. Reichstein and A. Grussner, tlelv. Chim. Acta., -17, 311,
(1934).
20. J. Bakke and 0. Theander Chemical Comm. , 175 (1971).
21. M. Luckner "Secondary Metabolism in Plants and Animals",
Science Paperbacks, p. 74, (1977).
22. E.G.C. Clarke "Isolation and Identification of Drugs",
The Pharmaceutical Press, London, (1971).
ASCORBIC ACID 77
23. T. Masaru, W. Sanae, M. Katsuko, T. Sachiko, F. Akji

(Biochem. Lab. Kagawa Nutr. Coll., Tokyo, Japan). Vila-
mins 45(3), 136, 1974, fJapan). Through
24. C.D. Wilson, 0. Gisvold and R . F . Doerge "Textbook o f Orga-
nic Medicinal and Pharmaceutical Chemistry", 7th Ed.,
J.B. Lippincott Co., Philadelphia, (1977).
25. E . Piyanowski, Prezem. Rony Spozywezy, 11,410 (1954).
26. G.A. Vaisman and S.G. Rozhntskaya Aptechnoc Delo. (3),
16. Through: Anal. Abstract, 2, 563 (1955).
27. S.R. Mohanty, K.R.K. Rao and L.V. Kannan, Anal. Chim.
Acta, 14 (6) 587 (1956). Through: Anal. Abstract, 2, 347,
(1956);
28. G.S. Deshmukh and M.G. Bapat, Z. Anal. Chem. 145(4), 256
(1955).
29. (a) M. Schmall, C.W. Pifer and E.G. Wollish, Anal. Chem.
25(10), 1486 (1953). Through Anal. Abstract, 1,370,
-
(1954).
(b) M.H. Hashmi, M.A. Shahid, M.A. Akhtar and N.A. Chugtani,
Mikrochim Acta, 5, 901 (1973).
30. H.. Freytag, Z. anal. Chem., 139(4), 263, 1953. Through.
Anal. Abstract, 1, 371, (1954).
31. J.A. Owen and B.Iggo, Biochem. J., 62(4), 675 (1956).
32. C. Mike, J. Deutch and C.E. Weeks, J. Assoc. Office Agr.
Chemists, 48(6), 1248 (1965). Through Chem. Abstract, 64,
72840 (1966).
33* H. Imai and T. Fugitani J. Chem. SOC., Japan, Pure Chem.
Sect., s(11), 1212 (1955). Through: Anal. Abstract 2,
2567 (1956).
34. M.X. Sullivan and H.G.N. Clarke, J. Ass. Off. Agric. Chem.,
38(2), 514 (1955). Through: Anal. Abstract, 2, 258 (1956).
-
35. J.H. Roc and C.A. Kuether, J. Biol. Chem. 147, 399 (1943).
Through: Chem. Abstract, 37, 31185 (1943).
36. W.J. Ralls, J. Agric. Food Chem. 23(3), 609, (1975).
Through: Chem. Abstract, 83, 41610 F (1975).
37. Yu-Tuan Cheng., F.A. Isherwood and L.W. Mapson, Biochem.
J., 55(5), 821 (1953). Through: Anal. Abstract, I, 372,
(1954).
38. V. Sanda Ceskosl. Farmac., 2(3), 79 (1954). Through: Anal.
Abstract, 2, 2563, (1955).
39. W. Hermann, R. Strohecker and F. Matt., Z. Lebensmitt. Un-
tersuch. U. Forsch., 97, 263, (1953). Through: Anal.
Abstract, 1, 369, 1954.
40. 2. Padre, E. Smid and V. Sicho, Naturwissenschaften, 42(8) ,
210, (1955). Through: Anal. Abstract, 2, 853 (1956).
41. J.E. Schlack, J. Ass. Office, Analyt. Chemist., 57, 1346,
(1974). Through: Reference 14.
42. R. Gerstl and K. Ranfft, Z. Lebensmitt-elunters. U. Forsch.,
154, 12, (1974). Through Reference 14.
-
43. L . A . Packla and P.T. Kissinger, Anal. Chem., 48, 3 6 4 ,

( 1 9 7 6 ) . Through Reference 1 4 .
4 4 . S.P. Sood, L.E. Sartori, D.P. Wittmer and W.G. Haney,
Anal., Chin., 4 8 , 796, ( 1 9 7 6 ) . Through Reference 1 4 .
4 5 . A. Marchesini, F . , Montauori, D. Muffats, D. Maestri, J.
Food Sci., 3 9 ( 3 ) , 5 6 8 , ( 1 9 7 4 ) . Through Chem. Abstract,
8 2 , 2717 F ( 1 9 7 5 ) .
4 6 . K S . Owen, F.W. Franklin, J. Food Technol., E ( 3 ) , 2 6 3 ,
( 1 9 7 5 ) . Through Chem. Abstract, 83, 1 3 0 1 0 2 J ( 1 9 7 5 ) .
4 7 . B. Roe, J . H . Bruemmer, Proc. Fla. State Hortic. SOC.,
87, 210, ( 1 9 7 5 ) . Through Chem. Abstract, 83, 130100 G
-
(1975).
CAPTOPRIL
Harold Kadin
1. Description 80
1.1 Name, Formula, Molecular Weight 80
1.2 Appearance, Color, Odor 80
2. History 80
3. Synthesis 84
4.2 Solid State Properties 97
4.3 Solution Data 106
5. Stability 107
5.1 Solid State Stability 107
5.2 Solution Stability 108
6. Analytical Tests and Methods 112
6.1 Elemental Analysis 112
7 Analysis in Biologic Fluids and Tissues and in Animal Rations 120
7.1 Thin Layer Radiochromatography (TLRC) 120
7.2 Gas Chromatography-Mass Spectroscopy (GC-MS) 122
7.3 Gas Chromatography-Flame Photometric Detection (GF-FPD) 123
7.4 High Performance Liquid Chromatography with UV Detection (HPLC-UVD) 124
7.5 Spectrofluorometry 124
7.6 Radioimmunoassay (RIA) 124
7.7 Semiautomated Ellman Colorimetry 125
7.8 High Performance Liquid Chromatography with 126
Electrochemical Detection (HPLC-ECD)
7.9 High Performance Liquid Chromatography with 128
Fluorescence Detection (HPLC-FD)
8. Drug Metabolism-Pharmacokinetics 128
8.1 Blood Level Studies 128
8.2 Urinary Excretion Studies 129
8.3 Miscellaneous Distribution Studies 134
9. Acknowledgments 131
10. References 131
11. Review Coverage Dates 137
Analylical Profilca of Drug Substances Copynghi 0 1982 by The American

Volume 11 79 Pharmaceutical Association
ISBN 0-12-260811-9
80 HAROLD KADIN
1. Description
1.1 Name, Formula, Molecular Weight
Captopril, Capoten@, or Lopirins
is l-(3-mercapto-2-D-methyl-l-oxopropyl)-L-proline
(S,S) with -
Chem. -
Abstr. Registry Number
62571-86-2.
The asterisks indicate the two S,S optically

active centers.
1.2 Appearance, Color, Odor
Captopril is a white to off-white crystalline
powder with a slight mercaptan odor.
2. History
The captopril story began in 1971 with a
report (1) on the isolation and synthesis of
teprotide, an antihypertensive nonapeptide from
the venom of a Brazilian Pit Viper. This venom
peptide was hypotensive through inhibition of an
exopeptidase, known as the angiotensin converting
enzyme (ACE). The latter performs dipeptide
scission at the carboxyl end of the decapeptide,
angiotensin I to yield angiotensin 11, the most
powerful natural vasoconstrictor known. ACE
further potentiates hypertension through
scission-inactivation of the nonapeptide
vasodilator, bradykinin. Clinical investigations
with both teprotide (2) and captopril ( 3 ) have
implicated ACE as the key enzyme in human
hypertensive diseases.
The first ACE inhibitor shown to be
clinically efficacious against hypertension was
the synthetic venom peptide, teprotide ( 2 ) .
CAPTOPRlL 81
However teprotide was expensive and only effective

parenterally. A simpler, orally effective ACE
inhibitor was desired.
Analogs of the snake venom hypotensive
peptides were quantitatively rated for their in
vitro inhibition of ACE (4) and for their effect
o n e contractile properties of guinea pig ileum
( 5 ) . The ratings aided construction of a
hypothetical active site for ACE (Figure 1). This
model was based primarily on the similarity of the
enzymatic properties of ACE to those of
carboxypeptidase A (CASE A) even though the latter
yields amino acids rather than the dipeptides of
the former. Further, the active site of CASE A,
like ACE contains zinc but unlike that of ACE had
been structurally characterized, in the
crystalline state, by x-ray diffraction (6). Thus
the elaboration of the hypothetical ACE active
site and the possibility of a simple ACE inhibitor
were stimulated by a 1973 report (7) on,
D-2-benzylsuccinic acid, a simple inhibitor, of
CASE A.
Specific points of inhibitor attachment
within the real CASE A site were extrapolated to
that within the hypothetical ACE site ( 8 ) . On the
basis of these extrapolations, synthesis of simple
ACE inhibitors were initiated in 1974. As each of
a great number of candidates was quantitatively
rated in the aforementioned --
in vitro screens
(previously developed for the venom peptide
studies), the hypothetical site was verified and
refined. In the refined model schematically
represented in Figure 1, three dimensional amino
acid configurations within the catalytic site
provided suitably spaced subsites or multiple
points of attachment to substrate or inhibitor.
The semi-circular clefts in the figure represent
hydrophobic subsites which may interact with the
lipophilic side chains of inhibitor or substrate.
The latter are also putatively bound to the ACE
site via the X-H group at their nonscissile
terminal peptide bond. As indicated, one of the
first simple ACE inhibitors was patterned after
all of the venom peptide inhibitors in sharing a
terminal proline (succinyl-L-proline). The choice
of proline was also influenced by the superior
biological stability exhibited by teprotide with
82 HAROLD KADIN
RELATIVE
ENZYME INHIBITOR IN VlTRO INHIBITION
CAABOXYPEPTIDASE A
D-2-EENZYLSUCCINIC ACID
-7 FH2 ?-
0 = CCH,-CH-C -C = 0
ANGIOTENSIN-CONVERTING ENZYME
SUCC1NYL.L-PROLINE 1
D-2-METHYLSUCCINYL.L-PAOLI" 15
CAPTOPRIL 14000
F i g . 1. Key steps i n t h e d e s i g n o f a s p e c i f i c i n h i b i t o r of
t h e a n g i o t e n s i n c o n v e r t i n g enzyme.
CAPTOPRIL 83
its two terminal prolines (9). In an emulation of

D-2-benzylsuccinic acid, but with appropriate
lengthening of the chain, the nitrogen and
carbonyl of the penultimate "venom peptide" amide
linkage were substituted, respectively, with a
peptidase-inhibitory methylene and a highly
anionic, zinc binding carboxyl. As indicated in
Figure 2 the analogy was then extended to the
better snake venom inhibitors bearing a
penultimate alanine in addition to the terminal
proline (D-2-methylsuccinyl-L-proline) . The
largest increase in inhibitory activity (about 14
thousand fold over succinyl-L-proline) was
obtained when the zinc binding carboxyl was
replaced with a thiol which has considerably
greater affinity for zinc. The snake venom
peptide, teprotide, does not have comparable
affinity for zinc, however its binding is enhanced
by additional interactions of its amino acid side
chains with "clefts" on the enzyme beyond the
active peptidase site (10).
In short, the orally active antihypertensive
drug, captopril, was announced to the scientific
community in 1 9 7 7 (11). It was made generally
available for treatment of hypertensive diseases
in 1 9 8 1 (12,841. Clinical investigations (13)
suggest it is also highly efficacious in the
treatment of congestive heart failure. Its
uniquely designed highly specific affinity for the
active site of ACE has resulted in a high ratio of
clinical success with a relatively low index of
side effects. It has been effective where
conventional antihypertensive therapies fail or
have an untoward number of side effects.
84 HAROLD KADIN
3. Synthesis
A process (14) is presented (Figure 2) in a
chemical reaction sequence which follows this
brief description.
Methacrylic acid (I) is condensed with thio-
lacetic acid (11) to give racemic 2-methyl-3-
acetylthiopropionic acid (111). L-proline is then
acylated with the acid chloride (IV) of the thio-
ester (111). The resulting proline thioester (VI)
is resolved from its - - isomer by aqueous cry-
R,S
stallization. Saponification of compound VI with
sodium hydroxide affords the sodium salt of
captopril which after acidification yields
captopril (VII) .
4.11 Infrared Spectra
The infrared spectrum of captopril in
chloroform is presented in Figure 3 and as a KBr
pellet in Figure 4. The infrared spectrum in the
latter indicates the presence of the following
frequencies and their structural assignments (15).
cm-1 Assignment
1750 C = 0 (COOH group)
1725
1640 C = 0 (amide)
2560 S - H
DiffereQfes in the fingerprint regions
(1350-900 cm ) of the mineral oil mull infrared
spectra of batches 3 and 4 (Figure 5 and 6,
respectively) indicate that the low melting batch
3 and the high melting batch 4 are polymorphs (see
Section 4.21) .
CAPTOPRIL 85
Figure 2
Chemical Reaction Schematic Diagram

I + I1 I11
CH2 = CCOOH
MW = 86.01
AcSH
MW 76.11
+ AcSCH2CHCOOH
MW = 162.20
-+
Methacrylic Thiolacetic Acetylthioisobutyric
IV
AcSCH2CHC
\ COOH
c1 COOH
MW = 180.65 MW = 115.13 MW = 259.32
Acid Chloride" L-Proline Proline Thioester
VI VI I
"'?
H3C 0
AcSCH2CHCN
COOH
d)
COOH
Proline Thioester MW = 217.28
Captopril
0
Ac = CH3C-
II
(a) Reflux (b) SOCl , DMF, Distillation
(c) H 0 + NaHCO Ci C12 Wash, HC1,
ci!ystallizaiG.on 41.
(d) H20 + NaOH, HC1, CH2C12 extraction
0
Ll
0
rl
c
u
c
.ti
a
k
ld
a
c
fd
4J
ul
a,
ro
5
0
X
d
.ti
Ll
a
0
4J
a
Id
u
w
0
5
Ll
LI
i,
a,
m
a
a c
a 0
4J
c
m
Q)
k
4J
ro
c
H
86
4J
a,
rl
r(
a,
PI
k
m
M
c
-4
ak
ld
a
c
ld
&
a,
[o
7
0
X
Li
a
0
4J
PI
ld
u
u-l a,
0 a
2
k 5
4J k
0
a,
a
2
rl
v) wI
a c
a, 4
k 2
id k
k a,
44 PI
G
H
..
lJ
k
&
(I)
c
H
WAVELENGTH (MICRONS)
3500 2500 2Ooo lsbo 1600 1400 1200 200

FREQUENCY (CM-’)
F i g u r e 5. I n f r a r e d S p e c t r u m of C a p t o p r i l , B a t c h 3 , Mineral O i l M u l l
Instrument: Perkin-Elmer, Model 6 2 1

WAVELENGTH (MKRONS)
F R E w € w (CM-')
Figure 6. Infrared Spectrum of Captopril, Batch 4, Mineral Oil Mull
Instrument: Perkin-Elmer, Model 621

90 HAROLD KADIN
4.12 Nuclear Magnetic Resonance Spectra

1
The 270 MHZ H-NMR spectrum of captopril in
CDCl is shown in Figure 7. The spectrum was
obtained from the University of Chicago courtesy
of Professor Josef Fried. Spectral assignments
are shown in Table 1.
The 13C-NMR of captopril in CD OD is shown in
Figure 8. The spectrum was obtained on a Varian
Associates XL-100 NMR spectrometer equipped with a
Nicolet TT-100 data system. Major peaks are
assigned in Table 2 . Minor peaks arise from the
presence of cis-trans isomerism at the amide bond
(16).
Table 1
Proton-NMR Data for Captopril
Proton Chemical Shift ( 6 )'PPM from TMS (ext.
COOH 9.81 (s)
CL-CH 4.60 (m)
B-CH2 2.03 (m)i 2.25 (m)
Y-CH,L 2.07 (m)
6 -CH2 3.63 (m)
9
-CH-C 2.44 (d,q) J=6,9
-STHA 2.82 (m)
-CH3 L 1.17 (d) J=6
SH 1.53 (ad) J=9,8
' multiplicities: d=doublet; q=quartet;
m=multiplet. J=proton-proton coupling
constants in Hertz.
0
F
CJ
I
X
5:
k
a,
x
ZJ
..k
m
4J
c
$
k
4J
(II
c
H
4 i
f *
0
0
4
I
I 4
X
c
a
-d
L!
rd
3
..
c,
d
$
k
4J
m
c
H
92
CAPTOPRIL 93
Table 2
Carbon-13 NMR Data f o r C a p t o p r i l i n CD30D.
Carbon # Chemical S h i f t ( 6 )
1 ppm from TMS
175.69
59.84
30.03
25.49
48.24
174.91
43.1
17.07
28.1
R e f e r e n c e d from c e n t e r peak o f t h e C D 3 0 D
m u l t i p l e t a t 4 9 . 0 ppm
4.13 Ultraviolet Spectra
S p e c t r a of c a p t o p r i l i n a q u e o u s m e t h a n o l ,
( F i g . 9 ) w a t e r , 0.1M sodium h y d r o x i d e and 0 . 1 M
h y d r o c h l o r i c a c i d ( F i g . 101, a r e p r e s e n t e d (17).
These s p e c t r a d e p i c t a n end a b s o r p t i o n s l o p e
w i t h o u t peak o r s h o u l d e r . The s l o p e s p e c t r u m i n
0 . 1 M sodium h y d r o x i d e was s h i f t e d c o n s i d e r a b l y
towards h i g h e r wavelengths. S i n c e weak s u l f h y d r y l
a b s o r p t i o n i s r e p o r t e d ( 1 8 ) i n t h e 220-230 nm
r e g i o n , t h i s a b s o r p t i o n s h i f t may b e d u e t o
i o n i z a t i o n o f t h e s u l f h y d r y l f u n c t i o n by t h e
a l k a l i . This s h i f t towards higher wavelengths
w i t h i n c r e a s e i n pH h a s been u s e d by O n d e t t i ( 1 9 )
t o d e t e r m i n e t h e pK o f t h e s u l f h y d r y l i n
captopril (Section 8.32).
HAROLD KADIN
. . . . . .
I ; 0 1 . . . . . . . . . . .0,
. !. !. ..
I '
. . ./ i ! .a,* , , , 4 , -
Fig. 9. U l t r a v i o l e t absorption s p e c t r a of C a p t o p r i l i n
10%aqueous methanol s o l u t i o n .
Instrument: Cary 15.
Fig. 10. U l t r a v i o l e t absorption s p e c t r a of C a p t o p r i l i n

H20, 0.1E H C 1 and 0.1N NaOH
Instrument: Cary 15
CAPTOPRIL 95
The spectra suggest that there is a maximum

at about 200 nm, attributable to the thiol
function. However, precise determination of the
peak absorbance was difficult because the
extremely large blank absorbances prevented
maintenance of a stable balance at this
wavelength. Consequently the peak maximum is
uncertain in Figure 9. However the peak
absorbance is valid in Figure 15 in which the
solvent was an HPLC mobile phase (System 4 - Table
8 - Section 6.32).
4.14 Mass Spectrum

The mass spectral pattern indicated in Figure
12 was obtained (20) on an AEI MS-9 Mass
Spectrometer.
The fragmentation, responsible for the spec-
trum in Figure 12+ is depicted schematically in
Figure 11. The M of m/z 217 and the other frag-
ments are consistent with a sulfur-containing
compound of the composition C 9 H1 5NO 3 S (Section
1.1).
Fiaure 11
Mass Spectral Fragmentation Schematic Captopril
170 103
J
172+m/z
H
m/z 70
m/z 199 + H20
M+ F
m/z 2 1 7 , 1 m / z 1{3
m/z 202 + CH3
+ C02 r m / z 140 + SH
b m / z 126 + CH2SH
I
L m / z 171 + S C H 2 4 m/z 127 + C02
96 HAROLD KADlN
6640 SQ14225 BFlTCH ttNNQ23NB
90.-
80-
>
F
U
II)
78.-
7
Z
W
t-
Z
H
50
W
>
H
t-
a
_I
w
L l
Figure 12. Mass Spectrum of Captopril
Instrument: AEI MS-902 Spectrometer Equipped with

Frequency Modulated Tape Recorder,
Spectrum Processed o n Digital
Equipment Corporation PDP-11 Computer
CAPTOPRIL 97
4.2 Solid State Properties

4.21 Polymorphism
An unstable, low (86OC) melting and a stable,
high (106OC) melting form of captopril have been
observed. These forms exhibited different unit
cells (Section 4.26) on single crystal X-ray
examination, differences in their powder X-ray
(Section 4.27), and differences in the solid state
infrared spectra (Figures 5 and 6). Agreement of
their optical rotations, infrared in solution and
bioassays established them as polymorphs.
4.22 Differential Thermal Analysis
(D.T.A. 1
DTA of the high melting polymorph ( 2 2 )
yielded a sharp, well-defined endotherm at 106OC
whereas the low melting polymorph produced a sharp
endotherm at 86OC. When the low melting polymorph
was allowed to resolidify and the DTA repeated,
the endotherm at 86OC had disappeared and an
endotherm at 106OC appeared. The latter suggests
that the high melting form is the stable
polymorph. A DuPont 900 Thermoanalyzer programmed
for a temperature rise of 15' per min was utilized
for these thermograms.
4.23 Melting Range
The U.S.P. (Class 1) melting range for the
high melting polymorph was 105.2 - 105.9' ( 2 1 ) .
This agrees well with its D.T.A. endotherm of
106OC. The low melting polymorph has a melting
range of 87-88', in agreement with its DTA
endotherm of 86O.
4.24 Differential Scanning Colorimetry
(D.S.C.)
Use of DSC as a purity index for captopril is
supported by titrimetric assays (17) of the
carboxyl function (alkalimetry) and of the
sulfhydryl function (iodimetry). For instance for
batch 4 these yielded 99.6% for the carboxyl and
99.2% for the sulfhydryl in very good agreement
with the DSC of 99.7% mole 8 ( 2 2 ) .
98 HAROLD KADIN
4.25 Hygroscopidity
Under ordinary conditions captopril is not
hygroscopic. Equilibrium moisture studies (23)
indicate no moisture pickup by captopril up to 50%
relative humidity at room temperature. Above 50%
R.H. it shows a tendency to cake after one to two
days.
Captopril did not exhibit any visual physical
changes and remained dry from 0 to 67% R.H. on
exposure for 14 days. Samples exposed to 81% R.H.
for 14 days appeared moist (24).
4.26 Single Crystal X-ray Diffraction
Single crystal X-ray analyses have been
completed (25) for both the low (melting range
86-87OC) and high (melting range 105-106O) melting
polymorphs. Both forms are orthorhombic with the
following crystal data:
A. High melting polymorph -
a = 6.834(2), -b =
0
" 3 space group
8.821(2), c = 17.982(4)A; V = 1084A,
P2 2 2 wiFh four molecules3per unit cell; cal-
culakeh density = 1.33 gcm- .Refined to R = 0.04
for the 745 observed single crystal intensities.
B. Low melting polymorph - -=
a = 9.496(3), b
0
03 space group
12.304(3), c = 19.282(5)A; V = 2253A;
P2 2121 witF eight molecules egr unit cell;
calculated density = 1.28 gcm .
Refined to R =
0.06 for the 1093 observed single crystal inten-
sities.
The structure in both has the S,S absolute
configuration with a 2 ( T r a n s ) conformation about
the N-C(0) amide bond (the O-C-N-C(2) dihedral
angles vary from -4 to +6O). The molecular
conformation differ in detail, most notably in the
conformation about the (S)C-C(C0) bond.
Atomic coordinates relative to orthogonal
axes for the high melting form are:
CAPTOPRIL 99
S -7.010 1.595 -5.766

N1 -6.499 -1.729 -1.938
c2 -5.799 -2.755 -1.152
c3 -6.274 -2.492 0.282
c4 -7.067 -1.228 0.252
c5 -7.511 -1.024 -1.144
C6 -6.188 -4.132 -1.626
06A -7.165 -4.364 -2.286
06B -5.394 -5.106 -1.204
c7 -6.215 -1.576 -3.227
07 -5.3 08 -2.253 -3.754
C8 -7.041 -0.624 -4.050
c9 -6.163 0.238 -4.947
c10 -8.052 -1.432 -4.821
It was predicted that salt formation with
resultant dissociation to a carboxylate anion
would influence Capoten to crystallize in its less
common E ( c i s ) conformation. This prediction was
tested (25) by performing single crystal analysis
on the dicyclohexylamine salt of captopril. The
analysis indicated that the salt was indeed in the
E conformation, i.e. the carbonyl groups of the
amide and carboxyl functions are cis to each
other.
4.27 Powder X-ray Diffraction
The stable, higher melting polymorph and the
lower melting, metastable polymorph are shown in
the powder X-ray patterns Figures 13 and 14
respectively (26). The values given in the
patterns are also listed in Tables 3 and 4 for the
high and low melters respectively. The tables
also show the relative intensities (based on peak
areas) of the various peaks.
A powder X-ray pattern taken on the low
melting polymorph after it was heated to about
95OC showed conversion to the stable form.
The X-ray pattern was taken with copper Ka,
nickel filtered X-radiation.
a k
a a,
u 4J
a,
w E
0 0
100
0
PI
P
I
L '
I
!
I
i
i
101
Table 3
Powder X-Ray Diffraction Data f o r Figure 13 (High Melting Polymorph)
2-13 (DEG.) D(ANGSTROMS) PEAK REL. PEAK AREA REL. AREA
9.99 8.85 22.7 0.208 88.1 0.270
11.35 7.80 37.9 0.347 138.4 0.424
14.24 6.22 15.2 0.139 122.3 0.374
16.45 5.39 21.0 0.192 77.5 0.237
17.21 5.15 57.1 0.523 169.6 0.519
17.98 4.93 47.5 0.435 166.8 0.511
19.25 4.61 34.4 0.315 146.3 0.448
19.85 4.47 109.1 1.000 324.5 0.994
20.78 4.27 37.7 0.346 130.1 0.398
22.23 4.00 49.8 0.456 161.7 0.495
24.52 3.63 24.1 0.221 86.4 0.265
25.03 3.56 15.3 0.140 60.2 0.184
25.97 3.43 62.6 0.574 326.6 1.000
26.56 3.36 13.7 0.126 68.2 0.209
28.26 3.16 68.7 0.630 243.1 0.744
29.79 3.00 10.6 0.097 102.0 0.312
30.81 2.90 8.4 0.077 42.5 0.130
31.66 2.83 9.1 0.083 43.9 0.134
33.45 2.68 8.3 0.076 47.9 0.147
34.28 2.61 17.9 0.164 98.2 0.301
Table 3 (Continued)
~ - ~ ( D E G . ) D(ANGSTROMS) PEAK REL. PEAK AREA REL. AREA
36.17 2.48 14.8 0.136 62.3 0.191

36.34 2.47 13.3 0.122 37.5 0.115
37.53 2.40 9.3 0.085 64.9 0.199
38.63 2.33 15.3 0.140 73.3 0.224
Sorted Data (Highest Peak First)
z-O(DEG.) D(ANGSTR0MS) PEAK REL. PEAK AREA REL. AREA
19.85 4.47 109.1 1.000 324.5 0.994

28.26 3.16 68.7 0.630 243.1 0.744
25.97 3.43 62.6 0.574 326.6 1.000
17.21 5.15 57.1 0.523 169.6 0.519
22.23 4.00 49.8 0.456 161.7 0.495
17.98 4.93 47.5 0.435 166.8 0.511
11.35 7.80 37.9 0.347 138.4 0.424
20.78 4.27 37.7 0.346 130.1 0.398
19.25 4.61 34.4 0.315 146.3 0.448
24.52 3.63 24.1 0.221 86.4 0.265
9.99 8.85 22.7 0.208 88.1 0.270
Table 4
Powder X-Ray Diffraction Data f o r Figure 1 4 (Low Melting Polymorph)
2 - 8 (DEG.) D (ANGSTROMS) PEAK REL. PEAK AREA REL. AREA
8.71 10.15 24.5 0.392 68.7 0.259

9.31 9.50 13.7 0.219 26.3 0.099
11.77 7.52 14.3 0.229 89.1 0.336
12.71 6.96 14.0 0.224 43.0 0.162
13.13 6.74 16.6 0.266 69.4 0.261
15.00 5.91 31.8 0.509 142.4 0.536
17.21 5.15 14.7 0.235 43.2 0.163
17.89 4.96 14.0 0.224 36.8 0.138
18.23 4.87 39.5 0.632 101.0 0.380
18.40 4.82 34.8 0.557 77.2 0.291
19.51 4.55 16.9 0.270 91.2 0.343
20.10 4.42 13.1 0.210 57.3 0.216
20.61 4.31 30.6 0.490 112.0 0.422
20.87 4.26 13.1 0.210 34.8 0.131
21.38 4.16 13.0 0.208 33.8 0.127
22.14 4.02 62.5 1.000 265.5 1.000
23.25 3.83 13.9 0.222 69.1 0.260
23.50 3.79 12.1 0.194 34.4 0.130
24.10 3.69 16.8 0.269 113.4 0.427
24.86 3.58 12.5 0.200 43.4 0.163
25.20 3.53 17.4 0.278 83.3 0.314
Table 4 (Continued)
~-WDEG.) D(ANGSTROMS) PEAK REL. PEAK AREA FEL. AREA
25.88 3.44 17.8 0.285 105.0 0.395

27.33 3.26 14.6 0.234 90.5 0.341
28.35 3.15 12.8 0.205 33.3 0.125
28.94 3.09 14.4 0.230 104.1 0.392
30.05 2.97 14.0 0.224 72.8 0.274
34.47 2.60 16.6 0.266 99.3 0.374
Sorted data (Highest Peak First)
2-B(DEG.) D(ANGSTR0MS) PEAK REL. PEAK AREA REL. AREA
22.14 4.02 62.5 1.000 265.5 1.000

18.23 4.87 39.5 0.632 101.0 0.380
18.40 4.82 34.8 0.557 77.2 0.291
15.00 5.91 31.8 0.509 142.4 0.536
20.61 4.31 30.6 0.490 112.0 0.422
8.71 10.15 24.5 0.392 68.7 0.259
25.88 3.44 17.8 0.285 105.0 0.395
25.20 3.53 17.4 0.278 83.3 0.314
19.51 4.55 16.9 0.270 91.2 0.343
24.10 3.69 16.8 0.269 113.4 0.427
34.47 2.60 16.6 0.266 99.3 0.374
13.13 6.74 16.6 0.266 69.4 0.261
17.21 5.15 14.7 0.235 43.2 0.163
106 HAROLD KADlN
4.3 Solution Data

4.31 Solubility
Captopril at 25OC is freely soluble (1 to 10
parts solvent to 1 part solute) in water,
methanol, ethanol (SD3A), isopropanol, chloroform,
or methylene chloride. However, it is only
soluble (10-30 parts solvent to 1 part solute) in
ethyl acetate (27). The solubility of captopril
in water, at 250CI is 160 mg/ml (28). A
solubility-temperature profile of captopril in
water obeyed a linear equation up to 4OoC (28).
Beyond this temperature captopril showed extra-
ordinarily high water solubility.
Solubility in sesame and corn oils was less
than 1 mg/ml at 25OC, whereas the solubility in
the synthetic oil triacetin (glyceryl triacetate),
at 25OC, was greater than 20 mg/ml (29).
4.32 pKa
-
The pK of the carboxyl of captopril (pK ) is
reported (23) to be 3.7. Whereas a carboxyl break
was readily observed with alkali potentiometry,
the sulfhydryl break could not be detected (17).
Therefore, the pK of the sulfhydryl in captopril
(pK ) was not estfmated by classical potentio-
metgy. It was, however, estimated at 9.8 (pK2) by
Ondetti (19) and Weiss (30) using sulfhydryl U.V.
shifts to higher wavelengths with increase in pH
(Section 4.13). The method utilized was adapted
from Benesch and Benesch (31).
4.33 Metal Complex Formation
Captopril was modelled (11) as a selective
and competitive inhibitor of the angiotensin
converting enzyme (Section 2 ) . Part of this
inhibition resides in the binding of the zinc
cofactor within the enzyme's active site by
captopril's thiol function. Constrained within
the active site by the multiple interactions of
site and inhibitor, captopril bars entry of
angiotensin I and thus prevents its conversion to
the most powerful natural pressor, angiotensin 11.
Since captopril lacks an amino group, it does not
CAPTOPRIL 107
complex metals in solution with the well-docu-

mented avidity (32,33) of amino group bearing
thiols like cysteine, glutathione, and in vivo
metal depletors like 2 - m e r c a p t o p r o p i o n y ~ g l y c i n e
and penicillamine. Indeed, Weiss (30) reported
that an alkali potentiometric study of the extent
of zinc ion complexation with cysteine (I), 2-
mercaptopropionyl glycine (11), and captopril
(111) indicates the order of binding, at pH 7.4,
to be I > I1 > 111.
Captopril binds mercuric ion (Section 7 ) to
block its colorimetric reaction with Ellman's
reagent, thus allowing a measurement of
non-sulfhydryl colorimetric interferences.
4.34 Optical Rotation
The optical rotation of the captopril in
absolute ethanol (34), using the Perkin-Elmer 155
Automatic Polarimeter, was determined to be: a
= -127.8'. The -
R,S
- isomer rotates at about +5'?
4.35 Partition Coefficients
A partition ratio (solvent/aqueous) after shaking
equal volumes of cosaturated aqueous (pH 2) and
methylene chloride was 1.39 ( 2 1 ) . A comparably
determined partition ratio between equal volumes
of cosaturated 0.1M HC1 and octanol was 1.9 (30).
When utilizing salcing out partition from aqueous
acid into methylene chloride at the captopril
concentrations prevalent in the urine analysis
(about 25-50 mcg/ml for a 100 mg dose) NaCl but
not Na2S04 was found to enhance captopril
oxidation (Section 7). This has been attributed
to trace chlorine generation from acidic chloride
plus oxygen.
5. Stability
5.1 Solid State Stabilitv
No significant decomposition was detected
(35) in SQ 14,225 bulk samples, stored at +5'c,
+33'C and +5OoC f o r up to 6 months or exposed to
900 foot-candles in a light box f o r 30 days, when
compared to -2O'C samples which served as the
108 HAROLD KADIN
c o n t r o l . Samples were examined f o r a p p e a r a n c e ,

c o l o r , o d o r , LD s a f e t y and by q u a n t i t a t i v e TLC
and IlPLC , i o d i m 2 p r i c t i t r a t i o n , i n f r a r e d , and
optical rotation.
5.2 Solution S t a b i l i t y
C a p t o p r i l i n aqueous s o l u t i o n undergoes an
oxygen f a c i l i t a t e d , f i r s t o r d e r , f r e e r a d i c a l
oxidation a t its t h i o l to yield captopril
d i s u l f i d e ( 2 8 ) . H y d r o l y s i s a t t h e amide l i n k a g e
o c c u r s o n l y u n d e r f o r c i n g c o n d i t i o n s (see S e c t i o n
5 . 2 5 ) . O x i d a t i o n w a s d e l a y e d by a d j u s t m e n t t o
lower pH, a d d i t i o n o f c h e l a t i n g a g e n t s , i n c r e a s i n g
captopril concentration, u t i l i z a t i o n of nitrogen
o r l o w oxygen h e a d s p a c e , and i n c o r p o r a t i o n o f
a n t i o x i d a n t s . O x i d a t i o n seems t o o c c u r l e s s
r e a d i l y i n methanol ( 3 6 ) . N o d e g r a d a t i o n o f
c a p t o p r i l w a s o b s e r v e d ( 4 0 mcg/ml) i n t h i s s o l v e n t
f o r up t o 2 weeks a t 5OC.
5.21 S t a b i l i t y and S o l u t i o n pH
O x i d a t i o n r a t e c o n s t a n t s a t v a r i o u s pli v a l u e s
( 2 8 ) i n Table 5 , s u g g e s t t h a t c a p t o p r i l i s
o p t i m a l l y s t a b l e below pH 3.5, t h e o x i d a t i o n r a t e
b e i n g e s s e n t i a l l y c o n s t a n t from pH 2 t o 3. The
r a t e c o n s t a n t s i n c r e a s e r a p i d l y above pH 4 . Using
HPLC and c o l o r i m e t r y ( 3 8 ) , c a p t o p r i l a q u e o u s
s t a b i l i t y w a s s t u d i e d , a t 50 mcg/ml, i n a r o t a t i n g
b a s k e t d i s s o l u t i o n a p p a r a t u s f o r up t o 1 8 0 m i n u t e s
a t 37OC i n d i s t i l l e d water, and a t pH 1, 2 and 3 .
E x c e l l e n t s t a b i l i t y a t pH 1 and 2 b u t a p p r e c i a b l e
d e g r a d a t i o n a t pH 3 , and i n d i s t i l l e d w a t e r w a s
observed. S u r p r i s i n g l y , t h e r a t e of d e g r a d a t i o n
a t pH 3 exceeded t h a t i n d i s t i l l e d water. The
more r a p i d o x i d a t i o n a t pH 3 w a s a t t r i b u t e d t o
c a t a l y s i s v i a g r e a t e r t r a c e m e t a l s o l u t i o n from
the dissolution baskets.
CAPTOPRIL 109
Table 5
Oxidation Rate Constant for Captopril (5 mg/ml)
in Citrate-Phosphate Buffers at Various pH
Values at 5OoC
PH
(day ) x 10f
Rate-Eonstan
2.13 8.38
2.59 9.01
2.89 8.22
3.13 8.31
3.53 9.92
3.88 9.13
4.23 12.94
4.67 19.43
5.16 28.93
5.63 42.03
5.22 Solution Stability, Metal Ions
and Chelating Agents
Transition metal ions most effectively cata-
lyze oxidation of captopril through a recycling of
oxygen free radicals (28). The most effective of
these catalysts are ubiquitous copper and iron, in
given order. As little as 1 ppm of copper has
been observed to catalyze captopril oxidation in
solution (28).
As has been demonstrated with cysteine (39)
lower levels of disodium edetate (EDTA Na2) may
enhance metal ion catalyzed thiol oxidation,
whereas higher levels retard oxidation.
Disodium edetate 0.1% (Na2EDTA 0.1%) best
stabilized 0.5 mg captopril per ml (of
citrate-phosphate buffer at pH 4, p = 0.5) in
Teflon-faced rubber sealed vials (37).
Analysis of urinary captopril was necessary
for dosage form bioavailability and dose titration
studies. The necessity for long term storage of
samples prior to analysis required development of
an acid-chelate stabilization (40). This
stabilization utilized diethylenetriamine
pentaacetic acid (DTPA) reputed (40) to be a more
effective metal chelator than Na2EDTA. A
110 HAROLD KADIN
relatively large amount of DTPA (about 4 0 0 mg in 5

ml) was mixed into the periodic urine voiding.
This was followed by an acidification to about pH
2 with a mixture of citric and oxalic acids and a
rapid refrigeration. DTPA, citric and oxalic have
been reported ( 4 1 ) to be effective sequestering
agents in the pH range 2 to 5 for Al, Cu, Fe, Ni
and Zn. Analysis of captopril in urine samples
stabilized in this manner, before and after 90-120
days of refrigeration, agreed remarkably well(40).
5.23 Concentration and Solution
Stability
The greater the captopril concentration, the
slower the oxidation ( 2 8 ) . For example, no signi-
ficant degradation (within + 3 % ) could be detected
by automated Ellman colorimgtry ( 4 2 ) at a concen-
tration of 2 5 0 mg captopril per ml of solution at
pH 1 2 . 5 - 1 4 after overnight room temperature
storage in uncovered beakers. At the opposite
extreme, captopril at an analysis concentration of
2 5 - 5 0 micrograms per ml of solution at pH 1 3 . 5
stored overnight at room temperature in open tubes
lost 8 4 % of its sulfhydryl activity ( 2 1 ) .
5.24 Oxygen Tension and Solution
Stability
The following 2 1 / 2 hour accelerated
stability at 4OoC on 2 5 ml of 1 mg captopril per
ml solutions in unstoppered 100 ml volumetric
flasks using air, oxygen, or nitrogen purging,
where indicated, was carried out ( 2 8 ) .
Table 6
Oxygen Tension and Solution Stability
M PPm 8 Captopril
Code Na2HP04 PH Cu Purge Recovered (HPLC)
1 0.08 7.9 2 None 73
2 0.08 7.9 2 Air 58
3 0.16 7.5 10 None 88
4 0.16 7.5 10 Air 62
5 0.08 7.8 2 N2 96
6 0.04 7.8 1 O2 0
(KH2P041
CAPTOPRIL 111
Under these conditions it is apparent that

oxygen causes rapid, complete degradation, air
facilitates degradation, and nitrogen protects
captopril in solution.
5.25 Amide Hydrolysis and Solution
Stability
Captopril solutions at 5 mg per ml in 0 . 5 M
-
HC1, containing 0.1 mg EDTANa /ml to minimize
oxidation , were heated at elegated temperatures.
The rate of hydrolysis was monitored by HPLC. The
data yielded first order linear plots from which
Wang (43) calculated rate constants (Table 7). An
Arrhenius plot yielded a heat of activation for
amide hydrolysis of 21.4 kcal/mole, comparable to
other amides.
Table 7
Rate Constants for Captopril Hydrolysis
-
k t90%
5.5 x lo,*-7 hr-l
-1 10 years
4OoC PH 3
4OoC PH 4 5.5 x hrml 10 years
22oc PH 3 6.7 x 10-lOhr -1 86 years
22oc PH 4 6.7 x 10 hr 86 years
It is clear from these t ' s (times for 90% amide
hydro1ysis1 , that hydro188f s contributes insigni-
ficantly to degradation.
112 HAROLD KADIN
6. Analytical Tests and Methods

6.1 Elemental Analysis
The following results were obtained:
Element Calculated Found for Batch 5
C 49.78 49.97
H 6.96 6.84
N 6.45 6.50
S 14.77 14.63
6.2 Spectrophotometric Methods

6.21 Colorimetry
Spectrophotometry for captopril has included
the widely utilized Ellman's reaction (44) in
which sulfhydryl reduction of 5,5'-dithiobis-2-
nitrobenzoic acid yields a mole of intensely
yellow 2-nitro-5-thiobenzoate anion per mole of
captopril. Manual (40,451 and automated colori-
metries have been utilized for captopril analysis
in chows (Section 7.71), urine (Section 7.72) and
in pharmaceutical formulations by TLC (Section
6.31).
Kinetic colorimetry vs pH (from pH 5 to 10)
studies (46) of a manual version of Ellman's
colorimetry of captopril established that the pH
optimum was at pH 7 (1 M phosphate, 0.05 M EDTA) .
Maximum color was attained within 2 min. and
stability was maintained for at least 45 min.
The S-nitroso-Bratton-Marshall colorimetry
(47) has been applied to analysis for captopril in
various formulations (48). It was adapted from a
method (47) for cysteine in biological materials.
In this method the thiol reacts with nitrous acid
to form a relatively stable S-nitroso derivative.
Excess nitrous acid is destroyed by sulfamic acid.
The S-nitroso derivative is then hydrolyzed by
mercuric ions to release nitrous acid. The latter
diazotizes sulfanilamide, presumably at a faster
rate than destruction of the nitrous acid by
CAPTOPRIL 113
excess sulfamic acid. The diazotized

sulfanilamide is then coupled to
N-(1-naphthy1)ethylenediamine to yield a stable
measureable red azo dye. The method was found to
be less suitable for analysis of captopril in
urine than Ellman's Reaction (Section 7.72).
Five simple, spectrophotometric identity
tests for captopril reported by Valatin (49)
include the observation of an evanescent red when
captopril reacts with nitrous acid. In addition
captopril color tests were described yielding
purple with nitroprusside, blue with ferric
chloride, red on addition of neutral N-ethylmale-
imide followed by strong alkali, and finally
orange-yellow (specific for proline) on acid
hydrolysis followed by neutralization then reac-
tion with ninhydrin.
6.22 Fluorometry
Captopril was reacted with
N-[p-(2-benzoxazoyl)- phenyl] maleimide in pH 6.85
buffer and the fluorescence of the
captopril-maleimide adduct was then measured at
310 nm excitation and 365 nm emission (49).
For coupling of captopril to

N-(7-dimethylamino-4-methyl coumarinyl) maleimide
to yield a highly fluorescent derivative see
Section 7.5.
6.3 Chromatosraphic Methods
6.31 Thin Layer Chromatography (TLC)
For quantitation of captopril (purity assay)

standard and sample solutions are each
chromatographed at 100 pg and 200 pg levels on
Analtech silica gel G plates using the solvent
system benzene-acetic acid (75:25). The captopril
zones on the plate are located by spraying a
guide-channel with a basic methanolic solution of
5,5'-dithiobis-2-nitrobenzoic acid. Captopril on
the guide-channel appears as a yellow zone. The
silica gel containing captopril zones on the
untreated portion of the plate are removed from
the plate, eluted with 5% aqueous trichloroacetic
acid, and reacted with a methanolic solution of
5,5'-dithiobis-2-nitrobenzoic acid, at an alkaline
114 HAROLD KADIN
pH, to form an intensely yellow 2-nitro-5-

thiobenzoate anion that is measured at 412 nm in a
spectrophotometer (50).
In a thin-layer semi-quantitative procedure
for measuring the individual impurities in
captopril, samples are chromatographed at 100 pg
and 200 pg levels and standards are
chromatographed at concentrations ranging from 0.5
pg to 4.5 pg. TLC separation is carried out on
Analtech Silica Gel G plates, developed in
conventional and in continuous development
chambers, using the solvent system benzene-acetic
acid (75:25). After development, the plates are
air dried and placed in a chamber saturated with
iodine vapors. A semi-quantitative estimate of
the concentration of each impurity in the sample
is made based on a visual comparison of the size
and intensity of each impurity zone with the
appropriate standard zones (50).
These TLC procedures, after appropriate
initial extractions, have been adapted for
formulations. They have also been applied to
semi-quantitative analysis of captopril disulfide
in tablets and as a TLC identity test for
captopril in tablets (50).
Streaked or spotted plates should be
introduced into the developing chamber (2 per
chamber) immediately after applying the solutions
to the plates. This minimizes the conversion of
captopril to its disulfide.
Placebo powders were spiked with known
amounts of captopril and assayed using the TLC
procedure. The results of 15 assays gave a
recovery of 100.6%, a standard deviation ( s ) of
1.39 and a coefficient of variation (C.V.) of 1.38
(50)
6.32 High-Performance Liquid Chroma-
tography ( HPLC)
Three HPLC systems were investigated (51) for
selective separation of captopril from pharmaceu-
tical excipients, synthetic intermediates,
degradation products and impurities. These
included anion exchange, amino, and
CAPTOPRIL 115
octadecylsilane (ODS) systems. The first and

second appeared not to be the systems of choice
when they did not achieve necessary separations of
pharmaceutical excipients from captopril. A
heavily loaded version of the third system ( 1 5 %
O D s , monolayer bonded on silica, Partisil ODS-2)
was selected as optimum for bulk and tablet
analysis. The third system was clearly superior.
Nevertheless, the first two systems had very
occasional usage when formulations contained
excipients which interfered with captopril or its
disulfide analysis in the heavily loaded C18
system.
The characteristics of these systems and of
several others investigated are summarized in
Table 8 below.
Since captopril has a U.V. absorption maximum
at about 2 0 0 nm with broad end absorption (see
Figure 1 5 ) U.V. detection was at 230 nm or lower,
compatible with a good baseline and low solvent
interference inherent in a good signal to noise
ratio. An alternative to U.V. detection, for
captopril per se (see Section 7.4 for captopril
U.V. derivative HPLC) is the electrochemical
detector (systems 8 and 9) introduced for sul-
fhydryl analysis by Saetre and Rabenstein ( 5 2 ) .
The electrochemical detector's near neutral
detection potential makes it especially thiol
selective. It has been reported (53) to have a
sensitivity at least 200 fold over that of U.V.
detection.
Fig. 15. U.V. Spectrum of C a p t o p r i l in Mobile Phase -
System 4 (Table 8)
Instrument: Beckman Acta C I11
116
Table 8
C a p t o p r i l HPLC S y s t e m s
System Flow Loop Remarks

No. Rate Injection and
ml/min Volume p 1 S t a t i o n a r y Phase Mobile Phase Detection Reference
1 0.6 20 250 x 4 . 6 mm 5 2 5 mg c i t r i c U.V. 51

P a r t i s i l SAX acid. H20 220 nm
10um S t r o n g +37.7mg N H 4 C I T
a n i o n Whatman to 1 liter with
CH30H a d j u s t t o
p H 3.30 w i t h U.V. 51
0.1 N HC1 220 nm
2 1.0 20 300 x 3 . 9 mm 0 . 0 1 M N a EDTA

u Bondapak NH2 i n 0.05% SAC- U.V. 51
1 0 um a m i n o CH3CN, 9 5 : 5 2 2 0 nm
Waters
3 1.0 20 250 x 4 . 6 mm CH,OH-H,O-85% U.V. 51

P a r t i s i l ODS 5% ~ ~ $ 20 7 : 7~
5: ' 2 2 0 nm
10um ODS mono- 0.1
l a y e r Whatman
System Flow Loop Remarks
No. Rate Injection and
ml/min Volume p 1 Stationary Phase Mobile Phase Detection Reference
4 1.0 20 250 x 4 . 6 mm CH OH-H 0-85% U.V. 51

Partisil ODS 15% H3$04, 3 0 :50: 220 nm Optimum
lOpm ODS mono 0.05 resolution
layer Whatman system
5 1.0 20 200 mm Hypersil U.V. 54
ODS +
TSIM 5um H go4, 47.5: 230 nm
ODS + trimethyl- 52.5:l. 0
silylimidazole
monolayers
Shandon
6 1.0 1.0 200 mm Hypersil CH OH-H 0 - 1 8 U.V. 54
(ml) ODS + TSIM 5pm H3$04, $3 :57 : 230 nm As say
ODS + trimethyl- 1.0 disulfide
silylimidazole in low
monolayers potency
Shandon captopril
formula-
tions
7 1.0 20 2 5 0 x 4 . 6 mm CH30H-(0.1 M Electrochem., 53
Partisil ODS 5 % KH PO + + 0.10 V , Hg
10pm ODS mono 152mMfi2P04) , Pool vs
layer Whatman 55% :456 Ag/AgCl
8 0.5 20 300 x 4.6 mm MC CH30H-(0.05 M Electrochem., 55
Chromegabond 209 KH PO + 30mM + O.lOV, Adapted
10um heavily H $0 f, Hg film for
loaded ODS 53% :15% vs. Ag/AgCl "total"
ES Industries urinary
captopril
analysis
9 1.0 20 250 x 4.6 mm CH OH-H 0-85% U.V. 56
Partisil ODS 5% H3d04, ?0:80: 214 nm
10pm ODS 0.1
Whatman
10 1-2 Waters 300 x 3 . 9 mm p CH C1 : HAc, U.V. 57
-
c
Variable Porasil 1Opm 9 T l2 240 nm Qua1 .
W
Volume silica gel nor- Sepn. of
mal phase Waters captopril
from its
R,S isomer
11 0.5-1 Waters 250 x 4.6 mm H O(to pH 3.0 U.V. 58
Variable Lichrosorb RP18 z6.5 M H3P04) 220 nm
Volume 5pm "capped" ODS - CH3CN,
Merck 65 : 35
12 100 -- 30 x 5.7 cm Prep- CH OH-H20-0.05 RI 59
PAK C18 ODS M dPO 45 : Prep.
Waters 55 ? 0?65 Sepn.
captopril
impurities
ODS = Octadecylsilyl
120 HAROLD KADIN
6.33 Gas Liquid Chromatography (GLC)

A c a p t ~ p r i l - ~synthesis
~c separated captopril
from its R,S optical isomer by GLC of its methyl
ester (after CH2N2 treatment) on 1.5% OV-17 at
18OOC (60). The gas chromatogram of the
pre-resolution isomer mixture showed two peaks of
almost the same peak area. After a dicyclohexy-
lamine resolution, GLC of the desired captopril
(S,S fraction) indicated a single peak at the
retention time of the second peak in the chromato-
gram of the mixture. The method is recommended,
over the more conventional, appreciably less
sensitive, measurement of optical rotations (Sec-
tion 4.34), for process control of the isomer
separation.
G.C.-mass spectrometry (Section 7.2) and
G.C.-flame photometry (Section 7.3) of the N-ethyl
maleimide adduct (Section 7 ) of captopril as
methyl and heptafluoroisopropyl esters, respec-
tively, have also been reported.
Acid iodate (Iodine) titration, before and
after chromatography through a Jones Reductor
Column was used to measure disulfide in captopril
(61). For small amounts of disulfide this
technique measures, relatively inaccurately, small
differences between two large numbers. Therefore
the subsequently developed, more accurate TLC
(Section 6.31) and HPLC (Section 6.32) direct
assays are preferred. However the iodine
titration, with starch indicator, is useful as a
thiol purity assay (61). A "Dead Stop" end point
indicator (62) makes the titration more amenable
to automation.
7. Analysis in Biological Fluids and Tissues
and in Animal Rations
Extreme instability of captopril in biologi-
cal media necessitated development (63) of an
immediate, quantitative thiol derivatization with
N- ethylmaleimide (NEM) followed by freezing.
Studies (64) with radioactive captopril in whole
blood had indicated that a 5 min delay before
addition of NEM resulted in a 10% l o s s of
CAPTOPRIL 121
captopril whereas a 30 min delay yielded a 65%

loss. The NEM derivative was found (65) to be
stable in frozen whole blood or in blood stored at
5OC for at least 3 months: it was unstable in a
frozen or 5OC phosphate buffer (pH 6 . 8 5 ) when
stored for more than 4 weeks.
Antioxidants and metal chelators were inef-
fective (63) as captopril stabilizers in whole
blood.
Long term stabilization, at the relatively
high concentrations of 1 to 100 mcg per ml pre-
valent in urine after captopril dosage, has been
achieved (40) with metal chelators, acid pH, and
5 O C storage (see Section 5.22).
Stability and homogeneity analysis of capto-

pril in animal feeds was required for multiple
long range toxicological studies. Despite a high
proportion of thiol reactive air, inorganics, and
proteins in the feeds, stabilization during the
analysis was achieved (46) by an isopropanolic
extractant fortified with trichloracetic acid and
metal chelators (see Section 7.71).
7.1 Thin Layer Radiochromatography (TLRC)
NEM stabilization allowed TLRC of captopril
in whole blood (63). Aliquots of whole blood were
analyzed for total radioactivity and NEM- treated
aliquots were extracted with methanol.
Reconstituted residues of the extracts were
applied to silica gel GF plates, developed with
chloroform/ethyl acetate/glacial acetic acid
(4:5:3) and analyzed for radioactivity associated
with captopril and its disulfide by zonal analy-
sis. The limit of detection was about 10 ng
captopril/ml of blood.
In TLRC analysis of the highly radioactive
captopril preparations used for drug metabolism
studies the thiol of captopril is particularly
susceptible to free radical degradation
(Section 4.2). Reaction of thiol across the
highly active carbonyl double bond of formaldehyde
(incorporated in the mobile phase) protects the
radioactive captopril during the chromatography
(66).
122 HAROLD KADIN
When dilute aqueous solutions of captopril

and an excess of formaldehyde are used the product
of the reaction has been shown by NMR and IR to be
the captopril hemithioacetal.
cp COOH
The hemithioacetal can be reverted to captopril,

for sulfhydryl analysis, by acid, base, or
bisulfite.
7.2 Gas Chromatography-Mass Spectroscopy
(GC-MS)
NEM-Captopril has been determined by GC-MS in
the selected ion mode (67) in whole blood, urine
(68), amniotic fluid (691, milk (69), and tissues
(68) (liver, kidney, lung, and placenta). For
determination in whole blood, proteins are
precipitated from vortexed whole blood plus
internal standard with freshly prepared 10%
metaphosphoric acid. The NEM-captopril is
absorbed from 0.45 pM membrane filtrates on
purified suction-dried Amberlite XAD-2 resin and
eluted with a freshly purified (neutral alumina
chromatography) ethyl acetate. The drug is
extracted from the ethyl acetate into 5% sodium
bicarbonate. After acidification and saturation
of the aqueous layer with sodium chloride, the NEM
derivative is extracted with ethyl acetate. After
evaporation of the solvent extract, samples are
methylated for GC-MS.
The G.C. was carried out with dried helium
gas at 6 ml/min on a 3 % OV-101 column with injec-
tor at 28OOC and column temperature programming at
200-280°C at 20°C/min. All glass surfaces had
been previously silanized with Sylon-CT. The
spectrometer was a modified Electronic Associates
Quad 300 quadropole. The samples (1 p l ) were
coinjected with 0.5 p 1 of N,O-bis(trimethylsily1)
trifluoroacetamide. The latter was used to
prolong column life. The peak height intensity
CAPTOPRIL 123
data of the m/z 230 and 248 ions was collected by

selected ion monitoring. The limit of detection
and the practical detection limit with a 90%
confidence limit are 5.5 and 16.5 ng per ml of
blood, respectively.
The internal standard, an NEM blocked,
deuterated or fluorinated derivative of captopril,
compensates for extraction and other possible
losses.
The procedure described (67) could be applied
to most of the biological media mentioned with but
slight revision. However milk required a greater
modification. The high fat content of milk made
XAD-2 chromatography unfeasible. Therefore
NEM-captopril was instead extracted from sodium
chloride saturated milk samples with ethyl
acetate.
7.3 Gas Chromatography-Flame Photometric
Detection (GC-FPD)
A GC method for captopril in blood and urine
utilizing a sulfur selective dedicated FPD has
been reported (70). Blood is treated with a s o h .
of N-ethylma1eimi.de in phosphate buffer s o h (pH
7.4) and with metaphosphoric acid (10% soln);
after centrifugation, the supernatant soln. is
extracted with ethyl acetate, and, after further
purification, the extract is evaporated, the
captopril-N-hexylmaleimide adduct is added as
internal standard, and the mixture is treated to
convert the two adducts into their
hexafluoroisopropyl esters for analysis by g.1.c.
at 215O on a glass column (1 m x 3 mm) packed with
2 % of OV-210-yn Gas-Chrom Q (80 to 100 mesh), with
N (50 ml min ) as carrier gas and
flame-photometric detection. Urine is analysed
similarly, but without deproteinisation. The
calibration graph (peak-height ratio vs. concn.)
is rectilinear for 1 to 5 ug ml- of I.
The sensitivity though not specified appears
to be in the microgram range. It is therefore not
satisfactory for human blood studies which are in
the nanogram range.
124 HAROLD KADIN
7.4 High Performance Liquid Chromatography

with U.V. Detection (HPLC-UVD)
An HPLC procedure for captopril in whole
blood and urine has been reported (71). Captopril
is thiol-blocked with p-bromophenacyl bromide
(~BPAB)or N-(4-dimethylamino-3,5-dinitrophenyl)
maleimide (DDPM), then the addition products were
separated and determined by HPLC on a reversed
phase column. Captopril in blood or urine could
be derivatized with pBPAB then excess pBPAB
removed by hexane extraction. The captopril
disulfides in the same samples were then reduced
by tributylphosphine to captopril which in turn
were thiol-blocked with DDPM. HPLC of the adduct
mixture thus allowed a separate analysis of
captopril or disulfides, before and after
reduction. The method is reported to be
sensitive, and precise down to 5 ng per ml of
whole blood and 0.1 mcg per ml of urine.
7.5 Spectrofluorometry
The drug, stabilized with dithioerythritol is
absorbed on a Brinkmann XAD-2 column from an
acidified diluted blood and eluted with pH 6.9
phosphate-30% dimethylformamide buffer. Captopril
in the eluate is reacted with
N-(7-dimethylamino-4-methyl coumarinyl) maleimide
(DACM) to form a fluorescent derivative (Section
6.22). After acidification, the derivative is
extracted into toluene. The fluorescence is
measured at 375 nm/425 nm-excitation/fluorescence.
The analysis was validated by the GC-MS procedure
described above in the range of 0.5 mcg to 10 mcg
per ml of blood (72).
7.6 Radioimmunoassay (RIA)
Development and application of a simple,
highly sensitive RIA for NEM-captopril in blood
plasma has been described (89). The lower
detection limit is 2 ng per ml of plasma.
CAPTOPRIL 125
7.7 Semiautomated Ellman Colorimetry,

(Figure 16)
7.71 Semiautomated Sulfhydryl Analysis
in Laboratory Animal Rations
Feeds were extracted with isopropanol
fortified with trichloroacetic, ethylenediamine
tetraacetic, and diethylenetetramine pentaacetic
acids. Pigments in the initial extract and
turbidity on aqueous dilution necessitated a
cleanup using isooctane after aqueous dilution of
the extract. Further aqueous dilution of the
isopropanol-water extract insolubilized trace
isooctane and more polar lipids. Finally
centrifugation in open top tubes removed the last
traces of isooctane. In the design of the
colorimetry from manual colorimetry ( 7 3 ) the
sequence of adding Ellman's reagent before the
alkali was reversed to maintain solubility of
Ellman's within the autoanalyzer lines. In
addition, methanol and aqueous buffer were added
to Ellman's reagent to enhance both stability and
solubility. Finally the pH of Ellman's reaction
was maintained at pH 8.5 rather than at its
optimum pH 7 (Section 6 . 2 1 ) to dissolve trace
protein haze. Due to the limited selectivity of
the colorimetry it was necessary to analyze for
and subtract the response of drug- unfortified
(blank) feed. The cleanup served to minimize this
blank such that captopril could be analyzed down
to 0.03% in a variety of animal feeds with good
accuracy.
7.72 Semiautomated Ellman Colorimetry
for Urinary Captopril and Its
Disulfides
The necessity for long term storage of
samples prior to analysis and the presence of an
oxidation-prone thiol of captopril required
development of an acid-chelate stabilization
method for urinary captopril (Section 5.22). Thin-
layer radiochromatography (Section 7 . 1 ) indicated
that human urinary captopril was primarily free
(unchanged) and, in almost equal proportion,
126 HAROLD KADIN
disulfide-conjugated with cysteine ( 7 4 ) . Rela-

tively small amounts of captopril disulfide were
observed. An electrochemical reduction (52) was
utilized to release disulfide-conjugated captopril
for thiol colorimetry. Of several rugged reduc-
tion cells evaluated, one with a VycorB Disc
separating the anode and the mercury pool cathode
was preferred. Methylene chloride partitions from
acidified salt-saturated urines, before and after
reduction, allowed the measurement of free and
disulfide-conjugated captopril. The drug
partitioned into the solvent whereas the aqueous
phase retained acid protonated, amino
group-bearing thiols like cysteine. Subsequent
solvent evaporation volatilized other potential
colorimetric interferences. An automated colori-
metry of 25 samples per hour was developed by
utilizing relatively thiol selective Ellman's
Reagent, 5,5'-Dithio-Bis-(2-Nitrobenzoic Acid)
(Sections 6.21 and 7.71). Results were confirmed
by an HPLC method with electrochemical detection,
developed while this method was in use (42).
with Electrochemical Detection (HPLC-
-
ECD)
Captopril in urine has been determined by an
HPLC-ECD procedure in which the drug is separated
from interferences on a monolayer bonded, heavily
loaded (about 20% carbon) octadecylsilane reverse-
phase column with a mobile phase of 55% methanol
and 45% aqueous phosphate buffer (55). A commer-
cially available thin layer mercury film detector
was used (Section 6.32). The anodic current,
resulting from the oxidation of Hg to Hg-captopril
complex, was a linear function of captopril con-
centration. The peak height was reproducible with
a relative standard deviation of 0.54% (n=5).
Captopril concentration as low as 0.5 mcg per ml
of urine can be quantitated. Sample treatment
involved simply centrifugation, filtration, and
deoxygenation with nitrogen. Recovery of capto-
pril from urine spiked at 2.0 mcg per ml was
better than 95%.
CAPTOPRIL 127
TECHNICON AUTOANALYZER
FOR ELLMAN COLORIMETRY
WASTE
P r
IFLOW CELL I
COLOR I M ETER
RECORDER
WASTE
NOMINAL
- FUNCTION
ML/MIN
~~
YELLOW HCI WASH 1.2

2 MIN
MIXING YELLOW' CELL 1.2
c 01LS
YELLoW' ,TEA-EDTA 1.2
J GREY
4 , AIR 1.o
HCI DIL 0.8
ELLMANS 0.8
WASTE
* = SOLVAFLEX SOLVENT TECHNICON
RESISTANT TUBING, SAMPLER I I
OTHER TUBING TYGON WITH 50 2/1 CAM
ELLMAN'S =
TEA-EDTA 0.08% ELLMAN'S
= 1.86% EDTA Nao 2H20 + IN 50% MEOH (COLD)
2Ooh TRIETHANOLAMINE + + 0.01 M
0.02% TWEEN 80 ACETATE pH 4.7
Figure 16
128 HAROLD KADIN
7.9 High-Performance Liquid Chromatography

with Fluorescence Detection (MPLC-FD)
Jarrott et a1 (75) describe an assay for
quantitating plasma captopril levels. Blood from
patients taking this drug was collected into tubes
containing edetate and ascorbic acid, and the
plasma was separated by centrifugation. After
addition of an internal standard, the plasma was
deproteinized and the supernate was adjusted to pII
6.5. N-(l-Pyrene)-maleimide was added to
derivatize captopril and an internal standard to
fluorescent adducts. These derivatives then were
extracted into ethyl acetate-benzene (1:l) and
separated from other derivatized thiols by high-
performance liquid chromatography. The sensi-
tivity of the assay was 150 pmoles/ml. The HPLC-
FD uses a mobile phase of methanol-potassium
phosphate buffer (5 mM, pH 6.5) (52:48) and the
flow rate was 2 ml/min through a radially com-
pressed octadecylsilane 10 cm x 8 mm (i.d.1 column
and a spectrofluorometer with a 20 p 1 flow cell.
Excitation was at 340 nm with emission taken at
390 nm.
8. Drug Metabolism - Pharmacokinetics
Two features salient to the metabolism and
pharmacokinetics of captopril include a) the
reversible transformation of its thiol function to
dimer and mixed disulfide (74,76-79) and b) the
biological stability of its amide linkage (80,
Section 3). The following will emphasize these
aspects.
8.1 Blood Level Studies
Captopril is absorbed rapidly as indicated by
measurable blood levels of the drug 15 min after
ingestion (81). These findings are compatible
with reports of the onset of antihypertensive
activity as soon a s 15 min after a single oral
dose (3) in hypertensive patients and with the
rapid onset of blockade of angiotensin I-induced
increases in blood pressure in healthy subjects
(91). Other studies revealed that the captopril
metabolites included its dimer disulfide and the
mixed disulfides with glutathione, cysteine, and
serum albumin (78,811 .
CAPTOPRIL 129
After single oral dosage with 100 mg

radioactive captopril, ten healthy subjects
attained a mean maximal concentration ( C ) in
blood of 800 + 76 ng/ml at a mean time (Fax) of
0.93 + 0.08 hours (8l,82). Methanol extr%%.on,
ultrariltration, and dithiothreitol reduction
studies (77) coupled with TLRC (Section 7.1)
established that about 5 0 % of the total blood
radioactivity was unchanged captopril, about 10%
dimer disulfide and the remainder other polar
metabolites including protein and non-protein
mixed disulfides (81,82). Thereafter the
captopril fraction of total radioactivity declined
rapidly with time whereas the mixed disulfide
fraction increased. The curvilinearity of
semilogarithmic plots of these captopril blood
levels with time indicated that half-life (t 1 ,
values could not be accurately calculated. #or
unchanged captopril levels, curvilinearity may be
due to the mixed disulfide fraction acting
reversibly to replenish captopril, possibly
extending the duration of pharmacologic activity
(34,76-79). However plots of total radioactivity
allowed calculation of t of 4.3 + 0.2 hours for
the 4-12 hour time inter$al and 1g.4 + 2.6 hours
for the 12-48 hour time interval (81): Mean
concentrations of total radioactivity in blood
(expressed as captopril equivalents) were
41 + 7 ng/ml at 24 hours and 20 + 5 ng/ml at
48 fiours after drug administration.
Blood level and urinary excretion studies in

6 hypertensive patients, dosed over a 10 day
perigd, indicated that the metabolic disposition
of C-captopril was the same at the beginning
and end of the study, and was comparable to the
disposition in healthy subjects (83).
Radiolabeled captopril was administered on days 1
and 10, and blood and urine samples were analyzed
for captopril and its metabolites by radiometric
assay procedures. Non-radioactive drug (100 mg
t.i.d.1 was used for dosage on Days 3 to 9.
8.2 Urinary Excretion Studies
In the fasting state, absorption averaged
7 0 - 7 5 % of an oral dose, as evidenced by human
urinary excretion studies (81,82,84). The
presence of food in the gastointestinal tract
130 HAROLD KADIN
reduces absorption of an oral dose by about 35 to

40% (84).
At least 95% of the radioactivity recovered
in urine after a 100 mg oral dose of radiolabelled
captopril to mildly hypertensive patients was
accounted for as captopril and specific urinary
metabolites. Captopril and captopril-cysteine
mixed disulfide each accounted for about 45% of
the urinary radioactivity (about 3 3 % of the dose)
and the disulfide dimer accounted for about 5% of
the radioactivity in urine (about 4% of the dose)
(74) .Additional minor urinary metabolites, in
rats and dogs, are S-methyl captopril,
captopril-S-methyl sulfoxide, N-acetyl
cysteine-captopril and glutathione-captopril mixed
disulfides (80). These minor metabolites have
not, as yet, been reported in man, but appear to
be present in monkeys (90).
Renal dysfunction, as measured by creatinine
clearance, decreased the excretion rate of
captopril after a single 100 mg oral dose (85).
The authors suggest a method of dosage reduction,
based on creatinine clearance measurements, in
cases of moderate to severe renal dysfunction.
8.3 Miscellaneous Distribution Studies
Biological stability of the amide function
was confirmed by studies (80) on rat dermal
collagen; uptake of radioactive proline was
considerable, whereas insignificant uptake of
captopril labelled in the proline moiety was
observed.
Whole body radioautography of rats indicated
that captopril ( 5 0 mg/kg-intravenously) did not
readily enter the central nervous system whereas
it readily entered the placenta of pregnant rats
(86)
Captopril did not readily enter the breast
milk of lactating women dosed with captopril at
100 mg t.i.d. for 7 days (87,88). Relatively
insignificant milk peak levels were found (4.7
ng/ml)
CAPTOPRIL 131
9. Acknowledgments
I would like to express my appreciation to
individuals who have been very helpful for the
contributions indicated: Drs. J. Fried and M.
Porubcan - NMR, Drs. Y.J. Wang and T. Prusik -
Stability, Mr. A. Restivo and Mr. D. Domina -
Synthesis and Solubility, Drs. A. Cohen and P.
Funke - MS and GC-MS, Ms. M. Malley and Dr. J.
Gougatas - Single Crystal X-Ray, Mr. F. Dondzila,
Mr. S. Perlman and Dr. J. Kirschbaum - HPLC, Mr.
H. Roberts - TLC, Mr. R. Poet and Dr. G. Brewer -
Proof Reading and Manuscript Clarity, Ms. D.
Walker - Word Processing, Dr. D. Cushman -
History, Drs. B. Migdalof and D. McKinstry - Drug
Metabolism - Pharmacokinetics.
10. References
1. Ondetti, M.A., Williams, N.J., Sabo, E.F.,
Pluscec, J., Weaver, E.R.; and Kocy, O.,
Biochemistry, -
10, 4033 (1971).
2. Gavras, H., Brunner, H.R., Laragh, J.H.,
Sealey, J.E. Gavras, I., and Vukovich, R.A.,
New Eng. J. Med., 291, 817 (1974).
3. Case, D.B., Atlas, S.A. Laragh, J.H., Sealey,
J.E., Sullivan, P.A., and McKinstry, D . N . ,
Progr. Cardiovasc. Dis.; 21, 195 (1978).
4. Cushman, D.W. and Cheung, H.S., Biochem.
Pharmac., -
20, 1637 (1971).
5. Rubin, B., Laffan, R.J. Kotler, D.G.,

O'Keefe, E.H., DeMaio, D.A., and Goldberg,
M.E., J. Pharmacol. Exp. Therap., -
204, 271
(1978).
6. Quiocho, F.A. and Lipscomb, W.N., Adv.

-
Protein Chem., 25, 1 (1971).
7. Byers, L.D. and Wolfenden, R., Biochem., 12,
2070 (1973).
8. Cushman, D.W., Cheung, H.S., Sabo, E.F. and
Ondetti, M.A., Biochemistry, 16, 5484 (1977).
132 HAROLD KADIN
9. Cushman, D.W., Cheung, H.S., Sabo, E.F., and

Ondetti, M.A., Fed. Proc., -38, 2778 (1979).
10. Cushman, D.W., Pluscec, J., Williams, N.J.

Weaver, E . R . , Sabo, E.F., Kocy, O., Chueng,
H.S., and Ondetti, M.A., Experientia, - 29,
1031 (1973).
11. Ondetti, M.A., Rubin, B., and Cushman, D.W.,

Science, -
196, 441 (1977).
12. New Drug Application No. 18-343 Submitted by

E . R . Squibb & Sons Inc., Approved April 6,
1981.
13. Levine, T.B., Franciosa, J.A., Cohn, J.N.,

Circulation, -
62, 35 (1980).
14. Ondetti, M.A. and Cushman, D.W., United

States Patent 4,105,776, August 8, 1978.
15. Toeplitz, B., Personal Communication, April,
1976.
16. Porubcan, M.A., Personal Communication, June,

1981.
17. Whigan, D.B., Personal Communication, April,
1976.
18. .
Karchmer , J H. , "Treatise on Anal. Chem. ' I ,
Part 11; Volume 13, Page 410 (1966), Edited
By Kolthoff, I.M. and Elving, P.J., John
Wiley Publishers.
19. Ondetti, M., Personal Communication, February
1976.
20. Puar, M.S. and Funke, P.T., Personal
Communication, February, 1976.
21. Kadin, H., Personal Communication, March,
1976.
22. Valenti, V., Personal Communication, March,

1976.
23. Wadke, D.W., Personal Communication, March,

1976.
CAPTOPRIL 133
24. Karten, J. and Kabadi, B., Personal

Communication, July, 1980.
25. Malley, M. and Gougatas, J.Z., Personal
Communication, May, 1976.
26. Jacobson, H. and Ochs, Q . , Personal
Communication, May, 1976.
27. Weiss, A.L. and Wang, Y.J., Personal
Communication, February, 1980.
28. Wang, Y.J., Personal Communication, December,
1980.
29. Weiss, A.L., Personal Communication, March,
1980.
30. Weiss, A.L. Personal Communication, May,
1976.
31. Benesch, R. and Benesch, R., J. Am. Chem.
- , 77,
SOC. - 5877 (1955).
32. Funae, Y., Toshioka, N., Mita, I., Sugihara,
T., Ogura, T., Nakamura, Y., and Kawaguchi,
S., 19, 1623 (1971).
Chem. Pharm. Bull., -
33. Jocelyn, P.C. , "Biochemistry of the
Sulfhydryl Group", Page 344 (19721, Academic
Press.
34. Dondzila, F., Personal Communication, May,
1981.
35. Shipkowski, E.R., Personal Communication,
October, 1980.
36. Roberts, H.R., Personal Communication, May,
1977.
37. Timmins, P., Personal Communication, January,
1980.
38. Poet, R., Personal Communication, May, 1980.
39. Hanaki, A. and Kamide, H., Chem. Pharm.
Bull., 26, 325 (1978).
134 HAROLD KADIN
40. Kadin, H., Poet, R.B., "Sequential

Electrochemical Reduction, Solvent Partition
and Automated Thiol Colorimetry for Urinary
Captopril and its Disulfides", J. Pharm.
Sci., In Press.
41. Chas Pfizer and Company, New York, "Chelating
Agent Usage Calculator".
42. Kadin, H., Personal Communication, March,
1979.
43. Wang, Y.J., Personal Communication,
September, 1979.
44. Ellman, G.L. and Lysko, H., J. Lab. and Clin.
Med., 70, 518 (1967).
45. Poet, R., Personal Communication, February,
1978, July 1979.
46. Kadin, H., Personal Communication, October,
1978.
47. Lidell, H. and Saville, B., Analyst, -
84, 188
(1959).
48. Whigan, D.B., Personal Communication,
January, 197 9.
49. Valatin, P., Personal Communication, July,
1980, Sept., 1980.
50. Roberts, H., Personal Communication, January,

1978, August, 1979.
51. Perlman, S. and Kirschbaum, J., J. Chromat.,
-
206, 311 (1981).
52. Saetre, R. and Rabenstein, D.L., Anal. Chem.,
-
50, 276 (1978).
53. Yeh, P., Abstracts, No. 60, Page 54, Eastern
Analytical Symposium, New York, N.Y.,
November, 198 0.
54. Tenneson, M.E., Personal Communication,
February-March, 1980.
CAPTOPRIL 135
55. Yeh, P., Personal Communication, October,

1980.
56. Perlman, S., Personal Communication, April,
1980.
1979.
1981.
59. Dondzila, F., Personal Communication,

December-January, 1980.
60. Hasegawa, M., Ohyabu, S., Ueda, T., and
Yamaguchi, M., J. of Labelled Compounds and
Radiopharmaceuticals, -
18 I 643 ( 1981).
61. Whigan, D.B., Willis, S.L., and Jenkins,
E.J., Personal Communication, October, 1976.
62. Rohmer, M., Personal Communication, February,
1980.
63. Migdalof, B.H., Singhvi, S.M., and Kripalani,
K.J., J. Liquid Chromat., 3(6), 857 (1980).
64. Yeh, P. and Roberts H., Personal
65. Ivashkiv, E. and Roberts, H., Personal
66. Egli, P., Personal Communication, October,
1979.
67. Funke, P.T., Ivashkiv, E., Malley, M.F., and
Cohen, A.I., Anal. Chem., -
52, 1086 (1980).
68. Ivashkiv, E., Personal Communication,
March-April, 1980.
69. Ivashkiv, E., Personal Communication, August,
1979.
136 HAROLD KADIN
70. Matsuki, Y., Fukuhara, K., Ito, T., Ono, H.,

Ohara, N . , and Yui, T., J. Chromat., -
188, 177
(1980).
71. Kawahara, Y., Hisaoka, M., Yamazaki, Y.,
Inage, A., and Morioka, T., Chem. Pharm.
-
Bull., - 150 (1981).
- 29(1),
72. Ivashkiv, E., Personal Communication, August,
1980.
73. Kadin, H., Personal Communication, January,
1977.
74. Kripalani, K.J., Meeker, F.S., Dean, A.V.,
McKinstry, D . N . , and Migdalof, B.H., Fed.
Proceedings, - 39, 307 (1980).
75. Jarrott, B., Anderson, A., Hooper, R. and

Louis, W.J., J. Pharm. Sci., -
70, 665 (1981).
76. Komai, T., Ikeda, T., Kawai, K., Kameyama,

E., Shindo, H., J. Pharm. Dyn., -
4, 677
(1981).
77. Wong, K.K., Lan, S.J., and Migdalof, B.H.,
Biochem. Pharmacology, -
30, 2643 (1981).
78. Migdalof, B.H., Wong, K.K., Lan, S.J.,

Kripalani, K.J. and Singhvi, S.M., Fed.
Proc.,
- --39, 757 (1980).
79. Shindo, H., Komai, T., Ikeda, T., Kawai, K.,
Kawamata, W., and Kameyama, E., J.
-
Pharmacobiodyn., -
30, S-9 (1980).
80. Ikeda, T., Komai, T., Kawai, K., and Shindo,

H., Chem. Pharm. Bull., -
29(5),
- 1416 (1981).
81. Kripalani, K.J., McKinstry, D . N . , Singhvi,

S.M., Willard D . A . , Vukovich. R.A. and
Migdalof , B. H. , Clin. Pharmacol. and Therap. ,
27,
- 636 (1980).
82. McKinstry, D . N . , Singhvi, S.M., Kripalani,
K.J., Willard, D.A., and Migdalof, B.H.,
"Recent Advances in Hypertension Therapy:
Captopril", Excerpta Medica, Page 3, (1981),
Amsterdam-Oxford-Princeton.
CAPTOPRIL 137
83. McKinstry, D.N., Kripalani, K.J., Migdaloff,

B.H., Willard, D.A., Clin. Pharmacol. &
Ther.. 2 7 . 2 7 0 ( 1 9 8 0 ) .
84. Singhvi, S.M., McKinstry, D.N., Shaw, J.M.,
Willard, D.A., and Migdalof, B.H., J. Clin.
Pharmacol. (in press).
85. Rommel, A. J. Pierides, A.M., and Heald, A.
Clin. Pharmacol. Ther., -
27, 282 ( 1 9 8 0 ) .
86. Heald, A.F. and Ita, C.E., Pharmacologist,

19,
- 129 (1977).
87. Devlin, R.G. and Fleiss, P.M., Clin.

Pharmacol. & Ther., -
27, 2 5 0 ( 1 9 ' 8 0 ) .
88. Devlin, R.G. and Fleiss, P.M., J. Clin

Pharmacol. , -
21, 1 1 0 ( 1 9 8 1 ) .
89. Pearson, F.M., Martin, V.I., Aldujaili,

E.A.S., Williams, B.C., and Edwards, C.R.W.,
Acta Endocrinologica, -97, 5 2 4 3 , 1 4 8 ( 1 9 8 1 ) .
90. Migdalof, B.H., Personal Communication,

January, 1 9 8 2 .
91. Ferguson, R.K., Brunner, H.R., Turini, G.A.,
Gavras, H. and McKinstry, D.N., Lancet, -
I,
775 I (1977) .
11. Review Coverage Dates
This review summarizes communications up to
June 10, 1 9 8 1 . However Sections 2 and 8 are'
updated to about January, 1 9 8 2 .
CEFOTAXIME
Farid J . Muhtudi and Mahmoud M . A. Hassan
1 Description 140
1.1 Nomenclature 140
1.2 Formulae 140
1.4 ElementalComposition 142
2.1 Solubility 142
2.2 Moisture Content 142
2.3 pH Range 142
2.4 Optical Rotation 142
3. Synthesis of Cefotaxime 152
4. Metabolism 152
5. Pharmacokinetics 156
6. Microbiological Activity 156
7.2 Non-Aqueous Titration 159
7.3 Chromatography 159
7.4 Spectrophotometry 165
7.5 Microbiological Assay 166
8. References 167
Analytical Profiles of Drug Substances Copyright 0 1982 by The American

ISBN &12-260811-9
140 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN
1. Description
1.1. Nomenclature
(a) Sodium 7-[2-(2-amino-4-thiazolyl)-2-

m e t hox y iminoa c e tam i d o ] c epha 1o s po r a n a t e .
(b) Sodium 3-acetoxymethyl-7-[ 2-(2-amino-
4- t h i a z o 1y 1) -2- m e t hox y i m i n o ] a c e t am id0 ]
-3-cephem-4-carboxyla te .
(c) (6 R-trans)-3-[ (Acetyloxy) methyl]-7-[ [
(2-amino-4-thiazolyl) (methoxy- imino)
a c e t y 11 amino]- 8-0xo-5 - t h ia-1 -azab i c yclo
[ 4 . 2 . 0 ] oct-2-ene-2-carboxylic acid
monosodium s a l t .
(d) (6-R, 7R)-7-[ 2-(2-Amino-4-thiazolyl)
glyoxylamido] -3-(hydroxy methyl) -8-0x0-
5-thia-1-azabicyclo [ 4 , 2 , 0 ] oc t-2-ene-
2-carboxylic a c i d a-(O-methyloxime),
a c e t a t e ( e s t e r ) monosodium s a l t .
(e) 5-Thia-1-azabicyclo [ 4.2.01 oc t-2-ene
2-2 c a r b o x y l i c a c i d , 3 - [ ( a c e t y l o x y )
methyl] -7-- [ [ (2-amino-4-thiazolyl)
(me thox y i m i n o ) y a c e t y l amino ] -8 -0xo- ,
[6R-[6a,78(Z)II-
1.1.2 Generic Name
Cefotaxime sodium; HR 756; RU-24,662;

RU-24,756
1.1.3 P r o p r i e t a r y Names
C l a f o r a n ; Primafen; Z a r i v i z ; T a r i v i d .
1.2. Formula
1 . 2 . 1 Empirical C16H16N507S2Na
1.2.2 Structural
m
?2
V
I
0 =. v
I
0
I
hl
X
V
1 . 2 . 3 CAS no.
[ 611846-23-31 (1) C16H16N507S2Na

[ 60846-21-11 (1) C H N 0 S (as f r e e acid)
16 1 7 5 7 2
[63527-52-61 (2)
1.3. M o l e c u l a r Weight
477.23
1.4. E l e m e n t a l Composition
C, 40.23;; H , 3.38%; N , 14.67%;
0, 23.47%; S , 13.44%; Ma 4.82%.
1.5. Appearance, C o l o r , Odor and T a s t e :
White t o creamy w h i t e c r y s t a l l i n e powder,
o d o r l e s s and h a s a s a l t y t a s t e a t the b e g i n n i n g ,
f o l l o w e d by b i t t e r n e s s.
2.1. Solubility
F r e e l y s o l u b l e i n water ( 0 . 5 g s o l u b l e i n 5 ml) ( 3 ) ,
s l i g h t l y s o l u b l e i n alcohol ( a b s o l u t e , 95%),
i n s o l u b l e i n chloroform (4)
2.2. M o i s t u r e C o n t e n t
Not more t h a n 6 p e r c e n t , d e t e r m i n e d by t h e Karl
F i s c h e r method, u s i n g a 0.2 g sample d i s s o l v e d
i n 2 m l methanol ( 3 ) .
0
Loss on d r y i n g a t 60 C u n d e r vacuum i n t h e
p r e s e n c e o f phosphorus p e n t o x i d e f o r 4 h r .
s h o u l d n o t exceed 6 % ( 3 ) .
2.3. pH r a n g e
The pH of c e f o t a x i m e a s a 10%a q u e o u s s o l u t i o n
i s 4 . 5 t o 6.5 d e t e r m i n e d p o t e n t i o m e t r i c a l l y ( 3 ) .
2.4. O p t i c a l R o t a t i o n
0
[ a]D ( c = l % aqueous s o l u t i o n ) + 58 t o + 64
(on d r i e d b ases) (3).
The o p t i c a l r o t a t i o n of c e f o t a x i m e ( c = l %
aqueous s o l u t i o n ) was d e t e r m i n e d u s i n g a
P e r k i n E l m e r 2 4 1 MC P o l a r m a t i c and found t o be:
[ a~D240 + 59.30.
CEFOTAXIME 143
2.5. Spectral Properties

2.5.1 Ultraviolet Spectra
The UV s p e c t r a o f c e f o t a x h e i n a n aqueous
s o l u t i o n and i n 0 . 1 N H C 1 a r e p r e s e n t e d i n
Fig. 1 and F i g . 2 r e s p e c t i v e l y . These were
scanned from 200 t o 400 nm u s i n g a Pye-
UnicumSP 8-100 U l t r a v i o l e t s p e c t r o -
photometer. The f o l l o w i n g t a b l e 1 shows
t h e UV d a t a .
Table 1. UV c h a r a c t e r i s t i c s of cefotaxime
solvent c onc en t r a t i o n max nm. E (lX, lcm)
water 0.5 mg/ml 234

0.1N R C l 0.05 mg/ml 205,262
0.1N HC1 0.01 mg/ml 263 420 (5)
2.5.2 Infrared Spectra

The I R s p e c t r a of c e f o t a x i m e a s KBr-disc
and a s n u j o l mull a r e shown i n F i g . 3 and
Fig. 4 r e s p e c t i v e l y . The KBr-disc w a s
recorded on a P e r k i n E l m e r 58OB i n f r a r e d
s p e c t r o m e t e r . The s t r u c t u r a l a s s i g n m e n t s
have been c o r r e l a t e d w i t h t h e f o l l o w i n g band
f r e q u e n c i e s (Table 2).
Table 2. I R c h a r a c t e r i s t i c s of c e f o t a x i m e
-1 Assignment
Frequency Cm
3420 -NH2
3340 (broad) -NH, -NH2
2940 -S-CH2
1 7 60 -C=O lactam 0
II
1730 -C=O e a r b o x y l i c , O-C-CH3
0
11
1650 -C-NH
0
1620 -C-NH,-C=N-,-C=C-
0
1540 -C-N-
1385-13 55 -0-CO-CH3
1180 C=O i n e s t e r
1050 C-0 stretching
230
240
250
304
350
400
Fig.1 The UV spectrum of Cefotaxtme in water
200
210
250
2 60
270
300
350
400
Fig.2 The UV spectrum o f C e f o t a x i m e in O . I N H C I .
144
3.0 40 5.0 MICRONS 6.0 70 8.0 9.0 10 12 14
I
4000 3500 3000 2500 2000 1800 1600 1400 1200 1000 1)oo
8
CD
0
0
a0
0
0
0
4
0
0
2
0
0
$
0
0
'0,
0
0
2
0
0
0
cu
0
0
v)
cy
a
0
0
c3
0
0
In
rn
CEFOTAXIME 147
2.5.3 Nuclear Magnetic Resonance S p e c t r a
2.5.3.1 Proton SDectra
The PMR s p e c t r a of c e f o t a x i m e i n
d e u t e r a t e d d i m e t h y l s u l f o x i d e (DMSOD6) and
d e u t e r i u m o x i d e w e r e r e c o r d e d on a v a r i a n
T-60A, 60-MHZ NMR s p e c t r o m e t e r u s i n g sodium -2,
2-dimethyl-2-silapentane-5-sulfonate (DSS) a s a n
i n t e r n a l r e f e r e n c e . These a r e shown i n F i g . 5
and F i g . 6 r e s p e c t i v e l y .
The f o l l o w i n g s t r u c t u r e a s s i g n m e n t s h a v e been
made ( T a b l e 3 ) ( 6 ) .
T a b l e 3. PMR c h a r a c t e r i s t i c s of c e f o t a x i m e
Group Chemical Sh Et ( P P )
DMSQD6
D2°
-0co CH, 2.00(s) 2.12(s)

2-G2 3 . 3 3 (9) 3 . 5 3 (9)
N - E 3 3 . 8 3 (s) 4.00(s)
0-CH
-3
AC . 4.97(9) 4.83 ( d )
6-H 4.97(q) 5.20(d)
7-H 5.60(2d) 5.82 ( d )
5-H 6.70(s) 6.97 (s)
2-Nl12 7.22 (bs) -
CONH
- 9.47 ( d )
s = s i n g l e t , d=doublet, q = q u a r t e t , 2d=doublet
of d o u b l e t s , bs=broad s i n g l e t .
13
2.5.3.2 C-NMR
13
C-NMR c o m p l e t e l y d e c o u p l e d and o f f r e s o n a n c e
s p e c t r a a r e p r e s e n t e d i n F i g . 7 and F i g . 8
respectively. Both were r e c o r d e d o v e r 5000 H,
r a n g e i n d e u t e r i u m o x i d e ( c o n c . 100 m g / l m l D20)
148
0
149
150 FARID J. MUHTADI AND MAHMOUD M . A. HASSAN
4 a
1
I
-TTJ_LLl- -J ' I ' -I

'_J _ ' ' '-11 I rI ' I '
F i g . 7. The 1 3 C NMR of Cefotaxime, completely decoupled

spectrum .
'I
I I II
Fig. 8 . The 1 3 C NMR of Cefotaxime, o f f resonance (Proton

coupled) spectrum
CEFOTAXIME 151
on FT-80A-8OMHz NMF. s p e c t r o m e t e r , u s i n g 1 0 mm sample

t u b e and d i o x a n e a s a r e f e r e n c e s t a n d a r d a t 2OoC.
T h e c a r b o n chemical s h i f t a r e a s s i g n e d on t h e b a s i s
of t h e a d d i t i v i t y p r i n c i p a l s and t h e o f f r e s o n a n c e
s p l i t t i n g p a t t e r n ( T a b l e 4) ( 6 ) .
12
NOCH3
H2N !?1 6
OCCH3
15
COONa
13
T a b l e 4. Carbon Chemical s h i f t s o f c e f o t a x i m e
Carbon no. Chemical s h i f t Carbon Chemical s h i f t

[ PPm 1 no. [ PPm 1
8-C=0 174.69(s) C-3 117.37 ( S )
1o-c=0 171.25(s) C-5 113.64 (d)
13-C=0 168.78 ( s ) c-7 65.07 (d)
15-C=0 165.08( s) C-6 59.59(d)
,
c-2 164.65(s) C-14 58.23 ( t )
c-11 148.62 (s) c-2 26.35( t )
,
c-4 141.40( s) C-12 63.55 ( 4 )
c-4 132.42 (s) C-16 21.22 ( 4 )
s=singlet, d=doublet, t = t r i p l e t , q=quartet.

152 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN
3. S y n t h e s i s of Cefotaxime
Cefotaxime i s a s e m i - s y n t h e t i c c e p h a l o s p o r i n . The
s t a r t i n g material f o r s u c h c e p h a l o s p o r i n s i s 7-aminoceph-
a l o s p o r a n i c a c i d (7-ACA) which o b t a i n e d e i t h e r by mild
a c i d o r enzymatic h y d r o l y s i s of c e p h a l o s p o r i n C . ( 7 , 8 , 9 , 1 0 )
P r e p a r a t i o n of t h e S i d e Chain: (5)
The s t a r t i n g m a t e r i a l e t h y l a c e t o a c e t a t e [ l ] w a s o x i -
mated t o produce oximinoethylacetoacetate [ 21. Methyla-
t i o n of [ 21 followed by bromination y i e l d e d syn-methoxy-
iminobromoketone [ 3 ] . Condensation of [ 3 ] w i t h t h i o u r e a
[ 4 ] i n aqueous medium and a t a low t e m p e r a t u r e gave syn-
a m i n o t h i a z o l y l d e r i v a t i v e [ 51. The a m i n o t h i a z o l y l r i n g
w a s p r o t e c t e d by t r i t y l a t i o n t o g i v e t h e N - t r i t y l d e r i v a -
t i v e [6]. S a p o n i f i c a t i o n of t h e l a t t e r by b o i l i n g w i t h
NaOH s o l u t i o n a f f o r d e d t h e c o r r e s p o n d i n g a c i d [ 7 ] . Acy-
l a t i o n of t h e amino group of 7-ACA w i t h t h e r e s u l t i n g
a c i d [ 7 ] proved d i f f i c u l t . T h i s h a s been overcome by t h e
u s e of symmetrical a n h y d r i d e [ 8 ] , which w a s o b t a i n e d by
condensing two m o l e c u l e s of [ 7 ] i n t h e p r e s e n c e of d i c y -
c l o h e x y l c a r b o d i i m i d e [ a ] . 7-ACA w a s a c y l a t e d by compound
[8] t o g i v e [ 91. Removal of t h e t r i t y l r e s i d u e under
a c i d i c c o n d i t i o n gave t h e f r e e a c i d form (R=H) of c e f o -
taxime [ l o ] .
P r e p a r a t i o n of a S t a b l e Sodium S a l t :
T h i s w a s prepared by adding t h e s o l i d f r e e a c i d t o
a n aqueous a l c o h o l i c s o l u t i o n of a n o r g a n i c sodium s a l t
t o g i v e t h e s t a b l e a c t i v e D-form. (5)
The s y n t h e s i s of c e f o t a x i m e i s p r e s e n t e d i n Scheme 1.
I t i s i n t e r e s t i n g t o n o t e t h a t t h e syn-isomer of
cefotaxime i s u p t o 100 times more a c t i v e a g a i n s t c e r t a i n
organisms t h a n t h e a n t i - i s o m e r .
4. Metabolism
The metabolism of c e f o t a x i m e i n r a t , dog and man

u s i n g 14C-labelled c e f o t a x i m e h a s been s t u d i e d by
Chamberlain e t a l . (11). Cefotaxime i s w e l l absorbed i n
t h e t h r e e species a f t e r intramuscular administration. It
i s e l i m i n a t e d mainly v i a t h e u r i n e . The major m e t a b o l i t e
being d e s a c e t y l c e f o t a x i m e . The amount of unchanged c e f o -
CEFOTAXIME 153
OH
Scheme 1: S y n t h e s i s of Cefotaxime N’
methylation
bromination
then I
N,0CH3
H2N<NH2 + ‘OC2H5
S CH*
/
[41 Br [31
condensation
i
H2N -(syl:*5
s
[ 51
tr i t y l a t ion
condensation
C 6H 1 1 N=C=NC6H1
[a1
/
N
H C-0
3
+ symmetrical anhydride
[81
COOH
7- A M
1 Acylation
155
CEFOTAXIME
Tr,n
H /
<N),/)[;'j
S
0-CH
3
0'
(4 CH 20Ac
C02H
1
acidification
0 -CH
3
N'
H 2N ~R=H acid
C02R ~
cH20Ac
R=Na Cefotaxime
[lo1
taxime and t h e major m e t a b o l i t e e l i m i n a t e d i n t h e u r i n e

i s s i m i l a r f o r each s p e c i e s . Two f u r t h e r m e t a b o l i t e s ,
UP1 and UP2 a r e d e t e c t e d i n dog and human u r i n e b u t n o t
i n r a t urine.
The f o r m a t i o n of b o t h UP m e t a b o l i t e s i s r e l a t e d t o
t h e r a t e of r e n a l e l i m i n a t i o n of d e s a c e t y l c e f o t a x i m e
( 11) *
The m e t a b o l i c pathway f o l l o w s t h e r o u t e i n dog and

human which may occur i n t h e l i v e r i s p r e s e n t e d i n Scheme
?
L.
5. P harmacokine t i c s
The p h a r m a c o k i n e t i c s of c e f o t a x i m e w a s r e p o r t e d t o
be d o s e independent f o r d o s e s up t o 2.0 g ( 1 2 ) . Following
t h e i n t r a m u s c u l a r (i.m.) i n j e c t i o n of a 0 . 5 g (13) and
1 . 0 g d o s e s ( 1 2 ) , t h e mean peak serum l e v e l of 11.7 \ig/ml
and 22 ug/ml a t 0.5 h were o b t a i n e d r e s p e c t i v e l y . A f t e r
i n t r a v e n o u s ( i . v . ) p o l u s i n j e c t i o n of 1 . 0 g c e f o t a x i m e ,
t h e mean peak l e v e l w a s 105 ug/ml and an a p p a r e n t volume
of d i s t r i b u t i o n of 25 l i t r e s w a s e s t i m a t e d ( 1 2 ) . Values
of 32 and 37 l i t r e s f o r t h e volume of d i s t r i b u t i o n were
a l s o r e p o r t e d ( 1 3 ) . Cefotaxime i s mainly e l i m i n a t e d by
r e n a l e x c r e t i o n and s e r u m - h a l f - l i f e ranged from 0.92 t o
1.35 h a f t e r 1 g -i.m. i n j e c t i o n , and from 0.84 t o 1 . 2 5 h
a f t e r i . v . a d m i n i s t r a t i o n ( 1 2 ) . The m a j o r i t y of t h e d o s e
being e x c r e t e d unchanged i n t h e u r i n e (-51% w i t h i n 8 h)
(12). I n a n o t h e r s t u d y (13) t h e serum c l e a r a n c e of 341
mlfmin. per 1.73 m2 and r e n a l c l e a r a n c e of 130 mlfmin p e r
1.73 m2 were o b t a i n e d . P r o t e i n b i n d i n g of c e f o t a x i m e
ranged from 35 t o 45%.
6. Microbiological Activity
Cefotaxime p o s s e s s e s a n e x t r e m e l y broad a n t i b a c t e r i a l
spectrum and i s v e r y a c t i v e a g a i n s t C positive bacteria
e s p e c i a l l y on E n t e r o b a c t e r i a c e a e , Haemophilus i n f l u e n z a e
o r Neisseria gonorrhoeae. I t is a l s o a c t i v e on Bacteroides
f r a g i l i s and Pseudomonas a e r a g i n o s a ( 1 4 ) . Cefotaxime
p e n e t r a t e s w e l l through t h e c e l l w a l l s , showing h i g h 6-
l a c t a m a s e s t a b i l i t y and s t r o n g a f f i n i t y t o t h e i m p o r t a n t
t a r g e t enzymes ( 1 4 ) .
CEFOTAXIME 157
Scheme 2 : Metabolism of Cefotaxime.

OCH3
N'
co-
Cefotaxime
Deacetylation
OCH3
'N
2
-<: D;k"oE(A
Desacetyl
NH
cefotaxime
co- CH OH
Desacetyl
cef o t a x ime l a c t o n e
ELo
09 0
OCH3
N’ H H
UP 2
CEFOTAXIME 159
7. Methods of A n a l y s i s
7 . 1 . I d e n t i f i c a t i o n Tests
The f o l l o w i n g i d e n t i f i c a t i o n t e s t s are
mentioned under c e p h a l e x i n i n t h e B.P. 1980
(15) :-
A) The i n f r a - r e d a b s o r p t i o n spectrum determined

i n t h e s o l i d s t a t e , i s concordant w i t h t h e
r e f e r e n c e spectrum of c e f o t a x i m e .
B) Mix 20 mg w i t h a few d r o p s of an 8 0 % V/V

s o l u t i o n of s u l f u r i c a c i d c o n t a i n i n g 1 % V/V
of n i t r i c a c i d ; a yellow c o l o r is produced.
C) Mix 20 mg w i t h 5 d r o p s of a 1 % V/V s o l u t i o n of
g l a c i a l a c e t i c a c i d and add 2 d r o p s of a 1 % W/V
s o l u t i o n of copper s u l f a t e and d r o p of 2M sodium
hydroxide; an o l i v e - g r e e n c o l o r i s produced.
7.2. Non-Aqueous T i t r a t i o n Assay
Weigh a c c u r a t e l y a b o u t 0.15 g of c e f o t a x i m e and

d i s s o l v e i n 50 m l g l a c i a l a c e t i c a c i d . Assay
potentiometrically with 0.1 N perchloric acid.
1 m l of 0.1 N p e r c h l o r i c a c i d c o r r e s p o n d s t o
23.87 mg of c e f o t a x i m e ( 3 ) .
7.3. Chromatography
7.3.1 Thin Layer Chromatography (TIX)
TLC of c e f o t a x i m e i s perforlned on s i l i c a
g e l c o a t e d , s e l f - i n d i c a t i n g chroma t o g r a p h i c
p l a t e s (60 F 2 5 4 , Merck).
The developing s o l v e n t system is:-
Ethylacetate/Ethanol/Water/Formic a c i d
(60 : 25 : 1 5 : 1) by volume.
Development i s c a r r i e d o u t i n a l i n e d t a n k ,
and t h e s o l v e n t i s allowed t o m i g r a t e a b o u t
1 5 cm from t h e s t a r t i n g p o i n t s .
The p l a t e s are v i s u l i z e d by examining under
u l t r a v i o l e t l i g h t a t 254 nm ( 3 ) .
The above method c a n be adopted f o r t h e

d e t e r m i n a t i o n of t o t a l i m p u r i t y c o n t e n t of
cefotaxime as f o l l o w s ( 3 ) :-
A 2 % s o l u t i o n of t h e sample t o be
determined i s d i s s o l v e d i n a m i x t u r e of
8 v o l . a c e t o n e and 2 v o l . water.
Cefotaxime r e f e r e n c e s u b s t a n c e (HR 756
s t a n d a r d ) i s d i s s o l v e d i n t h e same m i x t u r e t o
produce 0.02 %, 0.04 % and 0.08 % s o l u t i o n s .
Two s p o t s 0.01 and .0.02 m l of t h e t e s t e d

s o l u t i o n s are a p p l i e d t o t h e c h r o m a t o p l a t e
t h e s e r e p r e s e n t i n g 200 and 400 pg r e s p e c t i v e l y .
A 0.01 m l of e a c h s t a n d a r d s o l u t i o n is
a l s o applied t o t h e chromatoplate represent-
ing 2 , 4 , 8 p g r e s p e c t i v e l y .
The p l a t e i s developed a s above.
The t o t a l i m p u r i t y c o n t e n t should n o t
exceed 5 %.
Another TLC system i s recommended f o r

cefotaxime a s f o l l o w s : -
A s o l u t i o n of c e f o t a x i m e i s a p p l i e d i n t o
a s i l i c a g e l G c h r o m a t o p l a t e . The p l a t e i s
developed i n t h e s o l v e n t Methanol : Ammonia
(100 : 1 . 5 ) . A f t e r development, t h e p l a t e
i s a i r d r i e d , sprayed w i t h 0.5 % i o d i n e i n
chloroform. Cefotaxime developed a d a r k
brown s p o t which has Rf v a l u e of 0.83 ( 4 )
Descending paper chromatography w a s

accomplished u s i n g Whatman no. 1 paper.
The s o l v e n t system c o n s i s t e d of i s o p r o p a n o l -
p y r i d i n e - w a t e r (65 : 5 : 30, V / V ) . The
chromatogram was s u b j e c t e d over-night
f o r development. Examination was conducted
under t h e UV l i g h t a t 254 nm.
7.3.3 Gas Liquid Chromatography (GLC)
A GLC method f o r t h e d e t e r m i n a t i o n of
cefotaxime h a s been adopted i n our l a b o r a t o r y
CEFOTAXIME 161
u s i n g a Varian GC - 3700 g a s chromatograph

equipped w i t h Varian CDS 111 i n t e g r a t o r .
Column c o n d i t i o n s : 3 Z OV-17on chromosorb WHP
(80 - 100 mesh) g l a s s column (2 m x 2 mm)
The column w a s c o n d i t i o n e d i s o t h e r m a l l y
a t 120° f o r 1 0 min. and t h e n t h e temp-
e r a t u r e w a s programmed a t 10°/min up t o
2100.
C a r r i e r g a s : Helium, f l o w r a t e was a d j u s t e d
t o 20 m l / m i n .
D e t e c t o r : FID, hydrogen and a i r f l o w rates
were a d j u s t e d t o 40 ml/min. and 300 ml/min.
respectively .
Ethanol w a s used a s t h e s o l v e n t and t h e c h a r t
speed w a s a d j u s t e d t o g i v e 0.5 cm./min.
The r e t e n t i o n t i m e = 8.15 min.
The GLC of cefotaxime i s p r e s e n t e d i n F i g . 9.
Another GLC method h a s been d e s c r i b e d € o r

t h e d e t e r m i n a t i o n of r e s i d u a l s o l v e n t s i n
cefotaxime ( 3 ) .
Apparatus : A 1 . 5 m PORAPAK Q column programed a t 150'

with a flame i o n i z a t i o n d e t e c t o r was used.
I s o p r o p a n o l w a s used a s t h e i n t e r n a l s t a n d a r d .
Solution I : 70 mg cefotaxime s t a n d a r d (HR 756) and 0.08 mg

i s o p r o p a n o l were d i s s o l v e d i n water t o produce
1ml.
S o l u t i o n I1 : 0.05 mg methanol, 0.13 mg e t h a n o l and 0.08 mg

i s o p r o p a n o l were mixed w i t h water t o produce
1ml.
Procedure : S o l u t i o n s I and I1 w e r e i n j e c t e d and t h e

s o l v e n t l e v e l s w e r e determined from t h e
peak h e i g h t s c o r r e c t e d a g a i n s t t h e i n t e r n a l
standard.
Results : Methanol should n o t exceed 0.2 % e t h a n o l o r

any o t h e r o r g a n i c s o l v e n t should n o t exceed 1 % .
F i g 9. GLC of c e f o t a x i m e
A = cefotaxime
7.3.4 High Performance L i q u i d Chromatography (HPLC)
S e v e r a l HPLC s y s t e m s f o r t h e i d e n t i f i c a t i o n
and s e p a r a t i o n of c e f o t a x i m e and i t s metabo-
l i t e s have been r e p o r t e d . These s y s t e m s a r e
g i v e n i n T a b l e 5.
HPLC o f c e f o t a x i m e and i t s m e t a b o l i t e s
u s i n g system 1 (11) i s p r e s e n t e d i n F i g . 10.
T a b l e 5. HPLC Systems of Cefotaxime
System Column Mobile P h a s e Retention Time Detect ion Ref.

no. (min) ( nm)
1. A n u c l e o s ill Acetonitrile- 13.0 W region

ODS column wa t e r - a c e t i c a c i d
( 8 : 9 1 : 1 by v o l )
2. A Microbondapak, wa t er-me t h a n o l
C18 corasil (39 : 7 ) c o n t a i n i n g 30 .O 254 nm
0.005 M h e p t a n e
guard column
sulfonic acid
3. Si-C18, 10 Methanol-water- - 235 nm

(1 : 5)
microns
KH2 P O 0.06 %
4
N a 2 HPO 2H20
4
t o r e a c h pH 7 . 8
4. Li ch r os o r b Phosphoric acid- 17.0 310 nm cL7 1

RP-8 methanol
Table 5 contd.
System Column Mobile Phase R e t e n t i o n Time Detection Ref.

no. (min) (nm)
5. A Rever sed-phase 20 m m o l sodium 4.8 254 nm
Hibar RT 250-4 d i h y d r o g enp ho s pha t e
L i c h r o s o r b RP i n water-methanol-
18-7 um, c o r a s i l acetonitr ile
C18 37-50 Vm (83:7:10, V / V / V )
guard column
6. A 10-cm octade- 1 4 % methanol, 12.0 254 nm (19)

c y l s i l a n e column 1% acetic acid, ( s e t a t 0.005
a n d a 4-cm pre- in distilled absorbance
column c o n t a i n i n g water unit)
copellicular
oc t a d e c y l s i l a n e
CEFOTAXIME 165
A
I D
13
R e t e n t i o n time (min)
F i g . 1 0 HPLC of C e f o t a x i m e and i t s M e t a b o l i t e s
A , D e s a c e t y l c e f o t a x i m e ; B , UP1; C , UP2;
D, Desacetyl cefotaxime l a c t o n e ; E , cefotaxime.
7.4 Spectrophotometry
A PMR method f o r q u a n t i t a t i v e d e t e r m i n a t i o n
of c e f o t a x i m e and o t h e r c e p h a l o s p o r i n s i n b u l k
d r u g s and v a r i o u s d o s a g e f o r m s i s r e p o r t e d ( 2 0 ) .
The d e t e r m i n a t i o n i s based on t h e i n t e g r a t i o n
of t h e 6-H and 1 o r 7-H of t h e @-lactam r i n g
system r e l a t i v e t o t h a t of t h e n i n e p r o t o n s of
t-butanol. The method i s r a p i d , a c c u r a t e and
precise, with an average standard deviations
of 2 1 . 5 1 i n s t a n d a r d m i x t u r e s and k 1 . 1 5 i n
p h a r m a c e u t i c a l f o r m u l a t i o n s . The p r o c e d u r e
f u r n i s h e s a s p e c i f i c means o f i d e n t i f i c a t i o n
of e a c h i n d i v i d u a l c e p h a l o s p o r i n a s w e l l a s
d e t e c t i o n of t h e commonly e n c o u n t e r e d i m p u r i t i e s .
Procedure:
Weigh a c c u r a t e l y a p o r t i o n of t h e powder
e q u i v a l e n t t o 100 mg of t h e c e p h a l o s p o r i n i n t o
a g l a s s s t o p p e r e d sample t u b e . Add 1.0 m l
a c c u r a t e l y measured D20 c o n t a i n i n g a n a c c u r a t e
w e i g h t of t - b u t a n o l , s t o p p e r and s h a k e f o r 3 min.
T r a n s f e r a b o u t 0 . 5 m l of t h e r e s u l t i n g s o l u t i o n
i n t o a NMR t u b e and r e c o r d t h e s p e c t r u m , a d j u s t i n g
t h e s p i n r a t e t o r e d u c e t h e s p i n n i n g s i d e bands a s
much a s p o s s i b l e . I n t e g r a t e t h e peaks of i n t e r e s t
(The 6- o r 7-H of t h e B-lactam r i n g a p p e a r i n g a t
4.86 - 5.80 ppm and t h e 9 p r o t o n s of t - b u t a n o l
a p p e a r i n g a t 1.23 ppm) a t l e a s t t h r e e times and
determine t h e average i n t e g r a l s .
The amount of c e p h a l o s p o r i n i s t h e n c a l c u l a t e d
as follows:-
Where Ac i s t h e i n t e g r a l v a l u e of t h e c e p h a l o s p o r i n
signal, % t h a t of t h e t - b u t a n o l s f g n a l , E.Wc is t h e
m o l e c u l a r weight of c e p h a l o s p o r i n and E.W is one
b
n i n t h of t h e m o l e c u l a r weight of t - b u t a n o l (= 8 . 2 4 ) .
7.5 M i c r o b i o l o g i c a l Assay
C e f otaxime i s m i c r o b i o l o g i c a l l y assayed by t h e
a g a r w e l l d i f f u s i o n method of Grove and R a n d a l l (21)
a s modified by Chabbert and Boulinger ( 2 2 ) .
The f o l l o w i n g t a b l e 6 summarises t h e media and

t h e t e s t organisms a s r e p o r t e d .
T a b l e 6. Nedia and Test Organisms Used
Med iurn Test Organism -

Ref.
1. Agar a n t i b i o t i c Spores of B a c i l l u s s u b t i l i s (23)

no. 2 (Difco)
II 11
2. B a c i l l u s s u b t i l i s (ATCC 6633) (24)
II 11
3. S a r c i n a l e u t e a (ATCC 9341) (24)
4. II II
Escherichia coli 3989 (13)
It
5. 11
E. c o l i (ATCC 9637) (26)
I1 II
6. Proteus morganii (19)
7 . A n t i b i o t i c no. 3 Staphylococcus epidermid i s (25)
(Oxoid) Q 305
8. Mueller-Hinton P r o t e u s m i r a b i l i s (ATCC 14273) (18)
agar
Pseudomonas a e r u g i n o s a ( K 1118)
CEFOTAXIME 167
8. References
1. "Annual Drug Data Report", Ed. J . R . P r o u s , V 0 1 . 1 1 1 ,

J . R . P r o u s S . A . , B a r c e l o n a , S p a i n , p. 5 3 , ( 1 9 8 1 ) .
2. Chemical A b s t r a c t , I n d e x Guide S u p p l . , 1977-1979

c u m u l a t i v e , The American Chemical S o c i e t y , U.S.A.
3. C l a f o r a n " Q u a n t i t a t i v e and Q u a l i t a t i v e methods of

analysis" Roussel Uclaf, P a r i s , France.
4. S . Khawaja, U n i v e r s i t y of Riyadh, S a u d i A r a b i a
" P e r s o n a l Communication''.
5. R. Bucourt, D. Bormann, R. Heymes and ?I. P e r r o n n e t

6 , S u p p l . A , p. 6 3 , (1980).
J . A n t i m i c r o b i a l Chemotherapy, -
6. M.M.A. Hassan and F . J . Muhtadi, "Unpublished Data".
7. B. Loder, G.G.F. Newton and E.P. Abraham, Biochem.

J., 2,4 0 8 , (1961).
8. R.B. Morin, B . G . J a c k s o n , E . H . F l y n n , and R.W. Roeske,
J . Am. Chem. SOC., - 8 4 , 3400, ( 1 9 6 2 ) .
9. B. F e c h t i g , H. P e t e r , H. B i c k e l and E . V i s c h e r ,
Helv. Chim A c t a , 2, 1108 (1968).
10. E . H . F l y n n , Ed., C e p h a l o s p o r i n s and P e n i c i l l i n s , C h e m i s t r y ,

B i o l o g y , Academic P r e s s , New York, p. 27, ( 1 9 7 2 ) .
11. J . Chamberlain, J . D . Coombes, D. D e l l , J . M . Fromson,

R . J . I n g s , C.M. Macdonald and J . McEwen, J . A n t i -
m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 6 9 , ( 1 9 8 0 ) .
1 2 . F. Esmieu, J. G u i b e r t , H . C . R o s e n k i l d e , I. Ho and A.
L e Go, J . A n t i m i c r o b i a l Chemotherapy, - 6 , Suppl. A,
p. 83, ( 1 9 8 0 ) .
13. K. P . Fu, P . Aswapokee, I . H o , C . M a t t h i j s s e n and

H . C . Neu, J . A n t i m i c r o b . Agents Chemother, -1 6 , 592,(1979).
1 4 . E. S c h r i n n e r , M. L i m b e r t , L. P e n a s s e and A. Lutz
J. A n t i m i c r o b i a l Chemotherapy, -
6 , Suppl. A , p. 25, (1980).
15. B.P. (1980) "The B r i t i s h Pharmacopeia" Her M a j e s t y

S t a t i o n e r y O f f i c e , Cambridge ( 1 9 8 0 ) .
16. D.S. Reeves, L.O. White, H.A. H o l t , D . B a h a r i ,

M. J . Bywater and R . P . Bax, J. A n t i m i c r o b i a l Chemotherapy,
-
6, Suppl. A , p. 93, ( 1 9 8 0 ) .
1 7 . T. Bergan, R. S o l b e r g , Chemotherapy ( B a s e l ) , 27 (3),

155, (1981).
18. F. Kees, E. S t r e h l , K. S e e g e r , G . S e i d e l , P. Dominiak

and H. G r o b e c k e r , Arzneim. F o r s c h . , 31 (l), 3 6 2 , ( 1 9 8 1 ) .
19. R. Wise, N . Wright and P . J . W i l l s , J . A n t i m i c r o b .

Agents and Chemother., 9, 526, ( 1 9 8 1 ) .
20. F . J . Muhtadi, M.M.A. Hassan and M.M. Tawakkol,

Spectroscop. l e t t . I n P r e s s (1982).
21. D.C. Grove and W.A. R a n d a l l , Assay Methods of

A n t i b i o t i c s , A n t i b i o t i c Monograph, Vol. 2. M e d i c a l
E n c y c l o p e d i a , N e w York, p. 3 4 , ( 1 9 5 5 ) .
22. Y . Chabbert and H. B o u l i n g e r , Rev. F r a n c . E t . C l i n .

B i o l . (-
2 ) 636, (1957).
23. N . Lambert-Zechovsky, C . A u f r a n t , E . Bingen, C. Blunn,

M.C. Proux and H . M a t h i e u , J. A n t i m i c r o b i a l Chemo-
therapy, -6 , S u p p l . A, p. 235, ( 1 9 8 0 ) .
24. H. Lode, B. Kemmerich, G . G r u h l k e , G. D z w i l l o ,

P. Koeppe and I . Wagner, J . A n t i m i c r o b i a l Chemotherapy,
6, Suppl. A, p. 193, ( 1 9 8 0 ) .
-
25. W.M. Glb'ckner, U. H o f f l e r , J. K i n d l e r , G. P e t e r s and

H.G. S i e b e r t h , J . A n t i m i c r o b i a l Chemotherapy, 5,
Suppl. A , p. 219, ( 1 9 8 0 ) .
26. J . P . F i l l a s t r e , A. L e r o y , G. Humbert and M. Godin,

J . A n t i m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 1 0 3 ,
(1980).
Acknowledgment
The a u t h o r s would l i k e t o t h a n k R o u s s e l - U c l a f ,
P a r i s , France f o r kindly providing cefotaxime
sample.
CEFOXITIN, SODIUM
Gerald S. Brenner
I . Introduction 170
1.1 History 170
1.3 Definition 170
2.1 Infrared Spectrum 171
2.2 Magnetic Resonance Spectra 171
2.3 Ultraviolet Spectrum 179
2.4 Mass Spectrum 179
2.6 Solubility 182
2.8 Hygroscopicity 183
2.9 Acid Dissociation Constant 183
3. Synthesis 183
4. Stability-Degradation Products 185
4.1 Bulk Stability 185
4.2 Solution Stability 186
5 . Pharmacokinetics and Metabolism 187
5.1 Pharmacokinetics 187
5.2 Metabolism 187
5.3 Intramuscular Absorption and Bioavailability 187
5.4 Effect of Probenecid 188
6.2 Ultraviolet Spectrophotometric Analysis 189
6.3 Chromatographic Analysis 189
6.4 Perchloric Acid Titration 190
6.5 lodometric Assay 190
6.7 Hydroxylamine Assay 190
7. Determination in Biological Fluids 191
8. References 192
Analytical Profiles of Drug Substances Copyriaht Q 1982 by The American

Volumc I I I69 Pharmaceutical Association
ISBN &12-260(111-9
170 GERALD S. BRENNER
1. Introduction
1.1 Historv
Sodium cefoxitin, a semi-synthetic cephamycin anti-
biotic was synthesized, tested and developed by the Merck
Sharp and Dohme Research Laboratories. Cefoxitin is active
against Gram-positive and Gram-negative bacteria ( 1 ) and is
therapeutically important because of its resistance to de-
struction by bacterial 6-lactamase (2,3,4).
The Chemical Abstracts name for sodium cefoxitin
(MK-306) is 3-[: [Aminocarbonyl)oxylmethyl I-7-methoxy-8-0x0-
7~~2-thienylacetyl)aminol-5-thia-l-azabicyclo~4.2.Oloct-2-
ene-2-carboxylic acid sodium salt. The CAS registry no. is
33564-30-6.
Other names include 3-carbamoyloxymethyl-7a-methoxy-
7~-(2-thienylacetamido)-3-cephem-4-carboxylic acid sodium
salt, sodium 3-carbamoyloxymethyl-7-methoxy-7-[2-(2-thienyl~
acetamidol-3-cephem-4-carboxylate and sodium (6R,7R)-3-(hy-
droxymethyl)-7- -methoxy-8-oxo-7-[2-(2-thienyl)acetamidol-5-
thia-l-azabicyclo[4.2.Oloct-2-ene-2 carboxylate carbarnate
(ester) .
‘1 6H16N3Na07S2 Molecular Weight: 449.44

1.3 Definition
Sodium cefoxitin, unless specified otherwise, is
defined as the crystalline, non-solvated salt form of the
compound. Cefoxitin when specified refers to the free acid.
1 .4
Appearance, Color, Odor
White to off-white granules or powder having a
slight characteristic odor.
CEFOXITIN, SODIUM 171
2.1 Infrared Spectrum
The infrared spectrum of c e f o x i t i n presented i n
Figure 1 was taken i n a KBr p e l l e t using a P e r k i n Elmer
Model 621 infrared spectrophotometer. A list of t h e
assignments made f o r some of t h e c h a r a c t e r i s t i c bands is
given i n Table I ( 5 ) .
Table I
IR Spectral Assignments f o r Cefoxitin
Frequency (ern-') Assignment
3200-3500 various N-H s t r e t c h

3000 (broad) OH s t r e t c h
1780 B-lactam C=O s t r e t c h
1715 carboxyl C=O s t r e t c h
1680 carbamate C=O s t r e t c h
1660 amide C=O stretch
1420 various -CH2 bends
1080 carbamate C-0 s t r e t c h
The infrared spectrum of sodium c e f o x i t i n taken a s

a mineral o i l m u l l is shown i n Figure 2.
2.2 Magnetic Resonance Spectra

2.21 Proton Spectrum
The proton magnetic resonance spectrum of
c e f o x i t i n presented i n Figure 3 was obtained using a Varian
A-60A spectrometer with DMSO-d6 a s t h e solvent. The
s p e c t r a l assignments a r e given i n Table I1 ( 6 ) . The proton
magnetic resonance spectrum f o r sodium c e f o x i t i n i n
DMSO-d6 is shown i n Figure 4 .
Table I1
Proton Magnetic Resonance Assignments f o r Cefoxitin
ppm ( 6 ) Relative I n t e n s i t y Multiplicity Assignment
9.38 1 Singlet 0
II
-C-NH-
7.34 1 Mu 1ti p l et thienyl-a
G -85-7.05 2 Mu1t i p l e t thienyl-B
0
-% u
a,
0
-0 c
a0 .rl
U
x
C
0 'z
-O 0
9 -
ir
V
m
!-I
w
C
H
I I 1 I
0 0 0 0 0 0
g c o W c t N
O/o 3 3N V l1I W S NV t l l
172
0
0
P-
O
0
al
H
8
0
r
I
0
0
0
2
0
0
::
0
0
0
w
I73
174
A
1m
FIGURE 4
I
A
I
I . J . I .
I
. .
,
.
,
I
, ,
. . . . I ,
,
. .
,
. I
,
.
,
, .
,. 1
,
,
,
. .
,
.
,
1 .
,
. .
,
.
.
1 .
,
. . . I
8.0 7.0 6.0 5.0 4.0 3.0 2.0 10 0

PPM
Figure 4 . Proton Magnetic Resonance Spectrum of Sodium Cefoxitfn in DMSO-d 6 '

Table I1 (cont'd)
ppm (6 1 Relative Intensity Multiplicity Assignment
6.52 2 Singlet 0
I1
0-c-NH2
5.13 1 Singlet 6-H
4.49-5.0 2 Mu1tiplet 0
II
-CH20C-
0
3'83
3.08-3.80
3.40
} 6-7
Singlet
Mu1tiplet
Singlet
thien yl-CH2-C
b-CH
7-OC~;~
II
2.22 Carbon-13 Spectrum

The carbon-13 magnetic resonance spectra of
cefoxitin presented in Figures 5 and 6 were obtained using a
Varian CFT-20 spectrometer with DMSO-d as the solvent at
a concentration of 0.5 -
M. The spectra assignments are P
given in Table I11 (7).
Table I11
Carbon-13 Magnetic Resonance Assignments for Cefoxitin
ppm( 6 ) Assignment
170.4 amide C=O
162.5 carboxyl C=O
160.3 C8, lactam C=O
156.3 carbamate C=O
136.5 c21
126.5
126.4} C31 and C4l
c a r boxy I
\
onide
C
\
c
corbomok
7 ow3
I
J1
I c,
175 I60 PPM I25
Figure 6. C-13 WiA Spectrum of Cefoxitin in DMSO-d

6'
Table I11 (cont'd)
ppm ( 6 ) Assignment
125.8
125.5
124.9 c j1
95.1 c7
62.8 c6
61.9 C
52.5 OEB, -I
0
II
35.9 thien yl-CH2-C-
-
25.7 c4
2.3 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t absorDtion sDectrum o f c e f o x i t i n i n
pH 7.0 phosphate buffer is chabacterized by a peak a t about
2 5 nm and a shoulder a t about 262 nm (Figure 7) with
A?$m values of approximately 330 and 209 respec-
t i v e l y . The shoulder a t 262 nm is d u e t o t h e 3-cephem
chromophore and t h e maximum a t 235 nm is due to thienyl-
a c e t y l s i d e chain.
2.4 Mass Spectrum

The mass spectrum o f c e f o x i t i n was obtained u s i n g a
Finnigan 3200 mass spectrometer i n t h e e l e c t r o n impact moie
w i t h an ionizing energy of 70 eV and a probe temperature of
175OC. The output from t h e mass spectrometer was analyzed
(8) and presented i n t h e form of a bar graph shown i n Figure
8.
The ion of highest mass seen i n t h e spectrum o f
c e f o x i t i n f r e e acid (molecular weight 427) is m/e 366, which
0
corresponds t o t h e lactone formed from t h e
C H 2-CO N H OCH3 ~cr''

N N
0 CH2
0
d
Cefoxit i n 1ac tone
\
\ \
I
250 nm 300 nm
Figure 7. UV Spectrum of Cefoxitin in pH 7 Phosphate Buffer.

Concentration: 2.75 mg/100 ml.
180
“1
D
Ln
(3
c
.d
U
*r(
x
0
u
a
D CJ
D
(3 w
0
D
u)
N
c
0
.rl
U
D
D 3
N 4
0
rn
a
d
D
2
c 0
0, 0
+
D
Q D
d In
AlISN31NI 3 A l l V l 3 Y
181
carboxylic acid and carbamate group. The n e x t ion (m/e 322)

corresponds t o l o s s o f C02 from the l a c t o n e . Scission o f
t h e s i d e chain amide bond of t h e l a c t o n e g i v e s m/e 241,
which l o s e s CH20
J
to give m/e 210. A t lower mass one s e e s
m /e 124,
@CH=C=O +
m/e 112,
m/e 97,

The s p e c i f i c r o t a t i o n o f a 1% (w/v) sodium
c e f o x i t i n s o l u t i o n i n methanol determined a t 509 nm and
25OC i s +210° + 4' calculated on an anhydrous and
solvent f r e e b a s i s .
2.6 Solubility
The s o l u b i l i t y o f sodium c e f o x i t i n i n t h e following
s o l v e n t s a t room temperature is s t a t e d i n terms of t h e
U.S.P. d e f i n i t i o n s : v e r y s o l u b l e i n water, s p a r i n g l y
soluble i n methanol and dimethylformamide, s l i g h t l y s o l u b l e
i n ethanol and i n s o l u b l e i n e t h e r , chloroform, acetone
aromatic and a l i p h a t i c hydrocarbons.
The s o l u b i l i t y of c e f o x i t i n i n water is t y p i c a l of
an organic acid with l i m i t e d aqueous s o l u b i l i t y and
increases with increasing pH. A t pH 1 , t h e measured
s o l u b i l i t y is about 0.3 mg/ml and a t pH 4 , t h e observed
value is about 25 mg/ml ( 9 ) .
2.7 Crvstal ProDerties

2.571 C r y s t a l l i n i t y
Sodium c e f o x i t i n exists i n s e v e r a l solvated
and i n a desolvated c r y s t a l l i n e form. An amorphous form has
a l s o been i d e n t i f i e d . X-ray powder d i f f r a c t i o n p a t t e r n s and
polarized l i g h t microscopy can d i s t i n g u i s h c r y s t a l l i n e from
amorphous forms ( 1 0 ) .
2.72 X-Ray Powder Diffraction

The x-ray powder d i f f r a c t i o n p a t t e r n o f
c r y s t a l l i n e desolvated sodium c e f o x i t i n was obtained with a
Philips-Norelco d i f f r a c t o m e t e r , using copper Kcc r a d i a t i o n .
Samples of t h i s form show broad d i f f r a c t i o n l i n e s suggesting
t h a t t h e s o l i d is m i c r o c r y s t a l l i n e and composed of extremely
small c r y s t a l l i t e s ( 1 1 ) . The amorphous form shows no
d i s t i n c t X-ray powder d i f f r a c t i o n p a t t e r n .
2.73 D i f f e r e n t i a l Scanning Calorimetry

C r v s t a l l i n e sodium c e f o x i t i n e x h i b i t s a
decomposition exotherm a t approximately 19O0-2OO0C by
d i f f e r e n t i a l scanning calorimetry (DSC). The amorphous form
does not e x h i b i t any well-defined thermal t r a n s i t i o n s by DSC
below 35OoC. Gradual decomposition of t h i s s o l i d is
observed by DSC a s temperatures increase over 15OoC ( 1 1 ) .
2.8 Hygroscopicity
The water uptake o f sodium c e f o x i t i n a s a function
of r e l a t i v e humidity was s t u d i e d a t ambient temperature
( 1 1 ) . Equilibrium water levels were approximately I-2% a t
35% RH, 4-6% a t 47% RH and 15-17% a t 76% RH.
2.9 Acid Dissociation Constant

The d i s s o c i a t i o n constant o f c e f o x i t i n derived from
aqueous t i t r a t i o n (12) and s o l u b i l i t y d a t a (9) a r e i n good
agreement and i n d i c a t e t h a t c e f o x i t i n i s a r e l a t i v e l y s t r o n g
organic acid with a pKa of approximately 2.2.
3. Synthesis
Cefoxitin has been prepared by chemical modification of
cephamycin C , a n a t u r a l l y occuring a n t i b i o t i c produced by
Streptomyces lactamdurans (13, 1 4 ) . This r o u t e i s presented
i n Figure 9. Cepharnycin C (I) is tosylated t o t h e N-tosyl
d e r i v a t i v e (11) and then e s t e r i f i e d w i t h methyl chloromethyl
ether t o y i e l d t h e methoxymethyl ester (111). The ester i s
t h e n t r e a t e d with 2-thienylacetyl c h l o r i d e t o exchange t h e
aminoadipoyl s i d e chain f o r t h i e n y l a c e t y l . The ester
protecting group i s then removed w i t h acid t o y i e l d
c e f o x i t i n (IV).
r II:
I
1 CICH20C H3
Figure 9
S y n t h e s i s of C e f o x i t i n from Cephamycin C
C e f o x i t i n h a s a l s o been p r e p a r e d b y t o t a l s y n t h e s i s (15)
and v i a t h e a c y l a t i o n and m e t h o x y l a t i o n of 7-amino
c e p h a l o s p o r a n i c a c i d ( 1 6 , 17, 18, 19).
4. Stability-Degradation Products
4.1 Bulk S t a b i l i t v
A t room t e m p e k a t u r e sodium c e f o x i t i n i s s t a b l e for
a t l e a s t t h r e e y e a r s when p r o t e c t e d from m o i s t u r e . A t ele-
v a t e d t e m p e r a t u r e , t h e s o l i d e x h i b i t s a b i p h a s i c decomposi-
t i o n p a t t e r n t y p i f i e d by an i n i t i a l more r a p i d d e c o m p o s i t i o n
p e r i o d f o l l o w e d by a slower d e c a y p e r i o d ( 2 0 ) . T h i s phenome-
non may b e related t o d e g r a d a t i o n of c e f o x i t i n b y low l e v e l s
of w a t e r i n t h e sample. S i n c e 6 - l a c t a m c l e a v a g e is water-
consuming, t h e e x t e n t of t h i s d e g r a d a t i o n pathway is l i m i t e d
by a v a i l a b l e water i n t h e s o l i d . Amorphous sodium c e f o x i t i n
h a s been shown t o be c o n s i d e r a b l y less s t a b l e t h a n i t s
c o r r e s p o n d i n g c r y s t a l l i n e form ( 2 0 ) .
A t e m p e r a t u r e d e p e n d e n t d i s c o l o r a t i o n of t h e s o l i d
h a s been n o t e d . The d i s c o l o r a t i o n i s n e g l i b l e a t 5OC and
becomes greater a t e l e v a t e d t e m p e r a t u r e . I t h a s been shown
t h a t an i n e r t a t m o s p h e r e ( a r g o n or n i t r o g e n ) m a r k e d l y
d e c r e a s e s t h i s change. T h i s development of color is n o t
d i r e c t l y r e l a t e d t o l o s s of p o t e n c y , i . e . c o n s i d e r a b l e
d i s c o l o r a t i o n c a n o c c u r w i t h no m e a s u r a b l e loss of p o t e n c y .
S e v e r a l d e g r a d a t i o n p r o d u c t s of s o l i d sodium
c e f o x i t i n h a v e been i d e n t i f i e d . From material s t o r e d a t
6OoC for t h r e e d a y s , two compounds h a v e been i s o l a t e d and
identified.
I,p
4.2 Solution S t a b i l i t y
The s o l u t i o n s t a b i l i t y o f sodium c e f o x i t i n has been
studied i n aqueous b u f f e r s i n t h e pH range 3 t o 9 ( 2 0 ) . The
degradation of sodium c e f o x i t i n i n t h i s pH range follows
apparent f i r s t - o r d e r k i n e t i c s . Maximum s t a b i l i t y i n water
i s i n t h e pH range o f 5-7. Under these pH c o n d i t i o n s ,
sodium c e f o x i t i n undergoes 10% chemical l o s s i n two days a t
25OC. Ten percent l o s s a t pH 3 occurs i n about 40 hours
and a t pH 9 i n about 14 hours. TLC s t u d i e s were c a r r i e d o u t
during k i n e t i c r u n s . P a t t e r n s become complex suggesting
t h a t t h e i n i t i a l B-lactam hydrolysis product i s unstable and
s u s c e p t i b l e t o transformation t o a considerable number of
secondary products (20).
The s o l u t i o n s t a b i l i t y o f sodium c e f o x i t i n was a l s o

studied a f t e r c o n s t i t u t i o n with frequently used I . V .
infusions and admixture w i t h comnonly used I . V . and I.M.
a d d i t i v e s (21 1. S t a b i l i t y i n t h e s e systems was e s s e n t i a l l y
t h e same a s t h a t observed f o r unbuffered s o l u t i o n s . I n
t h e s e s t u d i e s , sodium c e f o x i t i n was shown t o maintain
potency i n s o l u t i o n f o r a t l e a s t 30 days a t 5OC and f o r 30
weeks when stored i n t h e frozen s t a t e .
I n an attempt t o i s o l a t e degradation products

formed i n s o l u t i o n , a 10% aqueous s o l u t i o n of sodium
c e f o x i t i n was heated f o r four days a t 8OoC and then
subjected t o preparative TLC. F r a c t i o n s i s o l a t e d were
examined by NMR, mass spectrometry and i n f r a r e d . The
following compounds were i d e n t i f i e d .
Thiophene-2-acetic acid Thiophene -2- acetamidr
CON+
N- ( 2'-mothoxyacotamido) thiophene-
2- ace t am ido
5. Pharmacokinetics and Metabolism

The pharmacokinetics and metabolism of sodium c e f o x i t i n
i n humans following parenteral administration have been t h e
subject of a number of i n v e s t i g a t i o n s and reviews
(22,31,32).
5.1 Pharmacokinetics
Followina intravenous administration (bolus o r
i n f u s i o n ) , c e f o x i t i n i s d i s t r i b u t e d r a p i d l y between serum
and t i s s u e and e x h i b i t s a terminal serum h a l f - l i f e of 30 t o
50 m i n u t e s . Total body clearance of c e f o x i t i n ranges from
approximately 250 m l t o 350 m l / m i n while r e n a l clearance i s
approximately 200 t o 300 m l / m i n . Urine contains a t l e a s t
90% of t h e dose a s unchanged drug and less than 5% of t h e
dose i s eliminated by metabolism and b i l i a r y clearance
(23,24,25). The d i s p o s i t i o n k i n e t i c s are f i r s t o r d e r ,
showing no e f f e c t of dose (0.25 t o 3 g.) or infusion r a t e .
Multiple dose regimens i n t h i s range given every f o u r hours
do not cause accumulation i n healthy volunteers. The volume
of d i s t r i b u t i o n i n t h e vascular compartment is about 8
l i t e r s (26-34). These d a t a a r e adequately described by a
two-compartment open model w i t h elimination occurring from
t h e c e n t r a l compartment.
5.2 Metabolism
Sodium c e f o x i t i n i s not metabolized appreciably i n
man. Urine samples from s e v e r a l human s t u d i e s were sepa-
rated by HPLC and TLC techniques. These s t u d i e s show t h a t
more than 90% of t h e c e f o x i t i n administered by e i t h e r t h e
intravenous o r intramuscular route i s recovered i n t h e u r i n e
a s i n t a c t drug. A microbiologically i n a c t i v e metabolite,
descarbamoyl c e f o x i t i n , was found t o t h e extent of 1-6% i n
some i n d i v i d u a l s , 2 t o 4 hours post dosing (26,271. This
metabolite was not found i n t h e u r i n e o f a l l s u b j e c t s .
Descarbarnoyl c e f o x i t i n
188 GERALD S . BRENNER
5.3 Intramuscular Absorption and B i o a v a i l a b i l i t y

Sodium c e f o x i t i n is raDidly and completely absorbed
following intramuscular administration. Peak serum levels
a r e a t t a i n e d i n 30 m i n u t e s o r less and 85 t o 95% of t h e
i . m . dose is recovered i n u r i n e within 12 hours of
administration.
Intramuscular administration o f sodium c e f o x i t i n

with either 0.5 o r 1.0% l i d o c a i n e hydrochloride a s a d i l u e n t
has no apparent e f f e c t on t h e b i o a v a i l a b i l i t y o f t h e
an tibiotic (32,35>.
5.4 E f f e c t o f Probenecid
The concurrent o r a l o r intravenous administration
of probenecid with i . m . o r i . v . i n j e c t i o n s of c e f o x i t i n has
a l a r g e influence on t h e time course o f t h e a n t i b i o t i c i n
serum (26,36). Probenecid administered intravenously
concurrent with c e f o x i t i n i n c r e a s e s t h e serum h a l f - l i f e o f
c e f o x i t i n from approximately 40 minutes t o 80 m i n u t e s and
reduces t h e r e n a l clearance of t h e drug from 200-300 ml/min
t o less than 100 m l / m i n .
6. Methods o f Analysis
6 . 1 I d e n t i f i c a t i o n Tests
U l t r a v i o l e t spectrophotometry i s used t o i d e n t i f y
sodium c e f o x i t i n . The spectrum o f a sample dissolved i n pH
6.0 phosphate buffer scanned from 220 t o 310 nm compares
q u a l i t a t i v e l y t o t h a t of a c e f o x i t i n standard s i m i l a r l y
tested.
The i d e n t i t y o f sodium c e f o x i t i n is a l s o
established by i n f r a r e d spectroscopy. The i n f r a r e d
absorbance spectrum of a s o l i d sample prepared e i t h e r a s a
potassium bromide d i s c o r mineral o i l m u l l is compared t o a
standard sample prepared i n an i d e n t i c a l manner.
A color test has been employed f o r t h e d e t e c t i o n of

c e f o x i t i n i n s o l u t i o n (37). To t h e r e s i d u e obtained by
drying a s o l u t i o n containing 10-50 mcg of c e f o x i t i n i s added
1-2 m l of 0.01% ninhydrin i n concentrated sulfuric acid and
t h e color is allowed t o develop a t room temperature. A
vivid blue c o l o r appears i n a few m i n u t e s . Other
cephalosporin a n t i b i o t i c s do not give t h e same c o l o r
response.
Cefoxitin and f i f t e e n other cephalosporins have

been i d e n t i f i e d by thin-layer chromatography coupled with
color r e a c t i o n s (38) and by spectroscopic methods (39).
6.2 U l t r a v i o l e t Spectrophotometric Analysis
I n t a c t sodium c e f o x i t i n e x h i b i t s a UV absorption
band near 262 nm a t t r i b u t e d t o t h e OX-N-C=C linkage i n t h e
molecule. Beta-lactam r i n g opening l e a d s t o disappearance
of t h i s absorption band. This observation is t h e b a s i s of a
q u a n t i t a t i v e assay f o r c e f o x i t i n which i s s t a b i l i t y
indicating. Calculation of i n t a c t compound i s based on t h e
net absorbance a t 262 nm of t h e sample and of t h e standard
i n pH 6.0 phosphate buffer a s determined by s u b t r a c t i n g a
base l i n e correction a t 262 nm from t h e maximum a t t h e same
wavelength. The correction i s found by extending t o 262 nm
t h e s t r a i g h t portion of t h e UV curve between 340 and 300 nm.
6.3 Chromatographic Analysis

6.31 Thin Layer Chromatography
Thin layer chromatography using t h e system
chloroform/acetone/formic acid (10:9: 1 ) w i t h 0.25 rnm s i l i c a
gel p l a t e s has been employed f o r both sodium c e f o x i t i n and
the f r e e acid. The a i r dried p l a t e is sprayed w i t h 0.2% p-
dimethylaminocinnamaldehyde i n methanol/conc. s u l f u r i c acid
( 4 : l ) and heated a t 105' f o r f i v e m i n u t e s . The Rf f o r
c e f o x i t i n i n t h i s sytem is approximately 0.45.
Cefoxitin has a l s o been chromatographed on

s i l i c a w i t h developing s o l v e n t s of n-butanol/water/acetic
acid ( 4 : l : l ) and benzene/methanol/acetic acid (50:10:6)
giving approximate Rf values of 0.7 and 0.2 r e s p e c t i v e l y .
Detection can be accomplished by fluorescence quenching or
iodine s t a i n i n g .

Several HPLC procedures have been developed
t o s e p a r a t e c e f o x i t i n from process i m p u r i t i e s and
degradates. A system frequently used is described below.
Mobile Phase: 20% a c e t o n i t r i l e i n water

containing 1% a c e t i c acid.
Column : Ten micron, microporous

oct,adecylsilane bonded reversed phase packing i n a 3.9 mn X
30 cm column. Column temperature and pressure a t 25OC
( o r ambient) and 1000-1500 p s i g , respectively. Flow r a t e of
I .O-l.3 ml/min.
Detection: U.V. a t 254 nm

Procedure: Aliquots (10 mcl) o f a sample

s o l u t i o n containing 0.25 m g h l i n 0.02 M pH 7 phosphate
buffer a r e i n j e c t e d . The r e t e n t i o n time
f o r c e f o x i t i n is
approximately 10-15 m i n u t e s .
6.4 Perchloric Acid T i t r a t i o n

Sodium c e f o x i t i n can be determined by non-aqueous
t i t r a t i o n . An accurately weighed sample of about 800 mg i s
dissolved i n about 100 m l of g l a c i a l a c e t i c acid and t i t r a -
t e d potentiometrically w i t h standardized 0.1N p e r c h l o r i c
acid i n s p e c t r a l grade dioxane. An e l e c t r o d z p a i r i s
employed c o n s i s t i n g of a calomel e l e c t r o d e which has been
r e f i l l e d w i t h 0.1N LiC104 i n a c e t i c anhydride a s t h e
i n d i c a t i n g e l e c t r o d e , and a platinum r i n g a s t h e r e f e r e n c e
electrode.
6.5 Iodometric Assay

Sodium c e f o x i t i n bulk chemical and formulations can
be determined by iodometric assay. I n t h e assay, a l i q u o t s
of t h e sample and o f a s u i t a b l e c e f o x i t i n standard s o l u t i o n
a r e hydrolyzed f o r 20 minutes with 1 N NaOH and then
a c i d i f i e d t o pH 3.0 with a c e t a t e bufTer and I N H C 1 . Then,
0.01N iodine s o l u t i o n is added and, a f t e r a 2 h i n u t e w a i t ,
t h e excess iodine is t i t r a t e d with 0.01N sodium t h i o s u l f a t e
s o l u t i o n t o a s t a r c h end p o i n t . A blank determination i s
made a t t h e same time on a l i q u o t s o f both sample and
standard s o l u t i o n s t r e a t e d only with buffer and 0.01N iodine
and allowed t o stand f o r 20 minutes.
An automated iodometric assay has been developed

based on the manual method described above w i t h t h e
exception t h a t excess iodine is measured c o l o r i m e t r i c a l l y
r a t h e r than by t h i o s u l f a t e t i t r a t i o n ( 4 0 ) .
6.6 Microbiological Assay

For bulk and formulated products an agar d i f f u s i o n
( p l a t e ) assay can be conveniently c a r r i e d out using
Staphylococcus aureua a s t h e t e s t organism ( 4 1 ) .
6.7 Hydroxylamine Assay
Sodium c e f o x i t i n can be determined by means of t h e
colored complex formed between f e r r i c ion and t h e hydroxamic
acid formed by t h e a c t i o n o f hydroxylamine on t h e beta-
lactam ( 4 2 ) . This method has been s u c c e s s f u l l y automated
and has been specified by t h e FDA a s t h e d e f i n i t i v e assay

method f o r c e r t i f i c a t i o n of t h e a n t i b i o t i c .
7. Determination i n Biological F l u i d s
High performance l i q u i d chromatography has been u t i l i z e d
f o r t h e determination of c e f o x i t i n i n b i o l o g i c a l f l u i d s .
Cefoxitin and i t s descarbamoyl metabolite have been
q u a n t i t a t i v e l y analyzed i n human urine employing an anion-
exchange column w i t h U.V. detection ( 2 3 ) . More r e c e n t l y
Wheeler, e t a l . (43) have employed HPLC u t i l i z i n g a C-18
reversed phase packing and a solvent system of
a c e t o n i t r i l e / a c e t i c acid/O. 005M potassium dihydrogen
phosphate (25/0.5/74.5, v / v / v ) f o r the q u a n t i t a t i o n of
c e f o x i t i n i n serum and s a l i v a .
An HPLC method has been developed f o r t h e determination

of c e f o x i t i n i n serum which l e n d s i t s e l f t o automation
( 4 4 ) . The system is described below:
Mobile Phase: 11% methanol i n pH 6.86 buffer
containing 1% a c e t o n i t r i l e .
Column: Ten micron C-8 reversed phase packed i n a

3.9 mn x 25 cm column with a disposable precolumn i n l i n e
and a precolumn i n the i n j e c t i o n loop. A flow r a t e o f 3.0
m l / m i n is used.
Detection: U.V. a t 254 nm.
Procedure: Serum samples (100 1.111 a r e t r e a t e d with

100 1 of i n t e r n a l standard (aqueous cefmetazole, 15 u l / m l
and 75 u 1 10% t r i c h l o r o a c e t i c a c i d . SarnDles a r e m i x e d ,
allowed t o stand for 15 m i n u t e s and centkifuged t o remove
p r e c i p i t a t e d protein. For automated a n a l y s i s , c l e a r
supernatant is t r a n s f e r r e d t o microcentrifuge tubes. A
standard curve i s generated by spiking blank serum with
appropriate levels of c e f o x i t i n and then processing a s
described above.
Cefoxitin i n b i o l o g i c a l f l u i d s has a l s o been determined

microbiologically by t h e cup-plate diffusion-technique using
e i t h e r Staphylococcus aureus MB2876 (26,27) o r B a c i l l u s
s u b t i l i s MB36 (35) a s t h e t e s t organism.
192 GERALD S.BRENNER
8. References
1 . H. R . Onishi, D. R. Daoust, S. B. Zimnerman, D. Hendlin

and E. 0. Stapley, "Abstracts, XI1 I n t e r s c i e n c e Confer-
ence on Antimicrobial Agents and Chemotherapy", A t l a n t i c
C i t y , N.J., 1972, p. 77 and preceding two a b s t r a c t s .
2. G. Darland and J . Birnbaum, Antimicrob. Agents Chemo-

ther. -
1 1 , 725 (1977).
3. H. R . Onishi, D. R . Daoust, S. B. Zimerman, D. Hendlin

and E. 0. Stapley, Antirnicrob. Agents Chemother., -
5 , 38
(1974).
4 . H. C. Neu, Antimicrob. Agents Chemother., 6, 170 (1974).
5. J . A. Ryan and J . Stevenson, Merck Sharp and Dohme

Research Laboratories, personal communication.
6. A. Douglas, Merck Sharp and Dohme Research Laboratories,

personal comnunication.
7. A. Douglas, Merck Sharp and Dohme Research Laboratories,

personal communication.
8. W. J . A. Vandenheuvel, Merck Sharp and Dohme Research

Laboratories, personal communication.
9. J . A. McCauley and A. Shah, Merck Sharp and Dohme

Research Laboratories, personal communication.
10. E. R. Oberholtzer and B. Singleton, Merck Sharp and

Dohme Research Laboratories, personal communication.
1 1 . E. R. Oberholtzer, Merck Sharp and Dohme Research Labora-

t o r i e s , personal communication.
12. G . Bicker, Merck Sharp and Dohme Research Laboratories,

personal communication.
13. S. Karady, S. H. P i n e s , L. M. Weinstock. F. E. Roberts,

G. S. Brenner, A. M. Hoinowski, T. Y. Cheng and M.
S l e t z i n g e r , J . Am. Chem. SOC. -
94, 1410 (1972).
14. L. M. Weinstock (Merck & Co., I n c . ) Fr 2,253,022; Ger
Offen 2,456,528; Jap. K75,105,687, Neth 7,414,820
15. R. W. R a t c l i f f e and B. G. Christensen, Tetrahedron Lett

4 6 , 4645 (1973); -
- 46, 4649(1973); 46, 4653 (1973).
16. G. G. Hazen (Merck and Co., Inc.) US 3,780,033.
17. B. C. Christensen and L. D. Cama (Merck & Co., Inc.) Fr
2,163,144; Ger Offen 2,258,278.
18. B. C. Christensen and R. A. Firestone (Merck & Co.,
I n c . ) US 3,775,410.
19. B. G . C h r i s t e n s e n e t . a].. (Merck & Co., Inc.) Brit

1,348,984; Ger Offen 2,143,331.
20. E. R . Oberholtzer and G . S. Brenner, J. Pharm. S c i . , -
68,
863 ( 1979).
21. M. J . O'Brien, J . B. Portnoff, and E. M. Cohen, Am. J .
Hosp. Pharm 36, 33 (1979).
22. K . C. Kwan and J . D. Rogers i n 6-lactam A n t i b i o t i c s

(Handbook o f Experimental Pharmacology) ed. A. L.
Demain. Springer-Verlag, Heidelberg, i n press.
23. R. P. Buhs, T. E. Maxim, N. Allen, T. A. Jacob and F.

J . Wolf, J . Chromatog -
99, 609 (1974).
24. A. M. Geddes, L. P. Schnurr, A. P. B a l l , D. McChie, G .
R. Brookes, R . Wise and J . Andrews, Br. Med. J . -
1 , 1126
( 1977).
25. M. N . Logan, R . Wise and R. P. Grimley, J . Antimicrob.

Chemother. -5 , 620 (1979).
26. C. S. Goodwin, E. B. Raffery, A. D. Goldberg, H. Skeggs,
A. E. T i l l , and C. M. Martin, Antimicrob. Agents
Chemother. - 6, 338 (1979).
27. P. F. Sonneville, R. R . Kartodirdjo, H. Skeggs, A. E.
T i l l and C. M. Martin, Europ. J . C l i n . Pharmacol. -
9,
397 (1976).
28,
28. C. Simon, E. Meyer and V. Malerizyk, Arm, Forsch. -
1541 (1978).
29. G . J . Pazin, S. N. Schwartz, M. Ho, J . A. Lyon and A.

W. Pasculle, Rev. I n f e c t . D.S. -
1 . 189 (1979).
30. J.J. Schrogie, R . 0. Davies, K . C. Yeh, D. Rogers, G .

I . Holmes, H. Skeggs and C. M. Martin, J . Antimicrob.
Chemother. -4 , :Suppl. B, 69 (1978).
31. J . J . Schrogie, J . D. Rogers, K . C. Yeh, R. 0. Davies,

C. I . Holmes, H. Skeggs and C. M. Martin, Rev. I n f e c t .
Disc. -1 , 90 (1979).
32. W. Brumfitt, J . Kosmidis, J . M. T. Hamilton-Miller and

J . N. G . G i l c h r i s t , Antimicrob. Agents Chemother. -
6 , 290
(1974).
33. A. M. Brisson, J.B. F o u r t i l l a n , D. Barthes, P. Courtois

and B. Bezq-Giraudon, Therapie 35, 209 (1980).
34. R . Wise, B. Cadge, A. P. Gillett and A. Bhamjee, J .

Infection 1 : Suppl. 1 , 49 (1979).
35. P. F. Sonneville, K . S. Albert, H. Skeggs, H. Centner,

K . C. Kwan and C. M. Martin, Europ. J . C l i n . Pharmacol.
-
12, 273 (1977).
36. P. H. Vlasses, A. M. Holbrook, J . J . Schrogie, J . D.

Rogers, R. K . Ferguson and W. B. Abrams, Antimicrob
Agents Chemo. Ther. 1, 847 (1980).
37. J . R. Carlin and T. A. Jacob, Merck Sharp and D o h e

Research Laboratories, personal comnunications.
40. J . M. Konieczny, Merck Sharp and Dohme Research Labora-

t o r i e s , personal communications.
41. Code of Federal Regulations, Sections 4 4 2 . 1 4 a ( b ) ( l ) ( i )

and 436.105.
42. Code o f Federal Regulations, Sections 4 4 2 , 1 4 a ( b ) ( l ) ( i i )

and 4 4 2 . 4 0 ( b ) ( l ) ( i i ) .
43. L. A. Wheeler, M. de Meo, B. D. Kirby, R. S. Jerauld and

S. M. Finegold, J . Chromatog. -
183, 357 ( 1980).
44. R. S h a f f e r , Merck Sharp and Dohme Research Laboratories,

personal comnunication.
L i t e r a t u r e Reviewed t o June 1981

Acknowledgement
The author wishes t o thank Mrs. E. Moyer f o r typing t h e

manuscript, Ms. F. R. Berg and Ms. A. M. Hendrick f o r
l i t e r a t u r e r e t r i e v a l work and Mr. C. Dillman and Ms. N .
G i l b e r t f o r preparation of the f i g u r e s .
CLOFIBRATE
Mahmoud M . A. Hassan and Aida A. Elaxxouny
1. Description 198
1.2 Formulae 198
1.4 Appearance, Color, Taste, Odor 199
2.1 Boiling Range 199
2.2 Density 199
2.3 Refractive Index 199
2.4 Solubility 199
3. Synthesis 207
4. Metabolism 209
5. Pharmacology 209
6. Methods of Analysis 21 1
6.1 Elemental Analysis 21 1
6.2 Identification Tests 21 1
6.3 Purity Tests 21 1
6.4 Official Methods 212
6.5 Ultraviolet Spectrophotometry 213
6.6 Thin Layer Chromatography 214
6.7 Thin Layer-Gas Liquid Chromatography 214
6.8 Gas Liquid Chromatography 214
6.9 Gas Chromatography-Mass Spectrometry 217
6.10 High-Performance Liquid Chromatography 218
7. Proton Magnetic Resonance Spectrometry 219
8. References 22 1
Analytical Profiles of Drug Substances Copyrilht 0 1982 by The American

Volume I I 197 Phlmuceuliul h o c u t i o n
ISBN & 1 2 - ~ l l - 9
198 MAHMOUD M. A. HASSAN AND AIDA A. ELAZZOUNY
1. Description
1.1 Nomenclature
1.11 Chemical Names
a. 2-(4-chlorophenoxy) -2-methylpropanoic acid

e t h y l ester.
b. E t h y l 2-p-chlorophenoxy isobutyrate
c. E t h y l 2-(p-chlorophenoxy)-2-methyl
propionate.
d. Propanoic a c i d , 2-(4-chlorophenoxy)-2-
methyl-, e t h y l ester.
1.12 Generic Name
Clof i b r a t e
1.13 Trade Names
Amotril ; Anparton ; Ateculon ; Atheropront ;

A t e r i o s a n ; Atromidin ; Atromid-s ; C l a r i p e x ;
Clobren-SF ; CPIB ; H y c l o r a t e ; Lipavion ;
Neo-Atromid ; Normet ; R e c o l i p ; Regelan ;
S e r o t i n e x ; Skleromexe ( 1 ) .
1.2 Formulae
1.21 Empirical
'1 2H15'3'
1.22 Structural
CLOFIBRATE 199
1.23 CAS R e g i s t r y No.
(637-07-0)
1.24 Wiswesser L i n e N o t a t i o n (2)
GR DOXVO 2
1.3 M o l e c u l a r Weight
242.71
1.4 Appearance, C o l o r , Taste, Odor
S t a b l e , c o l o r l e s s t o pale-yellow l i q u i d w i t h a f a i n t ,
c h a r a c t e r i s t i c o d o r and a c h a r a c t e r i s t i c t a s t e .
2.1 B o i l i n g Range
158-1 60
20
148-150
2.2 Density
d25 : 1.138 - 1.144

2.3 R e f r a c t i v e Index
n 2o
D
1.500 - 1.505
2.4 Solubility
Insoluble i n water, s o l u b l e i n acetone, a l c o h o l ,

benzene, and c h l o r o f o r m .
2.51 I n f r a r e d Spectrum
The I n f r a r e d Spectrum of C l o f i b r a t e i s r e c o r d -
ed a s a f i l m on a Unicam Sp-1025 S p e c t r o p h o t o -
meter and i s shown i n F i g . 1 . The a s s i g n m e n t s
f o r t h e c h a r a c t e r i s t i c bands i n t h e I n f r a r e d
Spectrum a r e l i s t e d i n T a b l e 1.
Fig. 1. I R Spectrum of Clofibrate as F i l m .
CLOFIBRATE 20 1
Table I
I R C h a r a c t e r i s t i c s of Clof i b r a t e
-1
Frequency Cm Assignments (3)
1750 C = 0 (ester).
1605
1590 C = C (aromatic).
1500
1150 c-0-c
1100 (C-0 s t r e t c h i n g ) .
1390
1375
(Symmetrical de-
format i o n ) .
830 a r o m a t i c para-
d i s u b s it u t i o n
770
68 0
c-c1
Other bands c h a r a c t e r i s t i c of C l o f i b r a t e are

3020, 2970, 1290, 1250, 1190, 1030, 1020, 980
and 920 Cm-l.
2.52 U l t r a v i o l e t Spectrum (UV)
The UV spectrum of c l o f i b r a t e i n methanol w a s

scanned from 400-200 nm u s i n g V a r i a n Cary 219
Spectrophotometer and i s shown i n F i g . 2 .
The spectrum e x h i b i t s t h r e e maxima a t 288
(708), 280 (10233) and 226 (8511).
2.53 Nuclear Magnetic Resonance Spectrum
2.531 Proton Spectrum
The p r o t o n NMR S p e c t r a of C l o f i b r a t e i n
d e u t e r a t e d chloroform and i n Acetone-&,
were recorded on a Varian T-60 A, 60 MHz
NMR S p e c t r o m e t e r , u s i n g t e t r a m e t h y l s i l a n e
as an i n t e r n a l reference. The PMR spec-

trum i n d e u t e r a t e d chloroform i s shown
i n F i g . 3. The PMR s p e c t r a l assignment
of c l o f i b r a t e a r e g i v e n i n T a b l e 2 ( 4 ) .
Table 2
PMR C h a r a c t e r i s t i c s of C l o f i b r a t e
Protons Chemical S h i f t s
C0Cl3 Acet one-D
6
-CH2CH3 1.20 1.23 t

-C-(CH ) 1.56 1.57 s
3 2
-CH CH 4.18 4.20 q
-
Four a r o m a t i c 6.96 7.03 g
pro t o n s
s = singlet; t - triplet; q = quartet
2.532 S N M R Spectrum
13C NMR Spectrum of c l o f i b r a t e i n deu-

t e r a t e d chloroform u s i n g t e t r a m e t h y l s i -
l a n e as a n i n t e r n a l r e f e r e n c e w a s r e c o r -
ded on a J e o l FX 100, 100 MHz i n s t r u m e n t
a t ambient t e m p e r a t u r e and u s i n g 10 mm
sample t u b e . The d a t a c o n s i s t of 8192
d a t a p o i n t s over a 5000 Hz S p e c t r a l Width.
The c o m p l e t e l y decoupled spectrum is
shown i n F i g . 4. The carbon c h e m i c a l
s h i f t v a l u e s , shown i n T a b l e 3 a r e d e r i v -
ed from b o t h a d d i t i v i t y p r i n c i p l e s and
t h e off-Resonance Spectrum F i g . 5 ( 5 ) .
CLOFI BRATE 203
Fig. 2. W Spectrum of Clofibrate in Methanol.
Fig. 3. PMR Spectrum of Clofibrate and Tetramethylsilane

in CDC13
F i g . 4. I3C-NMR Spectrum of C l o f i b r a t e i n CDC13
Fig. 5. 13C-NMR Proton-Coupled S p e c t r u m of C l o f i b r a t e i n

CDC13
CLOFIBRATE 205
Table 3
13C NMR C h a r a c t e r i s t i c s of C l o f i b r a t e
Carbon No. Chemical S h i f t Carbon No. Chemical S h i f t

p pm ppm
154.18 7 79.62
120.85 8 25.34
129.04 9 25.34
121.43 10 173.67
129.04 11 61.30
120.85 12 14.03
2.54 Mass Spectrum
The mass spectrum of c l o f i b r a t e ( F i g . 6 )

o b t a i n e d by e l e c t r o n impact i o n i z a t i o n shows
a molecular i o n M+ a t m / e 242 ( r e l a t i v e i n t e n -
s i t y 41.1%) and a b a s e peak a t 128. The pro-
posed f r a g m e n t a t i o n p a t t e r n i s p r e s e n t e d i n
T a b l e 4.
- -
180 190 200 210 220 230 240 250 260 270 280 290 300
Fig. 6. Mass Spectrum of C l o f i b r a t e .

CLOFIBRATE 207
Table 4
Mass F r a g m e n t a t i o n P a t t e r n of C l o f i b r a t e .
a
CB2 -
m/e -
RI% Ion
- m/e
- R I% Ion
-
243 100 M.H
+ 242 41.1 M+
169 27 a 169 33.1 a
129 10 - 130 33.0 b+2H
115 78 - 128 100.0 b
87 21.2 c+H
R I = Relative Intensity
Mass s p e c t r a l d a t a , b o t h by e l e c t r o n i m -
p a c t and by m e t h a n e c h e m i c a l i o n i z a t i o n have
a l s o been r e p o r t e d . ( 2 , 6, 7 ) .
3. Synthesis:
The s y n t h e s i s of C l o f i b r a t e h a s b e e n a c h i e v e d by
two main methods (8-15).
Method I : T h i s i n v o l v e s c o n d e n s a t i o n of p a r a - c h l o r o -
p h e n o l w i t h a c e t o n e and c h l o r o f o r m i n t h e p r e s e n c e o f
sodium h y d r o x i d e f o l l o w e d by e s t e r i f i c a t i o n of t h e
resulting acid, afforded clof ibrate.
Method I1 : By condensing phenol w i t h e t h y l 2-chloro-2-

methyl-propionate i n t h e p r e s e n c e of a s u i t a b l e dehydro-
c h l o r i n a t i n g a g e n t and t h e n c h l o r i n a t i o n of t h e r e s u l t a n t
product, afforded c l o f i b r a t e .
C~G OH + CH3COCH 3 + CHC13
,or +
dehydrochlor i n a t i n g
agent
OH
Clof i b r a t e
F-C00C2R5
cl- CH3
- CH3
I
c12
COOC2H5
Clof i b r a t e
CLOFIBRATE 209
4. Metabolism:
The metabolism of c l o f i b r a t e and d i f f e r e n t c l o f i b r i c
a c i d d e r i v a t i v e s i n s e v e r a l a n i m a l s p c e c i e s and i n man were
reported (16-22). I t h a s been shown t h a t c l o f i b r a t e i s
c o m p l e t e l y h y d r o l y s e d t o c l o f i b r i c a c i d (para-chlorophenoxy
i s o b u t y r i c a c i d ) which is t h e n c o n j u g a t e d and e x c r e t e d .
I n man t h e two c o n j u g a t e s of c l o f i b r i c a c i d found i n u r i n e
were p r e s e n t i n p l a s m a . The h i g h e s t plasma c o n c e n t r a t i o n s
of c l o f i b r i c a c i d m e t a b o l i t e s were g e n e r a l l y found i n pa-
t i e n t s w i t h r e n a l d i s e a s e . S i n c e c l o f i b r a t e undergoes
r a p i d h y d r o l y s i s i n v i v o and i n v i t r o , t h e r e s u l t i n g c l o -
f i b r i c a c i d i s persumed t o b e t h e a c t i v e d r u g . S t u d i e s i n
v i v o and i n v i t r o w i t h c l o f i b r a t e and c l o f i b r i c a c i d i n d i -
c a t e t h a t t h e l a t t e r may e x e r t i t s e f f e c t by m u l t i p l e modes
of a c t i o n ( 2 3 ) .
5. Pharmacolopy: (24-32)
Clof i b r a t e r e d u c e s e l e v a t e d t r i g l y c e r i d e and c h o l e s -
t e r o l c o n c e n t r a t i o n s i n serum; t h e e f f e c t on serum l i p o -
p r o t e i n s i s p a r t i c u l a r l y e v i d e n t on t h e v e r y l o w - d e n s i t y
f r a c t i o n . When a d m i n i s t e r e d t o r a t s , i t c a u s e d a d e c r e a s e
i n serum t r i g l y c e r i d e and a l t e r a t i o n s i n a d i p o s e t i s s u e
u p t a k e and release of L i p i d s ( 3 3 ) . The b i o c h e m i c a l changes
produced i n c l u d e a d e c r e a s e i n a d e n y l c y c l a s e a c t i v i t y ,
i n h i b i t i o n of acetyl-coenzyme. A carboxylase, i n h i b i t i o n
of c h o l e s t e r o l and t r i g l y c e r i d e b i o s y n t h e s i s . The i n h i b i -
t i o n of h e p a t i c t r i g l y c e r i d e f o r m a t i o n i s a n e a r l y meta-
b o l i c consequence of c l o f i b r a t e a d m i n i s t r a t i o n and p r e -
c e d e s t h e f a l l i n serum t r i g l y c e r i d e . I t s e f f e c t s on
blood c o a g u l a b i l i t y s u g g e s t t h a t i t may r e d u c e t h e hyper-
coagulability frequently associated with a t h e r o s c l e r o s i s .
Uric a c i d c o n c e n t r a t i o n s , where e l e v a t e d , h a v e f r e q u e n t l y
shown a t r a n s i e n t r e d u c t i o n , and t h e s h o r t e n i n g of t h e
r e c a l c i f i e d C l o t t i n g - t i m e , which o c c u r s d u r i n g post-pran-
d i a l lipacmia, i s prevented.
C l o f i b r a t e w a s f i r s t used i n c o n j u n c t i o n w i t h a n d e r o -
s t e r o n e b u t i t became e v i d e n t t h a t t h e e f f e c t s produced
were n o t enhanced by t h e s t e r o i d . I t i s used i n a t h e r o
s c l e r o t i c conditions manifested i n coronary h e a r t d i s e a s e
and i n c e r e b r a l and v a s c u l a r d i s e a s e s , i n f a m i l i a l hyper-
c h o l e s t e r o l em i a and i n x a n thoma t o u s c o n d i t i o n s . E x u d a t i v e
d i a b e t i c r e t i n o p a t h y h a s been improved by c l o f i b r a t e .
Samuel e t a 1 ( 3 4 ) r e p o r t e d t h e s i g n i f i c a n t r e d u c t i o n
of c h o l e s t e r o l l e v e l s by t h e combined o r a l a d m i n i s t r a t i o n
of neomycin and c l o f i b r a t e . Musa e t a 1 ( 3 5 ) s t u d i e d t h e
e f f e c t s of c l o f i b r a t e upon t h e d i s t r i b u t i o n , m e t a b o l i s m ,
210 MAHMOUD M. A. HASSAN AND AlDA A. ELAZZOUNY
t r a n s p o r t and plasma b i n d i n g of 1 3 1 1 - t h y r o x i n e i n e n t h y -
roid individuals. I t had no c o n s i s t e n t e f f e c t upon t h e
b i n d i n g c a p a c i t i e s of t h y r o x i n e - b i n d i n g g l o b u l i n o r
t h y r o x i n e b i n d i n g pre-albumin. The h e p a t i c d i s t r i b u t i o n
s p a c e and c o n t e n t of t h y r o x i n e - i o d i n e were lower a f t e r
c l o f i b r a t e . The h e p a t i c t h y r o x i n e c l e a r a n c e and plasma-
t o - l i v e r t h y r o x i n e f l u x were unchanged. C l o f i b r a t e d i d
n o t a l t e r t h e plasma p y r o x i n e i o d i n e , t h e d a i l y t h y r o -
x i n e degradation r a t e o r t h e t o t a l thyroxine d i s t r i b u t i o n
s p a c e . These f i n d i n g s f a i l e d t o s u p p o r t t h e h y p o t h e s i s
t h a t c l o f i b r a t e p r o d u c e s i t s h y p o l i p i d e m i c e f f e c t by
d i s p l a c i n g t h y r o x i n e from i t s b i n d i n g p r o t e i n s and s h u n t -
ing it i n t o t h e l i v e r .
H a r r i s o n and Harden ( 3 6 ) s t u d i e d t h e e f f e c t of- c l o -

f i b r a t e on 6 p a t i e n t s w i t h h y p o t h y r o i d i s m and i s c h e m i c
h e a r t d i s e a s e . The p a t i e n t s were m a i n t a i n e d on t h e maxi-
mum d o s e of t h y r o x i n e which t h e y c o u l d t o l e r a t e b u t a l l
s t i l l had e v i d e n c e o f h y p o t h y r o i d i s m and l e v e l s of cho-
l e s t e r o l i n t h e serum were e l e v a t e d . C l o f i b r a t e produced
a r a p i d f a l l i n serum c h o l e s t e r o l a v e r a g i n g 37% and w a s
m a i n t a i n e d up t o t h e two y e a r s t h e d r u g was c o n t i n u e d .
I n t h r e e p a t i e n t s i t was p o s s i b l e t o i n c r e a s e t h e d o s e of
t h y r o x i n e d u r i n g t h e r a p y w i t h c l o f i b r a t e ; i n two p a t i e n t s
w i t h u n t r e a t e d s e v e r e h y p o t h y r o i d i s m , c l o f i b r a t e was
w i t h o u t e f f e c t on serum c h o l e s t e r o l u n t i l t h y r o x i n e w a s
added .
Clof i b r a t e I n vivo
Hydrolysis
-
Clof i b r i c a c i d Glucuron i d e
Metabolism of Clof i b r a t e
CLOFIBRATE 21 1
6.1 Elemental Analvsis
c, 59.38% ; H, 6.232 ;
C 1 , 14.61:; ; 0 , 19.78%.
6.2 I d e n t i f i c a t i o n Tests
Those mentioned i n t h e B.P. (1980) ( 3 7 ) .
A. The i n f r a - r e d a b s o r p t i o n s p e c t r u m , Appendix I1
A , is c o n c o r d a n t w i t h t h e r e f e r e n c e s p e c t r u m of
clof ibrate.
B. The l i g h t a b s o r p t i o n , i n t h e r a n g e 220 t o 250nm,

of a 2-cm l a y e r of a 0.001 per c e n t w/v s o l u t i o n
i n a b s o l u t e e t h a n o l e x h i b i t s a maximum o n l y a t
226 nm; a b s o r b a n c e a t 226 nm, a b o u t 0.91, Appen-
d i x I1 B.
C. The l i g h t a b s o r p t i o n , i n t h e r a n g e 250 t o 350 nm,

of a 2-cm l a y e r of a 0.01 p e r c e n t w/v s o l u t i o n ,
i n a b s o l u t e e t h a n o l e x h i b i t s two maxima, a t 280
nm; and 238 nm a b s o r b a n c e a t 280 nm, aboilt 0.87,
and a t 283 nm, a b o u t 0.62, Appendix I1 R .
D. To 0.05 m l of a 1 0 per c e n t w/v s o l u t i o n i n

e t h e r add 0.05 m l of a s a t u r a t e d s o l u t i o n of
hydroxylammonium c h l o r i d e i n e t h a n o l (96 p e r
c e n t ) and 0.05 m l of a s a t u r a t e d s o l u t i o n of
potassium hydroxide i n e t h a n o l (96 p e r c e n t ) .
Heat f o r two m i n u t e s on a w a t e r - b a t h , c o o l ,
a c i d i f y w i t h 0 . 5 M h y d r o c h l o r i c a c i d , and add
0.05 m l of a 1 p e r c e n t w/v s o l u t i o n of i r o n
(111) c h l o r i d e ; a v i o l e t c o l o u r is produced.
6.3 P u r i t y Tests
(a) Water Content : n o t more t h a n 0.2%.
(b) Acidity: Hix 1 0 . 0 g w i t h 1 0 0 m l o f n e u t r a l i z e d

a l c o h o l , add 3 d r o p s of p h e n o l p h t h a l e i n T.S.
and t i t r a t e w i t h 0.1 N sodium h y d r o x i d e : n o t
more t h a n 0 . 9 m l is r e q u i r e d f o r n e u t r a l i z a t i o n
(c) Para-Chlorophenol:
The USP (XX) d e s c r i b e s a spectrophotomet-

r i c method f o r t h e d e t e c t i o n of p-chlorophenol.
The p e r c e n t a g e of p-chlorophenol i s n o t allowed
t o exceed 0.003%, w h i l e t h e B.P. (1980) d e s -
c r i b e s a g a s chromatographic p r o c e d u r e .
6.4 O f f i c i a l Methods: U.S.P. XX (38)
To a beaker c o n t a i n i n g 7 5 m l of 1 N sodium
hydroxide add a b o u t 3 g of a s t r o n g l y b a s i c
p o l y s t y r e n e anion-exchange r e s i n , and a l l o w t h e
mixture t o stand f o r about 15 minutes, with
o c c a s i o n a l s t i r r i n g . Wash t h e r e s i n w i t h water
u n t i l t h e l a s t washing i s n e u t r a l t o l i t m u s
paper, t h e n wash w i t h t h r e e 50 m l p o r t i o n s of
methanol. Place a plug of g l a s s wool i n t h e
b a s e of a 1-X 15 c m ion-exchange t u b e , and
t r a n s f e r t o t h e t u b e a s u f f i c i e n t amount of
Ion-exchange r e s i n , s l u r r i e d i n methanol, t o
produce a column bed h e i g h t of from6-cm t o
8-cm.
T r a n s f e r a b o u t 200 mg of C l o f i b r a t e , accu-
r a t e l y weighed, t o a 100 m l v o l u m e t r i c f l a s k ,
add methanol t o volume, and mix. T r a n s f e r 10.0
m l of t h i s s o l u t i o n t o t h e Ion-exchange column,
and c o l l e c t t h e e l u a t e i n a 100 m l v o l u m e t r i c
f l a s k . R i n s e t h e column w i t h 25 m l of methanol,
c o l l e c t t h e r i n s i n g i n t h e volumetric f l a s k ,
d i l u t e w i t h methanol t o volume, and mix. Trans-
f e r 5 . 0 m l of t h i s s o l u t i o n t o a 50 m l volume-
t r i c f l a s k , d i l u t e w i t h methanol t o volume,and
mix.
D i s s o l v e a n a c c u r a t e l y weighed q u a n t i t y
of USP C l o f i b r a t e RS i n methanol, and d i l u t e
q u a n t i t a t i v e l y and s t e p w i s e w i t h methanol t o
o b t a i n a s o l u t i o n having a known c o n c e n t r a t i o n
of about 2 0 pg p e r m l .
Concomitantly d e t e r m i n e t h e a b s o r h a n c e s of
t h e Standard p r e p a r a t i o n and t h e Assay p r e p a r a -
CLOFIBRATE 213
t i o n i n 1-cm c e l l s a t t h e wavelength of maximum

absorbance a t about 226 nm, w i t h a s u i t a b l e
s p e c t r o p h o t o m e t e r , u s i n g methanol a s t h e b l a n k .
C a l c u l a t e t h e q u a n t i t y , i n mg, of C12H15C103 i n
t h e p o r t i o n of C l o f i b r a t e t a k e n by t h e formula
lOC(Au/As), i n which C i s t h e c o n c e n t r a t i o n , i n
ug per m l , of USP C l o f i b r a t e RS i n t h e Standard
p r e p a r a t i o n , and Au and AS a r e t h e a b s o r b a n c e s
of t h e Assay p r e p a r a t i o n and t h e Standard pre-
.
para t i o n , r e s p e c t i v e l y
6. 5 U l t r a v i o l e t Spectrophotometry
A f t a l i o n e t a1 ( 3 9 ) r e p o r t e d t h a t e t h a n o l could
n o t be used a s a s o l v e n t f o r t h e u l t r a v i o l e t s p e c t r o -
photometry of c l o f i b r a t e i n g e l a t i n o u s c a p s u l e s w i t h
v e g e t a b l e o i l s because t h e l a t t e r i n t e r f e r e s t r o n g l y
a t t h e Xmax of c l o f i b r a t e ( 2 2 7 nm). However, by us-
ing dioxane, t h e a b s o r p t i o n c o e f f i c i e n t of v e g e t a b l e
o i l w a s found c o n s t a n t from 255 t o 280 nm w h i l e t h a t
of c l o f i b r a t e i n c r e a s e d from 0 . 2 4 a t 265 nm t o 0.48
a t 280 nm ( u s i n g a 0.01% s o l u t i o n ) . I n t h e a s s a y ,
t h e c o n t e n t of 5 c a p s u l e s w a s homogenized, 0 . 5 g d i s -
solved i n dioxane t o g i v e 25 m l s o l u t i o n , 1 m l d i l u t -
ed f u r t h e r w i t h d i o x a n e t o 100 m l , and t h e a b s o r p t i o n
w a s determined a t 280 nm and 265 nm w i t h r e s p e c t t o
a s t a n d a r d s o l u t i o n of 0.01 g v e g e t a b l e o i l i n 100 m l
dioxane measured a t 280 nm. The t r u e a b s o r p t i o n w a s
taken a s t w i c e t h e d i f f e r e n c e a t t h e two wave l e n g t h s
E r r o r s of ?4% were o b t a i n e d when a p p l i e d t o s y n t h e t i c
m i x t u r e of 1 :1.
A spectrophotometr i c a s s a y had been d e s c r i b e d

( 4 0 ) f o r t h e q u a n t i t a t i o n of c l o f i b r i c a c i d i n
plasma and u r i n e . T h i s i n v o l v e s s o l v e n t e x t r a c t i o n
of c l o f i b r i c a c i d from a c i d i f i e d plasma o r u r i n e and
subsequent measurement of t h e UV absorbance a t 2 2 6 n m .
The U.S.P. X I X ( 4 1 ) d e s c r i b e s an a s s a y procedure

f o r c l o f i b r a t e based on t h e p a s s a g e of t h e s o l u t i o n
of c l o f i b r a t e i n methanol on a column of s t r o n g l y
b a s i c p o l y s t y r e n e anion-exchange r e s i n . The a b s o r -
bances of t h e a s s a y p r e p a r a t i o n and a s t a n d a r d c l o -
f i b r a t e p r e p a r a t i o n are c o n c o m i t a n t l y determined i n
1-cm c e l l s a t 226 nm u s i n g methanol a s t h e blank.
6.6 Thin Layer Chromatography (42)
M i x t u r e s of c l o f i b r a t e and x a n t h i n o l n i c o t i n a t e
were , s e p a r a t e d on s i l i c a g e l l a y e r s . Using c h l o r o -
form a s s o l v e n t , t h e hRf of c l o f i b r a t e and x a n t h i n o l
n i c o t i n a t e a r e 82 and 3 r e s p e c t i v e l y . Using c h l o r o -
form a c e t i c a c i d 95:5, t h e hRf v a l u e s a r e :
Clof i b r a t e ( 7 8 ) , x a n t h i n o l (0) and n i c o t i n i c a c i d
(15) *
6.7 Thin Layer - Gas L i q u i d Chromatography:

A s p e c i f i c and s e n s i t i v e method f o r t h e d e t e r m i -
n a t i o n of c l o f i b r a t e i n b i o l o g i c a l f l u i d s w a s d e s c r i -
bed ( 43 ) . C l o f i b r a t e w a s s e p a r a t e d from a s s o c i a t e d
f a t t y a c i d s by TLC and t h e m e t h y l ester was q u a n t i -
f i e d by GLC.
6.8 Gas L i q u i d Chromatography:
Gas l i q u i d c h r o m a t o g r a p h i c methods occupy a pro-

minent p o s i t i o n i n t h e q u a n t i t a t i o n of c l o f i b r a t e i n
d r u g s , t i s s u e s and b i o l o g i c a l f l u i d s . I n t h e method
of S i l v e s t r i ( 4 4 ) , c l o f i b r a t e i s e x t r a c t e d from t a b -
l e t s w i t h e t h y l e t h e r and from t i s s u e s , blood o r u r i n e
by homogenization w i t h t h e a d d i t i o n of N a C l and 7%
HClO4 s o l u t i o n w i t h e t h e r - l i g h t p e t r o l e u m (1 :1).
The e t h e r e x t r a c t is washed w i t h water, d r i e d o v e r
anhydrous sodium s u l p h a t e and e v a p o r a t e d t o s m a l l
volume i n a stream of n i t r o g e n . GLC performed a t
19OoC on a column ( 6 f t . x 1 / 8 i n . ) of 5% b u t a n e - d i o l
s u c c i n a t e on Chromsorb P(100 t o 120 mesh), w i t h n i t -
r o g e n as c a r r i e r g a s and a f l a m e i o n i z a t i o n d e t e c t o r ,
methyl h e p t a d e c a n o a t e is used a s i n t e r n a l s t a n d a r d .
Clof i b r i c a c i d i s s i m i l a r l y d e t e r m i n e d a f t e r a d s o r p -
t i o n on A m b e r l i t e IRA-400 and e s t e r i f i c a t i o n by
treatment with methanolic HC1. Overall recoveries
were 98X ( + 5 % ) f o r t h e ester and 92% ( + l O Z ) f o r t h e
f r e e a c i d . The l i m i t of d e t e c t i o n i s 0 . 5 ug of t h e
ester o r a c i d p e r m l of u r i n e o r g . of t i s s u e .
Karmen and Haut ( 4 5 ) d e s c r i b e d a method f o r

a s s a y i n g c l o f i b r a t e i n serum based on GLC of t h e
methyl e s t e r . Two i n t e r n a l s t a n d a r d s similar i n
c h e m i c a l s t r u c t u r e t o c l o f i b r a t e (chlorophenoxy a c e -
t i c and chlorophenoxy p r o p i o n i c a c i d s ) a r e added; t h e
compounds a r e e x t r a c t e d , c o n v e r t e d t o m e t h y l e s t e r s
and s u b j e c t e d t o GLC u s i n g a n a l k a l i f l a m e i o n i z a t i o n
CLOFIBRATE 215
d e t e c t o r s e l e c t i v e l y s e n s i t i v e t o halogen.
Knuechel and Ochs ( 4 6 ) d e s c r i b e d a g a s chromato-

g r a p h i c method f o r t h e d e t e c t i o n of c l o f i b r a t e i n t h e
serum of t r e a t e d p a t i e n t s . The r e t e n t i o n time of c l o -
f i b r a t e w a s found between m e t h y l p a l m i t a t e and m e t h y l
stearate. C l o f i b r a t e was d e t e c t e d i n v e r y s m a l l
amounts i n serum g l o b u l i n s which were s e p a r a t e d by
f r a c t i o n a l p r e c i p i t a t i o n , w h e r e a s t h e amounts e x t r a c t e d
from albumin c o r r e l a t e d w e l l w i t h t h o s e of t h e serum.
F r a c t i o n s o f c h o l e s t e r o l ester from serum of c l o f i b -
r a t e - t r e a t e d p a t i e n t s , c o n t a i n e d s m a l l amounts of c l o -
f i b r a t e i n s p i t e of r e p e a t e d f r a c t i o n a t i o n on s i l i c a
g e l column, d u e t o e s t e r i f i c a t i o n of c l o f i b r a t e by
cholesterol.
Berlin ( 4 7 ) reported a q u a n t i t a t i v e gas c h r o m a t e

g r a p h i c method of c l o f i b r i c a c i d i n plasma u s i n g 2-(4-
chloro-3-methylphenoxy)-2-methylpropionic a c i d as in-
t e r n a l s t a n d a r d . To blood plasma (200 p l ) i s added
H3PO4 a c i d 2M (50 p l ) and i n t e r n a l s t a n d a r d s o l u t i o n in
t o l u e n e (0.5 m l ) . A f t e r s h a k i n g (10 min) t h e o r g a n i c
phase i s t r a n s f e r r e d t o a 3 m l t a p e r e d g l a s s t u b e and
t h e plasma i s r e - e x t r a c t e d w i t h 0 . 5 m l t o l u e n e . Diso-
dium hydrogen p h o s p h a t e 0.5M ( 0 . 5 ml) i s added t o t h e
combined o r g a n i c p h a s e s and t h e m i x t u r e i s s h a k e n f o r
1 0 min. The o r g a n i c p h a s e i s removed and t h e aqueous
phase i s made a c i d i c w i t h H3PO4 a c i d 5 M (50 p l ) CH2C12
(100 1-11>i s added and t h e m i x t u r e i s e x t r a c t e d on a
whirl-mixer f o r 30 s e c o n d s . A f t e r removal of t h e
aqueous p h a s e , diazomethane i n e t h e r (50 1-11>is added
and t h e s o l u t i o n i s e v a p o r a t e d under n i t r o g e n t o a b o u t
1 / 5 of i t s volume and one p l i s i n j e c t e d . Blank plasma
samples showed no p e a k s i n t e r f e r i n g w i t h t h e c l o f i b r i c
a c i d o r i n t e r n a l s t a n d a r d m e t h y l ester p e a k s . The
method i s l i m i t e d t o d e t e r m i n a t i o n of plasma c o n c e n t r a -
t i o n s down t o 1 pg ml-I d u e t o t h e c o n c e n t r a t i o n
chosen f o r t h e i n t e r n a l s t a n d a r d .
Another method f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n
of c l o f i b r i c a c i d i n blood p l a s m a had been d e s c r i b e d
( 45 ) . The s u b s t a n c e is e x t r a c t e d from a c i d i f i e d
plasma i n t o benzene, t h e e x t r a c t i s e v a p o r a t e d t o
d r y n e s s and t h e r e s i d u e i s m e t h y l a t e d and s u b m i t t e d
t o chromatography on a g l a s s column packed w i t h 3%O V 4 7
on chromosorb WHP, 100-120 mesh. The c o n d i t i o n s a r e
a s f o l l o w s : i n j e c t o r t e m p e r a t u r e , 18OoC; d e t e c t o r
t e m p e r a t u r e , 210°; column i n i t i a l t e m p e r a t u r e , 150°

f o r 6 min. t h e n programmed a t 195' a t 10°/min. and
h e l d a t t h e f i n a l t e m p e r a t u r e f o r 5 min. b e f o r e re-
c y c l e . The f l o w r a t e s were: helium c a r r i e r g a s , 25
ml/min. helium a u x i l i a r y g a s , 35 ml/min; a i r , 400 m l /
min. hydrogen, 40 m l / m i n . The r e t e n t i o n t i m e s of
c l o f i b r i c a c i d m e t h y l ester and of a c t a d e c a n e ( e x t e r -
n a l s t a n d a r d ) were 3 and 5 min. r e s p e c t i v e l y .
A r a p i d g a s c h r o m a t o g r a p h i c method i s d e s c r i b e d
( 4 9 ) f o r t h e d e t e r m i n a t i o n of c l o f i b r i c a c i d i n
p l a s m a and u r i n e . The a s s a y i n v o l v e s a n e x t r a c t i o n
i n t o t o l u e n e and b a c k - e x t r a c t i o n of c l o f i b r i c a c i d
and t h e i n t e r n a l s t a n d a r d ( 2 - n a p h t h o i c a c i d ) i n t o t h e
methylating agent (trimethylanilinium hydroxide).
The s i l a n i z e d g l a s s column ( 6 f t . x 4 mm i . d . ) was
packed w i t h 3% of SE-30 on 80-100 mesh Gas Chrom Q
0
and was o p e r a t e d a t 150 C w i t h a c a r r i e r g a s ( n i t r o -
gen) f l o w - r a t e o f 25 ml/min ; t h e i n j e c t i o n p o r t tem-
p e r a t u r e w a s 29OoC. The f l a m e i o n i z a t i o n d e t e c t o r
was o p e r a t e d a t 27OoC w i t h a hydrogen f l o w - r a t e of
20 mllmin. and a n oxygen f l o w - r a t e of 200 mllmin.
Under t h e s e c o n d i t i o n s , t h e r e t e n t i o n times were 1 . 5
min. f o r c l o f i b r i c a c i d and 2 . 5 min. f o r t h e i n t e r n a l
s t a n d a r d . The blood samples a r e drawn i n t o h e p a r i n i -
zed t u b e s and t h e plasma w a s s e p a r a t e d by c e n t r i f u g a -
t i o n . To 1 . 0 m l of plasma i n a 1 5 m l g l a s s t u b e were
added 1 m l of 0.4 M h y d r o c h l o r i c a c i d and 6 m l o f
t o l u e n e c o n t a i n i n g 120 pg of t h e i n t e r n a l s t a n d a r d .
The t u b e w a s shaken f o r 5 min. and c e n t r i f u g e d f o r
3 min. a t 4000 g . A 5 m l p o r t i o n of t h e o r g a n i c
phase i s t r a n s f e r r e d t o a p o i n t e d c e n t r i f u g e t u b e .
T r i m e t h y l a n i l i n i u m h y d r o x i d e ( 5 0 p l ) i s added and
t h e m i x t u r e i s e x t r a c t e d on a Vortex-mixer f o r 1 min.
A f t e r b r i e f c e n t r i f u g a t i o n , 1 p 1 o f . t h e aqueous
layer is injected directly. The d e t e r m i n a t i o n of
u r i n a r y c l o f i b r i c a c i d is c a r r i e d o u t e s s e n t i a l l y a s
d e s c r i b e d f o r plasma. For a n a l y s i s of g l u c u r o n i d e
m e t a b o l i t e of c l o f i b r i c a c i d i n u r i n e , t h e sample i s
d i l u t e d 1 : l O w i t h 0.2 M sodium a c e t a t e b u f f e r of pH
5.0 and 4 ml of t h e d i l u t e d sample are i n c u b a t e d o v e r
n i g h t w i t h 2000 Fishman u n i t s of a g l u c u r o n i d a s e -
a r y l - s u l p h a t a s e p r e p a r a t i o n from R e l i x Pomatia.
Wolf and Zimmerman ( 5 0 ) d e s c r i b e d a sumultaneous

GLC d e t e r m i n a t i o n of C l o f i b r a t e and i t s m e t a b o l i t e
c l o f i b r i c a c i d i n human plasma. T h i s h a s been ach-
i e v e d by u s i n g a g a s c h r o m a t o g r a p h i c column packed
CLOFIBRATE 217
w i t h Gas Chrom Q c o a t e d w i t h 10% S i l a r 1 0 C and n i t -

r o g e n a s c a r r i e r g a s . The method is r a p i d and do n o t
require a derivatization step, it is sensitive to
1 u g / d of e i t h e r compound i n b i o l o g i c a l s a m p l e s and
c o u l d b e used t o c h a r a c t e r i z e t h e i n v i v o c o n v e r s i o n
of c l o f i b r a t e ester t o t h e f r e e a c i d .
A c o m p a r a t i v e s t u d y of g a s - l i q u i d c h r o m a t o g r a -
p h i c b e h a v i o u r of t h e p e n t a f l u o r o b e n z y l esters and
t h e m e t h y l esters of t e n c h l o r o p h e n o x y a l k y l a c i d s
i n c l u d i n g c l o f i b r i c a c i d was a l s o r e p o r t e d ( 5 1 )
P a r a - c h l o r o p h e n o l and para-hydroxy b e n z o i c a c i d esters
which are added a s c a p s u l e p r e s e r v a t i v e s were d e t e r -
mined by g a s chromatography ( 52 ) . T h e s e were re-
a c t e d w i t h (EtO)2 P ( 0 ) C l and MeONa i n h e x a n e a t 50°
f o r 3 0 min. t o g i v e t h e d i e t h y l p h o s p h a t e esters.
These esters and c l o f i b r a t e were s e p a r a t e d and d e t e r -
mined on a column packed w i t h 3 . 5 % s i l i c o n e J X R on
Chromosorb G and HP a t 190' u n d e r N2 c a r r i e r . Flame
p h o t o m e t r i c and Flame t h e r m i o n i c d e t e c t o r s were u s e d .
The c o e f f i c i e n t s of v a r i a t i o n f o r 3 . 8 7 , 7.74 pg p a r a -
c h l o r o p h e n o l i n 1 0 m l were 0 . 8 7 , 0.56% w i t h FTB and
1.5, 0.56% w i t h FPD r e s p e c t i v e l y . P a r a c h l o r o p h e n o l
i n commercial p r e p a r a t i o n s was 1.6-5.1 ppm.
6.9 Gas Chromatography - Mass S p e c t r o m e t r y :

I m p u r i t i e s i n c l o f i b r a t e h a v e been s t u d i e d GC-MS
( 53 ) . T h r e e main i m p u r i t i e s were f o u n d , t h e m e t h y l
ester a n a l o g u e of c l o f i b r a t e , i t s d e s c h l o r o a n a l o g u e
and t h e d i c h l o r o a n a l o g u e .
J o h a n s s o n and Ryhage ( 5 4 ) have i d e n t i f i e d o t h e r

i m p u r i t i e s i n t h r e e c l o f i b r a t e p r e p a r a t i o n s . Samples
were o b t a i n e d by d i s s o l v i n g 0 . 5 m l of c a p s u l e - con-
t e n t i n 0.5 m l c h l o r o f o r m 5 p 1 of t h e s a m p l e w a s
i n j e c t e d i n t o t h e combined g a s c h r o m a t o g r a p h - mass
s p e c t r o m e t e r LKB 2091. The G . C . column u s e d w a s 3%
SE-30 g l a s s column 2.7 M x 2 mm ( i . d . ) . A constant
t e m p e r a t u r e of 16OoC f o r t h e f i r s t 8 min. w a s u s e d .
The c a r r i e r g a s f l o w - r a t e was 25 m l h e l i u m l m i n . The
mass s p e c t r a were o b t a i n e d w i t h a c o n s t a n t accelerat-
i n g v o l t a g e of 3 . 5 KV, a n e l e c t r o n e n e r g y of 70 eV
and a n i o n i z i n g c u r r e n t of 100 PA. R e p e t i t i v e scan-
n i n g of t h e m a s s r a n g e 1 0 t o 500 i n 25 w a s u s e d .
6.10 High-Performance Liquid Chromatography:
Bjornsson e t a 1 (55 ) developed a r a p i d , s e n s i -

t i v e and s p e c i f i c h i g h p r e s s u r e l i q u i d chromatogra-
p h i c method f o r t h e q u a n t i t a t i v e a n a l y s i s of c l o f i b -
r i c a c i d i n plasma, s a l i v a and u r i n e . Plasma (0.1 -
-1 m l ) , s a l i v a (1 ml) o r u r i n e d i l u t e d 1 : l O O w i t h
d i s t i l l e d water ( 1 . 0 ml) is p l a c e d i n a screw-capped
t u b e , and 100 p 1 of i n t e r n a l s t a n d a r d s o l u t i o n (con-
t a i n i n g 6.7 pg of t h e i n t e r n a l s t a n d a r d ) , 0.5 m l of
0 . 5 N s u l p h u r i c a c i d and 5 m l of t o l u e n e a r e added.
The samples a r e e x t r a c t e d by mixing f o r 10 min. f o l -
lowed by c e n t r i f u g a t i o n a t 1200 g f o r 10 min. t o
s e p a r a t e t h e o r g a n i c and aqueous p h a s e s . The lower
aqueous phase i s f r o z e n by immersing t h e t u b e i n a
d r y - i c e a c e t o n e b a t h , and t h e o r g a n i c p h a s e i s poured
i n t o a n o t h e r t u b e , which h a s a n e l o n g a t e d cone a t i t s
b a s e . Then 50 p 1 of 0 . 2 N NaOH are added, and t h e
m i x t u r e i s e x t r a c t e d on a Vortex-mixer f o r 2 rnin.
A f t e r b r i e f c e n t r i f u g a t i o n , t h e aqueous phase i s
drawn i n t o a s y r i n g e t h a t a l r e a d y c o n t a i n s 10 p 1 of
a s o l u t i o n of 5% g l a c i a l a c e t i c a c i d i n water and
t h i s m i x t u r e i s i n j e c t e d i n t o t h e chromatograph. For
t h e a n a l y s i s of t h e g l u c u r o n i d e c o n j u g a t e of c l o f i b -
r i c a c i d i n u r i n e , 5 m l of 6N h y d r o c h l o r i c a c i d were
added t o each sample, and t h e s o l u t i o n s were h e a t e d
a t 98OC f o r 30 min. b e f o r e t h e e x t r a c t i o n . The
samples were t h e n cooled and a n a l y s e d as d e s c r i b e d
above, e x c e p t t h a t t h e a d d i t i o n of d i l u t e s u l p h u r i c
a c i d i n t h e f i r s t s t e p w a s o m i t t e d . A V a r i a n Micro-
pak CH-10 r e v e r s e - p h a s e column ( 2 5 c m x 6 . 3 mm 0.d.
x 2.2 mm i . d . ) was used. The a b s o r b a n c e w a s measured
a t 235 11111. One pump of t h e dual-pump g r a d i e n t - e l u -
t i o n chromatograph c o n t a i n e d a c e t o n i t r i l e and t h e
o t h e r 0.5% a c e t i c a c i d i n d i s t i l l e d water; an i s o c r a -
t i c 42% a c e t o n i t r i t e m i x t u r e of t h e two s o l v e n t s was
used. The f l o w - r a t e of t h e s o l v e n t m i x t u r e w a s 70
me/hr w i t h a column i n p u t p r e s s u r e of 197 a t m (2900
p.s.i.). C o n c e n t r a t i o n s between 1 . 0 and 25 pg p e r
sample could be measured w i t h a c o e f f i c i e n t of v a r i -
a t i o n from 1-6%. 40-50 samples c a n e a s i l y be a s s a y e d
i n a day. The method d o e s n o t r e q u i r e p r i o r chroma-
tographic preparation o r m u l t i p l e extractions.
Woodhouse e t a1 ( 5 6 ) d e s c r i b e d a high-perfor-
mance l i q u i d chromatographic method f o r measuring
plasma c o n c e n t r a t i o n s of c l o f i b r i c a c i d a f t e r adminis-
t r a t i o n of c l o f i b r a t e t o humans. 50 pg of t h e i n t e r -
CLOFIBRATE 219
n a l s t a n d a r d (4-chloro-2-methylphenoxyacetic a c i d ) i n
methanol and 3M H C 1 (0.5 ml) were added t o plasma
(1 ml) i n a g l a s s s t o p p e r e d c e n t r i f u g e t u b e , s h a k e n ,
allowed t o s t a n d f o r 5 min. and t h e n e x t r a c t e d w i t h
6 m l e t h e r . Ether w a s evaporated t o d r y n e s s under
n i t r o g e n and t h e r e s i d u e d i s s o l v e d i n m e t h a n o l . 10
1~1p o r t i o n s of t h i s s o l u t i o n were i n j e c t e d i n t o t h e
chromatograph u s i n g a s t o p - f l o w i n j e c t i o n t e c h n i q u e .
The s t a i n l e s s s t e e l column (25 x 0.46 c m i . d . ) w a s
packed w i t h c18 P a r t i s i l (10 pm). The m o b i l e p h a s e
w a s 27% a c e t o n i t r i l e c o n t a i n i n g 0.4% o r t h o p h o s p h a t e
b u f f e r ( t o m a i n t a i n t h e pH a t 4.2) a t a f l o w - r a t e
of 2 mlfmin and a b a c k p r e s s u r e of 50 b a r . R e t e n t i o n
times of i n t e r n a l s t a n d a r d and c l o f i b r i c a c i d were
6 and 7 min. r e s p e c t i v e l y .
7, P r o t o n Magnetic Resonance S p e c t r o m e t r y :
Hassan and L o u t f y ( 4 ) h a v e d e v e l o p e d PMR

a n a l y t i c a l method f o r t h e q u a n t i t a t i o n of c l o f i b r a t e
a s a d r u g e n t i t y and i n c a p s u l e d o s a g e form. The
f o u r a r o m a t i c p r o t o n s q u a r t e t c e n t r e d a t 7 . 0 3 ppm
( F i g . 3) was chosen f o r q u a n t i t a t i o n of c l o f i b r a t e .
Malonic a c i d w a s employed a s a n i n t e r n a l s t a n d a r d ,
s i n c e i t e x h i b i t s two p r o t o n s m e t h y l e n e s i n g l e t a t
3.36 ppm ( F i g . 7) which i s w i d e l y s e p a r a t e d from
t h o s e of c l o f i b r a t e . Acetone was used a s t h e s o l v e n t ,
s i n c e c l o f i b r a t e and m a l o n i c a c i d a r e s o l u b l e i n i t
and i t s m e t h y l p r o t o n s s i n g l e t a t 2.07 ppm ( F i g . 7 )
d o e s n o t i n t e r f e r e w i t h t h e d o w n f i e l d p r o t o n s of
b o t h compounds. The p r o c e d u r e proved t o b e s i m p l e ,
r a p i d , a c c u r a t e and p r e c i s e . S t a n d a r d d e v i a t i o n s of
*1.07% and +1.34% were o b t a i n e d f o r p u r e d r u g and
c a p s u l e s r e s p e c t i v e l y . The PMR spectrum i n a d d i t i o n ,
p r o v i d e s a s p e c i f i c means of i d e n t i f i c a t i o n of c l o -
f i b r a t e , and d e t e c t i o n of i m p u r i t i e s .
Another PMR p r o c e d u r e have been a l s o r e p o r t e d

(57 1 f o r t h e d e t e r m i n a t i o n of c l o f i b r a t e . For t h e
b u l k d r u g , 100 t o 150 mg of sample i s shaken w i t h
5 m l of a s o l n . (10 mg ml-I of t h e i n t e r n a l s t a n d a r d
(hexamethylcyclotrisilazane) i n CCl4 and t h e m i x t u r e
is t r a n s f e r r e d t o a NMR t u b e . For c a p s u l e s , t h e
c o n t e n t s a r e e x t r a c t e d w i t h C C l 4 ( 4 x 5 m l ) , t h e com-
bined e x t r a c t s a r e made up t o 25 m l w i t h CCl4 and
5 m l of t h i s s o l n . i s p l a c e d i n t h e n.m.r. t u b e ,
t o g e t h e r w i t h 1 m l of t h e i n t e r n a l s t a n d a r d s o l n .
(40 t o 50 mg m l - I ) . The NMR spectrum i s t h e n
5 . . . . . l . . . . , . . . . , . . . . l . , . . , . . .
I ' I I I '
600 400 3OQ 2M
I
,JyI.11- 8.0 70 6.0 5.0 P H ( 6 ) 4 0
PARTS RFU MILLION, 6
F i g . 7 . PMR Spectrum of C l o f i b r a t e , Malonic a c i d and

T e t r a m e t h y l s i l a n e i n Acetone.
r e c o r d e d , and t h e c l o f i b r a t e concn. i s c a l c u l a t e d
by comparing t h e i n t e g r a l of t h e s i n g l e t peak a t
1 . 4 7 p.p.m. f o r c l o f i b r a t e w i t h t h a t f o r t h e i n t e r n a l
s t a n d a r d . The p r o c e d u r e i s s i m p l e , r a p i d and accu-
r a t e , and t h e r e c o v e r y i s 9 9 . 6 ? 3 . 2 0 X .
Acknowledgement :
The a u t h o r s would l i k e t o t h a n k Elr. T a n v i r A . B u t t o f t h e

Department of P h a r m a c e u t i c a l C h e m i s t r y , C o l l e g e of Pharmacy,
King Saud U n i v e r s i t y , f o r t y p i n g t h e m a n u s c r i p t .
CLOFIBRATE 22 1
9. References:
1. The Merck I n d e x , An E n c y c l o p e d i a of Chemicals and Drugs,

Martha Windholz -- e t a l , IX E d i t i o n , Merck and Co. I n c . ,
Rahway, N . J . , U.S.A. , P. 305 ( 1 9 7 6 ) .
2. Ateas o f S p e c t r a l Data and P h y s i c a l C o n s t a n t s of O r g a n i c

Compounds, e d i t e d by J . G . G r a s s e l l i and W . M . R i t c h e y ,
Vol. 3 , CRC P r e s s , Cranwood Parkway - C l e v e l a n d , Ohio,
P . 247 (P 2403) (1975).
3. K . N a k a n i s h i and P.H. Solomon, " I n f r a r e d A b s o r p t i o n Spec-

t r o s c o p y " Second E d i t i o n , Holden-Day, I n c , S a n f r a n c i c s c o ,
(1977).
4. M.M.A. Hassan and M.A. L o u t f y , Spec. L e t t . , 12 ( 3 ) , 177

(1979).
5. M.M.A. Hassan, Unpublished r e s u l t s .
6. B.S. F i n k l e , R.L. F o l t z and D.M. T y l o r , J . Chromat. S c i ,

-
1 2 , 304 ( 1 9 7 4 ) .
7. E . S t e n h a g e n , S . Abrahamsson and F.W. M c L a f f e r t y , " Re-

g i s t e r y of Mass S p e c t r a l Data" J o h n Wiley and S o n s ,
London, 2, 1447 ( 1 9 7 4 ) .
8. W.G.M. J o n e s , J . M . Thorp, and F7.S. Waring, B r i t . P a t .

860, 303 ( 1 9 6 1 ) .
9. J.M. Thorp, B r i t . a t . 898, 596 (1962).
10. M. J u l i a , M. B a i l l a r g e and G. T s c h e r n o f f , B u l l . SOC.

Chem. F r a n c e , 776 (1956).
11. G . B a r g e l l i n i , Gazz. Chem. I t a l . , 36, 334 ( 1 9 0 6 ) , Chem.

Z e n t r . 2, 326 ( 1 9 0 6 ) .
12. C.A. B i s h o f f , Ber., 33, 933 ( 1 9 0 0 ) .
13. D.T. W i t i a k , T . C-L HO, R.E. Hackney and W.E. Connor,

J . Med. Chem., 11,
1086 ( 1 9 6 8 ) .
14. V . N . Fedoseeva and B.M. Klebanov., K i m . Farm. Zh., -

3
( 1 0 ) 27 ( 1 9 6 9 ) .
15. J . E . Hoover , "Remington's P h a r m a c e u t i c a l S c i e n c e s "

F i f t e e n t h E d i t i o n , Mack P u b l i s h i n g Company, E a s t o n ,
P e n n s y l v a n i a , P. 796 ( 1 9 7 5 ) .
16. L. A l m i r a n t e , L. B r u s e g h i n i and L. F a z i , B u l l . Chim.

Farm, 108,292 (1969).
17. M.E. Laker and P.A. Mayes, Biorned. E x p r e s s . 2 ( 3 ) , 88

(1978).
18. M.E. Foed and E . G . Mcqueen, C l i n . Exp. P h a r m a m l . P h y s i o l ,

6 , ( 3 ) , 267 ( 1 9 7 9 ) .
-
19. B . J . Kudchodkar, H.S. S o d h i , L . H o r l i c k and D.T. Mason,

C l i n . Pharmacol. T h e r . , 22 ( 2 ) , 154 ( 1 9 7 7 ) .
20. D.T. W i t i a k and M.W. W h i t e h o u s e , Biomed. P h a r m a c o l . ,

-
18, 971 (1969).
21. D.T. W i t i a k , R . E . Hackney, and M.bJ, W h i t e h o u s e , J . Med.

Chem. __
1 3 , 607 ( 1 9 6 9 ) .
22. J.M. Thorp, L a n c e t , 1,1323 (1962).
23. D.T. W i t i a k , D.R. F e l l e r , E.S. S t r a t f o r d , R . E . Hackney ,

R. N a z a r e t h and G . Wagner, J . bled. Chem. 14,
1971) and
references therein cited.
24. J.M. Thorp, and W.S. \*Jaring, N a t u r e , 194 , 948 (1962).
25. M.F. O l i v e r , N . Engl. J. Med., 299, 1360 (1978).
26. R . I . Levy; D.S. F r e d r i c k s o n ; R. Shulman; D.W. B i l h e i m e r ;

J . L . Breslow; N . J . S t o n e , S . E . Lax; H . R . S l o a n ; R.M.
K r a u s s , and P.N. H e r b e r t , 77,
267 ( 1 9 7 2 ) .
27. S.M. Grundy; E.H. Ahrens, J r . G . S a l e n ; and E. O u i n t a o ;

J. C l i n . I n v e s t . , 48, 33a ( 1 9 6 9 ) .
28. P. S e g a l ; P.S. Roheim and H.A. E d e r ; J. C l i n . I n v e s t .

-
51, 1632 (1972).
29. B.M. Wolfe; J . P . Kane; R . J . Havel; and H.P. Brewster;

J. C l i n . I n v e s t . , 52, 2146 ( 1 9 7 3 ) .
30. N . A . D’Costa, F.C. Smigura, K. Kulhay and A . A n g e l ; J .

Lab. C l i n . Med., 90, 823 ( 1 9 7 7 ) .
CLOFIBRATE 223
31. M. Berman; M. Hall, R . I . Levy, S . E i s e n b e r g ; D.W.

B i l h e i m e r ; D , P h a i r ; and R.H. G r o e b e l . .J. L i p i d Res.,
1 9 , 38 (1978).
-
32. Goodman and Gilman's,"The P h a r m a c o l o g i c a l Basis of Thera-

p e u t i c s " , A . G . Gilman, W:S. Goodman, and A. Gilman, S i x t h
E d i t i o n , Macmillan P u b l i s h i n g Co., I n c . Mew York, P . 840
(1980).
33. H.J. F a l l o n , L.L. Adams and R . G . Lamb, L i p i d s , 1(2),

106 (1972).
34. P. Samuel, e t a l , C i r c u l a t i o n , 41, 109 (1970).
35. B.U. Musa, J . T . O g i l v i e , and J . T . Dowling, Metabolism

-
1 7 , 909 (1968).
36. M.T. 1 1 , 213

H a r r i s o n and R. McG. Harden, S c o t . Med. J . -
(1 966) .
37. B r i t i s h Pharmacopoeia, Vol. 1 ,
P r i n t e d i n England f o r Her M a j e s t y ' s S t a t i o n a r y O f f i c e
a t t h e U n i v e r s i t y Press, Cambridge, P . 1 1 6 , ( 1 9 8 0 ) .
38. The United S t a t e s Pharmacopoeia 2 0 t h R e v i s i o n , The
N a t i o n a l F o r m u l a r y , 1 5 t h E d i t i o n , U n i t e d S t a t e s Pharma-
c o p o i a l Convention, I n c . , R o c k v i l l , Md. , P . 157 (1980).
39. H. A f t a l i o n , R. S i m i n o v i c i and F.M. A l b e r t , Rev. Roum.

Chim., 5 ( 9 ) , 1195 (1968).
40. A.M. B a r r e t t and J . M . T h o r p . , B r . J . Pharmacol. Chem-

other. -3 2 , 381 (1968).
41. The United S t a t e s Pharmacopoeia, 1 9 t h R e v . , U n i t e d S t a t e s

Pharmacopoeia1 Convention, I n c . , R o c k v i l l e , Md. P . 9 6 ,
(1979).
42. A. d e J u a r e z , M. E s t r e l l a and G . C a r l o s , P r o - a n a l i s i s ,
2 ( 5 ) , 125 (1969).
-
43. A. S e d a r h a t , H . Nakamura and E.H. Ahrens, J . L i p i d R e s . ,

1 5 ( 4 ) , 352 (1974).
44. S. S i l v e s t v i , Famaco, Ed. P r v t . , g ( 3 ) , 197 (1970).
45. A. Karmen and H . Haut. , Biochem. Med. , 12 ( 2 ) , 154 (1975).

46. F. Knuechel and H . Ochs, Arzneim. Forsch. , 24 ( 4 ) , 576

(1974).
47, A. B e r l i n , J. Pharm. P h a r m a c o l . , 27, 54 ( 1 9 7 5 ) .
48. C.C. Tuong and A. Tuong, J . Chromat. , 106,97 (1975).
49. R. Gugler and C. J e n s e n , J . Chromat., 117,1 7 5 (1976).
50. M.S. Wolf and J . J . Zimmerman, J . Pharm. S c i -

6 9 ( I ) , 92
(1 9 8 0 ).
51. J. De Beer, C . V . Peteghem and A . H e y n d r i c k x , J . Chromato-
g r . , 157, 97 ( 1 9 7 8 ) .
52. M. K o i b u c h i and A. E j i m a , E i s e i Kagaku, 2 (6) 301

(1979).
53. E. D i d i n g , H. Sandstr'dm. H. O s t e l i u s , and R. K a r l e n ,

Acta Pharm. S u e c i c a , 13,
55 ( 1 9 7 6 ) .
54. E. J o h a n s s o n and R. Ryhage, J . Pharm. P h a r m a c o l . , 28,

927 ( 1 9 7 6 ) .
55. T.D. B j o r n s s o n and T.F. B l a s c h k e , J . C h r o m a t o g r . , 137,

145 ( 1 9 7 7 ) .
56. R.N. Woodhouse, D . G . Cresswell, L.F. Chasseaud and R.R.

B r o d i e , J . Chromatogr. 138,
218 ( 1 9 7 7 ) .
57. H.M. E l - F a t a t r y and H . Y . Aboul-Enein, Spec. L e t t . , 11

( 1 2 ) , 921 ( 1 9 7 8 ) .
John G . Hoogerhekk and Bruce E . Wyka
1. Description 226
1.1 Names, Formula, Molecular Weight, and Structure 226
1.2 Drug Properties 226
2.1 Nuclear Magnetic Resonance Spectra 227
2.5 Melting Range 235
2.6 Thermal Properties 235
2.8 Solubility 241
3. Synthesis 242
4. Stability 242
5 . Drug Metabolism and Pharmacokinetics 244
5.1 Drug Metabolism 244
6.3 Spectrophotometric Analysis 247
6.4 Titrimetric Analysis 248
6.6 Radiochemical Analysis 25 1
6.7 Microcalorimetric Analysis 252
6.8 Microbiological Analysis 252
7. Acknowledgements 252
8. References 253

Volume 11 225 Pharmaceutical Association
ISBN 0-12-260811-9
226 JOHN G. HOOGERHEIDEAND BRUCE E. WYKA
1. Description
1.1 Names, Formula, M o l e c u l a r Weight and S t r u c t u r e
C l o t r i m a z o l e was f i r s t s y n t h e s i z e d i n 1969 b y
Plempel e t a l . (1) and t e s t e d under t h e name Bay b5097.
The compound-iias been marketed w i d e l y under t h e t r a d e names
o f Canesten, L o t r i m i n , Gyne-Lotr imin, and Mycelex.
The chemical name o f c l o t r i m a z o l e i s 1 - ( 2 - c h l o r o -

pheny1)diphenylmethyl-1H-
- imidazole.
The m o l e c u l a r f o r m u l a o f c l o t r i m a z o l e i s
C 2 2 H i 7 C l N z and t h e m o l e c u l a r weight i s 344.8 g/mol.
The s t r u c t u r e i s g i v e n i n F i g u r e 1.
1.2 Drug P r o p e r t i e s
C l o t r i m a z o l e i s a broad-spectrum a n t i m y c o t i c agent
e f f e c t i v e a g a i n s t pathogenic dermatophytes, yeasts, and
s e v e r a l species o f Candida, T r i c h o hyton, Microsporum,
Epidermophyton, and Maa-i. & reparations o f the drug
are used b o t h i n t h e t o p i c a l t r e a t m e n t o f dermal i n f e c t i o n s
and t o combat v u l v o v a g i n a l c a n d i d i a s i s .
Results o f i n i t i a l c l i n i c a l studies w i t h t h i s
compound p u b l i s h e d i n 1969 ( 2 ) were f o l l o w e d b y more
d e t a i l e d r e p o r t s o u t l i n i n g t h e spectrum and mechanisms o f
clotrimazole activity (1,3,4). At therapeutic
c o n c e n t r a t i o n s d r u g a c t i o n i s f u n g i s t a t i c ; however, a t h i g h
c o n c e n t r a t i o n s ( 2 0 p g / m l ) some - in- vitro fungicidical
a c t i v i t y has been observed (1,4).
1.3 Appearance, C o l o r , Odor and T a s t e
Clotrimazole i s a colorless, odorless, tasteless,

c r y s t a l 1i n e s o l i d .
CLOTRIMAZOLE 227
2.1 Nuclear Magnetic Resonance S p e c t r a
2.1.1 P r o t o n Magnetic Resonance
The p r o t o n NMR spectrum ( F i g u r e 2) o f a 10% ( w / v )

solution o f clotrimazole i n deuterated chloroform a t
ambient temperature was o b t a i n e d b y u s i n g a V a r i a n CFT-20
spectrometer o p e r a t i n g a t a frequency o f 79.5 MHz. The
chemical s h i f t s g i v e n i n Table I a r e d o w n f i e l d from t h e
internal reference tetramethylsilane. P r o t o n assignments
are as i n d i c a t e d i n F i g u r e 1.
A 220 MHz p r o t o n NMR spectrum o f a s o l u t i o n o f

c l o t r i m a z o l e i n C 6 D 6 has a l s o been r e p o r t e d i n t h e
1i t e r a t u r e ( 5 ) .
Table I
PMR S p e c m s i g n r n e n t s
Protons Chemical S h i f t s (6) Intensity Multiplicity
5-H 6.75 1H triplet,

J=1.5 Hz
J=1.5 Hz
J=1.5 Hz
3'-H t o 6 ' - H
2"-H t o 6"-H 6.95-7.40 14H broad
8"-H t o 12"-H mu1t i p l e t s
228 JOHN G . HOOGERHEIDE AND BRUCE E. WYKA
10"
F i g u r e 1. S t r u c t u r e of C l o t r i m a z o l e .
I I 1 I I I I I I L
l " 1 " ' I " I I
9 8 7 6 s 5 4 3 2 1 0
8H 79.5 MHz
F i g u r e 2. Proton N u c l e a r Magnetic R e s o n a n c e S p e c t r u m of
C l o t r i m a z o l e in CDC13.
CLOTRIMAZOLE 229
2.1.2 Carbon-13 Magnetic Resonance
The carbon-13 p r o t o n decoupled NMR spectrum ( F i g u r e

3 ) o f a 20% ( w / v ) s o l u t i o n o f c l o t r i m a z o l e i n d e u t e r a t e d
c h l o r o f o r m a t ambient t e m p e r a t u r e was o b t a i n e d b y u s i n g a
V a r i a n XL-100 s p e c t r o m e t e r o p e r a t i n g a t a f r e q u e n c y o f 25.2
MHz. The c h e m i c a l s h i f t s a r e i n ppm ( 6 ) w i t h r e f e r e n c e t o
internal tetramethylsilane. The carbon-13 NMR spectrum
i n d i c a t e s t h e presence o f f i v e q u a r t e r n a r y carbons a t
6 = 75.0, 135.6, 139.1, 140.4, and 140.9, and seventeen
o l e f i n i c carbons a t 6 = 121.5, 127.0, 128.0 (4 e q u i v a l e n t
c a r b o n s ) , 128.1 ( 3 e q u i v a l e n t c a r b o n s ) , 128.5, 129.8, 130.1
( 4 e q u i v a l e n t c a r b o n s ) , 130.4, and 132.2 ppm.
2.2 Mass Soectrum
The mass spectrum ( F i g u r e 4 ) o f c l o t r i m a z o l e was

o b t a i n e d b y u s i n g a V a r i a n MAT CH5 medium r e s o l u t i o n s i n g l e
focusing instrument. The e l e c t r o n e n e r g y was 70 eJ, and
t h e probe and source t e m p e r a t u r e s used were 140 C and
25OoC, r e s p e c t i v e l y . The r e s u l t s a r e g i v e n i n T a b l e 11.
Table I1
m/e Ions
-
344 M'
309 (M-35)'
277 (M-67)'
242 (M-102)'
241 (M-103)'
239 (M-105)'
199
c)
t
165 H5C6CC6H 4
*C3H3N2 =
1111
' '~ " ' " " ' '
IIY)
!
I40
;':
'3-0 I , , , ,
190
I 2 , , I , # I
00
# I , , I I I 1
,,,,,
I I , , , / I ,
, , * , oI , , , ' .
20
P
4r"' '
' I ' 8 a I ' " " ' ' , l ' ' ' 1 1 ; ' ' , ~ ' ~ , ' ' , , , , , , . l , , , , , , , , . I . i l , I . I I
18. v v m l
Figure 3 . Carbon-13 Nuclear Magnetic Resonance Spectrum o f Clotrimazole in CDC13.

0
VI
m
0
O
m
0
In
(u
v)
0 0 :
O W
I
3
z
v)
v)
:a
- I
7
0
z
A 1 SN31NI 3AllWl3U
23 1
2.3 I n f r a r e d Spectrum
The i n f r a r e d spectrum ( F i g u r e 5 ) o f c l o t r i m a z o l e
as a d i s p e r s i o n i n m i n e r a l o i l was o b t a i n e d b y u s i n g a
P e r k i n Elmer Model 180 i n f r a r e d spectrophotometer . The
major a b s o r p t i o n bands a r e g i v e n i n Table 111.
The i n f r a r e d s p e c t r u m o f c l o t r i m a z o l e as a
d i s p e r s i o n i n potassium bromide has been r e p o r t e d i n t h e
1i t e r a t u r e ( 5 ) .
Table I 1 1
I n f r a r e d Band Assignments
Wavenumber ( c m - l ) Assignment
3170, 3115, 3085, 3075 (w) aromatic C-H ( s t r e t c h )
1585, 1570 (w) aromatic C=C, C=N

(stretch)
1510, 1500, 1450 (m) a r o m a t i c C=C, C=N

(stretch)
770, 760, 750 ( v s ) aromatic C-H o u t - o f - p l ane

bend
720, 700, 680 ( S - V S ) aromatic C-H o u t - o f - p l ane

bend
Notations: vs = very strong; s = strong; m = medium;

w = weak
2.4 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t s p e c t r a o f c l o t r i m a z o l e i n
methanol and methanol i c 0.1N h y d r o c h l o r i c a c i d ( F i g u r e 6 )
were o b t a i n e d b y u s i n g a C a G Model 118 spectrophotometer.
The maxima, m i n i m a , s h o u l d e r s and r e s p e c t i v e m o l a r
a b s o r p t i v i t i e s are g i v e n i n Table I V .
WAVELENGTH MICRONS
2.5 3 4 5 6 7 8 9 10 12 14 1822 3550
Figure 5. I n f r a r e d Spectrum o f C l o t r i m a z o l e i n Mineral O i l .

250 300 350

WAVELENGTH (nm)
Figure 6 . Ultraviolet Spectra o f Clotrimazole.

CLOTRIMAZOLE 235
Table I V
U 1t r av io 1e t Spectra 1 Character is t ics o f C 1o t r imazo 1e

2
Solvent A (nm) E x 10-
Methanol 274 (shoulder) 1.03

269 (shoulder) 2.40
265 (shoulder) 3.32
263 (shoulder) 3.21
259 (maximum) 3.40
253 (maximum) 2.94
249 (minimum) 2.64
Methanol i c 274 (shoulder) 1.75

0.1N 269 (shoulder) 3.28
hydrochloric 263 (maximum) 3.96
acid 261 (minimum) 3.84
259 (maximum) 3.93
254 (shoulder) 3.12
246 (minimum) 2.38
2.5 M e l t i n g Range
The f o l l o w i n g me1 t i n g ranges have been r e p o r t e d :
M e l t i n g Range Reference
143 t o 144OC 596

144 t o 145OC 7
141 t o 145OC a
2.6 Thermal P r o o e r t i e s
2.6.1 D i f f e r e n t ia1 Scann i n g C a l o r i m e t r y
The d i f f e r e n t i a l scanning c a l o r i m e t r y c u r v e
( F i g u r e 7 ) o f c l o t r i m a z o l e was o b t a i n e d b y u s i n g oa DuPont
Model 990 Thermal Analyzer a t a h e a t i n g r a t e o f 1 0 C/minute
under a n i t r o g e n atmosphere. A s i n g l e sharp endotherm was
observed w i t h an e x t r a p o l a t e d onset temperature o f 143OC.
236 JOHN G. HOOGERHEIDEAND BRUCE E. WYKA
P u r i t y a n a l y s i s b y d i f f e r e n t i a l scanning c a l o r i -
mgtry ( F i g u r e 8 ) was p e r f o r m e d a t a h e a t i n g r a t e o f
1 C/minute under a n i t r o g e n atmosphere. The p u r i t y o f t h e
sample was d e t e r m i n e d t o b e 99.5 m o l e p e r c e n t . The l a t e n t
h e a t o f f u s i o n AH^) was c a l c u l a t e d t o be 7540 c a l / m o l .
2.6.2 Thermograv i m e t r y
Thermogravimetric a n a l y s i s ( F i g u r e 9 ) o f c l o t r i -
mazole was p e r f o r m e d b y u s i n g a DuPont Modelo950 Thermo-
g r a v i m e t r i c A n a l y z e r a t a h e a t i n g r a t e o f 10 C/minute under
a n i t r o g e n atmosphere. No weighto loss was observed f r o m
ambient temper,ature t o about 180 C. The g r a d u a l w e i g h t
loss above 180 C i s due t o v a p o r i z a t i o n o f t h e m e l t .
2.7.1 X-Rav D i f f r a c t i o n
The X-ray powder d i f f r a c t i o n p a t t e r n ( F i g u r e 10)

o f c l o t r i m a z o l e was o b t a i n e d b y u s i n g a P h i l l i p s DP-3500
X - r a y D i f f r a c t o m e t e r and Cu K, r a d i a t i o n (1.5148 if
) . The
d a t a a r e g i v e n i n T a b l e V.
2.7.2 P o l ymorph ism
Borka e t a l . ( 6 ) have r e p o r t e d t h e f o r m a t i o n o f a
metastable f o f i o f clotrimazole from a cr%stal f i l m
p r e p a r a t i o n . They r e p o r t a m e l t i n g p o i n t o f 106 C f o r t h i s
m e t a s t a b l e f o r m and an i n f r a r e d spectrum t h a t i s d i f f e r e n t
from t h e s t a b l e form.
CLOTRIMAZOLE !37
'0
TEMPERATURE. "c
F i g . 7. D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y Curve of
Clotrimazole.
TEMPERATURE, *K
F i g . 8. D i f f e r e n t i a l S c a n n i n g C a l o r i m e t r y Curve of C l o t r i -
mazole f o r P u r i t y A n a l y s i s .
W
7
0
N
a
E
.r
L
+J
0
c
0
le
0
0)
w
L
3
0
1,
L
c,
a,
E
.I-
5
l-0
L
rn
0
E
L
a,
L:
I-
0,
W
L
S
m
*I-
L L
238
1
00
01 I I 1 1 i 1 1 i
40 36 32 20 24 20 16 12 8
28
Figure 10. Powder X-ray D i f f r a c t i o n P a t t e r n o f Clotrimazole.

JOHN G. HOOGERHEIDE AND BRUCE E. WYKA
Table V
X-Ray Powder D i f f r a c t m t e r n o f C l o t r i m e r o l e
-
28 d(!lr -
wb
9.172 9.641 80
9.398 9.410 10
9.817 9.009 47
10.203 8.669 10
12.361 7.161 100
14.178 6.247 19
15.159 5.345 7
15.641 5.665 7
16.645 5.326 32
l a . 517 4.791 a2
15.415 4.572 68
19.830 4.477 37
20.647 4.302 61
22.502 3.951 29
22.983 3.870 43
24.101 3.693 36
24.200 3.678 36
24.266 3.668 36
25.086 3.550 41
25.519 3.490 22
26.177 3.404 14
27.474 3.246 32
28.132 3.172 46
28.354 3.148 26
28.810 3.099 14
30.923 2.892 11
30.999 2.885 11
31.318 2.856 9
31.461 2.843 7
31.783 2.815 16
32.655 2.742 23
34.182 2.623 11
34.503 2.599 19
34.577 2.594 19
36.980 2.431 10
37.080 2.424 10
37.196 2.417 9
37.374 2.406 8
37.598 2.392 11
37.680 2.387 11
37.778 2.381 10
37.057 2.376 10
37.989 2.372 8
37.998 2.368 8
38.513 2.337 9
a d ( i n t e r p l a n a r distance) = n A/2 s i n
1/11 = r e l a t i v e i n t e n s t t y
CLOTRIMAZOLE 241
2.8 Solubilitv
The s o l u b i l i t y o f c l o t r i m a z o l e has been d e t e r m i n e d

i n solvents by using v i s u a l , g r a v i m e t r i c o r spectrophoto-
m e t r i c analysis (Table V I ) .
2.9 D i s s o c i a t i o n Constant
The pKa o f c l o t r i m a z o l e i n 50% aqueous e t h a n o l i s

r e p o r t e d t o be 4.7 ( 5 ) .
Table V I
S o l u b i l i t y o f C l o t r i m a z o l e i n Common S o l v e n t s
Measured
Solvent (m m ° C Met hod
Acetone 50 grav i m e t r i c
Benzene >loo visual
Chloroform >loo visual
Diethyl ether 14 g r av ime t r ic
Dimet h y l formamide >loo visual
Dimet h y l s u l f o x i d e 45 spec t r o p h o t omet r ic
E t h a n o l USP 95 grav imetr i c
Ethyl Acetate 45 g r av i m e t r i c
Met h ano 1 >loo visual
Mineral o i l 0.8 spec t r o p h o t omet r ic
Petroleum ether 1.1 spec t r o p h o t o m e t r ic
P o l y e t h y l e n e g l y c o l 400 60 spec t r o p h o t omet r ic
P r o p y l ene g l yco 1 35 spectrophotometr i c
Water <0.03 spectrophotometric
3. Synthesis
Clotrimazole i s synthesized by t h e r e a c t i o n o f
o - c h l o r o t r i t y l c h l o r i d e w i t h i m i d a z o l e i n t h e presence o f a
t e r t i a r y amine, as d e s c r i b e d b y Buechel, e t a l . ( 5 ) as
shown i n F i g u r e 11. The y i e l d i n this-syythesis is
solvent-dependent; reactions i n solvents w i t h h i g h
d i e l e c t r i c constants g i v e the higher y i e l d s .
Maul and S c h e r l i n g ( 9 ) used barium { 4C) c a r b o n a t e

as s t a r t i n g m a t e r i a l t o s y n t h e s i z e ' ' C - c l o t r i m a z o l e . They
made t h e i n t e r m e d i a t e s 2 - c t j l o r o - { c a r b o x y l - C ) benzoic
a c i d , 2-ch\ojo- { c a r b o x y l - C 1 benzoylchloride, 2-chloro-
C 1 benzophenone, ( 2 - c h l o r o p h e n y l d i p h e n y l -
{carboxyl-
{ 1 4 C ) methanol and (2-chloropheny1)diphenyl - { ' 1 C)-
methylchloride en route tq radiolabelled clotrimazole,
1-(2-~hlorophenyl)diphenyl-{ k1- methyl-1H-imidazole.
-
4. Stability
C l o t r i m a z o l e i s s t a b l e i n t h e s o l i d s t a t e unger
normal s t o r a g e c o n d i t i o n s . It i s u n a f f e c t e d b y h e a t (70 C)
and exposure t o d a y l i g h t f o r up t o two weeks (10).
I n s o l u t i o n , t h e s t a b i l i t y o f c l o t r i m a z o l e i s pH
dependent. I n an a l k a l i n e medium i t i s s t a b l e , b u t
h y d r o l y z e s i n an a c i d i c medium t o ( 0 - c h l o r o p h e n y 1 ) -
diphenylmethanol p l u s i m i d a z o l e . Buechel e t a l . r e p o r t on,
the relative hydrolytic stability of notrirnazole i n
s o l u t i o n i n e t h a n o l - w a t e r and i s o p r o p a n o l - w a t e r m i x t u r e s
under a c i d i c , n e u t r a l , and a l k a l i n e c o n d i t i o n s ( 4 ) .
Thermal and 1 i g h t pH-stabi 1 i t y s t u d i e s have been

performed. Known amounts o f c l o t r i m a z o l e were s e a l e d i n
g l a s s ampuls a f t e r t h e a d d i t i o n o f 10 m l o f an aqueous
b u f f e r . The pH range s t u d i e d was 1 t o 13. Table V I I g i v e s
t h e r e s u l t s f o r samples s t o r e d a t 7 5 0 , 850, and 95OC f o r
one week. R e s u l t s are g i v e n i n Table V I I I f o r samples
s t o r e d i n t h e d a r k and u n d e r 350 f o o t - c a n d l e s o f
f l u o r e s c e n t l i g h t f o r t h r e e months. Analyses were
performed by u s i n g t h i n - l a y e r chromatoqraphy and e l u t i o n
f o l l o w e d b y UV s p e c t r a l a n a l y s i s (10).
CLOTRIMAZOLE 243
Clp / PCI 3
CH2CI UV-light
CI CI
(1) (I[)
cm 1
J
(rn)
F i g u r e 11 S y n t h e t i c Pathway t o C l o t r i m a z o l e
I . 2-chlorobenzylchloride
11. 2-chlorobenzotrichloride
I1 I . 2-chlorotri tyl chloride
I V . Clotrirnazole
Table V I I
pH-Thermal S t a b i l i t y P r o f i l e
Recovery o f
C l o t r i r n a z o l e i n Percent*
pH -
- Buffer State -
75OC 85OC
-- 95OC
-
1 Hydroch 1o r i c a c i d solution 0 0 0
2 C it r a t e part solution 0 0 0
4 Citrate suspension 84 55 4
4 Acetate suspension 84 65 6
6 Phosphate suspension 101 98 90
10 Borate suspension 102 102 100
13 Sodium h y d r o x i d e suspension 101 105 102
* A f t e r one week.
5. Drug Metabolism and Pharmacokinetics
5.1 Drua Metabolism
After oral or topical administration clotrimazole

undergoes r a p i d b i o t r a n s f o r m a t i o n i n t o i n a c t i v e met a-
bolites. Duhm and c o - w o r k e r s (11) i s o l a t e d f i v e
c l o t r i m a z o l e m e t a b o l i t e s f r o m r a t u r i n e and b i l e b u t found
no c l o t r i m a z o l e . S t r u c t u r e s o f t h e metabol i t e s a r e shown
i n F i g u r e 12.
Human u r i n e and serum t e s t e d a f t e r o r a l doses

o f c l o t r i m a z o l e c o n t a i n o n l y t r a c e amounts o f t h e d r u g
substance. The two m a j o r m e t a b o l i t e s found i n u r i n e ,
serum, and b i l e a r e ( 2 - c h l o r o p h e n y l ) (4-hydroxypheny1)-
phenylmethane and ( 2 - c h l o r o p h e n y l ) bis-phenylmethane; a
s m a l l e r amount o f (2-chloropheny1)bis-phenylmethanol is
a1 so p r e s e n t .
5.2 Pharmacokinetics
E a r l y c l i n i c a l s t u d i e s employing o r a l adminis-
t r a t i o n o f c l o t r i m a z o l e p r o v i d e d d a t a on t h e pharmaco-
k i n e t i c s o f s y s t e m i c a l l y d i s t r i b u t e d drug substance i n
humans. As discussed above, metabolism of c l o t r i m a z o l e i s
e x t r e m e l y r a p i d , and r e 1 i a b l e pharmacokinetics d a t a have
been o b t a i n e d o n l y b y u s i n g a n a l y t i c a l m e t h o d s w h i c h
determine b o t h $tle d r u g substance and m e t a b o l i t e s . In
experiments w i t h C - l a b e l l e d drug, Duhm - et -a l . (11) found
t h a t no c l o t r i m a z o l e was d e t e c t e d i n serum f o r a 20 m i n u t e
i n t e r v a l a f t e r dosing. Peak serum l e v e l s o f up t o 4 u g h 1
were confirmed b y a number o f workers (1,12-15); these
l e v e l s were reached between two and f o u r h o u r s a f t e r d o s i n g
i n a d u l t s and a t about s i x hours a f t e r a d m i n i s t r a t i o n t o
children. Rosenkrantz and P u e t t e r (16) e s t i m a t e d t h a t up
t o 98% o f c l o t r i m a z o l e i n serum i s bound t o serum p r o t e i n s .
Several workers have f o l l o w e d systemic c l o t r imazole

c l e a r a n c e b y d e t e r m i n i n g t h e d r u g and m e t a b o l i t e s e x c r e t e d
i n urine. W e i n g a e r t n e r , e t a l . ( 1 7 ) f o u n d no d r u g
substance i n u r i n e u n t i l b e t w e e n 3 0 and 60 minutes a f t e r
a d m i n i s t r a t i o n ; t h e y r e p o r t e d peak u r i n e c o n c e n t r a t i o n s a t
between 6 and 1 2 h o u r s . Plempel and co-workers (1) have
r e p o r t e d peak u r i n e c o n c e n t r a t i o n s o f 30 t o 60 u g h 1 1 2 t o
14 hours a f t e r d o s i n g .
CLOTRIMAZOLE 245
Table V I I I
pH-L i g h t S t a b i l i t y P r o f i l e
Recovery o f
C l o t r i m a z o l e i n Percent**
pH -
- Buffer State Light Dark
-
1 Hydrochloric acid sol ut ion 9 12

2 C it r a t e part solution 20 41
4 Citrate suspension 98 102
6 Phosphate suspension 97 103
10 Borate suspension 99 103
13 Sod iurn h y d r o x i d e suspension 103 99
**Stored i n t h e dark and under 350 f o o t - c a n d l e s o f f l u o r e s c e n t

1 i g h t for 3 months.
I I
OH OH
CI
F i g u r e 12. C l o t r i m a z o l e M e t a b o l i t e s :
I . (2-chlorophenyl )diphenylmethanol
I I . ( 2-chl orophenyl ) d i phenylmethane
III. (2-chlorophenyl ) , (4-hydroxyphenyl ) phenyl-
methane
IV. (2-chlorophenyl ) , (4-hydroxyphenyl ) p h e n y l -
me t h a no1
V. 2-chlorobenzophenone
246 JOHN G. HOOGERHEIDE AND BRUCE E. WYKA
Pharmacokinetics o f t o p i c a l l y appl i e d c l o t r i m a z o l e
has been s t u d i e d i n s e v e r a l l a b o r a t o r i e s . Duhm a n d
co-workers ( 1 8 ) found t h a t w h i l e C - c l o t r i m a z o l e appl i e d
as a cream p e n e t r a t e d t h e s k i n t o a d e p t h o f 2000 bm, no
d r u g s u b s t a n c e o r m e t a b o l i t e s were d e t e c t e d i n t h e serum.
A s m a l l q u a n t i t y o f t h e m a t e r i a l ( u p t o 0.4%) was d e t e c t e d
i n t h e u r i n e over a f i v e - d a y p e r i o d . In a s i m i l a r study,
H o l t ( 1 9 ) used a m i c r o b i o l o g i c a l a s s a y w i t h a d e t e c t i o n
l i m i t o f 0.01 pg/ml; over a t h i r t y - d a y p e r i o d o f
c l o t r i m a z o l e c r e a m a p p l i c a t i o n h e o b s e r v e d no d r u g
s u b s t a n c e i n e i t h e r serum o r u r i n e . Wallace, - et -al. (20)
n o t e d t h a t 24 h o u r s a f t e r a t e n - d a y t r e a t m e n t w i t h
clotrimazole t o p i c a l preparations, drug l e v e l s i n t h e skin
were as h i g h as 2 pg/mg; t h i s v a l u e d e c r e a s e d t o a b o u t
0.4 vg/mg a f t e r f o u r d a y s .
In studies using c l o t r i m a z o l e vaginal t a b l e t s ,

100 mg, Duhm e t a l . ( 1 8 ) f o u n d peak serum l e v e l s o f
0.03 pg/ml a f t e r 2 4 h o u r s .
P h a r m a c o k i n e t i c s and p h a r m a c o l o g y o f c l o t r i m a z o l e
a r e f u r t h e r c o v e r e d i n a c o m p r e h e n s i v e summary b y Sawyer,
.
e t a1 ( 2 1 ) as we1 1 as i n r e v i e w s b y Meade ( 2 2 ) and Seneca
v3r
6. Methods o f A n a l y s i s
6.1 Elemental A n a l y s i s
Conventional procedures f o r t h e d e t e r m i n a t i o n o f
C,H,N, and C1 y i e l d e d t h e f o l l o w i n g r e s u l t s f o r a sample
c o n f o r m i n g t o USP XX s p e c i f i c a t i o n s .
%
-
E 1ement Found Theory
C 76.63 76.63
H 4.93 4.97
N 8.12 8.12
c1 10.18 10.28
CLOTRIMAZOLE 247
6.2 Identification
S e v e r a l methods have been proposed f o r t h e

identification o f clotrimazole. Kuhnert-Brandstaetter, e t
-
a l . ( 8 ) observed t h a t t h e d r u g s u b s t a n c e f o r m s e u t g c t i c s
w i t h p h e n a c e t i n and b e n z a n i l i d e w h i c h m e l t a t 110 and
115 C, r e s p e c t i v e l y . D a t a on e u t e c t i c m e l t i n g p o i n t s i s
used o to c o n f i r m t h e i d e n t i t y o f c l o t r i m a z o l e , w h i c h m e l t s
a t 143 C .
K r i h a r , e t a l . ( 2 4 ) have c h a r a c t e r i z e d t h e
u l t r a v i o l e t s p e c t r a o f T o t r i m a z o l e i n m e t h a n o l and i n 0.1N
HC1 as an a i d t o t h e i d e n t i f i c a t i o n o f t h i s d r u g substance,
The USP X X i d e n t i f i c a t i o n t e s t s f o r c l o t r i m a z o l e
( 2 5 ) i n c l u d e b o t h t h i n - l a y e r chromatography, i n w h i c h t h e
sample s p o t must appear a t t h e same R f v a l u e as r e f e r e n c e
s t a n d a r d m a t e r i a l , and i n f r a r e d s p e c t r o p h o t o m e t r y .
6.3 Spectrophotometric Analysis
L i m i t e d u s e h a s b e e n made o f u l t r a v i o l e t
s p e c t r o p h o t o m e t r y i n t h e a n a l y s i s o f c l o t r i m a z o l e due t o
i t s low a b s o r p t i v i t y . Kr6&nar, e t a l . ( 2 4 ) suggested t h a t
such a n a l y s e s s h o u l d be p o s s i b l e ; S z a b o l c s ( 2 6 ) used
m e a s u r e m e n t s a t 2 6 1 nm t o a s s a y c l o t r i m a z o l e i n
formulations.
Upon h e a t i n g w i t h t r i c h l o r o a c e t i c o r p e r c h l o r i c
a c i d s , s o l u t i o n s o f c l o t r i m a z o l e become b r i g h t y e l l o w ; t h e
c o l o r fades r a p i d l y w i t h t r i c h l o r o a c e t i c a c i d b u t p e r s i s t s
with perchloric acid (1). T h i s r e a c t i o n , which forms t h e
b a s i s f o r a c o l o r i m e t r i c assay o f t h e d r u g s u b s t a n c e , g i v e s
s o l u t i o n s which obey B e e r ' s l a w up t o c o n c e n t r a t i o n s o f 1 0
pg/ml when r e a d a t 436 nm ( 2 7 ) . T h i s method has been used
f o r t h e e s t i m a t i o n o f c l o t r i m a z o l e i n b i o l o g i c a l f l u i d s and
t i s s u e s a f t e r e i t h e r s o l v e n t e x t r a c t i o n (16,27) o r
extraction followed by thin-layer chromatography
(1,13,17,28).
248 JOHN G.HOOGERHEIDE AND BRUCE E. WYKA
6.4 T i tr i m e t r i c An a1y s is
Assay o f c l o t r i m a z o l e b u l k d r u g s u b s t a n c e i s
p e r f o r m e d b y nonaqueous t i t r a t i o n ( 2 5 ) . The s a m p l e
d i s s o l v e d i n g l a c i a l a c e t i c a c i d i s t i t r a t e d w i t h 0.1M
perchloric acid i n glacial acetic acid t o a green
e n d - p o i n t ; p - n a p h t h o l b e n z e i n i s used as i n d i c a t o r . Each
m i l l i l i t e r o f 0.1M p e r c h l o r i c a c i d i s e q u i v a l e n t t o 34.48
m i l 1i g r a m s o f c l o t T i m a z o l e .
A f t e r e x t r a c t i o n with c h l o r o f o r m , Szabolcs ( 2 6 )
assayed c l o t r i m a z o l e i n p h a r m a c e u t i c a l f o r m u l a t i o n s b y
t i t r a t i o n w i t h 0.1M - p e r c h l o r i c a c i d ; g e n t i a n v i o l e t was
used as i n d i c a t o r .
A two-phase t i t r a t i o n method developed b y

P e l l e r i n , e t a l . ( 2 9 - 3 1 ) s e r v e s as a b a s i s f o r t h e
t i t r a t i o n o f c T o t r i m a z o l e w i t h sodium l a u r y l s u l f a t e .
Samples o f c l o t r i m a z o l e f o r m u l a t i o n s a r e p a r t i t i o n e d
between c h l o r o f o r m and 2N s u l f u r i c a c i d . M e t h y l y e l l o w i s
a d d e d as i n d i c a t o r a n d t h e m i x t u r e t i t r a t e d w i t h
s t a n d a r d i z e d sodium l a u r y l s u l f a t e i n w a t e r . The end p o i n t
i s r e a c h e d when t h e c h l o r o f o r m l a y e r t u r n s g o l d - o r a n g e
( 3 2 ) . T h i s s t a b i l i t y - i n d i c a t i n g t i t r a t i o n has been used t o
assay c l o t r i m a z o l e i n f o r m u l a t i o n s (32,33) and t o f o l l o w
h y d r o l y s i s o f t h e drug substance ( 5 ) .
6.5 Chromatograph ic A n a l y s i s
C l o t r i m a z o l e c a n be s e p a r a t e d f r o m i t s i m p u r i t i e s
and d e g r a d a t i o n p r o d u c t s b y d e s c e n d i n g p a p e r c h r o m a t o g r a p h y
(10). The method uses paper i m p r e g n a t e d w i t h p r o p y l e n e
g l y c o l and a m o b i l e phase o f p r o p y l e n e g l y c o l - s a t u r a t e d
l i g r o i n . C l o t r i m a z o l e i s d e t e c t e d a t R f = 0.4 b y s p r a y i n g
w i t h Dragendorff reagent,
6.5.2 Th i n - L a y e r Chromatography
T h i n - l a y e r c h r o m a t o g r a p h y (TLC) o n s i l i c a g e l h a s
been used e x t e n s i v e l y t o s e p a r a t e c l o t r i m a z o l e f r o m
f o r m u l a t i o n and b i o l o g i c a l m a t r i c e s p r i o r t o q u a n t i t a t i o n .
A summary o f t h i n - l a y e r a d s o r b e n t s and m o b i l e phases used
i n these separations i s g i v e n i n Table I X .
Table I X
Th in-L ayer Chromatography Systems f o r C 1o t r imazol e
P l a t e Medium So 1vent Detect i o n R f Value Reference

( see be1ow) (see below) (see below)
1 s t Dimension : 0.57
2nd Dimens i o n : I 0.64 9
1 s t Development:
2nd Development: I1 - 17
1 s t Development:
2nd Development: I1 - 13
1 s t Development:
2nd Development: TI1 0.4 27
F I11 0.55 27
C I1 0.3 1,28
G IV 0.65 25
H IV,V 0.4 25
J IV,V 0.85 10
250 JOHN G. HOOGERHEIDEAND BRUCE E. W Y K A
Table IX (continued)
Plate Medium
a. Silica gel 60.
b. Silica gel foil (Polygram Sil NH.R, Machery-
Nagel ) .
c. Silica gel 60 F .
Solvent System
A. Benzene:methanol (4:l).
B. Methanol.
C. Chloroform.
D. Petroleum ether (40-60°):acetone:
benzene:ethanol :pyr id ine
(70:12:10:7:1).
E. Benzene.
F. Petroleum ether (40-60') :ethyl-
acetate:acetone:ethanol :
ammon i a ( 25%) (45:25 :25 : 5 :0.5 ) .
G. Xy1ene:n-propanol :ammonia (180:20:1).
H. Ether equilibrated with ammonia vapor.
J. Ethyl acetate:ammonia (25%) (98:2).
Detection Method
I. Autoradiography.
11. Trichloroacetic acid in n-butylacetate.
111. Spray sequentially with ethanolic iodine, sodium
carbonate, and sulfanil ic acid/sodium nitrite.
IV. Fluorescence quenching under short-wave
ultraviolet light.
V. Spray with Dragendorff reagent.
CLOTRIMAZOLE 25 1
Q u a n t i t a t i v e a n a l y s e s e m p l o y i n g TLC h a v e b e e n
c a r r i e d o u t i n s e v e r a l ways. R i t t e r , e t a l . (27) used
densitometry t o determine c l o t r i m a z o l e t h e TLC p l a t e
a f t e r formation o f a red s u l f a n i l i c acid derivative. Their
c a l i b r a t i o n c u r v e s were l i n e a r between 0.5 and 4 p g c l o t r i -
mazole per spot. O t h e r i n v e s t i g a t o r s have d e t e r m i n e d t h e
d r u g substance s p e c t r o p h o t o m e t r i c a l l y b y s c r a p i n g t h e bands
o f f t h e TLC p l a t e and r e a c t i n g e i t h e r w i t h b u t y l a c e t a t e
and p e r c h l o r i c a c i d (1,13,17,28) o r w i t h bromphenol b l u e
(10).
6.5.3 Gas Chromatography
C l o t r i m a z o l e i n human s k i n s a m p l e s h a s b e e n
d e t e r m i n e d b y gas c h r o m a t o g r a p h y w i t h e l e c t r o n - c a p t u r e
detection (20,34). Samples were e x t r a c t e d w i t h e t h e r ,
d r i e d , r e d i s s o l v e d i n benzene and c h r o m a t o g r a p h e d on 6',
1/8" g l a s s columns packed wdth 3% OV-17 on Gas Chrom Q.
Column t e m p e r a t u r e was 250 C and t h e c a r r i e r gas was
argon-methane a t 9 ml/min. S t a n d a r d c u r v e s were l i n e a r
o v e r t h e r a n g e o f 1-25 ng i n j e c t e d .
6.5.4 H i g h P e r f o r m a n c e L i q u i d Chromatography
S t a b i l i t y - i n d i c a t i n g assays o f c l o t r i m a z o l e i n t h e
b u l k d r u g s u b s t a n c e and i n f o r m u l a t i o n s have been c a r r i e d
o u t b y h i g h performance 1 i q u i d c h r o m a t o g r a p h y ( 3 5 ) . The
a n a l y s e s were p e r f o r m e d on a IA Bondapak C i a column w i t h a
m o b i l e phase o f methanol:O.O25M K 2 H P h ( 3 : l ) a t 1 . 0 m l / m i n .
Chromatographic r e s p o n s e was l i n e a r f r o m 2 t o 40 p g c l o t r i -
mazole i n j e c t e d . Average r e c o v e r y o f d r u g s u b s t a n c e f r o m
f o r m u l a t e d m a t e r i a l s r a n g e d f r o m 99.5 t o 100.0 p e r c e n t .
A n a l y s e s were r e p r o d u c i b l e , w i t h between-day r e l a t i v e
s t a n d a r d d e v i a t i o n s between 0.6 and 1.8 p e r c e n t .
6.6 Radiochemical A n a l y s i s
14
Duhm, e t a l . (18,36) used C-labelled c l o t r i -
mazole t o detEmTne drug d i s t r i b u t i o n s i n b i o l o g i c a l
samples. R a d i o a c t i v i t y was d e t e r m i n e d i n serum and u r i n e
b y t h e use o f l i q u i d s c i n t i l l a t o r s c o n t a i n i n g 8 g / 1 o f
b u t y l PBD i n t o l u e n e : d i o x a n e * e t h a n o l ( 1 : l : l ) . S k i n samples
were burned; t h e r e s u l t i n g I4C02 was absorbed i n base and
c o u n t e d b y means o f s c i n t i11 a t o r s .
252 JOHN G. HOOGERHEIDE AND BRUCE E. WYKA
6.7 Microcalorimetr i c Analysis
I n h i b i t i o n o f t h e r e s p i r a t i o n o f Saccharomyces
c e r e v i s i a e b y a n t i f u n g a l agents p r o v i d e s t h e b a s i s f o r a
s e n s i t i v e c l o t r i m a z o l e assay. Beezer and c o - w o r k e r s ( 3 7 )
m o n i t o r e d r e s p i r a t i o n m i c r o c a l o r i m e t r i c a l l y b e f o r e and
a f t e r a d d i t i o n o f c l o t r i m a z o l e ; degree o f i n h i b i t i o n o f
r e s p i r a t i o n was r e l a t e d t o t h e c o n c e n t r a t i o n of
c l o t r i m a z o l e . The minimum d r u g c o n c e n t r a t i o n d e t e c t a b l e b y
t h i s method was 3 x 10’5M.-
6.8 M i c r o b io 1og i c a l A n a l y s i s
M i c r o b i o l o g i c a l assays o f c l o t r i m a z o l e a r e a g a r
d i f f u s i o n assays based on a c o m p a r i s o n between t h e g r o w t h
i n h i b i t i o n zones p r o d u c e d b y s t a n d a r d s o l u t i o n s and t h o s e
produced b y t e s t samples. The t e s t o r g a n i s m C a n d i d a
p s e u d o t r o i c a l i s v a r . c a r s h a l t o n has been used b y several
*12,27,38) t o a s s a y c l o t r i m a z o l e i n serum,
u r i n e , and f e c e s . When t e s t i n g t h e s t a b i l i t y o f
++
c l o t r i m a z o l e i n c u l t u r e medium, H o e p r i c h and Huston ( 3 9 )
employed K l u v e r o m c e s f r a i l i s as t e s t o r g a n i s m .
s t u d y o f c o t r i m a z o l e s t a i i t v on samDle d i s c s . S a u b o l l e
and i o e p r i c h (40) used Candida” a l b i c a n s as t e s t ’ o r g a n i s m .
H o l t ( 4 1 ) has d i s c u s s e d s e o f s e v e r a l o r g a n i s m s and
In a
assay t y p e s i n t h e a n a l y s i s o f a n t i f u n g a l d r u g s , i n c l u d i n g
c l o t r i m a z o l e.
Two p a p e r s h a v e compared m i c r o b i o l o g i c a l a s s a y
r e s u l t s t o t h i n - l a y e r chromatographic determinations o f
c l o t r i m a z o l e (27,42).
7. Acknowledaements
The a u t h o r s w i s h t o t h a n k t h e S c h e r i n g C o r p o r a t i o n
Research L i b r a r y and P h y s i c a l and A n a l y t i c a l C h e m i s t r y
S t a f f s , i n p a r t i c u l a r Ms. Jean Nocka, Ms. M i c a e l a K a t z ,
D r . Henry S u r p r e n a n t , D r . Mohindar S. Puar, Ms. Jane
L i m p e r t , and Ms. L a u r e l Andersen, f o r t h e i r a s s i s t a n c e i n
the preparation o f t h i s analytical p r o f i l e .
CLOTRIMAZOLE 253
References
1. Plempel, M., Bartmann, K., Buechel, K. H., and Regel,

E., Deutsch. Med. Wschr. 94, 1356 ( 1 9 6 9 ) .
2. Oberste-Lehn, H., Baggesen, I., and Plempel, M.,

Deutsch. Med. Wschr. 94, 1365 (1969).
3. Marget, W. and Adam, D., -

Med.- n . 64, 1235 ( 1 9 6 9 ) .
K l i-
4. Buechel, K. H., Plempel, M., and Bartmann, K., E.

-
B e r . , 39 (1973).
5. Buechel, K. H., D r a b e r , W., Regel, E., and Plempel,

M., Arzneim. -
F o r s-
c h . 22, 1260 ( 1 9 7 2 ) .
6. Borka, L., and V a l d i m a r s d o t t i r , S., --

A c t a Pharm. -
Suec.
-
12, 479 ( 1 9 7 5 ) .
7. Schering-Plough L a b o r a t o r i e s , Qua1 i t y C o n t r o l Data.
8. K u h n e r t - B r a n d s t a e t t e r , M., Boesch, L., and E c k s t e i n ,

G., S c i . Pharm. 46, 54 ( 1 9 7 8 ) .
9. Maul, W. and S c h e r l i n g , D., -

J. Label. Comp.
Radiopharm. -
14, 403 ( 1 9 7 8 ) .
10. Schering-Plough Corporation, unpublished r e s u l t s .
11. Duhm, B., Medenwald, H., P u e t t e r , J., Maul, W.,

Med.- J .
Patzschke, K., and Wegner, L.A., P o s t g r a d . -
-
50, 13 (1974) S u p p l . 1.
12. Burgess, M. A. and Bodey, G. P., Antimicrob. Agents

Chemother. 2. 423 (19721.
13. W e i n g a e r t n e r , L . , G r u e n d i g , C., P a t s c h , R., and

S i t k a , U., Z e n t r a l b l . -
Pharm.- 111, 465 (1972).
14. Plempel, M., Postgrad. -

Med.- J-
. 55, 662 ( 1 9 7 9 ) .
15. Weuta, H., "Proc. 9 t h I n t e r n a t . Cong. Chemother.",
London, J u l y , 1975, i n Chemotherapy - 6, 179 ( 1 9 7 5 ) .
16. Rosenkranz, H. and P u e t t e r , J . , E.

2. D r u g . Metab.
Pharmaco. -
2, 73 (1976).
17. Weingaertner, L., Sitka, U., and G r u e n d i g , C., E.

-
J . -C l i n . Pharmacol. -
8, 131 (1973).
18. Duhm, B., Maul, W., Medenwald, H., P a t z s c h k e , K . ,

Wegner, L. A., and Oberste-Lehn, H., A r z n e i m . F o r s c h .
22, 1276 (1972).
-
19. J . -Cutan.
H o l t , R. J . , - - - P a t h o l . 3, 45 (1976
20. W a l l a c e , S. M . , Shah, V . P., E p s t e n, W. L.
Greenberg, J . , and Riegelman, S., 3. Dermatol,
-
113, 1539 (1977).
21. S a w y e r , P. R . , Brogden, R. N . , Pinder, R. M.,

S p e i g h t , T. M., and Avery, G. S., Drugs -
9, 424
(1975).
22. Meade, R. H., Am. -J -

- - - Pharrn. 36, 1326 (1979).
. Hosp.
23. Seneca, H., B i o l o g i c a l B a s i s o f Chemotherapy o f
I n f e c t i o n s and I n f e s t a t i o n s , P h i l a d e l p h i a , D a v i s , pp.
921-929 (1971).
24. KrsEmar, J . , S o t o l o n g o , M. A., and Kr6Emarov6, J.,

Cesk.
-- Farm. - 26, 345 (1977).
25. U n i t e d S t a t e s Pharmacopeia X X , p . 159.
26. Szabolcs, L., --

A c t a Pharm. Hung. -
46, 43 (1976).
27. Ritter, W., Plempel, M., and P u e t t e r , J., Arzneim.

F o r_
_ h . -24,
s c_ 521 (1974).
28. M a r g e t , W. and Adam, D., Acta Pediatr. - - 60, 341

Scand.
(1971).
29. P e l l e r i n , F., G a u t i e r , J . A., and Demay, D., Ann.

- - F-
Pharm. r a n c . 20, 97 (1962).
30. P e l l e r i n , F., G a u t i e r , J . A., and Demay, D., Ann.

Pharm. F r a n c . 22, 495 (1964).
CLOTRIMAZOLE 255
31. P e l l e r i n , F., G a u t i e r , J. A., and Demay, D., Talanta

12,
- 847 ( 1 9 6 5 ) .
32. U n i t e d S t a t e s Pharmacopeia XX, p . 160.
33. Kudo, A., Jap. -J. -C l i n . Rep. -

6, 85 (1972).
34. Wallace, S. M., Shah, V. P., Riegelman, S., and

E p s t e i n , W. L., -
Anal. - 611, 461 (1978).
Lett. -
35. Hoogerheide, J. G . , S t r u s i a k , S. H., Taddei, C. R.,

Townley, E. R., and Wyka, B. E., J. - Assoc.-O f f-
. Anal.
Chem. ( t o be p u b l i s h e d J u l y , 1 9 8 l r .
36. Duhm, B., Maul, W., Medenwald, H., P a t z s c h k e , K.,

Wegner, L . A. and P u e t t e r , J., P o s t g r a d . Med. J. 50,
13 (1974), Suppl 1. .
37. Beezer, A., Chowdhry, 6. Z., N e w e l l , R. D., and
T y r r e l l , H. J . V., -
A n a l-
. Chem.
- 49, 1781 ( 1 9 7 7 ) .
38. H o l t , R. J. and Neman, R . L., J. C l i n . P a t h o l . 25,
1089 (1972).
39. Hoeprich, P. D. and Huston, A. C., A b s t r a c t 104,
Proc. -
- 1 4 t h I n t e r s c i e n c e Conf. Antimicrob A X s
Chemother., San F r a n c i s c o , CA, S e p t . 11-13 (i97*
40. S a u b o l l e , M. A. and H o e p r i c h , P. D., A b s t r a c t 102,

Proc. -
- 14th I n t e r s c i e n c e Conf. A n t i m i c r o b . A q X s
Chemother., San F r a n c i s c o , CA, S e p t . 11-13 ( 1 9 7 4 ) .
41. H o l t , R. J . , J . C l i n . P a t h o l . 28, 767 ( 1 9 7 5 ) .
42. H o l t , R.J., Abs. P r o c . 1 0 t h I n t e r n a t . Cong.

.
M i c r o b i o l , Mexico, p . m , A C 9 - l m .
L i t e r a t u r e s u r v e y t e r m i n a t e d December, 1980.
DOPAMINE HYDROCHLORIDE
James E . Carter, John H . Johnson,
and David M. Bamke
1. Description 258
1.1 Chemical and Proprietary Names 258
1.2 Empirical Formula 258
1.3 Appearance, Color, and Odor 258
2.2 Solubility Profile 259
2.5 Proton Magnetic Resonance Spectrum 259
2.6 "C Magnetic Resonance Spectrum 263
2.7 Mass Spectra 265
2.8 Differential Scanning Calorimetry 266
3. Synthesis 268
4. Analysis 269
4.2 Colorimetric Assay 269
4.3 Nonaqueous Titration 269
4.4 Thin-Layer Chromatography (TLC) 269
4.5 Gas Chromatography 270
4.6 High Performance Liquid Chromatography (HPLC) 270
5. Stability 270
6. Analysis of Biological Samples 27 1
7. Metabolism and Excretion 27 1
8. Acknowledgement 27 1
References 272
Copyright 0 1982 by The American

Analytical Profiles of Drug Substances
ISBN 012-260811-9
258 JAMES E. CARTER E T A L .
1. Description
1.1 chemical and Proprietary Names
Dopamine hydrochloride is the mn-proprietary
name for 4-(2-aminoethyl)-1,2-benzenediol hydrochloride.
The free base (dopamine) has also been known in the
chemical literature as 3-hydroxytyramine and 3,4
dihydroxyphen~thylamine. The drug is available generically
for the correction of hamdynamic imbalances present in
the shock syndmm due to myocardial infarction, t r a m ,
endotoxic septicemia, open heart surgery, renal failure
and chronic cardiac decanpensation as in congestive heart
failure (1). Proprietary names include Cardiosteril, Docard,
Dopamine Fabre, Dopamine Gullini, Dopastat, Dynatra,
Intropin, Intropinject, Intropine and Orion.
1.2 Empirical F o d a
C8H12Cm2
Wlecular Weight
189.64
Structure
1.3 Appearance, Color & Odor

Dopamine hydrochloride is a white to off white
odorless crystalline pcxder.
2.1 Melting range
The Merck Index reports the mlting point of the
hydrochloride salt as 241OC with decanposition (2). The
DOPAMINE HYDROCHLORIDE 259
equilibrium melting range ( i n the absence of a i r ) has been

established a s 245OC - 246OC with deccenposition ( 3 ) .
2.2 Solubility Profile
Dopamine hydrochloride is f r e e l y soluble i n water:

soluble i n mthanol and hot ethanol: p r a c t i c a l l y insoluble
in ether, chloroform, benzene and toluene. Dopamine
hydrochloride is soluble in aqueous solutions of a l k a l i
hydroxides.
The KBr pellet infrared spectrum of 0.5% d o w e

hydrochloride obtained with a Perkin-Elmer 283 Infrared
Spectrophotomter is contained i n Figgie 1. The b r q d strong
absorption bands appearing a t 3350 cm and 3230 cm are due
to the O-H and N-H stretching vibrations. The i n t e m l e c u l a r
and i n t r a m l e c u l a r hydrogen bonding s h i f t s these absorption
bands to-lower frequencies than a f r e e h y d r o q l group (3700 -
3500 an ) and a f r e e a q n e (3500 - 3300 an ) . The -1
aromatic (3100 - 3000 cm ) and a l i p h a t i c (3000 - 2900 cm )
C-H stretching bands a r e prevent as expected. The N-H
bending vibrations 25 tile NH group shaw medium absorption
centered a t 1610 cm . The 2-H out of plane bending of
the 1 , 2 , 4 t r i s u b s t i t u t e d F e n e ring i indicated by
the sharp bands a t 876 cm and 812 cm . -B
2.4 Ul'zaviolet spectrum
Dopamine hydrochloride exhibits a single absorption
peak centered a t 280 nm w i t h a mlar absorptivity (E) of 2707
(Figure 2 ) . The spectrum was obtained with a Beclanan Acta
I11 double beam spectro@otometer.
2.5 Proton Maanetic Resonance S m t r u m
The 60 MHz proton magnetic resonance spectrum was

obtained with a Varian Associates T-60A spectrcneter. The
spectrum i n CD OD with tetramethylsilane (TMS) as internal
reference is d n t a h e d i n Figure 3. The s p l i t i n g patterns
are not simple. Howsver, t h e integration and qeneral
locations of psaks are consistent w i t h the structure. The
peaks a t 0.8 and 3.35 are due to mall amounts of CH30H
in the deuterated solvent.
Fig. 1 KBr Infrared Spectrum of Dopamjne Hydrochloride.
Instmnent: Perkin-Elmer MAel 283
Oopamfne H y d r o c h l o r i d e
UV Spectrum
Scan Speed 0 . 5 nmlscc

Chart Expansion 20 nmlinch
C o n c e n t r r t l o n Span 1.0
deuterium
280 nm
Concentration 2.109 x 10-4 n o l e / L
Absorbance 0.571
Nolar AbsorDtivlty 2707
Reference Cell 1% Sodium B i r u l f i t e
Operator U.UrekSv;&
Date t i - i 2 - # /
I I
240 240 200 SbO $0 nm
Fig. 2 Ultraviolet spectrum of IXpamhe Hydrochloride

Instrument: BeclaMn Acta 111
261
I
r :
I I I I I!
f--- I
li
I I I I I
1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . . . . . I . . . . I . . . . I . 1 . . . I
80 7 0 60 50 PPM:d: 4 0 30 20 10 0
Fig. 3 Proton Magnetic Resonance Spectnnn of Dopamine Hydrochloride in CD3OD

Instrument: Varian T-60A
Chemical s h i f t s (b) i n ppn r e l a t i v e to TMS are:
H O G C H ,-CH ,-NH ,.HCI
Proton # of Chemical
Assigment Protons S h i f t (b) Mu1t i p l i c i t y
a 4 3.0 multiplet
b 5 4.95 multiplet
C 3 6.75 multiplet
2.6 I3C Magnetic Resoname Spectrum
The "C analysis of dopamine hydrochloride was

taken on a JEOL FX-270 superconductiny NMR operating a t a
frequency of 67.83 IWz using a 45" (9 usec) pulse, a 3.6
second r e p e t i t i o n rate and 16384 data points ( 4 ) .
The sample was dissolved i n D20; P-pfoxane was

added. as a reference. Spectra of the e n t i r e C range were
obtained with ccmplete decoupling and gated decoupling with
Nuclear Overhauser Effect (NOE) to obtain the chemical s h i f t s
and coupling constants. The ccmpletely decoupled sFectrum is
sham i n F j w e 4.
The resolution of a l l a r m t i c carbons (not cham
in Figure 4 ) was obtained using a 2.5 kHz width. The
assignrrent of t h e a r m t i c carbon l i n e s was aided by
canparison to 3,4 dihydroxybenzene.
Fig. 4 l 3 C Magnetic Resonance Spectnnn of Dopamine Hydrochloride w i t h p-dioxane as reference.
Instrurtlent: JExlL FX 270
With the carbons n-red as indicated below the

chemical shifts and coupling constants are as s h m in
T a b l e I.
r
2
HO
Table I. I3C assignments and C-H

coupling constants for
Dopamine Hydrochloride
Carbon Number Shift (ppn) Jm(Hz)

129.1 -
116.3 159.3
143.9 -
142.7 -
116.4 156.7
121.1 160.0
32.0 128.3
4c.7 143.9
2.7 Mass Spectra
The electron inpact (EI) mass spectnrm at 70 eV

and the methane derived chemical ionization (CI) mass
spectrum were obtained with a Hewlett-Packard 5985 quadraple
mass spectrcaneter (5).
The CI spectrum contains t w prcaninent ions at n4e
137.0 and m/e 154.0. The m/e 154.0 corresponds to C H NO
which is protonated dopamine. Loss of m n i a leave8 &gg8;
which is the base peak i n the spec=tnrm at m/e 137.0.
266 JAMES E. CARTER ETAL.
The E I spectrum i s surprisingly simple. The base

peak a t m/e 124.1 corresponds to C H 0 The intact
dopamine a t m/e 153.0 i s seen in &e8s&ctrum. The m/e
77.0 and m/e 78.l+are due $0 the a m t i c r i n g and
correspond t o C H and C F respectively. The
hydrochloride s812 is no$ geen i n e i t h e r the E I o r C I
spectrum which js as expected.
2.8 Dif ferenti a1 Scanning Calorink=tq
Dopamine hydrochloride was heated a t a rate of 10'

per minute i n a Perkin-Elmr DSC-2 d i f f e r e n t i a l scanning
calorimeter frcan 460'K t o 530'K. A single sharp endothem
w a s observed with an onset temperature of 516'K (243OC) and
with the endothem maximum a t 519.8'K (246.8'C). An accurate
heat of t r a n s i t i o n bH) could m t be calculated because of
deccanposition upon rwlting.
2.9 C r y s t a l Properties
Crystals f r m a representative l o t of dopamine

hydrochloride are predaninantly triangular p l a t e s ( 3 ) . The
o p t i c a l crystallographic examination indicated t h a t the
crystals are triclinic or Fossibly m n o c l i n i c .
X-ray powder patterns w e r e obtained w i t h copper

radiation and a nickel f i l t e r . The d-spacings and r e l a t i v e
k t e n s i t i e s (I/I ) are listed i n T a b l e 11. The f i r s t column
shws the p a t t e r 8 f o r the drug a f t e r grinding to break up t h e
p l a t e s and therefore the preferred o r i e n t a t i o n of the
c r y s t a l s . Column 2 shows the d i f f r a c t i o n pattern of the
crystals without p r i o r grinding. The absence of several
key d i f f r a c t i o n l i n e s and the reduced influence of o t h e r s
i n this orientation is c l e a r l y d m n s t r a t e d by canparing
t h e two columns.
TABLE I1
X-ray Diffraction M e r Patterns of Dopamine Hydrochloride
Ground Sample Unground Sample

d-spcing I/Io d-spacing IDo
6.37 5
5.56 30 5.56 10
5.50 60
5.24 5 5.24 20
4.57 75 4.58 15
4.18 95 4.19 30
4.07 30 4.07 15
3.98 100 3.97 15
3.81 65 3.82 100
3.72 60 3.71 15
3.53 70 3.53 30
3.49 45 3.49 65
3.43 70 3.43 20
3.23 65 3.23 20
3.19 45
2.96 10 2.97 5
2.83 10 2.83 5
2.77 55 2.77 15
2.75 45
2.69 10
2.63 60
2.62 60 2.62 80
2.55 60
2.41 10 2.55 15
2.27 10
2.17 20 2.17 10
1.90 10 2.09 5
1.99 5
1.86 10 1.96 5
1.92 5
1.90 5
JAMES E. CARTER E T A L .
3. Synthesis
Dopamine hydrochloride i s available from a variety

of ccprmercial sources. Details of the synthetic process are
considered proprietary information. However, one workab1.e
process using known reactions and readily available materials
is shown in Figure 5 ( 6 ) .
CH30~cHoCH3N02
CH3O
-
cH30D
CH,O
CH=CHNOp
H2
-
cH30x3
CH 3O
CH2CH2NH2
H Br
HO
Fig. 5 Synthetic Scheme for Dopamine Hydrochloride
The d h t h o q p h e n e t h v l a r h e (knma s
homveratrylamine) has been converted to dopamine
hydrochloride in one step with w i d b e hydrochloride ( 7 ) .
4. Analysis
4.1 E l m t a l Analysis
Elerwntal analysis of a typical dopamine

hydrochloride sample is as follaws:
Element % Theoretical % Found
C 50.67 50.51
H 6.38 6.55
c1 18.70 I-
N 7.39 7.37
0 16.87 ---
4.2 Colorimetric Assay
A colorimetric assay f o r the analysis of d0pami.m

has been adapted f m The United S t a t e s Phaxmampeia
Epinephrine Assay ( 8 ) . "The Photarnetric Detection of
Adrenaline in P h a r m e u t i c a l Products" was f i r s t described by
Jhty in 1948 ( 9 ) .
4.3 Non-aqueous T i t r a t i o n
The purity of dopamine hydrochloride may be

assessed by non-aqueous t i t r a t i o n with crystal violet as
the indicator. The hydrochloride salt is dissolved in g l a c i a l
acetic acid, mercuric acetate is added to remove the chloride
as unionized mercuric chloride, crystal violet is added and
the amine is titrated to a green end-point with 0.1 N -
p r c h l o r i c acid.
4.4 Thin-layer Chromatoqraphy (TLC)
Thin-layer chrormtography on silica g e l is

p a r t i c u l a r l y helpful in assessing the purity of the r a w
drug. I n a solvent system of ethyl acetate-methanol-
m n i n u n hydrGxide (85:10 :5) a l l p o t e n t i a l
wthoxy-phenethylamine impurities are readily separated
and differentiated. The 3-hydroxy-4-methoxyphenethylamine and
dopamine fluoresce under s h o r t wave ultraviolet l i g h t .
After spraying w i t h ninhydrin and developing a t 110' f o r
5 minutes dopamine appears as a brown spot (R N 0.07) , the
f
3-hYdr0~-4-~thO analog
~ appears as a yellaw spot
(Rf N 0 . 2 2 ) , the 3-methoxy-4-hydroxy analog appears as
a pink spot (Rf N 0.26) and the dimethoxy compound a p p a r s
as a pink spot (Rf N 0.30).
270 JAMES E. CARTER ETAL.
I n a solvent system of n-buitanol-glacial acetic

acid-water (12:3:5), doparnine has an R of approximately 0.6.
f
The canpounds may also be visualized by spraying t h e silica
p l a t e with 0.5% iodine i n chloroform.
4.5 Gas ChmnatouraDhv
Because of the high-boilirLg, polar and oxidizable

nature of dopamine, it i s not readily amenable
to gas chrmtography.
4.6 High Performance Liquid Chrmtography (HFTC)
N o work has been reported for t h e HPLC analysis of

dopamine hydroFhloride i n pharmaceutical dosage forms as
might be expected f o r a non-patented product. One relevant
papr reports t h e chrmatographic behavior of dopamine and
related catecholamines i n a reverse phase system on t h e
ubiquitous octadecylsilane column (10) . The authors found
dopamine to be insensitive t o pH changes b e b e e n 2 and 5
w i t h n i t r i c acid but to be retained longer as t h e pH
increased in acetic acid. Doparnine appeared to form ion
pairs being retained f o r longer times with t r i c h l o r o a c e t i c
acid and octylsulfonic acid than with mineral acids.
5. Stability
Dopamine hydrochloride ( l i k e other catecholamines) i s

r e l a t i v e l y unstable t o heat, l i g h t and oxygen. The presence
of oxidizing agents or trace mtals such as copper or iron
also increases t h e rate of degradation. Properly stored i n
g l a s s containers t h e raw drug i s stable f o r three to f i v e
years.
Dopamine hydrochloride is generally f o m l a t e d with 1%

sodium b i s u l f i t e as an antioxidant. The r e s u l t a n t solution
has a pH of about 4 and when properly protected f r m a i r , heat
and l i g h t i s s t a b l e f o r three to f i v e years.
The s t a b i l i t y and compatibility of dopamine solutions

with c m n intravenous f l u i d s (11)various antibioiics (12)
and miscellaneous drugs and additives w i t h which it could be
potentially mixed (13) has been reported. Dopamine i s
unstable a t an aklaline pH. Thus dopamine solutions are not
stable i n sodium bicarbonate but are stable i n dextrose,
sodium chloride, sodium lactate, l a c t a t e d Ringer's solution,
and mannitol.
DOPAMINE HYDROCHLORIDE 27 1
6. Analysis of Biological Samples
Because of its rapid metabolism, methods f o r the

routine analysis of dopamine i n blood and urine have not
been developed. Methods which have been reported are
generally concerned with endogenous dopamine and its
mtabolism rather than the clinical pharmacology and
analysis of infused dopamine.
The analysis of l 4 C - d o m e and its radiolabelled

metabolities following an infusion i n human subjects has
been reported ( 1 4 ) .
A novel approach which may be applicable to analysis

following a dopamine infusion has recently been developed
(15). This method converts dopamine (and other catechols)
t o i t s mthoxy derivative i n the presence of catechol-c-
methyltransferase and S-adenosylmthionine- H methyl. The
t r i t i a t d amines are extracted from plasma with d i e t h y l
ether, separated by TLC and measured i n a s c i n t i l l a t i o n
counter. The method i s reported sensitive t o 0.1 mle/L.
7. Metabolism and Excretion
Dopamine metabolism and T g r e t i o n was studied follawing

intravenous administration of C material to six healthy
males ( 1 4 ) . Dopamine and its metabolites are excreted
primarily in the urine: the rsdioactive dose was
quantitatively recovered a f t e r 5 days. Approximately 75% of
the dose was converted i n t o dopamine-related lnetabolites.
The principal product was 3-methoxy-4-hydroxyphenylacetic
acid. Other p r h e n t metabolites were 3-methoxydopamine,
3,4-dihyroxyphenylacetic acid, 3,4-dihydroxyphenylethanol
and 3-methoxy-4-hydroxyphenylethanol. The remainbg 25%
of the infused dopamine w a s transformed i n t o norepinephrine
and a p a r e d i n the urine principally as metabolites of
norepinephrine.
The conjugates of doparnine arid its metabolites were

studied in cultured human skin fibroblastc and rat hepatam
c e l l s ( 1 6 ) . These studies along with references c i t e d
therein indicate the principal dopamine conjugates a s the
3-0 sulphate, and the 3+glucuronide.
8. Ackncwledgemnt
The manuscript was expertly typed by M s . Martine Bunting.

212 JAMES E. CARTER E T A L .
References
1. Anon., I n t r o p i n product literature, Awrican Critical

C a r e , WGaw Park, IL 60085.
2. The Merck I d a , 9 t h Edition, 3422, Merck & Co., Inc.,

F?ahway, N J , 1976.
3. S. P a l e d , Walter C. Mccrone Associates, Inc., Chicago,

I L , 60616. Personal Comnunication.
4. W i l l i a m J. McGranahan, Northwestern University,

D e p r t m n t of Chemistry, Evanston, I L 60201. Personal
Cammication.
5. Hoying L. Hung, Northvestem University, Departrent of
Chemistry, Evanston, I L 60201. Personal C m i c a t i o n .
6. Paul W. Erhardt, American Critical C a r e , McGaw Park, IL,

60085. Personal C a m m i c a t i o n .
7. P. Fabre, -
French P a t e n t 2332748-y36, (1977).
8. Anon., United States Pharmacopeia, XX Revision, 919,

Mack Publishing Co., Easton, PA, 1980.
9. J.R. Doty, Anal.

-- Chem., -
20, 1166 (1948).
10. P.A. A s r m s and C.R. meed, -

J. Chromatogr., 169, 303
(1979).
11. L.A. Gardella, J.F. Zaroslinski and L.H. Possley, Am.

-
J.
- Hosp. - -
Phann., 32, 575 (1975).
12. L.A. Gardella, H. Kesler, J.E. C a r t e r and J.F.

- -J. Hosp. Phann., 33, 537 (1976).
Zaroslinski, Am.
13. L.A. Gardella, H. Kesler, A.H. Amann and J.E. Carter,

- -J. Hosp. Pharm.,
Am. 35, 581 (1978).
14. M. Goodall and H. Alton, Biochem. Pharmacol., -
17, 905
(1968).
15. G. Koch, U. Johansson and E. Arvidsson, - -- l h . C

J. C -hm.
C l h Biochem., -
18, 367 !1980).
16. P.A. Crooks, X.O. Breakefield, C.H. Sulens, C.M.

C a s t i g l i o n e and J . K . Caward, Biochem. -
J., __ - 187
176,
(1978).
ERGONOVINE MALEATE
Van D.Rdj
1. Description 274
1.1 General Classification 274
2.1 Infrared Spectra 275
2.2 Ultraviolet Spectra 275
2.3 Fluroescence and Phosphorescence Spectra 275
2.6 Differential Scanning Calorimetry 283
2.9 Solubility 283
2.11 Circular Dichroism Configuration 289
3. Synthesis 289
3.1 Biosynthesis 289
3.2 Chemical Synthesis 290
4. Stability 290
5. Identification 293
6.2 Direct Spectrophotometric Analysis 293
6.3 Colorimetric Analysis 293
6.5 Automated Analysis 295
6.6 Fluorometric Analysis 295
6.8 Radiochemical Procedures 296
I. Metabolism 296
8. References 308

Analytical Profiles of Drug Substances
Volume 11 273 Pharmaceutical Aamiation
ISBN 012-260811-9
274 VAN D.REIF
Description
1.
1.1 General Classification
Ergonovine is a naturally occurring alkaloid
found in ergot (Claviceps purpurea). It is classed as one
of the water-soluble, amine ergot alkaloids, and is an
orally-active oxytocic (1,2). The maleate salt exhibits
greater stability than the free base and is the usual form
in which the alkaloid is utilized (3).
The name used by Chemical Abstracts for ergono-
vine maleate is [8,&(S)]-9,10-didehydro-N-(2-hydroxy-l-
methylethyl)-6-methylergoline-8-carboxam~deI (Z)-2-bute-
nedioate (1:l) salt. The Chemical Abstracts Registry
Number is 129-51-1. Ergometrine, ergostetrine, ergoba-
sine, ergotocine, and D-lysergic acid L-2-propanolamide
are other names that have been used for this alkaloid
(2,4).
H
I
HC-COOH
It
HC-COOH
c 1gH23N302 ' CqH404 Mol. Wt. = 441.48

1.3 Appearance, Color, Odor
Ergonovine maleate is a white to greyish white,
odorless, crystalline powder.
ERGONOVINE MALEATE 275
The infrared spectra of ergonovine maleate in
potassium bromide and in mineral oil are given in Figures
1 and 2 , respectively. Both were recorded on a Perkin-
Elmer 467 Grating Spectrophotometer. The KBr spectrum is
similar to that previously published (5). The KBr proce-
dure is also used for an official identity test (6).
Structural assignments (7,8) of some of the significant
bands are given in Table I. As reported (71, a carbonyl
band for the maleate moiety is not distinguished in the
KBr dispersion, and is seen only as a shoulder at 1690
cm-l in the mull.
Table I
Infrared Assignments for Ergonovine Maleate
(0.5% KBr Dispersion)
Frequency range (cm-l) Assignment

3260-3500 N-H, 0-H stretch
1650 amide C=O stretch
1570 carboxylate annion
1055 primary hydroxyl
750,775 indole C-H

The ultraviolet spectrum of ergonovine maleate in
ethanol is shown in Figure 3 . An absorptivity o f 20.5
(E=9160) was determined for the maximum at 311 nm in alco-
hol. A similar spectrum is obtained using water as sol-
vent; the absorptivity at the maximum at 311 nm was found
to be 18.7 (E=8260). The absorptivity values agree with
those previously published (9,101.
2.3 Fluorescence and Phosphorescence Spectra
A fluorescence emission spectrum in ethanol is
shown in Figure 4 . Excitation was at 3 2 5 nm. It was
obtained on a Perkin-Elmer Model 512 fluorescence spectro-
photometer. A similar spectrum was reported by Bowd
et al. (10) for ergonovine, and phosphorescence maxima
were found at 514,554, and 611 nm at 77OK in ethanol.
WAVELENGTH (Microns)
2.5 3.0 4.0 5.0

I
6.0
1
8.0 10.0 12.0 16.0 20.0 30.0 40.0 1.0
I
I I I I I I I I i i i i i
4OoO 3500 m 2500 2Ooo 1800 1600 1400 1200 loo0 800 600 400 200
WAVE NUMBER cm-4
Figure 1 - Infrared Spectrum of Ergonovine Maleate, USP Reference Standard,

Lot L(KBr Pellet)
WAVELENGM (Microns)
2.5 3.0 4.0 5.0 6.0 7.0 8.0
I
9.0
I
10
,
12
I
14
1
16 18 20
I I I
M 40
I
I 1 I I 1
1
w
V
z
U
E
h)
21
5,
z
21 U
e
I
-z
c
7
800 600 400
WAVE NUMBER(Crn-4
Figure 2 - I n f r a r e d S p e c t r u m o f E r g o n o v i n e Maleate, USP R e f e r e n c e S t a n d a r d ,

Lot L (Mineral O i l Mull)
278 VAN D.REIF
0.7
0.6
0.5
y 0.4
z
a
m
ac
0
Ln
$ 0.3
0.2
0.1 Fig. 3. Ultraviolet

Spectrum of Ergonovine
Maleate (USP Reference
0 1 1 1 1 1 1 1 Standard, Lot L ) . Sol-
210 235 260 285 310 335 360 vent-Alcohol.
WAVE LENGTH IomJ
Fig. 4. Fluorescence
E m i s s i o n Spectrum of Ergo-
I I 1 I 1 novine Maleate, 0 . 1 mg/ml
560 500 440 380 320 (USP Reference Standard,
NANOMETERS Lot L)

The proton NMR spectrum of ergonovine maleate
(USP Reference Standard, Lot L ) in deuterated dimethyl-
sulfoxide (d6) with tetramethylsilane as internal stand-
ard is shown in Figure 5. The spectrum was obtained with
a 100 MHz Varian XL-100 spectrometer (11). Spectral
assignments are given in Table 11. Bands corresponding
to side chain hydrogens were identified by irradiation of
the side-chain methine hydrogen (s=3.9). This resulted
in conversion of doublets at 1.12, 3.44, and 8.25 ppm to
singlets. The alcohol hydrogen was assigned to the shift
at 3.68 ppm because resonance was lost at this shift
after D20 exchange. The shift at 10.98 ppm, assigned to
the indole-NH, also showed a partial loss of resonance
after D20. Similarly, the only resonances above 7.5 ppm
for related indoles, 8.5 ppm (CDC13) for lysergic acid
dimethylamide and 11.4 ppm (deuteropyridine) for penni-
clavine, were attributed to the indole-NH (12,13). The
6-NCH3,H8,H9, and aromatic proton assignments are also
similar to those reported for penniclavine and lysergic
ac id dime thy1amide .
Table I1
Proton NMR Spectral Assignments for Ergonovine Maleate
Chemical Shift ( S ) Multiplicity Assignment
1.12 doublet -CH3 (side chain)
3.10 singlet 6-NCH3
3.44 doublet C-CH20
3.68 singlet C-OH
3.9 mu1 t ip let NCH-C
4.30 mu1 t ip let c(H-8
6.14 singlet maleate C-H
6.57 sharp multiplet H-9
7.6-7.4 mu 1tip le t H-12,H-13,H-14,H-2
8.25 doublet 8CNH
10.98 singlet indole-NH
ERGONOVINE MALEATE 28 1
The 13C NMR spectrum of ergonovine maleate (USP

Reference Standard, Lot L) in deuterated dimethylsulfoxide
is shown in Figure 6. It was recorded on a Varian
FT-80-A spectrometer at ambient temperature (11). The
spectral assignments are given in Table 111. The chemi-
cal shifts are similar to those reported by Bach et al.
(14) for ergonovine.
Table 111
13C NMR Assignments for Ergonovine Maleate
Carbon E
!
-C02H 169.3
-CON 168.1
HC=CH 136.2
c-10 134.6
C-15 131.8
C-16 126.0
c-11 125.7
C-13 123.3
c-9 121.0
c-2 120.5
C-14 112.5
c-12 111.6
c-3 106.8
OCH2 65 .O
c-5 61.8
c-7 53.6
HN-CH 47.7
N-CH3 41.7
C-8 40.8
c-4 24.7
-CH3( side chain) 17.7
2.5 Mass Spectrum

The mass spectrum of ergonovine maleate was
obtained with a Kratos DS-50s Data System coupled with a
MS-902 double focusing, high resolution mass spectrometer
(15). The ionizing electron beam energy was at 70eV and
the probe temperature was 250°. A line graph of the mass
spectrum is shown in Figure 7 and identification of the
pertinent masses are presented in Figure 8. The retro-
Diels-Alder reaction fragments at m/e 282 and 196 and the
fragments at m/e 220-223 were postulated by Bellman (16)
for other lysergic acid analogs. The identity of frag-
ments at m/e 167 and 154 have been postulated for other
ergot alkaloids (17,181.
2.6 Differential Scanning Calorimetry
The DSC thermogram (19) of ergonovine maleate
(USP Reference Standard, Lot L) is shown in Figure 9.
The thermogram was obtained at a heating rate of loo/
min in a nitrogen atmosphere using a Perkin-Elmer DSC-2.
No endotherms or exotherms were observed other than that
associated with the decomposition melt.
2.7 Melting Range
The melting range must be taken under a pro-
tective environment to limit decomposition. The follow-
ing melting point temperatures have been reported for
ergonovine maleate:
OC
- Reference
167 (with decomposition) 4
180-190 (in paraffin) 20
187-191 (silicone oil) 21
186-196 (under nitrogen) 21
185-190 (sealed capillary) 21
The X-ray diffraction pattern of ergonovine
maleate (USP Reference Standard, Lot L) as obtained with
a Philips diffractometer using C u K d radiation (191, is
presented in Figure 10. The calculated "d" spacings are
listed in Table IV.
2.9 Solubility
The following solubilities have been reported for
ergonovine maleate (4,221:
53111SNllNI 3hllVl3tl
-
m/e ASSIGNMENT RELATIVE INTENSITY
325 m+ 100
310 mt -CH3 0.4
307 mt-H$ 2.2
294 m t -CHflH 2 .o
282
223 25.5
222
221 6.8
196
167 [ &NH]' 7.3
154 [ & 1' 6.7
Figure 8 - Mass Spectrum Fragments
285
286 VAN D.REIF
t
0
c3
z
w
50 75 100 125 150 175 200 225

OC
F i g . 9. D i f f e r e n t i a l Thermal A n a l y s i s Spectrum of Ergo-

n o v i n e Maleate (USP R e f e r e n c e S t a n d a r d , L o t L)
loo -
90 -
80-
70 -
60-
c 50-
B
I-
z
40-
29
Figure 10 - X-ray Diffraction Pattern of Ergonovine Maleate (USP Reference Standard,
Lot L)
288 VAN D.REIF
Table I V
X-Ray Powder Diffraction Pattern
2 0 d (Ao) 1/10
39.6 2.28 4
38.7 2.33 4
36.5 2.46 11
34.4 2.61 4
32.8 2.73 4
29.6 3.02 7
28.9 3.09 13
26.4 3.38 26
24.8 3.59 19
23.9 3.72 24
22.9 3.88 20
22.1 4.02 32
21.6 4.11 98
19.9 4.46 16
18.6 4.77 18
17.7 5.01 13
16.2 5.47 64
15.1 5.87 40
14.2 6.24 89
8.8 10.00 100
7.5 12.00 36
Solvent Solub i 1ity (mg/ml)

water 28
alcohol 8
chloroform nearly insoluble ( <0.1)
ether nearly insoluble ( < 0.1)
The following optical rotations were reported
(23) for ergonovine maleate:
[d]i0= +53 (cz1.0 in water)
20
[a1578 = +57 (Cz1.O in water)
20
["(I546 = +74 (C=l.O in water)
20
[HI436 = +252 (C=l.O in water)
USP material ( 6 ) meets the specification,
[O( Ii5" = +51 to +56 ( G 0 . 5 in water).
2.11 Circular Dichroism-Configuration
A circular dichroism spectrum was reported (24)
for ergonovine maleate. Maxima were at approximately 218
and 318 nm, and a minimum was at 248 nm. Other isomers
containing the D-lysergic acid moiety gave similar spec-
tra; a compound containing the L-lysergic acid moiety
showed a reversed effect. This is consistent with the
positive Cotton above 300 nm reported for D-lysergic acid
(25). The rotatory dispersive effects are due to the
configuration at C-5; the configuration at C-8 has only
an additive or subtractive effect. In the case of D-
lysergic acid, and therefore, ergonovine, the hydrogen
at C-5 is in t h e 4 position (25) and the C-5 center is
in the S configuration (26).
3. Synthesis
3.1 Biosynthesis
Ergonovine was originally isolated from ergot
extracts (2,27-29). It has since been biosynthesized in
+
cultures of Claviceps species (30-351, and has been iso-
lated from seeds of I omoea violacea (36,371 and
Argyreia nervosa (38 The following pathway has been
constructed (39-44) for biosynthesis of the ergoline
skeleton by Claviceps species:
290 VAN D.REIF
mevalonic acid + tryptophan )

- 4-dimethyl-
allotryptophan
elymoclavine + agroclavine ,<- chanoclavine-I

5.
6-methyl-Ll 8,9-ergoline-8-carboxylic acid
\1
lysergic acid
The final step to produce ergonovine was shown to involve
incorporation of alanine with lysergic acid (44).
3.2 Chemical Synthes.is
Various semi-synthetic procedures for ergonovine
maleate involve addition o f the alkyl side chain to lyser-
gic acid. The D-lysergic acid starting material is
obtained by alkaline hydrolysis of an ergot alkaloid frac-
tion of biosynthetic origin (45). A relatively high-yield
sequence is shown in Figure 11. Lithium lysergate is
reacted with a sulfur trioxide-dimethylformamide complex
at O°C. L-2-amino-1-propanol is added, and the product is
isolated after addition of water and treatment with maleic
acid. Procedures for formation of the amide bond using
lysergic acid chloride ( 4 6 ) or phosgene (47) have been
patented. A complete fiftegn-step synthesis o f the
D-lysergic acid starting material from 3b-carboxyethylin-
dole has also been reported (48).
4. Stability
The maleate salt shows improved stability over ergono-
vine base, but the salt also oxidizes and darkens in the
presence of oxygen (3). The stability of ergonovine s o l u -
tions has been improved by the use of antioxidants such as
ascorbic acid (491, methionine, and d-mercaptopropionyl-
glycine (50). The known degradation products are shown in
Figure 12. Heat treatment of ergonovine (a dextrorotatory
isomer) under acid or alkaline conditions causes a reversi-
ble isomerization at the 8-position to form the dextrorota-
tory ergonovinine, also known as isoergonovine. More
stringent acid or base treatment eventually results in
hydrolysis to lysergic acid, isolysergic acid, and 2-amino-
1-propanol (51,521. When ergonovine is refluxed in the
LiOH-HZO
D-LYSERGIC ACID w
CH30H
CO NH -c -CH3
- DMF
CH3CHNH2
I
CH20H
I MALEIC A C I D
ERGONOV I NE MALEATE
Figure 11 - Synthesis of Ergonovine Maleate from Lysergic Acid

292 VAN D.REIF
y
CONH-C-CH~
A,
-
H+ or OH^
OH{ ERGONOVlNlNE
ERGONOVINE
(Iso-ergonovine)
d3 HN
OH *
._
LYSERGIC ACID ISO-LY SERG IC ACID
& &
HN
y
CONH-C-CH3
H;HzoH +
HN
y
CONH-C-CH3
H;HpH
LUMIERGONOVINE I LUMIERGONOVINE II
Figure 12 - Degradation of E r g o n o v i n e
presence of amines (53), the centers at C-8 and C-5 are

both racemized to give mixtures of ergonovine, ergonovi-
nine, L-lysergic acid L-2-propanolamide and L-isolysergic
acid L-2-propanolamide. When acidic solutions of ergono-
vine are irradiated, the 9,lO double bond is hydrated to
form lumiergonovine I and small quantities of lumiergono-
vine I1 (54-56). Degradation of the ergonovine indole
ring has been indicated by reports (57) of unidentified
fluorescent degradation products which do not react with
p-dimethylaminobenzaldehyde.
5. Identification
Alternately known as Van Urk's (58) or Erhlich's (59)
reagent, 1-dimethylaminobenzaldehyde has had widespread use
in ergonovine identity tests (59-61) and chromatographic
detection sprays (6,60,62). Indoles give a blue color with
this reagent (61); phenols and amines may also react,
usually to give other colors (58,591. Color reactions may
also be produced with aldehydes such as vanillin, piper-
onal, and paraldehyde (63). Iodinated potassium iodide
(brown flocculent) and ferric chloride in phosphoric acid
at 80° (blue-violet color) are two additional identity
test reagents (60).
The elemental analysis of ergonovine maleate
(USP Reference Standard, Lot L) is presented below.
Element % Calculated % Reported (64)
C 62.57 62.55
H 6.17 6.03
N 9.52 9.38
6.2 Direct Spectrophotometric Analysis
Bayer (65) described an ultraviolet spectrophoto-
metric method for ergonovine maleate tablets. The tablets
are extracted with 1% tartaric acid and the absorbance is
measured at 317 nm.
294 VAN D.REIF
Colorimetric Analysis
6.3
Official assays for ergonovine maleate drug sub-
stance, tablets, and injection (6,601 employ the p-di-
methylaminobenzaldehyde reagent. Drug substance or
injection samples are dissolved in water; tablet samples
are dissolved in aqueous tartaric ac id-benzalkonium chlor-
ide. The reagent is dissolved in aqueous sulfuric acid-
ferric chloride. After color development, absorbances are
read at 555 nm. Light or hydrogen peroxide (63,66,67) have
also been used in place of ferric chloride to catalyze
color development for quantitation. A similar quantitative
procedure using sodium nitrite t o catalyze color formation
was shown to have the advantages of speed, sensitivity, and
color stability (62).
The specificity of the reaction for indoles under
several conditions was studied by Kupfer (61). The speci-
ficity was very dependent on the particular conditions
employed and the development times. In general, for
indoles to be reactive, an unsubstituted 2- or 3- position
was required. The mechanism proposed by Pohm (68) involved
attachment of one aldehyde molecule at the 2-positions of
two indole molecules with release o f water.
An assay for ergonovine maleate tablets using
metaldehyde to produce a color at 550 nm has been published
(69). A colorimetric assay for tablets using ion-pairing
with bromocresol purple was reported (70).
6.4 Titrimetric Analysis
Ergonovine maleate can be determined titri-
metrically in glacial acetic acid with a 0 . 0 5 N perchloric
acid titrant and crystal violet indicator (71,721. The NF
XIV drug substance assay (73) is similar, except that ace-
tic anhydride is added to the system. Ergonovine was
determined in mixtures after thin-layer chromatography
with chloroform-methanol (97:3) on alumina. Spots were
eluted with chloroform, acetone was added, and the mixture
was titrated with 0.005 N perchloric acid in methanol (74).
The maleate moiety has been determined with 0.01 potas-
sium methoxide titrant, thymol blue indicator, and pyri-
dine as solvent (71).
6.5 Automated Analysis

Kirchhoefer and Wells (75) developed automated
methods for ergonovine maleate tablets and injections.
Samples were dissolved in pH 6.0 tartrate buffer. For
colorimetric quantitation, the sample stream was made
basic and extracted with n-butanol. The extract was mixed
with p-dimethylaminobenzaldehyde reagent, and after react-
ion, the stream was mixed with sodium nitrite for determin-
ation at 550 nm. For fluorometric detection the sample
stream was diluted with tartrate buffer. Excitation was
at 325 nm and the emission was read at 432 nm. The methods
were tested for interference from common tablet and injec-
tion excipients.
Robertson et al. (76) reported a general procedure
for basic drugs in which ergonovine was included. Brom-
thymol blue solution was mixed with an aqueous buffered
sample stream. The ergonovine-dye complex was extracted
into chloroform and determined at 410 nm.
6.6 Fluorometric Analysis
Fluorometry has been used to determine ergonovine
maleate in tablets and injections in an automated procedure
(75) (see section 6.51, and after column chromatography
(77) (see section 6.73). Samples were read in 0.1 tar-
tartic acid buffer with excitation and emission at 325 and
432 nm, respectively.
The thin-layer chromatographic systems used
to analyze ergonovine and ergonovine maleate are listed in
Table V. The applications, means of quantitation or
detection,and procedure specificity are listed when this
information was supplied in the literature sources. The
last five systems employ ion-exchange layers.
6.72 Paper Chromatography
Paper chromatographic systems for ergonovine
and ergonovine maleate are given in Table VI. Applications
and detection agents are also listed. Sprince (100) des-
cribed a variation of the Ehrlich reagent which gave rapid,
long-lasting color development for paper chromatography.
A p-dimethylaminobenzaldehyde in HCl spray was followed by
a 1% sodium nitrite spray.
296 VAN D.REIF
6.73 Column Chromatography

The column chromatographic
- . methods that have
been used to analyze ergonovine maleate or ergonovine base
are described in Table VII.
Sondack (103) analyzed ergonovine maleate in
tablets and injectables after derivatization with N-tri-
methylsilyldiethylamine and N-trimethylsilylimidazole. A
1% OV-1 on Gas Chrom Q column at 260° was used. The sep-
aration of ergonovinine and lumiergonovine I from ergono-
vine was demonstrated.
6.75 High-Performance Liquid Chromatography
High-Performance Liquid Chromatographic
systems for ergonovine and ergonovine maleate are listed in
Table VIII, along with applications and specificity infor-
mat ion.
6.76 Electrophoresis
The electrophoresis of ergonovine on cellu-
lose thin layers at 500V and 3000V was reported (112).
The following electrolytes were found to be the most suit-
able: formic acid-acetic acid-water (26:120:1000),
ammonia-water (2:98), and ammonia-water (1:9). A high
voltage paper electrophoretic separation of ergonovine
from its diasteromer, D-lysergic acid D-propanolamide, was
reported (113).
6.8 Radiochemical Procedures
A radioimmuno assay for ergonovine and other
ergot alkaloids in plasma was reported (114). Detection
was at the picomole level from 4 ml of plasma. The anti-
sera, produced by a conjugate of lysergic acid with human
serum albumin, reacted with lysergic acid derivatives, but
not with tryptophan. An antibody produced by a mixed
anhydride reaction of lysergic acid with bovine serum
albumin reacted with simple lysergic acid derivatives and
some clavines (115). A similar coupling using a Mannich
reaction yielded an antibody specific for lysergic acid.
7. Metabolism
Two metabolites of ergonovine, identified as the
6-glucuronides of 12-hydroxyergonovine and 12-hydroxy-
ergonovinine, were excreted in the bile of rats (99). Two
other metabolites were tentatively identified as glucur-
onides of ergonovine and ergonovinine. An earlier report
(116) demonstrated the formation of two unidentified polar
metabolites of ergonovine in rats.
Table V
Thin-Layer Chromatography Systems
Appl icat ion Separation of Ref.

-
(Quantita t ion/De tec tion Ergonovine from:
Toluene-Butanol Quantitation of ergono- Ergonovinine , 78

(4N HC1 saturated) vine in ergot mixtures ergot alkaloids
(6:4)/silica gel (colorimetric)
Chloroform-Methanol Quantitation of ergono- - 79
(3:l)/silica gel vine in tablets,injections
(in situ fluorometric)
--
Ch 1oro form-Methano1 Quantitation of ergot Ergotamine, 74
(97:3)/alumina mixtures (titrimetric) ergocryptine,
ergocrystine
Ethyl Acetate-n-Heptane- - Quantitation of ergot 8 Ergot alkaloids 80
Diethylamine (7:6:O .0005 ) / mixture s
Cellulose-formamide (in
-- situ fluorometric)
Chloroform-Ethano1- 23 Ergot alkaloid quanti- Ergonovinine , 81
Ace tone tation (colorimetric) 10 ergot alkaloids
(6:4:4)/silica gel G
Chloroform-E thanol 5 Ergot alkaloid quanti- Ergonovinine, 81
(9:l)/silica gel G tation (colorimetric) 10 ergot alkaloids
Table V (continued)
Solvent/Plate Rf x 100 Application Separation of Ref.

-
(Ouantitation/Detection) Ergonovine from:
Ethyl Acetate-DMF- 17 Ergot alkaloid quanti- 11 Ergot alkaloids 82
Ethanol tation (colorimetric)
(13:1.9:0.l)/silica gel
Benzene-DMF (13:2)/ 12 Ergot alkaloid quanti- 11 Ergot alkaloids 82
silica gel tation (colorimetric)
Methanol-Acetic Acid- 30 Ergot alkaloid quanti- Ergotamine, agrocla- 83
Water (2:1:2)/silica tation (colorimetric) vine, chanoclavin
ge 1
Methanol-Water-Tartaric 41 Ergot alkaloid quanti- Ergotamine, agrocla- 83
Acid (40:60:l)/silica tation (colorimetric) vine, chanoclavin
ge 1
Benzene-Ch lor0form- 11 Ergot extract quanti- 8 Ergot alkaloids, 84
Ethanol (2:4:1)/ tation (colorimetric) ergonovinine
silica gel
Chloroform-Methanol- - Purity determination
Water (75:25:3)/ and Identity for Ergo-
silica gel novine maleate (visual
after p-dimethylamino-
benzaldehyde)
Chloroform-Acetone- 45 Identification of Impuri- Ergonovinine,related 24
Methanol-Triethylamine ties in Ergonovine a 1kalo id s
15 :10:5 : 1 / s i 1ica ge 1 maleate (UV)
Table V (continued)
Application Separation of Ref.
(Quantitationstection) Ergonovine from:
~
6 Systems/silica - Ergot Alkaloid Identifi- Ergonovinine, 85

gel, alumina, cation ( 10% CuSO4-2% 5 ergot alkaloids
cellulose ammonium hydroxide, 5:l)
34 Systems /silica 0 to 80 Alkaloid Identification Ergonovinine, 86
gel, alumina in I. violacea extracts LSD, iso-LSD
(UV,p-dimethylamino-
benzaldehyde + sodium
nitrite
17 Systems/silica 0 to 66 Ergot Alkaloid Identifi- 14 ergot alkaloids 87
gel, alumina, cellulose cation ( W ,Ehrlich's
Reagent)
Chloroform-Methanol 10 LSD Identification Ergonovinine, 20 88
(9:l)/silica gel GF (Ehrlich's Reagent) ergot alkaloids
Chloroform (NH3 sat- 13 LSD Identification Ergonovinine, 20 88
uratedl-Methanol (Ehrlich's Reagent) ergot alkaloids
(18:l)/silica gel GF
Trichloroethane- 21 LSD Identification 9 alkaloids 89
Methanol (9:1)/ (Ehrlich's Reagent)
silica gel
Diisopropyl Ether- 0 Ergot alkaloid sepa- 15 ergot alkaloids 90
THF-Toluene-Diethyl- ration (w)
amine ( 7 0 : 15 :15:O. 1 ) /
si1ica ge1- formamide
Table V (continued)
Solvent/Plate Rf x 100 Application Ref.
-
(Quantitation/Detection) Ergonovine from:
Acetone/O.lM 78 Determination of Ergotoxine Ergonovinine, 13 91
Ammonium Carbonate/ Alkaloids (vani11in/H2SOq ) ergot a1kaloids
Ethanol (32.5:
67.5:l)/silica gel G
11 Systems/silica 0-83 methylsergide, methylsergide, 92
gel, alumina methergine detection methergine
Chloroform-Benzene- - Ergot alkaloid identifica- 4 Ergot alkaloids 93
Methanol (formation (0.5% chlorimino-2,6-
mide saturated,50 :4: dichloroquinone/methanol;
lO)/silica gel G 0.3% Congo Red, pH 7; 0.5%
quinone/aq. HC1)
Acetic Acid-HC1 36-4 Alkaloid identificat ion 30 alkaloids 94
(pH 0.6-2.35)/ (6M acetic acid + UV,
alginic acid-cellu- Dragendor ff ' s )
lose, Rexyn 102-
cellulose
1M Acetic Acid in 15 Alkaloid identification 30 alkaloids 94
Water-E thanol (6M acetic acid + UV,
(7:3)/Rexyn 102- Dragendorf f ' s )
cellulose
1M HC1 in Water- 21 Alkaloid identification 30 alkaloids 94
EFhanol ( 1 : 4 ) / (6M acetic acid + UV,
Dowex 50-X4-cellu lose Dragendor ff ' s )
Table V (concluded)
Solvent/Plate Rf x 100 Application
-- Separation of Ref.
-
(Quantitation/Detection) Ergonovine from:
0.5M Acetate/AG 1- 21 Alkaloid identification 30 alkaloids 95
~4 Tacetatel- (6E acetic acid + UV,
cellulose Dragendor f f ' s
0.5M Ammonium Ace- 49 Alkaloid identification 30 alkaloids 95
ta te/DEAE ce 1 lu 1o se (6M acetic acid + UV,
Dragendor f f ' s
Table VI
Paper Chromatography Systems for Ergonovine
Stationag Mobile
- - Application Separations Detection Reference
Phase
n-Butanol-Acetic
Whatman No. 1 - Estimation of Water-insoluble UV light 96
(descending) Acid-Water ergonovine from water-sol-
(4:1:5) maleate in ble ergot alka-
ergot ex- loids, ergometrine
tracts and from ergometrinine
injections
Whatman No. 3 Ace tone-5% determination - p-d imethyl- 97
5% octanol Aqueous Ammonia of ergonovine aminobenzaldehyde
(ascending) (2:3) in ergot extracts
A.H. Thomas n-Butylacetate- determination ergonovine from uv 98
NO. 3677- Nitromethane- of ergonovine ergonovinine
pH 6 sodium Ethyleneglycol- in water-sol-
hydroxide- monoethylether ble ergot
pot assium Ace tate-Pyr id ine extracts
pho spha te Water (100 :50: 5 0 :
8:lO)
Paper loaded Formamide-O.1N
- limit test for - p-d imethylamino- 60
with 10% Pota s s ium impurities in benzaldehyde
d imethy 1 Hydroxide (1:4) ergonovine maleate
phthalate in
chloroform
Table V I (continued)
Stationary Mobile Application

-- Separations
- Detection
~- Reference
Phase
Whatman No. 1 Butanol (water metabolite iden- Ergonovine from uv 9 99

or No. 3 saturated) t i f ication 12-hydroxy- Ehrlich ' s
ergonovine, Reagent
metabolites
Whatman No. 1 Butanol metabolite iden- Ergonovine from uv , 99

or No. 3 (ammonia, tification 12-hydroxy- Ehrl ich ' s
water saturated) ergonovine, Reagent
metabolites
W
E:
Table VII
Column Chromatography for Ergonovine
Packing Eluent App 1icat-

ion Separates Ergonovine Detec t ion Ref.
-
from
-
Silica gel Chloroform- Impurity Ergometrinine, D-LSA- TLC 24
Acetone-Meths- Isolation D-2-propranolamide
no 1-Tr iethyl- male ate
amine ( 15 : 10:
5:l)
Sephadex 96% Ethanol Plant Extracts 5 Ergot Alkaloids Van Urk 101
LH-20 or DMF or Reagent
Ace tone
Sephadex Water Plant Extracts Lysergic Acid Van Urk 101
G 25 Re agent
Ce 1ite I Ether Assay Tablets Fluorescence 77
Sodium Injections
Bicarbonate
Celitel Butanol-Ben- Degraded Lumiergonov ine I Van Urk 56
Water zene-20% Ace- Extracts and I1 Reagent
tic Acid
(1:1:2)
Cel itel (1)Chloroform Powdered Ergot Water-Insoluble p-Dimethyl- 98
Citric Acid (2)Sodium Bi- Extracts Ergot Alkaloids amino benzal-
carbonate dehyde
( 3 )Chloroform
Table VII ( c o n t i n u e d )
--
Packing Eluent Application
-- -S e_
parates Ergonovine Detection
~- Re€.
-
from
-
Cellulose/ (110.1% p y r i - Ergot Alkaloid Water I n s o l u b l e E r g o t p-Dimethyl- 102

pH 3 . 0 dinelether Mixtures A l k a l o i d s , Ergonovinine amino b e n z a l -
Citrate- (2110% d i e t h y l - dehyde
Phosphate amine / e t h e r
(3)ether
W
E:
Table VIII
High-Performance Liquid Chromatography Systems for Ergonovine
Co 1umn Mobile Application Ergonovine Separates Detection -
Ref.
from
-
microBondapak Acetonitrile- Injection, Ergonovinine,Lysergic 312 nm 104
C18 1% Acetic Acid Tablet Assay Acid
(1 :4)
LiChrosorb Acetonitrile- Identification Ergonovinine, 11 Ergot 320 nm 105
RP-2, Rp-8 0.01M Ammonium of Components of Alkaloids
and RP-18 Carbonate (2:3) Plant Extracts,
Fermentation
Mixtures
LiChrosorb n-Hexane-
- Identification Ergonovinine, 11 Ergot 320 nm 105
SI-60 Chloroform- of Components of A1 kaloid s
Ethanol(4:4:1) Plant Extracts,
Fermentat ion
Mixtures
Corasil C18 Methanol-0.1% Assay of Illicit Ergot Alkaloids Fluorometric 107
Ammonium Car- LSD Preparations
bonate (3:2)
Corasil 11 Acetonitrile- Assay of Illicit Ergot Alkaloids 254 nm 108
Isopropyl LSD Preparations
Ether (1:3)
microBondapak Methanol-Ace- Forensic Mixture Ergot Alkaloids 254 nm 109
C18 tic Acid-0.005 Identity
M Heptane Sul-
-
fonic Acid (40:1:59)
Table VIII (continued)
High-Performance Liquid Chromatography Systems for Ergonovine

Co 1umn Mobile App 1ic at ion
- Ergonovine Separates Detection Ref.
microPak NH2 Diethyl Ether- Separation o f Ergonovinine, 15 Ergot 310 nm 110
E thano1 Ergot Mixtures Alkaloids
(84:12)
Diethyl Ether- Separation of Ergonovinine, 15 Ergot 310 nm 110
Isopropanol Ergot Mixtures A1 ka loid s
(3:2)
Chloroform-Iso- Separation of Ergonovinine, 15 Ergot 310 nm 110
propano1 Ergot Mixtures Alkaloids
(9:l)
LiChrosorb Diethyl Ether- Separation of Ergonovinine, 15 Ergot 310 nm 110
NH2 Ethano 1 Ergot Mixtures Alkaloids
(93:7)
LiChrosorb Hexane-Chloro- Separation o f Ergonovinine, 10 Ergot 320 nm 111
SI-60 f o rm-Ac e to- Ergot Mixtures Alkaloids
ni tr ile-Methanol
(55 :20:25 :3)
308 VAN D. REIF
8. References
1. P. Brazeau in "The Pharmacological Basis of Thera-
peutics,"3rd ed., L.S. Goodman and A. Gilman, Eds.,
The MacMillan Co., New York (1965) p. 880-882.
2. A. Stoll and A. Hofmann in "The Alkaloids," vol.
VIII, R.H.F. Manske, Ed., Academic Press, Inc.,
New York (1965) p. 729,748.
3. G.E. Foster and G.A. Steward, Quart. J. Pharm.
Pharmacol., 21, 211 (1948).
4. "The Merck Iaex", 9th ed., Merck and Co., Inc.,
Rahway, N.J. (1976) p. 3575.
5. A.L. Hayden, O.R. Samuel, G.B. Selzer, and J.Caro1,
J . Assoc. Off. --Anal. Chem., 45, 822 (1962).
6. "The United States Pharmacopza," 20th rev., Mack
Publishing Co., Easton, PA (1980) p. 284.
7. R.J.Mesley and W.H. Evans, J. Pharm. Pharmacol.,
21, 713 (1969).
-
8. R.M. Silverstein and G.C. Bassler, "Spectrometric
Identification of Organic Compounds," 2nd ed.,
John Wiley & Sons, Inc., New York (1968 ) p.91,94.
9. M.F. Sharkey, C.N. Andres, S.W. Snow, A. Major,Jr.,
T. Kram, V. Warner, and T.G. Alexander, - J. -
Assoc.
-
Off.
--- Anal. Chem., 51,
- 1124 (1967).
10. A. Bowd, J.B. Hudson, and J.H. Turnbull, -- J. Chem.
S O ~ . ,Perkin 11, 1312 (1973).
-
11. Hoffman, B., Wyeth Laboratories, Inc., personal
communication.
12. K. Bailey and A.A. Grey, &. J. Chem., - 50, 3876
(1972).
13. R.G. Mrtek, H.L. Czespi, G . Norman, M.I. Blake,
and J.J. Katz, Phytochemistry, 7, 1535 (1968).
14. N.J. Bach, H.E. Boaz, E.C. Kornfeld, C. Chang,
H.G. Floss, E.W. Hagaman, and E. Wenkert, 2. Org.
Chem., 39 1272 (1974).
15. m s t e d t , J., Wyeth Laboratories, Inc. , personal
communic a t ion.
16. S.W. Bellman, - J.-Assoc.
- - -Off.Ana1. Chem., -51, 164
(1968).
17. M. Barber, J.A. Weisbach, B. Douglas, and G.O.
Dudek, - Chem.-Ind., 1072 (1965).
18. D. Voigt, S. Johne, and D. Groger, Pharmazie, 2,
10 (1974).
19. Sivieri, L., Wyeth Laboratories, Inc., personal
communication.
20. M. Kuhnert-Brandstatter, A. Kofler, R. Hoffmann, and

H.C. Rhi, --
Sci. Pharm.,33,
- 205 (1965).
21. M. Kuhnert-Brandstatter and L. Muller, Microchem. - J.,
13, 20 (1968).
-
22 "The United States Pharmacopeia," 20th rev., Mack
I
Publishing Co., Easton, PA (1980) p. 1133.

23. J. Sagel, Pharm. Weekbl., - 107, 119 (1972).
24. D.L.Sondack,J. Pharm. Sci., - 63, 1141 (1974).
25. A. Stoll and A. Hofmann, in "The Alkaloids," vol.VII1,
R.H.F. Manske, Ed., Academic Press, New York (1965)
p . 739-740.
2 6 . C. Chothia and P. Pauling, Proc. Natl. Acad.
1063 (1969).
s., 3,
27. M.S. Kharasch and R.R. Legault, - J. Am.
- - Chem.
- SOC., - 57,
1140 (1935).
28. H.W. Dudley, Proc. R. SOC. (London),
29. E.C. Kleidere-.
=, 478 (1935).
k . C h e m . SOC., 57,2007 (1935).
30. M. Abe, T. Yamano, S. Yamatodani, YTKozu, M.Kusumoto,
H. Komatsu, and S. Yamada, Nippon Nogei Kagaku Kaishi,
34, 580 (1960); through Chem. Abstr. 5933098f (1963).
-
31. Neth. Patent Appl. 6,409,764 (1965); through - Chem.
Abstr. 63:155096b(1965).
32. V.E. Tyler, Jr., U.S. Patent 3,224,945 (1965);
through --
Chem. Abstr. 6537330f (1966).
33. E. Borowski, K. Braun, K. Breuel, C. Dauth, and
D. Enge, Ger. (East) Patent 130,421 (1978); through
Chem. Abstr. 91318360f (1979).
--
34. Y. Yang, D. Yueh, S. Lu, 2. Huo,Q. Sun, X. Lin,Yao
Hseuh Hseuh Pao, 14,316 (1979); through --
--- Chem. A b s E
92:20497v (1980).
35. M.A. Sarkisova, A . Shalagina, and A. Bankovskaya,
Mikol. Fitopatol., 2,479 (1979); through - Chem. -Abstr.
92:126896h (1980).
-
36. K. Genest.' -J. Pharm. Sci.. 55. 1284 (1966).
--,-,
37. A. DerMarderosian and H.W. Youngken, Jr., Lloydia, - 29,
35 (1966).
38. J. Chao and A.H. DerMarderosian, J. Pharm. Sci.,62, -
588 (1973).
39. S . Agurell and E. Ramstad, Tetrahedron m., 501
(1961).
40. R.M. Baxter, S . I . Kandel, and A. Okany, -- Chem. Ind.,
1453 (1961).
41. H.G. Floss, U. Hornemann, and N. Schilling, Chem.
Comm., 105 (1967).
310 VAN D.REIF
42. J.E. Robbers and H.G. Floss, Arch. Biochem. Biophys.,

126, 967 (1968).
-
43. S . Agurell and J. Lindgren, Tetrahedron Lett., 5127
(1968).
44 U. Nelson and S . Agurell, ---
I Acta. Chem. Scand., -23, 3393
(1969).
45. W.L. Garbrecht, J. Org. - Chem., 24, 368 (1959).
46. I. Sas, L. Rosca, H. Gavrilescu, G. Tudor, I. Velea,
E. Nichiforescu, Ger. Patent 2,344,608 (1975); through
Chem. Abstr. 83:28436j (1975).
-~
47. L. Bernardi and B. Patelli, Fr. Patent 1,338,023 (1963);
through --
Chem. Abstr. 60:3026f (1964).
48. E.C. Kornfeld, E.J. Fornefeld, G . B. Kline, M.J. Mann,
D.E. Morrison, R.G. Jones, and R.B. Woodward, - J. -
Am.
Chem.
-- S O ~ . ,
3087 (1956).
49. A. Salomon and R.W. Spanhoff, Pharm. Weekblad, - 75, 798
(1938); through - Chem.
___ Abstr. 32:93979 (1938).
50. E. Noda, S . Izuhara, Y. Katayama, T. Umehara, Japan.
Patent 71 18,148; through -~ Chem. Abstr. 75:67497j(1971).
51. S. Smith. and G.M. Timmis, J. Chem. SOC., 1440 (1936).
52. Ibid., 1166 (1936).
53. M. Semonsky and A. Cerny, Chem. Listy,51, - 592 (1957);
through -~Chem. Abstr. 51:11366a (1957).
54. A. Stoll and W. Schlientz, - Helv. Chim.-Acta, 38,
- ~585
(19551.
55. H. Hellberg, Acta. Chem. Scand., g , 2 1 9 (1957).
Ibid.,
56. - -13, 1106959). -
57. W.E. Moore, Drug Stand., 27,187 (1959).
58. H.W. VanUrk, Pharm. WeekbGd., _- 66,101 (1929); through
Chem. Abstr. 23:1717 (1929).
-~
59. P. Cooper, -- Pharm. J., 495 (1956).
60. "European Pharmacopoeia," vol.1, Maisonneuve S.A.,
Sainte-Ruffine, France (1969) p. 289-291.
61. D. Kupfer, Anal. Biochem., 8, 75 (1964).
62. L.E. Michelon and W.J. Kelleher, Lloydia, - 26, 192
(1963).
63 N.L. Allport and T.T. Cocking, - Quart. --
J. Pharm.
Pharmacol., >,341(1932).
64. Sellstedt, J . , Wyeth Laboratories, Inc., personal
communication.
65 J. Bayer, Acta Pharm. Hung., 8, 35 (1958); through
Chem.
- ___ Abstr. 53:2541 (1959).
66. G.E. Foster, -J. __ Pharm.
__ Pharmcol., -
7, 1 (1955).
ERGONOVINE MALEATE 31 1
67. M.I. Smith, _ Public

____ _ _ Rep., 45, 1466-1481 (1930).
Health
68. M. Pohm, Arch. Pharm., 286, 509 7i953); - Chem. - Abstr.
49:8246c 1(9
5
5.)
69. H.J. VanDerPol, Pharm. Weekblad., 106, 515 (1971).
70. S. El-Masry, Manuf. Chem. & A e r o s o l e w s , 53 (1978).
71. I. Gyenes and K. Szasz, Magyar Kim. Folyoivat, 61,
356 (1955); through -- Chem. Abstr. 52:8457g ( 1 9 5 8 r
72. L. Safarik and V. Bumba, -- Pharm. Zentralhalle, - 96, 3
(1957); through -- Chem. Abstr. 52:14085c (1958).
73. "National Formulary," 14th rev., Mack Publishing Co.,
Easton, PA (1974) p. 254.
74. V.P.Georgievskii, ---Probl. Anal. Khim., 1, 94 (1970);
through -~
Chem. Abstr. 74:146428v (1971):
75. R.D.Kirchhoefer and C.E. Wells, -- J. Assoc. -- O f f . Anal.
Chem. .58, 879 (1975).
76. D.L. Robertson, F. Matsui, W.N. French, - Can.
. -J.Pharm.
-
Sci., 7, 47 (1972).
77. K. Kirchhoefer, J. Assoc. O f f . Anal. Chem., 9, 1433
(1978).
78. P. Horak Cesk. Farm., 17, 89 (1968); through Chem.
Abstr. 6 8 m 5 6 a 1 9 6 g .
79. J.M.G.J. Frijns, Pharm. Weekbl., 106, 865 (1971).
~~
80. M. Prosek, E. Kucan, M. Katic, an=. Bano, Chromato-

graphia, 9 , 273 (1976).
81. K. Roder ,-E. Mutschler, and H. Rochelmeyer, Pharm.
Acta Helv., -
-- 42, 407 (1967).
82. J.L. McLaughlin, J.E. Goyan, and A.G. Paul, - J._Pharm.
__
Sci.,-
- 53, 306 (1964).
83. S . Keipert and R. Voigt, - J. Chromatogr., 64, 327
(1972).
84. G.E. Ferraro, S.L. Debenedetti, J.D. Coussio, -- J.Pharm.
Pharmac., 28, 729 (1976).
85. K.C. Guvenand L. Eroglu, Eczacilik. Bul., 10, 53
(1968); through -- Chem. Abstr. 69:46680=1968).
86. J.M. Weber and T.S. Ma, ---Mikrochim.Acta, I, 217 (1976).
87. R. Fowler, P.J. G O ~ and , D.A. Patterson,J.Chromatogr.,
72, 351 (1972).
-
88. A.R. Sperling, J. Chromatogr. Sci., 266 (1974).
89. L .A. DalCort ivo7J.R. Broich , A. Dihrberg , B . E .Newman,
Anal.
- _ _ Chem., 38,1959 (1966).
90. J. Reichett and S . Kudrnac, - J. Chromatogr., - 87, 433
(1973).
91. G. Szepesi, J. Molnar, Sz. Nyiredy, Fresenius Z. Anal.
Chem.,
- - 294, 47 (1979).
312 VAN D.REIF
92. J.R. Bianchine, A. Niec, and P.V.J. Macaraeg, Jr.,

J. Chromatogr., 31, 255 (1967).
-
93. K.C. Guven.and TFAttinkurt, Eczacilik. Bul. , 20, 46
(1978); through -~ Chem. Abstr. 89:186116h (1978):
94 L. Lepri, P.G. Desideri, and M. Lepori, - J. Chromatogr.,
123, 175 (1976).
-
95. Ibid., 116, 131 (1976).
96. Foster, J. MacDonald, and T.S. G . Jones, - J.Pharm.
-
.
Pharmacol. .~ 1 .SO2 (1949).
97. P . Horak and-S. Kudrnac, Cesk. Farm. , 15,483 (1966);
Chem. Abstr. 66379622~(1967).
--
98. T.G. Alexander and D . Banes, J. Pharm. Sci. , 5 0 , 201
(1961).
99. M.B. Slaytor and S.E. Wright, ---- J. Med. Pharm. Chem.,
5 , 4 8 3 (1962).
-
100. H. Sprince, J. Chromatogr., 3 , 97 (1960).
101. A. Nikolin and B. Nikolin, Phytochem., 11, 1479 (1972).
102. J .E. Carless, _- J . Pharm. Pharmacol. , 5,8% (1953).
103. D.L. Sondack, -- J. Pharm. - Sci., 63, 584 (1974).
104. D.L. Sondack, -- J . C h r o m a t S . , E , 615 (1978).
105. L. Szepesy, I. Feher, G . Szepesi, and M. Gazdag, - J.
Chrom. , 149, 271 (1978).
106. J. Dolinar, Chromatographia, 10, 364 (1977).
107. I. Jane and B.B. Wheals, J- Chromatogr., 84, 181
(1973).
108. J.D. Wittwer, Jr., and J.H. Kluckhohn, - J. Chromatogr.
Sci., 11,
- 1.(1973).
109. I. Lurie, J . Assoc. Off. Anal. Chem., 60, 1035 (1977).
110. M. Wurst, M. Flieger, and Z. Rehacek, - Chromatogr.,
174, 401 (1979).
-
111. G. Szepesi, M. Gazdag, and L. Terdy, - J. Chromatogr.,
191, 101 (1980).
-
112 A.S.C. Wan, J. Chromatogr., 60, 371 (1971).
113 I.M. Jakovljzic , R.W. Soutec R.H. Bishara, Chromato-
graphia, 11,23 (1978).
114. T . T . Kleimola. Br. J. Clin. Pharmac.. 6. 255 (1978).
I - _ -
, I
115. H . Arens and M.H. Zenk, ~- Planta Med. , - 39, 336 (1980);
through --Chem. Abstr. 933173798q (1980).
116. M. Slaytor, J.N. Pennefather, and S.E. Wright,
Experientia, 15, 111 (1959).
FLUFENAMIC ACID
Enrico Abignente and Paolo de Caprarh
1. Description 314
1.1 Name 314
I .2 Formula, Molecular Weight 314
1.3 Elemental Composition 314
1.4 Appearance, Color, Odor, Taste 314
2.2 Nuclear Magnetic Resonance Spectrum 314
2.5 Fluorescence Spectrum 320
2.7 Solubility, Partition Coefficient 323
2.8 Crystal Properties, Polymorphism 325
3. Synthesis 327
4. Drug Metabolism and Pharmacokinetics 328
4.1 Metabolism 328
4.2 Absorption and Excretion 328
4.3 Protein Binding 33 1
5.4 Spectrophotometric Analysis 333
5.5 Fluorometric Analysis 334
5.6 Indirect Atomic Absorption Analysis 335
6. Determination in Body Fluids and Tissues 342
7. Acknowledgement 343
8. References 343
Analytical Protilcs of Drug Subsurncea Copyright 0 1982 by The American

Volumc I I 313 PharmaceuticalAssociation
ISBN 0-12-260811-9
314 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS
1. D E S C R I P T I O N
1. 1 . N a m e
F l u f e n a m i c a c i d is d e s i g n a t e d b y C h e m i c a l A b -
stracts s i n c e 1972 a s 2 - [ [ 3-(trifluoromethyl)phenyl]ami-
n o l b e n z o i c a c i d , w h e r e a s b e f o r e 19 7 2 i t w a s n a m e d N-
(a,a , a-trifluoro-m-toly1)anthranilic a c i d . O t h e r n a m e s
a r e : N - ( 3-trifluoromethylphenyl)anthranilic a c i d , a n d
3'-trifluoromethyldiphenylamine-2-carboxylic acid ( 1 ) .
T h e CAS R e g i s t r y N u m b e r is 530 - 7 8 - 9 .
1. 2. F o r m u l a , M o l e c u l a r W ei ght
COOH
4JF3
C H F NO2 M o l e c u l a r Weight: 281. 2 4
1 4 10 3
1. 3 . E l e m e n t a l C o m p o s i t i o n
C 59. 79%; €3 3 . 58%; F 20. 27%; N 4. 98%; 0 11. 3 2 %
1. 4. A p p e a r a n c e , C o l o r , O d o r , Taste
Pale y e l l o w n e e d l e s , p r a c t i c a l l y o d o r l e s s w i t h a
slight bitter t a s t e ,
2 . P HYS IC AL P R O P E R T I E S
2 . 1. I n f r a r e d S p e c t r u m
T h e IR s p e c t r u m of f l u f e n a m i c a c i d s h o w n in Fi-
g u r e 1 w a s obt ai ned w i t h a B e c k m a n M y c r o l a b 620MX
s p e c t r o p h o t o m e t e r i n a K B r p e l l e t c o n t a i n i n g 0. 4 m g of
f l u f e n a m i c a c i d / 1 0 0 m g of K B r . S o m e s p e c t r a l a s s i g n -
m e n t s a r e g i v e n i n T a b l e I.
T h i s s p e c t r u m is f n good a c c o r d a n c e w i t h t h e IR s p e c -
t r u m (1 8 0 0 - 600 c m - ) r e p o r t e d b y K u h n e r t - B r a n d s t ' a t t e r
e t al. ( 2 6 ) f o r the p o l y m o r p h i c m o d i f i c a t i o n I11 of f l u f e n a -
mic a c i d (see S e c t i o n 2. 8).
2. 2. N u c l e a r M a g n e t i c R e s o n a n c e S p e c t r u m
T h e 1 H NMR s p e c t r u m of f l u f e n a m i c a c i d is p r e -
92
80
70
50
30
10
10
C
1
TABLE I
IR Spectral Assignments
of Flufenamic Acid
Wavenumber, c m - ' Vibration Mode

1670 C=O stretching
1612, 1592, 1535 aromatic C=C stretching
1190, 1125 CF3 a s y m m . s t r e t c h i n g
803, 768, 758, 703 C-H out of plane bending
sented in F i g u r e 2. The s p e c t r u m was r e c o r d e d on a V a -

r i a n E M - 3 6 0 60 MIlz s p e c t r o m e t e r using a CDC13 solution
containing tetramethylsilane a s an i n t e r n a l standard. The
s p e c t r a l assignments are given in Table 11, and a r e in
accordance with the NMR s p e c t r u m of etofenamate, which
is the 2-(2-hydroxyethoxy)ethyl e s t e r of flufenamic acid,
reported by Boltze and Kreisfeld (2) a s r e g a r d s the a r o -
matic protons of the benzene rings.
TABLE I1
NMR S p e c t r a l Assignments
of Flufenamic Acid
Protons Chemical Shift, d Multiplicity

a 6.60-6.96 multiple t
b 7.05-7. 53 multiple t
C 8. 05 doublet
2. 3 . Ultraviolet Spectrum
The UV s p e c t r u m of flufenamic acid ( F i g u r e 3 )
w a s scanned f r o m 400 t o 210 nm on a C a r y 219 s p e c t r o -
START OF SWEEP EN0 OF SWEEP
Fig. 2. NMR Spectrum of Flufenamic Acid

F i g . 3. U l t r a v i o l e t Spectrum of Flufenamic Acid

FLUFENAMIC ACID 319
p h o t o m e t e r , u s i n g a solution of 9. 28 p g of f l u f e n a m i c a c i d
/ m l of 95% ethanol. T h e a b s o r p t i o n d a t a a r e l i s t e d in Ta-
ble 111.
T A B L E I11
UV Absorption Data of F l u f e n a m i c Acid in 95%
ethanol solution
L max' nm
1c m
219 4. 32 745
238 s houlde r -
2 88 4. 2 2 5 90
337 3. 8 8 269
The s p e c t r a l f e a t u r e s observed are substantially in a c -

c o r d a n c e with t h o s e p r e v i o u s l y d e s c r i b e d in the l i t e r a t u r e
(3, 4, 5, 6, 7, 8 ) . T h e log & vs. wavelength plot of a m e t h a -
nolic solution of flufenamic a c i d w a s r e p o r t e d by U n t e r -
halt ( 3 ) , w h e r e a s Ikeda e t al. ( 5 ) p r e s e n t e d the s p e c t r u m
of the d r u g in 0. 1 M phosphate buffer (pH 7. 0 ) . D e l l e t
al. (8) r e p o r t e d Amax v a l u e s of flufenamic a c i d and i t s
phenolic m e t a b o l i t e s in n e u t r a l , a c i d i c and a l k a l i n e m e -
thanol. In T a b l e l V are listed s o m e absorption data re-
p o r t e d in the l i t e r a t u r e .
~~
T A B L E IV
UV Absorption D a t a of F l u f e n a m i c Acid
1q"
Solvent Amax. n m log E
cm
Ref. -
Methanol 2 88 4.22 590 3
340.5 3. 8 9 276
0 . 0 1 N NaOH 289 4.10 448 3
0 . 1 NNaOH 288 4. 1 8 545 4
0 . 01 N HC1 287 4. 2 2 5 90 3
(in CH30H) 345 3. 94 310
2. 4. M a s s S p e c t r u m
T h e mass s p e c t r u m of f l u f e n a m i c a c i d is s h o w n
i n F i g u r e 4. T h e s p e c t r u m w a s r u n o n a H e w l e t t - P a c k a r d
Mod. 5982A s p e c t r o m e t e r w i t h a n i o n i z i n g e n e r g y of 70
e V , i n t e r f a c e d with a H e w l e t t - P a c k a r d Mod. 5934A d a t a -
h a n d l i n g s y s t e m . T h e c o m p u t e r c a l c u l a t e d i o n masses a n d
compared their peak intensities to the b a s e peak (m/e =
265). T h i s i n f o r m a t i o n w a s t h e n a u t o m a t i c a l l y p l o t t e d a s
a series of l i n e s w h o s e h e i g h t s a r e p r o p o r t i o n a l t o t h e
peak intensities. T h e molecular ion peak w a s observed
a t m / e = 281. S o m e c h a r a c t e r i s t i c p e a k s o b s e r v e d a r e
l i s t e d in Table V . T h e f r a g m e n t a t i o n p a t t e r n o b s e r v e d is
TABLE V
M a s s S D e c t r u m of F l u f e n a m i c A c i d
Mass (m/e) Species A b u n d a n c e 01,
281 M+ 44. 8
263 M+-H20 100.0
235 263-CO 22. 8
216 235-F 12. 3
166 216-CF2 14. 9
c o n s i s t e n t with mass s p e c t r a l d a t a p u b l i s h e d b y B o l t z e
a n d K r e i s f e l d ( 2 ) f o r e t o f e n a m a t e a n d C o t e l l e s s a et al.
( 43 ) for t h e m e t h y l e s t e r of f l u f e n a m i c a c i d .
2. 5 . F l u o r e s c e n c e S p e c t r u m
F l u f e n a m i c a c i d s h o w s n a t i v e f l u o r e s c e n c e i n so-
me o r g a n i c s o l v e n t s , e . g. d i o x a n a n d c h l o r o f o r m , w h e r e -
a s i n e t h a n o l f l u o r e s c e n c e is t o o w e a k t o be a n a l i t i c a l l y
u s e f u l (8. 9). M i l l e r et al. (10) r e p o r t e d t h a t f l u f e n a m i c
a c i d s h o w e d n o s i g n i f i c a n t f l u o r e s c e n c e at room t e m p e r a -
t u r e i n a c i d i c , n e u t r a l or a l k a l i n e e t h a n o l s o l u t i o n , b u t
w a s s t r o n g l y f l u o r e s c e n t at l o w t e m p e r a t u r e ( 7 7 0 K ) , p r e -
s u m a b l y b e c a u s e of t h e v i r t u a l a b o l i t i o n of b i m o l e c u l a r
q u e n c h i n g i n t h e latter c o n d i t i o n s . D e l l a n d K u t s c h b a c h
(11) i n v e s t i g a t e d t h e i n f l u e n c e on f l u o r e s c e n c e i n t e n s i t y of
t h e s o l v e n t a n d t h e a d d i t i o n of a h a l o g e n o a c e t i c a c i d , a s
00.
80.,
60,,
40.,
E!O#,
W
0,
c!
F i g . 4. Mass Spectrum of F l u f e n a m i c A c i d
well a s the influence of a t r e a t m e n t with a l k a l i a n d / o r a n

oxidant. T o obtain a f l u o r e s c e n t solution s o l v e n t s with a
d i e l e c t r i c c o n s t a n t = 0 a r e r e q u i r e d , and f l u o r e s c e n c e i n -
t e n s i t y is s t r o n g l y i n c r e a s e d by t h e addition of a haloge-
n o a c e t i c a c i d with a pKa value < 1 (e. g. t r i c h l o r o a c e t i c
a c i d ) . T h e s e findings a r e the b a s i s f o r t h e m o s t c o m m o n
m e t h o d of f l u o r o m e t r i c d e t e r m i n a t i o n of f l u f e n a m i c a c i d
(see Section 5. 5).
Dell e t al. (8) published the f l u o r e s c e n c e s p e c t r u m of
flufenamic a c i d in a C C 1 4 / t r i c h l o r o a c e t i c a c i d solution:
i n t h i s m e d i u m flufenamic a c i d s h o w s the e x c i t a t i o n m a x i -
m u m a t 372 n m with a s u b s i d i a r y m a x i m u m a t 296 n m ,
and t h e e m i s s i o n m a x i m u m a t 445 n m . T h e r e is a l i n e a r
r e l a t i o n s h i p between the f l u o r e s c e n c e i n t e n s i t y and the
c o n c e n t r a t i o n of f l u f e n a m i c a c i d u p t o 1 0 p g / m l .
S o m e f l u o r e s c e n c e d a t a r e p o r t e d in the l i t e r a t u r e f o r
flufenamic a c i d a r e l i s t e d in T a b l e VI.
T A B L E VI
F l u o r e s c e n c e Maxima of F l u f e n a m i c Acid
in v a ri o u s solvents
Solvent Excitation Emission Ref.

maximum, n m m a x i m u m , nm -
Methanol 305 400 8
Ethanola 335 510(420)b 10
Dioxan 3 34 410 9
Chloroform 340 430 9
CCl,/TCA 372 (296)b 445 8
a : a t 77OK.
b : subsidiary maximum.
2. 6. Melting r a n g e
In T a b l e VII a r e l i s t e d s o m e m e l t i n g point v a l u e s
r e p o r t e d by v a r i o u s a u t h o r s f o r flufenamic a c i d ,
T h e d i s c r e p a n c i e s a m o n g t h e s e v a l u e s a r e due t o t h e
d i f f e r e n t p o l y m o r p h i c m o d i f i c a t i o n s which c a n be p r e s e n t
i n c o m m e r c i a l p r o d u c t s (see Section 2 . 8).
FLUFENAMIC ACID 323
TABLE VII
M e l t i n g R a n g e of F l u f e n a m i c A c i d
M. P . , OC Crystallization Ref.
solvent
125 50% e t h a n o l 12
1 2 4 - 125, with r e s o l i d i f i c a -
tion a n d r e m e l t i n g a t 134-
136 ligroine 13
127-128 NR 3
132-133 cyclohexane 14,15
133 NR 16
1 3 3 - 134 NR 2
1 3 3 -1 3 4 o r 1 25- 126, r e s o -
l id ify in g a n d r e m e l t i n g at
1 3 4 -1 3 6 NR 17
134. 3 ethanol 18
1 3 4 - 136 cyclohexane 19
NR : not r e p o r t e d
2 . 7. S o lu b i l i t y, P a r t i t i o n Coef fi ci e n t
Flufenamic acid was reported to be soluble a t
r o o m t e m p e r a t u r e in m e t h a n o l , e t h a n o l , d i e t h y l e t h e r ,
c h l o r o f o r m , a c e t o n e , D M F , and p e a n u t oil ( 3 , 4 , 1 3 ) . T h e
s o l u b i l i t y in w a t e r a t 22OC is gi ven b y R o l t z e a n d K r e i s -
f e ld ( 2 ) a s 0 . 0067 mg/ml. In T a b l e VIII a r e l i s t e d s o m e
v a l u e s of s o l u b i l i t y in w a t e r a t v a r i o u s p H v a l u e s .
A s t u d y b y G h a n e m et al. ( 2 1 ) i n d i c a t e s t h a t t h e s o l u b i -
l i t y of f l u f e n a m i c a c i d is i n c r e a s e d b y n o n i o n i c s u r f a c t a n -
t s , u r e a a n d s o d i u m citrate. T h e e f f i c i e n c y of t h e s u r -
f a c t a n t s t o w a r d s s o l u b i l i z a t i o n is i n t h i s o r d e r : T w e e n
80 > R r i j 9 9 > T w e e n 40 > M y r j 53. T h e e f f e c t of u r e a ,
a m i d o p y r i n e , p h e n a z o n e a n d p a r a c e t a m o l on t h e s o l u b i l i t y
of f l u f e n a m i c a c i d a n d o t h e r a n t i r h e u m a t i c d r u g s w a s s t u -
d i e d b y D a a b i s e t al. ( 2 2 ) .
1- O c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t w a s e s t i m a t e d b y
Dunn (2 3 ) t a k i n g a d v a n t a g e of t h e a d d i t i v e - c o n s t i t u t i v e n a -
TABLE VIII
Solubility of F l u f e n a m i c Acid in
Water a t v a r i o u s pH V a l u e s
PH Solubility , Ref,
mg/ml
3 0. 003a 13
7 1. 8a 13
7 1 20
8 4. oa 13
a : a t 37OC
t u r e of l o g P, as follows:
= log 'anthranilic acid + nphenyl

+ n3-cF3 =
= 1. 21 + 2 . 60 + 1 . 0 7 = 4. 88
T e r a d a e t al. ( 2 4 ) have d e t e r m i n e d the t r u e p a r t i t i o n
coefficient, P, a n d the a p p a r e n t p a r t i t i o n coefficient, P',
of f l u f e n a m i c acid. P w a s m e a s u r e d by e q u i l i b r a t i n g a
1-octanol solution of the d r u g , the i n i t i a l c o n c e n t r a t i o n ,
C,, of which w a s 1 0 - 3 - 1 0 - 2 m o l / l , with 0. 01 N HC1:
u n d e r t h i s condition, d r u g m o l e c u l e s e x i s t e x c l u s i v e l y a s
the unionized f o r m . A f t e r e q u i l i b r i u m w a s a t t a i n e d , c o n -
c e n t r a t i o n i n the a q u e o u s p h a s e , Cw, was m e a s u r e d s p e -
c t r o p h o t o m e t r i c a l l y and P w a s c a l c u l a t e d b y the equation:
P = C,/Cw, s i n c e t h e c o n c e n t r a t i o n change in t h e o r g a -
nic p h a s e c a n b e n e g l e c t e d f o r s u c h a highly hydrophobic
compound, obtaining l o g P = 5 . 62.
PI w a s d e t e r m i n e d with the 1- o c t a n o l / p h o s p h a t e b u f f e r
(pH 8. 0) s y s t e m , u s i n g the equation:
w h e r e C a n d V a r e t h e e q u i l i b r i u m c o n c e n t r a t i o n and v o
l u m e of a q u e o u s ( s u b s c r i p t w) and o r g a n i c ( s u b s c r i p t 0 )
p h a s e s , r e s p e c t i v e l y . Ci is t h e i n i t i a l c o n c e n t r a t i o n i n
the a q u e o u s p h a s e . L o g PI w a s found t o b e 1 . 7 4 .
FLUFENAMIC ACID 325
Lombardino et al. (25) have a l s o determined the p a r t i -

tion coefficient of flufenamic acid with some 1-octanol/
buffer s y s t e m s .
2. 8. Crystal P r o p e r t i e s , Polymorphism
Flufenamic acid can exist a s s e v e r a l crystalline
modifications. Kuhnert-Brandstatter et al. have d e s c r i -
bed five different modifications and have reported t h e i r
melting points and infrared s p e c t r a , a s well a s the t h e r -
mogram obtained by differential scanning c a l o r i m e t r y f o r
the f i r s t four f o r m s (16, 26).
According to K r c flufenamic acid can exist a s at l e a s t
seven crystalline modifications with different melting po-
ints (27). K r c has reported the f r e e energy vs. t e m p e r a -
t u r e plot of seven crystalline f o r m s of flufenamic acid.
Modifications I, I1 and I11 were described in detail in
t e r m s of c r y s t a l morphology, optical p r o p e r t i e s , X - r a y
diffraction powder data and infrared s p e c t r a .
Other authors a l s o studied the polymorphism of flufena-
mic acid. Galdecki et al. (28) investigated the c r y s t a l l i -
zation of the d r u g f r o m boiling solvents, B u r g e r and
Ramberger ( 2 9 ) examined the applicability of s o m e t h e r -
modynamic r u l e s to the polymorphic s y s t e m of flufenamic
acid. These r u l e s c o r r e l a t e the heats of transition o r
fusion, IR s p e c t r a and densities of the modifications with
t h e i r stability behavior. In this study flufenamic acid w a s
investigated mainly by quantitative DSC and qualitative s o -
lubility determination (by thermomicroscopy) a s well a s
by IR spectroscopy to differentiate eight crystalline modi-
fications (Table IX). It w a s pointed out ( 2 9 ) that modifi-
cations I, I1 and I11 investigated by the various authors
a r e identical, whereas modification IV studied by Kuhnert
( 2 6 ) coincides with modification V by K r c ( 2 7 ) , who did
not describe Kuhnert's modification V. Since the l a t t e r
f o r m had the lowest melting point of all eight modifica-
tions, it was indicated by B u r g e r and R a m b e r g e r a s modi-
fication VIII.
F r o m the practical point of view, modifications I and
I11 a r e the most important, because they can be present
in the c o m m e r c i a l product. The transition point of these
two f o r m s is a t 42°C: modification I11 i s the stable f o r m
at room t e m p e r a t u r e ( b e l o w 42" C) , w h e r e a s m o d i f i c a t i o n
I is t h e s t a b l e form a b o v e 42OC ( 2 7 ) . M o d i f i c a t i o n I11
w a s o b t a i n e d by B u r g e r a n d R a m b e r g e r by s t i r r i n g f o r 1 2
h o u r s a t 2OoC a x y l e n e s u s p e n s i o n of a commercial p r o -
d u c t f o r m e d by m o d i f i c a t i o n I ( 2 9 ) .
T A B L E IX
M e l t i n g P o i n t s of c r y s t a l l i n e M o d i f i -
c a t i o n s of F l u f e n a m i c A c i d
Mo d ific a t i on M. P . , O C Ref.
I 133 16,26
134 27, 2 9
I1 128 1 6 , 26, 27, 2 9
I11 125 16,26
126 29
126. 5 27
IV 124 27
Va 122 1 6 , 26, 29
122.5 27
VI 120 27
VII 118 27
VIIIb 100-110 26
108 2 5 29
a : T h i s m o d i f i c a t i o n w a s i n d i c a t e d a s IV i n t h e p a -
p e r s 1 6 a n d 26.
b : T h i s modification was indicated as V in the p a p e r s
1 6 a n d 26.
2. 9. D i s s o c i a t i o n C o n s t a n t
T h e pKa of f l u f e n a m i c a c i d w a s r e p o r t e d to b e
3 . 9 by A g u i a r a n d F i f e l s k i ( 2 0 ) a n d 4 . 5 by F r e y a n d El-
S a y e d (30). Terada et al. ( 2 4 ) h a v e found a v a l u e of
3 . 8 5 u s i n g t h e pH -dependent s o l u b i l i t y m e t h o d ( 3 1 ) ; t h i s
value is c o n s i d e r a b l y d i f f e r e n t from t h e c o r r e s p o n d i n g
FLUFENAMIC ACID 327
value obtained by p o t e n t i o m e t r i c t i t r a t i o n i n 5 - 10% a q u e o u s

acetone ( 3 2 ) . In T a b l e X a r e l i s t e d s o m e pKa v a l u e s ob-
tained by p o t e n t i o m e t r i c t i t r a t i o n in v a r i o u s a q u e o u s s o l -
vent s y s t e m s .
TABLE X
pKa V a l u e s of F l u f e n a m i c Acid
obtained bv P o t e n t i o m e t r i c T i t r a t i o n
Solvent pKa Ref.
Water 7. 5 33
75% Aqueous methanol 5.75 3
50Vn Aqueous ethanol 5. 94 33
80% Aqueous 2-methoxy-
ethanol 6. 0 33
Dioxane: w a t e r ( 2 : 1) 6. 8 25
3 . SYNTHESIS
Wilkinson and F i n a r ( 1 2 ) f i r s t s y n t h e s i z e d flufenamic
a c i d by r e a c t i n g o-iodobenzoic a c i d with m - t r i f l u o r o m e -
thylaniline in p o t a s s i u m c a r b o n a t e a q u e o u s solution, i n t h e
p r e s e n c e of c o p p e r b r o n z e . T h e c r u d e p r o d u c t w a s p u r i -
fied via i t s a m m o n i u m s a l t .
Moffett and A s p e r g r e n (19) p r e p a r e d f l u f e n a m i c a c i d
s t a r t i n g f r o m o - c h l o r o b e n z o i c a c i d which w a s r e a c t e d
with m-trifluoromethylaniline i n 85070 aqueous p o t a s s i u m
hydroxide and a m y l alcohol with c o p p e r p o w d e r . Some
patented synthetic m e t h o d s follow the l a t t e r s c h e m e , as
i l l u s t r a t e d in F i g u r e 5.
Figure 5
Synthesis of F l u f e n a m i c Acid
F l u f e n a m i c a c i d w a s a l s o obtained via the r e a c t i o n b e t -
ween o-iodobenzoic a c i d and m-trifluoromethylphenylhydro-
xylamine ( 3 4 ) . Another method involves the r e a c t i o n of
m e t h y l o - c h l o r o b e n z o a t e with N-(3-trifluoromethylphenyl)-
f o r m a m i d e (35). F l u f e n a m i c a c i d w a s a l s o p r e p a r e d by
photolysis of the c o r r e s p o n d i n g b e n z o t r i a z i n o n e ( 3 6 ) .
4. DRUG METABOLISM AND PHARMACOKINETICS

4. 1. M e t a b o l i s m
T h e m e t a b o l i c t r a n s f o r m a t i o n s which f l u f e n a m i c
a c i d u n d e r g o e s in m a n and a n i m a l s a r e d e p i c t e d in F i g u r e
6 , a c c o r d i n g t o Glazko ( 3 7 ) and O b e r e t al. (38), which
d e m o n s t r a t e d that t h e d r u g is e x c r e t e d m a i n l y i n f o r m of
i t s m e t a b o l i t e s . T h e i r s t u d i e s w e r e c a r r i e d out by t r a c e r
m e t h o d s u s i n g [14C] - c a r b o x y l - l a b e l l e d f l u f e n a m i c acid.
T h e 4'-hydroxy- and 5 - h y d r o x y - d e r i v a t i v e and f l u f e n a m i c
a c i d itself a r e e l i m i n a t e d in u r i n e chiefly i n conjugated
f o r m , w h e r e a s t h e 4', 5 - d i h y d r o x y - d e r i v a t i v e is not conju-
gated. A l l f o u r compounds h a v e b e e n found i n t h e s t o o l
i n unconjugated f o r m .
4'-Hydroxy- and 5-hydroxyflufenamic a c i d s w e r e s y n t h e -
s i z e d by Bowman et al. (39).
4. 2. Absorption and E x c r e t i o n
4. 21. In A n i m a l s
T h e r a t e of p e r m e a t i o n of f l u f e n a m i c a c i d
t h r o u g h the gut m e m b r a n e and the amount of d r u g a b s o r -
bed w e r e studied i n v i t r o on s e g m e n t s of t h e small i n t e -
s t i n e of golden h a m s t e r s ( 2 0 ) . T h e p e r m e a t i o n of flufena-
m i c a c i d w a s pH-dependent, a c c o r d i n g t o t h e postulation
t h a t i t is only the unionized m o l e c u l e of t h e d r u g t h a t pas-
s e s through a cell, due t o i t s lipoid solubility. T h i s s t u -
d y showed t h a t a t pH 2. 5 the p e r m e a t i o n of f l u f e n a m i c
acid, which is a p p r o x i m a t e l y 96% unionized, is 20 t i m e s
f a s t e r t h a n a t pH 7. 2, at which only 0. 05% of t h e d r u g is
i n the unionized f o r m .
T h e a b s o r p t i o n and t h e u r i n a r y e x c r e t i o n of f l u f e n a m i c
a c i d w a s s t u d i e d i n r a b b i t s following both c u t a n e o u s and
o r a l application ( 4 0 ) of t h e same d o s e ( 3 0 m g / k g ) . A f t e r
4 8 h o u r s from cutaneous application, 5. 9% of t h e applied
d o s e w a s found i n the u r i n e ; t h e blood level of flufena-
m i c a c i d r e m a i n e d c o n s t a n t o v e r the f i r s t s i x h o u r s at
about 3 pg/ml, o v e r c o m i n g t h e blood l e v e l obtained o r a l -
l y a s f r o m the 4th hour.
m
u.
q
R
Erc
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m f
0
r
CD
4 c
R a, a,
lil
rcl
Erc I
rl
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Erc
R +I
I 0
E
(I)
0 d
.rl
/
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lu
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k
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V
3 30 ENRICO ABIGNENTE AND PAOLO DE CAPRARIIS
Rosenberg and Bates ( 4 1 ) compared the blood concentra-

tions produced in r a t s following o r a l administration of f l u -
fenamic acid alone o r together with cholestyramine. F l u -
fenamic acid strongly bound to the r e s i n , s o that a 6 0 -
70% d e c r e a s e in both the r a t e and the extent of absorption
of the d r u g was observed: following an o r a l dose of 5 0
m g / k g a peak plasma concentration of 8 9 . 2 p g / m l w a s
reached at 1 hour, whereas when cholestyramine and the
d r u g were coadministered the peak l e v e l dropped to 3 2 . 3
pg/ml. F u r t h e r studies on pharmacokinetics of flufena-
m i c acid in the r a t a r e reported. F r e y and El-Sayed ( 3 0 )
determined the flufenamic acid concentrations in s e r u m
and g a s t r i c mucosa a f t e r o r a l and subcutaneous admini-
stration. Lin et al. ( 4 2 ) determined p l a s m a l e v e l s a f t e r
intravenous administration, Cotellessa e t al. ( 4 3 ) d e t e r -
mined plasma and u t e r u s levels a f t e r intravenous and o r a l
administration.
A s r e g a r d s the excretion, Glazko ( 3 7 ) showed that dogs
eliminated only 2-770 of an o r a l dose in the urine and 53-
7 9 % in the f e c e s , w h e r e a s the corresponding values f o r
monkeys were 45-8070 and 12-2170, respectively, S i m i l a r
r e s u l t s were obtained by Ober et al. ( 3 8 ) in e x p e r i m e n t s
with dogs. Lombardino et al. ( 2 5 ) r e p o r t e d that the s e -
r u m half life a f t e r o r a l administration of the aluminium
salt of flufenamic acid w a s 3 h o u r s f o r rats and dogs,
and 4 hours f o r rabbits.
4 . 2 2 . In Humans
In addition to the r e s u l t s obtained in r a b -
bits ( 4 0 ) , P a n s e e t al. have a l s o studied the absorption
of flufenamic acid through the human skin ( 4 4 ) .
Glazko ( 3 7 ) reported that n e a r l y 100% of an o r a l dose
of flufenamic acid w a s absorbed; the r e n a l elimination of
the drug and i t s metabolites was 5170, of which only 2 . 6%
w a s unaltered drug. Such r e s u l t s were m o r e r e c e n t l y
confirmed by Dell e t al. ( 4 5 , 4 6 ) with two different meth-
ods for the determination of all fluorine-containing c o m -
pounds a s a group in the urine, which showed that the r e -
nal elimination of flufenamic acid and i t s metabolites w a s
4 9 . 470 within t h r e e days a f t e r o r a l administration. The
peak p l a s m a level w a s reached a f t e r two hours, and the
p l a s m a elimination half life w a s found t o be approximate-
FLUFENAMIC ACID 331
l y 3 h o u r s . D e l l e t al. ( 4 5 ) a l s o r e p o r t e d t h a t 3. 670 of
a n o r a l d o s e of flufenamic a c i d was e x c r e t e d unconjugated
into the u r i n e within 6 d a y s : however, f e m a l e s u b j e c t s
e l i m i n a t e d only 1. 9% and the m a l e o n e s 5. 370. On the
c o n t r a r y , no difference w a s o b s e r v e d between m e n and
women in the t o t a l amount of all m e t a b o l i t e s e x c r e t e d by
the r e n a l route. Another study on t h e sex-dependence of
the r e n a l e x c r e t i o n of flufenamic a c i d and o t h e r f e n a m a t e s
in m a n and a n i m a l s was r e p o r t e d by L o r e n z a n d D e l l (47).
T h e bioavailability of o r a l p h a r m a c e u t i c a l f o r m u l a t i o n s
of flufenamic a c i d w a s investigated by A r i a s a n d Cadorni-
ga ( 4 8 ) and Angelucci e t al. (49).
4. 3 . P r o t e i n binding
T h e bovine and human s e r u m a l b u m i n (BSA and
HSA, r e s p e c t i v e l y ) binding affinity of flufenamic a c i d w a s
investigated by Chignell b y c i r c u l a r d i c h r o i s m s t u d i e s
(50-52). T h e r o l e of hydrophobicity f o r the binding affini-
t y w a s investigated by Dunn on HSA (23) and by T e r a d a e t
al. on BSA (24). T h e hydrophobicity a s well a s the with-
d r a w i n g ability of the - C F 3 substituent c o n t r i b u t e signifi-
cantly t o the binding affinity, which w a s d e t e r m i n e d f o r
BSA by m e a s u r i n g the ability of flufenamic a c i d t o d i s p l a -
c e 2 - (4' - hydroxypheny1azo)benzoic a c i d c o m p e t i t i v e l y u n d e r
conditions of pH 7 . 0 and 25OC (24). T h e binding constant,
K, w a s d e t e r m i n e d : the value obtained f o r BSA by T e r a -
d a e t a l . , 6 . 5 l~o 5 1. m o l - l , s e e m s t o c o n f o r m t o a v a -
lue, 1. 3 x l o 6 1. m o l - l , obtained by Chignell with HSA at
pH 7 . 4 (51).
T h e i n t e r a c t i o n between BSA and s e v e r a l c a t i o n i c and
anionic d r u g s including flufenamic a c i d w a s studied by
B l a n c h a r d e t al. (53) u s i n g the e l e c t r o n s p i n r e s o n a n c e
s p i n labeling technique.
T h e binding of flufenamic a c i d t o HSA w a s s t u d i e d by
O t a g i r i e t al. ( 5 4 ) v i a m i c r o c a l o r i m e t r i c i n v e s t i g a t i o n s .
T h e h e a t flux g e n e r a t e d by the binding is p r o p o r t i o n a l t o
the amount of the d r u g bound t o the p r o t e i n . If only one
binding s i t e on t h e d r u g m o l e c u l e c o n t r i b u t e s t o the h e a t
flux, then the d a t a can r e a d i l y be i n t e r p r e t e d i n t e r m s of
the binding constant, AG, AH, and A S f o r binding t o that
site. If m a n y s i t e s a r e involved having d i f f e r e n t e n t h a l -
pies of binding, then unambiguous interpretation of the

data may be impossible. T h i s is the c a s e of flufenamic
acid, which h a s been reported t o have t h r e e v e r y high
affinity s i t e s f o r HSA and other s i t e s of lower affinity(51).
Sudlow et al. (55, 56) have c h a r a c t e r i z e d two distinct
binding s i t e s ( I and 11) f o r anionic d r u g s on HSA by the
u s e of fluorescent probes. Flufenamic acid binds s e l e c t i -
vely to s i t e 11, a s well a s ibuprofen, flurbiprofen and
ethacrynic acid , whereas phenylbutazone and warfarin
bind to s i t e I. Drugs which bind t o s i t e I1 a r e all a r o m a -
t i c carboxylic acids, which would be l a r g e l y ionized a t
physiological pH.
Kaneo et al. (57) examined the binding t o BSA of s i x
nonsteroidal antiinflammatory d r u g s including flufenamic
acid by the u s e of dialysis a t pH 7 . 4 and 37OC. It was
found that flufenamic acid strongly binds t o BSA and the
f r e e fraction of t h i s d r u g e x i s t s within 1% over the t h e r a -
peutic range.
5. METHODS OF ANALYSIS
5. 1. Identification T e s t s
Flufenamic acid can be identified by virtue of i t s
U V , IR, N M R , m a s s and fluorescence s p e c t r a (see Sec-
tion 2). Various chromatographic methods a r e a l s o s u i -
table f o r purposes of identification ( s e e Section 5 . 7 ) .
Devaux e t al. (58) described two color r e a c t i o n s and
one fluorescent reaction. The color r e a c t i o n s a r e due t o
the diphenylamine s t r u c t u r e , w h e r e a s the fluorescent r e a -
ction was explained by the formation of substituted a c r i -
dones ( s e e Section 5.5). T h e s e reactions can be c a r r i e d
out a s follows:
a ) Flufenamic acid (at l e a s t 1 m g ) and about 0. 5 g of
oxalic acid a r e heated into an oil bath at 180-200°C 4-5
minutes. After cooling the r e s i d u e is dissolved in 95%
ethanol t o obtain a stable, intense blue color. The a b s o r -
ption maximum is at 585-590 nm.
b ) Flufenamic acid ( a t l e a s t 100 p g ) is added in a mix-
t u r e of 1 m l CH3COOH:H2S04 (d.1.83)(98:2), 5 rnl CH3-
CO0H:HCl (d. 1.18)(50:50), and 1 m l 0. 10% aqueous levu-
lose. The mixture is heated a t 100°C 25 minutes t o ob-
FLUFENAMIC ACID 333
tain a violet color. The absorption maximum is at 597nm.

c ) Flufenamic acid is dissolved in conc. H2SO4 and
heated 10 minutes at 100°C: the solution exhibits an in-
tense green fluorescence when excited by white light, and
blue when excited by UV light.
5. 2. T i t r i m e t r i c Analysis
Flufenamic acid can be titrated in acetone with
0 . 1 N aqueous potassium hydroxide in the p r e s e n c e of
phenolphtaleine (3). A nonaqueous t i t r i m e t r i c method w a s
described by Walash and Rizk (59), which used 0 . 1 N s o -
dium methoxide as the t i t r a n t and DMF o r tetramethyl-
u r e a as the solvent, m e a s u r i n g the end point e i t h e r with
a thymol blue indicator o r potentiometrically. Various
potentiometric methods in aqueous solvent s y s t e m s a r e
cited in Section 2. 9.
5. 3. Colorimetric Analysis
Flufenamic acid and i t s metabolites w e r e d e t e r -
mined a s a group colorimetrically in urine a f t e r alkaline
hydrolysis and fusion with sodium peroxide: fluorine was
then distilled a s H2SiF6 in the p r e s e n c e of H2S04 and
Si02. A. solution of alizarine-3-methylamino-N, N-diace-
tato-cerium(II1) was added to the distillate to obtain a co-
l o r reaction, and a c o l o r i m e t r i c determination w a s effec-
ted at 617 nm. Measurements in the range h l p,g a r e
possible (46).
The color reactions cited in Section 5. 1 can a l s o be
used for quantitation.
5.4. Spectrophotometric Analysis
The f i r s t UV spectrophotometric method f o r fena-
m a t e s analysis w a s described by Carey (60). The W ab-
sorbance of flufenamic acid ( s e e Section 2. 3 ) e i t h e r in
methanol a t 288 nm (3,40,44) o r in 0. 1 N NaOH at 287-
290 nm (4, 30,411 can be used f o r quantitative analysis.
Beltagy (61) described a spectrophotometric method f o r
the determination of s e v e r a l acidic d r u g s including flufe-
namic acid which w a s determined obtaining the ion-pair
association complex of the d r u g and safranine in a pH 7. 4
buffer, then extracting the complex with chloroform and
measuring the absorbance of the e x t r a c t .
5. 5. F l u o r o m e t r i c A n a l y s i s
Mehta and Schulman ( 9 ) a f f i r m e d t h a t t h e native
f l u o r e s c e n c e exhibited by f l u f e n a m i c a c i d i n o r g a n i c s o l -
v e n t s (see Section 2. 5) could b e u s e f u l f o r i t s d e t e c t i o n
and d e t e r m i n a t i o n , N e v e r t h e l e s s the m e t h o d m o s t corn -
monly u s e d involves the f l u o r o m e t r i c d e t e r m i n a t i o n of flu-
f e n a m i c a c i d i n c a r b o n t e t r a c h l o r i d e a f t e r addition of a
CC14 solution of t r i c h l o r o a c e t i c a c i d which s t r o n g l y i n c r e -
ases the f l u o r e s c e n c e i n t e n s i t y (11). T h e f l u o r e s c e n c e
m a x i m a u n d e r t h i s condition a r e r e p o r t e d i n Section 2. 5.
T h i s method w a s u s e d by a n u m b e r of a u t h o r s t o a s s a y
flufenamic a c i d i n body f l u i d s and t i s s u e s (8, 37, 40, 49, 62,
63, 64).
T h e r e a c t i o n of f l u f e n a m i c a c i d with f o r m a l d e h y d e gives
1-(m-trifluoromethylphenyl)-4-oxo-1,2 - d i h y d r o - 3, 1- b e n z o -
xazine (4), which is s u i t a b l e f o r f l u o r o m e t r i c d e t e r m i n a -
tion of the p a r e n t d r u g (63). T h e r e a c t i o n s c h e m e is d e -
picted in F i g u r e 7. T h e m e t h a n o l i c s o l u t i o n of t h e b e n z o -
xazine d e r i v a t i v e s h o w s two e x c i t a t i o n m a x i m a at 278 and
0
COOH
NH
I
- HCHO
Figure 7
Reaction of F l u f e n a m i c Acid with F o r m a l d e h y d e
342 n m and a n e m i s s i o n m a x i m u m a t 440-450 n m ( 4 ) . T h e
c o r r e s p o n d i n g v a l u e s r e p o r t e d by D e l l e t al. ( 6 3 ) a r e 346
and 458 nm.
Another fluorogenic r e a c t i o n of f l u f e n a m i c a c i d , a l r e a d y
c i t e d i n Section 5. 1, w a s s t u d i e d by D e l l and K a m p ( 4 ) .
F l u f e n a m i c a c i d w a s h e a t e d with c o n c e n t r a t e d s u l f u r i c
a c i d to give a m i x t u r e of two i s o m e r i c a c r i d o n e s , I and
11, as i l l u s t r a t e d i n F i g u r e 8. T h e f l u o r e s c e n c e f e a t u r e s
of t h e s e compounds, which had b e e n p r e v i o u s l y s y n t h e s i -
zed by Wilkinson and F i n a r (12), a r e v e r y s i m i l a r s o that
FLUFENAMIC ACID 335
Reaction of F l u f e n a m i c Acid with conc. H2S04

the quantitative d e t e r m i n a t i o n c a n b e m a d e on t h e mixtu-
r e . In T a b l e XI a r e l i s t e d the wavelengths of excitation
and e m i s s i o n m a x i m a i n n e u t r a l , a c i d i c and a l k a l i n e m e -
thanol ( 4 ) .
T A B L E XI
F l u o r e s c e n c e Data of Trifluoromethylacridones
Solvent E x c i t a t i o n / e m i s s i o n m a x i m a , n m , of:
system 4 - C F 3 - a c r i d o n e (I) 2 - C F 3 - a c r i d o n e (11)
Methanol 400 / 420 400 / 4 2 1

Methanol- H C1 400/440 400/440
Methanol - NaOH 400 / 4 5 5 - 4 6 2 400/460
H a t t o r i e t al. ( 6 5 ) p r o p o s e d a f l u o r o m e t r i c method
which involves the t r e a t m e n t of a n ethanolic solution of
flufenamic a c i d with 0. 5% A1C13 solution i n a b s o l u t e e t h a -
nol t o obtain a n a l u m i n i u m c h e l a t e , which f l u o r e s c e s a t
440 n m following activation a t 358 n m . T h e m a x i m u m
f l u o r o m e t r i c s e n s i t i v i t y of flufenamic a c i d c l a i m e d f o r
t h i s method is 4 n g / m l .
5 . 6. I n d i r e c t Atomic Absorption A n a l y s i s
It w a s found that flufenamic acid, c o p p e r and
2-(2-hydroxyethyl)pyridine combined i n the r a t i o 1:1:1 t o
f o r m a c h e l a t e complex (18). T o obtain t h i s r e s u l t the
s a m p l e containing the d r u g w a s t r e a t e d with a r e a g e n t
p r e p a r e d adding 9. 0 m l of 0. 1% c u p r i c s u l f a t e solution t o
1.5 m l of 2-(2-hydroxyethyl)pyridine. The complex w a s

then extracted with propyl acetate, and the amount of flu-
fenamic acid p r e s e n t in the s a m p l e was obtained i n d i r e c -
tly f r o m the amount of copper determined in the organic
solvent by atomic absorption analysis.
5. 7. Chromatographic Analysis
5.71. P a p e r Chromatography
Flufenamic acid can be detected by p a p e r
chromatography (66) using the following solvent s y s t e m s :
a ) Methyl isobutyl ketone : f o r m i c acid : w a t e r (10 p a r t s
of ketone s a t u r a t e d with 1 p a r t of 470 f o r m i c acid); Rf =
0.95.
b ) Chloroform : methanol : f o r m i c acid : w a t e r (a mixtu-
r e of 1 p a r t of methanol and 1 p a r t of 470 f o r m i c acid
used t o s a t u r a t e 10 p a r t s of chloroform); Rf = 0. 95.
c ) Benzene : methyl ethyl ketone : f o r m i c acid : water
( a mixture of 9 p a r t s of benzene and 1 p a r t of ketone sa-
turated with 1 p a r t of 2’70 f o r m i c acid); Rf = 0. 94.
d ) Benzene : f o r m i c acid : water (10 p a r t s of benzene
s a t u r a t e d with 1 p a r t of 270 f o r m i c acid); Rf = 0. 91.
e ) Methyl ethyl ketone : diethylamine : w a t e r (921:2:77);
Rf = 0. 83.
f ) Methyl ethyl ketone : acetone : f o r m i c acid : water
(40:2:1:6); Rf = 0. 95.
Flufenamic acid can be visualized by UV light, or s p r -
aying the p a p e r with 0.470 p-nitrobenzenediazonium fluo-
borate solution in 1:2 dioxane:water o r with 2’70 aqueous
phosphomolybdic acid solution.
Schmollack and Wenzel (67) developed a method f o r the
detection and quantitative determination of flufenamic acid
using a c h a m b e r p a p e r a n a l y s i s apparatus. Flufenamic
acid w a s then determined fluorometrically a f t e r t r e a t m e n t
with formaldehyde vapor t o obtain the strongly fluorescent
benzoxazine derivative.
5.72. Thin L a y e r Chromatography
Several thin l a y e r chromatographic methods
have been developed f o r identification and quantitative de-
termination of flufenamic acid. Some d e t a i l s of these
methods a r e s u m m a r i z e d in Table XII. It h a s t o point
out that in all c a s e s s i l i c a g e l plates w e r e used.
T A B L E XI1
T h i n L a y e r C h r o m a t o g r a p h y of F l u f e n a m i c Acid
Ref. Solvent S y s t e m Plates Rf D e t e c t ion
3 Cyclohexane:CHC13:CH30H:CH3COOH (60:30:5:5) S. G. G F 0. 54 W(254nm)
3 B e n z e n e : e t h e r : CH3 COOH: CH3 OH ( 120 : 60: 18: 1) id. 0. 7 8 id.
4 Benzene:C2H50H:CH3COOH (20:2:1) S. G. H F NR UV following h e a -
t i n g with HCHO
8 Cyclohexane :e t h y l acetate : CH3 COOH ( 2 0 :30 :2 ) S. G. F 6 0 0.58 W ( 3 5 6 nm)
8 Cyclohexane: CHC13:CH3COOH (40:50: 10) S. G . H F NR id.
40 id. S.G. H F F 0.43 W
44 id. S. G. H F NR UV
63 id. id. 0. 58 U V ; heat. HCHO
+ U V ; iodine
63 B e n z e n e : m e t h a n o l (9: 1) id. , 0. 23 id.
63 Cyc1ohexane:ethyl acetate (1:1 0 ) id. 0. 23 id.
41 1sopropanol:ammonia:water (20: 1 : 2 ) NR 0. 64 NR
68 To1uene:acetic a c i d ( 9 : l ) S. G. G 0.73 H N 0 2 spray
68 To1uene:acetic a c i d (97. 5:2. 5 ) id. 0. 60 id.
69 Chloroform:methanol(7:3) i n NH3 atm. S.G. 60 0. 37 HCHO/HCOOH at
loooc + w
S. G . : silica gel. NR : not r e p o r t e d .
Unterhalt ( 3 ) h a s identified f l u f e n a m i c a c i d i n a m i x t u r e
with o t h e r n o n s t e r o i d a l a n t i i n f l a m m a t o r y d r u g s : the s p o t
v i s u a l i z e d by UV light w a s e l u t e d with m e t h a n o l and flufe-
n a m i c a c i d w a s d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t 288
n m with a m e a n r e c o v e r y of 88%.
Dell and K a m p ( 4 ) a s s a y e d f l u f e n a m i c a c i d in u r i n e and
s e r u m : t h e i r method involved a n e t h e r e a l e x t r a c t i o n f r o m
t h e biological fluids followed by T L C . T h e p l a t e s w e r e
then exposed t o f o r m a l d e h y d e v a p o r ( 2 h o u r s at 8OoC) and
t h e UV-fluorescent spot w a s eluted with m e t h a n o l and f l u -
f e n a m i c a c i d d e t e r m i n e d f l u o r o m e t r i c a l l y . An a l t e r n a t i v e
method p r o p o s e d by the s a m e a u t h o r s involved t h e v i s u a -
lization of flufenamic a c i d by UV light and a t r e a t m e n t of
the s c r a p e d off s i l i c a g e l with conc. H2SO4 followed by
f l u o r o m e t r i c d e t e r mi nat ion.
T h e method d e s c r i b e d by P a n s e e t a l . ( 4 0 ) f o r t h e d e -
t e r m i n a t i o n of flufenamic a c i d in u r i n e and p l a s m a w a s
s i m i l a r , involving e x t r a c t i o n f r o m b i o l o g i c a l m a t e r i a l ,
T L C and elution of the d r u g , followed by q u a n t i t a t i v e d e -
termination ei t h e r spectrophotometrically in methanolic
solution o r f l u o r o m e t r i c a l l y in a CC14 solution i n the p r e -
s e n c e of t r i c h l o r o a c e t i c a c i d . T h e l a t t e r m e t h o d w a s a p -
plied by s e v e r a l a u t h o r s ( a l r e a d y c i t e d in Section 5. 5) f o r
t h e d e t e r m i n a t i o n of f l u f e n a m i c a c i d in b i o l o g i c a l fluids
and t i s s u e s . P a n s e e t al. ( 4 4 ) d e s c r i b e d a l s o a method
f o r t h e d i r e c t d e n s i t o m e t r i c d e t e r m i n a t i o n of f l u f e n a m i c
a c i d on the s i l i c a gel thin l a y e r s .
F l u f e n a m i c a c i d w a s d e t e r m i n e d d i r e c t l y in p l a s m a by
G e i s s l e r e t a l . ( 6 9 ) by addition of m e t h a n o l t o p r e c i p i t a t e
p r o t e i n s , T L C , t r e a t m e n t of t h e d r i e d p l a t e with f o r m a l -
dehyde v a p o r in t h e p r e s e n c e of f o r m i c a c i d a t 100°C f o r
45 m i n u t e s t o f o r m t h e benzoxazine d e r i v a t i v e , and d i r e c t
f l u o r i m e t r y of t h e plate. Use of f o r m i c a c i d s h o r t e n s t h e
r e a c t i o n t i m e and e n h a n c e s t h e f l u o r e s c e n c e i n t e n s i t y and
the sensitivity (quantities I 2 ng/spot m a y be detected).
A method involving r e v e r s e d - p h a s e thin l a y e r c h r o m a t o -
g r a p h y w a s r e p o r t e d by B o l t z e and K r e i s f e l d (2), which
u s e d s i l i c a gel p l a t e s i m p r e g n a t e d with a 10'70 solution of
Dow-Corning 200 i n e t h e r . F l u f e n a m i c a c i d w a s c h r o m a -
t o g r a p h e d u s i n g buffer : dioxane : a c e t o n e (2: 1 : l ) a s the s o l -
vent s y s t e m , in which t h e buffer w a s a t pH 5. 2 , 6. 2, and
FLUFENAMIC ACID 339
7. 2, r e s p e c t i v e l y . In T a b l e XI11 a r e l i s t e d Rf and R M v a -
l u e s found f o r flufenamic a c i d .
T A B L E XI11
Reversed-Dhase T L C of F l u f e n a m i c Acid
pH of the buffer Rf RM= l o g ( l / R f - l )
-
5. 2 0. 78 - 0.55
6. 2 0. 76 - 0.5
7. 2 0. 69 - 0. 350
5.73. Gas Chromatography

Roseboom and Hulshoff ( 7 0 ) h a v e developed
a r a p i d and s i m p l e c l e a n - u p and d e r i v a t i z a t i o n p r o c e d u r e
that c a n be g e n e r a l l y applied t o the g a s c h r o m a t o g r a p h i c
d e t e r m i n a t i o n of a c i d i c d r u g s including f l u f e n a m i c a c i d i n
p l a s m a s a m p l e s . T h e d r u g w a s e x t r a c t e d f r o m acidified
p l a s m a with c h l o r o f o r m : i s o p r o p a n o l (95:5), which w a s
then e v a p o r a t e d . T h e r e s i d u e w a s d i s s o l v e d i n toluene,
then the d r u g w a s b a c k - e x t r a c t e d with a small v o l u m e of
a methanolic 20% t e t r a m e t h y l a m m o n i u m hydroxide solution
(TMAH). T h e solution obtained w a s added t o N , N - d i m e -
t h y l a c e t a m i d e . A f t e r t r e a t m e n t with n-butyl iodide the
d r u g w a s c h r o m a t o g r a p h e d a s i t s n-butyl ester. A gas
c h r o m a t o g r a p h equipped with a flame ionization d e t e c t o r
w a s u s e d . T h e g l a s s c o l u m n s (150 c m x 2 mm I. D. ) w e r e
packed with 3% OV-1, 3% OV-17 o r 3y0 SP-1000, all on
100-120 m e s h C h r o m o s o r b W HP. T h e c a r r i e r gas ( n i -
t r o g e n ) f l o w - r a t e w a s mantained a t 20 m l / m i n . T h e r e c o -
v e r y of flufenamic a c i d in the first e x t r a c t i o n s t e p with
ch1oroform:isopropanol w a s 69%, but with toluene, which
c a n a l s o be u s e d f o r the e x t r a c t i o n f r o m p l a s m a , a r e c o -
v e r y of 95% w a s achieved. Toluene h a s the advantage
that no e v a p o r a t i o n of the e x t r a c t is n e c e s s a r y , and it
can b e e x t r a c t e d d i r e c t l y with the TMAH solution. In t h e
b a c k - e x t r a c t i o n with TMAH a r e c o v e r y of 62Y0 w a s obtai-
ned. T h e r e t e n t i o n t i m e s of the n-butyl e s t e r of flufena-
m i c a c i d with v a r i o u s s t a t i o n a r y p h a s e s a r e l i s t e d i n Ta-
ble XIV.
TABLE XIV
G a s ChromatoaraDhic Data f o r Flufenamic Acid (701
Stationary Column Re tent ion
phase temperature, time,
OC sec
370 ov-1 2 00 2 34
370OV-17 210 271
370 sP-1000 230 218
Another g a s chromatographic method f o r quantitative de-

termination of flufenamic acid in p l a s m a w a s reported by
Cotellessa et al. (43). Flufenamic acid w a s extracted
f r o m plasma with benzene, a f t e r dilution of p l a s m a s a m -
ple with an equal volume of 0. 25 M acetate buffer (pH
4.35). The benzene e x t r a c t w a s evaporated to d r y n e s s
under vacuum. The r e s i d u e w a s dissolved in methanol
and methylated with diazomethane. The s a m e procedure
w a s a l s o applied t o r a t u t e r u s homogenates. The methy-
lated sample w a s dissolved in a n acetone solution of the
i n t e r n a l standard 3-chloro-6-aminobenzophenone. G a s
chromatographic analysis w a s c a r r i e d out using a gas
chromatograph equipped with a flame ionization detector.
The stationary phase w a s 370 OV-17 on Gas-Chrom Q
(100-120 m e s h ) packed into a g l a s s column ( 3 m x 2 m m
I. D. ). The column t e m p e r a t u r e w a s 23OoC and the c a r -
r i e r g a s (nitrogen) flow-rate w a s 40 m l / m i n . F I D s e n s i -
tivity was 1 p g / m l p l a s m a and 5 ,ug/g u t e r u s .
A m a s s s p e c t r o m e t e r coupled with the g a s chromato-
graph was employed to a s c e r t a i n the identity of the m e -
thyl e s t e r of flufenamic acid with the GC peak. A m a s s
s p e c t r u m of the methyl e s t e r is presented. The recove-
r i e s f r o m plasma in the range 10-100 ,ug/ml and f r o m
u t e r u s homogenates were 98. 270 and 9070, respectively.
A g a s chromatographic method f o r the detection of non-
s t e r o i d a l antiinflammatory d r u g s including flufenamic acid
in urine collected f r o m h o r s e s that had received these
compounds orally h a s been developed by Hunt et al. (71).
T h i s procedure involves the isolation of the d r u g s f r o m
FLUFENAMIC ACID 341
urine by solvent extraction and on-column methylation of

the carboxylic acid group.
5. 74. High P e r f o r m a n c e Liquid Chromatography
A method f o r the separation and d e t e r m i n a -
tion of some nonsteroidal antiinflammatory d r u g s including
flufenamic acid w a s described by Dusci and Hackett ( 7 2 ) .
This procedure, which can be applied to s e r u m s a m p l e s
of s m a l l volume (100 pl), involved the extraction of the
drugs with acetonitrile. The e x t r a c t w a s taken to d r y n e s s
at 5OoC under a s t r e a m of nitrogen. The r e s i d u e w a s re-
dissolved in 1 0 0 pl of the elution solvent (60% acetonitrile
in 45 mM KH2P04 adjusted to pH 3. 0 with H 3 P 0 4 ) . An
aliquot of 10-20 pl was injected in a high performance li-
quid chromatograph equipped with a variable wavelength
U V detector. The column (30 c m x 3. 9 m m I. D. ) w a s
packed with pBondapak c18. The conditions f o r individual
analysis of flufenamic acid were as follows: flow-rate of
the elution solvent 2. 0 ml/min, wavelength 282 nm. F o r
the separation of flufenamic acid f r o m the mixture of an-
tiinflammatory drugs (flufenamic and mefenamic acid, na-
proxen , ibuprofen , indomethacin, phenylbutazone, oxyphen-
butazone) a flow-rate of 0 . 8 m l / m i n and a wavelength of
225 nm was used: under these conditions the elution t i m e
of flufenamic acid w a s 10. 5 min.
Using the above -mentioned elution solvent , flufenamic
and mefenamic acid were not separated. A modified elu-
tion solvent (3570 acetonitrile in 0. 7% NHqC1 buffered to
pH 7. 8 with ammonia) allowed to obtain the separation of
all the drugs investigated. Using a flow-rate of 1. 0 m l /
min, the elution time of flufenamic acid w a s 10. 2 min
(mefenamic acid 7. 8 min). The r e c o v e r y of flufenamic
acid was 92t370 in a s e r i e s of ten plasma s a m p l e s e x a m i -
ned, in the range 1. 0-20 p g / m l .
Lin et al. ( 4 2 ) have developed a H P L C procedure f o r
the determination of flufenamic acid and mefenamic acid
in plasma, A single extraction s t e p i s followed by r e v e r -
sed-phase chromatography. Flufenamic acid and mefena-
mic acid can be internal standards f o r each other during
e i t h e r assay. The extraction of flufenamic acid f r o m a c i -
dified plasma s a m p l e s (1 m l ) , t o which 4 p g of mefenamic
acid had been added, w a s accomplished with carbon t e t r a -
chloride. T h e e x t r a c t w a s e v a p o r a t e d t o d r y n e s s u n d e r a
s t r e a m of nitrogen and t h e r e s i d u e w a s r e d i s s o l v e d i n t o
0. 5 m l of methanol, and a n aliquot w a s i n j e c t e d in t h e
c h r o m a t o g r a p h , which w a s equipped with a s t a i n l e s s steel
column ( 3 0 c m x 4 m m I. D. ) packed with a s t a b l e r e v e r -
s e d - p h a s e s t a t i o n a r y p h a s e of p o r o u s silica b e a d s c o a t e d
with c h e m i c a l l y bonded cyanopropylsilane m o n o l a y e r s . T h e
elution solvent w a s w a t e r : a c e t o n i t r i l e : a c e t i c a c i d (60:30:
10). T h e f l o w - r a t e w a s 1 m l / m i n with a n o p e r a t i n g p r e s -
s u r e of 1000 p s i at r o o m t e m p e r a t u r e . T h e effluent w a s
m o n i t o r e d continuously at 254 n m . Under t h e s e conditions
flufenamic a c i d had an elution t i m e of 10. 4 min, with a
m e a n r e c o v e r y f r o m p l a s m a of 100.7?3.4% in t h e 1-10 p g
r a n g e . T h e s e n s i t i v i t y l i m i t w a s 1 p g / m l of p l a s m a .
A method f o r t h e d e t e r m i n a t i o n of flufenamic a c i d a n d
i t s m a j o r m e t a b o l i t e s , 4 ' - h y d r o x y - and 5-hydroxyflufena-
m i c a c i d , w a s d e s c r i b e d by Kubo e t al. ( 7 3 ) : t h e a c i d i -
fied s e r u m w a s e x t r a c t e d with ethyl a c e t a t e . T h e o r g a n i c
e x t r a c t was e v a p o r a t e d and the r e s i d u e w a s r e d i s s o l v e d
i n ethanol and c h r o m a t o g r a p h e d , u s i n g a c o l u m n packed
with Bondapack c 1 8 . T h e m o b i l e p h a s e c o n s i s t e d of wa-
t e r : ethanol (52:48) containing 0. 1% N a 2 H P 0 4 and 0. 5%
t e t r a b u t y l a m m o n i u m b r o m i d e , a d j u s t e d t o pH 7. 81. T h e
r e c o v e r i e s f r o m p l a s m a w e r e 98. 870 f o r f l u f e n a m i c a c i d ,
97.0% f o r 4 ' - h y d r o x y - d e r i v a t i v e J a n d 98.070 f o r 5-hydro-
xy-derivative.
6. DETERMINATION IN BODY FLUIDS AND TISSUES

Many m e t h o d s a m o n g t h o s e outlined in t h i s a n a l y t i c a l
p r o f i l e have been applied t o t h e detection a n d quantitative
d e t e r m i n a t i o n of flufenamic a c i d in biological s a m p l e s of
a n i m a l o r human origin. Such a p p l i c a t i o n s w e r e r e p o r t e d
i n the p a p e r s l i s t e d below :
Colorimetry 46
Spe c t rophot o m e t r y 4, 30, 40,41, 44, 60, 61
Fluorimetry 4, 8, 11, 37, 40, 45, 47, 49, 62,
63, 64, 65, 67, 69
Indirect A A A n a l y s i s 18
PC 67
TLC 4, 8,40, 41, 44,45, 63, 64, 68, 6 9
FLUFENAMIC ACID 343
GC 43, 70, 7 1
HPLC 42, 72, 7 3
7. AC KNOWL ED G EM EN T
T h e a u t h o r s would l i k e t o t h a n k D r . M i c h e l e L i g u o r i
and M r . Vincenzo Migliaro, Ente F a r m a c o l o g i c o Italiano,
N a p l e s , f o r p r o v i d i n g IR, NMR a n d UV s p e c t r a of f l u f e -
namic acid.
8. R E F E R E N C E S
1. M e r c k Index, 9th e d . , 1976, M e r c k a n d C o . , R a h w a y ,
N. J . ; p. 4028.
-
2. K. H. B o l t z e a n d H. K r e i s f e l d , A r z n e i m . - F o r s c h . 27,
1 3 0 0 (1977).
-
3. B. U n t e r h a l t , A r c h . P h a r m . 303, 4 4 5 (1970).
4. H. D. D e l l a n d R. K a m p , A r c h . P h a r m . 303, 785 -
(1970).
5. K. Ikeda, K. U ekam a, M. O t a g i r i a n d M. H a t a n o , J.
-
P h a r m . Sci. 63, 1168 ( 1 9 7 4 ) .
6. J. K r a c m a r , M. A l v a r e z s o t o l o n g o , J. K r a c m a r o v a ,
B. M o r a v c o v a a n d H. D osl ova, P h a r m a z i e - 33, 659
(1978).
7. J. K r a c m a r and J. K r a c m a r o v a , C e s k . F a r m . - 29, 1 9
(1980). C . A . 93, 192094 ( 1 9 8 0 ) .
8. H. D. Del l , M. D o e r s i n g , W. F i s c h e r , J. F i e d l e r ,
-
H. J a c o b i a n d R. K a m p , A r z n e i m . - F o r s c h . 31, 9
(1981).
9. A. C. M e h t a a n d S. G. S c h u l m a n , T a l a n t a 20, 702-
(1973).
10. J. N . M i l l e r , D. L. P h i l l i p s , D. T. B u r n s a n d J.
W. B r i d g e s , T a l a n t a 25, 4 6 (1978).
11. H. D. D e l l a n d B. K u x c h b a c h , F r e s e n i u s ' Z.Ana1.
Chem. - 262, 356 (1972).
12. J. H. Wi l ki nson a n d I. L. F i n a r , J. C h e m . S o c . , 3 2
( 1948).
1 3 . P a r k e , D a v i s & Co. , F r . P a t e n t M1341, J u l y 2, 1962.
C . A . 58, 10130a (1963).
14. B r i s t o c M y e r s C o . , Neth.App1. 6, 604, 860, O c t . 13,
-
1966. C . A . 66, 55513 ( 1 9 6 7 ) .
15. P. F. J uby, T. W. H u d y m a a n d M. B r o w n , J . M e d .
C h e m . 11, 111 ( 1968).

16. M. K u h z r t - B r a n d s t g t t e r , A . K o f l e r a n d G. K r a m e r ,
S c i . P h a r m . 42, 150 ( 1 9 7 4 ) . C . A . - 82, 4 7 6 8 1 ( 1 9 7 5 ) .
17. E . M. J o n e s T P a r k e , D a v i s & C o . , B r i t . P a t e n t
953, 741, A p r . 2, 1964. C. A . 60, 1 5 6 8 8 b ( 1 9 6 4 ) .
18. T. Minamikawa, K. Sakai, N. H a s h i t a n i , E . F u k u s h i -
-
m a and N. Y a m a g i s h i , C h e m . P h a r m . B u l 1 . 21, 1 6 3 2
(1973).
19. R . B. Moffett and B. D. A s p e r g r e n , J. Am. C h e m .
SOC. 82, 1600 (1960).
20. -
A . J . A g u i a r a n d R. J. F i f e l s k i , J . P h a r m . S c i . 55,
1 3 8 7 (1 9 6 6) .
21. A . H. Ghanem, H. E l - S a b b a g h a n d H. A b d e l - A l i m ,
P h a r m . Ind. 42, 854 (1980).
22. N. A . D a a b i r S. A . K h a l i l a n d V. F. N a g g a r , Can.
J. P h a r m . Sci. 11, 1 1 4 ( 1 9 7 6 ) .
23. W. J. Dunn, J-Med.Chem. - 16, 4 8 4 ( 1 9 7 3 ) .
24. H. T e r a d a , S. M u r a o k a a n d T. F u j i t a , J . M e d . C h e m .
1 7 , 330 (1974).
25. 7 G. L o m b a r d i n o , I. G. O t t e r n e s s a n d E. H. W i s e -
-
m a n , A r z n e i m . - F o r s c h . 25, 1 6 2 9 ( 1 9 7 5 ) .
26. M. K u h n e r t - B r a n d s t a t t e r , L. B o r k a a n d G. F r i e d r i c h
-
- S a n d e r , A r c h . P h a r m . 307, 8 4 5 (1974).
27. J. K r c , J r . , M i c r o s c o p e 25, 31 ( 1 9 7 7 ) .
28. Z . Galdecki, M. L. G l o w k a a n d Z . G o r k i e w i c z , A c t a
P o l . P h a r m . 35, 77 (1978). C.A. 89, 1 5 5 7 6 3 ( 1 9 7 8 ) .
29. A . B u r g e r a n d R. R a m b e r g e r , M i k r o c h i m . A c t a - 1, 17
(1980).
30. H. H. F r e y a n d M. A . E l - S a y e d , A r c h . I n t . P h a r m a -
-
codyn. T h e r . 230, 300 (1977).
31. A . A l b e r t a n d E . P. S e r j e a n t i n "The D e t e r m i n a t i o n
of Ionization Constants", C h a p m a n a n d Hall, London,
1971, p. 72.
32. H. T e r a d a a n d S. M u r a o k a , M o l . P h a r m a c o 1 . - 8, 9 5
(1 9 7 2 ).
33. U. Jahn and T. W a g n e r - J a u r e g g , A r z n e i m . - F o r s c h .
24, 4 9 4 (1974).
34. H. M o r i y a m a , H. N a g a t a and T. T a m a k i , S u m i t o m o
C h e m i c a l Co. , L t d . , J a p a n . P a t e n t 7 1 14, 656, A p r .
20, 1971. C.A. 75, 48703 (1971 ) .
35. S. K o m u r a , K. K k a m u r a and M. T a k e n a k a , O t s u k a
FLUFENAMIC ACID 345
C h e m i c a l D r u g s C o . , L t d . , J a p a n . P a t e n t 7 3 26, 744,
A p r . 9, 1973. C.A. - 79, 31680 ( 1 9 7 3 ) .
36. G. R . Allen, J r . , and F. R . R . Church, A m e r i c a n
Cyanamid Co., S. A f r i c a n P a t e n t 71 00, 512, Sep. 3,
1971. C.A. 76, 140237 ( 1 9 7 2 ) .
37. A . J. Glazko, Ann. P h y s . Med. , Suppl. 9, 24 ( 1 9 6 7 ) .
38. R . E. O b e r , K. Ritchie and S. F. Chang, F e d . P r o c .
24, 547 ( 1 9 6 5 ) .
39. R. E. Bowman, K. D. Brunt, K. E. Godfrey, L.
K r u s z y n s k a , A . A . Reynolds, R . I. T h r i f t , D. Waite
and W. R . N. Williamson, J. Chem.Soc. P e r k i n I, 1
(1973).
40. P. P a n s e , P. Z e i l l e r and K. H. Sensch, A r z n e i m . -
F o r s c h . 21, 1 6 0 5 ( 1 9 7 1 ) .
41. H. A . R o s e n b e r g and T. R . B a t e s , P r o c . S o c . E x p .
-
Riol. Med. 145, 93 ( 1 9 7 4 ) .
42. C. K. Lin, C. S. Lee and .J. H. P e r r i n , J . P h a r m .
Sci. 69, 95 ( 1 9 8 0 ) .
43. L. C z e l l e s s a , R . Riva, P. Salva, F. M a r c u c c i and
E. Mussini, J. C h r o m a t o g r . 192, 4 4 1 ( 1 9 8 0 ) .
-
44. p. P a n s e , P. Z e i l l e r and K. H. Sensch, A r z n e i m . -
F o r s c h . 24, 1 2 9 8 ( 1 9 7 4 ) .
45. H. D. Dell, J. F i e d l e r , H. J a c o b i and B. Wasche,
A r z n e i m . - F o r s c h . 27, 1 3 2 2 (1977).
46. H. D. D e l l and J. F i e d l e r , F r e s e n i u s ' Z.Ana1. Chem.
-
270, 278 ( 1 9 7 4 ) .
47. D. L o r e n z and H. D. Dell, Naunyn-Schmiedeberg's
-
A r c h . P h a r m a c o l . 277, Suppl. , R 4 4 (1973).
48. J. A r i a s and R . Cadorniga, Boll. Chim. F a r m . - 112,
804 (1973).
49. L. Angelucci, €3. P i e t r a n g e l i , P. C e l l e t t i and S. F a -
villi, .J. P h a r m . Sci. 65, 455 ( 1 976).
50. C. F. Chignell, 1 , i f e S c i . 7, 1181 (1968).
51. -
C. F. Chignell, Mol. P h a r m a c o l . 5, 4 5 5 ( 1 9 6 9 ) .
52. C. F. Chignell and D. K. S t a r k w e a t h e r , M o l . P h a r -
m a c o l . 7, 229 ( 1 9 7 1 ) .
53. J. B l a n c h a r d , T . N. T o z e r , D. L. S o r b y and L. D.
-
Tuck, Mol. P h a r m a c o l . 11, 1 3 3 ( 1 9 7 5 ) .
54. M. Otagiri, G. E. H a r d e e and J. H. P e r r i n , Bio-
c h e m . P h a r m a c o l . 27, 1 4 0 1 (1978).
55. G. Sudlow, D. J. B i r k e t t and D. N. Wade, Mol.
P h a r m a c o l . 11, 824 (1975).

56. G. Sudlow, T J . B i r k e t t a n d D. N. W a d e , Mol.
-
P h a r m a c o l . 1 2 , 1052 ( 1976).
57. Y. Ka n e o, A. K ai , S. K i r y u a n d S. I g u c h i , Y a k u g a k u
Z a s s h i 96, 1412 (1976). C.A. - 86, 1 0 0 7 4 7 ( 1 9 7 7 ) .
58. G. D e v a G , P. M e s n a r d a n d A . M. B r i s s o n , Ann.
P h a r m . F r . 27, 239 (1969).
59. M. I. W a l a s r a n d M. R i z k , I nd i a n J . P h a r m . 39, 8 2 -
(1 9 7 7 ). C . A . - 87, 157253 (1977).
60. -
J. B. C a r e y , J. C l i n . I n v e s t . 40, 1 0 2 8 ( 1 9 6 1 ) .
61. Y. A . B e l t a g y , Z e n t r a l b l . P h a r m . , P h a r m a k o t h e r . La-
- -
b o r a t o r i u m s d i a g n . 116, 925 (1977). C. A , 88, 1 5 8 5 4 8
(1978).
62. R. A . B u chanan, C. J. E a t o n , S. T. Koeff a n d A . W.
K r i n k e l , C u r r . T h e r . R e s . 11, 5 3 3 ( 1 9 6 9 ) .
63. H. D. Del l , J. F i e d l e r a n T B . W z s c h e , A r z n e i m . -
-
F o r s c h . 27, 1312 ( 1977).
64. H. D. Del l , H. Jacobi, R. K a m p a n d J. K o l l e , A r z -
n e i m . - F o r s c h . 31, 2 1 ( 1981).
65. Y. H a t t o r i , T. Arai, T. M o r i a n d E. F u j i h i r a ,
-
C h e m . P h a r m . Bull. 18, 1 0 6 3 (19 7 0 ) .
66. L. R e io , J. C h r o m a t o g r . 68, 1 8 3 ( 1 9 7 2 ) .
67. -
W. S c h m o l l a c k a n d U. W e n z e l , P h a r m a z i e 29, 5 8 3
(1 9 7 4 ).
68. B. D e m e t r i o u a n d B. G. O s b o r n e , J. C h r o m a t o g r . - 90,
4 0 5 (1 9 7 4) .
6 9. H. E. G e i s s l e r , E. M u t s c h l e r a n d A . S c h u m a c h e r ,
J. C h r o m a t o g r . - 146, 1 6 9 ( 1978).
70. H. R o s e b o o m a n d A . Hulshoff, J. C h r o m a t o g r . 173, -
6 5 (1 9 7 9 ) .
71. J. P. Hunt, P. E. Haywood a n d M. S. M o s s , P r o c .
Int. S y mp . E q u i n e Med. C o n t r o l , 3 r d , 1979, 9. C. A .
94, 97262 (1981).
72. -
J. D u s c i a n d L. P. H a c k e t t , J. C h r o m a t o g r . 1 7 2 ,
5 1 6 (1 9 7 9) .
73. 0. Kubo, K. N i s h i d e a n d N. K i r i y a m a , J. C h r o m a -
-
t o g r . 174, 254 (1979).
L i t e r a t u r e surveyed through October, 1981.

HEXESTROL
Hassan X Aboul-Enein,
Essam A. Lo@, and
Mohumed E . Mohumed
1. Description 348
1.2 Formulae 348
I .4 Elemental Composition 349
2.2 Melting Point 349
2.3 Solubility 350
3. Synthesis 358
4. Stability and Decomposition Products 361
5 . Metabolism 361
6.1 Titrimetry 362
6.2 Colorimetry 362
6.3 Ultraviolet Spectrophotometry (Uv) 363
6.5 Mass Fragmentography 370
6.6 Biological Assays 370
References 372

Analytical Profiles of Drug Substances Pharmaceutical hsociation
Volume I I 347 ISBN 0-12-Mo811-9
348 HASSAN Y. ABOUL-ENEIN ET AL.
ANALYTICAL PROFILE-HEXESTROL
1. Descr i p t ton
1.1 Nomenclature
1.11 - Chemical N a m e s
(?) 3 , 4 - Di(p-hydroxy phenyl) n-hexane.

4 , 4 ' - (1, 2 d i e t h y l - 1, 2 - e t h a n e d i y l )
bis-phenol.
4 , 4 ' - (1, 2 d i e t h y l e t h y l e n e ) d i p h e n o l .
4 , 4 ' - dihydroxy - y,b-diphenyl hexane.
4 , 4 ' - dihydroxy - a ,B-diethyldiphenylethane.
p, p l - dihydroxy - d i p h e n y l hexane.
meso - 3 , 4 - b i s (p-hydroxyphenyl) -n-
hexane .
1 . 1 2 - Generic Name
H e x e s t r o l , H e x o e s t r o l , Dihydrodiethyl-
s t i l b e s t r o l , Hexanoestrol, Cycloestrol ,
Hormoestrol, S y n e s t r o l .
1.13 - Trade Name
S yn t r o g h e , Fo 11i p l e x , S yn t hovo .
1.2 Formula
1 . 2 1 - Emprical
'18 H22 '2
1.22 - Structural
C2R5
270.4
HEXESTROL 349
C , 79.96%; H, 8.2%; 0,11.84%
1.5 Appearance, Color, odor
w h i t e , o d o r l e s s , c o l o r l e s s c r y s t a l s or
c r y s t a l l i n e powder (1).
2. Physical properties
2.1 Crystal properties
The f o l l o w i n g t a b l e shows t h e c r y s t a l form of

h e x e s t r o l d e r i v a t i v e s and t h e i r m e l t i n g p o i n t s .
These d e r i v a t i v e s c a n be used f o r i d e n t i f i c a t i o n
purposes too ( 2 ) .
0
Derivative Crystalization Crystal M.P., C
solvent form
Meso Form
Di-Me e t h e r Me2CO-MeOH plates 146

Dipropionyl Pet. ether crystals 127-128
Dibutyryl Pet. ether crystals 106-107
Dicaproyl Pet. e t h e r crystals 96-97
Disuccinyl CHC13-pet. e t h e r crystals 150-153
DL(+) Form
Di-Me ether C&-pet. ether 56
2.2 Melting p o i n t
The f o l l o w i n g are t h e m e l t i n g p o i n t s f o r h e x e s t r o l
i n i t s meso, DL-forms and t h e i r a n t i p o d e s .
0
Form M.P., C Ref
Meso
-
185-188 3
184-185 4
186 5
DL (+)Form 128 2
L(-) Form 80 2
D(+) Form 80 2
2.3 Solubility
Freely soluble i n ether, soluble i n acetone,

a l c o h o l , methanol, s o l u b l e i n v e g e t a b l e o i l s
upon warmming a l s o s o l u b l e i n d i l u t e s o l u t i o n
of a l k a l i h y d r o x i d e . S l i g h t l y s o l u b l e i n
c h l o r o f o r m , benzene; i n s o l u b l e i n water (1, 3 ) .
2.4 Identification
The f o l l o w i n g i d e n t i f i c a t i o n tests a r e d e s c r i b e d
i n t h e P h a r m a c e u t i c a l Codex 1979 ( 5 ) .
1) To a b o u t 25Omg add l m l of a c e t i c a n h y d r i d e and

2 m l of d e h y d r a t e d p y r i d i n e , b o i l u n d e r a r e f l u x
c o n d e n s e r f o r 1 5 m i n u t e s , c o o l , add 5Oml of water
and s h a k e t h o r o u g h l y u n t i l a p r e c i p i t a t e is
produced which, a f t e r washing w i t h water and
d r y i n g , m e l t s a t a b o u t 138OC.
2) D i s s o l v e a b o u t lOmg i n 5 m l of H2SO4; t h e
s o l u t i o n i s c o l o r l e s s ( d i s t i n c t i o n from
s t i l b o e s t r o l , which g i v e s a g o l d e n - y e l l o w
color).
2.5 Spectral properties
2.51 Ultraviolet spectra
H e x e s t r o l i n e t h a n o l shows maxima a t 230nm

( E l % , l c m , 775) and 280 nm ( E l % , lcm, 1 4 0 ) ;
0.11NaOH h e x e s t r o l g i v e s maxima a t 242 nm
( E l % , l c m , 965) and 295 nm (El%, l c m , 1 7 5 ) .
A s shown i n f i g u r e ( 1 ) and i n agreement t o
t h e f i g u r e s p u b l i s h e d by C l a r k e ( 3 ) .
The i n f r a r e d spectrum of h e x e s t r o l i n K B r
d i s c i s g i v e n i n f i g u r e ( 2 ) . Major band
a s s i g n m e n t s are a s follows:
-1
Frequency Cm Assignment
3400 P h e n o l i c OH
1610, 1600 Aromatic r i n g
C=C s t r e t c h
HEXESTROL 35 1
L
200 nm 250 nm 300 nm 350 nm
F i g . 1. The u l t r a v i o l e t a b s o r p t i o n spectrum of H e x e s t r o l
i n ethanol.
I n s t r u m e n t : Pye Unicam SP8-100
Wavenumber
Figure 2. I R s p e c t r u m of H e x e s t r o l i n K B r .
Instrument: P e r k i n Elmer 567
Other f i n g e r p r i n t bands c h a r a c t e r i s t i c to-l

h e x e s t r o l a r e 1530, 1450, 1300 and 1110 cm .
2.53 Nuclear Mametic Resonance Spectrum
a) PMR
A t y p i c a l PMR spectrum of h e x e s t r o l i s shown
i n Figure (3).
The sample was d i s s o l v e d i n d e u t r a t e d CDC13

and a drop of d e u t r a t e d d i m e t h y l s u l f o x i d e
(DMSO-db) u s i n g TMS a s t h e i n t e r n a l s t a n d a r d .
The spectrum was determined on a V a r i a n

T60-A s p e c t r o m e t e r .
The f o l l o w i n g s t r u c t u r a l a s s i g n m e n t s have
been e l i c i t e d from F i g u r e ( 3 ) .
Chemical S h i f t (6) Assignment
T r i p l e t a t 0.50 CH CH -
-3 2
Multiplet centered a t 1.3 C H e 2
M u l t i p l e t c e n t e r e d a t 1.90 -CH-CH-
Doublet of d o u b l e t eight aromatic

c e n t e r e d a t 6.86 protons
character i s t i c
f o r para
substitution
of t h e r i n g .
Broad s i n g l e t exchangable p h e n o l i c OH
w i t h D20 a t 7.67 group.
b) 13C-NMR
*
The I3C-NMR of h e x e s t r o l has been d e t e r m i n e d ,
t h e off-resonance spectrum ( F i g . 4 ) shows
seven s i n g l e t s . The complete spectrum i s
shown i n F i g . 5. The spectrum was determined
on a Varian FT-80A i n DMSO-db a s s o l v e n t ,
t u b e d i a m e t e r 10 mm, s p e c t r a l width 5000 Hz
*H.Y. Aboul-Enein, unpublished d a t a

I . I I I . 1
1 . . . . 1 . . . . 1 . . . . 1 . . . . ~ . . . . ~ . . . . ’ ~ ” . ” ” ~
ao ZO 6.0 5.0 PPM( b) 4.0 3.0 2.0 1.0
F i g u r e 3. PMR s p e c t r u m of H e x e s t r o l i n C D C l -DMSO-d w i t h IT% a s

i n t e r n a l standard. 3 6
Instrument: V a r i a n T60-A
354
355
a c q u i s a t i o n t i m e : 1.638 sec.; p u l s e w i d t h :
4 Usec; number of d a t a p r i n t : 16384.
S p e c t r a l a s s i g n m e n t s a r e l i s t e d below:
Chemical S h i f t Assignments
( i n ppm r e l a t i v e t o TMS)
12.06 CH3
26.97 CH2
52.77 -
CH-
134. 4 2 c 1- c.1
115.05
c 2-c 6 02- c 6
128.82 c3-c 5 c--c
3 5
155.39 c4-c*4
2.54 Mass Spectrum
The mass spectrum of h e x e s t r o l , o b t a i n e d by

chemical i o n i z a t i o n w i t h i s o b u t a n e g a s , i s
shown i n P i g . 6. The spectrum w a s determined
by d i r e c t i n l e t t o Ribermag 10-10R m a s s
s p e c t r o m e t e r and e x h i b i t s c o m p a r a t i v e l y
l i t t l e fragmentation.
The f o l l o w i n g t a b l e g i v e s t h e most prominent

i o n s and t h e i r r e l a t i v e i n t e n s i t i e s .
Mass ( d e ) Relative Intensity %
27 0 4.6 (M+)
219 10.5
178 11.8
1 77 93
1 36 9.9
.
I
80 90 14011012Q 130 W 1 5 0 1601?01801902002102PO2303+0250260270
F i g u r e 6. Mass Spectrum of H e x e s t r o l ( C I - i s o b u t a n e ) determined

by d i r e c t i n l e t i n s e r t i o n .
135 100 ( b a s e peak)

134 36.9
107 42.6
+. +'
1 0 -@ CH CH
r - C Hl 2
CH2CH3
CH-CH I
m/e 177 mfe 178
+*
O
D
+*
CH2CH3 CH2CH3
H@ @
H! LH
m f e 135 m f e 134
3. Synthesis
S e v e r a l methods have been p u b l i s h e d and p a t e n t e d

f o r t h e s y n t h e s i s of h e x e s t r o l . I n 1938 Campbell
-
et-a1- (6) i s o l a t e d h e x e s t r o l i n p o o r y i e l d , from
t h e p r o d u c t s of d e m e t h y l a t i o n of a n e t h o l e .
Some of t h e s y n t h e t i c a p p r o a c h e s f o r h e x e s t r o l
a r e summerized a s f o l l o w s : -
1) C a t a l y t i c h y d r o g e n a t i o n of p s e u d o d i e t h y l -
s t i l b e s t r o l g i v e s h e x e s t r o l a l o n g w i t h some
d i e t h y l s t i l b e s t r o l (7).
=:
EtEt
H O G @OH
I
catalytic
hydrogenat i o n
0
Et
I
HO&- ,CH @H+HO c = c
I
I Et
Et
HEXESTROL 359
Hydrogenation of t h e d i m e t h y l e t h e r s of d i e t h y l s -
t i l b e s t r o l and pseudodiethylstilbestrol w i t h
subsequent d e m e t h y l a t i o n a f f o r d s t h e meso-isomer
of h e x e s t r o l which i s more p o t e n t t h a n t h e
DL-isomer ( 7 ) .
H
CH CH
HO
meso-f orm
2) Hydrogenation of 4 , 4 ' dihydroxy y-6 d i p h e n y l y-6

hexadiene g i v e s h e x e s t r o l i n q u a n t i t a t i v e y i e l d ( 7 ) .
HOO'L!!
CH CH
0 OH
Fd Icharcoa 1
>- . Hexestrol
3) H e x e s t r o l may be prepared by t h e a c t i o n of e t h y l
magnesium bromide on a n i s a l d a z i n e w i t h subsequent
d e m e t h y l a t i o n (8).
Hexes t r o l
The above methods f o r t h e s y n t h e s i s of h e x e s t r o l
i n v o l v e t h e u s e of i n t e r m e d i a t e s which a r e d i f f i c u l t
t o prepare.
360 HASSAN Y. ABOULENEIN ET AL.
4) B e r n s t e i n and Wallis d e s c r i b e d ( 4 ) a n a l t e r n a t e
i n e x p e n s i v e method f o r t h e s y n t h e s i s o f h e x e s t r o l
s t a r t i n g from p-hydroxypropiophenone a s shown
i n scheme ( 1 )
I I1
I1 -b
,CH30 N a -@ YHCH
alcohol OH2
I11
I11 F
HBr
C H 3 @'
0 CH-CH
Br 2 5
IV
Scheme 1 VI
5) Kharasch and Kleiman(9) p r e p a r e d h e x e s t r o l d i m e t h y l -
e t h e r from a n e t h o l e hydrobromide and G r i g n a r d r e a g e n t
i n t h e p r e s e n c e of a h a l i d e of c o b a l t , n i c k e l o r i r o n ,
t h e f r e e r a d i c l e g e n e r a t e d from t h i s r e a c t i o n
dimerizes t o give hexestrol dimethylether i n y i e l d s
r a n g i n g from 14-41%. The h i g h e r m e l t i n g p o i n t f o r
t h e meso-form a l l o w s i t s s e p a r a t i o n from t h e DL-
by-product formed i n t h i s r e a c t i o n .
r 1
HEXESTROL 361
4. S t a b i l i t y and Decomposition p r o d u c t s
H e x e s t r o l is a r e l a t i v e l y s t a b l e compound a t room
t e m p e r a t u r e ; however i t i s recommended t o b e k e p t
i n a w e l l c l o s e d c o n t a i n e r p r o t e c t e d from l i g h t .
5. Yetabolism
I l e x e s t r o l i s m e t a b o l i z e d i n a s i m i l a r way a s d i e t h y l -
s t i l b e s t r o l , i t i s excreted c h i e f l y as a glucuronide
conjugate ( 3 ) . This glucuronide i s mostly excreted
i n t o t h e b i l e which i s s u b j e c t e d t o h y d r o l y s i s by
i n t e n t i n a l g l u c u r o n i d a s e enzyme d u r i n g i t s p a s s a g e
i n t o t h e g u t . T h i s a l l o w s t h e d r u g be r eab so r b ed ,
r e c o n j uga t e d and r e - e x e r e t e d ( h e p a t i c c i r c u l a t i o n )
( 1 0 ) . O t h e r p o s s i b l e m e t a b o l i t e i n t e r m e d i a t e s which
should b e i n v e s t i g a t e d a r e shown in scheme ( 2 ) . This
i s i n a n a l o g y t o t h e p o s s i b l e m e t a b o l i t e s of d i e t h y l
s t i l b e s t r o l which h a s r e c e n t l y a t t r a c t e d t h e a t t e n t i o n
by b e i n g l i n k e d t o t h e o c c u r a n c e o f v a g i n a l a d e n o c a r c i -
noma i n a d o l e s c e n t d a u g h t e r s whose m o t h e r s had r e c e i v e d
d i e t h y l s t i l b e s t e r a l d u r i n g p r e g n a n c e (11, 1 2 , 13).
HO
HO * H o e o H
OH
Me0 Me0
HO Me0
Scheme 2. Expected M e t a b o l i t e s of H e x e s t r o l
HASSAN Y. ABOUL-ENEIN ET AL.
6.1 Titrimetry
- Aqueous (Bromometry)
The f o l l o w i n g method h a s been d e s c r i b e d f o r t h e
q u a n t i t a t i v e d e t e r m i n a t i o n of h e x e s t r o l d i a c e t a t e (14) :
To 70 mg of sample add 1 0 m l of 0.5N m e t h a n o l i c KOH,
and h e a t under r e f l u x f o r 30 m i n u t e s on a water-bath.
Cool, add 5Oml of a c e t i c a n h y d r i d e , shake u n t i l t h e
h y d r o l y s a t e i s d i s s o l v e d , t h e n add 2 m l of 30% K B r
s o l u t i o n , 2 ml of conc. H2SO4 and 20 m l of 0.1N-KBr03,
and set a s i d e i n t h e d a r k f o r 1 0 min. Add lgm of K I
and 100 m l of H 2 0 , and t i t r a t e w i t h O.lN-Na2S203 u s i n g
s t a r c h a s i n d i c a t o r . The e r r o r i s - +1% over t h e r a n g e
7 0 t o 90 mg of h e x e s t r o l d i a c e t a t e .
6.2 Colorimetry
H e x e s t r o l has been determined i n o i l y s o l u t i o n and

t a b l e t form a s f o l l o w s ( 1 5 ) :
For t a b l e t : e x t r a c t a sample c o n t a i n i n g 2 t o 3 mg of
h e x e s t r o l w i t h methanol, d i l u t e t h e e x t r a c t w i t h water
t o 100 m l , and f i l t e r . To a 20 m l a l i q u o t add b o r a t e
b u f f e r s o l u t i o n (pH 1 1 ) .
For o i l y i n j e c t i o n s : Mix a sample c o n t a i n i n g 0.4 t o

0.6 mg of h e x e s t r o l w i t h l i g h t petroleum (5ml) , shake
t h e s o l u t i o n w i t h methanol (6ml) , add b u f f e r s o l u t i o n
(13ml), shake, and c o l l e c t t h e aq. methanol phase i n
a lOOml f l a s h . Twice r e p e a t t h e e x t r a c t i o n w i t h methanol
(6ml) and b u f f e r s o l u t i o n (13ml) and t o t h e combined aq.
e x t r a c t s add methanol (2ml).
For d e t e r m i n a t i o n of h e x e s t r o l : h e a t t h e prepared
s o i u t i o n on a water b a t h f o r 30 min., c o o l i t t o room
temperature, add d i a z o t i z e d s u l p h a n i l i c a c i d s o l u t i o n
(12ml) and mix. A f t e r 40 minutes d i l u t e w i t h b u f f e r
s o l u t i o n t o 100 m l , and measure t h e e x t i n c t i o n a t 495 nm
a g a i n s t water. Beer's law i s obeyed f o r 0.5 t o 4 mg of
h e x e s t r o l p e r 1 O O m l . R e s u l t s a g r e e to. w i t h i n +lo% w i t h
t h e o r e t i c a l values.
HEXESTROL 363
B e l i k o v , d e s c r i b e d a s i m i l a r p r o c e d u r e (16) which
depends on d i a z o d i z a t i o n of h e x e s t r o l w i t h d i a z o -
s u l p h a n i l i c a c i d . However, t h e azo-dye formed i s
measured i n a l k a l i n e medium a t 420 nm.
Another c o l o r i m e t r i c method f o r d e t e r m i n a t i o n of
hexestrol i n feeds i s reported a s follows (17):
The ground f e e d i n g s t u f f (40gm) mixed w i t h lOgm of

sand i s s e t a s i d e w i t h c h l o r o f o r m o v e r n i g h t , t h e n
e x t r a c t e d w i t h CHC13 f o r 6 h o u r s . The e x t r a c t i s made
up t o 2 O O m l of CHC13. The r e s i d u e from e v a p o r a t i o n
of t h e f i n a l CHC13 e x t r a c t i s d i s s o l v e d i n t r i e t h y l a m i n e -
t e t r a h y d r o f u r a n - w a t e r (1:5 : 1 4 ) , t h e s o l u t i o n is r e t a i n e d
i n an o l e a t e d c e l l u l o s e column f o r 1 h o u r , t h e n t h e
impurities a r e eluted with triethylamine - tetrahydro-
f u r a n - water m i x t u r e . The column i s a c i d i f i e d w i t h
N-H2SO4 and e x t r a c t e d w i t h e t h y l e t h e r , t h e e x t r a c t
i s evaporated, the r e s i d u e i s dissolved i n ethanol,
and t o t h i s s o l u t i o n a r e added water, dil.HC1, a
molybdotungstophosphate r e a g e n t and t h e n Na2C03 s o l u t i o n .
The e x t i n c t i o n of t h e c e n t r i f u g e d s o l u t i o n i s measured
a t 750 run a g a i n s t a r e a g e n t b l a n k and compared w i t h
t h a t of s t a n d a r d s o l u t i o n of h e x e s t r o l t r e a t e d s i m i l a r l y .
6.3 U l t r a v o i l e t S p e c t r o p h o t o m e t r y (U.V.)
H e x e s t r o l w a s q u a n t i t a t i v e l y determined i n tablet
s p e c t r o p h o t o m e t r i c a l l y ( 1 8 ) . The d r u g was e x t r a c t e d
w i t h CHC13 from a n a c i d i f i e d powdered sample ( c o n t a i n i n g
a b o u t 5mg of h e x e s t r o l ) . The CHC13 e x t r a c t w a s con-
c e n t r a t e d and 2 , 2 , 4 t r i m e t h y l p e n t a n e w a s added and
h e x e s t r o l w a s e x t r a c t e d w i t h 0.1N NaOH. The combined
a l k a l i n e s o l u t i o n w a s a c i d i f i e d and r e - e x t r a c t e d w i t h
CHC13. The CHC13 s o l u t i o n was washed w i t h water and
d r i e d o v e r Na2S04 and e v a p o r a t e d t o d r y n e s s .
The r e s i d u e w a s d i s s o l v e d i n e t h a n o l and measured a t

280nm. The p e r c e n t a g e r e c o v e r y ranged from 94.4-98.9%.
Kovalenko r e p o r t e d a q u a n t i t a t i v e method f o r t h e a s s a y
of h e x e s t r o l ( 1 9 ) . About 25mg of h e x e s t r o l w a s weighed
and d i s s o l v e d i n 25-3Om1 of 0.1N NaOH i n a 5Oml v o l u m e t r i c
f l a s k . The volume w a s a d j u s t e d t o 5Oml w i t h 0.1N NaOH
s o l u t i o n . Then p i p e t t e 5ml of t h i s s o l u t i o n i n t o a
second 5Oml v o l u m e t r i c f l a s k and t h e volume a d j u s t e d
t o 5Oml w i t h 0.1N NaOH s o l u t i o n . The same s e r i a l d i l u t i o n

was r e p e a t e d a n d t h e c o n c e n t r a t i o n of h e x e s t r o l i n t h e
l a s t s o l u t i o n w a s d e t e r m i n e d by m e a s u r i n g t h e a b s o r p t i o n
a t 241 nm u s i n g 0.1N NaOH s o h t i o n f o r t h e b l a n k . S i n c e
t h e s e s o l u t i o n s f o l l o w t h e Lambert-Beer law 1.8 pg m l ,
t h e d e t e r m i n a t i o n c a n b e made from a s t a n d a r d c u r v e .
6.4 Chromatographic A n a l y s i s
6 . 4 1 P a p e r Chromatography
Tompsett h a s d e s c r i b e d a method f o r d e t e c t i o n
of h e x e s t r o l and o t h e r s t i l b e s t r o l d e r i v a t i v e s
a l o n g w i t h t h e p - h y d r o x y m e t a b o l i t e s of
p h e n o b a r b i t o n e and p h e n y t o i n i n u r i n e u s i n g
t h e 2-dimensional paper chromatography ( 2 0 , 2 1 ) .
The two s y s t e m s u s e d were i s o p r o p a n o l :

NH3 (0.99) :H20 (8 :1:1) and C6H6 :E t O A c :H 2 0 ( 2 :1: 1).
The d e t e c t i n g a g e n t s u s e d were P a u l y ' s r e a g e n t

(red-brown) , d i a z o t i z e d p - n i t r o - a n i l i n e (brown) ,
d i a z o t i z e d d i e t h y l a m i n o e t h y l p-aminophenyl
s u l p h o n e (brown) and 1 - n i t r o s o - 2 - n a p h t h o l
n i t r i c a c i d m i x t u r e (+ve). The s e n s i t i v i t y
r a n g e f o r t h i s method i s 5-80ug.
Column chromatography h a s b e e n a p p l i e d t o
p u r i f y the c a t t l e feed e x t r a c t s containing
h e x e s t r o l and o t h e r s t i l b e s t r o l s t o remove
i n t e r f e r i n g substances b e f o r e i t s determina-
t i o n . An example of t h e columns u s e d f o r t h e
p u r i f i c a t i o n of t h e e x t r a c t s i s A 1 0 column
2 3
(22).
Verbeke ( 2 3 ) used columns c o n t a i n i n g XAD-2,

C e l i t e , o r n e u t r a l A1203 (Brockman A c t i v i t y I )
f o r p u r i f i c a t i o n o f t i s s u e e x t r a c t s and u r i n e
u s i n g d i s t i l l e d water; 1 5 m l water-washed
e t h e r f o l l o w e d b y , lhl CgHg t h e n benzene-
i s o o c t a n e ( 1 : l ) ; and b e n z e n e : i s o o c t a n e (1:l)
a s e l u e n t s r e s p e c t i v e l y f o r d e t e c t i o n of
h e x e s t r o l and o t h e r a n a b o l i c s .
HEXESTROL 365
6 . 4 3 Thin Layer Chromatography
S e v e r a l r e p o r t s have been published on t h e

d e t e c t i o n , q u a n t i t a t i v e d e t e r m i n a t i o n of
h e x e s t r o l and o t h e r c h e m i c a l l y r e l a t e d d r u g s
f o r example d i e t h y l s t i b e s t r o l i n f e e d s (meat,
milk) and i n b i o l o g i c a l f l u i d s .
Table (1) summerizes t h e s o l v e n t systems

a n d t h e d e t e c t i n g a g e n t s used i n t h e
cited references.
6.44 Gas-Liquid Chromatography
Various g a s - l i q u i d chromatographic methods

have been developed f o r d e t e c t i o n of h e x e s t r o l
i n meat and o t h e r a g r i c u l t u r a l p r o d u c t s . Gain
and S c h o l l ( 3 1 ) d e s c r i b e d a method f o r
d e t e r m i n a t i o n of h e x e s t r o l i n m o l a s s e s - based
l i q u i d feed supplements, a f t e r t h e p r e p a r a t i o n
of t h e b i s ( t r i m e t h y l s i l y l ) acetamide d e r i v a t i v e .
However t h e method showed i n t e r f e r i n g peaks o r
low recovery due t o emulsion f o r m a t i o n . Most
of t h e g a s chromatographic d e t e r m i n a t i o n
r e q u i r e s d e r i v a t i z a t i o n of h e x e s t r o l b e f o r e
i n j e c t i n g i n t o t h e g a s chromatograph.
Table ( 2 ) summerizes t h e d a t a o b t a i n e d from

t h e l i t r a t u r e s t i l l 1980.
T a b l e 1. (contd.)
S o l v e n t system Stationary Detect i n p R-emarks Ref.

phase agent
2 d i m e n s i o n a l TLC 5%H2SO4- s e n s i t i v i t y 0.5- 23

a) CHC13:EtOH:C H induced f l u o r e lOppb
6 6
(36:1:4) scence a t
n-C H :Et20:CH2C12 366m
6 14
W
m (4:3: 2)
m
petroleum e t h e r Silica gel v a n i l li n a p p l i e d t o animal 27
(40-65OC) : ( a c t i v a t e d a t 120 OC reagent) feeding s t u f f
2 d i m e n s i o n a l TLC
using: Silica gel H 28
a) CHC13:EtOH(9:1)
b) C 6H :E t OAc ( 3 :1 )
t.1.c
CH2 C12:Me2C0(4 :1) K i e s e l g e l or ultraviolet a t s e n s i t i v i t y range
Kieselgel F 254m 0 . 2 . 2 up, 29
254
( t h e h i g h performance
t l c r a n g e 10-200ng
T a b l e 1.
S o l v e n t system Stationary Detecting Remarks Ref.

phase agent
Toleune: EtOAc ( 1 9 :1 ) A l u m i n ium Ultraviolet S e n s i t i v i t y 5,lOppb 24

ox i d e a t 254 nm
C6H6 :E t O A c (20: 1) ( i n f r e s h l i v e r and

kidney)
CH2C12:Me2C0(4: 1) Silica gel U 1 tr av i o l e t recommended € o r 25

a t 254 nm r o u t i n e test of
CHC13:EtOAc(4:1)
e s t r o g e n s i n food
2 d i m e n s i o n a l TLC w i t h
n C6H14:Et20:CH2C12
(4:3:2) i n b o t h d i r e c t i o n
or
EtOAc:C6H6(l:3)
a f t e r s o l v e n t system ( a )
n-C H :Et20:CH2C12 Silica gel G ultraviolet s e n s i t i v i t y of t h e 26

6 14
( 4 :3 :2) 254m. por 20' test i s 0 . 5 y g
T a b l e 1. (contd.)
S o l v e n t system Stationary Detecting Remarks

Ref.
phase agent
h.p. t.1.c.
CH C1 :Ple2C0(4:1) Kieselgel F d e n s itome- (t.1.c. range
2 2 254
W
trically at 200-2000ng) .
91
W 287 (h.p.t.1.c range
10-2 Ong )
C6H14:Me2C0(3 :2 ) o r Po 1yamid e t h e i n f l u e n c e s of
C6H6:Me2C0(3:1) chemical s t r u c t u r e
and m i g r a t i o n r a t e 30
are discussed.
T a b l e 2.
Column Carrier G a s Detector Remarks Ref.
3% OV-1 on Chromosorb Ar-CH electron d er i v a t i z e d by 32

4
hep t a f l u o r o b u t y r i c
WHP (80 t o 100 mesh) a t (19:l) capture a n hyd r i d e
var ious temperature
He electrolytic s e n s i t i v i t y €or electron
conductor c a p t u r e (40-400 p i c o g ) ; 32
and f o r e l e c t r o l y t i c
c o n d u c t o r (1-5 ng)
2% OV-17, 3%0V-1,3%0V-1 electron d e r i v a t i z e d by hepta-

N2
5% of n e o p e n t y l g l y c o l capture fluorobutyric anhydride; 33
s e b a c a t e o r 2% Carbowax s e n s i t i v i t y range
20M on s i l a n i s e d Chromosorb 6 t o 262 ng/ml
W (80 t o 100 m e s t ) a t
var i o u s temperatures .
3% QF-1 o n 100 t o 200 flame ioniza- i n j e c t e d as a dipropionate
mesh Gas. Chrom Q a t 170'. t ion d e t e c to r derivative; sensitivity 34
0.05 mg/ml
Ap i e z o n L-Ep i k o t e Electron- d er i v a t i z ed by
1001(5 :1) on s i l a n i s e d N2 capture trifluoroacetic
Chromosorb G a t 160Oc. anhydr iod e .
3 70 HASSAN Y. ABOUL-ENEIN ET AL.
6.45 High Performance L i q u i d Chromatography
H e x e s t r o l has been determined among o t h e r

a n a b o l i c s y n t h e t i c and n a t u r a l hormones i n meat
by HPLC. The columns used were Rp-2 (10 v m ) ,
Rp-18 ( 5 pm) o r Zorbax CN ( 5 pm) by g r a d i e n t
e l u t i o n w i t h a c e t o n i t r i l e : H 2 0 (1:9) p l u s 25%,
i n c r e a s i n g r e c t i l i n e a r l y i n 5 m i n u t e t o 45%, of
a c e t o n i t r i l e : H20 ( 9 : l ) . The f l o w r a t e w a s 2 m l
min.-l, and 100 mg e a c h of L i C l and LiC104 were
added t o each 1 0 h l of e l u e n t .
The e l u a t e w a s examined by voltammetry w i t h

v i t r e o u s carbon e l e c t r o d e s and on silver-AgC1
(3M-KC1) r e f e r e n c e e l e c t r o d e ; t h e p o t e n t i a l
sweep r a t e was 5 m ~ s - 1 .
Other columns used f o r t h e d e t e r m i n a t i o n of

h e x e s t r o l and o t h e r r e l a t e d d r u g s i s L i c h r o s o r b
RP-8 w i t h m i x t u r e s (35:13, t o 25:23) of methanol
and water ( c o n t a i n i n g i n a l l i n s t a n c e s 2 p a r t s of
a c e t o n i t r i l e ) as a mobile phase (0.8 t o 1.6 m l
min-l). The v o l t a m m e t r i c c u r v e of t h e e l u a t e was
recorded by t h e u s e of a v i t r e o u s - c a r b o n e l e c t r o d e ,
a p l a t i n u m c o u n t e r - e l e c t r o d e and a silver-AgC1
r e f e r e n c e e l e c t r o d e a t pH 3 , t h e peak p o t e n t i a l ( V )
f o r h e x e s t r o l w a s +.9. Amounts r a n g e from 1-4 ng
g-l of h e x e s t r o l i n meat could be determined by
HPLC .
6.5 Mass Pragmentography
H e x e s t r o l among o t h e r s t i l b e s t r o l w a s q u a n t i t a t i v e l y
determined by combined GC/MS ( 3 8 ) .
The s e n s i t i v i t y r a n g e i s 40 p a r t p e r 1 09 , t h e method
is s u c c e s s f u l l y used f o r d e t e c t i o n of e s t r o g e n s and
i n meat p r o d u c t s . I t i n v o l v e s e x t r a c t i o n of l i v e r ,
kidney and muscle t i s s u e i n a c e t o n i t i l e : water ( 9 : l ) .
The drug w a s c o n v e r t e d t o i t s t r i m e t h y l s i l y l d e r i v a -
t i v e f o r a n a l y s i s on a column packed w i t h 2%0V-17
on Chromosorb G and coupled t h r o u g h a Watson-Biemann
H e s e p a r a t o r t o t h e mass s p e c t r o m e t e r .
6.6 B i o l o g i c a l Assays
Heinert, i d e n t i f i e d hexestrol i n milk using the

mouse u t e r i n e weight b i o a s s a y method ( 3 9 ) . Rennet
HEXESTROL 371
c o a g u l a t i o n of t h e m i l k p e r m i t t e d d e t e c t i o n of
0.001 ppm of h e x e s t r o l which could a l s o be d e t e c t e d
i n c h e e s e a f t e r c o a g u l a t i o n of m i l k . L i e m - e t-
a1 (40)
d e s c r i b e d a b i o l o g i c a l a s s a y of e s t r o g e n i c s u b s t a n c e s
i n cosmetic i n c l u d i n g h e x e s t r o l , based on a p p l i c a t i o n
of t h e cosmetic p r o d u c t s t o t h e shaven s k i n of
c a s t r a t e d female mice; v a g i n a l smears were t a k e n
subsequently f o r a n a l y s i s .
Acknowledgements
The a u t h o r s wish t o t h a n k M r . Khalid N.K. Lodhi

f o r h i s technical a s s i s t a n c e i n determining t h e
NMR s p e c t r a of h e x e s t r o l and M r . Uday C. Sharma
f o r t y p i n g t h e manuscript.
References
1. a) R e m i n g t o n ' s P h a r m a c e u t i c a l S c i e n c e s , #ack
P u b l i s h i n g Co., E a s t o n , P a . , p. 920 ( 1 9 7 5 ) .
b) Merck I n d e x , N i n t h e d i t i o n , Merck & Co. , I n c . ,

Rahaway, N . J . , U.S.A., p . 615, 4571 ( 1 9 7 6 ) .
2. D i c t i o n a r y of o r g a n i c compounds Vol. 2 , E y r e S. S p o t t i s
woods - (London) P u b l i s h e r s L t d . , E . & F. M. Spon L t d . ,
New York, p. 1036 ( 1 9 6 5 ) .
3. E.G.C. C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n of Drugs",

The P h a r m a c e u t i c a l P r e s s , London, 1 9 6 9 , p. 363.
4. S. B e r n s t e i n and E.S. Wallis, J . Amer. Chem. S O C . ,

-
62, 2871, ( 1 9 4 0 ) .
5. The P h a r m a c e u t i c a l Codex, E l e v e n t h E d i t i o n , t h e
P h a r m a c e u t i c a l Press, London, p . 4 1 1 ( 1 9 7 9 ) .
6. N.R. Campbell, E . C . Dodds and W . Lawson, N a t u r e ,

142, 1 1 2 1 ( 1 9 3 8 ) .
-
7. E . C . Dodds, R . Robinson, e t a l . ( a ) N a t u r e , 141, 247

(1938), (b) P r o c . Roy. SOC. (London), B 7 , 1 4 0 (1939).
8. N . R . Campbell, E.C. Dodds and W. Lawson, P r o c . Roy.

SOC. (London), B 8 , 253 ( 1 9 4 0 ) .
9. M.S. K h a r a s c h and M. Kleiman, J . Amer. Chem. SOC.

65, 491,
- (1943).
10. B.N. La Du, H.G. Mandel and E . L . Way, "Fundamentals

of Drug Metabolism and Drug D i s p o s i t i o n " , The
W i l l i a m s & W i l k i n s Co., B a l t i m o r e , MD., 1 9 7 2 , p.263.
11. A.L. H e r b s t , H. U l f e l d e r and D . C . P o s k a n z e r ,

N e w Engl. J . Med., 284, 878 ( 1 9 7 1 ) .
12. A.L. H e r b s t , R . J . Kurman and R . E . Scully, Obstet.

GynecFl. , 9, 287 ( 1 9 7 2 ) .
13. a) J.A. McLachlan and R.L. Dixon, New C o n c e p t s i n

S a f e t y E v a l u a t i o n , ( e d i t e d by M . A . Mewhlman,
R.E. S h a p i r o and H. B l u m e n t h a l ) , Vol. 1, P a r t 1,
p . 423, Hemisphere, Washington ( 1 9 7 6 ) .
HEXESTROL 373
b) M. M e t z l e r , R. G o l t s c h l i c h and J . A . Mc L a c h l a n ,
E s t r o g e n s i n t h e Environment, ( e d i t e d b y J . A .
McLachlan), p. 293, Elsevier (1980).
14. I . C a l a f e t e a n u , E. Dumitrescu and P. G r i n t e s c u ,

Revta Chim., G, 41, (1965); t h r o u g h Anal. A b s t r . 13,
3786 (1966).
15. I. K e r e n y l , Acta Pharma. hung., 34, 261, ( 1 9 6 4 ) ,

t h r o u g h Anal. A b s t r . -
1 3 , 1980, ( 1 9 6 6 ) .
16. V.G. B e l i k o v , Med. Prom. SSSR, -

1 7 , 32, (1963).
17. The A d d i t i v e s i n Animal F e e d i n g - s t u f f s Sub-

committee of t h e A n a l y t i c a l Methods c o m m i t t e e of t h e
S o c i e t y f o r A n a l y t i c a l C h e m i s t r y . R e p o r t of t h e
Hormones P a n e l . A n a l y s t , 88, 925, ( 1 9 6 3 ) .
18. M.J. Matherne, J r . , J . A s s . o f f . a g r i c . chem.,

48, 613,
- (1965).
19. L. I. Kovdlenko, F a r m a t s i y a , g,
5 4 , (1969) ; t h r o u g h
Chem. A b s t r . -
7 2 , 59121e, (1970).
20. S.L. Tompsett, J. Fharm. Pharmacol. -

13, 747, ( 1 9 6 1 ) .
21. S.L. T o m p s e t t , J . Pharm. Pharmacol. -

1 6 , 207, (1964).
22. H. Van Waes, Rev. Agr. ( B r u s s e l s ) , 23, 1 1 3 5 , ( 1 9 7 0 ) ;

t h r o u g h Chem. A b s t r . , -
74, 632312, ( 1 9 7 1 ) .
23. R. Verbeke, J . Chromatogr., 1 7 7 , 6 9 , (1979); t h r o u g h

Chem. A b s t r . , 21, 206599q, (1979).
24. H. J . S t a n and F.Y. H o h l s , Z. Lebensm. - Unters .

F o r s c h . , 1 6 6 , 257, (1978); t h r o u g h Anal. A b s t r . -
35,
6F16, (1978).
25. H. Jare, 0. R u t t n e r , W. Kroeza, F l e i s c h w i r t s c h a f t ,

86, 3 6 3 1 t ,
56, 1326, ( 1 9 7 6 ) ; t h r o u g h Chem. A b s t r . ,
-
(1977).
I . Karkocha, Rocz. Fanstw. Z a k l . H i g . , 26, 497,

(1975); t h r o u g h Chem. A b s t r . , 8 4 , ?368w, ( 1 9 7 6 ) .
R. F e r r a n d o and A. R e n a r d , B u l l . SOC. Chim. b i o l . ,

50, 1855 ( 1 9 6 8 ) .
-
314 HASSAN Y. ABOULENEIN ET AL.
28. T. L a r s , J . Chromatgr., 48, 560, (1970).
29. H. J a r c , 0. R u t t n e r , and \J. Krocza, J . Chromatogr.,

-
1 3 4 , 351, (1977).
30. H.C. H s i n , J a p a n A n a l y s t , 23, 1 2 2 6 , (1974); t h r o u g h

Anal. A b s t r . -29, 2E16, (1975).
31. A.V. J a i n , E.D. S c h o l l , J . Assoc. O f f . Anal. Chem.,

-
58, 818, (1975).
32. F . J . Lawrence and J . J . Ryan, J . Chromatogr., 130,

97, (1977).
33. H. Ehrsson, T. Walle and H. B r o e t e l l , A c t a . Pharm.

Suec., -
8 , 319, (1971).
34. M. G a b r i e l l a . C. Guido and S. P a t r i z i a , A n n a l i 1st. Sup.

Sanita, - 5, 586, ( 1 9 6 9 ) ; t h r o u g h Anal. A b s t r . , 20,
2051;
(1971).
35. P . J . Cooper, M.J. d e F a u b e r t Maunder and G . J .

McCutcheon, A n a l y s t , 92, 382, ( 1 9 6 7 ) .
36. C.G.B. F r i s c h k o r n , M.R. Smyth, H.E. F r i s c h k o r n and

J . Golimowski. F r e s e n i u s ' Z. Anal. Chem. - 300, 407 (1980).
37. M.R. Smyth, C.G.B. F r i s c h k o r n , F r e s e n i u s ' 2. Anal.

Chem. -
301, 220 (1980).
38. G . H o e l l e r e r and D. J a h r , Z. L e b e n s m i t t e l u n t e r s .
U. - F o r s c h . , 157, 65, ( 1 9 7 5 ) ; t h r o u g h Anal. A b s t r .
- 4 D 83, (1975).
29,
39. H.H. H e i n e r t , Arch. L e b e n s m i t t e l h y g . , 26, 1 5 2 , ( 1 9 7 5 ) ;

t h r o u g h Chem. A b s t r . 8 3 , 162274h, (1975).
40. D.H. Liem, L.G. H u i s i n ' t Veld, G . J . R u n d e r v o o r t ,

J. R o o s e l a a r , J. Ten Have, J. SOC. Cosmet. Chem., 27,
307 (1976); t h r o u g h Chem. A b s t r . , -
8 8 , 41529q (1978).
MESTRANOL
Hum& A . El-Obeid and Abdulluh A. Al-Badr
1. Description 376
1.2 Formulae 316
1.5 Appearance 371
2.1 Crystal Data 377
2.2 Melting Point 377
2.3 Solubility 311
3. Synthesis 385
4. Absorption, Metabolism, and Excretion 387
5.2 Spectrophotometric Methoas 391
5.3 ChromatographicMethods 391
References 404
Analytical Profiles 01Drug Subslancea Copyright 01982 by The American

ISBN 0-12-260811-9
376 HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
1. Description
1.1 Nomenclature
1.11 Chemical Names
Ethinyloestradiol-3-methyl ether.
1 7 a-Ethnylestradiol-3-methyl ether
3-Plethoxy-19-nor-17 a-pregna-1,3,5(10)-trien-20-
yn-17-01.
19-Norpregna-1,3,5(1O)-trien-2O-yn-l7-ol,3-
methoxy- (17a)-
17a-Ethynyl-3-methoxy-1,3,5(lO)-estratrien-l7~-01.
17~-Ethynyl-3-methoxyoestra-ly3,5(lO)-trien-l7-ol.
17~t-Ethynyl-1,3,5(10)-estratriene-3, 17B-diol-3-
methyl ether.
1.12 Generic Name
Mestranol.
1.13 Trade Names
Mestranol is an ingredient of the following pro-

pietary oral contraceptive preparations:
Conovid, C-Quens, Enavid, Enovid, Metrulen,
Norinyl, Previson, Ortho-Novum, Ovanon, Ovulen,
Sequens, Syntex Menophase.
1 . 2 Formulae
1.21 Empirical
C21H2602
1 . 2 2 Structural
OH
MESTRANOL 377
1.23 CAS No.
72-33-3
310.42
C 81.25%, H 8.44%, 0 10.31%.
1.5 Appearance
White crystalline powder.
2.1 Crystal Data
Crystal data were reported by Ohrt et a1 (1) for some

esterone-related compounds. The data given for mes-
tranol was : a 6.998, b 39.737, c 6.8718., B117.58',
Pz1, 2 = 4.
2.2 Melting Point
Melts between 146' and 1 5 4 O with a range of 4'(2,3).
2.3 Solubility
Almost insoluble in water, soluble 1 in 44 of ethanol,

1 in 23 of ether, and 1 in 4.5 of chloroform, 1 in 12
of dioxane, 1 in 23 of acetone, slightly soluble in
methanol ( 2 , 4 , 5 ) .
2.4 Identification
2.41 Infrared Spectroscopic Test
B.P. (4) and U.S.P. XIX (2) make use of the in-
frared absorption spectrum of mestranol as me n
of identification of the drug. The infrared ab-
sorption spectrum of the sample exhibits maxima
which are only at the same wavelengths as, and
have similar relative intensities to, those in
the spectrum of a standard mestranol. The infra-
red spectrum of mestranol will be discussed later
in the spectral properties of the drug.
2.42 Ultraviolet Spectroscopic Test
U.S.P. X I X requires that for the identification

o f the drug, the ultraviolet absorption spectrum
of methanolic solution exhibits maxima and minima
at the same wavelengths as that of a similar
solution of a standard mestranol, concomitantly
measured. The ultraviolet spectrum of mestranol
will be discussed later in the spectral proper-
ties of the drug.
2.43 Thin Layer Chromatographic Test
B.P. and U.S.P. XIX describe a thin layer chro-

matographic method for the identification of mes-
tranol in which the principal spot in the chro-
matogram of the substance being examined is
compared with that of mestranol obtained under
identical conditions.
2.44 Color Test
According to B.P. a solution of mestranol in sul-

furic acid appears orange-red by transmitted
light, shows a yellowish-green fluorescence by
reflected light and produces a reddish-brown f l o -
cculent precipitate after addition of ferric
ammonium sulfate solution and a rose-red floccu-
lent precipitate when water is added.
2.45 Solubility Test
B.P. uses a solubility test to distinguish mes-

tranol from ethinylestradiol. The former is
insoluble in a 5 % wfv solution of potassium
hydroxide.
2 . 5 1 Ultraviolet Spectrum
The ultraviolet absorption spectrum of mestranol

obtained from a solution in neutral methanol in
the region of 200 to 350 nm using a Varian Cary
219 spectrophotometer is shown in Figure I. Two
absorption maxima at about 2 1 8 and 286 nm and
MESTRANOL 379
J
c
c
CI
F i g . 1. U l t r a v i o l e t spectrum of m e s t r a n o l i n n e u t r a l
methanol.
two minima at about 246 and 284 nm were observed.

Reported (5) ultraviolet absorption spectrum of
mestranol in methanol exhibited two maxima at
279 nm (E 1%, 1 cm 82) and 287.5 nm (E 1%,lcm
14.4).
2 . 5 2 Infrared Spectrum
The infrared spectrum of mestranol is presented in

Figure 2 . The spectrum was obtained from nujol
mull using a Unicam SP 3-300 infrared spectro-
photometer. The spectral assignments are presen-
ted in Table 1.
Table 1. Infrared spectral assignments for mestranol.
Band Frequencyl Structural assignment

Wavenumber cm
3500 0-H stretching.

3 300 ECH stretch.
1510, 1620 -C=C-stretch of the aro-
matic ring.
1460 C-H Deformation of -CH2-
1380 -C-H Deformation of -C-CH
3
1300 C-0-C stretch.
65 3 32-H Deformation.
2 . 5 3 Proton Magnetic Resonance (PIR) Spectrum.
The PMR spectrum of mestranol is presented in

Figure 3 . The sample was dissolved in CDCl and
3
the spectrum obtained on a Varian-T60A NMR
spectrometer with tetramethylsilane as the inter-
nal standard. The spectral assignments shown in
Table 2 , are in agreement with reported studies
(6) *
W.Vd#@P- 9 10 11 12 13 (4I S U
3 4 5 5 6 7
100
w
u
7u
60
50
44
34
20
10
Fig. 2. I n f r a r e d spectrum of mestranol; Nujol M u l l .

do0 200 ti 0 wa
axyl -H
F i g . 3. PMR spectrum of m e s t r a n o l i n CDC13 w i t h TMS i n -

t e r n a l standard
MESTRANOL 383
Table 2. PMR spectral assignments for mestranol.
Chemical Shift. Multiplicity Number of Species

PPd6) Protons.
0.92 singlet 3 -CH3

2.12 singlet 1 -OH
2.55 singlet 1 acetylenic
proton.
3.72 singlet 3 methoxy
protons.
6.87 multiplet. 3 Aromatic
protons.
2.54 I3C - Nuclear Magnetic Resonance (13C NMR)

Spectrum
The I3C NMR spectrum of mestranol in CDCl using

3
tetramethylsilane as an internal standard ref-
erence was obtained on a Jeol FX 100, 100 MHz
instrument at an ambient temperature using a lorn.
sample tube. The spectrum is presented in
Figure 4 and the chemical shift values, derived
from the off-resonance spectrum, is shown in
Table 3.
Table 3. I3C NMR spectral assignments for mestranol.
Carbon Chemical shift. Carbon Chemical shift.

No. No. (ppm)
1 126.2590 13 47.2199
2 113.9788 17 79.9181
3 157.5908 18 12.7185
4 111.5932 20 87.7616
5 137.9037 21 73.9221
10 132.6407 CH3-0- 55.2115
2.55 Mass spectrum and Fragmentometry
The mass spectrum of mestranol, obtained by elec-

tron impact ionization, using Nermag GC-Mass
spectrometer model R 1010, is presented in Figure
5. The spectrum shows a molecular ion PI? at m/e
310 (relative intensity 24.4%) and a base peak
384
Fig. 4.
J
1 3 C NMR spectrum of mestranol i n CDC13 with TMS
i n t e r n a l reference.
11
Fig. 5. Mass spectrum of mestranol (EI).

MESTRANOL 385
at mfe 227. Based on the data interpretation and

summary (INTSIJM) program presented by Smith et al,
(7) for the possible fragmentation of the basic
skeleton of estrogenic steroids, a proposed frag-
mentation pattern of mestranol is shown in
Table 4.
Table 4 . Proposed mass fragmentation pattern

of mestranol.
mle
- Relative intensity % ion
-
311 6.3 M+I
310 24.4 M+
242 15.7 C16H18d +

228 19 ‘1bH20q +
227 100
C15H150? -t
160 10
159 14.6
147 20.5
145 11.9 Cl0H93 +

129 20.7
128 26.8
116 13.9
3. Synthesis
Mestranol was prepared by Colton et al. (8) as f o l l o w s :

Estrone {I} i s converted to its 3-methoxy analog (11) by
reaction with methyl sulfate. The ethynyl group may then
be introduced at position 1 7 either through reaction with
sodium acetylide in liquid ammonia followed by hydrolysis
o f the sodoxy compound, or through Grignardization with
ethynyl magnesium bromide. Almost the sole product of the
ethynylation reaction is that which results from attack of
reagent from the least hindered a-side of the steroid,Fig.6.
3 86 HUMEIDA A. EL-OBEID AND ABDULLAH A. AL-BADR
3
t I11
&Br
J
&’
TECH &CH t
0
HCEC N&iq .NH3
ONa
CH30 CH30
Figure 6. Synthesis of mestranol.

MESTRANOL 387
4. Absorption, Metabolism and Excretion

Mestranol is a synthetic estrogen which is more potent than
estradiol. It is readily absorbed from the gastrointesti-
nal tract and is slowly metabolized and excreted in urine
and feces. The absorption, metabolism and excretion of
the drug has been extensively studied in animals and
humans I Fig. 7.
Wijmenga and Van der Molen (9) reported a biological half-

life of mestranol of 50 hours and that a small proportion
of the drug was excreted in milk of nursing mothers.
Mills et al, (10) showed that after the injection of tri-

tium-labelled mestranol to women, the radioactivity rep-
resenting the metabolites of the drug in the blood dis-
appeared with an average half-life of 45 hours (range
37-65 hours). Mahesh - al, (11) studied the metabolic
et -
clearance rate and blood half-life of mestranol. The me-
tabolic clearance rate of the drug after a single i.v.
injection to women was 1265Llday. The radioactivity half-
life after injection was 45.1 hours. No change in the
metabolic clearance rate of the drug was noticed for upto
7 months of oral contraceptive medication. Using the con-
stant infusion technique, Bird & Clark (12) measured the
metabolic clearance rate of mestranol in normal young
women and found it to be 1741L124 hours. The mean conver-
sion ratio for mestranol to ethynylestradiol was 0.236 and
to ethynylestradiol sulfate 3.369; that of mestranol to
the sulfate was 6.476. The mean transfer constant for
mestranol to ethynylestradiol was 0.182. The principal
circulating form of the drug was ethynylestradiol sulfate.
Mills - a1 (13) administered mestranol orally to normal

et -
women in order to measure the metabolic clearance rate
and to study the urinary excretion of the drug. Mestranol
was rapidly cleared from the plasma with an average meta-
bolic clearance rate of 1247Llday. About 30% of mestranol
administered was excreted in urine in 5 days, less than 4%
in an unconjugated form, 10% as sulfate conjugates and
about 52 as glucuronide conjugates. Comparison of the
metabolic clearance and urinary excretion rates made
before and again after six cycles of treatment with Ortho-
Novum SQ containing mestranol showed that prolonged admi-
nistration had no effect on the metabolic clearance rate,
rate of urinary excretion or mode of conjugation of the
drug or its metabolites.
In a study by Bolt and Remmer (14) following the i.v. ad-

ministration of 14C-Labelled mestranol to female rats; 45
and 3% of the radioactivity were found to be excreted in
the feces and urine, respectively, in 3 days. Only 2 . 5 %
of the radioactivity was expired as 14CO2 within 4 days
indicating no significant degradation of the steroid nuc-
leus. Some metabolites separated from fecal extracts had
an unaltered 3-methoxy group. Others, however, were de-
methylated to derivatives of ethynylestradiol.
The chronic i.v. injection of radioactive mestranol into

mice resulted, as reported by Bolt and Remmer ( 1 5 ) , in an
accumulation of radioactivity in organs to a greater ex-
tent than activity accumulated after radioactive estradiol
administration. The metabolites of mestranol were tightly
bound to the liver tissue and were not removed by solvent
extraction or by acid hydrolysis. The high demethylation
rate of mestranol in mice as compared to that in rats may
be due to a high activity of the microsomal oxidase in the
mouse liver. Following a single i.v. injection of the
steroid into female rats, 1.5% of the radioactivity was
recovered from urine collected in the first 3 days, and
55% of the activity was excreted in the feces within 3
weeks.
Hanasono and Fischer (16) studied the excretion of tritium-

labelled mestranol and other contraceptive steriods and the
enterohepatic circulation (EHC) of their metabolites in
female rats. Mestranol was rapidly and extensively elimi-
nated as metabolites in the bile after a single i.v. dose.
The cumulative percentage of administered radioactivity
appearing in the bile at the end of 8 hours was 6 9 % . Mes-
tranol appeared in the bile primarily as a glucuronide
conjugate and other polar materials which were not sulfate
conjugates. Intact female rats given single i.p. doses of
the labelled steroid eliminated radioactive metabolites in
the urine and feces at a much slower rate than that seen
in the bile of animals with biliary fistulas. Fecal excre-
tion was the major route of elimination in intact animals
for the steroid and accounted for 80% or more of the radio-
active dose by the end of 7 days. Experiments were also
conducted to assess the enterohepatic circulation of meta-
bolites of the steroid. 59% of the intraduodenally infused
radioactivity associated with the biliary metabolites of
mestranol, underwent enterohepatic circulation and appeared
in the bile during a 24 hour period. The glucuronide con-
jugate fraction of biliary metabolites was the most impor-
tant fraction undergoing enterohepatic circulation. The
MESTRANOL 389
T Glucuronide and
sulfate conjugates
17a-Ethynylestradiol
H O
HO 1
@PCH
2-Hydroxy-17a-ethynyles-
tradiol.
OH 0
17a-D-Homoestradiol D-Homoestrone
Figure 7. Metabolism of mestranol.

polar metabolites, which could not be hydrolysed by 6-glu-

curonidase, were not absorbed from the intestine and
re-excreted in the bile. The authors conclude that the
enterohepatic circulation is an important feature of the
disposition of the contraceptive steroid in rats and is
dependent on the excretion of glucuronide conjugates of
the steroid metabolites in the bile.
In a study using radioactive mestranol, Williams (17)

identified 17 a-ethynylestradiol as a urinary metabolite
using reverse isotope dilution technique.
Abdel-Aziz and Williams (18), studying the urinary metabo-

lites of mestranol in guinea pigs, identified 17 a-ethynyl-
esteradiol conjugates, D-homoestradiol-176 and D-homoes-
trone, as metabolites.
3 14
In their study of the metabolism of 4- H and 4- C-mestra-
no1 by women, Williams - et -
a1 ( 1 9 ) , reported that reactions
involving position 4 were no greater than 1.7 to 3% of the
dose as measured by the liberation of 3H into body water.
The extent of deethynylation in vivo was no greater than
1 to 2% of the dose as measured by urinary estrone metabo-
lites. Mestranol (0.7 and 0.32% of the dose), 17a-ethynyl-
estradiol ( 6 . 6 and 11.3%) and 2-hydroxy-17a-ethynylestra-
diol ( 0 . 6 4 and 0.7%) were identified as metabolite glycons
by reverse isotope dilution after ketodase hydrolysis of
the urine from two of the women.
(a) The B.P. ( 4 ) recommended a titrimetric method in

which the drug in tetrahydrofuran is treated with
silver nitrate and then titrated with standard
sodium hydroxide solution. The end point deter-
mined potentiometrically.
(b) An argentimetric determination of some acetylene

steroidal drugs, including mestranol, had been
developed by Roushdi _
et _
a1 (20). The assay method
depends on the precipitation of the steroid sil-
ver salt from alcoholic ammoniacal silver nitrate
solution followed by extraction with a suitable
organic solvent, evaporation of the solvent and
then applying Volhard's method.
MESTRANOL 391
(c) Roushdi et a1 (21) used a nonaqueous titrimetric

method for determination of acetylenic steroids.
The method involves the precipitation of the
steroid silver salt using ammoniacal silver nit-
rate solution. The salts extracted will chloro-
form or ether and titrated with perchloric acid in
acetic acid using Gentian Violet as indicator.
5.2 Spectrophotometric Methods
5.21 Ultraviolet Spectrometric Methods
a) Bastow (22) deternined mestranol from its ab-

sorption at 280 nm after elimination of the
interfering ketonic absorption by reduction
with borohydride. Residual interference was
allowed for by a three point correction.
b) Gorog and Csizer (23) described methods for the

determination of (i) mestranol as an impurity in
norethynodreal and norethisterone and (ii)
mestranol in contraceptive tablets. For (i)
the sample is dissolved in methanol and treated
with sodium borohydride. A solution of mestra-
no1 is treated sinilarly. The extinctions of
the two solutions are measured at 287, 290.5
and 294 nm, and the mestranol content is calcu-
lated from a given equation. For (ii) the pow-
dered tablets are heated under reflux with di-
chloromethane. After filtration and evapora-
tion of the solvent, mestranol is determined as
in (i). The mean recovery for the latter
method was 98.3 ? 2.3%.
c) A specific spectrophotometric method for the

determination of 17-ethynyl steroids was pub-
lished by Szepesi and Gorog (24). 17a-Ethynyl
steroids were determined by quantitative con-
version to 17-0x0 steroids by sodium t-but-
oxide at 81°, followed by spectrometric deter-
mination of the 16-glyoxalyl derivatives, Anlax.
294 nm, E 10,700, standard deviation 1.1%.
0
By the same principle, at 0 , the 17-0x0
impurity content in 17-ethynyl steroids was de-
termined. The method was not applicable to
+OX0 steroids.
392 HUMEIDA A. EL-OBEID AND ABDULLAH A. ALBADR
d) Shroff and Grodsky ( 2 5 ) described an ultravio-

let spectrophotometric method for the estima-
tion of mestranol in fresh tablets. In this
method the sample is shaken with water and me-
thylcyclohexane, centrifuged and the extinction
of the upper phase measured at 287.7 nm. Base-
line corrections applied are calculated from
(i) the extinction at 302 nm and (ii) the ex-
tinctions at 278 nm and 302 nm and the extinc-
tion ratio E to E for pure mestranol;
278 287.7
the two corrected values of for the
E287. 7
sample should agree within 2 0.006%. The con-
tents of mestranol is calculated from the mean
corrected value. The coefficient of variation
for ten samples is 1.14%. The method is not
preferred for aged tablets because on storage
an interfering complex is formed between poly
(vinyl pyrrolidone) and magnesium stearate ex-
cipients.
5.22 Colorimetric Methods
a) Chlorine o-tolidine reagent was used by

Huettenrauch ( 2 6 ) for the selective detection
of steroids with aromatic ring A and C13-OH
group whether this was free, esterified or
ether-linked. Mestranol gave positive reac-
tion with sensitivity of 1-2 pg/cc.
b) Shroff and Huettemann ( 2 7 ) developed a colori-

metric method for assaying mestranol in tab-
lets. The method is based on the formation
of a colored complex with phenol-sulfuric acid
reagent. This complex exhibits an absorption
maximum of 550 nm, obeys Beer's Law and is
stable for reasonable length of time. Accord-
ing to the method the tablets are moistened
with water and shaken with methylcyclohoxane.
An aliquot of the organic phase is evaporated
in an atmosphere of nitrogen and treated with
phenol-sulfuric acid reagent and the extinc-
tion measured at 5 5 0 nm against a reagent
blank. The error and precision are 2 1 . 6 % and
1 . 5 2 % respectively. The presence of nor-
ethisterone and excipients do not interfere.
MESTRANOL 393
The method is claimed to be superior to the

ultraviolet spectrophotometric method describ-
ed by Shroff and Grodsky and summarized above.
c) A direct colorimetric method for the assay of

mestranol in one tablet is reported by Comer
et -
- a1 (28). In this method one tablet is dis-
solved in sulfuric acidlmethanol (7:3) and the
extinction measured at 545 nm against a rea-
gent blank. To avoid interference by chlor-
madinone, the temperature must be kept below
5 O during the color reaction. The coefficient
of variation was 1 . 0 5 % . Very similar methods
have also been described by Templeton et a1
(29) for one tablet assay and by Wu (30) for
mestranol in combination with ethynodiol dia-
cetate. The latter procedure require prior
chromatographic separation of mestranol.
d) Beyer (31) developed an automated colorimetric

procedure for the quantitation of mestranol in
tablets according to which the tablets are sus-
pended in water and extracted with chloroform-
ethanol. In an automatic system, re-extraction
is effected with 10% ethanol solution in 90%
sulfuric acid. The extinction is measured at
538 nm. The coefficient of variation is 21%.
e) Rizk et a1 (32) determined mestranol colorime-

trically by means of silver ions. An ethanolic
solution is treated with aqueous ammonia and an
excess of aqueous silver nitrate and the pre-
cipitated silver compound is filtered off.
Excess silver ions in the filterate is deter-
mined by a dithizone spectrophotometric pro-
cedure and the amount of steroid is calculated
from the silver ions consumed.
5 . 2 3 Infrared Spectroscopic Methods
a) Mestranol was determined in chloroform solution

-1
by infrared spectrophotometry (33) at 3308 cm
-1
(Ethynyl vibrations) or 1 5 0 2 cm (aromatic
system vibrations).
b) Chatten et a1 (34) reported a quantitative

assay of antifertility agents by infrared spec-
troscopy. Two pure estrogenic substances and
eight pure progestogens were analysed in the
selected wavelength range and shown to obey
Beer-Lambert Law. Although progestogen ana-
lysis in compound tablets was accomplished in
several cases using the technique, the estro-
genic content in all compound tablets was too
low t o permit analysis.
c) Beyermann and Roder (35) analysed mestranol in

oral contraceptives by infrared spectrophoto-
metry after separation of the components by
thin-layer chromatography. The separation was
performed on silica gel with ethyl acetate as
a solvent. The appropriate spot extracted with
chloroform and the infrared spectrum in liquid
paraffin mull was recorded and mestranol deter-
mined from its extinction at 7.95 um.
5 . 2 4 Spectrofluorometric Methods
a) Cullen et a1 ( 3 6 ) developed a sensitive proce-

dure for the analysis of norgestrel and struc-
turally related steriods based on sulfuric acid
induced fluorescence. The selectivity of the
reaction and mechanism of fluorescence forma-
tion were studied. The reaction is specific
for A4 -3-ketosteroids which have both a 17B-
hydroxy and a 17a-alkyl or alkyne substitution
and A 1y3y5(10)-triene-3-01 steroids. A two-
step mechanism was tentatively explained on the
basis of the effects of temperature, time, ini-
tial acid concentration and subsequent dilution
with water on fluorogen development. The pro-
cedure has been automated to permit unit
dose analysis.
b) A spectrofluorometric procedure for the deter-

mination of mestranol in some oral contraceptive
tablets had been developed (37) which utilizes
the native fluorescence of mestranol. The
method is claimed to be rapid, reliable and
sensitive. No separations are required and the
method is applicable in the presence of norethy-
nodrel, ethynodiol diacetate and norethisterone,
MESTRANOL 395
but chlormadinone acetate interferes. The

method is applicable to single-tablet analysis.
According to the procedure one powdered tablet
is shaken with anhydrous ethanol and centrifu-
ged. An aliquot of the supernatant is diluted
with ethanol and the fluorescence at 327 nm
(excitation at 284 nm) is compared with that of
a standard solution. Anhydrous ethanol is used
as a reference solution and a correction appli-
ed for its fluorescence. The accuracy under the
conditions studied was 2 1.29% and the preci-
sion ranged from 2 0.896 to ?r 1.73.
c) Mariani and Mariani-Vicari (38) also described

a method for the assay of mestranol spectro-
fluorometrically. A sulfuric acid-methanol
(1:l) reagent is used and a previous separa-
tion of the two components (ethynylestradiol
and mestranol) in the compound preparation is
not required. For tablets the hydrolysis of
the starch by diastase is followed by extrac-
tion into chloroform. The wavelengths of emi-
ssion and excitation spectra are 515 nm and
475 nm respectively.
d) An automated fluorimetric method for one-tablet

assay of mestranol was carried out by Comer et
a1 (28). One tablet i s dissolved in sulfuric
-
acid-methanol (7:3) and the fluorescence mea-
sured with Kodak Wratten N0.58 ,& Corning 1-60
primary filters and a Kodak Wratten 22 second-
ary filter. A scheme for automation of the
fluorimetric method is illustrated. In twelve
replicate experiments, the coefficient of vari-
ation was 1.30%. The fluorescence reaction is
retarded by NO NO2 and H 0 Related ster-
3’ 2 2’
oids, such as estradiol methyl ether, esterone
methyl ether and esterone, cause little
interference.
e) Templeton et a1 ( 2 9 ) described a fluorimetric

assay method of mestranol in which one tablet
is allowed to disintegrate in 20% sodium hydr-
oxide solution and water, then extracted with
chloroform and an aliquot of the extract eva-
porated to dryness. The residue dissolved in
50% methanol solution in concentrated sulfuric
acid and the fluorescence measured at 498 nm,
with excitation at 468 nm.
f> A fluorimetric assay method for the determina-

tion of mixture of mestranol and norethynodrel
was reported by Pastor et a1 (39). For mestra-
no1 alone extracts (in 1 : 5 chloroform-methanol)
were evaported to dryness, dissolved in ethanol
cooled, and allowed to react with a mixture of
1 : 3 acetic acid-sulfuric acid at room tempera-
ture before measuring the fluorescence emission
at 560 nm after excitation at 545 nm. When
norethynodrel was present with mestranol, the
extracts were treated with borohydride in
methanol at room temperature before addition of
concentrated hydrochloric acid and processing
as above to estimate mestranol. Norethynodrel
was estimated in the mixture, from borohyd-
ride and acetic acid-sulfuric acid treatment,
by excitation at 485 nm and emission at 520 nm.
Variation in measurements were 3% for mestranol
and 2% for norethynodrel.
g) Mestranol in oral contraceptive tablets was also

determined fluorirnetrically ( 4 0 ) by disintegrat-
ing a tablet in dilute hydrochloric acid, ext-
racting with methylene chloride, treating with
0.2% hydroquinone (in 70:30 sulfuric acid-
ethanol) and measuring the fluorescence.
h) Dusinsky and Radejova (41) reported fluorime-

tric methods for the determination of mestranol
and some other estrogenic hormones. Methods,
based on the determination of 'native' fluore-
scence and that 'induced' by sulfuric acid,
for determining mestranol, ethinylestradiol,
estradiol benzoate and valerate and the total
estrogenic hormone content in mixtures of
natural conjugated estrogens are described. The
methods are claimed to be simple, highly sen-
sitive and rapid. There is no need €or pre-
liminary clean-up or hydrolysis.
5.25 Nuclear Magnetic Resonance (NMR) Spectrometric

Methods
Avdvich et al(6) used an NMR technique to quanti-

tate mestranol bulk drug. Diphenylacetic acid was
used as the internal standard and pyridine as the
solvent. The amount of mestranol was calculated
from the integrals of the peaks at 6.33 ppm
MESTRANOL 397
(methoxyl protons) and at 6.80 ppm (ethynyl proton

de-shielded by pyridine). The average deviation
was ? 0.6% and the results were in good agreement
with those obtained by an official method.
The literature describes several thin-layer chro-

matographic methods for the separation and analy-
sis of mestranol in mixtures and contraceptive
tablets. For quantitation, the plate is first
scraped and mestranol eluted before treatment with
a suitable reagent. Table 5 symmarizes thin-
layer chromatographic methods for mestranol.
a) Quantitative separation of progestins and es-

trogens from anovulatory formulations had been
performed (57) using gel filteration on a syn-
thetic polysaccharide (Sephade LH-20) and the
compounds were determined directly by U.V.
spectrophotometry.
b) Erunner and Kunze (58) published an analytical

method for mestranol in combination with nore-
thisterone (norethinadrone) or norethynodrel
by partition chromatography and ultra-violet
measurement. According to this procedure oral
contraceptive tablets were extracted with di-
methylformamide-formamide (1:l) and the ster-
oids separated by partition chromatography
with use of this solvent as stationary phase
on Celite with heptane as mobile phase. Mes-
tranol, in the relevant fraction, is deter-
mined by measuring the extinction at 287 nm
with base-line correction obtained from the
extinction at 302 and 315 nm. Interference
was noticed with ethynodiol diacetate and
chlormadinone acetate. The method had been
adopted as official first action after a
collaborative study was conducted (59).
c) A method was described (60) for the separation

of steroids in mixtures of pharmaceutical in-
terest by means of silicic acid column chro-
Table 5: Thin-Layer Chromatographic systems for mestranol analysis.
Stationary Phase Developing solvent Visualization Quantitative Reference
Silica Gel or 0.5% Vanillin 42

Neutral Allumina H2S04-EtON -
Kieselgel G Cyclohexane/EtoAc - Treat with 40% SbCl in

(7:3) HOAC measure extincgion 43
at 570 nm or fluoresence.
Silica Gel G/H Ether/Cyclohexane Iodine vabors Treat with 15% TiCl in 44
3
(8:2) RoAc-H2S04, measure
extinction at 530 nm.
W
W
00
Propylene glycol- Toluene, Cyclohexane- 20% p-MeC H4S03H - 45
impregnated Kaie- toluene ( 3 : 2 ) or pet- in 947: E t h , heat
selguhr G. roleum ether. at 120 for 10 min.
Paraffin oil-imp- 30,50 or 70% HoAC Same as above. - 45

regnated solutions.
-Kieselguhr G.
Silica Gel Benzenelethyl methyl - Treat with H SO -i"ieOH(2:1)46

2 3
ketone (9:l). measure extinction at
540 run.
contd......
Stationary Phase Developing solvent Visualization Quantitative Reference
Xieselgel G/HR
a) 1-Dimensional EtoAc/cyclohexane/Me2C0 85X Phosphoric- Use Icieselgel FIR 47,43
(5:1.5:2 ) MeOE (1:1) or Treat with 15% TiCl -
SbCl -EoAc (1:l conc. HC1. Pleasure 3
w/v>? heat at extinction.
b) 2-Dimensional Above solvent followed 12Oo-13O0.
by Cyclohexane/EtoAc or Iodine vabors. 47,48
(23:27).
Silica Gel GF 2-Dirfiensional :

Chloroform/methanol (9:l) H SO. and heat for Visual comparison of 49
4
or Benzene/acetone(95:1) 36 minutes at 1
00'. of the spots sizes
followed by benzene/ UV irradiation. and color intensi-
methanol (95:5) or ties with standard
roethylene chloride/raethanol spots.
/water (150:9:0.5)
Kiesel G Benzene Iodine vabors - 50
Silica gel G Heptane/acetone(4:1) EtOII-conc. HCl(49 :1) Extract in propanol 51

plus H SO -H 0(7:3)
2 4 2
measure extinction
at 545 nm.
Silica gel - HC1 gas and W - 52
irradiation.
contd... ....
Stationary phase Developing solvent Visualization Quantitative Reference
Silica gel G, Petroleum ether/benzene/ Iodine vabors, 2,4- - 53
A1 0 GF254 and He2C0 (5 :4 :1) dinitro-phenylhydra-
2 3 zine or 2% H SO in
Silica gel GF 2 4
254 EtOIi.
Silica gel G-MiJ Petroleum et'ner/?{e CO

2
50% H2S04 54
Silica gel 60 F254 2-Dimensional:
Toluene-95Z/ethanol(9:1) H SO 55
followed by BuOAcfLight 2 4
petroleum/AcOE(70:30:1)
Silica gel GF BenzenefEtoAc (4:l) 0.5% H3 SeO -conc. By spectrodensitome- 56

H SO and Aeat at try by scanning at
04 360 nm by reference
100 for 15 min.
to an internal standard.
MESTRANOL 401
matography. Association of estrogenic with

progestational steroids, in sone cases exten-
ded to androgens, were considered. The elution
was performed with a gradient of ether in pet-
roleum ether, obtained by means of two metering
pumps. The effluent was monitored continuously
by means of a hydrogen flame ionization detec-
tor, a part of the effluent being continuously
drawn off t o the detector. The quantitative
analysis of the steroids separated by the
method was performed by a U.V. spectrophoto-
metric or a colorimetric procedure.
5.33 Liquid Chromatography
Hara and Hayashi (61) studied the correlation of

retention behaviour of steroidal pharmaceuticals
in polar and bonded reverse-phase liquid column
chromatography. For the systematization of cor-
relation between the chemical structures of solu-
tes and retention behaviour in liquid column chro-
matography, the retention volume of the modified
steroids on silica (Corasil 11) and chemically
bonded reverse-phase columns (Bondapak C18/Corasil)
were studied using various binary solvent systems.
Retention parameters for the functional groups of
the steroids were calculated according to Martin's
additive rule. By comparing these values obtained
on normal and reverse-phase columns, characteristic
features of both packings with regard to solute
structures and solvent systems were elaborated.
5 . 3 4 Gas Chromatography
a) A comparison of an W spectrophotometric assay

and a gas-liquid chromatographic assay was
conducted (25) for mestranol in tablets. Both
methods gave results in good agreement and both
are suitable for fresh tablets. In the gas-
chromatographic method the column used was
stainless steel (21 in x 0.25 in) of 4% of
XE-60 on Diatoport S, maintained at 195
0
. Ni-
trogen was used as a carrier gas at 70-75 m l /
hour. The method is preferred for the assay of
aged tablets because on storage a complex bet-
ween poly (vinylpyrolidone) and magnesium
stearate excipients was produced which inter-
fered with the W methods and was identified by
gas-liquid chromatography.
b) Okuno and Higgins (62) reported a method for

the determination of residues of mestranol and
ethynylestradiol in foliage, soil and water
samples. The lower limits for the detection of
mestranol and its 3-hydroxy homolog, ethynyl-
estradiol were 0.05, 0.1 and 0.01 ppm for foli-
age, soil and water respectively. Samples were
extracted in an acid medium to free any conju-
gated ethynylestradiol and then cleaned up by
Florisil column chromatography. Water samples
were directly analysed by gas chromatography
using a flame ionization detector and a column
packed with OV 17 on Gas Chrom Q. Operating
0
temperatures were 260 for column, 275'for the
inlet and 290' for the detector. Further
cleaning up of the soil and vegetation samples
was carried out by gel permeation and Florisil
chromatography. Analysis was then carried out
by gas chromatography. The lower limit of
detection was lOOng for each steroid.
c) Mestranol and norethisterone were determined

in estrogen-progestin contraceptive tablets
containing both components (63) by extraction
with ethyl acetate and a single gas chromato-
graphy, with testosterone propionate as inter-
nal standard. The recoveries of mestranol and
norethisterone were 98.54 and 99.14% respec-
tively and the precision was 1.40 and 1.74%
respectively. The procedure could be applied
to single tablets containing 0.05 mg mestranol
and 1 mg norethisterone.
d) Templeton et a1 (29) performed a gas-liquid

chromatographic assay for mestranol. A tablet
is allowed to disintegrate in sodium hydroxide
in water, then extracted with chloroform. A
dilute cholestane solution is used as an inter-
nal standard. The column used is a glass
column (2 ft. x 4 mm) containing Diatoport S
(80 to 100 mesh) impregnated with 6%0of sili-
cone-gum rubber W-96 operated at 205 , with
He (60 to 70 ml per min.) as carrier gas and
flame ionization detector.
MESTRANOL 403
ACKNOWLEDGEMENT
The a u t h o r s wish t o t h a n k Mr. A l t a f Hussain Naqvi, f o r

t y p i n g t h i s manuscript. A sample of m e s t r a n o l was k i n d l y
donated by S e a r l e Research and Development, D i v i s i o n of G.D.
S e a r l e and Co., Chicago, I l l i n o i s , USA.
REFERENCE
1. J.M. Ohrt, B.A. Haner and D.A. Norton, Acta Cryst., 1 7 ( 2 ) ,

1 6 1 1 (1964).
2. The United-States Pharmacopeia, 19th ed., Mack Publishing
Co., Easton, Pa., p. 1405 (1975).
3. Remington's Pharmaceutical Sciences, 15th ed., Mack Pub-
lishing Co., Easton, Pennsylvania p.920 (1975).
4 . British Pharmacopoeia, Her Majesty's Stationery Office,
London, U.K., p. 291 (1973).
5 . E.G.C. Clarke, Isolation and Identification of Drugs, The
Pharmaceutical Press, London, p . 4 0 5 (1969).
6 . E.W. Avdovich, M. Bowron and B.A. Lodge, J. Pharm. S c i . ,
59(12), 1 8 2 1 (1970).
7. D.H. Smith, B.G. Buchanan, I?.C. White, E.A. Feigenbaum,
J. Lederberg and C. Djerassi, Tetrahedron, 2, 3117 (1973).
8 . F.B. Cotton, L.N. Nysted, B. Reigel and A.L. Raynold,
J. Amer. Chem. Soc., 79, 1 1 2 3 (1957).
9. H.G. Wijmenga and H.J. van der Molen, Acta endocr.,Copenh.,
61, 665 (1969), per
- , 209, 2106 (1969).
10. T.M. Mills, T.J. Lin, W.E. Braselton, J.E. Ellegood and
V.B. Mahesh, Am. J. Obstet. Gynecol., 1 2 6 ( 8 ) , 987 (1976).
11. V.B. Mahesh, T.M. Mills, T . J . Lin, J . O . Elegood and W. E.
Braselton, Pharmacol. Steroid Contracept. Drugs, 117,30
(1977).
12. C.E. Bird and A.F. Clark, J. Clin, Endocrinol. Metab.
3 6 ( 2 ) , 296 (1973).
13. T.M. Mills, T.J. Lin, S . Hernandez-Ayub, R.B. Greenblatt,
J . O . Ellegood and V.B. Mahesh, Am. J. Obstet. Gynecol.,
1 2 0 ( 6 ) , 773 (1974).
1 4 . H.M. Bolt and H. Remmer, Xenbiotica, 20, 77 (1972).
1 5 . H.M. Bolt and H. Remmer, ibid, 2 ( 5 ) , 489 (1972).
1 6 . G.K. Hanasono and L.J. Fischer, Drug Hetab. Dispos.,
2 ( 2 ) , 159 (1974).
1 7 . K . I . Williams, Steroids, 1 3 ( 4 ) , 539 (1969).
18. M . T . Abdel-Aziz and K . I . H . Williams, J . Drug Res., 60,
195 (1974).
1 9 . J.G. Williams, C. Longcope and K . I . H . Williams, Steroids,
2 5 ( 3 ) , 343 (1975).
20. I.M. Roushdi, A . I . El-Sebai and S . Belal, Egypt. J. Pharm.
Sci., 1 5 ( 1 ) , 8 1 (1974).
-
21. I.M. Roushdi, A . I . El-Sebai and S. Belal, ibid, 1 5 ( 2 ) ,
217 (1974).
22. R.A. Bastow, J. Pharm. Pharmacol., 1 9 ( 1 ) , 4 1 (1967).
23. S. Gorog and E. Csizer, Acta Chim. (Budapest), 6 5 ( 1 ) ,
41(1970).
24. G. Szepesi and S. Gorog, Analyst, 9 9 ( 1 1 7 7 ) , 218 (1974).
MESTRANOL 405
25. A.P. S h r o f f and J . Grodsky, J . Pharm. S c i . , 5 6 ( 4 ) , 460

(1967).
26. R. H u e t t e n r a u c h , P h a r m a z i e , 2 0 ( 1 1 ) , 740 (1965).
27. A.P. S h r o f f a n d R.E. Huettemann, J . Pharm. S c i . , 5 6 ( 5 ) ,
654 (1967).
28. J . P . Comer, P . Hartsaw a n d C.E. S t e v e n s o n , J . Pharm. S c i . ,
5 7 ( 1 ) , 147 (1968).
29. R . J . Templeton, W.A. A r n e t t and I . M . J a k o v l j e v i c , u,
5 7 ( 7 ) , 1168 (1968).
30. J.Y.P. Wu, J . Assoc. O f f . Anal. Chem., 5 8 ( 1 ) , 75 (1975).
31. W.F. Beyer, J . Pharm. S c i . , 5 7 ( 8 ) , 1415 (1968).
32. M. R i z k , J.J. V a l l o n and A. Badinand, A n a l y t i c a chim.Acta,
6 5 ( 1 ) , 220 (1973).
33. E . Pawelczyk, I. P l u t a and B. M a r c i n i e c , Acta P o l . Pharm.,
3 0 ( 3 ) , 289 (1973).
34. L,T.- C h a t t e n , E . J . T r i g g s and S . J . Glowach, J . Pharm. Belg.
3 1 ( 1 ) , 6 3 (1976).
35. K. Beyermann and E. Roder, Z. A n a l y t . Chem., 2 3 0 ( 5 ) , 347
(1967).
36. L . F . C u l l e n , J . G . R u t g e r s , P a . A. L u c c h e s i and G . J . Papa-
r i e l l o , J . Pharm. S c i . , 5 7 ( 1 1 ) , 185 (1968).
37. J . Theron, J . Pharm. S c i . , 4 9 ( 1 1 ) , 1648 (1970).
38. A. M a r i a n i and C. Mariani-Vicari, Z e n t r a l b l . Pharm. Phar-
makother. L a b o r a t o r u i m s d i a g n . , 111(1),1 9 (1972).
39. J . P a s t o r , A.M. P a u l i and N. P a p o c c h i a , Trav. SOC. Pharm.
i v l o n t p e l l i e r , 3 3 ( 3 ) , 411 (1973).
40. J . H . M . Miller and P. Duguid, P r o c . Anal. Div. Chem. SOC.,
1 3 ( 1 ) , 9 (1976).
41. G . Dusinsky and E. R a d e j o v a , Farm. Obz., 4 6 ( 5 ) , 225 (1977).
42. J . S . Hathews, Biochim. Biophys. Acta, 69, 1 6 3 (1963).
43. R. H u e t t e n r a u c h and I. K e i n e r , P h a r m a z z , 2 0 ( 4 ) , 242 (1965).
44. D. H e u s s e r , Dent. Apotheker-Ztg., 1 0 6 ( 1 3 ) , 4 1 1 (1966).
45. D. S o n a n i n i and L. Anker, Pharm. Acta Helv., 4 2 ( 1 ) 54
(1967).
46. P. Horak, Cesk. Farm., 1 6 ( 5 ) , 232 (1967).
47. E. Roder, Dent. Apotheker-Ztg., 1 0 7 ( 2 9 ) , 1007 (1967).
48. I. Szekacs a n d M. Klembala, Z . Klin. Chem. Klin. Biochem.,
8 ( 2 ) , 1 3 1 (1970).
49. M.B. Simard and B.A. Lodge, J . Chromatogr., 5 1 ( 3 ) , 517
(1970).
50. H. Sachweh, P r o c . Conf. Appl. Phys. Chem., 2nd., 1,289
(1971). E d i t e d by Buzas, I l o n a . Akad. Kiado: B u d a p e s t ,
Hung.
51. A. G r o c h u l s k i , Acta P o l . Pharm., 2 8 ( 2 ) , 185 (1971).
52. J. J e f f e r y , J . Chromatogr., 5 9 ( 1 ) , 216 (1971).
53. S. Enache, Rev. Chim., 2 4 ( 5 ) , 648 (1973).
54. B. M i j a t o v i c , L j . R i s t o v i c and J . G r u b o r , Arch. Farm.,
2 3 ( 2 ) , 9 1 (1974).
406 HUMEIDA A. EL-OBEIDAND ABDULLAH A . AL-BADR
55. G. Cavina, G . Moretti and M. P e t r e l l a , J . Chromatogr.,

1 0 3 ( 2 ) , 268 (1975).
56. A.P. Shroff and C . J . Shaw, J . Chromat. S c i . , 1 0 ( 8 ) , 509
(1972).
57. A. Alvarez Fernandez and N.V. T o r r e , J . Pharm. S c i . , 5 8 ( 6 ) ,
740 (1969).
58. C.A. Brunner and F.M. Runze, J . A s s . O f f i c . Anal. Chem.,
53(2).
. . _ 234 (1970).
59. C.A. Brunner, J . A s s . O f f i c . Anal. Chem., 54(3), 590
(1971).
60. G . Cavina, G . M o r e t t i , A. M o l l i c a and R. A n t o n i n i , J.,
Chromatogr., 60 ( 2 ) , 1 7 9 (1971).
61. S. Hara and S. Hayashi, J. Chromatogr., 142, 689 (1977).
62. I. Okuno and W.H. H i g g i n s , B u l l . Environ. Contam. T o x i c o l . ,
1 8 ( 4 ) , 428 (1977).
63. G. M o r e t t i , G . Cavina, G . C h i a p p e t t a , I. F a t t o r i , M.
P e t r e l l a and V . Pompi, B u l l . Chim. Farm., 1 1 6 ( 8 ) , 463
(1977).
NOSCAPN
Mohammed A. Al-Yahya and Mahmoud M . A . Hassan
1. Description 408
1. I Nomenclature 408
1.2 Formulae 408
2.1 X-Ray Diffraction 414
2.2 Solubility 415
3. Preparation 429
3.1 Isolation from Opium 429
4. Synthesis of Noscapine 429
4.1 Tissue Culture Method 429
4.2 Chemical Methods 429
5 . Biosynthesis of Noscapine 429
6. Metabolism 43 3
7.2 Microcrystal Tests 436
7.4 Complexometric Methods 441
8. References 45 6
Copyight 0 1982 by The AmdUn

Analytical Profilesof Drug Substances
Volume 1 I 407 phmn.ce~iiulAmciation
ISBN 0-12-260811-9
408 MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
1. D e s c r i p t i o n
1.1 Nomenclature
a- (S)-6,7-Dimethoxy-3-(5, 6, 7 , 8-
tetrahydro-4-methoxy-6-methyl-l ,
3-d i o x o l o [ 4,5-g] i s o q u i n o l i n - 5 - y l ) - l
(3 H) -isobenzof uranone. (1)
b- 1-a -2-methyl-8-methoxy-6,7-methylene
dioxy-1- ( 6 , 7-dimethoxy-3-phthalidyl) -
1, 2 , 3 , 4-tetra-hydroisoquinoline.
(1)*
c- 1 (3 H) Isobenzofuranone, 6 , 7 -
dimethoxy-3-(5, 6, 7 , 8-tetrahydro-4-
methoxy-6-methyl-1, 3-dioxolz-[4, 5-g]
isoquinolin-5-y1)-, [s-(R* s ) I . (2)
d- (3 S)-6, 7-dimethoxy-3-[(5R)-5, 6, 7 ,
8-tetra-hydro-4-methoxy-6-methyl-1,-
3-dioxolo [ 4 , 5-g] isoquinolin-5-y1]
phthalide. (3).
1.1.2 Generic Names
d-Gnoscapine; 1-6-Narcot i n e ; I-Narcot i n e ;

N a r c o t i n e ; Noscapine.
1.1.3 Trade Names
Capval; Coscopin; Coscotabs; Key-

t u s s c a p i n e ; Longatin; Lyobex; Fethoxy-
h y d r a s t i n e ; Narcompren; Narcosine;
Marcotussin; N e i t a c l o n ; Nicolane;
Nipaxon; Noscapal; Noscapalin; NSC 5366;
Opian; Opianine; Terbenol; Tusscapine;
Vadebex.
1.2 Formulae
1.2.1 Empirical
NOSCAPINE 409
1.2.2 Structural
OCH3
Nos c a p i n e
The a l k a l o i d n o s c a p i n e can be c l e a v e d
v e r y r e a d i l y i n t o two m o i e t i e s ; w i t h d i l u t e
s u l f u r i c a c i d , c o t a r n i n e and o p i a n i c a c i d a r e
g e n e r a t e d . Under a c i d i c r e d u c i n g c o n d i t i o n s ,
e.g., zinc i n hydrochloric acid o r s u l f u r i c
a c i d , h y d r o c o t a r n i n e and meconine a r e formed
(Scheme 1 ) . With t h e s t r u c t u r a l e l u c i d a t i o n
of c o t a r n i n e , o p i a n i c a c i d , h y d r o c o t a r n i n e
and meconine, and g i v e n t h e p r e s e n c e of a
l a c t o n e r i n g i n n o s c a p i n e , the structure o f
t h i s a l k a l o i d was e s s e n t i a l l y e s t a b l i s h e d ( 4 ) .
1.2.3 CAS No
(12 8- 62 - 1)
1.2.4 Niswesser L i n e N o t a t i o n
T C566 DO FO K N EH & & T J H

01 K J T56 B VO D H J HOL 1
0 1 "ALPHA" LV.
1.2.5 S t e r e o c h e m i s t r y and A b s o l u t e C o n f i g u r a t i o n
T h e s t e r e o c h e m i s t r y of n o s c a p i n e
h a s been s t u d i e d by many w o r k e r s (5-9). The
prolonged a c t i o n of h o t m e t h a n o l i c p o t a s s i u m
h y d r o x i d e on n a t u r a l (-)- a - n a r c o t i n e r e s u l t s
i n t h e f o r m a t i o n of a n e q u i l i b r i u m m i x t u r e
of t h e o r i g i n a l b a s e and a new o p t i c a l l y
'2
OCH3
'n' Noscapine
OCH 3 OC!I
Hydrocotarnine Cotarnine
+ +
CRO
OCH OCH
Meco n ine Opianic acid
Scheme 1
NOSCAPINE 41 1
a c t i v e d i a s t er eoisomer , (-) -6 -narc0 t i n e ,

which can be w r i t t e n a s shown i n Scheme 2.
Lithium aluminum h y d r i d e r e d u c t i o n of t h e
a- and 8-noscapines r e a d i l y a f f o r d s a-
narcotinediol 1 and 6 - n a r c o t i n e d i o l 2
respectively .
A c e t y l a t i o n of t h e s e d i o l s g i v e s r i s e t o
t h e corresponding d i a c e t a t e s 2 and k , b u t
subsequent c a t a l y t i c h y d r o g e n o l y s i s y i e l d s
one and t h e same d e x t r o r o t a t o r y benzyl-
i s o q u i n o l i n e 2. The f o r e g o i n g sequence
c l e a r l y e s t a b l i s h e s t h a t a- and 8-noscapine
must d i f f e r from each o t h e r o n l y i n t h e i r
s t e r e o c h e m i s t r y a t C-9.
The b e n z y l i s o q u i n o l i n e 5 shows a p o s i t i v e
Cotton e f f e c t near 295 m u , s o t h a t i t s C-1
hydrogen must b e a l p h a as i n d i c a t e d . I t
f o l l o w s t h a t t h e C-1 hydrogen i n (-)-a-
n a r c o t i n e and i n (-)- 6 - n a r c o t i n e must a l s o
be a l p h a (Scheme 2 ) .
A 1 t e r n a t i v e l y , a-narco t ined i o l w a s c y c l i z ed
v i a i t s monomesylate d e r i v a t i v e t o t h e N-
m e t h o t e t ra h y d r o p r o t o b e r b e r i n e s a l t 5. T h i s
material underwent N-demethylation on p y r o l y s i s
t o y i e l d t h e p r o t o b e r b e r i n e b a s e 7. Reductive
removal of t h e hydroxyl group w a s achieved
i n e t h a n o l i c p e r c h l o r i c a c i d o v e r a palladium
c a t a l y s t . The t e t r a h y d r o p r o t o b e r b e r i n e 8 t h u s
o b t a i n e d showed a s t r o n g n e g a t i v e r o t a t i o n , so
t h a t i t s C-14 hydrogen must be a l p h a .
The i d e n t i c a l sequence w a s c a r r i e d o u t
using p n a r c o t i n e d i o l t o y i e l d t h e tetra-
h y d r o p r o t o b e r b e r i n e b a s e ?. Hydrogenolytic
c l e a v a g e of t h i s s p e c i e s t h e n provided t h e
same 1evor o t a t o r y t e t r a hyd r o pro t o b er b er i n e 8.
The c o n c l u s i o n i s t h a t t h e C-1 hydrogens i n
b o t h a- and 6-noscapine a r e a l p h a (Scheme 3 ) .
Turning t o t h e s t e r e o c h e m i s t r y a t C-9 f o r
(-)-a - and (-)- B-narcotine, m o l e c u l a r models
i n d i c a t e d t h a t t h e d i h e d r a l a n g l e between t h e
p r o t o n s a t C-13 and C-14 of t h e 13-a-hydroxy
412 MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN
CH3
OCH3
(-)-a-Narcotine (-) -6-Narcotine
( n a t u r a l isomer)
1
--
LiA1H4
1L1A1H4
OCH~ 1
- 2
a-Narcotinediol B-Narcotinediol -
1. Ac 0, pyridine
1
--
-
Ac 20 , P Y ~
idine
OAc
CH20Ac
OCH3
0CH3
Scheme 2
NOSCAPINE 413
H ........OH pyridine
-
6
@CH3
d-Narcotinediol
7
-
as o u t l i n e d
9
-
OCH3
B-Narcotinediol
Scheme 3
b a s e 7 i s a b o u t 160'. On t h e o t h e r hand,
f o r the 13-6 -hydroxy b a s e 2 d e r i v e d from
6 - n a r c o t i n e , t h i s a n g l e is o n l y a b o u t 60'.
Following exchange of t h e h y d r o x y l i c p r o t o n s
f o r d e u t e r i u m , i t w a s determined t h a t t h e
s p l i t t i n g c o n s t a n t .J13,14 w a s 9 Hz f o r
species 1, and o n l y a b o u t 1 . 5 Hz f o r 9.
The l a r g e c o u p l i n g v a l u e of 9 Hz i s iz accord
w i t h a t r a n s arrangement of t h e C-13, 1 4
hydrogens i n I_, and t h e s m a l l c o u p l i n g
c o n s t a n t of 1 . 5 Hz a r g u e s f o r a c i s r e l a t i o n -
s h i p i n 9, t h u s s e t t l i n g t h e s t e r e o c h e m i s t r y
a t C-9 f& a- and 8-noscapine.
413.43
1.4 E l m e n t a l Composition
C, 63.91%; H, 5.61%; N , 3.39%; 0 , 27.09%
1.5 Appearance, C o l o r , Odor and Taste
Noscapine o c c u r s i n t h e form of Orthorhombic

b i s p h e r o i d a l p r i s m s , t a b l e t s from d i a c e t o n e o r
a s f i n e , almost w h i t e c r y s t a l l i n e powder.
T r i b o l u m i n e s c e n t d 1.395. I t i s o d o r l e s s and
tasteless.
2.1.1 X-ray d i f f r a c t i o n
Crystallographic d a t a f o r noscopine are

s c a r c e . The o n l y r e p o r t e d d a t a is due t o
Love11 (10) and Steward and P l a y e r (11).
These a r e a s f o l l o w s :
Long needle-shaped c r y s t a l s were o b t a i n e d by
r e c r y s t a l l i s a t i o n of t h e commercial n o s c a p i n e
from e t h a n o l o r methanol. Weissenberg
photographs t a k e n w i t h Cu Ka (1.5418 A)'
r a d i a t i o n revealed the following systematic
absences:
hOO, h = 2n + 1
OKO, K = 2n -I-1
001, 1 = 2n + 1
NOSCAPINE 415
d e f i n i n g unambiguously t h e space group

P212121. C e l l dimensions were o b t a i n e d
from 28 v a l u e s of 32 r e f l e x i o n s from n o s c a p i n e
u s i n g two a x e s i n each c a s e , measured w i t h a
counter diffractometer. The f o l l o w i n g d a t a
were o b t a i n e d :
M.W. 413.41
M.p. (OC) 178
C r y s t a l system O r t hor homb i c
r
Space group
P212121
Cell 15.398(12)
Dimensions b 32.686(36)
(8) c ( p r i m ) 8.022(8)
d 3 ) 4037(11)
z 8
Qcalc (g. ~ m - ~ ) 1.360
.
Qexp (g ~ m - ~ ) 1.38
2.1.2 Melting P o i n t
174-176OC(3)
176OC s u b l i m e s a t 15OoC-16O0C under 11 mm
p r e s s u r e a t 2 mm d i s t a n c e (1)
2.2 Solubility
I t i s i n s o l u b l e i n water; s l i g h t l y s o l u b l e i n
a l c o h o l ( 9 5 % ) , i n e t h e r and i n carbon t e t r a c h l o r i d e .
S o l u b l e i n chloroform, benzene and v e r y s o l u b l e i n
a c e t o n e (12).
2.3 D i s s o c i a t i o n Constant
I t i s a v e r y weak b a s e , pKa 7.8 ( 1 ) and

4.85 i n 80% m e t h y l c e l l o s o l v e ( 1 3 ) .
[a], + 42' to + 48'

(2% w/v i n 0 . 1 M h y d r o c h l o r i c
a c i d ) (3)
[ u I D - 198O (1% w/v i n c h l o r o f o r m ) ,
[a], - 146 (2% w/v i n t o l u e n e ) ,

[a], - 147' (1.59% i n b e n z e n e ) ,
[a], + 50 (1%w/v i n h y d r o c h l o r i c a c i d ) (14)
2.5.1 U l t r a v i o l e t Spectrum
The W spectrum of n o s c a p i n e i n methanol

w a s scanned from 200 t o 400 nm u s i n g V a r i a n
Carry 119 Spectrophotometer. I t e x h i b i t s a
c h a r a c t e r i s t i c UV spectrum ( F i g . 1) w i t h
two maxima:
1%
Xmax
cm
310.2 114.9 (C, 9.42 mg p e r 100 ml)
290.6 106.4 (C, 9.42 mg p e r 1 0 0 ml)
Other UV s p e c t r a l d a t a of n o s c a p i n e have
a l s o been r e p o r t e d :
Xmax Log E
209 4.86 )
291 3.60 ) i n e t h a n o l (1, 15)
309-310 3.69 )
Xmax (E)
291 a b o u t 1.1 )
310 a b o u t 1.4 ) i n a l c o h o l 95% (3)
291 3981 1
3 09 4898 ) i n methanol (12)
2.5.2 I n f r a r e d Spectrum
The I R s p e c t r a of n o s c a p i n e a s K B r d i s c
and n u j o l m u l l were r e c o r d e d on a P e r k i n
E l m e r FT-680B s p e c t r o p h o t o m e t e r and shown in
F i g . 2 t?, F i g . 3 respectively. The s t r u c t u r a l
a s s i g n e m e n t s have been c o r r e l a t e d w i t h t h e
f o l l o w i n g band f r e q u e n c i e s (Table 1)
NOSCAPINE 417
Fig. 1. W Spectrum of N o s c a p i n e i n M e t h a n o l .
Wavelmgt h F
*O 5.0 6.0 70 8.0 s.0 f0 12 !4
Fig. 2. I R Spectrum of Noscapine as K B r d i s c .

1 .
2500 2000 800 700
Fig. 3 . I R Spectrum of Noscapine as Nujol Mull.

420 MOHAMMED A . AL-YAHYA A N D MAHMOUD M. A . HASSAN
T a b l e 1. I R C h a r a c t e r i s t i c s of Moscapine
-1
Frequency c m Assignement
3000, 2945, 2880, Methylened i o x y and

2845, 2800 C-H and -CH frequencies.
3
17 60 ( y - l a c t o n e ) 3-C=O g r o u p
1625 -c=c-
1600,1505,1480, Aromatic
1280-1 22 5 Aromatic m e t h o x y - a r y l C-0
stretching vibrations.
790, 815, 8 3 5 , 885 2 a d j a c e n t H atoms, i s o l a t e d
H atom C-H o u t of p l a n e
d e f o r m a t i o n . Tetra and
.
pen t a s u b s t i t u t e d b e n z e n e s
Other c h a r a c t e r i s t i c a b s o r p t i o n bands are:

1460, 1430, 1405, 1 3 9 0 , 1 3 8 0 , 1 3 6 5 , 1 3 3 0 , 1310, 1 2 0 0 ,
1120, 1 0 8 5 , 1040, 1 0 1 0 , 980, 930, 900, B O O , 765, 750,
735, 725, 715, and 700 cm-'.
O t h e r I R d a t a a r e a l s o r e p o r t e d (16)
2.5.3 N u c l e a r Magnetic Resonance S p e c t r a
2 . 5 . 3 . 1 P r o t o n Spectrum
The PMR s p e c t r u m of n o s c a p i n e i n
d e u t e r a t e d chloroform w a s r eco r d ed on
a V a r i a n XL200, 2 0 0 MHz NMR s p e c t r o -
meter u s i n g t e t r a m e t h y l s i l a n e a s a
r e f e r e n c e s t a n d a r d ( F i g . 4 ) . The
following s t r u c t u r a l a s s i g n m e n t
h a v e been made ( T a b l e 2 ) .
I,
0
7 6 5 4 3 2
F i g . 4. PMR Spectrum of Noscapine and T e t r a m e t h y l s i l a n e i n Deuterated Chloroform.

422 MOHAMMED A . AL-YAHYA AND MAHMOUD M.A. HASSAN
Table 2. PMR C h a r a c t e r i s t i c s of Noscapine
Ass ignemen t (Group) Po s i t i o n Chemical S h i f t ( 6 )
-CH2-CH2 3 , 4 of i s o q u i n o l i n e 2.32 (m)

N- CH3 2 of i s o q u i n o l i n e 2.53 ( s )
OCH3 8 of i s o q u i n o l i n e 3.84 ( s )
OCH3 &'of p h t h a l i d y l 4.02 (s)
OCH3 5'of phthalidyl 4.08 ( s )
-CH- 1 of i s o q u i n o l i n e 4.37 (d)
-CH- 9 of p h t h a l i d y l 5.55 (d)
-CH2- methylened i o x y 5.92 ( s )
-CH- 2'0f phthalidyl 6.05 (d)
-CH- 5 of i s o q u i n o l i n e 6.29 ( s )
-CH- 3'of phthalidyl 6.94 (d)
s = s i n g l e t , d = doublet, m = multiplet
Other PMR s p e c t r a l d a t a was a l s o
r e p o r t e d ( 1 7 an.d 5 5 ) .
2.5.3.2 I3C-NMR Spectra
I3C-NMR c o m p l e t e l y decoupled and

o f f - r e s o n a n c e s p e c t r a are shown i n
Fig. 5 and Fig. 6 r e s p e c t i v e l y . Both
were r e c o r d e d o v e r 11001.1 HZ r a n g e ,
i n d e u t e r a t e d c h l o r o f o r m (CDC13) on
XL-200,200 MHz NMR s p e c t r o m e t e r .
Using 10 mm sample t u b e and tetra-
methylsilane as reference standard
a t 25OC.
The c a r b o n chemical s h i f t s a s s i g n e d
on t h e b a s i s of t h e a d d i t i v i t y p r i n c i -
p a l s and o f f - r e s o n a n c e s p l i t t i n g
p a t t e r n (Table 3) (18).
9 a
Fig. 5.
7 6 5 4
1 3 2 f
l3C-NMR Spectrum of Noscapine i n Deuterated Chloroform.

0
1
3
424
50 60 70 80 90 100 110 120 130 1 4 0 150 160 170 180
1 1 1
190 200 210 220 230 240 250 260 270 280 290 300 310
T 1
1
2
320 330 340 350 360 370 380 390 400 410 420 430 440 4 5 0
Fig. 7. EI-Mass Spectrum of Noscapine.

426 MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN
’
OCH3
22
Table 3. Carbon Chemical S h i f t s of Noscapine
Carbon No. Chemical S h i f t Carbon No. Chemical S h i f t

PPm Ppm
c-1 60.84 (d) c-12 46.29 (q)

c- 2 49.99 (t) C-13 81.83 (d)
c-3 28.03 (t) C-14 120.17 (s)
c-4 134.03 (s) C-15 118.19 (d)
c-5 117.65 (d) C-16 102.29 (d)
C-6 140.45 (s) C-17 147.67 (s)
c-7 100.73 (t) C-18 148.37 (s)
C-8 141.14 (s) c-19 117.11 (s)
c- 9 152.18 (s) c-20 168.06 (s)
c-10 132.09 (s) c-21 56.78 (4)
c-11 59.36 (q) c-22 62.22 (q)
2.5.4 Mass Spectrum
The mass spectrum of n o s c a p i n e obtained

by e l e c t r o n impact i o n i z a t i o n and which w a s
recorded on Ribermag R-10-10 mass spectrometer
equibbed w i t h d i r e c t i n l e t probe. The spectrum
( F i g . 7) shows m o l e c u l a r i o n peak and shows
a b a s e peak a t m / e 220.
The mass spectrum of n o s c a p i n e o b t a i n e d by

butane chemical i o n i z a t i o n ( F i g . 8) shows a
m o l e c u l a r i o n peak PI+ a t m / e 413 w i t h a
r e l a t i v e i n t e n s i t y of 2.8% and a b a s e peak
a t m / e 220. The most prominent f r a g m e n t s ,
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2t
1
290 300 31 0 320 330 340 350 360 370 380 390 4-00410 420 430 440 450 460 470
Fig. 8. CI-Mass Spectrum of Noscapine.
t h e i r r e l a t i v e i n t e n s i t i e s and s t r u c t u r e s
a r e l i s t e d i n Table 4 .
Other mass s p e c t r a l d a t a f o r p h t h a l i d e i s o -
q u i n o l i n e s was a l s o r e p o r t e d (19, 2 0 ) .
Table 4 . Mass Fragments of Moscapine
m/e Relative Intensity 96 Fragment
413 2.8 M+
221
220
195 8.0
OH
193
OCH3
NOSCAPINE 429
3. P r eDara t i o n
3.1 I s o l a t i o n from Opium
Noscapine o c c u r s up t o 11%n a t u r a l l y i n
opium (Papaver sornniferum L . (Fam. papaveraceae) .
I t w a s f i r s t d i s c o v e r e d by Derosne i n 1803 ( 2 0 ) ,
and i s o l a t e d by Robinquet i n 1817 ( 2 1 ) .
Noscapine can be s e p a r a t e d from o t h e r opium
a l k a l o i d s by t h e procedure o u t l i n e d i n Scheme 4 (90).
Another p a t e n t method h a s been a l s o d e s c r i b e d

f o r i t s i s o l a t i o n on a n i n d u s t r i a l scale ( 2 2 ) .
4. S y n t h e s i s of Noscapine
4.1 By T i s s u e C u l t u r e Method
Khanna e t a 1 (23) d e s c r i b e d a method f o r t h e

s y n t h e s i s of noscapine a l o n g w i t h o t h e r a l k a l o i d s
by t i s s u e c u l t u r e of Papaver somniferum Linn.
4.2 By Chemical Methods
P e r k i n and Robinson (24) d i s c o v e r e d t h a t h e a t i n g

a m i x t u r e of c o t a r n i n e 1 and meconine 1i n e t h a n o l
r e s u l t e d i n a s m a l l y i e l d of noscapine 3 .
The expected second isomer of noscapine-(because
of t h e presence of 2 a s s y m e t r i c c e n t r e s ) w a s n o t
found. The s y n t h e t i c noscapine was t h e n
r e s o l v e d and t h e n o s c a p i n e o b t a i n e d shown
t o b e i d e n t i c a l w i t h t h e n a t u r a l p r o d u c t . (Scheme 5).
A q u i t e e f f i c i e n t s y n t h e s i s of noscapine
w a s developed by Hope and Robinson i n 1914 ( 2 5 ) , i n
which c o t a r n i n e L i s condensed w i t h iodomeconine 2
and t h e adduct w a s reduced w i t h sodium amalgum t o
g i v e t h e d e s i r e d p r o d u c t , corresponding t o t h e
n a t u r a l s e r i e s (Scheme 6 ) .
5. B i o s y n t h e s i s of Noscapine
I t has been p o s t u l a t e d t h a t t h e ph t h a l i d e i soqu i n o 1i n e s

a r e formed i n n a t u r e by o x i d a t i v e m o d i f i c a t i o n of t e t r a -
h y d r o p r o t o b e r b e r i n e s , and p r e v i o u s work w i t h l a b e l e d
precursors supports t h i s hypothesis (26).
Powdered Op i um
+
+
Shake w i t h warm calcium c h l o r i d e s o l u t i o n
F i 1t e r
Insoluble matter C F i ltrate
(discard) (hydroch l o r i des o f a 1ka l o i ds)
Reduce volume (evaporate under

4
reduced p r e s s u r e ) t o syrupy 1 i q u i d
4
Add 10% NaOH s o l u t i o n
Precipitates p k a l ine solution

(noscapine, papaverine, thebaine) (morphine, codeine,
4
Dissolve i n d i l u t e a l c o h o l E x t rn
aac rt cw
e i nt he ) ci h l o r o f o r m
$.
Add a c e t i c a c i d t o make s l i g h t l y 0
C h 1 o r o f orm
Aqueous
acidii
Add 3 volumes o f b o i l i n g water
extract
(contain-
alkaline
solution
A i n g codeine) (morphine,
Precipitate Solution
(papave r ine, (theba ine) 4 narceine)
4
noscap ine)
4 Further
purification Make a c i d i c
-
4
\ Further
pur i f ic a t i o n
Dissolve i n b o i l i n g
Aqueous a c i d i c
s o l n . ( s a l t s of
morphine and
rz 1
0.33%(aqueous)oxali c a c i d na r c e i ne)
soln.
4
A l l o w t o stand
4
Make s l i g h t l y
a l k a l i n e w i t h ammonia
Precipitate Solution
Bring t o b o i l i n g Repeat (morphine) (nar-
ceine)
C rys t a 1 s
(papave r ine ac id
A l l o w t o stand
Sol u t i o n
(noscap ine
I
Further
i
Further
p u r i f ic a t ion pur i f i ca-
oxalate)
+
oxa 1 a t e )
Make a l k a l i n e w i t h ammonia
t ion
Precipitate
(noscapine)
1i s o l u t on
(discard)
+
+
Dissolve i n b o i l i n g alcohol
Crystallization
Scheme 4: Isolation of from powdered op i um .
NOSCAPINE 43 1
OCH3 OCH
I 2
Cotarnine Plecoriine
D C2H50H
0
0ch3
3
(2)-a-Narco t ine
Scheme 5
432 MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN
1
Cotarnine - Iodomeconine 2
\ \
CH 3 CH 3
CH 3O
Na/Hg
H.. ... *..s . 0
OCH3 OCIl
3
(+) -a-Narcotine
Scheme 6
NOSCAPINE 433
S e v e r a l f e e d i n g e x p e r i m e n t s ( 6 , 8 , 94 ) have been r u n
t o e l u c i d a t e t h e b i o g e n e s i s of n o s c a p i n e i n Papaver
somnif erum L . (Papaveraceae) . When l a b e l e d (+) -
t y r o s i n e was f e d t o t h e p l a n t , r a d i o a c t i v e n a r c o t i n e
l a b e l e d s p e c i f i c a l l y and e q u a l l y a t C-1 and C-3 was
o b t a i n e d . The b e n z y l i s o q u i n o l i n e s y s t e m of n o s c a p i n e
i s t h u s d e r i v e d b i o l o g i c a l l y from two Ar-C-C u n i t s which
c a n a r i s e from t y r o s i n e .
The c a r b o n atoms t h a t a r i s e from t h e S-methyl of

m e t h i o n i n e were c l e a r l y p i n p o i n t e d when, a f t e r f e e d i n g
radioactive methionine, noscapine labeled a t t h e
l a c t o n e c a r b o n y l , t h e m e t h y l e n e d i o x y g r o u p , and t h e
N- and 0-methyl c a r b o n atoms w a s o b t a i n e d ( 2 7 , 2 8 ) .
Progressing f u r t h e r along the biogenetic locus, t h e
b e n z y l i s o q u i n o l i n e (+)- n o r l a u d a n o s o l i n e l a b e l e d C-1
l e d t o n o s c a p i n e a l s o l a b e l e d C-1 ( 2 9 ) . Even more
s i g n i f i c a n t l y , when q u a d r u p l y l a b e l e d (+)- and (-) -
r e t i c u l i n e were f e d s e p a r a t e l y t o P. somniferum, i t
w a s found t h a t b o t h enantiomers were i n c o r p o r a t e d i n t o
n o s c a p i n e , b u t w i t h t h e (+)-isomer d o i n g so s l i g h t l y
more e f f i c i e n t l y . E v i d e n t l y e p i m e r i z a t i o n of t h e
wrong b e n z y l i s o q u i n o l i n e p r e c u r s o r must o c c u r , p r o b a b l y
by o x i d a t i o n - r e d u c t i o n a t C-1. I n keeping w i t h t h i s
c o n c l u s i o n c o n s i d e r a b l e l o s s of t r i t i u m o c c u r e d i n t h e
c o u r s e of i n c o r p o r a t i o n of b o t h r e t i c u l i n e s . Another
i m p o r t a n t o b s e r v a t i o n i s t h a t t h e l a c t o n e c a r b o n y l of
t h e p h t h a l i d e i s o q u i n o l i n e must b e d e r i v e d from t h e
N-methyl group of t h e b e n z y l i s o q u i n o l i n e p r e c u r s o r
( 2 7 , 29, 3 0 ) .
F i n a l l y , i t h a s been found t h a t t h e f e e d i n g of l a b e l e d
( - ) - s c o u l e r i n e r e s u l t s i n t h e f o r m a t i o n of r a d i o a c t i v e
noscapine. Protoberberines are, therefore, t h e precursors
f o r t h e phthalideisoquinolines i n plants. S i g n i f i c a n t l y ,
( - ) - s c o u l e r i n e , which p o s s e s s e s t h e same a b s o l u t e
c o n f i g u r a t i o n a s ( + ) - r e t i c d i n e and ( - ) - a - n a r c o t i n e ,
w a s more t h a n one hundred t i m e s more e f f i c i e n t t h a n
i t s enantiomer a s a p r e c u r s o r f o r ( - ) - a - n a r c o t i n e . The
b i o g e n e t i c sequence i n p l a n t s i s , t h e r e f o r e , b e n z y l i s o -
qu i n o l i n e s +- t e t r a h y d r o p r o t o b e r b e r i n e s + p h t h a l i d e i s o -
q u i n o l i n e s . The b i o s y n t h e s i s of (-)-cC-narcotine i s
shown i n Scheme 7 .
6. Metabolism
The m e t a b o l i s m of n o s c a p i n e was r e p o r t e d
&:
Labeled t y r o s i n e
bCH3
*<:w
Labeled noscapine
H
1 Papaver
$H3-S-(CH ) -C-COOH
2 2 1 somn i f e r u m ) I
NH2 N\*
CH 3
Labeled methionine CH 3O
- 0
OgH3
L a b e l e d nos cap i n e
Laheled ( a - n o r l a u d a n o s o l i n e L a b e l e d n‘bscapine
Scheme 7
NOSCAPINE 435
Scheme 7 ( c o n t i n u e d )
(4)
"H3
Labeled (+) - r e t i c u l i n e R = H o r T
(Labeled (-) - r e t i c u l i n e somewhat Labeled n o s c a p i n e
less e f f i c i e n t ) ( a p p r e c i a b l e l o s s of
tritium)
CH30
HO
OCH3
Labeled (-) - s c o u l e r i n e Labeled noscapine

(Some t r i t i u m l o s s )
436 MOHAMMED A . AL-YAHYA AND MAHMOUD M . A . HASSAN
(31, 3 2 ) . Oral a d m i n i s t r a t i o n of to
male r a b b i t s and e x a m i n a t i o n o f t h e 24 h o u r s u r i n e by
p r e p a r a t i v e TLC and methane c h e m i c a l i o n i z a t i o n mass
s p e c t r o m e t r y r e v e a l e d t h e p r e s e n c e of two O-monodeme-
t h y l a t e d compounds a s f r e e m e t a b o l i t e s 2 and 5, One
0 - d i d e m e t h y l a t e d d e r i v a t i v e 2 o r 5 and t h e i r c o n j u g a t e d
forms. Noscapine g i v e n orally to rats was
m e t a b o l i s e d t o di-0-demethyl-noscapine 3 or 4 , cotarnine
5 , h y d r o c o t a r n i n e 1,o x y c o t a r n i n e 2, a n d 0-demethyl-
-
meconine 5. T h e s e m e t a b o l i t e s were i s o l a t e d from u r i n e .
A l l p o s s i b l e m e t a b o l i t e s of n o s c a p i n e a r e
shown i n Scheme 8.
7.1 I d e n t i f i c a t i o n Tests
The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e
d e s c r i b e d by t h e B r i t i s h Pharmacopoeia ( 1 9 8 0 ) .
a) The l i g h t a b s o r p t i o n , i n t h e r a n g e 230 t o
350 nm, of a 0.005 p e r c e n t w/v s o l u t i o n i n
m e t h a n o l e x h i b i t s two maxima, a t 291 nm and
310 nm and a minimum a t 263 nm; r a t i o of t h e
a b s o r b a n c e a t t h e maximum a t 310 nm t o t h a t
a t t h e maximum a t 291 nm, a b o u t 1 . 2 .
b) To 1 0 mg add 0 . 5 m l of s u l f u r i c a c i d and mix;

a g r e e n i s h - y e l l o w s o l u t i o n i s formed which
t u r n s r e d and f i n a l l y v i o l e t o n h e a t i n g .
c) S o l u t i o n s i n organic s o l v e n t s , such as
methanol and c h l o r o f o r m , a r e l e v o r o t a t o r y ;
aqueous a c i d i c s o l u t i o n s are d e x t r o r o t a t o r y .
7-2 M i c r o c r y s t a l Tests
a) According t o t h e method of C l a r k e and W i l l i a m s

(33) , i n potassium chromate s o l u t i o n , n o scap i n e
f o r m s f e a t h e r y r o s e t t e s o r b u n c h e s of b l a d e s ,
s e n s i t i v i t y b e i n g 1 i n 1500 ( F i g . 9 ) .
b) I n sodium c a r b o n a t e s o l u t i o n r o s e t t e s and
b u n c h e s of n e e d l e s a r e s e e n a t t h e same
s e n s i t i v i t y (Fig. 10).
NOSCAPINE 437
Fig. 9 . C r y s t a l s of Noscapine with Potassium Chromate S o l u t i o n .
Fig. 10. C r y s t a l s of Noscapine with Sodium Carbonate S o l u t i o n .

43 8 MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN
Scheme 8 . P o s s i b l e M e t a b o l i t e s of (-)-a-narcotine
‘
0
J
be@ 0
OCH3
nos c a p i n e
1
CH3
0-demezhyla ted
5 meconine
cotarnine
1 hydrocotarnine
CH3 c o t a r n i n e
(pseudo b a s e form)
OCH3
ox yc o t a r n i n e
0-d i d eme t h y l a t e d m e t a b o l i t e s
NOSCAPINE 439
7.3 T i t r i m e t r i c Methods
The o f f i c i a l methods of d e t e r m i n i n g Noscapine

a r e d e s c r i b e d by t h e B.P. (3) and U.S.P. ( 3 4 ) .
7.3.1 Non-Aaueous T i t r a t i o n
The B.P. (3) d e s c r i b e s t h e f o l l o w i n g

method :-
D i s s o l v e 0.5 g i n 40 m l of anhydrous
g l a c i a l a c e t i c acid previously neutralised
t o c r y s t a l v i o l e t , warming g e n t l y . T i t r a t e
w i t h 0.1 M p e r c h l o r i c a c i d u s i n g 0.25 m l
of c r y s t a l v i o l e t s o l u t i o n a s i n d i c a t o r .
Each m l of 0.1 M p e r c h l o r i c a c i d i s
e q u i v a l e n t t o 0.04134 g of C22H23 NO,.
The U.S.P. describes the following

met hod :-
D i s s o l v e about 1 . 5 g of Noscapine,
a c c u r a t e l y weighed, i n 25 m l of g l a c i a l
a c e t i c a c i d . Add 25 m l of d i o x a n e and
5 d r o p s of c r y s t a l v i o l e t T . S . , and t i t r a t e
with 0.1 N perchloric acid i n g l a c i a l a c e t i c
a c i d t o t h e end-point change from p u r p l e t o
b l u e . Perform a b l a n k d e t e r m i n a t i o n , and
make any n e c e s s a r y c o r r e c t i o n . Each m l of
0.1 N perchloric acid is equivalent t o
41.34 mg of C22H23N07.
Another method w a s d e s c r i b e d by T u t h i l l
e t a l . (35) u s i n g m a l a c h i t e g r e e n a s b e t t e r
indicator than c r y s t a l v i o l e t .
7.3.2 Polarographic T i t r a t i o n
a) Using a dropping mercury e l e c t r o d e a s

i n d i c a t o r n o s c a p i n e h y d r o c h l o r i d e c a n be
t i t r a t e d w i t h Cadmium I o d i d e i n s o l u t i o n
of n e u t r a l s a l t s ( 0 . 1 t I KNO N a C l or
3’
Na2S04) ( 3 6 ) .
b) Dusinsky (37) d e s c r i b e d a method where

noscapine c a n be t i t r a t e d i n a l k a l i n e
s o l u t i o n (1.25 N NaOH) showing

depression i n t h e polarographic curve
a t 1.5V.
Sodium a l i z a r i n s u l p h o n a t e h a s been
used f o r t h e d e t e r m i n a t i o n of n o s c a p i n e
(38). A p o t e n t i a l of - 0 . 6 5 V was used
and t h e t i t r a t i o n medium was 0.3 N K C 1
a d j u s t e d t o a pH of 4 - 6 .
SoucKova and S;ka (39) s t a t e d a method

using t u n g s t o s i l i c i c acid. This acid
w a s found e s p e c i a l l y u s e f u l s i n c e t h e
r e a c t i o n i s v e r y s e n s i t i v e and pre-
c i p i t a t i o n i s immediate. The p o l a r o -
graphy a l s o gave t h e c o m p o s i t i o n of
t h e t u n g s t o s i l i c i c acid organic base
complex. The poor s e l e c t i v i t y of
T u n g s t o s i l i c i c a c i d i s due t o i t s
h i g h s e n s i t i v i t y which a l l o w s a c c u r a t e
d e t e r m i n a t i o n of 10-20 mg of b a s e . A
0.01 M aqueous s o l u t i o n of t u n g s t o -
s i l i c i c a c i d i s used w i t h a dropping
mercury c a t h o d e and S . C . E . anode a t
0 . 6 5 V . The pH of s o l u t i o n a d j u s t e d
w i t h HC1 ( 0 . 1 t o 0 . 6 N ) . P l o t of
c u r r e n t vs a c i d used c o n s i s t s of two
s t r a i g h t l i n e s and t h e i n t e r s e c t
c o n s i d e r e d as t h e e q u i v a l e n c e p o i n t .
Another method f o r p o l a r o g r a p h i c
t i t r a t i o n was a l s o r e p o r t e d ( 4 0 ) .
T h i s method is based on t h e f o r m a t i o n
of complex mercury compounds. S o l u t i o n
of K2HgI4 c o n t a i n i n g a n e x c e s s of i o d i d e
i s t h e most s u i t a b l e f o r t h e determina-
t i o n . The t i t r a t i o n i s c a r r i e d o u t
w i t h a d r o p p i n g mercury e l e c t r o d e a t
t h e p o t e n t i a l -0.8 V t o - 0.9 V (VS.
t h e S . C . E . ) w i t h 0 . 1 PI KN03 o r 0 . 1 M
H SO a s s u p p o r t i n g e l e c t r o l y t e .
2 4
7.3.3 P o t e n t iometr i c T i t r a t i o n
Tungsten rod w a s used a s i n d i c a t o r

e l e c t r o d e i n t h e p o t e n t iometr i c t i t r a t i o n
of n o s c a p i n e i n a 1 : 6 m i x t u r e of a c e t i c a c i d :
a c e t i c anhydride ( 4 1 ) .
NOSCAPINE 441
7.4 Complexometric
Noscapine is p r e c i p i t a t e d from 0 . 5 N H C 1
w i t h 0.028 M Bi-EDTA and 0.112 M K I forming
iodobismuthate complexes and EDTA i s being
set f r e e (42). After c e n t r i f u g a t i o n t h e f r e e
EDTA i s determined i n a n a l i q u o t of t h e super-
n a t e n t l i q u i d w i t h 0.01 M ZnSO4 i n pH 9 . 1
b o r a t e b u f f e r and Eriochrome b l a c k T as
i n d i c a t o r . T e r t i a r y amines, q u a t e r n a r y
ammonium s a l t s o r analogous sulphonium,
phosphonium and arsonium compounds i n t e r f e r e
i n the determination.
7.5 Spectrophotometric
7.5.1 Colorimetric
a ) Yoichi and Sano (43) d e s c r i b e a method f o r

a n a l y s i s of noscapine i n mixed pharmaceutical
preparations. The sample is mixed w i t h a
s o l u t i o n of chromotropic a c i d 0.2% i n 70%
(v/v) H3PO4 a c i d and h e a t e d a t 100°C f o r
30 m i n u t e s and t h e e x t i n c t i o n i s measured
a t 570 nm. A c a l i b r a t i o n c u r v e i s r e c t i -
l i n e a r f o r 30 t o 150 pg of noscapine
h y d r o c h l o r i d e per m l .
b) Solochrome Green V 150 ( C . I . Mordant

Green 15) h a s been used a s aqueous 1 mM
s o l u t i o n . The complex formed by noscapine
is e x t r a c t e d i n t o chloroform and t h e absorb-
ance i s measured a t 520 nm ( 4 4 ) .
c ) Another method f o r t h e q u a n t i t a t i v e
s e p a r a t i o n of p a p a v e r i n e from n o s c a p i n e i n
m i x t u r e s w a s a l s o r e p o r t e d (45). T h i s
method i s based on t h e f o r m a t i o n of a n
insoluble papaverine r e i n e c k a t e i n acid
s o l u t i o n i n t h e p r e s e n c e of e x c e s s
chloroform.
Procedure
T r i t u r a t e t h e sample (4.5 g) w i t h g l a c i a l
a c e t i c a c i d (25 ml) followed by H 2 0 ( 20 ml)
and f i l t e r . E x t r a c t a 1 0 m l a l i q u o t w i t h
CHC13 (8 X 1 0 ml) and wash e a c h e x t r a c t

i n t u r n w i t h H20 (15 m l ) , H20 (15 ml) p l u s
NaOH soln. ( 1 : 1) containing a l i t t l e
NaHS03 (15 m l ) , H 2 0 ( 1 5 m l ) , 0 . 1 M H2SO4
(15 m l and 1 0 ml) and 0.05% N a H C 0 3 s o l n .
(10 m l ) . E v a p o r a t e t h e combined washed
e x t r a c t s t o d r y n e s s o n a water b a t h .
D i s s o l v e t h e r e s i d u e i n CCl4 (50 ml) ,
s t r a i n through cotton-wool and p a s s t h r o u g h
a column of Ca(OH)2. Wash t h e column w i t h
C C l 4 (2 X 1 0 ml) and e x t r a c t t h e combined
CCl4 f r a c t i o n s w i t h 0 . 1 N H C 1 ( 2 X 1 0 m l ) .
Shake t h e H C 1 s o l n . w i t h CHC13 (10 ml) f o r
10 m i n . , add 2% ammonium r e i n e c k a t e s o l n .
( 1 0 m l ) , shake f o r 3 0 min. and f i l t e r
through s i n t e r e d g l a s s . To d e t e r m i n e
papaverine d i s s o l v e t h e ppt. i n acetone
and measure t h e e x t i n c t i o n a t 525 mu. To
d e t e r m i n e n o s c a p i n e , shake t h e CHC13 l a y e r
of t h e f i l t r a t e w i t h 0.25% AgN03soln.
( 4 0 m l ) , s e p a r a t e and f u r t h e r e x t r a c t w i t h
CHC13 (2 X 1 0 m l ) ; s t r a i n t h e combined CHC13
f r a c t i o n s through cotton-wool, d i l u t e t o
250 m l , and e i t h e r measure t h e e x t i n c t i o n
a t 310 mp o r e v a p o r a t e and t i t r a t e w i t h
0.05 N H C l O 4 i n g l a c i a l a c e t i c a c i d .
d) Thomas d e s c r i b e d a method f o r d e t e r m i n a t i o n
of some d r u g s c o n t a i n i n g a t e r t i a r y - a m i n e
group ( 4 6 ) . The d r u g i s h e a t e d w i t h 10%
malonic a c i d i n a c e t i c a n h y d r i d e a t 800
f o r 1 5 min. and, a f t e r d i l u t i o n w i t h e t h a n o l ,
t h e e x t i n c t i o n i s measured a t 333 nm. t h e
l i m i t of d e t e c t i o n f o r n o s c a p i n e hydro-
c h l o r i d e was 1 0 t o 30 ng m l - 1 . Dosage
forms r e q u i r e p r e l i m i n a r y e x t r a c t i o n of
t h e drug.
7 . 5.2 Infra-red
Bakre - et - a1 (47) d e s c r i b e d a n i n f r a - r e d
s p e c t r o s c o p i c method f o r t h e d e t e r m i n a t i o n
of t h e o r i g i n of opium as w e l l a s a s i m u l t a n e -
o u s a s s a y of n o s c a p i n e , t h e b a i n e and papa-
v e r i n e . 4.5 g f i n e l y ground sample w a s
t i t u r a t e d f o r 20 min. i n 25 m l water w a s
s l o w l y added w i t h c o n t i n u o u s s t i r r i n g and t h e
NOSCAPINE 443
resulting solution was filtered. 10 m l

A l i q u o t of f i l t r a t e w a s e x t r a c t e d f o u r t i m e s
i n t o 1 0 m l c h l o r o f o r m and e a c h e x t r a c t w a s
washed w i t h 1 0 m l water, 25 m l of 0.2% sodium
b i s u l p h i t e i n 30% aqueous sodium h y d r o x i d e ,
1 0 m l water and a g a i n 1 0 m l water. The combined
c h l o r o f o r m s o l u t i o n was f i l t e r e d t h r o u g h
c o t t o n wool and e v a p o r a t e d . The r e s i d u e i s d r i e d
i n a d e s i c a t o r t h e n mixed w i t h anhydrous c a r b o n
t e t r a c h l o r i d e , f i l t e r e d through s i n t e r e d g l a s s
and d i l u t e d t o 25 m l . The I R i s examined from
1100 cm-1 t o 1900 c m - l i n a 1 mm sodium c h l o r i d e
c e l l , noscapine b e i n g measured a t 1767 cm-l.
The a b s o r b a n c e i s compared w i t h a b s o r b a n c e s
of s o l u t i o n s of known c o n c e n t r a t i o n s .
Other I R methods f o r d e t e r m i n a t i o n of
i s o q u i n o l i n e a l k a l o i d s were a l s o r e p o r t e d
(48, 4 9 ) .
7.5.3 Ultra-Violet
T e t r a p o n a m i x t u r e of t h e h y d r o c h l o r i d e s
of Morphine, Noscapine, c o d e i n e and p a p a v e r i n e
w a s a n a l y s e d by J e n s e n ( 5 0 ) . Morphine w a s
s e p a r a t e d by e x t r a c t i o n w i t h c h l o r o f o r m from
s t r o n g a l k a l i n e s o l u t i o n and d e t e r m i n e d
s p e c t r o p h o t o m e t r i c a l l y w i t h NaN02 a t 440 run.
The o t h e r a l k a l o i d s were s e p a r a t e d by T L C
on K i e s e l g e l CF 254 w i t h e t h a n o l : benzene
1 : 4 a s s o l v e n t . The s p o t s ( l o c a t e d i n U.V.
r a d i a t i o n ) were e x t r a c t e d w i t h methanol and
determined a t 215 run f o r c o d e i n e , a t 279 nm
f o r p a p a v e r i n e and 312 nm f o r n o s c a p i n e .
7.5.4 Atomic A b s o r p t i o n
An i n d i r e c t method f o r t h e a n a l y s i s of
Noscapine i n d r u g s w a s r e p o r t e d ( 5 1 ) . A
complex i s formed between Noscapine and
Reinecke s a l t i n t h e p r e s e n c e of t a r t a r i c
a c i d a t pH 1 . 7 . This is extracted i n t o
c h l o r o f o r m and Noscapine i s d e t e r m i n e d
i n d i r e c t l y by measuring chromium c a t i o n by
a t o m i c a b s o r p t i o n s p e c t r op ho tome t er y .
7.5.5 Spectrofluorimetdc
a ) Noscapine h a s been determined i n m i x t u r e s

of opium a l k a l o i d s (52) by measuring t h e
f l u o r e s c e n c e a t 375 run ( e x c i t a t i o n a t 315 nm).
The sample i s b u f f e r e d a t pH 9 i n 0 . 1 N
s u l p h u r i c a c i d and 0 . 1 N sodium hydroxide.
Noscapine i s e x t r a c t e d i n chloroform and a
p o r t i o n of t h i s e x t r a c t i s t r e a t e d w i t h
t r i c h l o r o a c e t i c a c i d i n chloroform t o
quench t h e f l u o r e s c e n c e of papaverine.
A s t a n d a r d s o l u t i o n of 2-aminopyridine
i n 0 . 1 N s u l p h u r i c a c i d is a l s o measured a t
375 nm ( e x c i t a t i o n a t 315 nm). For t h e
c a l c u l a t i o n each f l o u r e s c e n c e r e a d i n g
on t h e t e s t s o l u t i o n i s c a l c u l a t e d a s a
p e r c e n t a g e of t h a t f o r t h e s t a n d a r d and
r e f e r r e d t o c a l i b r a t i o n g r a p h s prepared
s i m i l a r l y f o r Noscapine. Sub-microgram
amounts of Noscapine c a n b e determined
without preliminary separation.
b) Vedso s t a t e d a method (53) f o r t h e

d e t e r m i n a t i o n of Noscapine i n plasma and
urine. I t involves e x t r a c t i o n of 1 m l
sample a t pH 1 0 i n t o e t h y l e t h e r and
r e - e x t r a c t i o n w i t h d i l u t e HC1. The a c i d
i s n e u t r a l i s e d and t h e s o l u t i o n was a d j u s t e d
t o PH 9.2 w i t h a borax b u f f e r . Fluorescence
i s measured through 480 t o 580 mu f i l t e r
( e x c i t a t i o n a t 365 mu) b e f o r e and a f t e r
a u t o c l a v i n g a t 120OC f o r 3 0 min. S t a n d a r d s
( 0 t o 2.5 pg cm-3) are a l s o measured i n t h e
same way. I t was s t a t e d t h a t c o n c e n t r a t i o n s
from 0.05 ug p e r m l can be determined and
a l t h o u g h t h e p r e s e n c e of morphine gave an
i n c r e a s e i n f l o u r e s c e n c e about h a l f t h a t
f o r n o s c a p h e , c o d e i n e , n a r c e i n e and
papaver i n e d i d n o t i n t e r f e r e .
c ) A r e a c t i o n m i x t u r e of 10%malonic a c i d i n
a c e t i c a n h y d r i d e was used i n a method
r e p o r t e d by Rao and Tandon ( 5 4 ) . I n t e r -
f e r e n c e was caused by T e r t i a r y amines,
glucose,magnesium a c e t a t e and some i n o r g a n i c
s a l t s , b u t n o t by d i e t h y l a m i n e , a n i l i n e ,
benzoic a c i d , a s p i r i n and s a c c h a r i n .
NOSCAPINE 445
7.5.6 Nuclear Magnetic Resonance
A known amount of t - b u t y l a l c o h o l w a s added

as a s t a n d a r d t o n o s c a p i n e i n e t h a n o l f r e e -
c h l o r o f o r m and t h e peaks a t 3 . 8 3 , 4.00 and
4 . 0 5 ppm; c o r r e s p o n d i n g t o t h e n i n e methoxy
group p r o t o n s of n o s c a p i n e were i n t e g r a t e d
a l o n g w i t h t h e peak a t 1 . 3 ppm c o r r e s p o n d i n g
t o t h e n i n e methyl-group p r o t o n s of t - b u t y l
a l c o h o l ( F i g . 11). The amount of n o s c a p i n e
i s c a l c u l a t e d from t h e i n t e g r a t i o n r a t i o and
t h e known amount of s t a n d a r d ( 5 5 ) .
7.5.7 Mass
-
Noscapine w a s i d e n t i f i e d i n opium (92)
w i t h o u t any p r i o r s e p a r a t i o n . Samples a r e
introduced d i r e c t l y i n t o t h e ion s our c e us ing
a s o l i d sampling probe. Reagent g a s e s were
i s o b u t a n e and w a t e r , mass s p e c t r a l measurement
was a t m / e 220.
Arnold (93) d e s c r i b e d a G C /M S method

f o r t h e d e t e r m i n a t i o n of n o s c a p i n e i n opium
preparations.
7.6. Chromatographic
The paper-chromatographic method f o r t h e

d e t e c t i o n of a l k a l o i d s , e . g . , c o d e i n e , v e r a t r i n e ,
q u i n i n e and n o s c a p i n e i n s e v e r a l f o o d s ( a 100 g
sample) i s d e s c r i b e d . For t h e p r e l i m i n a r y
e x t r a c t i o n of t h e a l k a l o i d s , add t o t h e sample
300 m l of e t h a n o l a c i d i f i e d t o l i t m u s p a p e r w i t h
H C 1 . D i g e s t o n a water b a t h a t 400 f o r 24 h r . ,
f i l t e r and r e t a i n t h e f i l t r a t e . Add 100 m l of
acidified ethanol to the residue, d i g e s t f o r
a n o t h e r 1 2 h r . and f i l t e r a g a i n . Combine t h e
f i l t r a t e s and remove t h e a l c o h o l by e v a p o r a t i n g
on a w a t e r b a t h a t 4 0 0 . Add 30 m l of water
and e x t r a c t w i t h d i e t h y l e t h e r ( 5 X 5 0 m l ) .
Add NaOH s o l n . t o t h e a q . l a y e r till i t is
a l k a l i n e t o l i t m u s and e x t r a c t w i t h d i e t h y l
e t h e r ( 5 X 50 ml). Evaporate t h e e t h e r e x t r a c t
t o 50 m l , t r a n s f e r i t t o a s e p a r a t i n g - f u n n e l
and wash w i t h 2N HCl(3 X 20 m l ) . To t h e

combined a c i d washings add NaOH till t h e y a r e
a l k a l i n e t o l i t m u s and e x t r a c t w i t h d i e t h y l
e t h e r ( 3 X 50 m l ) . E v a p o r a t e t h e e t h e r
c o m p l e t e l y and d i s s o l v e t h e extract i n 5 m l
of e t h a n o l . R e t a i n t h i s a l c o h o l i c s o h . f o r
chromatography by t h e d e s c e n d i n g t e c h n i q u e on
Whatman No. 1 p a p e r . Apply d r o p s of t h e t e s t
and s t a n d a r d s o l n . t o t h e p a p e r and impregnate
i t s whole area above t h e s t a r t i n g l i n e w i t h a
f r e s h l y p r e p a r e d e t h a n o l i c s o h . of formamide
(1 : 1). Dry t h e paper between two f i l t e r -
p a p e r s , t h e n a t 400 f o r 3 0 min. Spray t h e
s t a r t i n g p o i n t s w i t h t h e e t h a n o l i c s o l n . of
formamide (1 : 1) and a f t e r 10 min. t r a n s f e r
t h e paper t o a g l a s s c o n t a i n e r c o n t a i n i n g
CHC13 a t 250. The development i s complete
i n 2.5 h r . Dry t h e chromatogram a t 1050
and s p r a y w i t h a s o h . of potassium i o d o p l a t i -
n a t e , washing o f f t h e excess w i t h water i n
o r d e r t o o b s e r v e t h e s p o t s on t h e w h i t e
background. The f o l l o w i n g amounts of a l k a l o i d s
can b e d e t e c t e d - c o d e i n e 0.01 t o 0.3 mg,
q u i n i n e 0.005 t o 0.015 mg, v e r a t r i n e 0.1 t o
0.15 mg and noscapine 0.05 t o 0.075 mg. ( 5 6 ) .
T a b l e 5 d e s c r i b e s methods used i n noscapine

a n a l y s i s . A n a l y s i s of noscapine i n opium by
paper chromatography and s p e c t r o p h o t o m e t r y
involved e x t r a c t i o n of noscapine and measure-
ment of t h e e x t i n c t i o n of t h e s p o t a t 290 mu
and comparison w i t h s t a n d a r d s ( 5 7 ) . Other
method was a l s o d e s c r i b e d ( 5 8 ) .
7.6.2 Thin Layer Chromatography
T h i s t e c h n i q u e h a s been used e x t e n s i v e l y
f o r a n a l y s i s of Opium and i t s p r e p a r a t i o n s
(63 - 7 1 ) .
T a b l e 6 - g i v e s a resume of t e c h n i q u e s and
T a b l e 7 - shows s p r a y r e a g e n t s and methods
used.
S t a h l and co-workers have proposed a

s t a n d a r d p r o c e d u r e f o r t h e s e p a r a t i o n of
opium a l k a l o i d s ( 6 3 ) . Dried opium ( 0 . 1 g)
...
I '
. . . . . - . .
I
. . . . I . . . . ~ " . . ~ . . . . ~ . . " . ~' I" '
I I
S O 400 300 zbo I
100
. o ni
H3
1 .
1 . 1 I
l . . l . . . ' l . . . ' l . . . . i . . - . l
I I .
1 . . .
1
. I . . . I I . . .. I
II
T a b l e 5. Paper Chromatography Used € o r Noscapine.
1 2 3 4 5 6
S t a t i o n a r y Phase Technique Mobile P h a s e Comment rferenct
Paper Two d i m e n s i o n a l 1. Water s a t . b u t a n o l - a c e t i c Alkaloids i n

acid 5:l Tetrapon
2. e t h e r - 0 . 1 M a c e t i c a c i c 11
5: 2
11
Paper Two d i m e n s i o n a l 1. Dioxan-Formic a c i d - w a t e r
90:0.5 : 9 . 5
11
2. n - B u t a n o l - a c e t i c a c i d 5:l
11
Paper One d i m e n s i o n a l 1. Dioxan-Formic acid-water
90:0.5:9.5
Paper S & S Ascending o r 25% (NH4)2 SO4 i n 0 . 5 N H C 1
204 3b. descending
Whatman No. 1 D e s c end i n g Isobutylalcohol - toluene
paper b u f f e r e d s a t u r a t e d w i t h water 1 : 1.
a t pH 3.5
Paper One d i m e n s i o n a l Butyl acetate - butanol Separation
- acetic acid - water of n o s c a p i n e
47:9:28:16 from papave-
r i n e enhancec
b e c a u s e of
u s e of b u t y l
acetate.
T a b l e 5. (contd ... .)
c
I 1 I 2 3 4 5 6
Paper One d i m e n s i o n a l Upper l a y e r of a m i x t u r e

n-butanol-acet i c a c id-water
5:1:4
Wha tman N o . 1 D esc end Chloroform Starting points (56)
impregnated w e r e sprayed
above s t a r t i n g w i t h f ormamide
l i n e with i n e t h a n o l 1:l
f ormamide- a f t e r drying
e t h a n o l 1:l t h e paper
\D
then d r i e d
between
f i l t e r papers
t h e n a t 40°C
f o r 30 min.
T a b l e 6. TLC Techniques Used f o r Noscapine
S t a t i o n a r y Phase Technique Mobile P h a s e Rf
K i e s e l g e l HF Normal chamber Tolune-acetone 95% e t h a n o l

254
25% aq. NH3
20 : 20 : 3 : 1
S i l i c a g e l G-Na2C03 tI
Chloroform - Ethanol
4 : l
II
Silica gel G. E t h y l acetate
P
VI
0
K i e s e l g e l 60 It
Chloroform Benzene -
Acetone 3 : 3 : 1
I1
Silicagel G Benz ene-methanol
4 : l
I1
Silica gel G B u t a n o l - A c e t i c acid-H 0
2
3 : l : l
S i l i c a g e l G impreg. 11
Chloroform - Ethanol
4% Na2C03 4 : l
11
Silica gel Chloroform-isopropyl
a l c o h o l 10% aq.NH3
30 : 10 : 1
Silica gel G Benzene-methanol 4 : 1
Table 7. TLC S p r a y R e a g e n t s and Methods Used € o r D e t e c t i o n of Noscapine
Reagent Procedure Ref.

~ ~~
5% 3,5-dichloro-p-benzoqui- A f t e r s p r a y i n g s p r a y w i t h aqueous
nonechlorimine i n i s o p r o p y l NH3 1 : 1 and o b s e r v e i n d a y l i g h t
alcohol and u l t r a v i o l e t .
4% Hg(N03)2 i n 3% HN03 After spraying t h e p l a t e i s heated

15 min. a t l l O O C and o b s e r v e d i n
d a y l i g h t . D e t e c t i o n l i m i t 2 vg
1. 3% H202 s o l u t i o n P l a t e i s f i r s t d r i e d 1 0 min. a t 100°C

2. 5% K4Fe(CN)6 s o l u t i o n t h e n s p r a y e d w i t h 1. t h e n d r i e d 1 0 min.
a t 100°C and s p r a y e d w i t h 2 and d r i e d
1 0 min. a t 100°C. Brown s p o t s i n t e n s i f i e d
t o r e d . D e t e c t i o n l i m i t 1 0 pg.
2.6 g C@(N@3)2 d i s s o l v e d i n After spraying, t h e p l a t e is heated a t

2 m l anhydrous a c e t i c a c i d 105 OC f o r 10 min. S p o t s a r e s t a b l e f o r
i s added t o 4 . 4 g o f NaN02 several h o u r s . Noscapine a p p e a r s a s
d i s s o l v e d i n 1 0 m l H20 t h e n b l u e - g r e e n f l o u r e s c e n t s p o t when viewed
20 ml a c e t i c a c i d and 50 m l under u l t r a - v i o l e t l i g h t .
H 0 i s added t o m i x t u r e .
2
P l a t e i s h e a t e d a t 80 O C f o r 1 0 min. and
4% c i t r i c a c i d i n
viewed i n d a v l i g h t ( d e t e c t i o n l i m i t 5 pg)
a c e t i c anhydride
and i n u l t r a - v i o l e t r a d i a t i o n ( d e t e c t i o n
l i m i t 0.5 Up).
452 MOHAMMED A. AL-YAHYA AND MAHMOUD M. A . HASSAN
w a s powdered and t r i t u r a t e d w i t h 5 m l of
70% e t h a n o l . The m i x t u r e w a s warmed a t
50 - 600 f o r 30 min. t h e n f i l t e r e d and d i l u t e d
t o 1 0 m l w i t h 70% e t h a n o l . T i n c t u r e of opium
(1 m l ) was d i l u t e d w i t h 9 m l 35% e t h a n o l .
Three p o r t i o n s ( 5 , 1 0 and 20 u l ) of opium
s o l u t i o n and similar p o r t i o n s of s t a n d a r d
s o l u t i o n were a p p l i e d t o a l a y e r of K i e s e l g e l
H F254 and chromatograph was developed t o
1 5 c m w i t h t o l u e n e - a c e t o n e - 95% e t h a n o l
25% a q . NH3 (20 : 20 : 3 : 1 ) . For d e t e c t i o n ,
t h e p l a t e w a s h e a t e d a t 110% f o r 1 0 min.
and t h e s e p a r a t e d zones l o c a t e d i n u l t r a v i o l e t
r a d i a t i o n . The s p o t s a r e t h e n sprayed u s i n g
a modified D r a g e n d o r f f ' s r e a g e n t and t h e n
w i t h 0.05 N t o 0 . 1 N s u l p h u r i c a c i d .
A l s o polyamide h a s been used a s l a y e r f o r

chromatography ( 7 6 ) . Where poly-E-caprolactam
r e s i n (Amilan CM 10075) was used. Develop-
ment f o r 2 h r . i n cyclohexane-ethyl a c e t a t e -
p r o p y l a l c o h o l - Me2NH 3 0 : 2.5 : 0.9 : 0 . 1
showed n o s c a p i n e a t Rf 0 . 6 1 and development i n
H20-ethanol - Me2NH 88 : 1 2 : 0 . 1 n o s c a p i n e
had Rf 0.00.
7.6.3 Gas Liquid Chromatography
D e r i v i t i z a t i o n of samples i n c l u d e d
a c e t y l a t i o n using acetic anhydride i n p y r i d i n e
(77) and t r e a t i n g sample w i t h t r i m e t h y l s i l y l
acetamide and t r i m e t h y l c h l o r o e t h a n e ( 6 8 ) .
I n t e r n a l s t a n d a r d s used were h i s t a p y r r o d i n e
h y d r o c h l o r i d e , o e s t r a d i o l v a l e r a t e (78)
Phenazone (79) and S q u a l i n e ( 7 7 ) . Another
method was a l s o r e p o r t e d ( 8 0 ) . Column t y p e s
e t c . a r e r e p o r t e d i n T a b l e 8.
7.6.4 HiPh Performance Liquid Chromatography
S e p a r a t i o n s of p h a r m a c e u t i c a l s combined
i n v a r i o u s f o r m u l a t i o n s by HPLC on S e p h e r o s i l
5 Um i s o c r a t i c a l y h a s been r e p o r t e d ( 8 1 ) .
Noscapine i n a n t i - c o l d p r e p a r a t i o n s w a s
s e p a r a t e d on a column of H i t a c h i g e l 3011-0(82)
T a b l e 8. GLC ConditionsUsed f o r Noscapine
Column S t a t i o n a r y Phase Flow-ra t e Temperature I e t e ct o r Ref,

Type
3 -1 24OoC Flame
4 f t x 4 m m D i a p o r t S (80-100) 3 0 cm min
glass mesh SE-30 i o n i z at i o r
3 -1
1 . 5 m x 2.3 Chromosorb G-HP 40 cm min 15OoC tg 235OC 11
mm g l a s s AW-DMCS (80-100) HI-EFF a t 1 . 2 5 C min-1

mesh 8 BP
Supelcoport - 2 2 5OC-2 7 O°C 11
(80-100 mesh) a t 2 0 min-1

~
3 -1
4 f t x 3mm 2% OV
11
Gas-Chrom 0 30 cm min 1 8 O o C 5 min.
N2
od g l a s s (100-120 mesh) 101 then 7 ' ~
min-l t o 25OoC
454 MOHAMMED A. AL-YAHYA AND MAHMOUD M. A . HASSAN
u s i n g methanol : 28% aqueous NH3 9 9 : l a s

e l u t i n g s o l v e n t and d e t e r m i n a t i o n by
spectrophotometry a t 230 nm o r 250 nm.
Paracetamol, p h e n a c e t i n , d i p y r o n e , a s p i r i n ,
c a f f e i n e , etenzamide and m e t h y l e p h e d r i n e
d i d n o t i n t e r f e r e . Other methods were a l s o
d i s c r i b e d (83, 8 4 ) .
7.6.5 Ion-Exchange Chromatography
Knox and Jurand r e p o r t e d a method (85) f o r

t h e s e p a r a t i o n of n o s c a p i n e on d r y packed
column of Zipax SCX o r SAX (37-44 um) b o r a t e
b u f f e r s a t pH 9.2-9.8 c o n t a i n i n g 4% a c e t o n i -
t r i l e and 1%propanol were used a t 500 t o
1500 l b p e r s q . i n . t o e l u t e n o s c a p i n e .
7.6.6 Ligand-Exchange Chromatography
P o r a g e l P.T. r e s i n was used t o s e p a r a t e

a l k a l o i d s and n o s c a p i n e h a s been a n a l y s e d on
t h i s r e s i n . 0.06 M-aqueous NH3 i n 33% e t h a n o l
w a s used as e l u a n t and t h e e l u t i o n volume
f o r n o s c a p i n e w a s 6.8 times t h e bulk column
volume ( 8 6 ) .
7.6.7 P a r t i t i o n Chromatography
I n t h e a s s a y of T e t r a p o n by p a r t i t i o n
chromatography t h e s t a t i o n a r y phase i s a
phosphate b u f f e r ( 8 7 ) . 5 m l of 0 . 2 N NaOH
i s added t o 0.4 g of papaveretum i n 20 m l
H 2 0 . The m i x t u r e i s e x t r a c t e d twice w i t h a
m i x t u r e of 1 0 m l of c h l o r o f o r m i n 3 0 m l e t h e r
and t h e n w i t h 1 0 m l of chloroform. The
f i l t e r e d e x t r a c t s a r e e v a p o r a t e d t o 0.5 -
1 . 0 m l and d i l u t e d w i t h 25 m l of e t h e r b e f o r e
t r a n s f e r t o t h e p r e p a r e d column, 200 m l water
s a t u r a t e d e t h e r is used t o e l u t e n o s c a p i n e .
Other a s s a y w a s a l s o r e p o r t e d ( 8 8 ) .
7.6.8 Paper E l e c t r o p h o r e s i s
Due t o f o r m a t i o n of m o l e c u l a r complexes
between n o s c a p i n e and 7-(2-hydroxyethyl)
t h e o p h y l l i n e , t e t r a m e t h y l u r i c a c i d and
7-carboxymethyltheophyline, n o s c a p i n e h a s
NOSCAPINE 455
been separated from other isoquinoline

alkaloids by reversed - phase paper
chromatography and electrophoresis.
Britton - Robinson buffer pH 3.5 to 4
was used as the mobile phase and o-xylene
as stationary phase ( 8 9 ) . Another method
was also reported (1).
ACKNOWLEDGEMENTS
The authors would like to thank the technicians,

Robert Hutchison, K.N. Ludhi and the Research
assistant Syed Rafatullah o f the College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia for their
kind technical assistance for the preparation of the
manuscript.
8. References
1. M. Windholz, "The Merck Index", 9th e d . , Merck and Co.

I n c . , Rahway, U.S.A. , p.872, (1976).
2. Chemical A b s t r a c t s , I n t r o d u c t i o n t o Volume 66
Subj ect Index, (1967).
3. " B r i t i s h Pharmacopoeia'', Vol. 1,Her M a j e s t y ' s

s t a t i o n a r y o f f i c e , London, p.311, ( 1 9 8 0 ) .
4. M. Shamma, "The I s o q u i o n o l i n e A l k a l o i d s " , Academic

P r e s s London, p.360, (1972).
5. A.R. B a t t e r s b y and H. S p e n c e r , J . Chem. SOC., 1087,

(1965).
6. M. O h t a , H. T a n i , S . Mavozumi, a n d S . Kodaira,Chem.
Pharm. B u l l . 2, 1080, ( 1 9 6 4 ) .
7. M. O h t a , H. T a n i , and S. Movozumi, Chem. pharm. B u l l -

12,
1072, (1964).
8. 1, 11,
A.R. B a t t e r s b y , and H. S p e n c e r s , T e t . L e t t . -
(1964).
9. S. S a f e , and R.Y. Moir, Can. J . Chem., 42, 160, (1964).
10. F.M. L o v e l l , Acta C r y s t . , A. 8 6 9 , (1953).
11. E.G. Steward and R.B. P l a y e r , Acta C r y s t . , my

1313, (1972).
12. J . G . Grasselli, and W.M. R i t c h e y , " A t l a s of S p e c t r a l

Data a n d P h y s i c a l C o n s t a n t s for Organic CompoundsI1
-
2nd ed. Vol.111 CRC P r e s s I r c . , p.670, ( 1 9 7 5 ) .
13. .
R. H. F. Manske, "The A l k a l o i d s " , Vol X I 1 , Academic
P r e s s , N e w York, London, 396, ( 1 9 7 0 ) .
14. J. S . G l a s b y , " E n c y c l o p e d i a of The A l k a l o i d s " , Plenum

P r e s s , New York and London, p.988, ( 1 9 7 5 ) .
15. C.G. F a r m i l o , " B u l l e t i n o n N a r c o t i c s " , (18-70) ,

S e p t . -December (1954).
NOSCAPINE 451
16. L.H. B r i g g s and L.D. Colebrook, Anal. Chem. 9
2 9 ( 6 ) , 904, (1957).
-
17. M. Shamma and V. S t . GeorRier, T e t . Lett., 2339 ( 1 9 7 4 ) ;

and T e t . -
32, 211, (1976).
18. M.M.A. Hassan and M.A. Al-Yahya ( u n p u b l i s h e d r e s u l t s ) .
19. M. Ohashi, J . M . Wilson, H. B u d z i k i e w i c z , 7". Shamma,

W.A. S l u s a r c h y k and C.Djerassi, J . Am. Chem. S OC., - 85,
2807, (1963).
20. G . A . C o r d e l l , " I n t r o d u c t i o n t o A l k a l o i d s t 1 , J h o n Wiley

and Sons I n c . , New York, 499, ( 1 9 8 1 ) .
21. R a b i q u e t , Am. Chem. Phys. -

2 (5), 275, (1817).
22. B a r t o k , A n t a l , Gaal, Gyorgy, Kaskoto, Z o l t a n , Nagy,

Sandor, Gynogy, I s t v a n , Kaczko, Gabor, K e r c k e s , Peter
and S e l m e c i , G y o r g y , Hung, 155,
407, ( c l . C 0 7 d ) ,
23 Dec. ( 1 9 6 8 ) .
23. Khanna Pushpa, and Khanna Renu, I n d i a n J . Exp. B i o l .

1 4 , 5 , 628, (1976).
-
24. W.H. P e r k i n , J r . , and R. Robinson, J . Chem. S O C . ,

London, - 99, 775, ( 1 9 1 1 ) .
25. E . Hope, and R . Robinson, J. Chem. S O C . , London,

105, 2085 (1914).
-
26. R. Robinson, "The S t a n d a r d R e l a t i o n s of N a t u r a l

P r o d u c t s " , Oxford U n i v e r s i t y P r e s s , Clarendo$
London and N e w York, (1955).
27. A . R . B a t t e r s b y , M. Hirst, D. McCaldin, R. S o u t h g a t e ,

and J . S t a u n t o n , J. Chem. SOC. C . , 2163, ( 1 9 6 8 ) .
28. R . N . Gupta and I . D . S p e n c e r , Biochem. Biophys.

Res. Commun. 13,
1 1 5 , (1963).
29. A.R. B a t t e r s b y , M. Hirst, T e t . L e t t . , 11,669, (1965).
30. A.R. B a t t e r s b y , R . J . F r a n c i s , M. H i r s t , R. S o u t h g a t e
and J. S t a u n t o n , "Chem. Commun". 12, 602, (1967).
31. N. Tsunoda, H. Yoshimura, and H. Kozuka, E i s e i

Kagaku, 2 2 ( 5 ) , 290, ( 1 9 7 6 ) .
45 8 MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN
32. B. Goeber, K.P. B r a n d t , S. P f e i f e r , and A. O t t o ,

Pharmazie, 32 ( 8 - 9 ) 5 4 3 , ( 1 9 7 7 ) .
33. E . G . C . C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n of

Drugs", Vol. I , The P h a r m a c e u t i c a l P r e s s , London, p .
451, (1978).
34. U.S.P. X V I I , "The United S t a t e s Pharmacopeial',

Mack P u b l i s h i n g Co. E a s t o n Pa., ( 1 9 6 5 ) .
35. S.M. T u t h i l l , O.W. K o l l i n g , and K.H. R o b e r t o , Anal.

3 2 (12), 1 6 7 8 , ( 1 9 6 0 ) .
Chem. -
36. V . S c h i l l e r o v a , and J. Zyka, C e s k o s l . Farm.,

6 ( 2 ) , 93, (1957).
-
37. G. Dusinsky, Ceskosl. Farmac. 4-(8), 400, (1955).
38. J. Zyka, Ceskosl. Farmac. 5 ( 6 ) , 301, (1955).
39. M. Souckova and J. Zyka, C e s k o s l . Farmac. 4 (4),

181, (1955).
40. M. Cihakova and J . Zyka, Cekosl. Farm.,? (lo), 572,

(1956).
41. T. P a s t o r and V. Vajgand, Mikrochim. Acta,

I1 ( 1 - 2 ) , 8 5 , ( 1 9 7 6 ) .
42. 5
B. Budesinsky, C e s k o s l . Farm. - (lo), 579, (1956).
43. Y. Y o i c h i and A. Sano, J a p a n A n a l y s t , 1 6 ( 7 ) , 7 0 8 ,

(1967).
44. R. Rao and S.N. Tandon, I n d i a n J . Chem. S e c t . A . ,

1 6 (11) 1000, ( 1 9 7 8 ) .
45. L. Kum-Tatt, and C . G . F a r m i l o , 3. Pharm-Pharmacol.

-
10, 4 2 7 , ( 1 9 5 8 ) .
46. A.D. Thomas, J . Pharm. Pharmac., 28 (11) 8 3 8 , ( 1 9 7 6 ) .
47. V. J. Bakre, Z . Karaata, J . C . Bartlet and C . G . Farmilo,

J. Pharm. Pharmacol. 11 ( 4 ) , 234, (1959).
48. M.E. P e r e l ' s o n , Kh-Sh. Baisheva, B.K. R o s t o t s k i i and

A.A. Kiryanov, Lek. Rast., 15,
382, (1969).
NOSCAPINE 459
49. L-Kum-Tatt, R.A. Rockerbie and L. Levi, J. Pharm.

Pharmacol., 10 ( l o ) , 621, (1958).
50. K. J e n s e n , Arch. Pharm. Chem. , 78 ( 8 ) , 249, (1971).
51. T. Minamikawa, and K. Matsumura, Yakgaku Z a s s h i ,

96 (4) , 440, (1976).
-
52. R.A. Chalmers and G.A. Wadds, A n a l y s t , Lond., 95,
234, (1970).
53. S. Vedso, Acta Pharm. Tox., Kbh., 18 ( 2 ) , 1 1 9 , (1961).
54. N. Rao and S. Tandon, Anal. L e t t . P a r t B y 2 (6),

477, (1978).
55. A.E. Aboutabl and M.M.A. Hassan, S p e c t r o s c . L e t t . ,

-
12 ( 3 ) , 231, (1979).
56. J. Kolankiewicz and M. Nikonorow, Acta Polon. Pharm.,

-
1 6 (Z), 115, (1959).
57. H . Asahina, and M. Ono, U . N . S e c r e t r i a t , Publn.

ST/SOA/SER.K 50, 9pp., (1957).
58. A.A. Abdel Rahman, Arch Pharm., B e r l i n , -

288 ( 2 ) ,
53, (1955).
59. V.E. Krogerus, I . R a u t i a i n e n , and B. Westerlund,

Medd. Norsk Farm. S e l s k . , 2 (4-5), 198, (1955).
60. H. Haussermann, Arch. Pharm. B e r l i n , 289 (6),

303, (1956).
61. J. Buchi and H. Schumacher, Pharm. Acta Helv.,

31 ( 9 ) , 417, 1956.
-
62. H. T h i e s and F.W. R e u t t e r , N a t u r w i s s e n s c h a f t e n ,

-
42 (16), 462, (1955).
63. E. S t a h l , H. J o r k , E. Dumont, H. Bohrmann and

H. Vollmann, A r z n e i m i t t e l - F o r s c h , 19
( 2 ) , 1 9 4 , (1969).
64. T.R. Baggi, N. Rao, and H. Murty, F o r e n s i c S c i . ,

-
8 ( 3 ) , 265, (1976).
460 MOHAMMED A . AL-YAHYA A N D MAHMOUD M . A . HASSAN
65. N. Rao, Curr. S c i . , 46 ( 1 8 ) , 637, (1977).
66. K. Guven, and N . Guven, E c z a c i l i k B u t t . , 14 ( 5 ) ,

75, (1972).
67. V. S h o s t e n k o , V. D a n e l ' y a n t s , and L. Chernysh,

F a r m a t s i y a , Mosk., 25 ( 3 ) , 74, (1976).
68. T. Vu. DUC, T. Vernay, and C. N i c o l e , Pharm.

Acta Belv., 2 ( 5 ) , 126, (1976).
69. K. Roeder, E. E i c h , and E. M u t s c h l e r , Arch. Pharm.

Berl., 304
( 4 ) , 297, (1971).
70. F. Reimers, Arch. Pharm. Chemi., 2 (7), 201, (1971).
71. V . Vukcevic-Kovacevic, B u l l . S c i e n t . Cons. Acad.

RSF, Yougosl., 4 , 13 ( 9 - l o ) , 306, (1968).
72. J . W . F a i r b a i r n and S. El-Masry, J. Pharm.

Pharmac., 2 ( s u p p l . ) , ( 1 9 6 7 ) .
73. S . H a s h i b a , TI. Tatsuzawa, and A. E j i m a , Bunseki

Kagaku, 26 (ll), 8 0 4 , ( 1 9 7 7 ) .
74. N. Rao, H. Murty, and T . Baggy, C u r r 45 (9),

Sci. -
332, (1976).
75. N. Rao, and S. Tandon, F o r e n s i c S c i . -

9 ( 2 ) , 1 0 3 , (1977).
76. Jen-Tzaw Huang, Hsing-Chien H s i u , and Kung-Tsung

Wang, J. Chromatogr. 2
( 2 ) , 391, (1967).
77. G . F i s h e r and R. G i l l a r d , J . Pharm. S c i . , 66 (3),

421, ( 1 9 7 7 ) .
78. E. Nieminen, Farm. A i k a k . , 3 (lo), 3 4 2 , ( 1 9 7 1 ) .
79. A. B e c h t e l , Chromatographia, 5 ( 7 ) , 404, (1972).
80. M. Ono, M. Shimamine, and K. T a k a h a s h i , E i s e i

Shikensho Hokoku, 95, 4 , ( 1 9 7 7 ) .
81. M. Caude and L e Xuan Phan, Chromatographia, 2 (l),

20, ( 1 9 7 6 ) .
82. M. Tatsuzawa, S. H a s h i b a , and A. E j i m a , Bunsekikagaku,

26
L (lo), 706, (1977).
NOSCAPINE 46 1
83. C . L . G u i l l e m i n , J . P . Thomas, S. T h i a u l t , and

J . P . Bounine, J . Chromatogr., 142,
321, ( 1 9 7 7 ) .
84. M. Tatsuzawa, T. Yamamiya, A. Ejima, and N. Takai,

Bunseki Kagaku, 27 ( 1 2 ) , 753, ( 1 9 7 8 ) .
85. J . H . Knox and J. J u r a n d , J. Chromat. 82 (2), 398,

(1973).
86. E. Murgia, and H. Walton, J . Chromat. 104 ( 2 ) ,

417, ( 1 9 7 5 ) .
87. C.G. L i n d b l a d , and A. Agren, Farm. Revy, 53 (4),

69, (1954).
88. C. S t a n l e y , E l l i s t o n , and C.L. Maureen, J . A s s . o f f .

a n a l y t . chem., 53 ( 3 ) , 585, ( 1 9 7 0 ) .
89. M. S t u c h l i k , and L. K r a s n e c , J . Chromat.

36 ( 4 ) , 522,
- (1968).
90. S.K. Sim " M e d i c i n a l P l a n t A l k a l o i d s " , 2nd ed.

Univ. of T o r o n t o P r e s s , 6 2 , ( 1 9 7 0 ) .
91. M. S i n i b a l d i , and B . R i n a l d u z z i , A n a l y t . L e t t . ,
4 ( 3 ) , 125, (1971).
-
92. S. Z i t r i n and J . Yinon, Anal. L e t t . 10 ( 3 ) , 235,

(1977) .
93. W. A r n o l d , Beitr. G e r i c h t l . Med. -
32, 199, (1974).
94. A . R . B a t t e r s b y , J . S t a u n t o n , H.R. W i t t s h i r e ,
B . J . B i r c h e r , and C. F u g a n t i , J . Chem. SOC. P e r k i n
Trans. 1. ( 1 2 ) , 1162, ( 1 9 7 5 ) .
PENICILLIN-G BENUTHINE
Franz Kreuzig
1. Description 464
1. I Name, Formula, Molecular Weight 464
1.3 Compendia1 References 465
1.4 Definition of International Unit 465
1.5 Content 465
2. Production 465
3.1 Solubility 466
4. Stability, Degradation, Artefacts 472
5. Biopharmaceutics 472
5.2 Metabolism 474
6.2 Iodometric Titration 475
6.3 Nonaqueous Titration 476
6.4 Spectrophotometric Assay 476
6.6 Thin-Layer Chromatography 478
6.7 High-Performance Liquid Chromatography 479
8. References 480
Analytical Profiles of Drug Substances Copyrighl 0 1982 by 'The American

ISBN 0-12-260811-9
464 FRANZ KREUZIG
i. DESCRIPTION
- -
I. I. Name, Formuia, Moiecuiar Weight
-----
Chemi c a i Names
---------
4-Thia-i -azabi c y c i o 1-3.2.0. -7 heptane-2-carboxyi ic a c i d ,
3,3-dimethyi-7-oxo-6 [-(phenyiacetyi)amino - 7 - /-- 2 S-(2a,
5a,6B)-7-, compd. w i t h N,N'-bis(pheny methyi ) - i Y 2 - e t h a n e -
diami ne ( 2 : i ) , t e t r a h y d r a t e .
Generic-names
Benzathi ne Peni c i iiin G, D i benzyi e t h y enedi ami ne-Di peni -
c i i i i n G, Benzathine B e n z y i p e n i c i i i i n , P e n i c i i i i n G Ben-
z a t h i ne.
-----------
Trade names
Tardociiiin, Extenciiiine ,
P a n s t r i i i i n e , Penidurai
C i t o c y i iina , Diami n o c i i i i n a , Wyci iii n a , Beaci i i in , Pen-
duran, B i c i i i i n , Moidamin, Retarpen, Pronapen, T r i - P e n i -
i e n t e , Debeci i7 i n , Retaci iii n , Penadur.
S
~ c H ~ - c o - N H - C H- CH/ 4\ P 3
' 'f'CH3
CH2 CH2
4
C-N- ' 7 'CHCOO 2
+I 1 2 I+
HN-CH2-CH2-NH.4 H20
C48H56N608S.4 H20 Moiecuiar Weight: 9 8 i , i 9
CA-Registry no. 1-1538-09-6-7
i. 2 . Appearance, Color, Odor
White, o d o r i ess , c r y s t a i iine powder.

PENICILLIN-G BENZATHINE 465
i.3. Compendiai References
P e n i c i i i i n G benzathine i s i i s t e d i n t h e f o i i o w i n g compen-
d i a : The European Pharmacopoeiai, t h e U n i t e d S t a t e s Pharma-
copoeia w i t h t h e Code o f Federai Regulations' and t h e B r i -
t i s h Pharmacopoeia. 3
i .4. D e f i n i t i o n o f I n t e r n a t i o n a i U n i t
One mg of pen c i i i i n G benzathine r e p r e s e n t s i 2 i i pen

c i i i i n units.
7.5. Content
The US Pharmacopoeia demands a c o n t e n t o f n o t i e s s t h a n

57.9 % and n o t more than 77.6 % of p e n i c i i i i n G and n o t
i e s s than i 0 5 0 and n o t more t h a n i 2 7 2 U/mg.
The European and B r i t i s h Pharmacopoeias r e f e r t o t h e sub-

stance w i t h o u t water. I t s h o u i d c o n t a i n a t i e a s t 96.0 %
t o t a i p e n i c i i i i n and 24.0 t o 27.0 % benzathine.
2. PRODUCT I ON
Szabo Edwards and Bruce4 d e s c r i b e d a f r a c t i o n a t e d a d d i -

t i o n o f a s o i u t i o n o f benzathine a c e t a t e i n w a t e r t o a
s o i u t i o n o f p e n i c i i i i n - N a i n water. When 60 % o f t h e benza-
t h i n e a c e t a t e i s added, t h e p r e c i p i t a t e i s f i i t e r e d and
washed w i t h w a t e r . To t h e f i i t r a t e a r e added 35 % o f t h e
benzathine a c e t a t e and t h e p r e c i p i t a t e t r e a t e d i i k e b e f o r e .
F i n a i i y 5 % o f t h e benzathine a c e t a t e a r e added t o t h e fii-
t r a t e . T h i s f r a c t i o n a t i o n method y i e i d s a m i c r o c r i s t a i i i n e
powder. Dropping b o t h m o i e t i e s simui t a n e o u s i y i n t o w a t e r ,
a p r o d u c t w i t h c r y s t a i s o f about i 0 0 um s i z e w i i i r e s u i t
/
a f t e r washing w i t h acetone, w a t e r and d r y i n g .
When t h e p r e c i p i t a t i o n medi um c o n t a i n s formamide the for-

466 FRANZ KREUZIG
mation o f needies i s prevented, which i s i m p o r t a n t f o r

p a r e n t e r a i appi i c a t i o n .
5
F o r o r a i a d m i n i s t r a t i o n a product o f inhomogeneous c r y s t a i
form i s o b t a i n e d by r e c r y s t a i i i s a t i o n from w a t e r and ace-
4
tone.
3. PHYSICAL PROPERTIES
3.7. S o i u b i i i t y
P e n i c i i i i n G benzathine i s p r a c t i c a i i y i n s o l u b i e i n c h i o r o -
form and e t h e r . The s o i u b i i i t y i n w a t e r i s a t h e r iow.
4
Szabo, Edwards and Bruce determined t h e so u b i i i t y i n f o r -
mamide, acetone , ethanoi , benzene and water a t d i f f e r e n t
temperatures .
Soi u b i 1 it y (mg/mi ) o f Peni c i i7 i n G Benzathi e i n
Formami de/Water M i x t u r e s a t D i f f e r e n t Temperatures
Formami de [-%-7 273K( O°C) 298K( 25OC) 3 i 5K( 4 2 O C ) 333K( 6OoC)
i00 2 i .2 28.0 92.5 7 58.0

95 3.2 3.8 72.0 43,O
90 3.2 3.2 i0.3 3 i .6
75 1.4 7.4 3.6 i3.2
50 7 .o i .o 2.5 5.7
25 0.3 0.4 0.7 2.0
S o i u b i i i t y (mg/mi) o f P e n i c i i i i n G Benzathine i n
D i f f e r e n t So7 vents a t D i f f e r e n t Temperatures
Temp. [-K(OC)-i acetone ethanoi benzene water
qnc 1 1 9 \ i c c') n 90 n i c
_.- I, -L- 3I 1
LYO I .3 3- ..L
- U . J.~
O u. 13
3 i 3 (40) 3.8 16.2 0.55 0.i7

322 (49) 4.3 23.9 0.72 0.27
334 ( 6 i )
I ,
--- 52.9 i. i o 0.46
3.2. M e i t i n g Range
A decomposition p o i n t o f 383 K (iiO°C) i s given6, t h e de-

termination o f the m e i t i n g point, according t o L. K o f l e r ,
g i v e s an i n t e r v a i from 385 - 388 K ('172 - ii5OC)? Crystais,
embedded i n i drop of i i q u i d p a r a f f i n e , w i l l decompose
between 403 and 409 K (130 and i36OC).
5
Other authors found 383 - 408 K (710 - i35OC).
3.3. O p t i c a i R o t a t i o n
P e n i c i i i i n G benzathine i s d e x t r o r o t a t o r y :
6
!'a-7i5 = t 206' ( c = i , methanoi ) .
3.4. C r y s t a i P r o p e r t i e s
These p r o p e r t i e s depend on t h e method o f p r e c i p i t a t i o n .

The c r y s t a i s shouid have a shape so as t o be suspended
e a s i i y i n a i i q u i d c a r r i e r f o r i n j e c t i o n . Adding forrnamide
t o the i i q u i d f o r p r e c i p i t a t i o n prevents the formation o f
needies and forms p i a t e - i i k e c r y s t a i s , which can be broken
t o an average p a r t i c i e s i z e o f 750 urn. 5
I
A method f o r t h e d e t e r m i n a t i o n o f t h e f r i a b i i i t y ( i n g/sec)
was given.8 The importance o f t h e a n g i e o f n a t u r a l s l o p e 9 ,
d i s p e r s i o n c h a r a c t e r i s t i c s , t h e cohesiveness and w e t t a -
b i i i t y have been discussed
i o.
3.5. U i t r a v i o i e t Spectrum
A spectrum of a m e t h a n o i i c s o l u t i o n o f p e n i c i l i i n G benza-
t h i n e (500 ug/mi), o b t a i n e d w i t h a Z e i s s DM 4 s p e c t r o -
I
photometer, i s shown i n f i g u r e i: t h e r e l e v a n t parameter
i s t h e absorbance o f t h e s h o u l d e r a t 263 nm, which can be
measured f o r spectrophotornetric assay.
250 300nm
Figure i : UV-Spectrum (see chapter 3.5.)
3.6. Infrared Spectrum

The infrared spectrum of p e n i c i i i i n G benzathine, obtained
as a KBr-tabiet ( 1 . 2 mg i n 300 mg), was r u n on a Perkin
Eimer i 7 7 . I t i s shown i n figure 2.
The most s i g n i f i c a n t s t r e t c h i s t h a t a t i780 cm-l which is
assigned t o the l3-iactam structure.
3.7. Nuciear Magnetic Resonance Spectrum

The proton NMR spectrum was obtained i n DMF-d7 solution
containing TMS as internal reference, u t i l i z i n g a Varian
EM-390 NMR spectrometer operating a t a frequency of 90 MHz
(see figure 3 ) .
Chemicai s h i f t s Muitipiicity Intensity Assignment
( 6 Y ppm)
8.60 d ( J = 7 Hz) iH -CO-NH--
7.30 m i0 H Phenyi -H-
5.60 S 6H tNH t H20
-2
5.50 2 d 2 H H-5’ -6 H
4.20 S iH H
-3
4. i o S 2H -
Phenyi -CH2-N
3.70 S 2 H Phenyi-CJ2-C0
3.20 S 2 H -2 -CH
N-CH -2 - N
i .60 S 3H 2R-CH-3
i .50 S 3H 2a-CH
-3
d = doubiet, s = s i n g i e t , m = muitipiet, J = coupling constant
These data a r e in good aqreement w i t h the paper of Wii-

ii
son e t a i .
3.8. Mass Spectrum

The mass spectrum of p e n i c i i i i n G benzathine was run on
a Varian MAT 3-11 in the f i e i d desorption mode (heating
W
W
v)
470
A
*---
--L- 7 - I I I
END OF SWEEP
I
600 JUO 150 0
I
3+ 1 .n
1 I20 , CO
1
.- f--
t- - I .
, !
-t - --
.... .-
. .. . --
I !I :.
_c
Figure 3: NMR-Spectrum (see chapter 3 . 7 . )

472 FRANZ KREUZIG
current i 0 - 20 mA). I n t e n s i v e i o n s w i t h t h e mass number

o f 5 7 5 can be a t t r i b u t e d t o p e n i c i i i i n G + benzathine + H+,
w h i i s t 9iO corresponds t o an a s s o c i a t e composed o f two
peniciiiin G + benzathine + Ht ( f i g u r e 4 ) .
4. STABILITY, DEGRADATION, ARTEFACTS
P e n i c i i i i n G benzathine i s very s t a b i e because o f i t s iow

s o i u b i i i t y i n w a t e r and b i o i o g i c a i media, r e s p e c t i v e i y .
Being d i s s o i v e d i n aqueous media, t h e p e n i c i i i i n G moiety
wiii degrade i n a r a t h e r compiex manneri2, which c o u i d be
i3
e l u c i d a t e d by NMR s t u d i e s .
These degradation p a t t e r n s may be compiicated by t h e amino-

i y s i s o f p e n i c i i i i n G by t h e benzathine c a t i o n . I t c o u i d
be demonstrated t h a t t h e r e a c t i o n of i,Z-diami noethane
(monocation) w i t h p e n i c i i i i n G g i v e s an increased r e a c t i o n
r a t e compared w i t h t h e monoamine o f s i m i i a r b a s i c i t y . This
i s a t t r i b u t e d t o i n t r a m o i e c u i a r generai a c i d c a t a i y s i s
which i n t u r n i n d i c a t e d t h a t n u c i e o p h i i i c a t t a c k takes
p i a c e from t h e l e a s t hindered a - s i d e i n disagreement w i t h
i4
t h e p r e d i c t i o n o f t h e t h e o r y o f s t e r e c e i e c t r o n i c control: .
5. BIOPHARMACEUTICS
5. i Pharmacokinetics
Because o f i t s iow s o i u b i i i t y i n w a t e r p e n i c i i i i n G benza-

thine i s the iongest acting p e n i c i i i i n ; the resultinq
c o n c e n t r a t i o n i n serum and u r i n e r e s p e c t i v e i y are, o f
course, r a t h e r low. On t h e o t h e r hand t h i s drug o f f e r s ,
according t o t h e amount administered, t h e r a p e u t i c p e n i -
c i i i i n G i e v e i s f o r about one month.
I- I00
I
P
w
I
Y
Q:
50
W
L
I-
4
w
a
500 600 700 800
MASS NUMBERS
Figure 4 : Mass-Spectrum (see chapter 3 . 8 . )

414 FRANZ KREUZIG
Peni c i iii n G benzathi ne, administered intramuscui a r i y , i s

s u i t e d f o r t h e p r o p h y i a x i s and l o n g term therapy o f syphi-
i i s and rheumatic diseases. E i i a s e t aiei5 f o l i o w e d serum
p e n i c i i i i n i e v e l s i n man a f t e r i n j e c t i o n o f t h i s compound,
and conciuded t h a t i t showed no s i g n i f i c a n t t o x i c i t y and
gave serum p e n i c i i i i n i e v e i s o f g r e a t e r d u r a t i o n than any
reported w i t h o t h e r forms o f r e p o s i t o r y peni c i i1i n s .
When 2.400.000 I U o f p e n i c i i i i n G benzathine are i n j e c t e d ,

the bioassay showed t h e existence of mean l e v e l s above
0.036 I U / m i serum and above 20 I U / m i u r i n e f o r t h e d u r a t i o n
o f 26 days. The corresponding values f o r one hour a f t e r
16
t h e i n j e c t i o n were 0.3 and 234 I U / m l r e s p e c t i v e l y .
The i n j e c t i o n o f p e n i c i i i i n G benzathine suspensions may

cause pain, t h e r e f o r e i o c a i a n e s t h e t i c s are added.
5.2. Metaboi ism
The p e n i c i i i i n G moiety of t h e drug decomposes i n p r i n c i -

pa7 as mentioned i n chapter 4.
6. METHODS OF ANALYSIS
6.i. Identification
The sampiei y3 i s shaken w i t h 0.1 N NaOH, e x t r a c t e d w i t h

ether, evaporated, t h e residue i s d i s s o i v e d i n ethanoi.
A f t e r the addi t i on o f p i c r i c acid, h e a t i ng and cooi ing ,
the p r e c i p i t a t e d c r y s t a i s a r e separated; t h e i r me7 ti ng
p o i n t i s 487 K (2i4OC).
Simoesi7 d i s s o i v e d t h e sampie i n 0.2 M NaOH and e x t r a c t e d
t h e benzathine moiety w i t h e t h e r . A f t e r evaporating t h e
residue, H C i (-10 % ) and NaN03 (10 % ) are added, t h e mix-
t u r e i s e x t r a c t e d w i t h e t h e r , t h e e t h e r i s evaporated and
t h e r e s i d u e mixed w i t h H2S04. A green c o i o r i s observed.
A d d i t i o n o f w a t e r g i v e s a p i n k c o i o r , a f t e r adding NaOH
t h e c o i o r changes t o b i u e .
6.2. Iodometric T i t r a t i o n
The f i r s t method was d e s c r i b e d by Aiicinoi8. Underivatized

p e n i c i i i i n i s i n e r t t o i o d i n e i n n e u t r a i aqueous s o i u t i o n .
A f t e r h y d r o i y s i s w i t h a7 k a i i , t h e r e s u i t i n g p r o d u c t s con-
sume 8 t o 9 moies o f i o d i n e . The d i f f e r e n c e o f t h e i o d i n e
consumption b e f o r e and a f t e r h y d r o i y s i s i s p r o p o r t i o n a i t o
t h e q u a n t i t y o f p e n i c i i i i n . P e n i c i i i i n G consumes 8.97
e q u i v a i e n t s o f i o d i n e p e r moi; t h i s r a t i o depends on t h e
c o n d i t i o n s o f assay, t h e r e f o r e a b i i n d sampie has t o be
taken i n t o account. The h y d r o i y s i s t i m e i s i 5 - 30 m i n u t e s .
Ei-Sebai e t a i . i 9 d e s c r i b e d t h e a n a i y s i s o f p e n i c i i i i n G
benzathine i n 0.067 M Na2HP04 s o i u t i o n w i t h JCi i n H C i -
s o i u t i o n ; t h e p e n i c i i i i n l i b e r a t e d was t i t r a t e d w i t h
0.05 M KJOg i n CHCi3; t h e e n d - p o i n t was t h e disappearance
o f t h e c o i o r f r o m t h e CHCi3-iayer.
The i n f i u e n c e o f i o d i n e consuming i m p u r i t i e s a r e discussed

by L e B e i i e e t a i . 2 0 who proposed a m o d i f i c a t i o n o f t h e
i o d o m e t r i c method and found t h a t benzathine and i o d i n e
2i
form a benzimidazoiinium s a i t
which i n t e r f e r e s i n t h e i o d o m e t r i c assay.
476 FRANZ KREUZIG
6.3. Nonaqueous T i t r a t i o n
F o r t h e d e t e r m i n a t i o n o f t h e b e n z a t h i n e m o i e t y 3 t h e sub-
stance i s d i s s o i v e d i n a s o i u t i o n o f NaCi and NaOH, ex-
t r a c t e d w i t h e t h e r , t h e combined e x t r a c t i s washed w i t h
water, t h e combined washings a r e e x t r a c t e d w i t h e t h e r ,
t h e e x t r a c t s a r e evaporated t o dryness, t h e r e s i d u e i s
d i s s o i v e d i n anhydrous g i a c i a i a c e t i c a c i d and t i t r a t e d
w i t h 0 . i M HCi04, u s i n g i - n a p h t h o i - b e n z e i n as i n d i c a t o r ;
t h e o p e r a t i o n i s repeated w i t h o u t t h e substance t o be
t e s t e d ( b i ank v a i ue) .
i m i o f 0 . i M HCi04 i s e q u i v a l e n t t o O.Oi202 g o f benza-
thine.
6.4. Spectrophotometri c Assay
P e n i c i i i i n G i n p e n i c i i i i n G benzathine i s determined by
measuring t h e e x t i n c t i o n o f a s o i u t i o n o f 50 mg o f sampie
i n -100 m i a b s o l u t e methanoi a t 263 nm i n comparison t o a
r e f e r e n c e s t a n d a r d2 .
HoibrookZ2 determined p e n i c i i i i n G b e n z a t h i n e a f t e r hea-

t i n g t h e substance w i t h a Na-acetate/CH3COOH s o i u t i o n
(pH = 4 . 6 ) , c o n t a i n i n g a t r a c e of CuS04. The absorbance
o f t h e r e s u i t i n g p e n i c i i i i n a c i d i s measured. The method
has a s t a n d a r d e r r o r o f 3 % and i s a p p i i c a b i e t o prepara-
t i o n s w i t h a potency of a t l e a s t 0.5 U/rng.
I t has been found t h a t benzathine forms a w a t e r s o i u b i e

~ . absorbance i s measu-
compiex w i t h B i e b r i c h ~ c a r i e t ~The
r e d a t 5-16 nm and i s r e l a t e d t o t h e p e n i c i i i i n G benza-
t h i n e c o n t e n t . The same a u t h o r s 2 4 s t a t e d t h a t p e n i c i i i i n
forms a compiex w i t h methyiene b i u e , which i s measured
a t 655 nm and shows c o r r e l a t i o n w i t h t h e p e n i c i i i i n G
b e n z a t h i ne c o n t e n t .
For the determination o f p e n i c i i i i n G benzathine i n o i n t -

ments t h e r e a c t i o n w i t h n i t r o p r u s s i de-Na and KMn0424 i n
a i k a i i n e s o i u t i o n produces a b i u e c o i o r . The s e n s i t i v i t y
i s 2000 U / m i o f ointment.
6.5. M i c r o b i o i o g i c a i Assay
The sampie i s d i s s o i v e d i n methanoi and f u r t h e r d i l u t e d

w i t h i % phosphate b u f f e r pH = 6.4 so as t o g i v e a con-
c e n t r a t i o n o f i . 0 U o f p e n i c i i i i n G/m7. The a g a r d i f f u s i o n
assay i s performed w i t h Staphylococcus aureus (ATCC
29727)*.
6.6. Thin-Layer Chromatography
Layer Soivent Systems V i suai ization Ref
i. ceiiuiose CH30H/2-butanoi/CHCi 3/HCi 70 % =
J2 25
= iO/iO/iO/i
2. siiica gei + CH30H/i-butanoiiCHCi3/CH3COOH =

J2 26
cei i ui ose = iO/lO/iO/i
3. si i i ca gei butyi acetate/CH3COOH/MeOH/i-butanoi / FeCi3 + K3!-Fe(CN)6-7 + HCi 27

phosphate buffer pH=7.3 = 80/4/5/i 5/20
4. siiica gei acetone/HCOOH = 95/5 FeCi3 + K3!-Fe(CN)6-7 + H2S04 28
5. siiica gei isoamyi acetate/CH30H/HCOOH/H20 NaN02 + NH4 suifamate + 28
(upper phase) = 65/20/i0/5 N-( i-naphthyi )ethyiendiamine
6. siiica gel CH3COOH/ butyi acetatel 0.i M phosphate KJ + H2PtCi6 -1. HCi - acetone 29
buffer pH=5.6/i-butanoi/CH30H =
= 20/40/i2/5/7,5
Comments :
ad 1: The d e t e c t i o n l i m i t s were i n t h e range o f 5-30,ug,

i t was s t a t e d , t h a t c e i i u i o s e has advantages o v e r
s i i i c a gei.
ad 2: The a u t h o r s c l a i m t h e ease o f t h e p r o d u c t i o n o f
t h i s p i a t e and t h e s p e c i f i t y o f t h e c o l o r o f t h e
spots a f t e r s p r a y i n g .
ad 3: With t h i s method i m p u r i t i e s and decomposition p r o -

ducts were i d e n t i f i e d and q u a n t i t a t e d v i s u a l l y .
ad 4 and 5: Aithough t h i s method i s used f o r p e n i c i i i i n G pro-

c a i n , i t r e f e r s t o t h e decomposition o f p e n i -
c i i i in G i n aqueous s o l u t i o n s t o benzyi peni c i iiic
a c i d and b e n z y i p e n i c i i i o i c a c i d . F u r t h e r decompo-
s i t i o n o f p e n i c i i i i n G occurs when a i c o h o l i c so-
i u t i o n s a r e used f o r s p o t t i n g t h e chromatograms,
t h e corresponding a1 k y i -a-D-peni c i i io a t e b e i ng
formed. The usefuiness o f d i f f e r e n t spray reagents
i s discussed.
ad 6: T h i s method a l i o w s f o r t h e d e t e c t i o n of p e n i i i o i c
a c i d and p e n i c i i i o i c a c i d amongst t h e main compo-
nents o f p e n i c i i i i n G benzathine.
6.7. High Performance L i q u i d Chromatography
The paper o f L e B e i i e e t a i .30, c o n c e r n i n g t h e HPLC d e t e r -

m i n a t i o n o f p e n i c i i i i n V benzathine, enabies t h e separa-
t i o n o f p e n i c i i i i n G from benzathine too, so i t can be
used f o r t h e i n d i r e c t d e t e r m i n a t i o n o f p e n i c i i i i n G ben-
z a t h i n e . The compounds a r e s e p a r a t e d on a RP 8-coiumn
w i t h t h e e i u e n t CH30H/0.05 M phosphate b u f f e r pH=3.5 =
53/47, t-h-e UV-detector was s e t t o 274 nm. Nachtmann and
G s t r e i n ” have m o d i f i e d t h i s method i n t h e f o i i o w i n g
480 FRANZ KREUZIG
aspects :
Eiuent: CH30H/phosphate b u f f e r 0.06 M t 7 % t r i e t h y i a m i n e
pH= 5.5 = 30/70, T = 323 K ( 5OoC) , h = 220 nm.
With i n c r e a s i n g pH o f t h e e i u e n t t h e r e t e n t i o n time o f
benzathine increases, This method o f f e r s t h e p o s s i b i i i t y
o f the q u a n t i t a t i v e determination o f both m o i e t i e s o f t h e
moi ecui e.
Puttemans e t have shown t h a t t h e e i u t i o n o r d e r o f

benzathine and p e n i c i i i i n can be changed by means o f ion-
p a i r HPLC, a p p i y i ng t h e e i u e n t CH30H/phosphate b u f f e r
4
pH=3.0 = 30/70, adding 5 .
i 0 M tetrabutyiammonium-
b romi de .
7. ACKNOWLEDGMENTS
I am indebted t o D r . B. Prager, Biochemie GmbH, Kundi ,

f o r performing and i n t e r p r e t i n g t h e NMR-spectrum, t o
D r . G. Schuiz, Sandoz F o r s c h u n g s i n s t i t u t GmbH, Wien, and
D r . A. N i k i f o r o v , Organisch-Chemisches I n s t i t u t der Uni-
v e r s i t a t Wien, f o r performing and i n t e r p r e t i n g t h e mass-
spectrum.
a. REFERENCES
7 ) European Pharmacopoeia, Council o f Europe, I 1 1 - i975

2) The United States Pharmacopoeia, Twentieth Revision,
7980, w i t h t h e N a t i o n a i Formulary, F i f t e e n t h E d i t i o n ,
-1980, United States Pharmacopoeia7 Convention, I n c .
3) B r i t i s h Pharmacopoeia 7980, London, Her M a j e s t y ' s
S t a t i o n e r y O f f i c e , 7980
4 ) J.L. Szabo, C.D. Edwards and F.W. Bruce, i n R. Brunner,
G. Machek: Die A n t i b i o t i k a , i/i Aiigemeiner T e i i -
PENICILLIN-G BENZATHINE 48 1
P e n i c i i i i n , V e r i a g Hans C a r i , Nurnberg, 7962, p. 344

5 ) D.P. 947 826
6 ) J.L. Szabo, C.D. Edwards and F.W. Bruce, i n R. Brunner,
G.Machek: D i e A n t i b i o t i c a , i/i A i i g e m e i n e r T e i l -
P e n i c i i i i n , V e r i a g Hans C a r i , Nurnberg, 7962, p . 259
7) U s t e r r e i c h i s c h e s Arzneibuch, 9. Ausgabe, 11. Band,
U s t e r r e i c h i s c h e S t a a t s d r u c k e r e i , Wien, i 9 6 9 , p . 376
8 ) M.L. E z e r s k i i : Khim.-Farm. 4, 54 (7970)
Zh. -
9 ) M.L. E z e r s k i i : Khim.-Farm. Zh. -
4, 47 (1970)
i 0 ) M.L. E z e r s k i i , G.M. Pismennaya: Khim.-Farm. 9, 9 i
Zh. -
( i 976)
__
1 1 ) W.L. W i i s o n , H.W. A v d o v i c h and D.W. Hughes: J. AOAC -
57,
i300 (i974)
i 2 ) M.A. Schwartz and F.H. B u c k w a i t e r : J. Pharm. S c i . -
5i,
i i i 9 (i962)
i 3 ) F. M i t s u m o r i , Y . A r a t a , S. F u j i w a r a , M. Muranaka and
Y . H o r i u c h i : B u i i . Chem. SOC. Jap. -
50, 3764 ( i 9 7 7 )
i 4 ) A.F. M a r t i n , J.J. M o r r i s and M . I . Page: J.C.S. Chem.
6, 298 ( i 9 7 9 )
Comm. -
i 5 ) W. E i i a s , A.H. P r i c e and H.J. M e r r i o n : A n t i b i o t . and
i, 4 9 i ( i 9 5 i )
Chemother. -
i 6 ) H. Fiamm, A. Luger, F. P a v i i k and M. R o t t e r : Wien.
K i i n . Wschr. -
88, 384 (1976)
i 7 ) R. Simoes, i n Vogei: Textbook o f P r a c t i c a i O r g a n i c
Chemistry , Longmans, London , 7 948 , p. 627
i 8 ) J. A i i c i n o : I n d . Eng. Chem., A n a i . Ed. -
i8, 6 i 9 (i946)
79) I. E i - S e b a i , S.M. Rida, Y.A. B e i t a g y , M.M.A. Ei-Khaiek:
Pharmazie -
29, i 4 3 (1974)
20) M.J. L e B e i i e and W.L. W i i s o n : J. Pharm. S c i . -
67, 1495
( i 978)
27) M.J. L e B e i i e and W.L. 68, i 3 2 2 ,
W i l s o n : J. Pharm. S c i . -
( i 979)
22) A. H o i b r o o k : J. Pharm. Pharmacoi. -
i 0 , 762 ( i 9 5 8 )
482 FRANZ KREUZIG
23) H.M. Nour Ei-Din, Y.M. Dessouky, M. E l - K i r d a n i : B u l l .

Fac. Pharm., Cairo Univ., -
i 0 , 779 (797'1)
24) G. Miihaud, L. P i n a u i t , J.P. Moretain: Am. F a i s i f .
Expert. Chim. -
68, i 9 i (1975)
25) G.S. Chung, R.T. Wang Tai-Wan K ' o Hsue, -
27, 27 (1973)
26) G.S. Chung, R.T. Wang Hua Hsueh -
4, 122 (7977)
27) J.J. Cruceanu, M. Med anu, E. Aiteanu and A. Moldovan:
Zen t r a 7 b 7 a t t P ha r m . Pha rmak o t he r . Labo r a t o riums d ia g n .
-
776, 25i (1977)
28) J.R. Fooks and G.L. Mattok: J. Pharm. Sci -
58, i357
( i969)
29) W.J. Wilson, M.J. L e B e i l e and K.G Graham: J. Pharm.
Sci. -
i 4 , 27 (7979)
30) M. LeBeile, K. Graham and W.J. W i son: J. Pharm. S c i .
-
68, 555 (7979)
3i) F. Nachtmann and K. G s t r e i n : J. Chromatogr., submitted
f o r pubiication
32) M. Puttemans, L. Dryon and D.L. Massart: J . Chromatogr.,
i n press
PHENYLBUTAZONFI
Syed Laik Ali
1.History 484
2.Description 484
2.2 Appearance, Colour, Odour 484
3. Synthesis 484
4.2 Solubility 486
4.4 Loss on Drying 487
4.9 Differential Scanning Calorimetry (DSC), 493
Differential Thermal Analysis (DTA),
Thermogravimetry (TG)
4.10 X-Ray Diffraction Spectrometry 497
4.11 Scanning Electron Microscopy 499
5. Colour Reactions 499
6. Degradation and Stability 502
7. Dissolution 507
References 518
Analylical Profiles of Drug Subslances Copyright 0 1982 by The American

Volume I I 483 PharmaceuticalAssociation
ISBN 0-12-260811-9
484 SYED LAIK ALI
1. History
After second world war a German Chemist Hans
Stenzl synthesised phenylbutazone, 1,2-diphenyl-
4-n-butylpyrazolidin-3,5-dion in the research
laboratories of J.R. Geigy in Basel, Switzerland
(1). This compound formed water-soluble salts and
was used in combination with aminophenazone in in-
jection solutions. Phenylbutazone was brought in
the market in 1952 as a single preparation under
the trade-name of Butazolidin. It has remarkable
antirheumatic and antiphlogistic properties.
2. Descr iption
2.1. Name, Formula, Molecular Weight
4-Butyl-1,2-diphenyl-3,S-pyrazolidinedione
C19H20N202 308.38
2.2. Appearance, Colour, Odour

A white or almost white, crystalline powder,
practically odourless, with a slightly bitter
taste.
3. Syn thesi s
The first synthesis was carried out by heatincl
n-butyl diethyl malonate with hydrazobenzene and
sodium alcoholate ( 3 ) .
PHENY LBUTAZONE 485
n-CCLHS-CH-COOC2Hg + HN-c&
I I
COOC2H5 HN-C6H5
Another method of synthesising phenylbutazone (3)

consists of reacting diethyl malonate with
hydrazobenzene which is further treated with
crotonaldehyde and subsequently hydrogenated
catalytically.
I
486 SYED LAIK ALI
I
C6H5
H2
I
C6H5
4.1.Melting Range (4): Phenylbutazone melts
between 104 and 107OC.
4.2. Solubility (5) : The solubility o f phenylbu-
tazone in various solvents at 2 0 0 ~is given in
Table 1 as 1 part per specified parts-solvent.
TABLE 1
Solubility of Phenylbutazone at 2OoC (5)
Solvent Solubility
Water practically insoluble
Ethanol 28 parts
Methanol 18 parts
Ether 15 parts
Chloroform 1.3 parts
Aceton 2.5 parts
Benzene 3.5 parts
PHENYLBUTAZONE 481
4.3. D i s s o c i a t i o n C o n s t a n t
P h e n y l b u t a z o n e h a s a n a c i d i c h y d r o g e n atom a t C -
4 . The P k a d e t e r m i n e d i s r e p o r t e d t o be 4.89
(6). P h e n y l b u t a z o n e is c o n s i d e r e d a c a r b o n a c i d
(carbon acids are acids i n which t h e d i s s o c i a t i n g
p r o t o n is bound t o a c a r b o n atom i n s t e a d of h e t r o -
atoms s u c h a s oxygen or n i t r o g e n ) a n d t h e P k a
v a l u e b e t w e e n 4.5 - 4.7 h a s a l s o b e e n g i v e n
( 7 , l l ) . The d e p e n d e n c e o f Pka v a l u e s o f p h e n y l -
b u t a z o n e on t h e s o l v e n t medium h a s a l s o b e e n
reported ( 8 ) . Pka v a l u e s i n pure m e t h a n o l , e t h a -
n o l and water a r e g i v e n a s 5.42, 5.76 a n d 5.07
respectively (8).
4.4. Loss on Dryin

When d r i e d i n vacuu: a t 8OoC a n d a t a P r e s s u r e
o f 30 2 1 0 mm o f m e r c u r y f o r 4 h o u r s ii looses n o t
more t h a n 0.5 % of i t s w e i g h t ( 9 ) .
4.5. U l t r a v i o l e t Spectrum (10)

Phenylbutazone i n s o l u t i o n absorbs u l t r a v i o l e t
r a d i a t i o n to produce a spectrum w i t h d i f f e r e n t
maxima i n d i f f e r e n t s o l u t i o n s . I n m e t h a n o l , 0 . 1 N
HC1 a n d 0 . 1 N NaOH i t h a s a b s o r p t i o n maxima a t
243, 235 a n d 263 nm r e s p e c t i v e l y . The corres o n -
d i n g molecular e x t i n c t i o n c o e f f i c i e n t s a n d Ale,, !?%
a r e r e p o r t e d t o be ( 1 0 ) :
Methanol 0 . 1 N HC1O.l N N a O H
Absorption 243 nm 235 nm 263 nm

Maximum
A%
A I'wl 482 440 669
€ 14860 13570 20630
The U V s p e c t r a a r e g i v e n i n F i g . 1. T h e a b s o r p t i o n
maxima i n a c i d a n d a l k a l i n e s o l u t i o n s a t 232 nm
a n d 264 nm h a v e a l s o b e e n r e p o r t e d (37).
4.6. I n f r a r e d Spectrum (10,11,62)
The i n f r a r e d spectrum o f p h e n y l b u t a z o n e is g i v e n
i n F i g . 2 . The spectrum was o b t a i n e d w i t h a
P e r k i n - E l m e r 257 s p e c t r o p h o t o m e t e r from a KBr
p e l l e t . T h e r e is a good a g r e e m e n t w i t h t h e s p e c t r a
r e p o r t e d i n l i t e r a t u r e ( 1 0 , 1 1 , 6 2 ) . The s t r u c t u r a l
YO
F i g . 1. UV Spectrum of P h e n l y b u t a z o n e in -(Methanol),
--- ( ) .1N NaOH) , - - (0.1N HCL)
488
Microns
25 3a Lo 50 60 70
03
P
W
-r (01- 1 1
F i g . 2. I R Spectrum of P h e n l y b u t a z o n e , KBr P e l l e t , P e r k i n -
E l m e r 257 S p e c t r o p h o t o m e t e r
490 SYED LAIK ALI
assignments may be correlated with the following

band frequencies .
Frequency (cm-1 ) Assignment
1720 and 1755 Characteristic stretching
vibrations of C = 0 group.
1600 and 1490 Characteristic skeletal
stretching vibrations of the
aromatic ring.
1300 Characteristic band of
dioxopyrazolidine compounds.
760 - 695 Bands of monosubstituted
phenyl.
4.7. Nuclear Magnetic Resonance Spectrum
The nuclear magnetic resonance spectrum of
phenylbutazone as shown in Fig. 3 was obtained
on a Varian T-60 NMR spectrometer in deuterated
chloroform containing 1 % tetramethylsilane as the
internal standard. The following spectral
assignments are made for Fig. 3:
Chemical Shift ( & ) Assignment
0.95 -
2 ppm
doublet and multiplet
Protons of n-butyl rest.
3.35 ppm Proton at C - 4 position.

triplet
7.30 ppm Aromatic phenyl protons.
mu1 t iple t
There is a good agreement with the reported values
in literature (62).
4.8. Mass Spectrum (121
Mass spectrum is given in Fig. 4
Instrument: Varian CH5
Sample temperature (direct inlet): 8OoC
Source temperature: 18OOC
Electron energy: 70 eV
The prominent ions of this spectrum can be
correlated to the structure as following:
m/e 308 = M 183 = C6H5NHNC6H5
265 = M -
C3H7 105 = CgHtjN2
252 = M -
C4H8 77 = CgH5
I . . ..I . . ..
I , . . . l . . . . 1 . . . * l , * ,
1 . . , , ' i ., . , " i . . " . i ~ " . ~ i ~ ' ~ ~ . i 1 . ' ~ * '
I
8.0 7.0 61) 5.0 m(6) 6.0 3.0 20 I .o 0
Fig. 3. N M R Spectrum of Phenlybutazone, Varian T60 Spectrometer.

103
0
10: 70eVV1180.C. Diddinlet: 8O'C
Fig. 4. Mass spectrum of P h e n l y b u t a z o n e

PHENY LBUTAZONE 493
The elemental compositions of these ions have been

determined by high resolution mass spectrometry
(instrument: CEC 21 - 110) and are in agreement
with the assignments made above (12).
The fragmentation pathways of Phenylbutazone and
oxyphenbutazone were established by means of deu-
terium labeling, metastable peaks and accurate
mass determinations. The major pathways are the
McLafferty rearrangement of the molecular ion and
formation of azobenzene and substituted azobenzene
ions (13). The mass spectra of the methyl deriva-
tives have also been discussed (13,14).
4.9. Differential Scanning Calorimetry (DSC),
Differential Thermal Analysis (DTA), Thermogravi-
metry (TG)
The solid-solid transition of phenylbutazone poly-
morphs by heating was investigated by DSC and DTA
(15). Three polymorphs of phenylbutazone were ob-
served. Form I melted at 103O, Form I1 twice at
93O and 103O and Form I11 at 93O (15). Form
I1 melted to an opaque paste at 93O, solidifying
immediately and melting again at 103OC. Further-
more, the mutual transition phenomenon among these
three polymorphs was observed by heating through
DSC (15). In another investigation solid-solid and
solid-liquid transitions of phenylbutazone poly-
morphs were investigated by DSC, DTA and TG (16).
Four polymorphs were observed with transition
points at 93.4, 95.1, 106.0 and 107.5OC (16).
The curves are reproduced in Fig. 5. In addition
two pseudopolymorphs from cyclohexane and isobu-
tanol were determined containing one mole solvent
to three moles phenylbutazone. The mutual transi-
tion behaviour of the polymorphs was investigated,
the melting enthalpies determined and the energy
of activation calculated from DSC-data. The
DSC-curves of the different crystalline modifica-
tions were recorded on a Dupont 990 thermal analy-
zer, equipped with a DSC-cell 910 and calibrated
with Indium (16). Thermal data of phenylbutazone
polymorphs is given in Table 2, DSC and DTA/TG
curves are reproduced in Fig. 6 (16).
494 SYED LAIK ALI
MODIFICATION
if i f
2- PROPANOL
RECRYST. FUSION OF 4
80 90 100 T(YC
F i g . 5. DSC-curves of p o l y m o r p h i c forms of p h e n l y b u t a z o n e
and t h e i r m i x t u r e s o b t a i n e d by c r y s t a l l i z a t i o n f r o m d i f f e r e n t
s o l v e n t s ( h e a t i n g r a t e 10°C/min, s e n s i t i v i t y 0.5 mcal/s i n ,
sample w e i g h t 2-3 mg).
PHENYLBUTAZONE 495
I \
a) from cyclohcxane; b) from isohutanol
TG7
cycloherane
Fig. 6. Simultaneous DTA/TG-curves of phenylbutazone

solva tes
496 SYED LAIK ALI
TABLE 2
Thermal Data of Phenylbutazone Polymorphs (16)
Polymorph Transition Enthalpy of
temperature OC transition
J/9
w 93.4 79.3
P 95.1 71.1
t 106.0 -
b 107.5 72.3
The thermal behaviour of the polyrnorphs under
different treatment conditions has also been
investigated (17). Compression of the
thermodynamically unstable forms at a*compression
force of 1590 - 2040 Kg induced polymorphic changes
in the crystals. Similar changes were also produced
through grinding. The apparent equilibrium
solubilities of 4 polymorphs at their transition
temperatures were determined (17).
In Table 3 different transition temperatures and
peak solubilities of the different polymorphs are
given .
TABLE 3 (17)
Differential Scanning Transition Temperatures and

Peak Solubilities of the Different Polymorphs (171
Form Solvent O F Transition Peak Solubility in
Crystalli- Temperature Phosphate buffer,
zation pH 6.95, at
36OC,
mg/100 ml
I Isobuty 1 8OoC 288.7

alcohol
I1 -
Cycloh e 90% 279.9
xane
I11 n- Heptane 93% 233.6
IV 2- Propa- 1050C 213.0
no1-Water
PHENY LBUTAZONE 491
Polymorphism o f p h e n y l b u t a z o n e b y a s p r a y - d r y i n g
method h a s a l s o b e e n i n v e s t i g a t e d r e c e n t l y ( 1 8 ) .
DTA-curves o f a s p r a y - d r i e d p h e n y l b u t a z o n e from 5 %
methylene c h l o r i d e s o l u t i o n are given i n Fig. 7
(18)
The i n v e s t i g a t i o n o f a r e f e r e n c e s u b s t a n c e o f
p h e n y l b u t a z o n e t h r o u g h DSC i n a c l o s e d s y s t e m g a v e
following results (19):
Impur i t i e s 0.2 - 0 . 1 Mol%
Melting p o i n t 105.5OC
L a t e n t h e a t of f u s i o n 6. 8 K cals./Mol
4.10. X-Ray D i f f r a c t i o n S p e c t r o m e t r y
The c r y s t a l s t r u c t u r e o f p h e n y l b u t a z o n e h a s b e e n
d e t e r m i n e d w i t h x - r a y d i f f r a c t i o n method ( 2 0 , 2 1 1 .
T h i n n e e d l e - l i k e c r y s t a l s o f p h e n y l b u t a z o n e , ob-
t a i n e d a f t e r c o n t r o l l e d e v a p o r a t i o n of a n a l c o h o l i c
s o l u t i o n were u s e d f o r measurements ( 2 1 ) . The u n i t
c e l l d i m e n s i o n s o f t h e c r y s t a l s were d e t e r m i n e d
from o s c i l l a t i o n s a nd W e i s s e n b e r g p h o t o g r a p h s t a k e n
about c r y s t a l l o g r a p h i c a x e s u s i n g n i c k e l - f i l t e r e d
c o p p e r r a d i a t i o n . The c e l l d i m e n s i o n s o f t h e
c r y s t a l s o f p h e n y l b u t a z o n e were r e f i n e d o n a com-
p u t e r - c o n t r o l l e d H i l g e r a n d Watts 4-circle d i f f -
r a c t o m e t e r . The c r y s t a l d a t a for p h e n y l b u t a z o n e i s
given i n Table 4 (21).
TABLE 4
C r y s t a l Data f o r P h e n y l b u t a z o n e ( 2 1 1
Space group p21/ c

a i n Ao 21.695 + 0.004
b i n Ao 5.823 T 0.002
c i n Ao 27.861 7 0.004
i n degrees 108. 06 0.10
Volume o f t h e
u n i t cell i n ~ 0 3 3348.565
Molecular f o r m u l a ClgHlg02Nz
Form u l a we i g h t 307.168
No. o f f o r m u l a w e i g h t s
in the unit cell 8
Measured d e n s i t y
i n gm/cc 1.211 - + 0.020
Calculated density
i n gm/cc 1.218
498 SYED LAIK ALI
90 100 110
Fig. 7 . DTA-curves of s p r a y - c r i e d phenylbutazone o b t a i n e d

a t v a r i o u s d r y i n g t e m p e r a t u r e s . Numbers i n t h e f i g u r e i n d i c a t e
d r y i n g t e m p e r a t u r e . A b s c i s s a : t e m p e r a t u r e , OC.
PHENY LBUTAZONE 499
Crystal structures of phenylbutazone and a 2:l

complex between phenylbutazone and piperazine are
also reported (22).
X-ray powder diffraction patterns of phenylbutazone
polymorphs and phenylbu tazone solvates have been
also investigated (16). The diffraction patterns of
three modifications are given in Fig. 8 (16).
X-ray diffraction patterns of the three peaks of
phenylbutazone polymorphs are given in Table 5 (16).
TABLE 5
X-ray diffraction D a D
9f the Dhenvlbutazone PolvmorDhs (16)
Polymorph 02 theta 1/10
8.7 + 0 . 2 100
4 19.4-
6.9
70
60
B 8.35
20.2
7.05
+
- 100
70
60
7.9 + 0.2
- 100
20.7 70
7.05 60
4.11. Scanning Electron Microscopy (16,17)
Photomicrographs were made using a Jeol JSM - U3
scanning electron microscope. Prior to investiga-
tions the substances were coated with gold using a
direct coating sputter technique giving a coating
layer of about 500 Ao. A photograph of polymorph
modification is given in Fig. 9 (16).
5 . Colour Reactions
When phenylbutazone is heated with glacial acetic
acid and hydrochloric acid, hydrazobenzene is for-
med immediately which rearranges itself to benzi-
dine. The blue or violet colour yielded is due to
oxidation of hydrazobenzene. After addition of
sodium nitrite and pouring into a solution of so-
dium carbonate and R-naphthol a red product is
formed, which mainly consists of 4,4'-bis (2-hydro-
xyl-1-azonaphthyl) biphenyl and 2-hydroxynaphthyl-
1-azobenzene (23). Red colouration is produced when
SYED LAIK ALI
L
;I,
i;.-
1
MODlFlKATlONd
Fig. 8. Powder X-ray diffraction of phenlybutazone polymorphs.

PHENY LBUTAZONE 501
Fig. 9
Scanning Elektron Photomicrograph of
Phenylbut azon ;
Polymorph s magn. 1200 x
502 SYED LAIK ALI
sulfuric acid and potassium nitrate are treated

with phenylbutazone which turns to orange-red on
reaction with ammonia solution (24).
Vanadium-sulfuric acid solution reacts with
phenylbutazone to give a dark green colouration
(25). An alcoholic solution of iron (111) chloride
and 2,2'-dipyridyl reacts with phenylbutazone to
give a red colouration (26). Hyrolysis of drug with
phosphoric acid followed by the diazotization and
coupling of the resulting products with R-Naphtol
gives a colour reaction (27).
6 . Degradation and Stability
Aqueous solutions of phenylbutazone sodium
decompose in course of hydrolysis and oxidation,
for example with sodium hydroxide and hydrogen
.
peroxide (28,29) Degradation occurs also in
oxygen-free basic solutions and in oxygenated
solutions (30,31). Degradation pathways in other
solvents such as N, N-dimethylformamide, N,
N-dimethylacetamide, diethyl carbonate and
propylene glycol-water have also been described
(32). Four mainly occuring decomposition products
of phenylbutazone are the following (29):
1. N- (2-Carboxycaproyl)-hydrazobenzene (Car-
boxylic acid of phenylbutazone) (I)
2. 4-Hydroxyphenylbutazone (11)
3 . N-(2-Carboxy-2-hydroxycaproyl)-hydrazoben-
zene
(0(-hydroxycarboxylic acid of
Phenylbutazone) ( 1 1 1 )
4. n-Caproylhydrazobenzene (IV)
PHENY LBUTAZONE 503
C6H5\ 0 C6 H5
N-N
0 9 0
HO pC4HCJ
I1
C C H5
\
N-N
O\COOH
HO -n-CbHg
I11
The oxidation product of phenylbutazone sodium in

aqueous solution is 4-hydroxyphenylbutazone (11),
the product of hydrolysis is N-(2-carboxcaproyl)-
hydrazobenzene (I). Under the action of hydroxyl
ions on 4-hydroxyphenylbutazone N-(2-carboxy-2-hy-
droxycaproyl)-hydrazobenzene (111) is formed which
leads after further hydrolysis to n-carproylhydra-
zobenzene (IV). This could be further oxidised
giving a mixture of cis and trans-azobenzenes
( 2 9 ) . Phenylbutazone forms in aqueous and
partially aqueous solutions by a reversible
reaction the compound I ( 3 3 ) . The reaction rate
and the position of equilibrium depend on the
solvent, b u t practically not on the pH ( 3 3 ) . The
temperature dependence of rate constants is given
in Table 6 ( 3 3 ) .
TABLE 6
Temperature Dependence of Rate constants; Phenylbutazone

concentration 0.6 M (33)
Medium Temp. k x 106 k2x10 6 K k3x10 6
0
C (S-5 (S-5 (5-5
*20 70.5 1.39 0.20 6.9 0.030

80.1 3.55 0.58 6.1 0.124
90.8 9,94 1.70 5.8 0.53
100.0 22.8 4.39 5.2 1.98
56% Tr iethylene- 60.4 0.087 0.076 1.1 -
glycol 70.9 0.297 0.33 0.9 0.33
78.9 0.750 0.81 0.9 0.95
89.7 2.12 - - 3.83
100.6 5.84 - 12.4
35% N-Methyl-2- 60.2 0.061 0.080 0.77

pyrrolidon + 70.1 0.198 0.319 9.62
15% Tr ie thylene- 80.7 0.620 1.20 0.51
glycol 91.0 1.55 3.56 0.44 7.8
100.8 4.06 10.9 0.37 23
PHENY LBUTAZONE 505
The first three compounds are colourless and may

be extracted with ether from acidified solution.
The coloured product n-caproylhydrazobenzene may
be isolated after extraction with chloroform (28).
The preparation and separation of decomposition
products of Phenylbutazone has been reported in
detail by Pawelczyk and Schmid (28,29,33). The
UV-spectra of Phenylbutazone and its decomposition
products in 0.001% ethanol solution are given in
Fig. 10 (28).
The degradation patte!:n of Phenylbutazone has been
given by Awang as following (38):
(+KOH)
C , H r N =N-C6Hs
1x
506 SYED LAIK ALI
The oxidative degradation of phenylbutazone with

an acidic potassium permanganate solution and an
alkaline hydrogen peroxide solution has also been
reported (34). Mass spectral behaviour of decom-
position products of phenylbutazone has also been
investigated (35,13).Two previously reported but
unidentified phenylbutazone degradation products
were isolated by chromatography and identified by
mass and nmr spectrometry (36).
Beckstead (37) has examined the decomposition of
phenylbutazone in solid dosage forms. Awang (38)
has tested 56 samples of phenylbutazone tablets
and 15 samples of phenylbutazone-antacid formula-
tions in the form of capsules and tablets. The
degradation of phenylbutazone bulk drug was ob-
served under conditions of accelerated hydrolytic
and oxidative decomposition. In presence of mag-
nesium carbonate phenylbutazone is unstable to
heat due to chemiesorption ( 3 9 ) . The photodegrada-
tion of phenylbutazone in aqueous solution leads
to 2-oxocapronic anilide and other compounds. In
the methanolic solution n-butyl (methoxy) malonic
dianilide is obtained. In aqueous solution in
presence of diethylamine the reaction leads to
n-butyl-diethylaminomalonic dianilide and other
compounds ( 4 0 ) .
Phenylbutazone formulations showed no evidence of
chemical instability when stored at ambient tem-
perature, 37OC and 37OC with 75% relative
humidity. Measurable chemical degradation occured
only at 6OoC, with several formulations showing
more than 50 % degradation. The extent of degrada-
tion can vary among the tablets of the same bottle
and between bottles of the same lot (41).
Chemical degradation occurs at 37OC also in some
phenylbutazone-antacid formulations and was common
at 50° and 6OoC (41). Phenylbutazone tablets
are also subject to physical instability which is
manifested in a decrease of dissolution rate
probably due to polymorphism of phenylbutazone
(17) or to change of the properties of excipients
such as gelatine and or acacia subcoats of sugar-
PHENY LBUTAZONE 507
c o a t e d t a b l e t s (42). The s t a b i l i t y o f p h e n y l b u t a -
zone i n s o l i d d i s p e r s i o n s c o n t a i n i n g p o l y e t h y l e n e
g l y c o l 6 0 0 0 , u r e a a n d p o l y v i n y l p y r r o l i d o n e was
i n v e s t i g a t e d by s u b j e c t i n g t h e samples t o accele-
r a t e d s t o r a g e c o n d i t i o n s . The d e c o m p o s i t i o n o f
p h e n y l b u t a z o n e i n c r e a s e d i n p r e s e n c e o f PEG 6000
a n d urea, w h e r e a s p o l y v i n y l p y r r o l i d o n e h a d n o
s i g n i f i c a n t e f f e c t on t h e s t a b i l i t y o f t h e d r u g .
The e f f e c t o f h u m i d i t y on t h e s t a b i l i t y o f p h e n y l -
b u t a z o n e f o r m u l a t i o n s was less p r o n o u n c e d t h a n t h e
t e m p e r a t u r e (43,44). T h e s t a b i l i t y o f p h e n y l b u t a -
z o n e is a l s o i m p a i r e d a t 5OoC when f o r m u l a t e d
w i t h p o l y s o r b a t e 80 (44). I n s u p p o s i t o r y f o r m u l a -
t i o n s d e g r a d a t i o n p r o d u c t s o f p h e n y l b u t a z o n e were
a l s o i d e n t i f i e d and t h e process was d e s c r i b e d a s
m a i n l y o x i d a t i v e (45,46).
7. Dissolution
I n r e s p e c t o f d i s s o l u t i o n r a t e of p h e n y l b u t a z o n e
PEG 6000 and u r e a had a n a d v e r s e e f f e c t b u t p o l y -
v i n y l p y r r o l i d o n e was c o n s i d e r e d t o be a s u p e r i o r
c a r r i e r (43). The i n i t i a l d i s s o l u t i o n r a t e o f
phenylbutazone and d e u t e r a t e d p h e n y l b u t a z o n e
( d - p h e n y l b u t a z o n e ) i n t o a 25 % ( v / v ) e t h a n o l - w a t e r
s o l v e n t a t 25OC a n d c o n s t a n t i o n i c s t r e n g t h from
a c o n s t a n t - s u r f a c e area p e l l e t was s t u d i e d a s a
f u n c t i o n o f a p p a r e n t pH, b u f f e r c o n c e n t r a t i o n a n d
s t i r r i n g r a t e ( 4 7 ) . The same a u t h o r ( 4 7 ) h a s
p o s t u l a t e d t h a t p h e n y l b u t a z o n e may h a v e a h i n d e r e d
d i s s o l u t i o n due to simultaneous r e v e r s i b l e nonin-
s t a n t a n e o u s c h e m i c a l r e a c t i o n which must take
place i n t h e a q u e o u s d i f f u s i o n l a y e r i f t h e d i s s o -
l u t i o n involves rate-determining d i f f u s i o n through
t h e aqueous d i f f u s i o n l a y e r . O t h e r a u t h o r s (48)
have i n v e s t i g a t e d t h e behaviour o f phenylbutazone
i n i t s t r a n s f e r t h r o u g h a d i m e t h y l s i l o x a n e mem-
b r a n e as a f u n c t i o n o f p H v a l u e .
The p r e s e n c e o f s u r f a c t a n t s i n p h e n y l b u t a z o n e f o r -
mulations caused an increase i n t h e i n i t i a l disso-
l u t i o n r a t e . The d i s s o l u t i o n d e c r e a s e d a f t e r
S t o r a g e (44).
508 SYED LAIK ALI
The dissolution rates of various polymorphic modi-

fications of phenylbutazone has been subject of
number of studies (15,16,17,16). Dissolution stu-
dies were conducted in a 0.2 m aqueous phosphate
buffer of pH 7.5.with the disc method.
when a forms show a slight transition into 6

and t ey are compressed into discs. The dissolu-
tion rate of the p-modification is higher than all
other modifications (16).
The dissolution rates of various phenylbutazone
polymorphs in 0.2 m phosphate buffer,pH 7.5 at
37OC with disc method at 100 rpm are given in
Fig 11 (16). In another study (18) the dissolution
rates of spray-dried phenylbutazone samples at
various drying temperatures were measured at
37OC in the USP dissolution test solutions. The
solubility of the samples obtained at 3OoC was
1.5 times higher than that of the sample obtained
at 12OoC and the bioavailability of the new form
crystals would be expected to compare with the
other known forms (18).
8 . Methods of Analysis
Titr imetr
Titration'of Phenvlbutazone dissolved in aceton
and with an ihdicator bromthymol blue with sodium
hydroxide has been adopted as the official method
of analysis in european pharmacopea ( 4 9 ) . In USP
XX ( 4 ) titration of phenylbutazone is performed
with 0.1 N tetrabutyl ammonium hydroxide. Deter-
mination of end-point is done potentiometrically
using a glass-calomel electrode system. These
methods are not specific, as the common decomposi-
tion products such as the carboxylic and hydroxy
carboxylic acids are also titratable. Bromometric
assay with a potentiometric end-point determina-
tion has also been performed ( S O ) . Volumetric
determinationfof phenylbutazone with chloramin-T
(51) and iodine-chlorine solutions are also known
(52). The official BP method involves a non-
aqueous titration with acetone as solvent (53). A
non-aqueous titration in tetramethyl urea a s sol-
vent has also been reported (54). The use of
509
1.0-\
I\
F i g . 10. W s p e c t r a o f p h e n l y b u t a z o n e and i t s d e c o m p o s i t i o n
p r o d u c t s i n 0.001% e t h a n o l s o l u t i o n s , p h e n l y b u t a z o n e , I , 11, 111.
1 2 3 4 5
T I M E (h)
F i g . 11. Comparison of d i s s o l u t i o n rates of p h e n l y b u t a z o n e

polymorphs i n 0.2m p h o s p h a t e b u f f e r p H 7.5 a t 37OC and 100 r p m .
510 SYED LAIK ALI
borhydrides of alkaline metals for the volumetric

analysis of phenylbutazone is also known (55).
Grav imetry
Phenylbutazone has been determined gravimetrically
after precipitation with xanthydrol (56,57).
Colorimetric Analysis
Determination at 750 nm afte:: reaction with phos-
phor-molybden-tungstic acid ( 5 8 ) , at 520 nm after
reaction of phenylbutazone with iron (111) chloride
and 2,2'-dipyridyl have been reported (26). Hydro-
lytic conversion of phenylbutazone to benzidine and
its subsequent diazotization and coupling reaction
has also been utilised for its colorimetric deter-
mination (59).
UV-Spectrophotometry
Phenylbutazone can be determined spectrophotometri-
cally after oxidation to azobenzene at 320 nm (60).
A uv-spectrophotometric measurement of phenylbuta-
zone in presence of decomposition products, other
medicinal agents and interfering dyes involves an
acid and base shake-out followed by measurement at
232 and 264 nm (37). Molar absorptivity values of
phenylbutazone and its decomposition products in
neutral and alkaline solutions are given in Table 7
(37)
Chroma tog r aph ic Methods
Paper Chromatography
Paper of Schleicher and Schull Company as statio-
nary phase and butanol, formic acid and water
(12+1+7) or butanol, ammonia 25% and water (84+8+8)
as mobile phases have been suggested for the paper
chromatographic separation of phenylbutazone (61).
Detection was performed with Dragendorff's reagent,
diazotized sulfanilic acid and 1% mercury (11) ni-
trate solutions (61).
Thin Layer Chromatography
Several systems for the TLC analysis of phenylbuta-
zone and its decomposition products are available.
Backstead (37) has reported cyclohexane-chloro-
form-methanol-acetic acid (60+30+5+5) as solvent
system and silica gel GF thin layer plates a s
stationary phase to be ideal for resolving and
semiquantitatively estimating
TABLE 7
Molar A b s o p t i v i t y Values of Phenylbutazone and i t s
Decomposition P r o d u c t s (37)
Compound Neutral Solution 0 . 0 1 N N NaOH 0 . 1 N NaOH

~~ ~~
h max h max 264 nm

nm t max nm L max
Phenyl
butazone 24 7 15360 264 20925 20925
Decornp. Prod. I 236 188 10 232 15425 3550
Decomp. Prod. I1 236 17385 232 16400 3835
Decornp. Prod. I11 236 19000 232 16550 3915
Decornp. Prod. I V 236 18455 236 14890 3080
* Decomposition P r o d u c t s I , 11, I11 and I V a r e t h e same a s
mentioned under 6 ( D e g r a d a t i o n and S t a b i l i t y )
512 SYED LAIK ALI
phenylbutazone and its decomposition products. A

solvent system of cyclohexane-methyl ethyl ketone-
chloroform-acetic acid (39+62+3+10) and silica gel
GF 254 thin layer plates were also found to be
suitable ( 6 2 ) . Several other solvent systems and
silicagel HF and GF254 thin layer plates have
been recommended for the TLC of phenylbutazone
(61,63,64,65). Silica gel TLC plates previously
treated with a 2% solution of sodium hydrogensul-
fite deters the formation of 4-hydroxyphenylbuta-
zone during the process of development of thin
layer plates in solvent system ( 6 6 ) . On-plate oxi-
dation of phenylbutazone was effectively suppressed
by utilising a 1 : 1 mixture of kieselgur and
silica gel and impregnating the TLC support with
Mcllvaine buffer, pH 6.0 (37). Silica gel TLC
folies and benzene - acetone (80+20) solvent have
been used to separate phenylbutazone from
ketophenylbutazone (67) .
Detection of phenylbutazone and its decomposition
products can be achieved by viewing the plates
under uv-light. Spraying the TLC plates with chlo-
rine-o-toluidine reagent gives blue to violet co-
loured spots with a sensitivity well below 1 micro-
gram (37). Folin-ciocalteu reagent gives also co-
loured spots and is useful in indicating the pre-
sence of components which could be overlooked by
both uv-light and chlorine-otoluidine reagent (37).
Phenylbutazone can also be detected with iron ( 1 1 1 )
chloride in hydrochloric acid, Dragendorff's rea-
gent and dimethylaminobenzaldehyde solution or sub-
jecting the TLC plates to iodine or chlorine va-
pours ( 6 2 ) . Another spray reagent used was 0 . 5 %
potassium dichromate in 20% sulfuric acid (38). A
quantitative densitometric determination of decom-
position products of phenylbutazone at 232 nm with
a Zeiss KM 3 Chromatogramm-Spektrometer has also
been reported (66).
Gas Chromatography
Anachrom ABS/OF-1 qlass column at 200oC isotherm
with nitrogen-as carrier gas were used for analy-
sing phenylbutazone through gas chromatography
( 6 2 ) . Perego (68) has given a gas chromatographic
method of determination of phenylbutazone in biolo-
gical fluids in presence of its metabolites
PHENY LBUTAZONE 513
l-phenyl-2-p-hydroxyphenyl-3,5-dioxo-4n-butylpyra-
zolidine and 1,2-diphenyl-3,5-dioxo-4-(3-hydroxy-
buty1)-pyrazolidine. A 2.5% SE 30 on Gas Chrom Q,
100-120 mesh glass column was used with nitrogen as
carrier gas and flame ionization detector.
Quantitation was done by using promazine as an
internal standard. Phenylbutazone was extracted
from the rat serum or urine after acidifying with 1
N HC1 and extracting with n-heptane (68). Ali (69)
has separated phenylbutazone and three of its de-
composition products I, I1 and I11 on a 2 m steel
column, packed with 3% SE 30 on Varaport 30,
100-120 mesh. Linear temperature programme was run
between 19O-25O0C, 6OC/min. Injector and detec-
tor temperatures were 230° and 27OoC respecti-
vely and nitrogen was used as a carrier gas and
flame ionization detector. Mostly decomposition
product I was found in phenylbutazone formulations
(69). A derivatization procedure for phenylbutazone
and other acidic drugs after extraction from plasma
and prior to its gas chromatographic determination
has been given by Roseboom (70). Phenylbutazone has
been chromatographed as its n-butyl ester after
treating it with n-butyl iodide in presence of N,
N-dimethylacetamide in a methanolic tetramethyl
ammonium hydroxide solution. 1.5 m glass columns
packed with 3% OV 1, 3% OV 17 and 3% SP 1000, all
on chromosorb WHP, 100-120 mesh, were used for the
determination of phenylbutazone at 230, 250, and
27OoC column temperatures isotherm respectively.
The injection port and detector (FID) temperatures
were 3OoC higher than the column temperature. The
retention times of phenylbutazone on these three
columns were found to be 220, 280 and 202 seconds
respectively. Mefenamic acid was used as an inter-
nal standard (70). The oxidation of phenylbutazone
was studied gas chromatographically on 2 m glass
column, packed with 10 % 1,4-butandiolsuccinat on
chromosorb W, 80-100 mesh at 13OoC column
temperature isotherm (71). It was shown that with
alkaline peroxide and acidic permanganate valeric
acid is obtained as oxidation product, while with
alkaline permanganate butyric acid. 5 % OV 7 on Gas
Chrom Q at 15OoC was used for the estimation of
intact phenylbutazone and of total degradation im-
purities in raw material and commercial dosage form
with diphenyl phthalate as an internal standard
(72). Phenylbutazone has been
514 SYED LAIK ALI
determined in plasma without interference from the

metabolites oxyphenbutazone and hydroxyphenylbuta-
zone on 3 % Apiezon L on Chromosorb W-HP, 80-100
mesh at 23OoC isotherm (73). The drug was extrac-
ted from plasma after addition of 1 N HC1 with
n-heptane. About 9 6 % of phenylbutazone was reco-
vered from plasma through this method. Phenylbuta-
zone and its metabolite oxyphenbutazone have been
determined in plasma on a 5 % OV 7 column by flash
methylation with trimethylanilinium hydroxide using
1-(o-chloropehnyl)-1-(p-chlorophenyl)-2,2,2- tri-
chloroethane as an internal standard (74). The
method described is of sufficient sensitivity to
determine plasma levels in humans after a 200 mg
dose of phenylbutazone. Oxyphenbutazone and phenyl-
butazone have been determined in plasma and urine
of dogs and horses by GLC on a 3% OV 210 on Gas
Chrom Q column at 185OC isotherm (75). A GLC de-
termination of phenylbutazone in human plasma down
to 10 ng/ml is reported using 5% OV 17 on Chromo-
sorb WHP column, 80-100 mesh and a 63Ni-electron
capture detector (76). A phenylbutazone analog
4-butyl-1,2-bis (p-tolyl)-3,5-pyrazolidine was
chosen as an internal standard (76). Fluoranthens
as the internal standard and N,O-bis (trimethyl-
silyl) trifluoroacetamide as the silylating agent
have been used to determine phenylbutazone and its
metabolites in human or rabbit plasma (77). Another
sensitive and specific method for the detection of
phenylbutazone in biological samples is its oxida-
tion with permanganate to azobenzene and subsequent
gas chromatographic determination on a 10% DC-200
on Gas Chrom Q, 80-100 mesh column with a flame
ionization detector ( 7 8 ) .
High Per formance !Liquid Chromatography
Phenylbutazone and oxyphenbutazone were determined
in plasma on a Sil-X-adsorption column using a mo-
bile phase of 0.002% glacial acetic acid and 10-23%
tetrahydrofurane in n-hexane with a flow-rate of 1
ml/min at 35OC column temperature and 254 nm
detection wavelength (79,SO). 2,4-dinitrophenyl-
hydrazone of benzaldehyde was used as an internal
standard. Detection limit for phenylbutazone was
found to be 0.2 )Ig/ml. Phenylbutazone and its meta-
bolites oxyphenbutazone and 'y-hydroxyphenylbutazone
were determined in plasma and urine through HPLC on
PHENY LBUTAZONE 515
Bondapack C i a column u s i n g a m o b i l e p h a s e o f m e -
t h a n o l - 0.01M sodium a c e t a t e b u f f e r (pH 4 ) i n a
l i n e a r g r a d i e n t (50 t o 1 0 0 % m e t h a n o l a t 5%/min
w i t h a f l o w - r a t e o f 2.0 m l / m i n ) a n d a d e t e c t o r wave-
l e n g t h o f 254 nm ( 8 1 ) . C a l i b r a t i o n c u r v e s f o r a l l
t h r e e compounds i n t h e r a n g e s o f 0.5 - 5 g/ml a n d
5 - 50 g/ml were found t o be l i n e a r . P1 sma a n d
B P
u r i n e amples were a c i d i f i e d w i t h H C l , e x t r a c t e d
w i t h b e n z e n e - c y c l o h e x a n e (1:l) a n d a f t e r
e v a p o r a t i n g t h e s o l v e n t r e s i d u e was t a k e n i n
methanol and chromatographed. D e t e c t i o n l i m i t f o r
p h e n y l b u t a z o n e a n d i t s m e t a b o l i t e s was 0.05 g/ml
P
( 8 1 ) . Same a u t h o r s ( 8 2 ) h a v e a l s o r e p o r t e d h e HPLC
determination o f phenylbutazone occuring as
metabolite o f a n t i - i n f l a m m a t o r y a g e n t s u x i -
b u t a z o n e i n p l a s m a and u r i n e . c18 Bondapack co-
lumn was u s e d w i t h methanol-0.5M KH2PO4 a s mo-
b i l e phase i n a l i n e a r g r a d i e n t (from 0 t o 100 %
m e t h a n o l a t 8 %/min w i t h a f l o w - r a t e o f 2.0 ml/min)
a t 254 nm ( 8 2 ) . M i n u t e q u a n t i t i e s o f p h e n y l b u t a z o n e
(50-100 ng/ml) h a v e b e e n d e t e r m i n e d i n plasma,
u r i n e , s a l i v a a n d sweat o f h o r s e s t h r o u g h HPLC on a
C1a-Bondapack r e v e r s e d - p h a s e column w i t h a m o b i l e
phase o f 2 % g l a c i a l acetic i n water-methanol
(35+65) a n d a f l o w - r a t e o f 2 ml/min. D e t e c t i o n was
a c h i e v e d a t 240 nm ( 8 3 ) . A n o t h e r p r o c e d u r e f o r t h e
determination o f phenylbutazone and oxyphenbutazone
is b a s e d on a m i c r o p h a s e e x t r a c t i o n from plasma a n d
i t s s u b s e q u e n t d e t e r m i n a t i o n t h r o u g h HPLC. A
L i c h r o s o r b RP 8 column a n d a m o b i l e p h a s e m e t h a -
n o l - w a t e r - f o r m i c a c i d (75+250+0.6) were used a t
25OC column t e m p e r a t u r e a n d 1 . 5 ml/min flow-rate.
D e t e c t i o n was d o n e a t 238 nm. 0 . 5 g compounds per
m l plasma c o u l d b e e a s i l y d e t e r m i l ed r (84).
Polarogr aphy
The p o l a r o g~-r a p h i c i n a c t i v i t y o f p h e n y l b u t a z o n e a n d
o x y p h e n b u t a z o n e a t t h e d r o p p i n g m e r c u r y electrode
l e d t o t h e i n t r o d u c t i o n o f a d e r i v a t i z a t i o n proce-
d u r e ( 8 5 ) which i n v o l v e s h y d r o l y s i s o f t h e d r u g i n
a m i x t u r e o f acetic and h y d r o c h l o r i c a c i d s , d i a z o -
t i z a t i o n , c o u p l i n g w i t h 2-naphthol and d i f f e r e n -
tial-pulse-polarography o f t h e r e s u l t i n g azo-dyes.
P h e n y l b u t a z o n e was d e t e r m i n e d i n t h e r a n g e o f
lO-7M. P o l a r o g r a p h i c parameters u s e d were f o r c e d
drop time: 2 s , p u l s e h e i g h t : 50 mv a n d s c a n - r a t e o f
2 mv/s. Two-electrode o p e r a t i o n was u s e d w i t h a
l a r g e s u r f a c e calomel electrode as a n o d e . The
516 SYED LAIK ALI
p o l a r o g r a m was recorded b e t w e e n -0.6 t o -0.8 V

( 8 5 ) . L i n e a r - sweep a n d d i f f e r e n t i a l - p u l s e
v o l t a m m e t r i c method for t h e d e t e r m i n a t i o n of
phenylbutazone and oxyphenbutazone i n
p h a r m a c e u t i c a l d o s a g e forms i s a l s o r e p o r t e d (86).
The method i s based on t h e e l e c t r o c h e m i c a l
o x i d a t i o n of b o t h d r u g s a t a g l a s s y c a r b o n
e l e c t r o d e i n 0.1 M sodium acetate-acetic a c i d i n
98 % e t h a n o l . An i n t e r r u p t e d - s w e e p p r o c e d u r e is
g i v e n f o r t h e d e t e r m i n a t i o n of p h e n y l b u t a z o n e i n
t h e presence o f oxyphenbutazone (86).
9. Drug Metabolism a n d P h a r m a c o k i n e t i c s
S e v e r a l a u t h o r s (.8 7 ,.8 8 . 8 9 , 9 0 1 h a v e d e m o n s t r a t e d
t h a t p h e n y l b u t a z o n e u n d e r w e n t aromatic a n d s i d e -
c h a i n o x i d a t i o n , b u t t h e q u a n t i t i e s i n which t h e
i d e n t i f i e d m e t a b o l i t e s were p r e s e n t i n human u r i n e
a c c o u n t e d f o r n o t more t h a n a f e w p e r c e n t of t h e
dose. T h e s e a m o u n t s i n c r e a s e d o n l y i n s i g n i f i c a n t l y
upon c l e a v a g e w i t h p - g l u c u r o n i d a s e . U n a l t e r e d
p h e n y l b u t a z o n e d i d n o t c o v e r more t h a n a b o u t 1 %
( 8 7 , 8 8 ) . T h e e l i m i n a t i o n of t h e d r u g from blood i s
l a r g e l y d e t e r m i n e d by b i o t r a n s f o r m a t i o n , s i n c e o n l y
a v e r y small amount is removed as u n c h a n g e d p h e n y l -
b u t a z o n e by s t r a i g h t forward r e n a l e x c r e t i o n
(87,88). P h e n y l b u t a z o n e u n d e r g o e s b i o t r a n s f o r m a t i o n
i n humans t o o x y p h e n b u t a z o n e , - h y d r o x y , p- r - d i h y -
1
d r o x y a n d r - o x o d e r i v a t i v e s . T e C-4 g l u c o r o n i d e s
of p h e n y l b u t a z o n e a n d v - h y d r o x y p h e n y l b u t a z o n e a r e
a l s o known ( 9 1 ) . T h e a b s o r p t i o n f r o m t h e g a s t r o -
i n t e s t i n a l t r a c t was f o u n d t o be r a p i d a n d com-
plete. A f t e r a d m i n i s t r a t i o n of 1 4 C - l a b e l l e d
p h e n y l b u t a z o n e t o a male v o l u n t e e r t h e i n t e g r a t e d
c o n c e n t r a t i o n of unchanged p h e n y l b u t a z o n e i n
plasma, a s estimated from t h e a r e a u n d e r t h e c o n -
c e n t r a t i o n c u r v e (AUC b e t w e e n 0 a n d 336 h o u r s was
6 3 % of t h a t of t o t a l 1 4 C - s u b s t a n c e s ( 9 2 ) . T h e
c o r r e s p o n d i n g AUCs of t h r e e s p e c i f i c a l l y d e t e r m i n e d
I!
metabolites o x y p h e n b u t a z o n e , - h y d r o x y p h e n y l b u t a -
z o n e a n d p - p i h y d r o x y p h e n y l b u a z o n e were 2 3 %, 2 %
a n d 0.5 % r e s p e c t i v e l y ( 9 2 ) . A s i n g l e o r a l dose was
s l o w l y e x c r e t e d from t h e o r g a n i s m , s i n c e w i t h i n 21
d a y s o n l y 88 % was r e c o v e r e d , 6 1 % from u r i n e a n d
27 % from faeces ( 9 2 ) . The sum o f s p e c i f i c a l l y
measured m e t a b o l i t e s ( o x y p h e n b u t a z o n e , r - h y d r o x y -
p h e n y l b u t a z o n e , p-r-dihydroxypehnylbutazone) a n d
p h e n y l b u t a z o n e i n u r i n e d i d n o t c o v e r more t h a n
PHENY LBUTAZONE 517
a b o u t 1 0 %. About 4 0 % a n d 1 2 % o f t o t a l u r i n a r y
r a d i o a c t i v i t y was d u e t o C - 4 - g l u c u o r o n i d e s o f
p h e n y l b u t a z o n e a n d r - h y d r o x y p h e n y l b u t a z o n e respec-
t i v e l y ( 9 2 ) . Direct g l u c u o r n i d a t i o n of p h e n y l b u t a -
z o n e i s t h e p r e d o m i n a n t b i o t r a n s f o r m a t i o n process
.
( 9 2) The time -cour se o f t h e plasma c o n c e n t r a t i o n
o f u n a l t e r e d d r u g is c h a r a c t e r i s e d by a n e a r l y
maximum o f 36.0 pg/ml a t 3 h o u r s a n d slow d e c a y
b e t w e e n 7 a n d 336 h o u r s c o r r e s p o n d i n g t o a n e l i m i -
n a t i o n h a l f - l i f e o f 88 h o u r s ( 9 2 , 9 3 , 9 4 ) .
Ackknowledgement: The a u t h o r t h a n k s D r . K.
S c h e i b l i , Ciba-Geigy, Basel, S w i t z e r l a n d f o r
p r o v i d i n g some u s e f u l i n f o r m a t i o n .
518 SYED LAIK ALI
References
1) J . R . G e i g y (Basel, S w i t z e r l a n d , German P a t .
No 814150 ( 1 9 4 9 ) ;
see a l s o R e c h e n b e r g , H.K., Phenylbutazone,
G e o r g e Thieme Ver l a g , S t u t t g a r t.
2) J . R . G e i g y ( B a s e l ) , S w i t z e r l a n d , P a t . No.
267222 ( 1 9 5 0 ) .
3) J . R . G e i g y ( B a s e l ) , S w i t z e r l a n d , P a t . No.
3 0 8 1 4 5 ( 1 9 5 5 ) ; C.A. 51, 7 4 2 9 a ( 1 9 5 7 ) .
4) U n i t e d S t a t e Pharmacopeia XX, Page 617
( 1 9 8 0 ) ; R o c k v i l l e , Md. 2 0 8 5 2 , USA
5) E u r o p a i s c h e s A r z n e i b u c h , Kommentar, P a g e
1073, W i s s e n s c h a f t l i c h e V e r l a g s g e s e l l s c h a f t ,
S t u t t g a r t (1976).
6) W a l l e n f e l s , K. a n d H. Sund, Drug R e s e a r c h , 9 ,
83 ( 1 9 5 9 ) .
7) M a u l d i n g , H.V. a n d M.A. Z o g l i o , J. Pharm.
Sic., 60, 309 ( 1 9 7 1 ) .
8) E l - F a t a t r y , H.M., M.M.K. S h a r a f e l - D e e n and
M.M. Amer, P h a r m a z i e , 34 1 5 5 ( 1 9 7 9 ) .
9) United S t a t e s Pharmacopeia XX, (1980)
R o c k v i l l e , Md. 2 0 8 8 , USA
10) D i b b e r n , H.W., UV and I R s p e c t r a o f some
important D r u g s , E d i t i o C o n t o r , A u l e n d o r f
(1979).
11) G i r o d , E., R. D e l l r e y a n d F. H a f l i g e r , H e l v .
Chim. Acta - 40 4 0 8 ( 1 9 5 7 ) .
1 2 ) Ciba-Geigy , P r i v a t e Communication.
13) Locock, R.A., R.E. M o s k a l y k , L.G. C a h t t e n a n d
L.M. Lundy, J. Pharm. S c i . , 63 1 8 9 6 ( 1 9 7 4 ) .
1 4 ) U n t e r h a l t , B., Arch. P h a r m a z . , 305 334 ( 1 9 7 2 )
1 5 ) M a t s u n a g a , J . , W. Nambu a n d T. N a g a i , Chem.
Pharm. B u l l . , 24 1 1 6 9 ( 1 9 7 6 ) .
1 6 ) M u l l e r , B.W., Pharm. Acta H e l v . , 5 3 333 ( 1 9 7 8 ) .
1 7 ) I h r a h i m , H.G., F. P i s a n o and A . B r u n o , J.
Pharm. S c i . , 66 669 ( 1 9 7 7 )
1 8 ) M a t s u d a , J . , S . K a w a g u c h i , H. K o b a y a c h i a n d
J. N i s h i j o , J. Pharm. P h a r m a c o l . , 32 579
(1980).
1 9 ) Ciba-Geigy, P r i v a t e Communication.
2 0 ) S i n g h , T.P. a n d M. V i j a y a n , C u r r . S c i . , 44
153 (1975).
21) V i j a y a n , M. , C u r r . S c i . , 40 262 ( 1 9 7 1 ) .
2 2 ) S i n g h , T.P. a n d M. V i j a y a n , J. Chem. SOC.,
P e r k i n 11, 6 9 3 ( 1 9 7 7 ) .
2 3 ) A u t e r h o f f , H. a n d H.P. P a u l i , A r c h . Pharm.,
309 538 ( 1 9 7 6 ) .
PHENY LBUTAZONE 519
B r e n g e l m a n s , J. and G. B r a u n , J. Pharmac.
B e l g i q u e , 11 309 ( 1 9 5 5 ) .
DAB 7 - D D F Akademie V e r l a g , B e r l i n 1 9 8 0 .
D e f f n e r , M. a n d A. I s s i d o r i d e s - D e f f n e r , Chim.
A n a l y t i q u e , 3 460 ( 1 9 5 8 ) : C.A. 53 1 0 6 6 6
(1959).
H e o h l e i n , H., P h a r m a z i e , 21 464 ( 1 9 6 6 ) .
P a w e l c z y k , E., R. Wachowiak a n d A.
Romanowski, D i s s e r t . Pharm. P h a r m a c o l . , 19
567 ( 1 9 6 7 ) .
P a w e l c z y k , E. a n d R. Wachowiak, Dissert.
Pharm. P h a r m a c o l , ?o 6 5 3 ( 1 9 6 8 ) .
P a w e l c z y k , E. a n d R. Wachowiak, D i s s . Pharm.
P h a r m a c o l . , 21 4 9 1 ( 1 9 6 9 ) .
P a w e l c z y k , E. a n d R. Wachowiak, D i s s . Pharm.
.
P h a r m a c o l . , 22 4 7 5 ( 1 9 7 0 )
S l i n g s b y , J. a n d D.A. Zuck, Can. J. Pharm.
Sci., 2 115 (1972).
Schmid, R.W., Helv. Chim. Acta 53 2239 ( 1 9 7 0 ) .
Awe, W. a n d H . J . K i e n e r t , Pharm. Acta H e l v . ,
-
38 8 0 5 ( 1 9 6 3 ) .
Awang, D.V.C. a n d A. V i n c e n t , J. Pharm. S c i . ,
-
6 5 68 ( 1 9 7 6 ) .
M a t s u i , F., D.I. R o b e r t s o n , M.A. P o i r i e r a n d
E.G. L o v e r i n g , J. Pharm. S c i . , 69 4 6 9 ( 1 9 8 0 ) .
Beckstead, H.D., K.K. K a i s t h a a n d S.J. S m i t h ,
J. Pharm. S c i . , 57 1 9 5 2 ( 1 9 6 8 ) .
Awang, D.V.C., A. V i n c e n t and F. M a t s u i , J.
Pharm. S c i . , 62 1 6 7 3 ( 1 9 7 3 ) .
Monkhouse, D.C. a n d J . L . L a c h , Cand. J.
Pharm. S c i . , 7 29 ( 1 9 7 2 ) .
R e i s c h , J . , KTG. Weidmann and J. T r i e b e ,
Arch. Pharm., 310 811 ( 1 9 7 7 ) .
M a t s u i , F . , D.L. R o b e r t s o n , P. L a f o n t a i n e , H.
K o l a s i n s k i a n d E.G. L o v e r i n g , J. Pharm. S c i . ,
-
67 646 ( 1 9 7 8 ) .
B a r r e t t , D. a n d J.J. F e l l , J. Pharm. S c i . , 64
335 ( 1 9 7 5 ) .
E l - G a m a l , S.S. , V.F.B. Naggar a n d A.M.
Motawi, S c i . Pharm., 49 20 ( 1 9 8 1 ) .
N a g g a r , V.F., S . S . E l q a m a l a n d M.A.
Shams-Eldeen, S c i . Pharm. , $3
- 335 ( 1 9 8 0 ) .
P a w e l c z y k , E. a n d R. Wachowiak, Acta P o l .
Pharm. , 26 4 2 5 ( 1 9 6 9 ) .
P a w e l c z y k , E. a n d M. Z a y a c , Acta P o l . Pharm.,
-
27 104 ( 1 9 7 0 ) .
S t e l l a , J.V. , J. Pharm. S c i . 64 706 ( 1 9 7 5 ) .
L o v e r i n g , E.G. and D.B. B l o c k F J . Pharm.
S c i . , 63 6 7 1 , 1 3 9 9 ( 1 9 7 4 ) .
SYED LAIK ALI
European P h a r m a c o p o i a , V o l u m e 11, Page 342

(1971), Maisonneuve S.A., F r a n c e .
J a n c i k , F., E. Kraus, B. B u d s i n s k y a n d 0.
C i n k o v a , Czekoslov.Farmac., 5 105 (1957);
C.A. 51 11936 (1957).
F e c k o T J . , Acta P o l . Pharm., 21 155 (1964) ;
C.A. 62 8942d (1965).
G e n g r G o v i c h , A.Y. a n d A.V. S e r d e s h n e v ,
Aptechn. Delo 2 13 (1960); C.A. 57 1519a
(19621.
B r i t i s h Pharmacopoeia p a g e 364 , (1973),
U n i v e r s i t y P r i n t i n g House, Cambridge
Walash, M . I . and M. R i z k , I n d i a n J . Pharm.,
-
39 453 (1977).
B a c h r a t , A.M., Z. BczakovaO L. Knazko and J.
B u b e r t , P h a r m a z i e , 32 398 (1977).
F r a n c h i , G., Farmaco ( P a v i a ) 12 650 (1957).
Mitra, R.K. and G.K. Ray, 1nd.J. Pharm., 25
262 (1963).
B r u n o , S. a n d G. L u p o l i , Farmaco ( P a v i a ) , Ed.
Sci. 11 462 (1956); C.A. 53 8540 (1959).
P u l v e r , R.
(1950).
Schweiz. Med.Wschr , 80 308 .
S t a j e r , G., P h a r m a z i e 25 748 (1970).
I b e , K . , K.H. Beyer, H T B u r m e i s t e r a n d K.D.
Grosser, Arch. T o x i k o l . , 22 349 (1967).
E i d e n , F., D e u t s h e A p o t h e k e r Z e i t u n g 107 1522
(1967).
G a n s h i r t , H. and A. M a l z a c h e r , Arch. Pharm.,
293 925 (1960).
A w e , W. and H.G. T r a c h t , Pharm. Ztg. - 108 1365
(1963).
Zarnack, J. and S. P f e i f e r , P h a r m a z i e 19 216
(1964).
A l i , S.L. a n d Th. S t r i t t m a t t e r , Pharm. Z t g . ,
-
123 720 (1978).
Thielemann, H. , Sci. Pharm., 41 336 (1973).
P e r e g o , R., E. M a r t i n e l l i a n d P.C. Vanoni, J .
Chromatog., 54 280 (1971).
A l i , S.L., Pharm. Z t g . , 117 383 (1972).
Roeseboom, H. and A. H u l s h o f f , J. Chromatog.,
-
173 65 (1979).
S z a b o , A.E.O G. S t a j e r a n d E. V i n k l e r , Arch.
Pharm., 307 960 (1974).
Watson, J . R . , F. M a t s u i , R.C. Lawrence a n d
P.M. McConnell,
J. Chromatog., - 76 141 (1973).
McGilvery, I.J., K.K. Midha, R. B r i e n a n d L.
W i l s o n , J. Chromatog., 2 17 (1974).
PHENY LBUTAZONE 52 1
Midha, K . K . , I.J. M c G i l v e r a y a n d C. C h a r e t t e ,
J. Pharm. S c i . 6 3 1 2 3 4 ( 1 9 7 4 ) .
B r u c e , R.B., W . R . Maynard Jr. a n d L.K. D u n n i g ,
J. Pharm. S c i . , 63 447 ( 1 9 7 4 ) .
S i o u f i , A . ? F. C a u d a l a n d F. M a r f i l , J. Pharm.
S c i . , 67 2 4 3 ( 1 9 7 8 ) .
T a n i m u r a , J . , J. S a i t o h , F. Nakagawa a n d T.
S u z u k i , Chem. Pharm. B u l l . , 23 6 5 1 ( 1 9 7 4 ) .
Bogan, I . A . , J. Pharm. P h a r m a c . , 2 1 2 5 ( 1 9 7 7 ) .
Pound, N . J . , I.J. M c G i l v e r a y a n d R.W. S e a r s ,
J. Chromatog., 8 9 23 ( 1 9 7 4 ) .
Pound, N . J . andR.W. S e a r s , J. Pharm. S c i . , 64
284 ( 1 9 7 5 ) .
Marunaka, T., T. S h i b a t a , Y. Minami a n d J.
Umeno,
J. Chromatog., 183 3 3 1 ( 1 9 8 0 ) .
Marunaka, T., T. S h i b a t a , Y. Minami, J. Umeno
a n d T. S h i n d o ,
J. Pharm. S c i . , 69 1 2 5 8 ( 1 9 8 0 ) .
A l v i n e r i e , M., J. C h r o m a t o g . , 181 1 3 2 ( 1 9 8 0 ) .
S p a h n , H. a n d E. M u t s c h l e r , A r z n e i m . - F o r s c h . ,
-
3 1 495 ( 1 9 8 1 ) .
Fogg, A.G. and Y.Z. Ahmed, A n a l y t . Chim. Acta
-
94 4 5 3 ( 1 9 7 7 ) .
Chan, H.K. and A.G. Fogg, A n a l y t . Chim. Acta
-
109 341 (1979).
B u r n s , J.J., R.K. Rose, T. C h e n k i n , A.
Goldman, A. S c h u l e r t a n d B.B. B r o d i e , J.
Pharmac. Exp. T h e r . , 109 346 ( 1 9 5 3 ) .
B u r n s , J.J., R.K. Rose, S. Goodwin, J.
R e i c h e n t h a l , E.C. H o r n i n g a n d B.B. Brodie, J.
Pharmac. Exp. T h e r . , 111 4 8 1 ( 1 9 5 5 ) .
Wagner, J . , H. S t i e r l i n a n d R. P u l v e r ,
A b s t r a c t s V I I Europ. R h e u m a t o l o g y C o n g r . ,
B r i g h t o n , England (1971).
M c G i l v e r a y , J.J., K.K. Midha and N. Mousseau,
P h a r m a c o l o g i s t 1 6 218 ( 1 9 7 4 ) .
Midha, K.K., J . P h a r m . S c i . , 63 279 ( 1 9 7 8 ) .
D i e t e r l e , W., J . W . F a i g l e , F. F r u h , H. Mory,
W. T h e o b a l d , K.O. A l t a n d W . J . Richter,
Arzneim.-Forsch., 26 572 ( 1 9 7 6 ) .
D a v i e s , D.S. a n d S.S. T h o r g e r i s s o n , Acta
P h a r m a c o l . , 2 S u p p l . 3 , 181 ( 1 9 7 1 ) .
H v i d b e r g , E.F., P.B. A n d r e a s e n a n d L. Ranek,
C l i n . P h a r m a c o l . T h e r . , 15 1 7 1 ( 1 9 7 4 ) .
SULFADIAZINE
Henry Stober and Wayne DeWitte
1. Description 524
2.5 Melting Range 53 1
2.6 Differential Thermal Analysis 53 1
2.7 Thermal Gravimetric Analysis 533
2.8 Microscopy 533
2.9 Polymorphism 534
2.10 X-Ray Powder Diffraction 534
2.1 1 Density and Contact Angle 536
2.13 Partition Coefficients 531
2.14 Solubility 531
3. Synthesis 539
4. Inorganic Compounds 539
4.1 Sodium Salt 539
4.2 Other Inorganic Compounds 539
5. Chemical Stability 541
6. Methods of Analysis 54 1
6.2 Volumetric Methods 54 1
6.4 Column Chromatography 542
6.5 High Performance Liquid Chromatography 543
6.6 Gas Chromatography 543
6.7 Paper Chromatography 543
6.8 Thin Layer Chromatography 544
7. Pharmacology 545
References 546
Analytical Profilcsof Drug Subrtanccr Copyright 0 1982 by The American

Volume I I 523 Pharmsccuiid Association
ISBN 0-12-260811-9
524 HENRY STOBER AND WAYNE DEWITTE
1. Description
Generic name - Sulfadiazine; Sulfapyrimidine;

Pyrimal; Debenal (1).
Nomenclature - The following nomenclature i s used

in Chemical Abstracts:
4-amino-N-2-pyrimidinyl-benzenesulfonamide
- [68-35-91
Synonyms
N - 2 -Pyrimidiny 1sul fani1amide

e-Amino-N-- (2-pyrimidyl)benzenesulfonamide
Structure
0
1 OH 1 ON402S Molecular Weight.: 250.27
1.2 Appearance? Color, Odor
White or slightly yellow powder. It is odorless

or nearly s o , and is stable in air but slowly darkens on ex-
posure to light ( 2 ) .
2.0 Physical Properties
The infrared spectrum (Figure 1) was determined

for a KBr pellet preparation o f Sulfadiazine USP (2) using a
Perkin-Elmer Model 621 IR spectrophotometer. The assignments
for important absorption bands are presented in Table I. As-
signments were made using general sources ( 3 ) and literature
references ( 4 , 5).
2.2 Nuclear Magnetic Resonance (NMR) Spectrum
The NMR spectrum of sulfadiazine was obtained in

DMSO-dG containing TMS as internal reference using a Perkin-
Elmer Model R-24B NMR spectrometer (Figure 2). The spectral
assignments are given in Table I1 (6).
i
525
526
SULFADIAZINE 527
Table I
Infrared Assignments for Sulfadiazine
-1
Wavenumber (cm ) Assignment
3450
3410
N-H symmetric stretching
1650 NH2 deformation
1580
1490
Ring skeletal vibrations
1440
1410
1325 SO2 asymmetric stretching
1155 SO2 symmetric stretching
Table I1
NMR Assignments for Sulfadiazine
Multiplicity No. of P r o t o n s Assignment
6.0 Singlet 2
6.7 Doub et 2 H2N&
7.0 Trip et
7.7 Doublet
8.5 Doublet
11.3 Singlet
Turczan and Medwick (7) reported similar assignments

for sulfadiazine and a classification scheme for the identifi-
cation of sulfonamides based on their NMR spectra.
The natural abundance 13C magnetic resonance

spectrum of sulfadiazine has been reported by Chang and
Floss ( 8 ) .
The ultraviolet spectra for sulfadiazine were de-

termined in 0.1M HC1, 0.1M NaOH and USP Simulated Intestinal
Fluid, pH 7 . 5 (without enzyme). Solutions were scanned from
350 nm to 200 nm using a Cary 1 4 spectrophotometer ( 9 ) . A
summary of the data obtained is presented in Table I11 along
with data obtained from the chemical literature.
Table I11
Ultraviolet Spectral Values for Sulfadiazine
Solvent A 1%
1 cm
0.1M- HC1 215 548

242 579
0.1M
- NaOH 242 821
254 794
USP Simulated Intestinal 240 82 1

Fluid, pH 7 . 5 (without 254 794
enzyme)
E thano1 270 844 ( 1 0 )
The ultraviolet spectrum for sulfadiazine in 0.1M

-
NaOH is presented in Figure 3 .
2.4 Mass Spectrum
The mass spectrum of sulfadiazine (Figure 4 ) was

obtained with a Kratos Model MS-25 mass spectrometer ( 1 1 )
The mass spectra of sulfapyrimidines have been

studied by Cambon and co-workers ( 1 2 ) . For sulfadiazine, pre-
ferential fragmentation occurs to eliminate SO, resulting in
the fragments observed at m/e = 186 and 1 8 5 . The m/e ass gn-
ments for sulfadiazine are presented in Table IV.
SULFADIAZINE 529
0.8
Solvent: 0.lM NaOH

Pathlength: 1.0 cm
0.6
-
Concantration: 0.008 mg/rnl
0.4
lu
0
z
a
m
a
8a
0.2
0.0
'20 240 260 280 300 320 340
WAVELENGTH (nml
Fig. 3. UV Spectrum o f S u l f a d i a z i n e
?;
20 40
Fig. 4. Mass Spectrum of S u l f a d i a z i n e

1
I
-
140
SULFADIAZINE 53 1
Table I V
Mass Spectral Assignments for Sulfadiazine
~~~ ~~ ~
m/e Relative Intensity (%) Assignment

(M+l)Ot
25 1 0.5
250 0.2 IP
186 90.9
185 100.0 Loss of H from m/e = 186
156 5.3
140 3.2
108 22.8
Q
U
92 59.4
' t,'
65 49.6
2.5 Melting Range
The USP melting range specification for sulfa-

diazine is 251-254OC (2) u s i n g LISP melting point procedure
l a (13). For a sample of Sulfadiazine USP, melting was
observed from 253-254OC followed by decomposition (14).
2.6 Differential Thermal Analysis (DTA)
The thermogram for Sulfadiazine USP exhibits a

sharp melting endotherm at about 26OOC followed by decomposi-
tion (15). A typical thermogram is shown in Figure 5.
Sunwoo and Eisen (16) used DTA to determine the
melting point and heat of fusion of sulfadiazine recrys-
tallized from acetone. Values of 265.6OC and 7 4 6 4 2 180
cal/mole, respectively, were obtained.
I
/
- I 26loC
-I--
I
ir'
0
3
x3
Du Pont 900 DTA
N, Atmosphere
Heating Rate: 10°C/min.
-
Sample Size: 3 mg
A T: 0.5OC/inch
L - L l L
50 100 150 200 250 300
Fig. 5. DTA Thermogram of S u l f a d i a z i n e

SULFADIAZINE 533
2.7 Thermogravimetric Analysis (TGA)
The thermogravimetric behavior of Sulfadiazine

USP in a N Z atmosphere was determined with a Perkin-Elmer
TGS-1 thermobalance at a scan rate of lO'C/minute (17). For
a 3 mg sample a weight l o s s of approximately 0.2% was observed
from RT up to 22OoC, followed by a gradually increasing weight
l o s s attributed to decomposition at higher temperatures.
Cook and Hildebrand (18) have employed TGA for the
identification of several sulfa drugs, including sulfadiazine.
Scans were conducted at a rate of 5OC/min up to about 800'C.
At this temperature no residue of the sulfa drug was left in
the sample pan. At lower temperatures SOz is suspected as the
major product of pyrolysis.
2.8 Microscopy
Sulfadiazine USP powder is composed of transparent

rod-like crystals which exhibit extinction and birefringence
under crossed polars. The microscopic crystallographic
properties of sulfadiazine reported in the literature have
been summarized by Tillson and Eisenberg (19) and are pre-
sented in Table V.
Table V
Microscopic Crystallographic Properties of Sulfadiazine
1 I
I Crystal
System
N
B Ny
Optic
Sign 2V Ext. Elong. Habit
onoclinic 1.596 1.675 1.830 + 76' P,i 2 lath

shaped
nclassified 1.615 1.663 >1.734 - -- P 2 rods
NOTE: The results reported for the unclassified system are

presumed to represent intermediate data which are quite
often obtained for some commercial samples and are
probably related to hydration.
Sulfadiazine forms characteristic "burrs" of very

fine needles when mixed with an acidic solution of gold
chloride (20).
Treatment of sulfadiazine with a solution of po-
tassium triiodide produces rosettes of blades or needles ( 1 0 ) .
2.9 Polymorphism
An evaluation of polymorphism in sulfonamides has

been conducted by Yang and Guillory (21). Sulfadiazine re-
crystallized from five different solvents did not exhibit
polymorphism under the experimental conditions employed.
2.10 X-ray Powder Diffraction
The X-ray powder diffraction pattern o f USP sulfa-

diazine is presented in Table VI (22). Strong lines are ob-
served at 12.8, 21.3, 22.9 and 29.4 degrees 28 for copper Ka
radiation. The instrumental and experimental conditions are
given below.
Instrumental
Conditions:
G.E. XRD-5: Spectrogoniometer

Genera tor : 30 kV, 13 mA
Tube Target: Cu 0
Radiation: Cu, Ni Filtered, Ka = 1.542A

Optics : lo Beam Slit
MR Soller Slit
0.2' Detector Slit
3 O Take-Off Angle
Goniometer: Scan Rate: 2 degrees 28/minute (5O/inch)
Detection: SPG-4 Detector
Rate Meter, 2000 cps full scale
Pulse Height Selection, E = 0.4V, E = 1.5V
Sample L U
Preparation : Sample was ground and back-packed into
an aluminum sample holder (opening of
3 x 1 x 0 . 2 cm) without sieving. Compound
acquires a high static electric charge
upon sieving.
SULFADIAZINE 535
Table VI
X-ray Powder Diffraction Pattern of Sulfadiazine
Major Lines
28 Degrees$;
7.0 12.63 10
11.7 7.56 15
12.8 6.92 87
14.1 6.28 26
14.6(s) 6.07 6
16.2(s) 5.47 10
16.5 5.37 14
19.0 4.67 20
19.8 4.48 12
20.6(s) 4.31 5
21.3 4.17 81
22.9 3.88 100
24.5 3.63 7
25.9 3.44 25
27.4 3.26 22
28.2 3.16 18
29.4 3.04 52
30.5 2.93 3
33.2 2.70 3
34.6 2.59 5
36.0 2.50 16
;?2@ degrees read to nearest 0 . 1 degrees
interplan planar Distance: d = 2 nh

sin 8
“““Relative Intensity in Per Cent Based on Strongest Signal.

Signals less than 3% relative intensity were excluded
based on 3 (S.D.) o f noise level at 5’ 28.
Under the experimental conditions employed, the relative

intensities are subject to change due to variations in
sample handling and particle size and are only included
as a guide for identifying strong lines.
(s) Shoulder or peak poorly resolved from stronger signals.
2.11 Density and Contact Angle
The density and contact angle, 8, of sulfadiazine

have been reported by Lerk and co-workers (23). Using sulfa-
diazine conforming to the specifications of Pharmacopeia
Nederlands, values of 1.48 g/cm3 and 28O (powder porosity
of 0.168) were obtained.
2.12 Dissociation Constant:
Sulfadiazine i s an ampholyte, and in aqueous

solutions can exist in the protonated, neutral and anionic
forms as illustrated below.
6 - H
I
- H
4
o=s=o
"=S=O
&J
\
+ H 0
7
@
\
+ H 0
Ng
- I
Salvesen and Schroder-Nielsen (24), using spectro-

photometric techniques, reported a pKa value of 2.21 for the
anilinium ion associated with sulfadiazine. These authors
also stated that the pKa of the pyrimidinium ion
of 2 - s u l f a n i l a m i d o p y r i m i d i n e s is less than zero. Krebs and

Speakman (25) used a solubility method to determine the acid-
ity o f the sulfonamide group. A pKa of 6.28 was obtained by
these workers, while Willi and Meier (26) obtained a pKa of
6.35 using titrimetry. A summary of the pKa values reported
in the literature for sulfadiazine is presented in Table VII.
SULFADIAZINE 537
Table VII
Reported pKa Values for Sulfadiazine
I
I Group
CI 2.21
S02-N-
--
Referencc
24
Method
Spectrophotometry, 0.5M
T = 24OC
- NaC1,
2.00 6.48 27 Experimental details not

stated.
25 - T = 38OC
Solubility, p = O.lM,
26 Titrimetry, p = 0.1M
- (KCl),
T = 2OoC
2.13 Partition Coefficient
The partition coefficient o f sulfadiazine was de-

termined for the chloroform/O.lM HC1 and chloroform/O.lM NaOH
systems at ambient temperat~res-(s25~C). The partition-co-
efficient presented in this monograph is defined as K where:
P
K = [S organic]/[S aqueous]
P
and [S] is the concentration of sulfadiazine in each phase.
System K
4
CHC13/0.1M- HC1 0.13
CHC13/0.1M- NaOH 0.0005
Sulfadiazine reportedly is extracted from dilute

aqueous acid solutions with ethyl ether (10).
2.14 Solubility
2.14.1 Equilibrium Solubility

The following equilibrium solubilities
were determined at room temperature (-25OC) and 37OC for a
sample of Sulfadiazine USP (28). An equilibration period o f
a b o u t 24 h o u r s was u s e d f o r t h e 3 7 O C c o n d i t i o n ( a g i t a t i o n
p r o v i d e d by r o t a t i o n a t 15 rpm i n a c o n s t a n t t e m p e r a t u r e b a t h )
and 72 h o u r s f o r t h e RT c o n d i t i o n ( a g i t a t i o n p r o v i d e d by a
w r i s t a c t i o n s h a k e r ) . A n a l y s i s o f t h e c l e a r s o l u t i o n was
performed by UV s p e c t r o p h o t o m e t r y .
S o l u b i l i t y (mg/ml)
Solvent RT 37°C
-
- HC1
0.1M 0.61 0.75
Water 0.074 0.10
0.1M p h o s p h a t e b u f f e r 0.35 0.67
(FH 7 . 4 )
The s o l u b i l i t y o f s u l f a d i a z i n e i n w a t e r
has been r e p o r ted i n t h e l i t e r a t u r e (29) a s approximately 1 g
i n 1300 m l ( 0 . 0 8 mg/ml) a t RT and 1 g i n 60 m l ( 1 6 . 7 mg/ml)
of b o i l i n g w a t e r . S u l f a d i a z i n e i s a l s o r e p o r t e d a s b e i n g
s p a r i n g l y s o l u b l e i n a l c o h o l and a c e t o n e , f r e e l y s o l u b l e i n
d i l u t e m i n e r a l a c i d s and s o l u t i o n s o f p o t a s s i u m and sodium
hydroxides ( 2 ) .
The s o l u b i l i t y o f s u l f a d i a z i n e i n w a t e r
and b i o l o g i c a l f l u i d s a t 37°C was r e c e n t l y r e p o r t e d i n t h e
Federal Register (30).
Solvent S o l u b i l i t y (mg/ml) @ 37°C
Water (pH 5 . 5 ) 0.14

Serum 1.60
U r i n e (pH 5 . 5 ) 0.18
U r i n e (pH 8 . 0 ) 4.50
The s o l u b i l i t y of s u l f a d i a z i n e i n s e v e r a l
normal a l c o h o l s was r e p o r t e d by Mauger and co-workers ( 3 1 ) .
The h i g h e s t s o l u b i l i t y o c c u r r e d i n m e t h a n o l . The H i l d e b r a n d
s o l u b i l i t y p a r a m e t e r s o f s u l f a d i a z i n e and o t h e r s e l e c t e d
s u l f o n a m i d e s were d e t e r m i n e d by Sunwoo and E i s e n i n s e v e r a l
a l c o h o l - w a t e r - g l y c o l s y s t e m s ( 1 6 ) . E l w o r t h y and W o r t h i n g t o n
determined t h e s o l u b i l i t y o f s u l f a d i a z i n e i n w a t e r , dimethyl-
formamide and a r a n g e o f m i x t u r e s o f t h e s e s o l v e n t s ( 3 2 ) .
2.14.2 Dissolution Rate
The p o o r s o l u b i l i t y o f s u l f a d r u g s , s u c h
a s s u l f a d i a z i n e , i n w a t e r was a f a c t o r i n p r o m p t i n g t h e Food
and Drug A d m i n i s t r a t i o n t o e s t a b l i s h b i o e q u i v a l e n c e r e q u i r e -
SULFADIAZINE 539
ments for dosage forms of these compounds. For oral solid

dosage forms of sulfadiazine, twelve tablets must be evaluated
using the USP Apparatus 11, 50 rpm, 37OC and 0.1M HC1 as the
dissolution medium. Specifications of 50% released in 30
minutes and 80% released in 60 minutes were indicated ( 3 0 ) .
The intrinsic dissolution rate of sulfa-

diazine in water was measured by Nogami and co-workers ( 3 3 )
using the rotating disk method. The dissolution of sulfa-
diazine under these conditions was found to be in accord with
the Noyes-Nernst equation concerning transport-controlled
processes. In a subsequent publication these authors used
the same technique to study the dissolution of sulfadiazine
in different aqueous solutions ( 3 4 ) .
3. Synthesis
The synthesis of sulfadiazine has been described by
Roblin ( 3 5 ) . Northey ( 3 6 ) has described the manufacturing
procedure used for sulfadiazine. The synthetic reactions
consist of condensing 2-aminopyrimidine with p-acetamido-
benzenesulfonyl chloride, followed by hydrolysis of the
N4-acetyl group with sodium hydroxide. Scheme I depicts
-
the reactions used by Roblin and co-workers.
4. Inorganic Compounds
4.1 Sodium Salt

The sodium salt of sulfadiazine is listed in the
USP XIX ( 3 7 ) and is employed medically when parenteral therapy
is indicated. Exposure of this compound to humid air results
in the gradual absorption of carbon dioxide with the corre-
sponding liberation of sulfadiazine. About 1 gram of sulfa-
diazine sodium dissolves in 2 ml of water.
4.2 Other Inorganic Compounds

Silver sulfadiazine has found use as an anti-
bacterial agent in the treatment of extensive burns. Spectro-
scopic data for silver sulfadiazine have been reported in the
literature ( 3 8 , 3 9 ) . Bult and Klasen ( 4 0 ) investigated the
structure of silver sulfadiazine and indicate that the silver
coordinates with both the sulfonamide group and the nitrogens
of the 2-aminopyrimidine substituent. A 1 : l complex is
formed.
Other metallic compounds of sulfadiazine reported
in the literature include those of the divalent metals Zn,
Cd, Hg, Ni and Mn as well as trivalent Fe ( 4 1 ) .
Scheme I
Synthesis of Sulfadiazine
p-Acetamidobenzene- 2-aminopyrimidine N4 -Acetyl sul fadiaz ine

sulfonyl chloride
/
hydro 1yze
Sulfadiazine
SULFADIAZINE 54 1
5. Chemical Stabilitv
Sulfadiazine is stable i n the solid state upon exposure

to air, humidity and temperature up to 100°C for two weeks
( 4 2 ) . It darkens upon exposure to light ( 2 ) . Upon pyrolysis
sulfadiazine yields 2-aminopyrimidine ( 4 3 ) and sulfur dioxide
(18). In solution sulfadiazine undergoes acid-catalyzed
hydrolysis via two pathways. The first pathway yields
sulfanilic acid and 2-aminopyrimidine ( 4 3 , 4 4 , 4 5 ) , whereas
the second pathway produces sulfanilamide and 2-hydroxy-
pyrimidine ( 4 3 , 4 6 ) . The kinetics of the autoxidation of
sulfadiazine i n solution at pH 2 . 0 - 9 . 2 have been reported
by Zajac ( 4 7 ) .
The results from an elemental analysis of sulfa-

diazine are listed below ( 4 8 ) .
Elemental Analvsis of Sulfadiazine

Element % Theory % Found
C 47.99 47.80
H 4.03 3.92
N 22.39 22.23
6.2. Volumetric Methods
The routine assay of sulfonamides can be accom-

plished using any of a variety of titrimetric methods. The
most widely used assay procedure is titration with sodium
nitrite solution to determine the aromatic amine function
(49) -
Several sulfonamides, including sulfadiazine, were
determined by titration with KBr03, the end point being indi-
cated by a biamperometric system with platinum-graphite
electrodes (50).
Agarwal, _ _ (51) have described a spectro-

et al.
photometric titration procedure for sulfadiazine and other
sulfonamides. The drug is dissolved i n a mixture of hydro-
chloric acid and glacial acetic acid and is then titrated w th
a 0.1N bromate-bromide mixture. The endpoint is determined
by monitoring the absorbance at 345 nm spectrophotometrical Y.
Greenhow and S p e n c e r ( 5 2 ) have r e p o r t e d a c a t a l y -

t i c t h e r m o m e t r i c t i t r a t i o n f o r s u l f o n a m i d e s . The d r u g i s
d i s s o l v e d i n dimethylformarnide and i s t i t r a t e d w i t h t e t r a -
n-butylammonium h y d r o x i d e . The e n d p o i n t i s d e t e c t e d
by m o n i t o r i n g t h e h e a t e v o l v e d from t h e a l k a l i - c a t a l y s e d
a n i o n i c p o l y m e r i z a t i o n o f a c r y l o n i t r i l e , which i s used a s
the indicator.
6.3 SDectroDhotometric Methods
The B r a t t o n - M a r s h a l l r e a c t i o n ( 5 3 ) , i n which s u l f a -
d i a z i n e i s d i a z o t i z e d and t h e n c o u p l e d w i t h N-(1-naphthy1)-
ethylenediamine dihydrochloride t o give a corored product,
i s one o f t h e most commonly used t e s t s f o r s u l f o n a m i d e s .
Davis and co-workers ( 5 4 ) r e p o r t a method f o r

d e t e r m i n a t i o n of sulfonamides f o l l o w i n g formation of an
i n d o p h e n o l dye and measurement o f a b s o r b a n c e a t 725 nm.
A method s p e c i f i c f o r s u l f a d i a z i n e i n t h e p r e s e n c e
o f o t h e r s u l f a n i l a m i d o p y r i m i d i n e s h a s b e e n r e p o r t e d ( 5 5 ) . The
d r u g i s r e a c t e d w i t h 2 - t h i o b a r b i t u r i c a c i d and i s d e t e r m i n e d
by measurement of t h e a b s o r b a n c e a t 305 nm.
Sulfadiazine present i n t a b l e t s , solutions f o r

i n j e c t i o n , e y e d r o p s , b l o o d o r u r i n e may be d e t e r m i n e d by
d i a z o t i z a t i o n , f o l l o w e d by c o u p l i n g w i t h p h l o r o g l u c i n o l u n d e r
a c i d c o n d i t i o n s . The r e s u l t a n t y e l l o w c o l o r i s m o n i t o r e d a t
415 nm ( 5 6 ) .
A method f o r t h e s e p a r a t i o n and q u a n t i t a t i o n o f
sulfonarnides h a s been r e p o r t e d b y Rader ( 5 7 ) . The p r o c e d u r e
i s b a s e d on f o r m a t i o n of i o n p a i r s between t h e s u l f o n a m i d e s
and tetrabutylammonium i o n , f o l l o w e d by s e p a r a t i o n on p a r t i -
t i o n c h r o m a t o g r a p h i c columns. The s e p a r a t e d s u l f o n a m i d e s a r e
t h e n d e t e r m i n e d by u l t r a v i o l e t s p e c t r o p h o t o m e t r y .
Another method f o r s e p a r a t i o n of s u l f o n a m i d e s , i n -
c l u d i n g s u l f a d i a z i n e , i n v o l v e s u s e of t h r e e columns. C e l i t e
s u p p o r t m a t e r i a l i s c o a t e d w i t h p h o s p h a t e b u f f e r (pH 1 . 7 o r
7 . 8 ) o r b o r a t e b u f f e r (pH 8.7), t h e sample a p p l i e d and
e l u t e d w i t h c h l o r o f o r m . The s e p a r a t e d s u l f o n a m i d e s a r e t h e n
d e t e r m i n e d c o l o r i m e t r i c a l l y f o l l o w i n g d e r i v a t i z a t i o n by t h e
Bratton-Marshall reaction (58).
SULFADIAZINE 543
Cobb and Hill (59) have reported a normal phase

high performance liquid chromatographic separation of sulfon-
amides. Sulfadiazine was separated from a number of other
sulfonamides using a silica column and a mobile phase con-
taining cyclohexane ( 8 5 . 7 % ) , anhydrous ethanol (11.4%) and
glacial acetic acid (2.9%).
Other separation methods for sulfadiazine in the

presence of other sulfonamides and of other antibacterial
drugs have been reported, utilizing a wide range of stationary
phases. These include reverse phase octadecylsilane packing
( 6 0 ) , reverse phase octylsilane ( 6 1 ) , ion pairing chroma-
tography ( 6 2 ) , cation exchange ( 6 3 ) , and anion exchange ( 6 4 ) .
Sulfadiazine has been separated from a number of

other sulfonamides by taking advantage of the differing com-
plexation reactions with cadmium(I1) and zinc(I1) ions ( 6 5 ) .
The stationary phases were either n-propylethylenediamine
bonded to silica, or commercially prepared C18 and C8 columns
loaded with 4 - d o d e c y l d i e t h y l e n e t r i a m i n e (in the mobile phase).
The effects of metal ion concentration and type are discussed.
Sulfapyrimidines have been analyzed by gas chroma-

tography following acid hydrolysis to give sulfanilic acid
and the respective aminopyrimidines. The aminopyrimidine
is then analyzed using a column containing 5% SE-30 and 5%
Carbowax 20M on Chromosorb W at a column temperature of
15OOC (66).
Sulfadiazine and its principal metabolite, N4-

acetylsulfadiazine, in plasma or urine, have been analyzed by
gas chromatography with electron capture detection following
de r vatization with azomethane ( 6 7 ) .
6.7 Paper Chromatography
Sulfadiazine and other sulfonamides have been

ana yzed by paper chromatography using several developing
solvents and detection methods ( 6 8 ) . The visualization method
most suitable for sulfadiazine was reported to be reaction
with Ehrlich reagent (p-dimethylaminobenzaldehyde in 1M HC1).
The chromatography was performed on Whatman No. 1 filter
paper. The R values €or sulfadiazine were 0 . 8 2 , 0 . 7 6 , 0 . 4 5 ,
f
0 . 0 0 , 0 . 2 4 , and 0.87 in solvent systems A-F, respectively.
The s o l v e n t s y s t e m s used were:
A. Methyl i s o b u t y l k e t o n e - f o r m i c a c i d - w a t e r
(10 p a r t s k e t o n e s a t u r a t e d w i t h 1 p a r t 4%
formic a c i d ) .
B. Chloroform-methanol-formic a c i d - w a t e r (10 p a r t s
chloroform s a t u r a t e d w i t h a mixture of 1 p a r t
methanol and 1 p a r t 4% f o r m i c a c i d ) .
C. Benzene-methyl e t h y l k e t o n e - f o r m i c a c i d - w a t e r
( a m i x t u r e o f 9 p a r t s b e n z e n e and 1 p a r t k e t o n e
s a t u r a t e d w i t h 1 p a r t 2% f o r m i c a c i d ) .
D. Benzene-formic a c i d - w a t e r (10 p a r t s benzene

s a t u r a t e d w i t h 1 p a r t 2% f o r m i c a c i d ) .
E. Methyl e t h y l k e t o n e - d i e t h y l a m i n e - w a t e r (921:2:
77).
F. Methyl e t h y l k e t o n e - a c e t o n e - f o r m i c a c i d - w a t e r
(40:2:1:6).
6.8 T h i n Layer Chromatography
S u l f a d i a z i n e c a n be d e t e r m i n e d i n m i x t u r e s o f
sulfonamides u s i n g a t h i n l a y e r chromatographic s e p a r a t i o n
f o l l o w e d by u l t r a v i o l e t s p e c t r o p h o t o m e t r i c a n a l y s i s (69).
The samples a r e s p o t t e d on f l u o r e s c e n t s i l i c a g e l H p l a t e s
and developed i n chloroform-methanol (88:12). The s p o t s o f
i n t e r e s t a r e s c r a p e d o f f t h e p l a t e a f t e r d e l i n e a t i o n u n d e r UV
l i g h t and e x t r a c t e d w i t h 1M NaOH. The a b s o r b a n c e s o f t h e
c e n t r i f u g e d e x t r a c t s a r e read w i t h a r e c o r d i n g s p e c t r o p h o t o -
meter.
Walash and Agarwal ( 7 0 ) r e p o r t a s e p a r a t i o n o f

s u l f a d i a z i n e from o t h e r s u l f o n a m i d e s . S u l f a d i a z i n e h a s a n Rf
o f 0.56 u s i n g s i l i c a g e l p l a t e s d e v e l o p e d w i t h a m i x t u r e o f
e t h y l a c e t a t e - m e t h a n o l (9:l). The s p o t c a n b e l o c a t e d u s i n g
any o f t h e f o l l o w i n g r e a g e n t s :
(1) s a t u r a t e d c o p p e r a c e t a t e i n methanol
(brown s p o t )
( 2 ) s a t u r a t e d c o p p e r a c e t a t e i n a c e t o n e (brown)
( 3 ) 5% c o p p e r s u l f a t e i n w a t e r (brown)
(4) 2% c e r i c s u l f a t e i n w a t e r w i t h 5 m l o f con-
c e n t r a t e d H2S04 ( y e l l o w ) .
SULFADIAZINE 545
The chromatographic behavior of a variety of

sulfonamides including sulfadiazine has also been studied
on strong and weak cation and anion exchange TLC plates
with several matrices using both aqueous and nonaqueous
solvent systems (71).
Clarke and Humphreys (72) describe methods for

identification of twenty-five sulfonamides using silica gel
G plates coated with NaOH, KHSO,, HzO and NaOH and solvent
systems 1-4, respectively. The spots are located using
N-(1-naphthy1)-
copper sulfate, p-dimethylaminobenzaldehyde, -
ethylenediamine-2HC1 or fluorescein.
(1) chloroform-methanol (4:l)

(2) chloroform-carbon tetrachloride-methanol
(7:2:1)
( 3 ) ethylacetate-methanol (9:l)
(4) acetone-methanol (4:1)
The separation of sulfadiazine from two of its
known hydrolysis products, sulfanilamide and sulfanilic acid,
has been reported using silanized silica gel layers impreg-
nated with triethanolamine dodecylbenzenesulfonate or
N-dodecylpyridinium chloride (73). Their behavior and that
-
of other sulfonamides was also studied on layers not im-
pregnated with detergents.
Cieri (74) describes a method for the detection

of sulfadiazine and other sulfonamides in animal feeds. The
sulfonamides are extracted from feed samples with alcohol or
acetone and are cleaned up by a Celite column chromatographic
technique. The eluate is spotted on an Adsorbosil-1 TLC
plate, which is developed in chloroform-methanol (95:5) and
then sprayed with an alcoholic solution of p-dimethylamino-
benzaldehyde.
7. Pharmacology
Sulfadiazine is used for the cure of infections orig-

inating from susceptible Gram-positive bacteria. It is
absorbed readily from the gastrointestinal tract to yield
reproducible blood levels. The solubility of sulfadiazine
in urine makes it suitable for the treatment o f E.
- -coli
infections in the urinary tract. The systemic toxicity of
sulfadiazine is low, and relatively few side effects are
associated with its use ( 7 5 , 76). The chronic toxicity of
sulfadiazine has been reported by Schmidt (77).
The metabolism of sulfadiazine in humans involves acetyl-

ation ( i n the liver), oxidation and hydrolysis. I n addition
to unchanged sulfadiazine, which accounted for over 50% of the
products excreted, Uno and Sehine ( 7 8 , 7 9 ) identified the
following metabolites in human urine:
N4-acetylsulfadiazine, sulfadiazine
N4-glucuronide,
- sulfadiazine sulfonate and
sulfanilamide.
The analytical method employed by these authors involved

separation of the metabolites by paper chromatography followed
by elution of each component and analysis using Ehrlichs re-
agent. The Bratton-Marshall reaction ( 8 0 ) and gas chromato-
graphy with electron capture detection ( 6 7 ) have also been
employed for the analysis of sulfadiazine in biological fluids.
The protein binding of sulfadiazine and other sulfa drugs

has been investigated by Hsu and co-workers ( 8 1 ) . They found
that binding affinity increased with the number of methyl
substituents in the 2-pyrimidine ring.
References
1. Merck Index, 9th ed., Merck and Co., Rahway, N.J., ( 1 9 7 6 )

page 1151.
2. The United States Pharmacopeia XIX, Mack Printing Co.,

Easton, PA., ( 1 9 7 5 ) page 4 7 6 .
3. L.J. Bellamy, The Infra-red Spectra of Complex Molecules,

Wiley, New York, 1958.
4. T. Uno, Chem. Pharm. Bull., 11,7 0 4 (1963).
5. Chem. Abs., -
3 9 , 2513e ( 1 9 6 2 ) .
6. G. Carter, CIBA-GEIGY Corp., Personal Communication
7. J . Turczan and T. Medwick, J. Pharm. Sci., 61, 434

(1972).
8. C. Chang and H . Floss, J . Med. Chem., 18,505 (1975).
9. D. Madrid, CIBA-GEIGY Corp., Personal Communication.

SULFADIAZINE 541
References (Continued)
10. E.G.C. Clarke (ed.), Isolation and Identification of

Drugs, The Pharmaceutical Press, London, England (1969)
page 547.
11. G. Carter, CIBA-GEIGY Corp., Personal Communication.
12. A. Cambon, R. Guedj, P. Robert, J. Soyfer and M. Azzaro,

Bull. SOC. Chim. Fr., Part 2 , 567 (1970).

Easton, PA., (1975) page 652.
15. J. Quitasol, CIBA-GEIGY Corp., Personal Communication.
16. C. Sunwoo and H. Eisen, J. Pharm. Sci., -

60, 238 (1971).
17. J. Quitasol, CIBA-GEIGY Corp., Personal Communication.
18. R.E. Cook and D.A. Hildebrand, Thermochimica Acta, 9,

129 (1974).
19. A.H. Tillson and W.V. Eisenberg, J. Amer. Pharm. ASSOC.,

Sci. Ed., -
4 3 , 760 (1954).
20. G.L. Keenan, J. Amer. Pharm. ASSOC., Sci. Ed., -

37, 202
(1948).
21. S . S . Yang and J.K. Guillory, J. Pharm. Sci., 61, 26

(1972).
22. J. Quitasol and R. Morris, CIBA-GEIGY Corp., Personal

Communication.
23. C.F. Lerk, A.J.M. Schoonen and J.T. Fell, J. Pharm. Sci.,
65, 843 (1976).
-
24. B. Salvesen and M. Schroder-Nielson, Medd. Norsk. Farm.

Selskap., 32, 87 (1971).
25. H.A. Krebs and J.C. Speakman, Brit. Med. J., 1,47
(1946).
26. A.V. Willi and W. Meier, Helv. Chim. Acta, -

39, 54
(1956).
27. T.Koizumi, T. Arita and K. Kakemi, Chem. Pharm. Bull.,

1 2 , 413 (1964).
-

29. Remington's Pharmaceutical Sciences, 1 4 t h ed., Mack
Publishing Co., Easton, PA., (1970) page 1200.
30. The Federal Register, -

44, 60320 (1979) .
31. J.W. Mauger, A.N. Paruta and R.J. Gerraughty, J. Pharm.

Sci.,-
- 6 1 , 94 (1972).
32. P.H. Elworthy and H.E.C. Worthington, J. Pharm.

Pharmacol., -
20, 830 (1968).
33. H. Nogami, T. Nagai and A. Suzuki, Chem. Pharm. Bull.,

1 4 , 329 (1966).
-
34. H. Nogami, T. Nagai and A. Suzuki, -

Ibid.,
- 1 4 , 339 ( 1966) .
35. R. Roblin, P. Winnek, J . Williams and J . English,

J . Amer. Chem. S O C . , 62, 2002 (1940) .
36. E . H . Northey, Ind. Eng. Chem., -

3 5 , 829 ( 1943) .

Easton, PA., (1975) page 477.
38. B. Sandman, R. Nesbitt and R. Sandman, J. Pharm. Sci.,

-
63, 948 (1974).
39. D.S. Cook and M.F. Turner, J . Chem. SOC., Perkin 11,
1021 (1975).
40. A. Bult and H. Klasen, J . Pharm. Sci., 67,284 ( 1978) .
41. Chem. Abs., -

9 0 , 214419a, (1979).

SULFADIAZINE 549
R e f e r e n c e s (Continued 1
43. H . A u t e r h o f f and U . S c h m i d t , D t s c h . A p o t h . - Z t g . , g4,

1581 ( 1 9 7 4 ) .
44. 5 7 , 1151 ( 1 9 6 8 ) .
V.S. V e n t u r e l l a , J . Pharm. S c i . , -
45. 2 2 , 455 ( 1 9 7 0 ) .
M . Z a j a c , Diss. Pharm. P h a r m a c o l . , -
46. M . Z a j a c , P o l . J . Pharmacol. P h a r m . , -
2 9 , 689 ( 1 9 7 7 ) .
47. I b i d . , 2 9 , 445 ( 1 9 7 7 ) .
M . Z a j a c , ~-
48. G . R o b e r t s o n , CIBA-GEIGY C o r p . , P e r s o n a l Communication.
49. The U n i t e d S t a t e s Pharmacopeia X I X , Mack P r i n t i n g C o . ,

E a s t o n , P A . , (1975) page 6 2 6 .
50. B . B . P r a s a d , G.D.Khandeliva1 and T . B . S i n g h , Acta P o l .

5,2 0 6 5 8 2 ~( 1 9 7 7 ) .
Pharm., 3 4 , 177 ( 1 9 7 7 ) ; Chem. A b s t r . ,
~-
51. S . P . Agarwal, M.I. Walash and M.I. B l a k e , J . Pharm.

S c i . , 6 1 , 779 ( 1 9 7 2 ) .
~-
52 E . J . Greenhow and L . E . S p e n c e r , Anal. Chern., 47, 1384

(1975).
53 A . C . B r a t t o n and F.K. M a r s h a l l , J . B i o l . Chem., 128,

537 ( 1 9 3 9 ) .
54. D.R. Davis, A.G. Fogg and D. T h o r b u r n B u r n s , A n a l y s t ,

9 9 , 12 ( 1 9 7 4 ) .
-
55. H.W. Conroy, J . Assoc. O f f . A g r i c . Chem., 2 , 679 (1954).
56. R . R . K r i s h n a and C . S . P . S a s t r y , I n d i a n Chem. J . , 13, 27

(1978).
57. B . R . R a d e r , J . Pharm. S c i . , 6 2 , 1148 ( 1 9 7 3 )
58. J . Crommen, J . Pharm. Belg., 32, 128 ( 1 9 7 7 ) ; Chem. A b s t r .

8 7 , 90789n ( 1 9 7 7 ) .
-
59. P.H. Cobb and G . T . H i l l , J . C h r o m a t o g r . , 123,444 (1977).

60. T . J . Goehl, L . K . Mathur, J . D . Strum, J.M. J a f f e , W.H.

P i t l i c k , V . P . Shah, R . I . P o u s t and J . L . C o l a i z z i ,
J . Pharm. S c i . , - 67, 404 ( 1 9 7 8 ) .
61. P . Helboe and M . Thomsen, Arch. Pharm. Chemi, S c i . E d . ,

5, 453 (1977);
- Chem. A b s t r . , -
87, 90800j (1977).
62. S.C. S u , A . V . Hartkopf and B . L . K a r g e r , J . Chromatogr.,

119, 523 (1976).
63. R.B. P o e t and H . H . Pu, J . Pharm. S c i . , 62, 809 (1973).
64. T.C. Kram, J . Pharm. S c i . , 61, 254 (1972).
65. N . H . C . Cooke, R . L . V i a v a t t e n e , R . E k s t e e n , W.S. Wong,

G . Davies and B . L . K a r g e r , J . Chromatogr., __ 149, 391
(1978).
66. J.W. T u r c z a n , J . Pharm. S c i . , -

57, 142 (1968).
67. A . Bye and G . Land, J . Chromatogr., 139, 181 (1977)
68. L . R e i o , J . Chromatogr., -
88, 119 (1974)
69. U.R. C i e r i , J . Chromatogr., -

49, 493 (1970).
70. M.I. Walash and S . P . Agarwal, J . Pharm. S c i . , -

61, 277
(1972).
71. L. L e p r i , P . G . D e s i d e r i and G . T a n t u r l y , J . Chromatogr.,

93, 201 (1974).
-
72. E . G . C . C l a r k e and D . J . Humphreys, J . Pharm. P h a r m a c o l . ,

22, 845 (1970).
-
73. L . L e p r i , P.G. D e s i d e r i and D . Heimler, J . Chromatogr.,

169, 271 (1979).
74. U . R . C i e r i , J . Assoc. O f f . A n a l . Chem., 59, 56 (1976).
75. W . Model, H . O . S c h i l d and A . Wilson, Applied Pharma-

c o l o g y , W.B. S a u n d e r s & C o . , P h i l a . (1976) Chapt. 42.
SULFADIAZINE 55 1
R ef e r e nc e s ( Co n tin u ed )
76. A . Burger ( e d . ) , M ed icin al C h em is tr y, I n t e r s c i e n c e ,

N e w York, ( 1 9 6 0 ) Chapt. 41.
77. L . H . Schmidt, J . Pharmacol. & E x p l . T h e r . , -

81, 17 ( 1 9 4 4 ) .
78. T . Uno and Y . S e h i n e , Chem. Pharm. B u l l . , -

1 1 , 872 ( 1 9 6 3 ) .
79. I b i d_. , -1 4 , 687 ( 1 9 6 6 ) .

T . Uno and Y . S e h i n e , _
80. W. A . R i t s c h e l , G . R i t s c h e l , C . R . Buncher and

J . Rotmensch, Drug I n t e l l . C l i n . Pharm., - 1 0 , 402 ( 1 9 7 6 )
81. P . Hsu, J . K . H . Ma, H.W. Ju n and L . A . L u z z i , J . Pharm.

S c i . , 63, 27 ( 1 9 7 4 ) .
~-
LEVARTERENOL BITARTRATE
Terry D.Wilson
1. Foreword 556
2. Description 556
2.2 Formula 556
2.4 Structure 556
2.5 CA Registry Number 556
3.1 Nuclear Magnetic Resonance 556
3.3 Dissociation Constants 560
3.4 Electron Spin Resonance 562
4. Stability 562
4.1 Oxidation 562
4.2 Dosage Form Stability 562
5 . Methods of Analysis 564
5.1 Colorimetric Titration 564
5.2 UV Spectrophotometric 564
5.3 Fluorescence 564
5.4 Radioenzymatic 565
5.5 lmmunoassay 565
5.6 Chromatography 568
6. Metabolism and Pharmacokinetics 577
7. Acknowledgement 578
8. References 581

Volume I I 555 PharrnacculicalAssocialion
ISBN 0-12-260811-9
556 TERRY D.WILSON
1. Foreword
L e v a r t e r e n o l b i t a r t r a t e is a s t r o n e a and
6, a d r e n e r g i c a g e n t h a v i n g p e r i p h e r a l v a s o -
c o n s t r i c t i o n and coronary a r t e r y d i l a t a t i o n
Dronerties. It is indicated i n t h e treatment
o f a c u t e h y not e ns i ve s t a t e s and cardiac a r r e s t .
A d m i n i s t r a t i o n i s done I.V. i n a 5% d e x t r o s e
s o l u t i o n i n d i s t i l l e d water o r s a l i n e a t a d o s e
o f l - l O a g p e r m i n u t e . 1 - 4 The p r e s e n t s u p p l e m e n t
follows the o r i g i n a l Analytical P r o f i l e s
review. 5
2. Description
2 . 1 Nomenclature
L e v a r t e r no1 B i t a r t r a t e
Levophed8 B i t a r t r a t e
( 2 ) -4- (2-amino-1 - h y d r o x y e t h y l ) - 1 , 2 -
b e n z e n e d i o l [R- (R*, R*)] -2 ,j-dihydroxybutane-
d i o a t e (1:l) s a l t , monohydrate
1-Norepinephrine Bitartrate
2 . 2 Formula
- _.
C8H11r403- C4H606
--+
2 . 3 M o l e c u l a r Wei h t
h y d r a t e 337 2
a n h y d r i d e 319.27
2.4 S t r u c t u r e
Anhydride r51-40-1]
3.1 Nuclear. M a g n e t i c Kesonance
The 1 H - n u c l e a r m a g n e t i c r e s o n a n c e s p e c -
t r u m o f l e v a r t e r e n o l b i t a r t r a t e monohydrate i s
shown i n F i g u r e 1. The s p e c t r u m was t a k e n f r o m
a 1 0 % s o l u t i o n i n 920 on a V a r i a n HA 1 0 0 s p e c t -
r o m e t e r w i t h a TMS e x t e r n a l s t a n d a r d . A s s i g n -
m e n t s o f c h e m i c a l s h i f t s a r e shown i n Table I .
P I
I
t
I
I/ I
I
Figure 1. 'H nuclear magnetic resonance spectrun of levarterenol

bitartrate monohydrate.
558 TERRY D. WILSON
Table I
I H n . m . r . C h e m i c a l S h i f t Data f o r
Levarterenol B it a r t r a t e
Chemical S h i f t
ppm (TKS) rio. I-! Assignments
7.02-7.3 3 aromatic H
5.11 11 OHx7, IdH,H2C ( e x c h a n g e )
GCH
4.84 2 ClCH
3~ 3 - 3 . 6 2 NCH2
The e a g r e e w i t h p r e v i o u s r e s u l t s o f R e i s c h et
-
a1 '97 who a l s o u s e d Tr-S a s e x t e r n a l s t a n d a r d . It
was n o t p o s s i b l e however t o c o n v e r t t h e p r i m a r y
a m i n e o f l e v a r t e r e n o l t o a TMS d e r i v a t i v
EMDS f o r p u r p o s e s o f a n n . m . r . s p e c t r u m .
expressed chemical s h i f t d a t a a s T v a l u e s r e l a t i v e
t o a p-dio ane i n t e r n a l standard f o r l e v a r t e r e n o l
s o l u t i o n s . $ He was a b l e t o show e v i d e n c e f o r
i n t e r m o l e c u l a r s e l f - a s s o c i a t i o n by v a r y i n g c o n -
c e n t r a t i o n s i n D20 a n d DDriSO s o l v e n t s . Upfield
a n d d o w n f i e l d s h i f t s were f o u n d r e s p e c t i v e l y f o r
t h e phenyl p r o t o n s with i n c r e a s i n g c o n c e n t r a t i o n s .
3.2 [(lass S p e c t r u m
A mass m e c t r u m o f l e v a r t e r e n o l b a s e i s
shown i n F i g u r e 2 t a k e n on a HP 5980A. Xxten-
s i v e fragmentation of the b i t a r t r a t e s a l t i n the
i o n source prevented acquisi.tion of a u s e f u l
s p e c t r u m . A weak m o l e c u l a r i o n (M') car; be
s e e n a t m/e 169. The b a s e peak a t m/e 139 c a n be
a t t r i b u t e d t o M+- CH2NH2. C t Q e r p e a k s s e e n a r e
in/e 151, $+ - H 0 ; m/e 111, N; - CHOHCH2NH2 a n d
m/e 93, M - H28 - CHOHCH2NH2.
Lass s p e c t r o m e t r y combined w i t h g a s chrom-
a t o g r a n h y (GC-MS) on l e v a r t e r e n o l h a s b e e n r e p o r t e d
by s e v e r a l a u t h o r s . T h e s e Tlrocedures i n v o l v e d e r -
i v i t i z a t i o n a n d m o n i t o r i n g s p e c i f i c m/e Deaks
commonly e m p l o y i n g m u l t i p l e i o n d e t e c t o r s ( M I D ) .
P e n t a f l u o r o roD'onyl d e r i v a t i v e s have been n r e -
n a r e d l l t 1 2 ~ f ) 3 s 1 h a n dt h e i o n s o f we753, m o l e c u l a r
i o n , 577, M+ - CH2NHCOC2F5; 549, M+ -CH~NHCOC~FS-
SfmPLE 533s SPF CTPlJf'l 1C2 RLTENTiOW TIFIE 2 6
LARCST 4 139 1 l o @ 3 33 2 5 3 s 65 3 24 ? 140 1. 23 4
LAST 4 152 1 . 1 7 161 I i? 169 1 . 6 4 170 1 7
U I N 273-4 N-150-KN LE'J3P-CD DZRECT IFLET €1
. __ PACE i ie0s
7- - - -. . - -
00.
60..
20..
80.,
60.,
48.,
28.
Fig. 2. Mass spectrum of l e v a r t e r e n o l base.

560 TERRY D. WILSON
CO and 176, 'CHZNHCOC~F~have been monitored. N-

pentafluorobenzyl-O-trimethylsilyl d e r i v i t i -
z a t i o n h a s a l s o been c a r r i e d o u t w i t h t h e f o l l o w -
i n g i o n s r e s u l t i n g : 563, m o l e c u l a r i o n ; 548, M+ -
CH 355, +M+ - CH2N=CHC6FS; 208, CHz=N+=CHC6F5and
173; C7F5
3.3 D i s s o c i a t i o n C o n s t a n t s
No g e n e r a l agreement h a s been reached on
t h e v a l u e s of d i s s o c i a t i o n c o n s t a n t s f o r l e v a r t e r -
e n o l . The r e s u l t s of s e v e r a l r e p o r t s a r e shown
i n Table 11.
Table I1
Dissociation Constants
PKal PKa2 ph3 reference
8.72 9.72 12 4
9.3 10.3 13 9
8 57 9.73 11.13 16
While pKa3 r e s u l t s from t h e i o n i z a t i o n of t h e

second p h e n o l i c group, pKai and pKa as determined
by t i t r a t i o n p r o c e d u r e s a r e a s s i g n e 5 t o t h e f i r s t
p h e n o l i c and t h e ammonium i o n o r v i c e v e r s a .
I t h a s been p o i n t e d o u t 9 t h a t t h e i o n i z a t i o n of
t h e s e two g r o u p s does n o t o c c u r i n d e p e n d e n t l y and
c a n be t h o u g h t o f as s i m u l t a n e o u s r e a c t i o n s a s
p i c t u r e d i n Scheme 1. I i s t h e c a t i o n i c form, I1
i s t h e n e u t r a l , I11 t h e z w i t t e r i o n i c , I V t h e
monoanionic and V t h e d i a n i o n i c form.
The c o r r e c t s t a t e m e n t s f o r t h e r e l a t i o n
between t h e macro- and m i c r o - i o n i z a t i o n c o n s t a n t s
are:
I
(u
X
0
I
X I
v-0
w
H W
W
5I 21
0
'cu
I
0
@
00
X I
Qo -
X
0
562 TERRY D. WILSON
Ganellin w a s a b l e t o derive a tautomeric

e q u i l i b r i u m c o n s t a n t , $, from t h e m i c r o c o n s t a n t s
u s i n g t h e r e l a t i o n : K t = a n t i l o g (pkl - p k 2 ) .
T h i s gave t h e r a t i o o f t h e z w i t t e r i o n t o t h e un-
v
c h a r g e d s c i e s w i t h a v a l u e of 1.8 a t 2 5 O C
i n water.
3.4 E l e c t r o n Spin Resonance

A h i g h r e s o l u t i o n E.S.R. spectrum h a s
been o b t a i n e d f o r l e v a r t e r e n o l semiquinone a n i o n -
r a d i c a l and was e s c r i b e d a s i d e n t i c a l t o t h a t
o f e p i n e p h r i n e .I' The p h o t o o x i d a t i o n g i v i n g r i s e
t o t h e a n i o n - r a d i c a l i s p i c t u r e d i n Scheme 2.
E.S.R. p a r a m e t e r s f o r t h e a n i o n - r a d i c a l
a r e : A(5-H) = 3.58 G, A(7-H) = 3.03 G, A ( 6 - H )
= 0 . 9 6 G, A(3-H) = 0.48 G and g= 2.0044.
4. Stability
4.1 Oxidation
The o x i d a t i o n o f l e v a r t e r e n o l h a s been
shown t o proceed v i a pH-dependa t&ays in c lu d -
i n g i n t r a m o l e c u l a r c y c l i z a t i a e f5,Ba' and
external nucleophilic attack a s s e e n i n Schemes
3a and 3b. These r e a c t i o n s were f o l l o w e d by
c y c l i c voltammetry.
Whereas t h e c y c l i z a t i o n r e a t i o n h a s a n
o v e r f b l r a t e c o n s t a n t of 0.066 s e c - E a t pH
6.0 , the nucleophilic r e a c t i o n rate v a r i e s with
t h e n u c l e o p h i l e from 0 . 2 2 t o 2100 sec'l f o r t h e
amino a c i d s c y s t i n e t o c y s t e i n e r e s p e c t i v e l y a t
pH 7.4.
4.2 Dosage Form S t a b i l i t
__ry
Stabilization of evarterenol i n solution
dosage forms h a s been a t t a i n e d by a d d i t i o n of
a n t i o x i d a n t s i n v a r i o u s combinations. These
i n c l u d e sodium b i s u l f i t e and t h e formula shown i n
Table 111. H r e a s c o r b i c a c i d i s t h e p r i n c i p l e
a n t i o x i d a n t . 25
T T
I
0 I
z
0 0 00
X X I
0
M T u
m 11
w -I N
if, 0 + N
g62
0 0
z
0
0
I
0 0
I
41 Y
W
A1 t
I-
I I
cu w
n
B'
I w
I I
0 0
"h;
\ /
0 0
X I
00
X I U
564 TERRY D.WILSON
T a b l e I11
L e v a r t e r e n o l S o l u t i o n Dosage Form
% by W t .
IJo r e p i ne ph r ine 1.00
Boric a c i d 1.50
Ascorbic a c i d 0.50
Pi- a c e t y 1-P - c y s t e ine 0.50
Sodium c a r b o n a t e q . s . pH 6 . 0
P u r i f i e d water, q.s.
5. Methods of Analysig
-T-
5.1 Colorimetric T i t r a t i o n
The U.S.P. a s s a y f o r n o r e p i n e p h r i n e b i t a r t -
r a t e i s c a r r i e d o u t by t i t r a t i n g a s o l u t i o n made
by a d d i n g a p p r o x i m a t e l y 500 mg n o r e p i n e p h r i n e b i -
t a r t r a t e t o 20 m l g l a c i a l a c e t i c a c i d . The t i t r a n t
used i s 0 . 1 N p e r c h l o r i c a c i d . 'JJhen t h e c r y s t a l
v i o l e t e n d p o i n t i s r e a c h e d , each m l o f 0 . 1 N
perchloric acid i s equivalent t o
epinephrine b i t a r t r a t e anhydride. 3*93 mg O f nor-
5.2 -
UV S p e c t r o p h o t o m e t r i c
Norepinephrine b i t a r t r a t e i n t h e i n j e c t a b l e
dosage form i s determined a c c o r d i n g t o t h e U.S.P.
by a s p e c t r a l method. Absorbance o f t h e sample
e l u t e d from a s i l i c e o u s e a r t h column i s compared
t o t h a t o f t h e s t a n d a r d a t k O ~ g / m l i n 1 i n 350
d i l u t e s u l f u r i c a c i d . The absorbance i s d e t e r -
mined at>max o f 278 m and a l s o a t two inima:
250 and 300 nm t o c o r r e c t f o r b a s e l i n e . 2 t
The p r e s e n c e of sodium s u l f i t e a s a n t i -
o x i d a n t i n l e v a r t e r e n o l s o l u t i o n dosage forms was
shown t o c a u s e i n t e r f e r e n c e i n t h e UV23ssay
u n l e s s i t w a s c a r r i e d o u t a t pH 7.0.
5.3 Fluorescence
W a n t i t a t i o n o f l e v a r t e r e n o l by f l u o r e s -
cence measurement p r o v i d e s a s e n s i t i v e " means o f
a n a l y s i s . R e a c t i o n s p r o d u c i n g f l u o r e s c e n t Dro-
ducts i n c l u d e o x i d a t i o n t o a n ethylenediamine
c o n d e n s a t i o n p r o d u c t (EDA) and a t r i h y d r o x y i n d o l e
LEVARTERENOL BITARTRATE 565
p r o d u c t (THI) ,gown i n Schemes 4a and 4 b

respectively.
Zarly d e s c r i p t i o n s of e s e methods a r e
a t t r i b y & e d t o : Weil-Malherbe @ and Anton and
Sayre. E l u t i o n from a c a t i o n ex hange r e s i n
a t pH 6 i s used w i t h *9 o r without?’ p r i o r alum-
inum oxide a d s o r p t i o n with h e x a c y a n o f e r r a t e ( I I 1 )
o x i d a t i o n i n t h e f o r m e r method. The THI method
was o r i g i n a l l y developed u s i n g t h e same o x i d i z i n g
a g e n t b u t h a s bee ed t o i n c l u d e an3&odine/
i o d i d e o x i d a t i o n 91y9’f43 and automation.
5.4 Radioenzymatic
S e n s i t i v e methods f o r l e v a r t e r e n o l have
been developed u t i l i z i n g a n enzymatic t r a n s f o r m a t -
i o n of t h e compound t o a r a d i o a c t i v e p r o d u c t which
i s measured by l i q u i d s c i n t i l l a t i o n . T y p i c a l
s i n g l e isotope d e r i v i t i z a t i o n techniques a r e
i l l u s t r a t e d i n Schemes V a and V b . These r e a c t i o n s
a r e c a t a l y z e d by t h enzymes catechol-0-methyl-
t r a n s f e r a s e (COMT) ang6ph e ne thanolami ne -N-
m e t h y l t r a n s f e r a s e (PNMT) r e s p e c t i v e l y . These
g i v e r i s e t o t r i t i a t e d p r o d u c t s when an 3H-labeled
coenzyme (S-adenosylmethionine) is p r e s e n t .
E a r l y methods were v e r y t e d i o u s and t i e consuming
r e q u i r i n g up t o f o u r days p e r sample 3f however
they have been s i m p l i f i e d and commercialized. 37
Products of t h e enzymatic r e a c t i o n were s e p a r a t e d
by TLC a f t e r e x t r a c t i o n w i t h to1uene:i-amyl a l c o -
h o l ( 3 : 2 ) , o x i d i z e d t o v a n i l l i n w i t h p e r i o d a t e and
counted b li u i d s c i n t i l l a t i o n i n t h e o r i g i n a l
methods. 35938,39 L t m t h o d s however e l i m i n a t e d
t h e o x i d a t i o n s t e p , go:f1st2 w h i l e a n o t h e r modi-
f i c a t i o n was a double i s o t o p e d e r i v i t i z a t i o n
which allowed fg$ concomitant p e r c e n t a g e r e c o v e r y
determination.
5.5 Immunoassay
An immunoassay method w a s used by b 5 w a
et g
- t o measure n o r e p i n e p h r i n e . N-maleylnor-
e p i n e p h r i n e was c o n j u g a t e d t o bovine serum a l -
bumin by a Nannich r e a c t i o n . Following removal
of t h e maleyl group, immunization of r a b b i t s gave
antiserum s p e c i f i c f o r t h e conjugate.
SCHEME 40
H
HO o\ e N H , -2HI ;2HI
+NH2CH2CH2NH2 0
H
-
- NH2CH2CHO
-2H
H
+NH2CH2CH2NH2
-2H
-2H20 H
SCHEME 4 b
-
FERRICYANIDE
PH 6 H ASCOREATE H
NORADRENOC HROME NORADRENOLUT IN
SCHEME 543
COMT
levarterenol + 3H-nonnetanephrine
3H-rne thy c a d e n o sylrnethionine
SCHEME 5b
levarterenol h - - f * 3H-epinephrine
3H-methyl-S-adenosylme thionine
568 TERRY D.WILSON
5.6 Chromato ra h y
5 d y e r and P a p e r Chromatography
Thin l a y e r and p a p e r chromatographic
systems f o r t h e s e p a r a t i o n a n d - d e t e r m i n a t i o n of
l e v a r t e r e n o l are l i s t e d i n Table I V .
S p e c i f i c and s e n s i t i v e methods f o r
l e v a r t e r e n o l have been developed u s i n g gas chrom-
a t o g r a p h y . Table V i n c l u d e s i n f o r m a t i o n on c o l -
umn m a t e r i a l , d e r i v i t i z a t i o n , d e t e c t i o n and
d e t e c t i o n l i m i t s . Reference 48 c o n t a i n s a
review of p r e v i o u s g . c . methods.
5.63 High-Performance L i q u j d Chromatography
Developments i n t h e f i e l d o f h i g h -
performance l i q u i d chromatography have supplemented
former methods f o r l e v a r t e r e n o l a n a l y s i s , e s p e c i a l -
l y i n b i o l o g i c a l samples where picogram l e v e l s a r e
r o u t i n e l y e n c o u n t e r e d . Ease, low c o s t and r a p i d -
i t y o f o p e r a t i o n i n a d d i t i o n t o s e n s i t i v i t y and
s p e c i f i c i t y a r e a f f o r d e d by t h e s e methods. T h i s
s e c t i o n i s a r r a n g e d a c c o r d i n g t o d e t e c t i o n methods
w i t h a t a b u l a t i o n of column, mobile phase and
d e t e c t i o n l i m i t i n f o r m a t i o n i n Table V I .
5.631 Ultraviolet
U . V . d e t e c t i o n was used e a r l
i n normal phase i o n - p a i r i n g p a r t i t i o n s t u d i e s . 5 6 9 3
d h i l e measurements are u s u a l l y made a t 280 nm, 52 ,53
250 nm was used i n a s t u d y i n which n o r e p i n e p h r i n e
enantiomers were s e p a r a t e d u s i n g t e t r a a c e t y l -
glucopyranosylisothiocyanate and t r i a c e t a r a b i n o -
3'
pyrano s y l i s o t h i o c y a n a t e d e r i v i t i z a t i o n . 0t h e r
s t u d i e s were done o p t i m i z i n g t h e e f f e c t of t h e
a c i d , t h e o r g a n i c m o d i f i e r and t h e pH o f t h e
mobile phase 55 and d t e r m i n i n g t h e r e v e r s e phase
r e t e n t i o n mechanism. 5%
5.632 Fluorescence
A comprehensive r e v i e w o f
f l u o r e s c e n c e d e t e c t i o n i n catecholamine
by H.P.L.C. h a s r e c e n t l y been p u b l i s h e d . 3rfalysis
B39,
includes d r o x y i n d o l e , 52 ,
escamine, k2 '!@' and o - p h t h a l a l d e h y d e ,
p r e and post-column. While T H I
d e t e c t i o n was used i n a d i r e c t i n j e c t i o n c o t i n -
uous f l o w double ion-exchange column s y s t e mz8 ,
TABLE N
THIN LAYER AND PAPER CHROMATOGRAPHY OF LEVARTERENOL
Medium Solvent D e t e c t i on R ef e r e n ce
Avicel 1st d i m e n s i o n : Diazotized 43
( m i c r o c r y s t a l - 1 - b u t a n o l :MeOH: p-nitroan-
l i n e c e l l u 1 o s e ) l N formic a c i d iline
(60 :20 :20)
2nd d i m e n s i o n :
CHC13 :MeOH : 1 N
NaOH ( 6 0 : 3 5 : 5 )
C e l l u l o s e MN 1 - b u t a n o l :EtOH: aqueous e t - 44
300Ecteola O.5N HOAc (38: hylened i a m -
( a n i o n ex- 8.5 :20) i n e (1:4)
changer )
Cellulose 1 - b u t a n o l :pyr- 0.5% p o t a s - 45

phosphate i d i n e :H20 sium f e r r i -
paper ( 1 4 :3:30) cyani de
1 - b u t a n o l :EtOH ferric
(95%):H20 (1:1: chloride
1)
TABLE V
GAS L I Q U I D CHROKATOGRAPHIC ANALYSIS o f LEVARTEKNOL

Column Detector -
De r i v i t i z a t i on Detection L i m i t s Reference
1%SE-30 electron pentafluoroben- 100 Pg 15

capture zylimine -
1%O V - 1 7 trimet h y l s i l -
Yl
5% SE-30 electron Dentafluoro - 1.4x1 0-'6rnol/sec 46
capture benzaldehyde
flame i o n - b i s- t rime t h y l 2.1x10-13mol/sec
ization sil y l a c et a m i d e
3% O V - 1 7 flame i o n - trimethylsil- 2 5 ng 47
iz a t i o n ylimidazole
7'% D C - 1 1 d u a l flame b i s- t rime t h y l 0 . 1 Pg 48
ionization silylacetamide
trif l u o r o a c e t -
amide
3% ov-1 electron 2,6-dinitro- 0.5 Pg 49

capture 4- t r i f l u o r o -
me t h y l b e n z e n e -
s u l f o n i c acid
TAELE V I
HIGH-PERFORb ANCE L I Q U I D CHRGh ATOGRAPHIC SYSTEP S for LEVARTERENOL
1. Ultraviolet Detection
Column L o b i l e Phase Detect i o n Limit Reference
S i l i c a gel Bu0H:Hexane ( 1 : l ) 0.1AAg 50
Et0Ac:Hexane (9:l)
BuOH:MeC12(2:3)
( 0 . 1 M HCPQ4 s t a t -
io n a r y )
LiChrosorb S I 1 0 0 Bu0H:NeCl (4:6) 1 . 0 pmole 51
VI ( 0 . 2 5 M H&O4 s t a t - ml
2 io n a r y )
AA Bondanak c18 0 . 1 7 ~HOAC PH 2.6 5 ng 52
Nucleosil C 1 8 0.67M p h o s p h o r i c a c i d 40 ~g 53
ml
O.D.S. 1 0 mM p h o s p h a t e b u f - - 54
f e r pH 2 . 8 : MeOH
S p h e r i s o r b 55 S i l i c a aqueous p e r c h l o r i c , - 55
Porasil- 6 0 S ~ilica acetic, chloroacetic,
dichloroacetic acids
LiChrosorp RP-8 0.1M H3PO4 pH 3 . 0 , - 56
N a o c t y l s u l f a t e : 1.15
$ pentanol
TABLE V I ( c o n t i n u e d )
HIGH-PERFORKANCE L I Q U I D CHROKATOGRAPHIC SYSTEGS for LEVARTERENOL

2. Fluorescence D e t e c t i o n
Column E o b i l e Phase Detection L i m i t Reference
Zipax SCX 0.08M NaH2Po4 pH 4.3 20 Pg 58
Zorbax ODS 0.1K RaH2POq pH 3.15
Zipax SCX 0.15M NaH2P04 1 Pg 59
Zipax SCX 60
4
CII Zipax SCX 61
hl
H i t a c h i 3011 g e l Me0H:Tris H C 1 pH 8 (7:3) 0 . 1 nmol 62

301 0-OH MeOH:O.lSM b o r a t e pH 8
(7:3)
TSK g e l 160 Me0H:Tris H C 1 0.5M pH 8 100 ng 63
TSK-LS 160 T r i s HCl:CH3CN (90:lO) 50 ‘ y g 64
pH 8.4
Zipax SCX O . l 5 K NaH2PO4 pH 4.37 1 Pg 65
Bondapak SCX C i t r i c acid/HOAc/NaOAc/ 7.5 ng 67
NaOH pH 2.8
Zipax SCX- 0.07M NaH2PO4 10 Pg 68
CDR-20 a n i o n exchange
TABLE VI (continued)
H 1GH-PERFOXti.ANCE LIQUID CHROI..ATOGRAPHIC SYSTEP?S for LEVARTERENOL
2. Fluorescence Detection
Column Nobile Phase Detection Limit Reference
TSK-LS 160 CH CN:O.OSM imida- 25 fmol 69
zo?e HC1 gH 7.3:
Na2EmA 0 mg/L
3. Electrochemical Detection
m
Zipax SCX 0 . 1 ~~ ~ 1 0 4 74
4
W
Vydac SCX 0.01M H2SO4./ 0.04 75
M NaS04
Carasil/CX acetate/citrate 76
buffer pH 5.2
c18 0.1M citric acid: 77
O.lN! NazHPOh (3:2)
0 . 3 mM Na octane
s u l fate
Nucleosil SA citrate/acetate 78
buffer pH 5.2
Zipax SCX- citrate/acetate 79
Vydac SCX buffer pH 5.1
TABLE V I (continued)
HIGH- PERF0 RIk.ANCE L I Q U I D E O G i ATOG RAPH I C SYSTEMS for LEVARTERENOL
Column Kobile Phase D e t e c t i o-
n --
Limit Reference
A Bondapak C18 0.1M HNO3 pH 3.0 - 80
octane s u l f a t e
M Bondapak c18 citrate/phosphate 81
b u f f e r : r eOH (85:15)
pH 3.3 Na o c t a n e sul-
f o n a t e 2 . 5 ~10-3M
EDTA
ABondapak c18 O.lMNaH2P04 0.lM 82
EDTA
Vydac SCX citrate/acetate 83
buffer
Bondapak Phenyl N a H PO4 b u f f e r pH 5.5 84
-A.( Bondapak C18 MeOfirH20 pH 4.2
0 . 0 0 2 M PIC B7 o r l38
Uhatman SCX 0.008M c i t r i c a c i d / 85
O.Ol3M a c e t a t e pH 5.2
0.01 mM EDTA
N u c l e o s i l SA c it r a t e / a c e t a t e buf - 0.25 ~ O I / L 86
f e r pH 5.2
H IGH-PERFORMA NCE L I Q U I D CHRONATOG RAPHIC SYSTEDS for LEVARTERENOL
Column N o b i l e Phase Detection L i m i t Re f e r e n c e
MBondapak c18 0.lK p h o s p h a t e b u f f e r 25 Pg 87
U l t r a s p h e r e ODS pH 2.8 N a o c t a n e s u l -
S p h e r i s o r b ODS f a t e 9mg/~ o r 0 . 1 5 ~
Biophase ODS c h l o r o a c e t i c a c i d pH
3.l/l mN;: EDTA/Na o c t a n e
4
VI s u l f a t e 25 mg/L
v,
4Bondapak C 1 8 0.1M c i t r a t e : O . l N 1 A4 g/L 88
phosphate b u f f e r ( 3 0 0 :
160) N a octane s u l f a t e
0 . 0 1 g/L
M Bondapak c18 N a O A c pH 4.8/ 7%

CH C N , N a h e p t a n e
4
su f o n a t e
U l t r a s p h e r e ODS citrate/phosphate buf-
f e r pH 4.85/ 14% MeOH
3 mN: N a o c t a n e s u l f a t e
--
H I GH -PERF0 RMA NC E L I QU I D CHXOKATOGRA PH I C S Y STEP!,S for LEVARTERENOL
3. Electrochemical_ D e t e c t i o n
Column Kobile Phase Uetection L i m i t Reference
Zipax SCX 0 . 1 ~~ ~ 1 0 4 0 . 0 3 pmol 96
Yanapak ODs- B r i tton-Robinson 40 ng 97
A1203 b u f f e r pH 1.8/0.5
mM N a h e p t a n e s u l f o n -
a t e and
T r i s pH 8.8/0.25% EDTA
N u c l e o s i l C18 c i t r a t e/phosphat e buf - 5 0 0 Pg 98
f e r pH 6.5fiteOH
N u c l e o s i l c18 NeOH:citrate/phosphate 1 ng/mL 99
b u f f e r pH 5.2 (1:20)
LEVARTERENOLBITARTRATE 577
a chemiluminescent f l u o r e s c e n c e d e t e c t o r h a s been
u t i l i z e d by r e a c t i n g bis(2,4,6-trichlorophenyl)
ox a la te with fluorescamine l a b e l e d l e v a r t e r e n o l .
T h i s method gave a 20-fold i n c r e a s e i n s e n g b t i v i t y
over conventional fluorescamine l a b e l i n g .
5.633 E l e c t r o c h e m i c a l
The f i e l d of e l e c t r o c h e m i c a l
d e t e c t i o n of o x i d i z a b l e compounds s e p a r a t e d by
H.P.L.C. h a s s e e n a r a p i d expansion i n r e c e n t
y e a r s f o l l o w i n g e a r l y work of P . T . K i s s i n g e r
-
et a -l . Recent reviews o f e l e c t r o c h e m i c a l d e t e c t -
i o n o f catecholamines i n c l u d e t h e following:70-73.
The e l e c t r o c h e m i c a l d e t e c t o r i n g e n e r a l c o n s i s t s
of a t h i n - l a y e r o r w a l l - j e t s e c t i o n connected
t o a r e s e r v o i r c o n t a i n i n g t h e r e f e r e n c e ( Ag/
AgCl o r S.C.E.) electrode plus an auxiliary
e l e c t r o d e , The working e l e c t r o d e o r anode p l a c e d
i n t h e t h i n - l a y e r ( 0,002-0.015 i n . ) s e c t i o n
c o n s i s t s of a w e l l f i l l e d with c a r b o n p a s t e ,
a g l a s s y c a r b o n e l e c t r o d e o r a gold/mercury
e l e c t r o d e . A number o f s t u d i e s have been con-
d u c t e d u s i n g each of t h e s e e l e c t r o d e s f o r t h e
d e t e c t i o n of l e v a r t e r e n o l . R e f e r e n c e s u s i n g
carbon p a s t e e l e c t r o d e s include3 74-90,
w h i l e g l a s s y c a r b o n e l e c t r o d e s were sed i n 91-
95. I n a d d i t i o n d u a l c a r b o n p a s t e $ and d u a l
g l a s s y carbon e l e c t r o d e s 97 have been u s e d . A
r e c e n t development i s t h e u s e of a r o t a t i n g d i s c
e l e c t r o d e w i t h which it i s p o s s i b l e t o d e c r e a s e
the d i f f u s i o n l a y e r thickness, t h u s i n c r e a s i g t h e
e f f e c t i v e d e t e c t i o n volume and s e n s i t i v i t y . $9 99
5.634 Radioenzymatic
The p r o d u c t s o f t h e r a d i o -
enzymated r e a c t i o n of l e v a r t e r e n o l i n t h e system
d e s c r i b e d i n 5.4 above u s i n g catechol-0-methyl-
t r a n s f e r a s e have been s e p a r a t e d by H.P.L.C.
with ormal phase 51 and r e v e r s e d - p h a s e systems.
53,1& Following f r a c t i o n c o l l e c t i o n , samples
were counted by l i q u i d s c i n t i l l a t i o n .
6. Metabolism and Pharmacokinetics
The metabolism o f l e v a r t e r e n o l h a s been o u t -
l i n e d i n t h e o r i g i n a l monograph. 5 L i t t l e was
known of t h e pharmacokinetics of t h i s d r u g how-
e v e r u n t i l t h e v e r y s e n s i t i v e methods p r e s e n t l y
a v a i l a b l e were developed. One d i f f i c u l t y encount-
578 TERRY D.WILSON
e r e d i n t h e s e measurements i s a d i s t i n c t i o n which
must be made between endogenous n o r e p i n e p h r i n e and
i t s m e t a b o l i t e s and t h e exogenous d r u g . T h i s i s
normally done by r a d i o a c t i v e l a b e l i n g . E a r l y
human s t u d i e s were done w i t h t h e a d m i n i s t r a t i o n
o f 3H-d,l-norepinephrine and t h e c o l l e c t i o n o f
u r i n a r y e x c r e t i o n data. A t r i e x p o n e n t i a l e x p r e s -
s i o n with a t h r e e compartment model e x p l a i n e d t h e
data when 0.02.ug/kg was i n f u s e d i n one h o u r .
The h a l f - l i v e s o b t a i n e d from t h e t h r e e s e c t i o n s of
t h e u r i n a r y s p e c i f i c a c t i v i t y / t i m e c u r v e were:
1.19 h r . , 5 . 2 2 h r . , and 23.43 h r . The r a t e o f
endogenous n o r e p i n e p h r i n e u t p u t was found t o be
0.022kc g/mg c r e a t i n i n e . lot A s i x compartment
model w i t h a b i e x p o n e n t i a l e q u a t i o n was used t o
e x p l a i n g t h e r u r i n a r y e x c r e t i o n data. lC2 Here
8 . 3 ~10- , u g / k g min d , l - n o r e p i n e p h r i n e was a d -
m i n i s t e r e d f o r 48 h o u r s . An a v e r a g e o f 2 7 a g o f
f r e e Ilor-epi appeared i n t h e u r i n e w i t h 5 8 1 4 g~
of m e t a b o l i t e s i n 24 h o u r s . I n t h e s e two s t u d i e s
f l u o r e s c e n c e and l i q u i d s c i n t i l l a t i o n measurements
were used.
Nore r e c e n t l y 3 H - l e v a r t e r e n o l i t s e l f h a s been
a d m i n i s t e r e d by i n f u s i o n and plasma k i n e t i c
data o b t a i n e d u s i n g t h e s e n s i t i v e r a d i o e n z y m a t i c
methods. R e s u l t s o b t a i n e d a r e shown i n T a b l e
VII. L e v a r t e r e n o l l e v e l s have been d e t e r m i n e d
a l o n g with m e t a b o l i t e s i n human u r i n e , plasma and
CSF u s i n g s e v e r a l o f t h e methods d i s c u s s e d above.
These a r e summarized i n Table V I I I .
7. Acknowledgement
The a u t h o r wishes t o e x p r e s s h i s t h a n k s t o
Mr. A l l a n G. Hlavac o f S t e r l i n g - W i n t h r o p Research
I n s t i t u t e f o r c o n t r i b u t i o n s t o t h e Nuclear Magnet-
i c Resonance and Mass Spectrum s e c t i o n s .
TABLE VII
LEVARTERENOL PHARNAC 0 K I N E T I C S
ZERO-ORDER INFUSION PLASMA DATA

--
Radio- Infusion Infusion Average Total Endogenous Average Reference
enzyme Rate Duration t 1/2 Clearance Production Plasma
Method m in L/mi n Rate Level pg/mL
C OMT 0.002 90 min - 2.8 0 9 54 %/ 240 103

AAg/min m 2 min
PNNT 0.01- 1 0 min 1.45 4-5 0.83-0 - 8 5 190 104
0.075 A g/min
0.06~g/ 10 h r 2.09
kg min
C ONT 0.1 - 5 60 min 2.4 3.07 0 . 7 fig/ 228 105
AAg/min min
TABLE V I I I
REFERENCES for LEVARTERENOL A N A L Y S I S in HLJhAN F L U I D S
Nethod Urine Plasma CSF

f1uore scence 33 33
GLC 48 48
radio-enzym- 36, 40, 41 40
atic
HPLC
REFERENCES for LEVARTERENOL M E T A B O L I T E A N A L Y S I S HUMAN F L U I D S
ETe thod Urine Plasma CSF

GC -MS 106, 107, 108 107
uv lo9
HPLC 111
LEVARTERENOL BITARTRATE 58 1
8. References
1. P h y s i c i a n ' s Desk Reference, C h a r l e s E. Baker,
J r . , Kedical Economics C o . , O r a d e l l , N . J . , p .
691 (1981).
2. O s o l , A . , e d . , Remington's Pharmaceutical
ence, 1 6 t h e d i t i o n , Back P u b l i s h i n g Co., East-
s-
on, P a , , p. 826 (1980).
3. Goodman, L.S. and Gilman, A , , e d , , The Phar-
macolo i c a l Basis of Thera e u t i c s , 5 t h e d i t -
~ T k E ' i T l ~ Ye w h . 491 (1975).
4. Foye, d.O., e d . , P r i n c i p l e s of h e d i c i n a l Chem-
i s t r y , 2 n d e d i t i o n , Lea and F e b i g e r , P h i l a d e l -
p h i a , P a . , p.377 (1981).
5. Schwender, C . F . , A n a l y t i c a l P r o f i l e s of Drug
Substances, Vol. 1, Academic P r e s s , New York,
N . Y . , p. 149 (1972).
6. Reisch, J . , A l f e s , H . , and lkollmann, H . ,
Anal. Chem., 238, 29 (1968).
z.
7. M i w a , A , , Yoshicka, M., S h i r a h a t a , A . , and
Tamura, Z., Chem. Pharm. Bull., 25, 1904
(1977)
8. F o r r e s t , J . , Heacock, R . , and F o r r e s t , T . ,
J. Pharm. Pharmac., 22, 512 (1970).
9. E r a n o t , J . , FEBS L e t t e r s , 67, 271 (1976).
10. Loon, B., P a l , V . , and Waynert, E . , Molec.
PCol
-' , 19, 44 (1981).
11 I Koslow, S., C a t t a b e n i , F . , and C o s t a , E.,
S c i e n c e , 176, 177 (1972).
12. Koslow, S., F r o n t i e r s in Catecholamine
Research, Usdin, E.and Snyder, S . , e d . ,
Pergamon P r e s s , New York, N . Y . , p . 1085
(1973)
13. Koslow, S., Racagni, G . and Costa, E . ,
Neuropharm., 13,-1123 (1974).
14. Hashimoto, Y., and F i y a z a k i , H . , 2. Chromat.
168, 59 (1979)-
15. Lhuguenot, J . C . , and Maume, B., J . Chromat.
S c i . , 12, 411 (1974).
16. m a n , K., Davis, J . , Colburn, R . , and J a r k e ,
F., J . Neurochem., 19, 1099 (1972).
17* G a n e l l i n , C . , J . E. Chem., 20, 579 (1977).
18. Yoshioka, b.., K i r i n o , Y . , Tamura, Z . , and
Kwan, T . , Chem. ma&. - B u l l . , 25, 7.5 (1977).
19. Hawley, M., Tatawawadi, S . , P i e k a r s k i , S . , and
Adams, R . , J . &. Chem. SOC., 89, 447 (1967).
20. Tse, D., IkcCreery, R . , and Adams, R . , J. E.
Chem., 19, 37 (1976).
582 TERRY D.WILSON
21. Chavdarian, C . , Karashima, D . , and C a s t a g - noli,

N . , J . P’ed. Chem., 2 1 , 548 ( 1 9 7 8 ) .
22. HechT, G T a n d Bigelow, N . , U.S. P a t e n t
number 3 , 8 0 8 , 3 1 7 , -
23. The U.S. Pharmacopeia,20th e d i t i o n , U.S.P.
Convention, H o c k v i l l e , Did., p. 554 (1980).
24. -
i b i d , p. 5 5 5 .
25 J u h a s z , F. and P a a l , T . Gyogyszereszet,
2 5 , 2 1 1 ( 1 9 8 1 ) . RINSDOC 41673N.
26. T i e t z , N . , e d i t o r , Fundamentals of C l i n i c a l
Chemistry, 2nd ed .. d . B . Zaunders. P h i l a -
delDhia. P a . , D. 809 (1976).
27. Weil-Palherbe ,-H, and Bone, A . , Biochem. J.,
51, 311 ( 1 9 5 2 ) .
28. Anton, A . , and S a y e r , D . , 2. Pharm. Exp. T h e r .
138, 360 ( 1 9 6 2 ) .
29 9 &ki,-T., 2. Chromat., 155, 415 ( 1 9 7 8 ) .
30 Ogasahara, S . , Kandai, T . , Yamatodani, A . ,
Watanabe, T . , Wada, H . , and S e k i , T . , J .
Chromat., 1 8 0 , 119 (1979).
31 O’Hanlon, J . , Campuzano, H . , and Horvath, S . ,
Anal. Biochem., 34, 568 ( 1 9 7 0 ) .
32 T e t c a l f , G . , Anal. Biochem., 57, 316 (1974).
33 Campuzane, H . , Wilkerson, J . , and Horvath,
S . , Anal. Biochem., 64, 578 (1975).
34 9 Westerink, B . , and K o r f , J . , J. Neurochem.,
29, 697 (1977).
35 Coyle, J . , and Henry, D . , J . Neurochem.,
21, 61 ( 1 9 7 3 ) .
36 Henry, D . , Starman, B., Johnson, D . , and
Williams, R . , - Sci
L i f e -” 16, 375 (1975).
37 * Up john D i a g n o s t i c s , C a t - A - K i t TM
38 * P a s s o n , P, and P z u l e r , J . , Anal. Biochem.,
51, 618 ( 1 9 7 3 ) -
39 0 Engelman, K . , and Portnoy, B . , C i r c . Kes.,
2 6 , 53 ( 1 9 7 0 ) .
40. P e u l e r , J . , and Johnson, G . , L i f e S c i . ,
21, 625 (1977).
41. Bauce, L . , T h o r n h i l l , J . , Cooper, K . , and
-
Veale, vJ., L i f e - S c”i 2 7 , 1921 ( 1 9 8 0 ) .
42. S a a r , h . , Bachmann, A . , and Gordon, R . ,
C l i n . Chem., 2 7 , 626 (1981).
43 * E G i n g , R . , and C l a r k , d . , 2. Chromat.,
5 2 , 305 ( 1 9 7 0 ) .
LEVARTERENOLBITARTRATE 583
45
46.
9
Levin, J
bio f f a t ,A
.. Anal. Biochem. , 51, 42 ( 1 9 7 3 ) .
a n d H o r n i n g , E. , B i o c h , B i o p h .
47
A c t a , 222 248 (1970):~
Rlaruyama , Y . , a n d Takemori , A , , Anal. u-
-
9
chem., 4 9 240 (1972).

48. L o v e l a d y , H . , a n d F o s t e r , L .’ * -
J . Chromat.,
108, 43 ( 975)
49 D o s h i , P. a n d Edwards, D. , -J . Chromat.,
210, 505 1981).
50 0 P e r s s o n , B . , a n d K a r g e r , B. , ?I. Chromat.W.,
1 2 , 521 ( 1 9 7 4 ) .
51 Eriksson, B., Andersson, I . ,
P e r s s o n , B . , A c t a Pharm. Suec. , 1 Barf,, 451
K * ’ and
(1977
52 I t e l l , L . , and Gustafson, A . , Clin. Chem.,
23, 473 (1977).
53 9
Appel, E . , B a y e r , P . , H a j d u , P., Palm, D.,
S c h o f e r , J . , a n d U i h l e i n , L’,, Nau-Schm. Arch.
Pharmac., 315, 233 (1981).
54 9
Nimura, N . , K a s a h a r a , Y . , a n d K i n o s h i t a , T . ,
-
J . C h r o m a t . , 213, 327 ( 1 9 8 1 ) .
55. Svendsen, H . , and Greibrokk, T . , J . Chromat.,
212, 153 (1981).
56 9
J o h a n s s o n , N . , 2. I&.C h r o m a t . , 4, 1435
(1981)
57 Anderson, G . , a n d Young, J . , L i f e S c i . ,
28, 507 (1981).
58 Okamoto, K . , I s h i d a , Y . , a n d Asai, K . ,
J . C h r o m a t . , 167, 205 (1978).
-
59 Yui, Y . , Kimura, h.. , I t o k a w a , Y. , a n d K a w a i ,
C . , J . C h r o m a t . , 177, 376 ( 1 9 7 9 ) .
60. Yui,-Y., F u j i t a , T . , Yamamoto, T . , I t o k a w a ,
Y . , a n d K a w a i , Ch., C l i n . Chem. , 26, 194
(1980).
61. Nimura, N . , I s h i d a , K . , a n d K i n o s h i t a , T . ,
-
J . C h r o m a t . , 221, 249 ( 1 9 8 0 ) .
62. Imai, K . , 2. C h r o m a t . , 105, 135 ( 1 9 7 5 ) .
63 Imai, K . , Tsukamoto, N . . , a n d Tamura, Z . ,
J . S h r o m a t . , 137, 357 (1977)
-
64. Imai, K . , and Tamura, Z . , Clin. Chem. Acta,
85, 1 ( 1 9 7 8 ) .
65 * Yui, Y . , a n d K a w a i , C . , J . C h r o m a t . , 206, 586
(1981) *
66. D a v i s , T . , Gehrke, C . , G e h r k e , C . , J r . , Cun-
ningham, T . , Kuo, K . , G e r h a r d t , K . , J o h n s o n ,
H . , a n d Willians, C . , C l i n . Chem., 24, 1317
(1978) I
584 TERRY D.WILSON
67 F r o e h l i c h , P . , and Cunningham, T . , Anal. Chim.

Acta, 97, 357 (1978).
68. Yui, Y . , Itokawa, Y . , and K a w a i , C . , Anal.
Biochem., 108, 11 (1980).
69 Kobayashi, S., S e k i n o , J . , Honda, K . , and
Imai, K . , Anal. Biochem,, 112, 99 (1981).
70 K i s s i n g e r , P . , An-em., 49, 447A (1977).
71 K i s s i n g e r , P.! B r u n t l e t t , C . , Davis, G . , Fel-
i c e , J . , Riggin, R . , and Shoup, R . , C l i n .
Chem., 23, 1449 (1977).
72 * Mefford, I . , Ward, If., F i l e s , L . , T a y l o r ,
B . , Chesney, b . , Keegan, D . , and Barchas,
J . , L i f e S c i . , 28, 477 (1981).
73 K i s s i n g e r , P . , B r u n t l e t t , C . , and Shoup,
R., L i f e S c i * I 28, 455 (1981).
74 Refshauge, C., K i s s i n g e r , P . , D r e i l i n g ,
R . , Blank, L . , Freeman, R . , and Adams, R . ,
Life a., 14, 311 (1974).
K i s s i n g e r , P . , Kiggin, R . , Alcorn, R . , and
75
Rau, L . , Biochem. Med., 13, 299 (1975).
76 Keller, R X A Y K e f f o r d , I . , and Adams,
R e , Life Sci
- 0 , 19, 995 (1976).
77 9 F e l i c e , L . , F e l i c e , J . , and K i s s i n g e r , P . ,
-
J . Neurochem., 31, 1461 (1978).
78 Allenmark, S., and Hedman, L . , J. Liq. Chro-
mat
-* , 2, 277 (1979).
79 S a s a , S . , and Blank, L . , Anal. Chim. Acta,
134. 29 (1979).
80. Asmus, P . , and F r e e d , C . , J . Chromat., 169,
303 (1979).
81. Wagner, J . , Palfreyman, IN,, and Z r a i k a , M . ,
J . Chromat., 164, 41 (1979).
82. Foyer, T . , and J i a n g , N., J . Chromat., 153,
365 (1978).
83 Anderson, G . , B a t t e r , D., Young, J., Shay-
2.
9
w i t z , B . , and Cohen, D . , Chromat., 181,

453 (1980).
84. Kempf, E , , and b a n d e l , P . , Anal. Biochem.,
112, 223 (1981).
85. -
Watson, E . , L i f e -‘I
Sci 28, 493 (1981).
86. Allenmark, S,, Hedman, L . , and S o d e r b e r g , A . ,
Microchem. J 25, 567 (1980).
87 Davis, G., ani’Kissinger, P. , Anal. Chem.,
_ _ 156
_53, - (1981).
80. Shoup, R . , and Kissinger, P . , C l i n . Chem.,
23, 1268 (1977).
LEVARTERENOL BITARTRATE 585
89. higgin, R., and K i s s i n g e r , P . , Anal. Chem.,

40, 2109 ( 1 9 7 7 ) .
90. Koyer, T., J i a n g , N . , Tyce, G . , and Sheps,
S., C l i n . Chem., 2 5 , 256 (1979).
91, Hashimoto, r a n d Paruyama, Y . , J. Chromat.,
152, 387 (1978).
92. Wenk, G . , and Greenland, H . , 2. Chromat.,
183, 261 (1980).
93 9
L'enner, D . , Brown, I " . , and L h o s t e , F . ,
J . Chromat., 224, 507 (1981).
94 G o l d s t e i n , D., F e u e r s t e i n , G., I z z o , J . ,
Kopin, I . , and K r e i s e r , H . , L i f e S c i . , 28,
467 (1981).
95 K r s t u l o v i c , A . , Dziedzic, S., Bertani-Dzied-
zic, L . , and DiHico, D . , 2. Chromat.,
217, 523 (1981).
96 Blank, C., 2. Chromat., 117, 35 (1976).
97 Goto, M., Nakamura, T . , and I s h i i , D . ,
J . Chromat., 226, 33 (1981).
-
98. d e s t e r i n k , B . , and Mulder, T . , J. Neuro-
chem
-' ' 36, 1449 (1981).
99 9 O o s t e r h u i s , B . , Brunt, K . , i d e s t e r i n k , B . ,
and Doornbos, D . , Anal, Chem. , 52, 203
(1980)
100. Uchikura, K., Horikawa, R . , and Tanimura,
T . , J . Chromat., 2 2 3 , 41 (1981).
101. G i t l o w , S., Mendlowitz, M . , B e r t a n i , L . ,
Wilk, S., and ' d i l k , E . , 2. C l i n , I n v e s t . ,
50, 859 (1971).
102. Maas, J . , and L a n d i s , D., 2. Pharm. Exp.
Ther., 177, 600 (1971).
103. mer, b.., Jackman, G . , Bobik, A , , K e l l e h e r ,
D., J e n n i n g s , G . , Leonard, P . , Skews, H . ,
and Korner, P . , L - i f e -S* )c i 25, 1461 (1979).
104. F i t z G e r a l d , G., Hossmann, V . , Hamilton, C . ,
Reid, J . , Davies, D . , and D o l l e r y , C . ,
C l i n . -- Pharm. T h e r . , 26, 669 (1979).
105. S i l v e r b e r g , A , , Shah, S . , Haymond, M . ,
and C r y e r , P . , &I. 2. P h y s i o l . , 234,
E252- (1978).
. _ .
106. I\!.aas, J . , Hattox, S , , Greene, N * , and
Landis, D., S c i e n c e , 205, 1025 (1979).
107. Gordon, E., 0-J., Black, K O , and
Kopin, I . , Biochem. Med ' 11, 32 (1974).
108. S j b q u i s t , €3. Lindstrom,B. , and Anggard, E . ,
"-0
I.
J * Chromat*, 105, 309 (1975).

-
586 TERRY D.WILSON
109. Mann, D., L i n c o l n , L . , a n d Yates, P . ,

L a n c e t , 2 0 , 1366 ( 1 9 8 0 ) .
110. Jackman, G . , C l i n . Chem., 2 6 , 1623 (1980).
111, Anderson, G . , Young, J . , Cohen, D . , Shay-
w i t z , B., a n d B a t t e r , D . , J . C h r o m a t . , 2 2 2 ,
112 (1981).
The p r e s e n t l i t e r a t u r e r e v i e w i n c l u d e s mat-
e r i a l p u b l i s h e d t h r o u g h December, 1981.
MEPROBAMATE
Charles M . Shearer

7. References 591
Analytical Profilesof Drug Substances Copyright Q 1982 by The American

Volume I I 587 Phumafeuticd Associalion
ISBN 012-260811-9
588 .
CHARLES M SHEARER
The following supplement contains updated informa-

tion pertaining to the analytical chemistry of meproba-
mate. A literature survey was conducted and is complete
up to January 1981. The numbering system for topics
discussed is the same as that in the original profile
(Volume 1 , pp. 207-232).
The carbon-13 NMR spectrum of a Wyeth In-House
Reference Standard in deuterated dimethyl sulfoxide as
obtained on a Varian FT-80-A spectrometer is presented
in Figure 1. The assignment of t e individual signals
is given in the following table( 17 .
Carbon Chemical Shift (ppm)

a 158.39
b 68.31
C 37.73
d 36.84
e 16.42
f 15.17
g 18.99

2.71 Polymorphism
Three crystal modifications were investi-
gated by Burger and Schulte (2) and two modifications
were investigated by Clements and Popli ( 3 ) . Both ref-
erences discussed thermal properties, infrared spectra,
X-ray diffraction patterns and dissolution studies.
I
150
I
100
wm
JI
50
I
0
Figure 1 - 13C NMR Spectrum of Meprobamate

590 CHARLES M . SHEARER
6.5 Titrimetric
Meprobamate, upon hydrolysis in acidic solution
forms in a stoichiometric amount ammonia, which can be
determined, after making ~$7 solution alkaline, by an
ammonia sensing electrode . Likewise when meprobamate
is hydrolyzed in a basic solution carbon dioxide is
formed, which can e determined by a carbon dioxide
sensing electrode0) .
6.64 Column chromatograph
Meprobamate has been'determined by HPLC(')
using as the eluant varying proportions of chloroform
mixed with carbon tetrachloride, hexane o r butyl ether
and a column consisting o f small particle fully porous
silica packing material or a monomolecular layer of a
cyanopropylsilane chemically bonded to a small particle,
fully porous silica support. The eluted peaks were
monitored using differential refractive index detection.
Meprobamate has also been assayed by HPLC

after alkaline hydrolyses, to 2-methyl-2-propyl-l,3-
propandiol and preparing the benzoyl ester of the
di~l(~). An eluant consisting of 60 parts acetonitrile,
30 parts methanol and 30 parts water eluted the benzoyl
derivative from a 15 cm x 4.6 mm Ultrasphere RP 18 col-
umn in 6 minutes at a flow rate of 2.5 ml/min. This
procedure was u s e d for analyses of meprobamate in
plasma.
MEPROBAMATE 59 1
7. References
1. Personal communication, B. Hofmann, Wyeth
Laboratories, Inc.
2 . A. Burger and K . Schulte, Arch. Pharm.
(Weinbaum), 314, 398 (1981).
3. J . A . 3 e m e n t s and S.D. Popli, Can. J . Pharm.
Sci.,
- - 8, 88 (1973).
4. Y. Michotte, D.L. Massart and L. Dryon, Pharm.
Acta Helv., 52, 152 (1977).
5 . S. T z a m i , Chem. Pharm. Bull.,I, 1820 (1979).
6. I.L. Honigberg, J.T. Stewart and M. Smith,
J. Pharm. Sci., 67, 675 (1978).
7 . R.N. G u p E and F. Eng., J. High Res. Chromatogr.
and Chromatogr. Comm., 3, 419 ( 1 9 8 0 ) .
TRIAMCINOLONE
David H.Sieh
1. Description 594
2.9 SolubilityData 597
3. Synthesis 598
5.2 Pharmacokineticsand Bioavailability 599
6.3 Colorimetric Andysis 600
6.4 PolarographicAnalysis 602
6.6 Fluorimetric Analysis 609
6.7 TitrimetricAnalysis 609
6.8 DifferentialBorohydride Analysis 609
6.9 Radioimmunoassay 609
7. Determinationin Body Fluids and Tissues 610
8. Acknowledgements 61 1
9. References 612
Analytical PloAlcs of Drup Substances Copfight 0 1981by The A m d w

Volume 11 593 PhUm.ceutial AUodBIion
ISBN 0.12.2601)11-9
594 DAVID H. SIEH
The following supplement contains updated

information pertaining to the analytical chemistry
of triamcinolone. A literature survey was conduct-
ed and is complete up to July 1981. The numbering
system for topics discussed is the same as that in
the original profile (Volume 1, pp. 367-396).
1. Description. Triamcinolone is a glucocorticoid
used primarily in the treatment of adrenocortical
and rheumatic disorders.
1.13 The CA registry number for triamcinolone
is 124-94-7.
The original profile] contained a
comparison of infrared spectral assignments of tri-
amcinolone in mineral oil with that of the compound
in the solid state. Bellomonte2 published the
infrared spectra (KBr disc) of 13 fluorinated
steroids, including triamcinolone and triamcinolone
acetonide, and discussed the special influence of
the fluorine atom. The major assignments, which
are in excellent agreement with Floreyl, are
supplemented with the assignments of the C-F
str tching frequency; i.e., 1065 (lo) and 979 (2O)
cm-f for triamcinolone and 1080 (lo) and 972 (2O)
cm-' for triamcinolone acetonide.
The improved 1H-NMR and the 13C-NMR
spectra3 of triamcinolone are shown in Figures 1
and 2, respectively (please refer to Figure 4,
Section 2.2 of the original profile] for comparison
and assignments of the 'H-NMR spectrum). The 13C-
spectrum was determined on a JEOL FX60Q spectro-
meter using a lOmm C/H dual probe. A complete
assignment for all the carbon atoms in the 13C-NMR
spectrum is listed in Table I. The assignments are
based on relative chemical hifts (dimethylsulfox-
ide-d6 = 39.5 ppm) and l3C-IgF coupling constants
and are consistent with literature values for
closely related compounds.4 The assignments were
simplified by the utilization of polarization
transfer to selectively enhance and phase alter
individual carbon types.
Figure 1. 'H-Nuclear Magnetic Resonance Spectrum of Triamcinolone ( S Q 9670) in
Dimethylsulfoxide-d6. Instrument: Varian XL-100A.
Figure 2. 13C-Nuclear Magnetic Resonance Spectrum of Triamcinolone (SQ 9670) in
Dimethylsulfoxide. Instrument: JEOL FX60Q.
TRIAMCINOLONE 597
TABLE I
13C-NMR Spectral Assignmentsa of Triamcinolone
Carbon Chemical Carbon Chemical
Number Shiftb Number Shift
1 152.7 12 35.9
2 129.1 13 46.2
3 185.4 14 43.2
4 124.2 15 33.6
5 167.0 16 71.4
6 30.3 17 87.6
7 27.3 18 16.7
8 33.3 (19.6) 19 22.1(5.8)
9 lOl.l(l74.8) 20 211.6
10 48.0 (22.4) 21 66.6
11 70.6(37.1)
aAll chemical shifts are in ppm from tetramethyl-
silane (TMS) with internal reference dimethyl-
sulfoxide = 39.5 ppm. bAll spectra were run in
DMSO-d6. 13C-19F coupling constants shown in
parentheses.
2.4 Mass Spectra
The low resolution mass spectra of 28
corticosteroid 21-esters and related compounds of
pharmaceutical interest have been determined by
Toft and co-workers6 on an AEI MS-12 via direct
probe. The mass spectra of triamcinolone and
triamcinolone diacetate are in excellent agreement
with those published in the original profile.
2.9 Solubility Data
2.91 Solubility
Solubilization of 19 steroid hormones,
including triamcinolone, triamcinolone acetonide
and triamcinolone diacetate by polyoxyethylene
lauryl ether was reported by Tomida7 who also
concluded that the solubilization of steroids by
polyoxyethylene lauryl ether micelles is directly
dependent upon their lipophilicity. Triamcinolone
was found to have an aqueous solubility of
2.07 x 10-4 M.
2.92Partition Coefficients
The role of crystal structure (as reflect-
ed by the melting point and the entropy of fusion)
598 DAVID H.SIEH
and of the activity coefficient (as reflected by

the octanol-water partition coefficient) in
controlling the aqueous solubility of either liquid
or crystalline organic nonelectrolytes, including
triamcinolone, triamcinolone acetonide and tri-
amcinolone diacetate was discussed by Yalkowsky and
Valvani.lo Structural relationships between a
large group of steroids, including triamcinolone
and triamcinolone acetonide, and their ether-water
partition coefficients were explored by F1ynn.l’
Correlation with biological activity was also
discussed.
The partition coefficients of triamcin-
olone in several systems have been reported by
Tomida.7 He determined that the aqueous-micellar,
octanol-water and ether-water partition coeffi-
cients were 96.3, 10.8 and 0.757, respectively.
These values have been corroborated by several
other investigators.8‘11
The mean diamagnetic susceptibility of
20 corticosteroids, including triamcinolone,-was
determined by the Faraday method.l2tl3 The
orientation of the principal molecular axes with
regard to the steroid skeleton was also given.
3. Synthesis
Barton and Heese14 developed and patented a
procedure for the chemical conversion of A1-de-
hydrotriamcinolone to triamcinolone using tri-
phenylmethyllithium in combination with lithium
aluminum hydride or LiH2Al(OCH2CH2OCH3)2 at - 7 8 O C.
Numerous microbiological dehydrogenations have
been published or patented on the conversion of A l -
dehydrotriamcinolone to triamcino1one.l Maximiz-
ation of this conversion was reported by Yoshida
and co-workers15 using a mixed culture system.
Modification and further improvement of the dehydre
genation was published16 and patented17 by Ryu and
co-workers using a semicontinuous enzymatic process
where the concentration of enzyme, active 3-keto-
steroid Al-dehydrogenase, is increased in parallel
with that of the steroid. Any water-soluble 16,17-
cycloborate of a steroid of the l6afl7a-dihydroxy-
3-keto-A4-pregnene series will work in this system.
TRIAMCINOLONE 599

5.1 Drug Metabolism
YamashitaIu studied the metabolism of
seven synthetic corticoids, including triamcinolone
in rat liver. He found that the metabolism of the
corticoids by rat liver slices or homogenates
proceeded at rates inversely proportional to the
anti-inflammatory potency of the compounds. The
major metabolic pathway for triamcinolone was found
to be 6B-hydroxylation. In addition, the liver
material also metabolized triamcinolone to the 20-
dihydro and the 11-dihydro derivatives.
5.2 Pharmacokinetics and Bioavailability
The permeability of the cornea to topi-
cally applied drugs, including triamcinolone, has
been extensively reviewed by Benson.19 Triamcin-
olone was usually determined by the blue tetra-
zolium assay (section 6.31). Local changes in the
skin and subcutaneous tissues are known to occur
as a result of percutaneous absorption of corti-
costeroids. This was the subject of an extensive
review article by Keipert20 and included discus-
sions of triamcinolone and triamcinolone acetonide.
The vasoconstrictor assay was the assay method of
choice in these studies.
The interaction between different corti-
costeroids, including triamcinolone, and commonly
administered antacids and its implication on bio-
availability was studied by Naggar, et a1.21
Triamcinolone was found to be quantitatively ad-
sorbed on charcoal and magnesium trisilicate and
not adsorbed at all on magnesium or calcium
carbonates.
The role of bioavailability of steroids
on their lipophilic character is of extreme import-
ance. Biagi and co-workers22 showed that the
chromatographic Rm value of triamcinolone and
several other steroids could be correlated with its
lipophilic character. One important conclusion
arising from this study is that the dependence of
protein binding absorption and biotransformation on
lipophilic character might strongly influence the
availability of steroids at the site of action.
The effects of fourteen steroids, includ-
600 DAVID H . SIEH
ing triamcinolone, on the NMR spectra of liposomes

derived from egg yolk phosphatidyl cholines were
studied by Ahmad and Mel101-s~~using continuous
wave and Fourier-transform measurements at 60 MHz.
The steroids were compared for their ability to
broaden the acyl methylene resonances of phospha-
tidylcholine, when incorporated into liposomes at
25% molar ratio. This study confirmed the import-
ance of the steroid side-chain at C-17 as a
requirement for sterol-phospholipid interaction.
6.3 Colorimetric Analvsis
.’
6.31 Occhipinti and c o - ~ o r k e r sreport-
~~
ed significant improvements in the tetrazolium
blue method for the determination of steroids
containing an a-ketol side chain. They reported
optimal concentration and experimental conditions
for rapid quantitative determinations of seventeen
representative steroids including triamcinolone and
triamcinolone acetonide, occurring in pharmaceu-
tical formulations. Tsuchikura and c o - ~ o r k e r s ~ ~
found that the 1,2-double bond, 16a-hydroxy and
16,17-acetonide groups inhibited the blue tetra-
zolium reactions of steroids.
The stoichiometry and mechanism of
the reaction of blue tetrazolium with triamcino-
lone was studied by Gorag and Horvath.26 On the
basis of previous literature data27r28 and experi-
mental results, they concluded that, initially,
the side chain was oxidized to the 20-keto-21-
aldehyde. This oxidation was followed by the base-
catalized D-homo rearrangement and subsequent
oxidation by blue tetrazolium to form an a-di-
ketone (D ring), which existed in the enolic form.
6.32 Tsuchikura and c o - ~ o r k e r sfound
~~
that the 1,2-double bond, 16a-hydroxy and 16,17-
acetonide groups inhibited the isonicotinic acid
hydrazide reaction of steroids. Yamashita and co-
w o r k e r ~investigated
~~ the qualitative and quanti-
tative determination of synthetic corticoids and
reported the isonicotinic acid hydrazide determina-
tion of triamcinolone and triamcinolone acetonide.
6.33 The colorimetric determination of
triamcinolone using its reaction with dinitro-
TRIAMCINOLONE 601
phenylhydrazine has been reported.29

6.34 The reaction of a 1 mg/ml aqueous
solution of sulfuric acid with triamcinolone
produced a yellow color in daylight and no color
when viewed with an ultraviolet lamp at 366 nm.30
Triamcinolone did not give a color reaction when
treated with vanillin and sulfuric acid.31
6.35 The characterization of glucocorti-
coids, including triamcinolone, with Dische's
reagent (diphenylamine in acetic acid and sulfuric
acid) or with a modified Dische's reagent (N-methyl-
diphenylamine in acetic acid and sulfuric acid) was
described by Rioux-Lacoste and Vie1.32 The
steroids were dissolved in 2 0 % acetic acid solu-
tions, reacted with the reagent at 85O C. and the
absorbance determined spectrophotometrically
between 600-650 nm.
6.36 The presence of substituents at
C-16 greatly reduces the chromogenicity of 17a-
corticosteroids in the Porter-Silber test (phenyl-
hydrazine and sulfuric acid).l Although this test
has been used for the determination of triamcino-
1 0 n e 1 2 5 ~ 2it
9 is not recommended because of the
low molar absorptivity (Ei:m = 37 @ 4 2 0 nm) 3 0
of the chromogen produced.
6.37 Triamcinolone has been quantitated
by its reaction with 2,3,5-triphenyltetrazolium
chloride followed by absorbance measurement.33
Triamcinolone was also determined spectrophoto-
metrically with 2,3,5-triphenyltetrazolium chloride
by Smoczkiewicz and Jasicziak.34 They found that
the absorption of light at 490 nm was much higher
€or compounds with neighboring OH groups; i.e.,
16a-17u-dio1, than for steroids without an OH group
at C-16. A 5 % solution of 2,3,5-triphenyltetra-
zolium chloride in methanol has been used as a
spray reagent for the detection of triamcinolone
in thin layer chromatographic systems.34
6.38 A process method has been developed
by Ivashkiv3= for rapidly estimating the conversion
of 9a-fluoro-16a-hydroxyhydrocortisone to triamcin-
olone in fermentation broths. The conversion of
the steroid was estimated by measuring the absorb-
602 DAVID H. SIEH
ance of the aqueous steroid borate complex at

241.5 and 271 nm and calculating the ratio.
6.4 Polarographic Analysis
Differential pulse polarography was used
to study the electroanalytical behavior of tri-
amcinolone.37 The half wave potentials vs. the
Ag/AgCl electrode of triamcinolone were determined
as -1.5OV (C-3 keto reduction) and -1.7OV (C-3 keto
reduction) in Britton-Robinson buffer pH 10 (50%
v/v in methanol), -1.56V (C-3), -1.75V (C-3) and
-1.95 ((2-20 keto reduction) in 0.03M tetramethyl-
ammonium hydroxide in methanol and -1.7OV (C-31,
-1.98V (C-3) and -2.20V (C-20) in 0.02M tetramethyl-
ammonium hydroxide in aqueous DMF (87% v/v). This
electroanalytical technique was then used to
determine triamcinolone in single-component
tablets3* and later in multicomponent and complex
pharmaceutical preparations.39
6.5 Chromatosraphic Analvsis
6.52 Thi; liyer chroAatographic analysis
A compilation of the chromatograph-
ic identification and-separation of triamcinolone
and other corticosteroids of similar structure is
shown in Table 11. This data has been published
after 1970.
The retention characteristics of 53
steroids on an acetonitrile-diatomaceous earth
column are given for elution with n-he tane
followed by stripping with chloroform.!i3 Triam-
cinolone was recovered nearly quantitatively (98.4%)
in the chloroform.
6.54 High Performance Liquid
Chromatography
A compiliation of the high perform-
~~
ance liquid chromatographic separation systems for

triamcinolone published after 1970 is shown in
Table 111.
6.55 Gas-Liquid Chromatography
Several gas liquid chromatographic
systems for the separation and identification of
triamcinolone with and without prior derivatization
are shown in Table IV. In addition, a gas chromato-
TABLE I1
Thin Layer Chromatographic Systems for the Detection and Determination of Triamcinolone
Solvent System Adsorbent Detection Rf Reference
-
Methylene ch1oride:diethyl Kieselgel GF254 UV at 254 nm 0.15 47
ether:methanol:water
(77: 15 :8 :1 .2)
1,-1-Dichloroethano1:acetone: 0.16
acetic acid (160:40:10)
Chloroform 0.03
Stationary phase-Formamide:
acetone (10:90)
Methylene ch1oride:p-dioxane: Silica gel Spray with 2,3,5-triphenyl 0.30 36
water (1 0:5 :5 ) -lower layer -2H-tetrazolium chloride
in methanol, heat at 110' C.
for 10 minutes.
Red-pink color
Chloroform saturated with Kieselgel GF254 Spray with 5 0 % ethanolic 0.06 48

ammonia-methanol (18:l) sulfuric acid spray, heat
at 120' C. for 5 minutes,
UV quench or iodine
chamber
Acetone:12N ammonia (99:l) 0.75
TABLE I1 (continued)
Thin Layer Chromatographic Systems €or the Detection and Determination o f Triamcinolone
Solvent System Adsorbent Detection R€

- Reference
Ethano1:SN ammonia ( 9 : l ) 0.58
Methylene ch1oride:dioxane: 0.40

water (10 :5 :5) -lower layer
Ethylene ch1oride:methanol: 0.05

water (95:5:0.2)
Methylene ch1oride:dioxane: Silica gel 60 UV at 254 nm 0.22 49

E water (10:5:5)-lower layer
Methylene ch1oride:dioxane: 0.07

water (1 2 :3 :5 ) -lower layer
Butyl acetate or Kieselgel 60 F254 Spray with 20% anhydrous Identity 50

Dich1oromethane:methyl acetate: zinc chloride in methanol; Test
water (2 :1 :1) -lower layer heat at llOo C. for 20
Stationary phase - Dioxane minutes. W at 254 and
366 run
Ch1oroform:methanol:glacial Silica gel UV at 254 nm 0.20 51

acetic acid (90:10:2)
TABLE II (continued)
Thin Layer Chromatographic Systems for the Detection and Determination of Triamcinolone
Solvent System Adsorbent Detection Rf

-
Reference
Chloroform Kieselguhr G Spray with sulfuric acid 0.24 30
Stationary phase - (1 mg/ml)
Formamide:acetone (90:10) W at 366 - violet color
Cyc1ohexane:ethyl acetate: 0.15 -

water (25:75:1) 0.21
Methylene ch1oride:diethyl- 0.04
ether:water (77:15:8 :1.2)
Ch1oroform:methanol: Silica gel Fluorescence at 2537 Ao 0.20 52

acetic acid (9O:lO:Z)
TABLE I11
High Performance Liauid ChromatoeraDhic Seuaration Svstems for Triamcinolone
Flow Rate Retention Time Detection

Co1umn Mobile Phase (ml/min) (min) or relative (nm) Ref.
Diol (polar coated n-hexane:isopropanol 1.3 20.0 254 54
silica-5~) (75 : 25)
250 x 4 . 5 m ID
Bondapak C18 1Ou 3.3g potassium phosphate 2.0 1.74 254/280 55

30cm x 3.9mm ID dibasic
4.2g potassium phosphate
monobasic
2.8L methanol
1.2L water
lop Silica gel 95% ethano1:methylene 1.55 4.90 254 56

25cm x 0.2cm ID chloride (5:95)
Spherisorb S10-ODS methanol :water (56 :44) 1.3 to 1.5 4.5 254 57
Corasil I1 n-hexane:ethyl acetate 1.0 9.05 254 58

2 ft. x 2.3mm ID (3:2)
Bondapak c18/ methanol :water (2:3) 1.5 0.4

Corasil acetonitri1e:water (1:9) 1.0
2 ft. x 2.3mm ID acetonitri1e:water (1 :4) 0.4
TABLE 111 (continued)
High Performance Liquid Chromatographic Separation Systems for Triamcinolone
Flow Rate Retention Time Detection

Co 1umn Mobile Phase (ml/min) (min) or relative (nm) Ref.
p Bondapak C18 methanol :water ( 7 0 : 3 0 ) 1.5 1.14 254 59
30cm x 4.0mm ID methanol :water (5O:SO) 1.86
acetonitri1e:water (60:40) 1.00
relative to
acetone 1.00
Bondpak C18/ methanol :water (60:40) 1.5 1.06
Corasi1 methanol :water (40:60) 1.76
2 61cm x 2.3mm ID acetonitri1e:water (40:60) 0.94
acetonitri1e:water (20:SO) 1.59
relative to
acetone 1.00
Bondapak Phenyl/ methano1:water (60:40) 1.0 0.98 254

Corasil methanol :water (40:60) 1.56
61cm x 2.3mm ID acetonitri1e:water (40:60) 1.04
relative to
acetone 1.00
Zorbax SIL cyc1ohexane:methylene 50 kg/cm2 28 254 60

3.8mm ID ch1oride:ethanol (9:4:1)
TABLE IV
Gas Liquid Chromatographic Systems
for the Separation and Identification of Triamcinolone
Detection
Column T Carrier Gas,
Column (OC1 Flow Rate Derivatization Rf
I
Ref.
2 . 5 % SE 30 on 250 FID None 1.13 61

80/100 N2, 40 cc/min Relative to
Chromosorb G cholesterol
2 ft. x 4mm 1.00
1% OV-17 on 260 EC trimethyl- 23.6 m i n . 62

100/120 silanized N2, 100 cc/min silyl
Supasorb
9 ft. x 0.25 in.
3% OV-17 on 260 F ID trimethyl- 3.53 min 63

Chromosorb WHP He, 60 cc/min silyl
1.8m x 2.0mm
TRIAMCINOLONE 609
graphic analysis of steroids, including triam-

cinolone, in pharmaceutical products has been
reported by Garzo and co-workers.64
6.6 Fluorimetric Analysis
Kadinqu has described a fluorimetric
determination of several corticosteroids, including
triamcinolone and triamcinolone acetonide, contain-
ing the A1r4-3-keto group. These A ring dienone-
containing steroids were reacted with zinc dust and
40% sulfuric acid in n-butyl ether. The fluores-
cence emission and activation maxima were then
determined at 390 and 340 nm, respectively.
Triamcinolone has been determined by
combustion followed by titration of the resulting
fluoride ion with thorium nitrate solution and
alizarin red S indicator.41
Steroids with a 16a, 17a-diol group can be
titrated with lead tetraacetate in acetic acid to a
potentiometric endpoint. The micro version of this
method using a 0 . 0 0 1 N lead tetraacetate solution
enabled triamcinolone contaminant to be determined
in its 1 6 , 1 7 - a ~ e t o n i d e , ~ ~ r ~ ~
6.8 Differential Borohydride Analysis
The differential borohydride assay develop
ed by Gor6g44 is an excellent method for the deter-
mination of A 4 - and A1t4-3-ketosteroids in pharma-
ceutical preparations. Addition of propylene
glycol to the reaction mixture to complex sodium
metaborate allowed K i r ~ c h b a u mto
~ ~ improve the
reduction of A 4 - and A4r6-3-ketosteroids. However,
A1r4-3-ketosteroids, i.e. triamcinolone,were less
than 10% reduced with sodium borohydride. By the
use of lithium borohydride instead of sodium boro-
hydride, Chafetz and c o - ~ o r k e r swere
~ ~ able to
determine triamcinolone.
6.9 Radioimmunoassay
A radioimmunoassav
.’ for the determination
of picogram quantities of triamcinolone in plasma
was reported by Loo and Jordan.65 The antibody
against triamcinolone was obtained by immunizing
rabbits with triamcinolone 21-hemisuccinate coupled
to bovine serum albumin. The problem of antiserum
cross reactivity (5%)with hydrocortisone was
TABLE V
Determination of Triamcinolone in Biological Fluids and Tissues
Chromatographic Biological Internal

Method Extraction System Standard Ref.
-
GLC with Ethyl acetate followed by Rat muscle 3H-triamcinolone 62
derivatization partitioning between hexane
and 70% methanol
GLC with Ethyl acetate Culture media of Progesterone 63

derivatization mouse and human
dermal fibroblasts
GLC Chlor0form Blood, urine and Cholesterol 61

body tissues
HPLC Diethylether Human serum Prednisone 54
5
TLC with Dichloromethane Plasma and urine H-triamcinolone 51,52
fluorescence
TRIAMCINOLONE 611
circumvented by the transformation of the steroids

to their Girard T hydrazones in ethanol denatured
plasma. Triamcinolone reacted very slowly with
Girard T.
7. Determination in Body Fluids and Tissues
Triamcinolone has been determined in bioloqical
fluids and tissues by several different methods of
analysis (see Section 6 . 9 ) in combination with
various extraction techniques. Table V summarizes
the results that have been published since 1 9 7 0 .
In addition, Saito and co-workers60 have described
a high pressure liquid chromatographic method for
the separation and quantitation of 12 synthetic
corticosteroids in blood and urine. Triamcinolone
administration produced serum peaks at 2-5 hours
with t+ = 4 hours.
8. Acknowledgements
The author would like to express his apprecia-
tion to Mr. Steve Highcock for performing the
literature search, to Dr. Michael Porubcan for
determining and analyzing the nuclear magnetic
resonance spectra, to Dr. John Dunham for his
critical review of the manuscript, and to
Marie Bruno for typing the manuscript.
612 DAVID H.SIEH
9. References
1. Florey, K., Analytical Profiles of Drug

Substances, Vol. 1, Academic Press, New York,
N.Y., pp. 367-396 (1972).
2. Bellomonte, G., Ann. 1st. Super. Sanita, g(2-31,
121-128 (1976).
3. Porubcan, M., Squibb Institute for Medical
Research,. personal
- communication.
4. Blunt, J.W.; Stothers, J.B.,Org. Magn. Res.,
.
9 (8), 439-444 (1977)
5. Doddrell, D.M.; Pegg, D.T.,J. Amer. Chem. SOC.,
102, 6388-6391 (1980).
6. Toft, P.; Lodge, B.A.; Simard, M.B., Can.J.
Pharm. Sci. , 7(2) , 53-61 (1972)
7. Tomida, H.; Yotsuyanagi, T.; Ikeda, K., Chem.
Pharm. Bull. , 26(9) , 2832-2837 (1978).
8. Grady, L.T.; Hayes, S.E.; King, R.H.,
Klein, H.R.; Mader, W.J.; Wyatt, D.K.;
Zimmerman, R.O., Jr., J. Pharm. Sci., 62(3) ,
456-464 (1973).
9. Verma, S.C.; Runikis, J.O.; Stewart, W.D.,
Indian J. Hosp. Pharm. , lO(5) , 167-173 (1973).
10. Yalkowsky, S.H.; Valvani, S.C.; J. Pharm. Sci.,
69 (8), 912-922 (1980).
11. Flynn, G.L. , ibid. , 60(3), 345-353 (1971).
12. Van den Bossche, G.; Sobry, R., Acta Crystallo.,
Section A. , A31(3) , 318-322 (1975).
13. Van den Bossche, G., _Zl Kristallogr. Kristall-
geom., Kristallphys., Kristalchem., 136(5-6) ,
402-410 (1972); C.A. 78:148114.
14. Barton, D.H.R.; Heese, R.H., Ger. Offen.
2329729 (1974); C.A. 80:108758.
15. Yoshida, T.; Sueki, M.; Taguchi, H.;
Kulprecha, S.; Nilubol, N.; Eur. J. Appl.
Microbiol. Biotechnol., 11, 81-88 (1981).
16. Ryu, D.Y.; Lee, B.K.; Thoma, R.W.; Humphrey,
A.E., Chem. Eng. Progr., Symp. Ser., 67(108) ,
80-84 71971)
17. Ryu, D.Y.; Lee, B.K.; Thoma, R.W., U.S. Patent
3585110 (1971).
18. Yamashita, O., Nippon Naibumpi Gakkai Zasshi,
47(12) , 1033-1045 (1972); C.A. 77:83767.
19. Benson, H., Arch.Ophthalmol., 91, 313-327 (1974).
20. Keipert, J.A., Med. J. Aust., 1(19), 1021-1025
(1971).
21. Naggar, V.F.; Gouda, M.W.; Khalil, S.A.,
Pharmazie, 32(2), 778-781 (1977).
TRIAMCINOLONE 613
22. Biagi, G.L.; Barbaro, A.M.; Gandolfi, 0.;

Guerra, M.C.; Cantelli-Forti, G., J. Med. Chem.,
18(9) , 837-838 (1975).
23. Ahmad, P.; Mellors, A., J. Membr. Biol., 41(3),
235-247 (1978).
24. Occhipinti, E.; Rigamonti, G.; Calati, M.,
Farmaco, Ed.Prat., 29(11) , 611-620 (1974).
25. Tsuchikura, H.; Shimano, H.; Uete, T.,
Horumon To Rinsho, 18 (6), 501-504 (1970);
C.A. 73:66805.
26. Gzrog, S.; Horvath, P., Analyst, 103(1225),
346-353 (1978).
27. Graham, R.E.; Biehl, E.R.; Kenner, C - T - ;
Luttrell, G.H. ; Middleton, D.L. ; J. Pharm. Sci.,
64, 226-230 (1975).
28. Graham, R.E.; Biehl, E.R.; Kenner, C.T., -ibid.,
-
65, 1048-1051 (1976).
29. Yamashita, 0.;Mineo, Y.; Nakagawa, T.;
Matsuoka, K.; Nakamura, M.; Mishina, Y.;
Marumoto, S.; Okada, K., Horumon To Rinsho,
19 (7), 531-535 (1971): C.A. 77:16241.
30. Vanderhaeghe, H.; Hoebus, J.; J. Pharm. Belg.,
31(1) , 25-37 (1976).
31. Hiai, S.; Oura, H.; Nakajima, T.; Planta Med.,
29 (2), 116-122 (1976).
32. Rioux-Lacoste, C.; Viel, C., Ann. Pharm. Fr.,
33 (3-4), 163-170 (1975).
33. Heintz, B.; Kalusa, R., Dtsch. Apoth.-Ztg.,
118 (27), 1000-1006 (1978).
34. Smoczkiewiczowa, A. ; Jasiczak, J. , Chem.Anal.,
16 (5), 1091-1099 (1971).
35. Ivashkiv, E., Biotechnol. Bioeng., 13(4) ,
561-567 (1971).
36. Byrne, J.A.; Brown, J.K.; Chaubal, M.G.;
Malone, M.H., J. Chromatogr., 137 (2),
489-492 (1977).
37. DeBoer, H.S., Den Hartigh, J.; Ploegmakers,
H.H.J.L., Van Oort, W.J., Anal.Chim.Acta., 102,
141-155 (1978).
38. DeBoer, H.S.; Lansaat, P.H.; Kooistra, K.R.,
Van Oort, W.J., ibid., 111(1) , 275-279 (1979).
39. DeBoer, H.S.; Lansaat, P.H., Van Oort, W.J.,
ibid., 116 (11, 69-76 (1980).
40. Kadin, H., Microchem. J., 20(2), 236-241 (1975).
41. Kofman, M.D.; Arzamastsev, A.P., Farmasiya,
21(3) I 25-27 (1972).
42. Czizier, E.; Garb'g, S.; Tzen, T., Microchim.
Acta, 966 (1970).
614 DAVID H.SIEH
43. Gorijg, S., "The Analysis of Steroids,"

CRC Crit.Rev.Anal.Chem. , 9 ( 4 ) , 3 3 3 - 3 8 3 ( 1 9 8 0 ) .
44. Ggrbg, S., J. Pharm. Sci., 5 7 , 1 1 3 7 - 1 1 4 0 ( 1 9 6 8 ) .
45. Chafetz, L.; Tsilifonis, D.C.; Riedl, J.M.,
ibid., 61, 1 4 8 - 1 5 0 ( 1 9 7 2 ) .
46. Kirschbaum, J. , ibid, 6 7 ( 2 ) , 2 7 5 - 2 7 6 ( 1 9 7 8 ) .
47. Cavina, G.; Chemello, N.; Loberto, D.;
Rocchi, I.; Romaniello, E.; Schweiger, L.;
Zanni, G., Ann. 1st. Super. Sanita, 9 ( 4 ) ,
261-309 (1973).
48. Bailey, K.; By, A.W.; Lodge, B.A., J.Chromatog.,
166(1) I 299-304 (1978).
49. Lanouette, M.; Lodge, B.A., ibid., 1 2 9 ,
475-477 ( 1 9 7 6 ) .
5 0 . Martin, J.L.; Duncombe, R.E.; Shaw, W.H.C.,
Analyst, 100 ( 1 1 8 9 ) , 2 4 3 - 2 4 8 ( 1 9 7 5 ) .
51. Kusama, M.; Sakauchi, N.; Kumaoka, S.,
Metab. , Clin. Exp. , 2 0 ( 6 ) , 5 9 0 - 5 9 6 ( 1 9 7 1 ) .
52. Kusama, M.; Sakauchi. N.: Kumaoka. S . . Nitmon
Naiburnpi Gakkai Zasshi, 4 6 ( 6 ) , 6 5 4 - 6 5 8 (1'970).
53. Graham, R.E.; Kenner, C.T., J. Pharm. Sci.,
6 2 (11), 1 8 4 5 - 1 8 4 9 ( 1 9 7 3 ) .
54. Schoeneshoefer, M.; Skobolo, R.; Dulce, H.J.,
J. Chromatogr. , 2 2 2 ( 3 ) , 4 7 8 - 4 8 1 ( 1 9 8 1 ) .
55. Baker, J.K.; Fifer, E.K., J. Pharm. Sci., 6 9 ( 5 ) ,
590-592 ( 1 9 8 0 ) .
56. Gaetani, E.; Laureri, C.F., Farmaco, Ed.Prat.,
2 9 ( 2 ) I 110-118 ( 1 9 7 4 ) .
5 7 . Gordon, G.; Wood, P.R., Analyst, 1 0 1 ( 1 2 0 8 ) ,
876-882 ( 1 9 7 6 ) .
58. Hara, S.; Hayashi, S., J.Chromatogr., 1 4 2 ,
689-703 ( 1 9 7 7 ) .
59. Tymes, N.W., J.Chromatogr. Sci., 1 5 ( 5 ) ,
151-155 (1977).
60. Saito, Z . ; Amatsu, E.; Ono, T.; H i h u m i , S.;
Mimou, T.; Hashiba, T.; Sakato, S . ; Miyamot0,M.i
Takeda, R., Nippon Naibumpi Gakkai Zasshi,
55(10) , 1296-1306 (1979).
61. Finkle, B.S.; Cherry, E.J.; Taylor, D.M.,
J.Chromatogr.Sci. , 9 ( 7 ) , 3 9 3 - 4 1 9 ( 1 9 7 1 ) .
- 7 7 ( 1 ) , 161-174
6 2 . Simpson, P.M., J.Chromatogr.,
(1973).
63. Au, D.S.-L.; Runikis, J.O.; Abbott, F . S . ;
Burton, R.W.; J.Pharm.Sci. , 7 0 ( 8 ) , 9 1 7 - 9 2 3 ( 1 9 8 1 ) .
64. Garzo, G.; Blazso,M.; G6rijg, S., Proc.Conf.App1.
Phys.Chem. ,2nd. , 1, 3 0 1 - 3 0 6 ( 1 9 7 1 ) i CA 7 6 : 9 0 0 8 8 .
6 5 . Loo, J.C.K.; Jordan, N., Res.Commun.Chem.Patho1.
Pharmacol. , 2 3 ( 3 ) , 4 9 3 - 5 0 4 ( 1 9 7 9 ) .
TRIAMCINOLONE ACETONIDE
David H. Sieh
1. Description 616
2.9 Solubility Data 620
2.10 Crystal Properties 62I
3. Synthesis 622
5.2 Pharmacokinetics and Bioavailability 624
6.2 Direct Ultraviolet Analysis 627
6.4 Polarographic Analysis 629
6.6 Fluorimetric Analysis 641
6.8 Differential Borohydride Analysis 641
6.9 Radioimmunoassay 642
7. Determination in Body Fluids and Tissues 642
9. References 644

615 Pharmaceutiul Amcialion
ISBN 012-260811-9
616 DAVID H . SIEH

of triamcinolone acetonide. A literature survey
was conducted and is complete up to July 1981.
The numbering system for topics discussed is the
same as that in the original profile (Volume 1,
pp. 397-422).
1. Description
Triamcinolone acetonide is a glucocorticoid
used mainly in the treatment of adrenocortical and
rheumatic disorders.
1.13 The C A Registry Number for triamcinolone
acetonide is 76-25-5.
The original prof ilel contains a comparison
of infrared spectral assignments of triamcinolone
acetonide in mineral oil with t3at of the compound
in the solid state. Bellomonte has published the
infrared spectra (potassium bromide disc) of 13
fluorinated steroids, including triamcinolone and
triamcinolone acetonide, and discussed the special
influence of the fluorine atom. The major
assignTents, which are in excellent agreement with
Florey , are supplemented with the assignments of
the C-F streFching frequency i.e., 1080 (1') and
972 (2') cm for triamcinolone acetonide.
Triamcinolone acetonide has been identified
by a combinaf ion of optical rotation and infrared
spectroscopy .
The improved 'H-NMR and the I3C-NMR spectra4
of triamcinolone acetonide are shown in Figures 1
and 2, respectively (refer $0 Figure 2, Section
2.2 of the originallprofile for comparison and
signments of the H-NMR spectrum). The
"C-spectrum was determined on a JEOL FX60Q
spectrometer using a 10 mm C/H dual probe. A
compi5te assignment for all the carbon atoms in
the C-NMR spectrum is listed in Table I. The
assignments are based on relative chemical shifts
a,
a
-d
d
0
c,
a,
u
a
a,
k
Figure 2. 13C-Nuclear magnetic resonance spectrum of triamcinolone acetonide
(SQ 9727) in dimethylsulfoxide-d6. (Instrument: JEOL FX60Q).
TRIAMCINOLONE ACETONIDE 619
(dimethylsulfoxide - d6 = 39.5 PPM) and 13C-”F

coupling constants and are consistent with
literature values for closely related compounds
5
.
The assignments were simplified by the utilization
of polarization transfer to selectively6enhance
and phase alter individual carbon types .
Table I. I3C-NMR Spectral Assignmentsa of
Triamcinolone Acetonide
Carbon Chemigal Carbon Chemical
Number Shift Number Shift
1 152.4 13 44.7
2 129.0 14 42.8
3 185.1 15 33.0
4 124.2 16 80.8
5 166.5 17 97.2
6 30.1 18 16.3
7 27.5 19 22.7 (5.8)
8 32.5 (16.6) 20 209.7
9 100.9 (175.8) 21 d 65.9
10 47.3 (22.4) -C-e 110.4
11 70.4 (36.1) CH,J 26.3
12 36.6 25.3
a All chemical shifts are in PPM from tetramethyl-
silane with intepal reference dimethylsulfoxide-
d = 39.5 gPy3 19All spectra were determined in
DhSO-d .
parentkeses.
g- F coupling constants shown in
Methylene carbon of the acetonide.
Methyl groups of the acetonide.
2.4 Mass Spectra
The low resolution electron impact mass
spectra of several fluorinated corticosteroidal
1,4-giene-3-ones have been determined by Lodge and
Toft . The mass spectral fragmentation pattern
found for triamcinolofe acetonide is in excellent
agreement with Florey .
In addition, the mass
spectra of triamcinolone acetonide
21-t-butylacetate was determined for comparison
purposes.
The gas chromatographic-mass spectral
characteristics of silanized triamcinolone
620 DAVID H. SIEH
acetonide were determined by Painter8 . Under the

reaction conditions employed (see section 6.55 for
reaction conditions and gas chromatographic
details), triamcinolone acetonide was initially
silanized at the C-21 position (m/z 506) and much
slower at the C-11 position (m/z 5 7 8 ) . An intense
fragment at m/z 4 4 7 [ l o s s of COCH OSi(CH ) 3 from
the disilanized material (m/z 578f helped 2onfirm
the structure.
2.9 Solubility Data
2.91 Solubility
and triamcinolone diacetate, by polyoxyethylene
lauryl ether was reported by Tomida who also
concluded that this solubilization is directly
dependent upon the lipophilicity of the steroids.
Triamcinolone acetonide yas found tg have a
solubil$.by of 4 . 9 5 x 10- M in water , 20 mg/g in
acetone , f1012 mg/ml in water and 1.44 mg/ml in
4 0 % ethanol .
Dissolution rates of triamcinolone acetonide
in aqueous media werf2determined at 28OC via a
rotating-disc method . Triamcinolone acetonide
solubility in distilled water ranged from 21
mcg/ml at 28OC to 33.6 mcg/ml at 5OOC. The
dissolutio -9 rat? con tant at maximum agitation was
4 . 5 1 x 10 hr- cm-9. Solubilities and
dissolution rates were markedly lower in potassium
chloride solution. The blue tetrazolium assay
(section 6.31) was used to determine the
triamcinolpge acetplqide. When the same
laboratory used C-triamcinolone acetonide to
determine solubilities, lower values were
obtained. Triamcinolone acetonide solubility in
distilled water ranged from 1 7 . 5 mcg/ml at 28OC to
26.5 mcg/ml at 5OOC.
The partition coefficients of triamcinolone
acetonhde in 3 systems have been determined by
Tomida . He determined that the aqueous-micellar,
octanol-water and ether-water partition
coefficients were 569, 2051 and 14.6,
respectively. The values of the octanol-water and
'TRIAMCINOLONEACETONIDE 62 1
ether-water partition coefficients are in

excellent agrffyf@flyith those published by other
investigators
The role of crystal structure (as reflected
by the melting point and the entropy of fusion)
and of the activity coefficient (as -reflected by
controlling the aqueous solubility of either
liquid or crystalline non-electrolytes, including
triamcinolone, triamcinolone acetonide and
triamcinolone diacetaff, was discussed by
Yalkowsky and Vaiyani .
Fairbrother found that a partition system
between acetonitrile and n-hexane gave
quantitative separation of triamcinolone
acetonide, found in the acetonitrile phase, from
petrolatum. Structural relationships between a
large group of steroids, including triamcinolone
acetonide, and their ether-water paftition
coefficients were explored by Flynn .
Correlation with biological activity was also
discussed.
The correlation of partitioning and
percutaneous absorption of topically applied
triamcin93one acetonide has been studied by Lien
and Tong . Partition coefficients of
triamcinolone acetonide on f6diatomaceous earth
column have been determined (see section 6.56).
Powder X-ray diffraction d a
t
a'' indicate
that triamcinolone acetonide may exist in two
polymorphic forms. For example, polymorphic form
1 exhibited a characteristic peak at 6.1 A', often
split with a smaller peak at 5.95 A'. Polymorphic
form 2, on the other hand, showed a large 6.3 A'
peak but none at 5 . 9 5 or 6.1 A'. Several other
differences in the diffractograms were also noted.
Infrared spectroscopy and differential scanning
colorimetry were unable to differentiate between
the two polymorphs.
Several parameters of grain size distribution
and particle nature of triamcinolone acetonide
crystal suspensions were determined by
polarization, electrof8transmission and electron
scanning microscopies .
622 DAVID H. SIEH
The sizes of drug particles in eye ointment

suspensions, including triamcinolone acetonide
preparationggwere measured microscopically by List
and Groenig using a membrane filtering
technique.
Murata and co-workers2' have reported the
observation of crystal forms of triamcinolone
acetonide, triamcinolone diacetate and other
glucocorticoids in sterile aqueous suspension by
electron microscopy.
3. Synthesis
Acetylation of 9a-fluoroprednisolone-21-
acetate followed by deacetylation of the
triacetate using potassium acetate in
dimethylformamide at 11O-12O0C for 4 hours under a
nitrogen atmosphere gave 9a-fluoro-pregna-1,4,16-
triene-11~,21-diol-3,2O-dione diacetate. The
resulting diacetate was oxidized with aqueous
potassium permanganate in acetone-formic acid for
3 seconds in a flow system at -5OC to give
1511-hydroxyprednisolone in 90% yield.
Ketalization with acetone followed by treatment
with sodium hydroxide in methanol at O°C for 3
hours in 91:itrogen atmosphere gave triamcinolone
acetonide
Toth and A o - ~ o r k e r spatented
~~ a process
where triamcinolone acetonide 21-nitrate was
treated with hydrogen fluoride in chloroform f o r 3
hours at O°C to give triamcinolone acetonide in an
83% yield.24
Reyba has patented a general procedure for
the synthesis of acetal fluoro derivatives of
steroids. For example, 5 grams of triamcinolone
in 8 ml of acetone was treated with 2 ml of
perchloric acid and stored at 15-17OC with
agitation for 2 hours to give 3.31 grams of
triamcinolone acetonide.
Castelli and A ~ c h e r ideveloped
~~ and patented
a one-step transformation of 16a,l7a-dihydroxy-
9,11-epoxy-3-oxo-pregn-1,4-diene to triamcinolone
acetonide. In a typical synthesis, one equivalent
of acetone was added to a 50% hydrogen fluoride
solution cooled to -3OOC followed by the addition
of one equivalent of the epoxide.
Microbial transformation of 16a,l7a-dihy-
droxy-3-oxo-pregn-4-ene-16,17-acetonide to
triamcinolone acetonide was accomplished by

incubatign with Arthrobacter simplex for 12 hours
at 28OC .
A procedure for the synthesis of 16,17-
dihyroxy steroid acetal and ketal derivatives,
including triamcinolone acetonide and2friam-
cinolone diacetate, has been patented .
5.1 Drug Metabolism
Since 68-hydroxylation was found to be the
major metabolic pathway for triamcinolone in vitro
and in vivo, this pathway was suggested for the
metabolisy80f triamcinolone acetonide. Kupfer and
Partridge found that the post mitochondria1
supernate of rat liver homogenate, supplemented
with NADPH, hydroxylates triamcinolone acetonide
with 68-hydroxytriamcinolone acetonide being the
major metabolite. Inducers of the hepatic
microsomal monooxygenase system, phenobarbital and
l-benzyl-2-thio-5,6-dihydrouracil, enhanced the
6B-hydroxylatiyj.
Yamashita found that the metabolism of
triamcinolone acetonide by rat liver slices or
homogenates proceeded at rates inversely
proportional to the antiinflammatory potency of
the compound. The liver material metabolized
triamcinolone acetonide to 68-hydroxytriamcinolone
acetonide.
Since esterification of steroid alcohols is
known to have a marked influence on their
fgsorption and excretion, the metabolism of
C-triamcinolone acetonide 21-phosphate in dogs,
monkeys angorats was studied by Kriaelani and
co-workers .
They found that the C-labelled
steroid was completely hydrolyzed to triamcinolone
acetonide following intramuscular or intravenous
injection. The radioactivity was eliminated
rapidly (t = 1 to 2 hours) from plasma and
tissues an8 the major route of excretion was via
the bile. 68-Hydroxytriamcinolone acetonide was
found to be the major metabolite in urine of all
three species. A l s o , metabolites present in the
urine as glucuronides and sulfates accounted for
approximately 10-15% of the radioaSiivity.
Recently, Gorqgn and Morrison studied the
metabolic fate of C-triamcinolone acetonide in
624 DAVID H.SIEH
rabbits, dogs, monkeys and rats. In the dog, rat

and monkey the major excretory route was the feces
irrespective of the mode of administration. In
the rabbit the excreted radioactivity was equally
distributed between urine and feces. The meta-
bolites were isolated by preparative TLC, located
by autoradiography and eluted and analyzed by MS,
IR, UV and NMR. The major metabolites of triam-
cinolone acetonide were identified as the C-21
carboxylic acids of triamcinolone acetonide and of
6B-hydroxytriamcinolone acetonide and the pre-
viously identified 6B-hydroxytriamcinolone ace-
tonide. In addition, MS and W data indicated the
presence of 9a-fluoro-11B,16a,l7-trihydroxy-3,20-
dioxo-1,4,6-pregnatrien-21-0ic acid cyclic 16,17-
acetal. They were unable to find conjugation as a
major mechanism of excre3i:gj
Zimmerman and Bowen studied the
teratogenic effects of triamcinolone acetonide on
mice by monitoring the incidence of cleft palate.
Since practically all of the radioactivity found
in the embryos and placentas was unmetabolized
drug, it was concluded that the triamcinolone
acetonide and not its metabolites was the
teratogenic agent.
5.2 Pharmacokinetics and Bioavailability
Since the desired site of activity for
topical corticosteroids is the dermis or the
epidermis, percutaneous absorption of these
materials has been extensively studied. Local
changes in the skin and subcutaneous tissue caused
by percutaneous absorption was the Qybject of an
extensive review article by Keipert and included
discussions of triamcinolone and triamcinolone
acetonide.
Kukita and c o - ~ o r k e r sreported
~~ that
triamcinolone acetonide was absorbed
percutaneously mainly by3the transfollicular
route. Hartmann and Ude studied the effect of
triamcinolone acetonide treatment with occlusive
foil by monitoring the plasma cortisol, urinary
cortisol and urinary triamcinolone acetonide
through quantitative thin layer chromatography.
Plasma cortisol and urSqary cortisol values were
also used by Rasmussen to study the percutaneous
absorption of triamcinolone acetonide in a 0.1%
ointment. Serum cortisol values were measured by

a radioallergosorbent test.
Bioavailability and activity of triamcinolone
acetonide using a novel drug delivery system, the
aeresol qyAck-break foam, was studied by Woodford
and B35ry . In another study, Woodford and
Barry compared the bioavailability and activity
of 0.1% triamcinolone acetonide and 0.1%
amcinonide preparations to a 0.025% fluocinoline
acetonide gel using a xasoconstrictor assay.
Vericat and co-workers evaluated the
bioavailability of triamcinolone acetonide
following topical application as a suspension in
oleoaqueous cream, suspension in fat excipient or
polyalcoholic solution by a vasoconstrictor assay.
Greatest bioavailability was observed with the
alcoholic solutions of the compound. A method
using the vasoconstrictive properties of
corticoids as indicated by thermal conductivity to
measure the amount and speed of these compounds,
including triamcinolone acetonide, penetrating the
skin aftfly topical application was described by
Tronnier . Elimination biokinetics of 0.01, 0.10
and 1.0% triamcinolone acetonide creams using a
vasoconstgjctor assay has been reported by Barry
and Brace .
Quantitative determination of percutaneous
3bsorption of radiolabeled drugs, including
H-triamcinolone acetonide, was described by
Schaeffet3agq co-workers in a series of
articles .
Using autoradiographic techniques,
a rapid penetration into the living layers of the
skin was observed. However, total excretion in
the urine took more than 72 hours after removal of
the excess of substance from the skin.
Autoradiographic techniques were also used to
guantitate the percutaneous absorption o i 9 - 5 1
H-triamcintg:gG acetonide in human skin I
animal skin and in huma32;5jn from lanolin

alcohol-ethyl cellulose film . Penetration of
triamcinolone acetonide through epidermal
membranes was ggeatly enhanced by the addition of
salicylic acid .
The influence of keratolytics and
moisturizers on the bioavailability of
triamcinolone acetonide using the blanching effect
as the parameter of5getection was examined by
Gloor and Lindemann .
626 DAVID H. SIEH
Verma and co-workers'' compared the potency

and effectiveness of fluorinated steroids like
triamcinolone acetonide and nonfluorinated
steroids such as 16a-hydroxyprednisolone acetonide
by studying solubility, partition coefficients,
vasoconstrictor ability and penetration across
human epidermis. They concluded that the
9a-fluoro group has no effect on the potency of
triamcinolone acetonide. The correlation of
physicochemical properties i.e., lipophilicity as
measured by partitioning in suitable solvent
systems, molar refraction, Taft's polar
substituent constant ( a o ) , molecular weight and
water solubility, and percutaneous absorption of
topically applied drugs, including triamcino39ne
acetonide, has been studi9j by Lien and Tong .
Biagi and co-workers showed that the
chromatographic R value of triamcinolone
acetonide could bg correlated with its lipophilic
character. Therefore, the dependence of protein
binding absorption and biotransformation on
lipophilic character strongly influences the
availability of steroids at&he sight of action.
Hackney and co-workers demonstrated that
mouse fibroblasts growing in vitro contain a
binding component for triamcinolone acetonide
which is apparently distributed intracellularly,
largely as a cytoplasmic soluble macromolecule.
Furthermore, the structure-activity relationships
of steroids active in growth inhibition were found
to be exacjtly paralleled by their ability to
displace H-triamcinolone acetonide from this
binding component.
A new, selective drug delivery system, which
uses liposomes as drug carriers, for the topical
route of administratiog-,was recentljy described by
Mezei and Gulasekharam . Using C-triamcinolone
acetonide, they found that liposomal encapsulation
increased the concentration of the drug at the
site where its activity is desired i.e., epidermis
and dermis. Because of the selectivity of the
liposomal dosage form, it could provide increased
efficacy and decreased toxicity of topically,
applied drugs.
6.2 Direct Ultraviolet Analysis
In the ultraviolet spectrum of a mixture of
triamcinolone acetonide and chlorhexidine, the
overlapping absorption maxima were approximated by
the use of Gauss-Lorenz function paraEtters for
calculations on a programmed computer .
Poly-
sorbate 80 and methyl- and propyl-p-hydroxy-
benzoates were found to interfere in the deter-
mination of corticosteroids such as triamcinolone
acetonide in dermatological preparatigfs by the
ultraviolet spectrophotometric method .
6.3 Colorimetric Analysis
6.31 Ochipinti and co-workers62 reported
significant improvements in the tetrazolium blue
method for the determination of steroids contain-
ing an a-ketol side chain. They reported optimal
concentration and experimental conditions for
rapid quantitative determinations of 17 represen-
tative steroids, including triamcinolone and
triamcinolone acetonide, occuring in pharma-
ceutical fggmulations. Tsuchikura and
co-workers found that the 1,2-double bond, the
16a-hydroxy and the 16,17-acetonide groups
inhibited the blue tetrazolium reaction.
Notable interference was shown by
polyethylene glycols, propylene glycol and lanolin
in the determination of corticosteroids such as
triamcinolone acetonide in dermatological 61
preparations by the blue tetrazolium method
Sorbitol and squalene caused only slight
.
interference.
A reaction rate method using a modified blue
tetrazolium reaction for the determination68f
triamcinolone acetonide has been described .
This method depends on the mixing of a 5% tetra-
methylammonium hydroxide in absolute ethanol
solution with a blue tetrazolium solution of
triamcinolone acetonide by an automatic stopped-
flow system and monitoring the absorbance at 525
nm during selected measurement time.
6.32 Tsuchikura and c o - ~ o r k e r sfound
~~
that the 1,2-double bond, the 16a-hydroxy and the
628 DAVID H . SIEH
16,17-acetonide groups inhibited the isonicotinic

acid hydrazide6seaction of steroids. Yamashita
and co-workers investigated the quantitative and
qualitative determination of synthetic corticoids
and reported the isonicotinic acid hydrazide
determination of triamcinolone and triamcinolone
acetonide. Substances including carbonyl,
reducing,acidic and basic compounds interfered
with the determination of triamcinolone acetonide
in ointment prepggations by the isonicotinic acid
hydrazide method . Girard’s fractionation method
was used to remove the interfering substances
prior to measurement.
acetonide produced a faintly yellow color in
daylight and no color when@ewed with an
ultraviolet lamp at 366 nm .
6.34 Although the Porter-Silber test
(phenylhydrazine and sulfuric acid) has been used
for the dtfermination of triamcinolone
acetonide , it is not6yecommended because of the
low molar absorptivity of the chromogen
produced.
6.35 The reaction of triamcinolone
acetonide with 2,3,5-triphenyltetrazolium chloride
instead of tetrazolium blue followed by absorbance
measurement according to the European Pharmacopeia
was used to quantitate the $&eroid in
pharmaceutical preparations .
6.36 A new method based on the oxidation
of the C-17 side chain with cupric acetate,
condensation of the resulting 20-keto-21-aldehyde
with 4,5-dimethyl-o-phenylenediamine and spectro-
photometric measurement of the quinoxaline deriva-
tive gbtained has been described by Szepesi and
Gdrdg for the determination of 21-hydroxycor-
ticosteroids, including triamcinolone acetonide,
in pharmaceutical preparations. This procedure
was used for the analysis of bulk material
(purification on a cation exchange column was
required after the oxidation step) or ointment (no
purification required).
Chafetz and Tsilif~nis’~ also used a cupric
acetate oxidation step to determine triamcinolone
acetonide. The oxidation step was followed by

chromogen formation with aqueous acidic
phenylhydrazine and absorption measurement at
370 nm. This procedure is claiE5d to be twice as
sensitive as STfpesi and Gdrdgs .
Bundgaard quantitatively determined triam-
cinolone acetonide by cupric acetate oxidation
followed by condensation with 3-methyl-benzo-
thiazol-2-one hydrazone in alkaline solution to
form the highly absorbing azine (Xmax = 400 nm).
This method was used to determine triamcinolone
acetonide in the presence of its 21-acetate ester.
6.4 Polarographic Analysis
Differential pulse polarography was used to
study the elecf5oanalytical behavior of triamcino-
lone acetonide . The half-wave potentials vs
Ag/AgCl electrode of triamcinolone acetonide were
determined as -1.46 V (C-3 keto reduction) and
-1.70 V (C-3 keto) in Britton-Robinson buffer pH
10 ( 5 0 % v/v in methanol), -1.60 V ((2-3 keto),
-1.79 V (C-3 keto) and -2.03 V (C-3 keto) in
0.03 M tetramethylammonium hydroxide in methanol
and -1.71 V (C-3 keto) , -1.98 V (C-3 keto) and
-2.15 V (C-20 keto) in 0.02 M tetramethylammonium
hydroxide in 87% aqueous dimethylformamide. This
electroanalytical technique was then used to
determine triamcinolone acetonide in multicom-
ponent and complex pharmaceutical preparations73 .
6.52 Thin Layer Chromatographic Analysis
A compilation of the thin layer chromato-
graphic properties and identification of triam-
cinolone acetonide published after 1970 is shown
in Table 11.
IdentificatifB30f triamcinolone acetonide in
a 0.025% ointment was accomplished by thin
layer chromatography on silica gel GF plates using
a developing solvent of methanol: chloroform:
benzene (20:100:40) and a 0.2%7Q1ue tetrazolium in
methanol spray reagent. Massa identified
triamcinolone acetonide by thin layer
chromatography on silica gel using a developing
630 DAVID H.SIEH
solvent of benzene:methanol ( 8 5 : 1 5 ) and

fluorescence at 366 nm.
Chromatography
Recently, a high-performance liquid
chromatographic (HPLC) procedure involving the use
of adsorption chromatography on Corasil I1 hasg4
been adopted by the United States Pharmacopeia
for the determination of triamcinolone acetBgide
in 0.1 and 0.025% creams. A similar method with
a silica gel column and a solvent system of 95%
ethano1:methylene chloride (5:95) has been used
for the analysis of triamcinolone acetonide
creams. Triamcinolone acggonide has also been
determined in rice starch using adsorption HPLC.
These methods, which do not involve the use of an
internal standard, and numerous other methods for
the detection and determination of triamcinolone
acetonide with internal standards are listed below
in Table 111.
Table 11. Thin Layer Chromatography of Triamcinolone Acetonide
Methylene Chloride: Silica gel Radioactive scanner 0.91 79

methanol (15:6)
A-Methylene Chloride:
methanol ( 9 3 : 7 )
B-Cyclohexane:
dioxane:water
( 5 0 :5 0 :10)
Equal volumes of 0.36

A and upper phase
of B
Benzene:acetone 0.22
(2:l)
Equal volumes of 0.64
A and upper phase
of B:methanol
(9:2)
Methano1:ethyl Silica gel Radioactive scanner 0.45 30

(2:98) H
Table 11. Thin Layer Chromatography of Triamcinolone Acetonide (Continued)
Solvent System Ad sorbe nt Detection Reference

Rf
To1uene:ethyl- Kieselgel UV at 254 nm 0.38 78

acetate:85% F-254
formic acid
(50:45: 5)
To1uene:isopropanol: 0.59
37% ammonium
hydroxide (70:29 :1)
To1uene:dioxane: 0.84
methanol:37%
ammonium hydroxide
(20:50 :20: 10)
Methylene chloride: Silica gel UV at 254 nm 0.45 79

diethylether: GF 254
methanol :water
(77:15 :8 :l. 2)
1,l-dichloroethanol: 0.42
acetone:acetic acid
(160:40 :lo)
To1uene:chloroform
(75:25)
Stationary phase-
F0rmamide:acetone
(10:9 0 )
Ethylene dichloride: 0.13
methano1:water
m
( 9 5 :5:0.2)
w
Ethylene chloride: Silica gel 5 0 % ethanolic 0.65 80

methano1:water GF 254 sulfuric acid, relative
( 9 5 :5 :0.2) 12OOC f o r 5 to 168-
minutes methylpredni-
solone acetate
= 1.00
Butylacetate or Kieselgel 6 0 Spray with 20% Identity 81

Dichloroethane: F 254 anhydrous zinc Test
methyl acetate : chloride in
water (2:1:1), methanol, llO°C
lower layer for 20 minutes,
Stationary phase - UV at 254 and
dioxane 3 6 6 nm
Chloroform: Silica gel W at 2 5 4 nm 0.47 82

methano1:glacial
acetic acid
(90:10:2)
To1uene:chloroform Kieselguhr Spray with 1 mg/ml 0.39 67

(75:25) Stationary G sulfuric acid,
phase-Formamide: violet color at
acetone (10:90) 366 nm
Methylene chloride: Silica gel G 0.13-0.15

diethylether:
methano1:water
(77:15:8 :l.2)
Chloroform: Silica gel Fluorescence at 0.47

methano1:acetic 2537AO
acid (90:10:2)
Table 111. High Performance Liquid Chromatography of Triamcinolone
Acetonide
Column Mobile Phase Flow Rate Retention Time Detection Reference
(ml/min) (min) (nm)
16 reverse Acetonitrile: 2.0 Variable 254 87
phase ODs, R P water (30:70)
and phenyl
columns
Hypersil ODS Methanol: 1.0 250 88

20 cm water (60:40)
10 p silica 95% ethanol: 1.55 1.28 254 85

Gel 25 cm x methylene
0.2 cm chloride (5:95)
u Bondapak C18 Methano1:water 1.5 1.50 254 89

30 cm x 4.0 nun (70:30)
Methano1:water 4.93
( 5 0 :50)
Acetonitrile: 1.22
water (60:40)
Acetonitrile: 2.21
water (40:60) Re1ative to
acetone 1.00
Acetonide (Continued)
(ml/min) (min) (nm)
Bondapak C18/ Methano1:water 1.5 1.22
Corasil (60:40)
61 cm x 2.3 mm Methano1:water 6.41
(40 :60)
Acetonitrile: 1.11
water (40:60)
Acetonitrile: 8.12
water (20:80) Relative to
acetone 1.00
E
m
Bondapak Methano1:water 1.0 1.28
Phenyl/Corasil (60:40)
Methano1:water 5.40
(40:60)
Acetonitrile: 1.17
water (40:60)
Acetonitrile: 4.56
water (20:80) Relative to
acetone 1.00
Z ipax water- 0.33 7.2 254 86

l m x 2 m m saturated
methy1ene
chloride
T a b l e 111. High Performance L i q u i d Chromatography o f Triamcinolone
Column Mobile Phase Flow Rate R e t e n t i o n T i m e D e t e c t i o n Reference

(ml/min) (min) (nm)
S p h e r i s o r b ODS Methano1:water 1.3-1.5 11.0 2 54 90,91
5-10 (56:44)
2 5 c m x 4.6 mm
Biosil A Petroleum 1.0 19.0 254 92

20-44 e t h e r :c h l o r o - or
5 0 cm x 2 . 1 mm form :methanol 250
( 6 0 :39:2) kg/cm2
8
I
.
M et h y l e n e 26.6
ch1oride:meth-
a n o l :water
( 9 7 :1:2)
M et h y l e n e 13.3
chloride:
isopropanol
(98:2)
(ml/min) (min) (nm)
10% ODS p - Acetonitrile: 2.0 12.0 254 93
Bondapak water (30:70)
5% ODS 6.0
Partisil
3-Rever sed Acetonitrile: 2.0 6.0 254 94

Phase ODS water ( 6 0 : 4 0 )
columns
Quantitative determination of triamcinolone

acetonide in a 0.0152% aeresol spray $8 HPLC has
been reported by Gibbs and Kirschbaum .
Using
halcinonide as the internal standard, separation
and quantitation was accomplished on a reverse
phase Partisil ODS 25 cm x 4.6 mm column with a
mobile phase of acetonitri1e:water (40:60), a flow
rate of 1.0 to 1.5 ml/min and UV detection at
254 nm. 88
A procedure has been developed by Tenneson
for the determination of triamcinolone acetonide
in human serum by HPLC with a detection limit of
1 ng/ml. The triamcinolone acetonide is extracted
from potassium chloride saturated serum samples
into diethylether. After evaporation, the residue
is taken up in the mobile phase (methanol:water,
60:40) and then separated (retention time 10
minutes) and quantified on a 5 micron Hypersil ODS
HPLC column monitoring the absorbance of the
eluate at 250 nm.
6.55 Gas Liquid Chromatography
Three gas liquid chromatographic (GLC)
systems for the separation and identification of
triamcinolone acetonide are shown below in Table
IV. In addition to those listed below, the gas
chromatographic properties of triamcinolone
acetonide that had been silanized at C-11 and
disilanized at C-11 and C-21 have been studied by
Painter . Triamcinolone acetonide was silanized
by reaction with trimethylsilylimidazo1e:bis-
trimethylsilyl acetamide:trimethylchlorosilane
(3:3:2) and chromatographed on a 5 ft. x 0.020 in
OV-1 stainless steel support coated open tubular
column held at 220OC. The direct injector and
flame ionization detector temperatures were 220
and 3OO0C, respectively. The helium carrier gas
pressure was 5.0 psig. The disilanized product
eluted at 2.5 minutes. The mass spectral
characteristics of the silanized products were
also determined (section 2.4).
Table IV. GLC Systems for the Separation and Identification of
Triamcinolone Acetonide
Column T Detection, carrier Derivati-
Column ("C) gas, flow rate zation R, Reference
1% OV-17 on 100/120 260 EC, nitrogen, tri- 31.5 96
silanized Supasorb 100 cc/min methyl- min
9 ft. x 0 . 2 5 in silyl
3% OV-17 on 260 FID,helium, tri- 4.38 97

Chromosorb WHP 60 cc/min methyl- min
1.8 m x 2.0 mm silyl
3% OV-3 on 250 EC, nitrogen, tri- 16 98

Chromosorb G 60 cc/min methyl- min
silyl
TRIAMCINOLONEACETONIDE 641

The versatile solvent system of n-hexane:-
ch1oroform:dioxane:water (90:10:40:5) was used for
the partition chroygtographic isolation of triam-
cinolone acetonide . The partition coefficients
were determined on a diatomaceous earth column.
6.6 Fluorimetric Analysis
Kadin74 has described a fluorimetric de e -
mination of corticosteroids containing the A f 1 5 -
3-keto group. These A-ring dienone-containing
steroids were reacted with zinc dust and 4 0 %
sulfuric acid in n-butyl ether and the fluore-
scence emission and activation maxima determined
at 390 and 340 nm, respectively. This simple
method allowed the quantitation of triamcinolone
acetonide in liquid formulations with a sensi-
tivity of about 2 micrograms. Under the condi-
tions employed only steroidal A-ring dienones
fluoresce.
Fluorescent silica gel chromatography and
photodensitometry have been used to determine
triamygnolone acetonide in pharmaceutical formula-
tions .
Steroids with a 16a117a-diol group can be
titrated with lead tetraacetate in acetic acid to
a potentiometric endpoint. The micro version of
this method using a 0.001 N lead tetraacetate
solution enabled triamcinolone contam
determined in triamcinolone acetonide%:3f to be
6.8 Differential Borohydride Analysis
Thel@,fferential borohydride assay developed
by Gorag is an exc 1 ent method for the deter-
mination of A - and A'1a-3-ketosteroids in pharma-
ceutical preparations. Addition of propylene
glycol to the reaction mixturfO$o complex sodium
metaborate allgwed Kir c baum to improve the
reducti n of A - and A"'-3-ketosteroids. How-
ever l AP'4-3-ketosteroids i.e., triamcinolone were
less than 10% reduced. By the use of lithium
borohydride insbyad of sodium borohydride, Chafetz
and co-workers were able to determine
642 DAVID H . SIEH
triamcinolone. This method should also allow the

determination of triamcinolone acetonide.
6.9 Radioimmunoassay
A radioimmunoassay for triamcinolone
acetonide &j plasma was described by Ponec and
co-workers .The antibody against triamcinolone
acetonide was obtained by immunizing rabbits with
triamcinolone acetonide-21-hemisuccinate coupled
to bovine serum albumin. The minimum detectable
amount was 200 pg.
7. Determination in Body Fluids and Tissues
Triamcinolone acetonide has been determined
in biological fluids and tissues by several
different methods of analysis in combination with
various extraction techniques. Table V summarizes
the results that have been published since 1970.
Table V. Determination of Triamcinolone Acetonide in Biological Fluids and
Tissues
Chromatographic Biological Internal
Method Extraction System Standard Re ference
GLC Ethyl acetate Rat muscle 3H -triamcino- 96
followed by lone acetonide
partitioning
between hexane
and 7 0 % aqueous
methano1
GL.C Ethyl acetate Mouse and Progesterone 97

human dermal
fibroblasts
TLC with Dichloro- Plasma and H-triamcino- 82,83

fluorescence methane urine lone acetonide
~~
HPLC Diethylether Human serum 88

644 DAVID H. SIEH
8. Acknowledgements
The author would like to express his
appreciation to Mr. Steve Highcock for conducting
the literature search, to Dr. Mike Porubcan for
resonance spectra, to Dr. John Dunham for his
critical review of the manuscript and to Diane
Walker for typing the manuscript.
9. References
1. Florey, K., "Analytical Profiles of Drug
Substances", Vol. 1, Academic Press,
New York, N.Y., pp. 397-422 (1972).
2. Bellomonte, G., Ann. 1st. Super. Sanita,
9(part 2-31, 121-128 (1973).
3. Hahdlos, M., Arch. Pharm. Chemi., 82 (25-26) ,
1392-1395 (1975).
4. Porubcan, M . , Squibb Institute for Medical
Research, personal communication.
5. Blunt, J.W,, J.B. Stothers, Org. Magn. Res.,
9(8),
- . _ 439 (1977).
6. Doddrell, D.M.,.D.T. Pegg, J. Amer. Chem.
SOC., 102, 6388 (1980).
7. Lodge, B.A., P. Toft, J. Pharm. Pharmacol.,
23 (31, 196-199 (1977).
8. Painter, J.L., Squibb Institute for Medical
9. Tomida, H., T. Yotsuyanagi, K. Ikeda, Chem.
Pharm. Bull., 26(9), 2832-2837 (1978).
10. Grady, L.T., S.E. Hays, R.H. King, H.R.
Klein, W.J. Mader, D.K. Wyatt, R.O. Zimmerer,
Jr., J. Pharm. Sci., 62(3), 456-464 (1973).
11. Verma, S.C., J.O. Runikis, W.D. Stewart,
Indian J. Hosp. Pharm., 10(5), 167-173
(1973)
12. Block, L.H., R.N. Patel, J. Pharm. Sci.,
62 (4), 617-621 (1973).
13. Behl, C.R., L.H. Block, M.L. Borke, ibid.,
.
65 (3), 429-430 (1976)
14. Yalkowsky, S.H. S.C. Valvani, ibid., 69(8),
912-922 (19801.
-
15. Flynn, GiL., ibid. , 60(3), 345-353 (1971).
16. Weber, D.J., T.R. Ennals, H. Mitchner, ibid.,
61(5), 689-694 (1972).
17. Fairbrother, J.E., Methodol. Dev. Biochem.,
5, 141-144 (1976).
18. Moellman, H . , B. Von Klot-Heydenfeldt,

D.H. Miemeyer, H. A l f e s , I n t . 2 . K l i n .
Pharmakol., Ther. Toxikol., 5(4), 434-443
(1972): C.A. 77:39117.
19. L i s t , . P . H . , J . M . Groenig, Pharm. I n d . ,
38 (12), 1159-1171 (1976) .
20. Murata, R . , A. Tanuma, N . H i k i c h i , H . N i w a ,
Tohoku Yakka Daigaku Kenkyu Nempo, 25, 57-61
71978) ; CA 91:16297.
21. P a n i c h i , F . , S. A f r i c a n P a t e n t , 72-06559
(1973)i CA 80:37396.
22. P a n i c h i , F . , G e r . Offen., 2,243,480 (1974);
CA 80:108759.
23. Toth, J . , A. Boor, M. K o v a t s i t s , 2 . Komesz,
P. Major, T. Kovats, K . S . Gorgenyi, E.
C z a j l i k , e t al, G e r . Offen., 2,246,203
(1973); CA 78:148133.
24. Reyba, S . A . , Spain P a t e n t , 414181 (1976); CA
85:177793.
25. C a s t e l l i , P . P . , A. A s c h e r i , G e r . Offen., 2,
448,548 (1975); CA 84:90423.
26. Rolland, G . I . , L. Mantica, R. Ciceri, G e r .
Offen., 2,016,353 (1971); CA 75:47508.
27. Compania Espanola d e E s t e r o i d e s S.A., Spain
P a t e n t , 483701 (1980); CA 93:9781.
28. Kupfer, D . , R. P a r t r i d g e , Arch. Biochem.
Biophys., 140(1), 23-28 (1970).
29. Yamashita, O., Nippon Naibumpi Gakkai Z a s s h i ,
47 (12), 1033-1045 (1972) .
30. K r i p a l a n i , K . J . , A . I . Cohen, I. Weliky, E.C.
S c h r e i b e r , J. Pharm. S c i . , 64(8), 1351-1359
(1975).
31. Gordon, S . , J. Morrison, S t e r o i d s , 32(1),
23-35 (1978).
32. Zimmerman, E . F . , D. Bowen, T e r a t o l o g y , 5(3),
335-343 (1972).
33. Zimmerman, E . F . , D. Bowen, i b i d . , 57-70
(1972).
34. K e i p e r t , J . A . , Med. J. Aust., 1(19),
1021-1025 (1971).
35. Kukita, A.. K . Yamada, T . Matsuzawa.
Y. Takada, . Taehan Pibukwa Hakhoe Chapchi. ,
15(2), 115-122 (1977); CA 87:141187.
36. Hartmann, F . , P. Ude, Verh. Dtsch. G e s . I n n .
Med., 80, 1548-1551 (1974); CA 83:6764.
37. Rasmussen, J . E . , Arch. Dermatol., 114,
1165-1167 (1978).
38. Woodford, R . , B.W. Barry, J. Pharm. S c i . ,
66 (1), 99-103 (1977) .
646 DAVID H.SIEH
39. Woodford, R., B.W. Barry, Curr. Ther. Res.,

Clin. Exp., 21(6), 877-886 (1977).
+
40. Vericat, C.F., C.R. Beaus, C.J. Coll, Con r
Nac. Biofarm. Farmacocinet., (Actas),
323-336 (1977); CA 89:135736.
41. Tronnier, H., Arch. Klin. Exp. Dermatol.,
237, 769-773 (1970).
42. Barry, B.W., A.R. Brace, J. Pharm.
Pharmacol., 26(Supple), 67-68 (1974).
43. Schaefer, H., W. Schalla, Percutaneous
Absorpt. Steroids (Int. Symp.) , 53-66 (1980) .
44. Schaefer, H., G. Stuettgen, W. Schalla,
E. Bauer, J. Gazith, Ad;. Pharmacol. Ther.,
Proc. Int. Cong. Pharmacol., 7th, 9, 223 -235
71979).
45. Schaeffer, H., G. Stuettgen, A. Zesch,
W. Schalla, J. Gazith, Curr. Probl.
Dermatol., 7, 80-94 (19’78).
46. Schaeffer, H., G. Stuettgen, Mykosen, Suppl.,
1, 164-170 (1978).
47. Schaeffer, H., A. Zesch, G. Stuettgen, Arch.
Dermatol. Res., 258(3), 241-249 (1977).
48. Baker, J.R.J., R.A. Christian, P. Simpson,
A.M. White, Br. J. Dermatol., 96(2), 171-178
(1977).
49. Lewis, J.D., B.D. Cameron, D.R. Hawkins,
L.F. Chasseaud. E.R. Frankline.
Arzneim.-Forsch., 25(10), 1646-1650 (1975).
50. Ponec, M., M.K. Polano, Arch. Dermatol. Res.,
265, 101-104 (1979).
51. Rimbau, V., F. Lleonart, Ar2neim.-Forsch.,
25 (7), 1042-1044 (1975) .
52. Iyer, B.V., R.C. Vasavada, Int. J. Pharm.,
3 (4-5), 247-260 (1979).
53. Iyer, B.V., Diss. Abstr. Int. B., 40(1), 173
(1979); CA 91:128981.
54. Polano, M.K., M. Ponec, Arch. Dermatol.,
112 (5), 675-680 (1976) .
55. Mezei, M., V. Gulasekharam, Life Sci.,
26 (18), 1473-1477 (1980).
56. Gloor, M., J. Lindemann, Dermatol.
Monatsschr., 116 (2), 102-106 (1980) .
57. Lien, E.J., G.L. Tong, J. SOC. Cosmet. Chem.,
24 (6), 371-384 (1973).
58. Hackney, J.F., S.R. Gross, L. Aronow,
W.B. Pratt, Mol. Pharmacol., 6, 500-512
(1970).
59. Biagi, G.L., A.M. Barbaro, 0. Gandolfi,

M.C. Guerra, G. Cantelli-Forti, J. Med.
Chem., 18 (91, 837-838 (1975).
60. Kakac, B., V. Rejholec, Cesk. Farm., 26(4) ,
124-127 (1977).
61. Halot, D., M. Lanson, Ann. Pharm. Fr.,
29 (11), 533-539 (1971) .
62. Occhipinti, B., G. Rigamonti, M. Calati,
Farmaco, Ed. Prat., 29(11), 611-620 (1974).
63. Tsuchikura, H., H. Shimano, T. Uete, Horumon
To Rinsho., 18 (6), 501-504 (1970).
64. Koupparis, M.A., K.M. Walczak,
H.W. Malmstadt, J. Pharm. Sci., 68(12),
1479-1482 (1979).
65. Yamashita, O., Y. Mineo, T. Nakagawa,
K. Matsuoka, M. Nakamura, Y. Mishina,
S. Marumoto, K. Okada, Horumon To Rinsho.,
19 (7), 531-535 (1971); CA 77:1624 1.
66. Nakaji, Y., M. Ota, T. Hayakawa, J. Kawamura,
Iyakuhin Kenkyu, 7 (1), 17-25 (1976); CA
88:141771.
67. Vanderhaeghe, H., J. Hoebus, J. Pharm. Belg.,
31(1), 25-37 (1976).
68. Heintz, B., R. Kalusa, Dtsch. Apoth.-Ztg.,
118(27), 1000-1006 (1978): CA 89:135905.
69. Szepesi; G., S. Gbrdg, Boll. Chim. Farm.,
.
114 (2), 98-106 (1975)
7 0 . Chafetz, L., D.C. Tsilifonis, J. Pharm. Sci.,
66 (8), 1145-1148 (1977) .
71. Bundgaard, H., Arch. Pharm. Chemi. Sci. Ed.,
6(3), 127-140 ('1978).
72. DeBoer, H.S., J. DanHartigh,
H.H.J.L. Ploegmakers, W.J. Van Oort, Anal.
Chim. Acta., 102, 141-155 (1978).
73. DeBoer, H.S., P.H. Lansaat, W.J. VanOort,
ibid, 116 (1), 69-76 (1980) .
74. Kadin, H., Microchem. J., 20(2), 236-241
(1975).
75. Massa, V., Labo-Pharma., 18(186), 80-81
(1970).
76. GbrOg, S. "The Analysis of Steroids", CRC
Crit. Rev. Anal. Chem. , 9(4), 333-383 m 8 0 ) .
77. Czizier, E. S. Gorag, T. Szen, Microchim.
Acta., 966 (1970).
78. Egli, R.A., S. Tanner, Fresenius 2. Anal.
Chem., 295(5), 398-401 (19/9).
79. Cavina, G., N. Chemello, D. Loberto,
I. Rocchi, E. Romaniello, L. Schweiger,
648 DAVID H.SIEH
G. Zanni, Ann. 1st. Super. Sanita, g(pt.41,

261-309 (1973).
80. Bailey, K., A.W. By, B.A. Lodge, J.
Chromatogr., 166(1), 299-304 (197v.
81. Martin, J.L., R.E. Duncombe, W.H.C. Shaw,
Analyst, 100 (1189), 243-248 (1975).
82. Kusama, M., N. Sakauchi, S. Kumaoka, Metab.,
Clin. Exp., 20(6), 590-596 (1971).
83. Kusama, M. N. Sakauchi, S. Kumaoka, Ni
Naibumpi Gakkai Zasshi, 46(6), 654-6
(1970); CA 74 :109918.
P
84. United States Pharmacopeia, XX, 812 (1980).
85. Gaetani, E., C.F. Laureri, Farmaco, Ed.
Prat., 29 (2), 110-118 (1974).
86. Higgins, J.W., J. Chromatogr., 115(1),
232-235 (1975).
87. Kirschbaum, J., R. Clay, R. Poet, "Symposium
on the Analysis of Steroids", Eger, Hungary,
May 1981, in press.
88. Tenneson, M.E. International Development
Laboratory - E.R. Squibb and Sons, Inc.,
Personal communication.
89. Tymes, N.W., J. Chromatogr. Sci., 15(5),
151-155 (1977).
90. Gordon, G . , P.R. Wood, Proc. Anal. Div. Chem.
SOC., 14(2), 30-32 (1977).
91. Gordon, G., P.R. Wood, Analyst, 101(1208),
876-882 (1976).
92. Cavina, G., G. Moretti, B. Gallinella,
R. Alimenti, R. Barchiesi, Boll. Chim. Farm.,
117 (9), 534-544 (1978).
93. Kirschbaum, J., J. Pharm. Sci., 69(4),
481-482 (1980).
94. KirschbaG, J,, R. Poet, K. Bush, G. Petrie,
J. Chromatogr., 190(2), 481-485 (1980).
95. Gibbs, V., J. Krischbaum, Squibb Institute
for Medical Research, personal communication.
96. Simpson, P.M., J. Chromatogr., 77(1), 161-174
(1973).
97. Au, D.S.-L., J.O. Runikis, F.S. Abbott,
R.W. Burton, J. Pharm. Sci., 70(8), 917-923
(1981).
98. Goebbeler, K.H., J. Brienlich, Pharm.-Ztg.,
28, 1037-1040 (1972) .
99. Ponec, M., M. Frolich, A. DeLijster,
A.J. Moolenar, Arch. Dermatol. Res., 259(1),
63-70 (1977).
100. GOrOg, S . , J. Pharm. Sci., 5 7 , 1137-1140
(1968).
101. Chafetz, L., D.C. Tsilifonis, J.M. Riedl,

ibid, 61, 148-150 (1972).
102. Kirschbaum, J., ibid, 67(2), 275-276 (1978).
103. Lerner, H., Squibb Institute f o r Medical
104. DeVincentis, J., Squibb Institute f o r Medical
TRIAMCINOLONE DIACEMTE
David H.Sieh
1. Description 652
2.2 Nuclear Magnetic Resonance 652
2.9 SolubilityData 655
3. Synthesis 656
8. References 677
AnalyticalProfiles of Drug Substances Copyright 0 1982 by The Americdn

Volume I 1 65 1 Phumaceutid Association
ISBN 012-2M8l1-9
652 DAVID H. SIEH

of triamcinolone diacetate. A literature survey
was conducted and is complete up to July 1981.
The numbering system for topics discussed is the
same as that in the original profile (Volume 1,
pp. 4 2 2 - 4 4 2 ) .
1. Description
Triamcinolone diacetate is a glucocorticoid
used primarily in the treatment of adrenocortical
and rheumatic disorders.
1.13 The CA Registry Number for triamcinolone
diacetate is 67-78-7.

2
The improved 'H-NMR and the 13C-NMR spectra
of triamcinolone diacetate are shown in Figures 1
and 2 , respectively (please refer ty Figure 3
section 2 . 2 of the original profile for compari-
fyn and assignments of the 'H-NMR spectrum). The
C-spectrum was determined on a JEOL FX60Q
spectrometer using a 10 mm C/H dual probe. A
compi9te assignment for all the carbon atoms in
the C-NMR spectrum is listed in Table I. The
assignments are based on relative chemfSallghifts
(dimethylsulfoxide-d = 39.5 PPM) and C- F
coupling constants akd are consistent wit3 litera-
ture values for closely related compounds .The
assignments were simplified by the utilization of
polarization transfer to selectively enhance and
phase alter individual carbon types .
Figure 1. '€I-Nuclear magnetic resonance spectrum of triamcinolone 16,21-
diacetate (SQ 9465) in CDC13. Instrument: Varian XL-100A.
Figure 2. 13C-Nuclear magnetic resonance spectrum of triamcinolone 16,21-
diacetate (SQ 9465) in dimethylsulfoxide-d6. Instrument: JEOL FX60Q.
TRIAMCINOLONE DIACETATE 655
Table I. I3C-NMR Spectral Assignmentsa of Triam-

cinolone 16,21-diacetate
Carbon Chemigal Carbon Chemica1
Number Shift Number Shift
1 152.7 13 47.4
2 129.0 14 43.1
3 185.3 15 31.6
4 124.2 16 75.1
5 166.9 17 87.8
6 30.3 18 16.3
7 27.2 19 23.0 (4.9)
8 33.2(18.5)' 20 203.4
9 lOl.O(l75.8) 21 67.5
10 48.0 (20.5) c=0 169.8
11 70.4(36.1) CH3 20.8
12 35.3 20.3
aAll chemical shifts are in PPM from tetramethyl-

silane with internal reierence dimethylsul-
foxide-d6 Z ~ ~ ~ . ~ ~ PAll
P Mspectra
. were run in
DMSO-d . C- F coupling constants shown in
parentfieses.
2.4 Mass Spectra
The low resolution mass spectra of 28 corti-
costeroid 21-esters and related compounds of
pharmaceutical intefest have been determined by
Toft and co-workers on an AEI MS-12 via direct
probe. The mass spectra of triamcinolone and
triamcinolofe diacetate are in excellent agreement
with Florey .
2.9 Solubility Data
2.91 Solubility
and triamcinolone diacetate by polyoxyethylene
lauryl ethgr has been reported by Tomida and
co-workers who also concluded that the solubili-
zation of steroids by polyoxyethylene lauryl ether
micelles was directly dependent upon their
lipophilicity. Triamcinolone diacetate was found
656 DAVID H. SIEH
to have a solubility of 7.417x M in water 6

and 13.2 mg/g in 70% ethanol .
The role of crystal structure (as reflected
by the melting point and the entropy of fusion)
and of the activity coefficient (as reflected by
controlling the aqueous solubility of either
liquid or crystalline organic nonelectrolytes,
and triamcinolone diaGetate was discussed by
Yalkowsky and Valvani .
The partition coefficients of triamcinolone
diacetgte in two systems have been reported by
Tomida . He determined that the aqueous-micellar
and octanol-water partition coefficients were 299
and 83.7, respectively. The value for the
octanol-water partition coefficient is in excel-
lent agreemeni-rath that published by other
investigators
Murata and co-workers have reported the
observation of crystal forms of triamcinolone
diacetate and other glucocorticoids in sterile
aqueous suspensions by scanning electron micro-
scopy.
3. Synthesis
Higashikawa12 has patented a procedure for
the preparation of triamcinolone diacetate by
reacting the 21-hydroxy precursor with acetic
anhydride in the presence of metal carbonate or
hydroxide. A procedure for the synthesis of
16,17-dihydroxy steroid acetal and ketal deriva-
tives anrj3acetylated compounds has been
patented .
6.3 Colorimetric Analysis
diacetate produced a very faint color in daylight
TRIAMCINOLONE DIACETATE 657
anf4no color when viewed in the ultraviolet at 366

nm .
6.37 Triamcinolone diacetate has been
quantitated by its reaction with 2,3,5-triphenyl-
tetrazoliumlt,jhloride followed by absorbance
measurement .
6.52 Thin Layer Chromatographic Analysis
Four systems used for the separation and
identification of triamcinolone diacetate by thin
layer chromatography are shown in Table 11.
Chromatography
A compilation of the liquid chromatographic
separation systems for triamcinolone diacetate
published after 1970 is shown in Table 111. K' is
defined as the capacity ratio.
7. Acknowledgements
The author would like to express his
appreciation to Mr. Steve Highcock for performing
the literature search, to Dr. Mike Porubcan for
resonance spectra, and to Dr. John Dunham for his
critical review of the manuscript.
8. References
1. Florey, K., "Analytical Profiles of Drug
Substances", Vol. 1, Academic Press, New
York, N.Y., pp. 422-442 (1972).
2. Porubcan, M., Squibb Institute f o r Medical
3. Blunt, J.W., J.B. Stothers, Org. Magn. R e s . ,
9(8), 439-444 (1977).
4. Doddrell, D.M., D.T. Pegg, J. Amer. Chem.
SOC., 102, 6388-6391 (1980) .
5. Toft, P., B . A . Lodge, M.B. Simard, Can. J.
Pharm. Sci., 7(2), 53-61 (1972).
6. Tomida, H., T. Yotsuyanagi, K. Ikeda, Chem.
Pharm. Bull., 26 (9), 2832-2837 (1978).
TABLE 11. Thin Layer Chromatography of Triamcinolone Diacetate
Solvent System Adsorbent Detection R,

&
Reference
Methylene chloride: Silica gel Spray with 50% 0.88 16
dioxane: water (10:5:5)- GF 254 sulfuric acid,
lower layer heat at 12OOC
for 5 min.
Ethylene chloride: 1.00
methanol: water Re1ative
(95:5:0.2) to 168-
methyl
predniso-
lone ace-
tate-1.00
Chloroform Spray with 0.18 14

Stationary phase - Kieselguhr G 1 mg/ml sul-
Formamide: acetone furic acid,
(10:90) uv at 366 nm
Methylene chloride: Silica Gel G 0.27
diethy1ether:methanol:
water (77:15:8 :l.2 )
Table 111. High Performance Liquid Chromatographic Systems for Triamcinolone
Diacetate.
Flow Retention Detection
Rate Time
Column Mobile Phase (ml/min) (min) (nm) Reference
Zipax Water saturated methy- 0.33 4.8 254 17
lm x 2mm lene chloride
IJ Bonda- Acetonitrile: water 1.0 K'=8.48 240 18

apak c18 (30:70)
m
30cm x
$ 3.9 mm
l~ Bonda- Methanol :water (70 :30) 1.5 1.30 254 19

pak c18 Methano1:water (50:50) 3.38
30 cm x Acetonitrile:water(60:40) 1.25
4.0mm Acetonitrile:water(40:60) 2.65
Relative to
acetone 1.00
Bonda- Methanol :water ( 60:40) 1.33
pak C / Methano1:water (40:60) 4.88
Corasif Acetonitrile:water(40:60) 1.11
61 cm x Acetonitrile:water(20:80) 9.06
2.3 mm Acetone = 1.00
Table 111. High Performance Liquid Chromatographic Systems for Triamcinolone
Diacetate.
Flow Retention Detection
Rate Time
Column Mobile Phase (ml/min) (min) (nm) Reference
Bonda- Methano1:water (60:40) 1.0 1.21
pak- Methanol :water ( 4 0 : 60) 5.60
Phenyl/ Acetonitrile:water(40:60) 1.21
Corasil Acetonitrile:water(20:80) 6.31
61 cm x Acetone = 1.00
2.3 mm
TRIAMCINOLONEDIACETATE 66 1
7. Grady, L.T., S.E. Hays, R.H. King, H.R.

Klein, W . J . Mader, D.K. Wyatt, R.O. Zimmerer,
Jr., J. Pharm. S c i . , 6 2 ( 3 j , 456-464 ( 1 9 7 3 ) .
8. Verma, S.C., J . O . Runikis, W.D. Stewart,
Indian J. Hosp. Pharm., 1 0 ( 5 ) , 167-173
(1973).
9. Yalkowsky, S . H . , S.C. Valvani, J . Pharm.
Sci., 6 9 ( 8 ) , 912-922 ( 1 9 8 0 ) .
1 0 . Flynn, G.L., ibid., 6 0 ( 3 ) , 345-353 ( 1 9 7 1 ) .
11. Murata, R . , A. Tanuma, N. Hikichi, H. Niwa,
Tohoku Yakka Daigaku Kenkyu Nempo, 2 5 , 57-61
( 1 9 7 8 ) ; C.A. 91:16297.
1 2 . Higashikawa, T., Patent 5 5 4 0 6 3 0 6 5 , 5 5 4 0 6 3 0 6 6
( 1 9 7 9 1 , Derwent 4- 48160B.
1 3 . Compania Espanola de Esteroides S.A., Spain
Patent, 4 8 3 7 0 1 ( 1 9 8 0 ) ; C.A. 93:9781.
1 4 . Vanderhaeghe, H., J. Hoebus, J. Pharm. Belg.,
31 (1), 25-37 ( 1 9 7 6 ) .
15. Heintz, B., R. Kalusa, Dtsch. Apoth.-Ztg.,
1 1 8 ( 2 7 ) , 1000-1006 ( 1 9 7 8 ) ; C.A. 8 9 : 1 3 5 9 0 5 .
1 6 . Bailey, K., A.W. By, B.A. Lodge, J.
Chromatogr., 1 6 6 (1), 299-304 ( 1 9 7 8 ) .
1 7 . Higgins, J.W., ibid., 1 1 5 ( 1 ) , 232-235 ( 1 9 7 5 ) .
1 8 . Van Dame, H.C., J. Assoc Off. Anal. Chem.,
6 3 ( 6 ) , 1184-1188 (1980).
19. Tymes, N.W., J. Chromatogr. Sci., 1 5 ( 5 ) ,
151-155 (1977).
CUMULATIVE INDEX
Bold numerals refer to volume numbers
Acetaminophen, 3, 1 Chloroprothixene, 2, 63
Acetohexamide, 1, 1; 2,573 Chlortetracycline hydrochloride, 8, 101
Allopurinol, 7, 1 Clidinium bromide, 2, 145
Alpha-tocopheryl acetate, 3, 111 Clindamycin hydrochloride, 10, 75
Aminophylline, L1, 1 Clofibrate, 11, 197
Aminosalicylic acid, 10, 1 Clonazepam, 6, 61
Amitriptyline hydrochloride, 3, 127 Clorazepate dipotassium, 4, 91
Amoxicillin, 7, 19 Clotrimazole, 11, 225
Amphotericin B, 6, 1; 7, 502 Cloxacillin sodium, 4, 113
Ampicillin, 2, I; 4, 518 Codeine phosphate, 10, 93
Ascorbic acid, ll, 45 Colchicine, 10, 139
Aspirin, 8, 1 Cyanocobalamin, 10, 183
Azathioprine, 10, 29 Cyclizine, 6, 83; 7, 502
Bacitracin, 9, 1 Cycloserine, 1, 53
Bendroflumethiazide, 5, I; 6, 597 Cyclothiazide, 1, 66
Benzyl benzoate, 10, 55 Cypropheptadine, 9, 155
Betamethasone dipropionate, 6, 43 Dapsone, 5.87
Bretylium tosylate, 9, 71 Dexamethasone, 2, 163; 4,519
Bromocriptine methanesulfonate, 8, 47 Diatrizoic acid, 4, 137; 5, 556
Calcitriol, 8, 83 Diazepam, 1, 79; 4, 518
Captopril, ll, 79 Dibenzepin hydrochloride, 9, 181
Carbamazepine, 9, 87 Digitoxin, 3, 149
Cefaclor, 9, 107 Digoxin, 9,207
Cefamandole nafate, 9, 125; 10, 729 Dihydroergotoxine methanesulfonate, 7, 81
Cefazolin, 4, 1 Dioctyl sodium sulfosuccinate, 2, 199
Cefotaxime, ll, 139 Diperodon, 6, 99
Cefoxitin, sodium, 11, 169 Diphenhydramine hydrochloride, 3, 173
Cephalexin, 4,21 Diphenoxylate hydrochloride, 7, 149
Cephalothin sodium, 1, 319 Disulfiram, 4, 168
Cephradine, 5,21 Dobutamine hydrochloride, 8, 139
Chloral hydrate, 2, 85 Dopamine hydrochloride, 11, 257
Chloramphenicol, 4,47, 518 Doxorubicine, 9, 245
Chlordiazepoxide, 1, 15 Droperidol, 7, 171
Chlordiazepoxide hydrochloride, 1, 39; 4, 518 Echothiophate iodide, 3,233
Chloroquine phosphate, 5, 61 Emetine hydrochloride, 10, 289
Chlorpheniramine maleate, 7.43 Epinephrine, 7, 193
663
664 CUMULATIVE INDEX
Ergonovine maleate, 11, 273 Methadone hydrochloride, 3, 365; 4, 520;

Ergotamine tartrate, 6, 113 9,601
Erythromycin, 8, 159 Methaqualone, 4,245, 520
Erythromycin estolate, 1, 101; 2, 573 Methimazole, 8, 351
Estradiol valerate, 4, 192 Methotrexate, 5, 283
Ethambutol hydrochloride, 7, 231 Methoxsalen, 9, 427
Ethynodiol diacetate, 3, 253 Methyclothiazide, 5, 307
Fenoprofen calcium, 6, 161 Methylphenidate hydrochloride, 10,473
Flucytosine, 5, 115 Methyprylon, 2, 363
Fludrocortisone acetate, 3, 281 Metronidazole, 5, 327
Flufenamic acid, 11, 313 Minocycline, 6, 323
Fluorouracil, 2, 221 Nabilone, 10,499
Fluoxymesterone, 7, 251 Nadolol, 9,455; 10, 732
Fluphenazine decanoate, 9,275; 10, 730 Nalidixic acid, 8, 371
Fluphenazine enanthate, 2, 245; 4, 524 Natamycin, 10, 513
Fluphenazine hydrochloride, 2. 263; 4. 519 Neomycin, 8, 399
Flurazepam hydrochloride, 3, 307 Nitrazepam, 9,487
Gentamicin sulfate, 9, 295; 10; 731 Nitrofurantoin, 5, 345
Glibenclamide, 10, 337 Nitroglycerin, 9, 519
Gluthethimide, 5, 139 Norethindrone, 4, 268
Gramicidin, 8, 179 Norgestrel, 4, 294
Griseofulvin, 8, 219, 9, 583 Nortriptyline hydrochloride, 1,233; 2, 573
Halcinonide, 8, 251 Noscapine, 11,407
Haloperidol, 9, 341 Nystatin, 6, 341
Halothane, 1, 119; 2, 573 Oxazepam, 3,441
Heroin, 10, 357 Oxytocin, 10, 563
Hexestrol, 11, 347 Penicillamine, 10, 601
Hexetidine, 7, 277 Penicillin-G benzothine, 11, 463
Hydralazine hydrochloride, 8, 283 Penicillin-V, 1,249
Hydrochlorothiazide, 10,405 Phenazopyridine hydrochloride, 3,465
Hydroflumethiazide, 7, 297 Phenelzine sulfate, 2, 383
Hydroxyprogesterone caproate, 4, 209 Phenformin hydrochloride, 4, 319; 5,429
Hydroxyzine dihydrochloride, 7, 319 Phenobarbital, 7, 359
Iodipamide, 3, 333 Phenoxymethyl penicillin potassium, 1, 249
Isocarboxazid, 2,295 Phenylbutazone, ll,483
Isoniazide, 6, 183 Phenylephrine hydrochloride, 3, 483
Isopropamide, 2, 315 Piperazine estrone sulfate, 5, 375
Isosorbide dinitrate, 4, 225; 5, 556 Primidone, 2, 409
Kanamycin sulfate, 6, 259 Probenecid, 10, 639
Ketamine, 6, 297 Procainamide hydrochloride, 4, 333
Ketoprofen, 10,443 Procarbazine hydrochloride, 5, 403
Khellin, 9, 371 Promethazine hydrochloride, 5,429
Leucovorin calcium, 8, 315 Proparacaine hydrochloride, 6,423
Levarterenol bitartrate, 1, 49; 2, 573; 11, 555 Propiomazine hydrochloride, 2,439
Levallorphan tartrate, 2, 339 Propoxyphene hydrochloride, 1,301; 4,520;
Levodopa, 5. 189 6, 598
Levothyroxine sodium, 5, 225 Propylthiouracil, 6, 457
Lorazepam, 9.397 Pseudoephedrine hydrochloride, 8,489
Meperidine hydrochloride, 1, 175 Reserpine, 4, 384; 5, 557
Meprobamate, 1, 209; 4, 520; 11, 587 Rifampin, 5, 467
6-Mercaptopurine, 7, 343 Salbutamol, 10, 665
Mestranol, 11, 375 Secobarbital sodium, 1, 343
CUMULATIVE INDEX 665
Spironolactone. 4,431 Triamcinolone diacetate, 1, 423; 11, 651

Sodium nitroprusside, 6, 487 Triamcinolone hexacetonide, 6, 579
Succinylcholine chloride, 10,691 Triclobisonium chloride, 2, 507
Sulfadiazine, 11, 523 Trifluoperazine hydrochloride. 9, 543
Sulphamerazine, 6, 515 Triflupromazine hydrochloride, 2, 523; 4, 521;
Sulfamethazine, 7, 401 5,557
Sulfamethoxazole, 2, 467; 4, 521 Trimethaphan camsylate, 3. 545
Sulfasalazine, 5, 515 Trimethobenzamide hydrochloride, 2, 551
Sulfisoxazole, 2, 487 Trimethoprirn, 7,445
Testolactone, 5, 533 Trioxsalen, 10,705
Testosterone enanthate, 4, 452 Triprolidine hydrochloride, 8, 509
Theophylline, 4, 466 Tropicamide, 3, 565
Thiostrepton, 7,423 Tubocurarine chloride, 7,477
Tolbutamide, 3, 513; 5, 557 Tybamate, 4, 494
Triamcinolone, 1, 367; 2, 571; 4, 521, 524; Valproate sodium and valproic acid, 8, 529
11,593 Vinblastine sulfate, 1,443
Triamcinolone acetonide, 1, 397, 416; 2, 571; Vincristine sulfate, 1,463
4, 521,7, 501; 11,615

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Analytical Profiles

ACADEMIC PRESS 1982

Norman W. Atwater Salvatore A. Fusari

COI’YRIGHT @ 1982, BY ACADEMIC I’RESS, INC.

Uiiifed Kirigdoni Edition pirblished b y

LlBRhRY O F CONGRESS CATALOG CARD N U M B E R : 70-187259

ISBN 0-12-26081 1-9

PRINTED IN THE UNITED STATES OF AMERICA

E . Abignente, University of Naples, Naples, Italy

J. H . Johnson,American Critical Care, Chicago, Illinois

Analytical Profilcs of Drug Substances Copyright 01982 by The American

1.11 Chemical Name

Aminophylline is chemically known as 1H-

1.12 Adopted Names

Aminophylline was also known as theophylline

1.13 Trade Names

Aminophylline is known as carena;

Anhydrous: C16H24N1004 420.44

Dihydrate: C16H28N1006 456.44

1.3 Appearance, Color and Odor

Aminophylline is available in the anhydrous form

The infrared spectrum of aminophylline in

Spectral characteristics of aminophylline

Figure 2 shows the ultraviolet spectrum of

2.13 Nuclear Magnetic Resonance

2.13.1 Proton NMR

An 80 MHz proton magnetic resonance

2.13.2 Carbon-13 NMR

The 20 MHz proton-noise decoupled

Fig. 1. IR Spectrum of Aminophylline

240 260 280 300 320 340 36

Fig. 2. W Spectrum of Aminophylline

Fig. 3 . H' NMR of Aminophylline

Fig. 4. I 3 C NMR of Aminophylline

-CH2- 10,ll 2.75

aIntensity of signal is proportional to the concentration.

-CH2 on ethylenediamine is buried in the solvent signal as

2.2 Other Properties

2.21 Differential Scanning Calorimetry and

The thermogram of aminophylline2 shows two

Aminophylline is soluble in water

(1 g/5 ml) .l It is insoluble in dehydrated alcohol and in

Aminophylline was first prepared by Gruter' by

4.1 Stability in Solution

Solutions of aminophylline become turbid on

4.2 Stabilitv in Solid State

Aminophylline crystals, in the presence of

Mixtures containing aminophylline and ephedrine

and 30" as a result of decomposition16 . Increase in the in

Decomposition of aminophylline suppositories is

5.1 Identification Tests

Spectral identification tests include IR, W,Mass

The following color reactions are also useful

(i) Ethylenediamine present in aminophylline

(iii) Aminophylline develops an orange color

(iv) Reaction between dimethylglyoxime and

(V) Reaction of theophylline with potassium

(vi) Bratton-Marshall: Aminophylline in acid

(vii) Addition of 1-2 drops of a 5% solution of