(Analytical Profiles of Drug Substances 9) Klaus Florey (Eds.) - Academic Press (1981) PDF

Analytical Profiles
of
Drug Substances
Volume 9
Edited by
Klaus Florey
The Squibb Institute for Medicd Research
New Brunswick, New Jersey
Contributing Editors
Jerome I. Bodin Hans-Georg Leemann
Rafik Bishara Gerald J . Papariello
Glenn A. Brewer, Jr. Bruce C. Rudy
Milton D. Yudis
Compiled under the auspices of the
Pharmaceutical Analysis and Control Section
Academy of Pharmaceuticul Sciences
ACADEMIC PRESS 1980

A Subsidiary of Harcourt Brace Jovanovich, Publishers
New York London Sydney Toronto San Francisco
EDITORIAL BOARD
Norman W. Atwater Salvatore A. Fusari

Rafii Bishara Boen T. Kho
Jerome I. Bodin Hans-Georg Leemann
Glenn A. Brewer, Jr. Gerald J. Papariello
Lester Chafetz Bruce C. Rudy
Edward M. Cohen Bernard Z. Senkowski
John E. Fairbrother Milton D. Yudis
Klaus Florey
Academic Press Rapid Manuscript Reproduction

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Main entry under title: (Revised)
Analytical profiles of drug substances.
Compiled under the auspices of the Pharmaceutical

Analysis and Control Section, Academy of Pharmaceuti-
cal Sciences.
Includes bibliographical references.
1. Drugs-Analysis-Collected works. 2. Chemis-
try, Pharmaceutical-Collected works. I. Florey,
Klaus, ed. 11. Brewer, Glenn A. 111. Academy
of Pharmaceutical Sciences. Pharmaceutical Analysis
and Control Section. [DNLM: 1. Drugs-Analysis-
Yearbooks. QV740 AAl A551
RS189.AS8 615l.1 70- 187259
ISBN 0-12-260809-7 (V.9)
PRINTED I N THE UNITED STATES OF AMERICA
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AFFILIATIONS OF EDITORS,
CONTRIBUTORS, AND REVIEWERS
H . Y . Aboul-Enein, Riyadh University, Riyadh, Saudi Arabia

E. A . Abourubl, Faculty of Pharmacy, Cairo University, Cairo, Egypt
A . A . Al-Budr, Riyadh University, Riyadh, Saudi Arabia
A . H . Amann, American Critical Care, McGraw Park, Illinois
N. W. Afwurer, E. R. Squibb and Sons, Princeton, New Jersey
D. M . Baaske, American Critical Care, McGraw Park, Illinois
S . A . Benezru, Burroughs Wellcome Company, Research Triangle Park, North
Carolina
R . Bisharu, Eli Lilly and Company, Indianapolis, Indiana
J. I. Bodin, Carter Wallace, Inc., Cranbury, New Jersey
G. A . Brewer, The Squibb Institute for Medical Research, New Brunswick, New
Jersey
J . E . Carrer, American Critical Care, McGraw Park, Illinois
L. Chuferz, Warner-Lambert Research Institute, Moms Plains, New Jersey
G . Clarke, The Squibb Institute for Medical Research, Moreton, Wirral, England
E. M . Cohen, Merck Sharp & Dohme, West Point, Pennsylvania
A . Egli, Sandoz Limited, Basel, Switzerland
J. Fairbrorher, Department of Pharmacy, University of Nottingham, Nottingham,
England
K . Florey, The Squibb Institute for Medical Research, New Brunswick, New Jer-
sey
P. R. B . Foss, Burroughs Wellcome Company, Research Triangle Park, North
Carolina
H . L. Fung, School of Pharmacy, S.U.N.Y. at Buffalo, Amherst, New York
S . A . Fusari, Parke-Davis, Inc., Detroit, Michigan
J. R. Greco, Schering Corporation, Bloomfield, New Jersey
M . M . A . Hassun, Riyadh University, Riyadh, Saudi Arabia
J. G. Hoogerheide, Schering Corporation, Bloomfield, New Jersey
A. I. Judo, Riyadh University, Riyadh, Saudi Arabia
vii
viii AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS
C . A . Janicki, McNeil Laboratories, Fort Washington, Pennsylvania

B. T. Kho, Ayerst Laboratories, Rouses Point, New York
C. Y. KO, McNeil Laboratories, Fort Washington, Pennsylvania
H. G. Leemunn, Sandoz Limited, Basel, Switzerland
L . J . Lorenz, Eli Lilly and Company, Indianapolis, Indiana
M, A, Lou&, Riyadh University, Riyadh, Saudi Arabia
E. F. McNif, School of Pharmacy, S.U.N.Y. at Buffalo, Amherst, New York
W . R . Michaefis, Sandoz Limited, Basel, Switzerland
E. M . Oden, Schering Corporation, Bloomfield, New Jersey
G. Paparieflo, Wyeth Laboratories, Philadelphia, Pennsylvania
A. Posr, Smith Kline & French Laboratories, Philadelphia, Pennsylvania
E. C. Rickard, Eli Lilly and Company, Indianapolis, Indiana
B. E. Rosenkranrz, Schering Corporation, Bloomfield, New Jersey
B. Rudy, Burroughs Wellcome Company, Greenville, North Carolina
I. G. Rutgers, Wyeth Laboratories, Philadelphia, Pennsylvania
B. Senkowski, Alcon Laboratories, Forth Worth, Texas
C. M . Shearer, Wyeth Laboratories, Philadelphia, Pennsylvania
L . Sfusarek, Eastman-Kodak, Rochester, New York
A. Vigevani, Pharmitalia-Carlo Erba SPA, Milan, Italy
R . J. Warren, Smith Mine & French Laboratories, Philadelphia, Pennsylvania
M . J. Williamson, Adria Laboratories, Columbus, Ohio
P . S . K. Yap, School of Pharmacy, S.U.N.Y. at Buffalo, Amherst, New York
M . D . Yudis, Schering Corporation, Bloomfield, New Jersey
J. E. Zarernbo, Smith Kline & French Laboratories, Philadelphia, Pennsylvania
M . U. Zubair, Riyadh University, Riyadh, Saudi Arabia
PREFACE
Although the official compendia list tests and limits for drug substances related to
identity, purity, and strength, they normally do not provide other physical or chemi-
cal data, nor do they list methods of synthesis or pathways of physical or biological
degradation and metabolism. For drug substances important enough to be accorded
monographs in the official compendia, such supplemental information should also
be made readily available. To this end the Pharmaceutical Analysis and Control
Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative ven-
ture to compile and publish Analytical Profiles of Drug Substances in a series of
volumes of which this is the ninth.
The concept of analytical profiles is taking hold not only for compendial drugs
but, increasingly, in the industrial research laboratories. Analytical profiles are
being prepared and periodically updated to provide physicochemical and analytical
information of new drug substances during the consecutive stages of research and
development. Hopefully, then, in the not too distant future, the publication of an
analytical profile will require a minimum of effort whenever a new drug substance is
selected for compendial status.
The cooperative spirit of our contributors has made this venture possible. It is
gratifying to note that increasingly profiles are being written not only in industrial
laboratories but also academic institutions worldwide.
All those who have found the profiles useful are requested to contribute a mono-
graph of their own. The editors stand ready to receive such contributions.
The goal to cover all drug substances with comprehensive monographs is still a
distant one. It is up to our perseverance to make it a reality.
Klaus Florey
ix
BACITRACIN
Glenn A . Brewer
1. Introduction 2
2. Chemistry 4
2.1 structure 4
2.2 Biosynthesis 8
3. Description 10
3.1 Composition, Formula, Molecular Weight 10
4. Physical Properties 12
4.1 Spectra 12
4.2 Crystal Properties 16
4.3 Solubility 18
4.4 Physical Properties of Solutions 19
5. Production 20
5.1 Microbiological 20
5.2 Isolation 24
6. Stability 25
6.1 Stability of Solid 25
6.2 Stabliiy of Solutions 26
6.3 Light Stability 27
6 . 4 Formulation Stability 27
6.5 Stability of Metal Salts 21
7. Analytical Methods 28
7 . 1 Identity Tests 28
7.2 Microbiological Assays 30
7.3 Chemical Methods 34
7.4 Chromatographic Methods 35
8. Mode of Action 42
9. Derivatives of Bacitracin 43
10. Reviews 44
References 45
Copyright @ 1980 by Academic Press. Inc.

Analytical Pmfiles of Drug Substances, 9 1 All rights of reproduction in any form reserved.
ISBN: 0-12-260809-7
2 GLENN A . BREWER
1. Introduction
The organism which produces bacitracin was
isolated by Miss B.A. Johnson in June 1943 from the
debrided tissue removed from a compound fracture of
the tibia of a seven year old girl named Margaret
Traceyl. Miss Johnson was working on a project di-
rected by Dr. Frank L. Meleney. These workers
thought that it might be possible to isolate an
antibiotic producing organism from the mixed bacte-
rial flora present in a severe wound.
A crude concentrate was soon produced, and in
October 1943 the first human clinical trial was
started2. The process for the manufacture of
bacitracin was scaled up and the first large scale
clinical studies were reported in 19473. Bacitracin
was approved as a certifiable antibiotic in July
1949.
In 1944, Magargo and co-workers isolated a
strain of Bacillus subtilis which had in vitro
activity toward Mycobacterium tuberculosis4. The
culture was studied in England and it was found that
the culture no lonqer showed activity aqainst
Mycobacterium tube;culosis. Subsequentiy , a strain
of Bacillus licheniformis was isolated from the cul-
ture and this isolate was found-to produce an anti-
biotic which was called Ayfivin5. When the compo-
sition of bacitracin was better understood, it was
realized that it and Ayfivin were probably identical,
and the latter name was no longer used6.
Although bacitracin was known to be active

primarily against Gram positive organisms, it was
widely used in all types of infections. It was ad-
ministered topically, by intramuscular injection, as
lozenge for infections of the mouth and throat,
intervaginally and as an ophthalmic preparation.
Apparently, as more potent preparations of
bacitracin were produced, the material also in-
creased in nephrotoxicity7. In a review on bacitra-
cin published in 1952, the author states8:
"The side effects resulting from the adminis-
tration of any therapeutic agent are of secondary
importance in assessing the clinical value of the
drug. They assume importance only if they limit
either the dosage or duration of treatment because
BACITRACIN 3
of harmful effects on any organ or tissue of the

body or any body function." This statement is in-
teresting in the present era in which the importance
of side effects practically eclipses the therapeutic
activity and a potent therapeutic agent may be dis-
carded because of relatively minor side effects.
Today, the U.S.P. recognizes bacitracin oint-
ments for topical and ophthalmic use and sterile
bacitracin for intramuscular injection9. In addi-
tion, the C.F.R. provides for the certification of
bacitracin oral dosage forms and bacitracin combina-
tion products with other antibiotics and steroids
for ophthalmic and topical uselo.
It is probable that the veterinary use of
bacitracin is more economically important than the
clinical use,although volume figures are not readily
available. The C.F.R. provides certification for
bacitracin powder, the manganese and zinc salts and
unrefined feed grade zinc bacitracin powder. In
addition, bacitracin methylene disalicylate oral
dosage forms, combination oral products with strep-
tomycin sulfate, implantation pellets and a large
variety of ophthalmic and topical dosage forms are
monographed.
It is interesting to note that the number of
publications on bacitracin chemistry and production
have not waned in the thirty four years since the
discovery of the antibiotic. It is rare to find a
year in which a patent was not issued on the produc-
tion of bacitracin, apd the literature on the chemis-
try of the antibiotic continues to grow.
2. Chemistrv
2.1 Structure
The key to the establishment of the
structure of a natural product is the isolation of
the pure substance. Counter-current distribution
analysis was used by Craig and co-workers to demon-
strate that at least three components were present
in commercial bacitracin12. The major component was
hydrolyzed and the following dipeptides were found:
phenylalanine and leucine
phenylalanine and ornithine.
In addition, phenylalanine, leucine, isoleucine,
4 GLENN A. BREWER
glutamic acid, aspartic acid, lysine, histidine,

cystine and ammonia were found by amino acid analy-
sis using starch column chromatography. It was rec-
ognized by Craig and co-workers that some of the
amino acids probably had the D-configuration, as
racemic mixtures were isolated in some cases.
Newton and Abraham also used countercurrent
distribution to study the purity of the antibiotic
ayfivin6. They demonstrated that there were at
least seven components in the mixture with the three
major components being present in the ratio 4:1:4.
Two'components were shown to be identical to compo-
nents in bacitracin and the name ayfivin was dropped
(see section 1).
The same workers showed that at least ten
components were present in crude bacitracinl3.
They were designated bacitracins E , D, B, A l l A, C,
G I F1 and F2. Bacitracins E, D, B and A showed a
broad absorption band in the U.V. at 253 nm. Compo-
nents C and G showed a sharper band at 250 nm while
the three F components had a broad maximum at 288
nm. They established that all the components con-
tained cysteine, ornithine, lysine, histidine,
aspartic acid, glutamic acid, phenylalanine and
leucine (or isoleucine). Bacitracin C also con-
tained a component which was not separable from
glycine in the chromatographic system used, while
the bacitracins B, D and E yielded valine. Bacitra-
cins D and E apparently do not contain amide group-
ings while A, B, C, G and the F components do.
Newton and Abraham continued their examina-
tion of the structure of acitracin A, the major
component of the complex19 . They established that
the antibiotic had three basic centers, one amide
and had a unit molecular weight of 1500.
In addition, they established that each unit
contained two carboxyl, one a-amino, one 6-amino and
one histidine glyoxaline as ionizable groups. Baci-
tracin A did not contain a disulfide linkage, but a
thiol group was liberated on acid hydrolysis. An
amide group was also liberated and the ultraviolet
absorbance at 254 nm disappeared on acid hydrolysis.
On hydrogenation with Raney nickel, the group which
contained the cystine residue was converted to an
alanine residue. The authors postulated that
BACITRACIN 5
bacitracin A contains a thiazoline ring.

Craig and co-workers used their newly devel-
oped ion exchange amino acid analyzer to establish
the amino acid composition of bacitracin A 15. The
same group established a molecular weight of 1470
for bacitracin A using a partial substitution meth-
odI6. They also proposed a cyclic structure for the
molecule.
Ingram reported that bacitracin A contained
no free amino end group based on methylation stud-
iesl7.
Porath, using partial acid hydrolysis, estab-
lished the amino acid sequence for the ring as glu-
tamic acid, cysteine, isoleucine, ornithine, histi-
dine and 2 moles of aspartic acidlg. He postulated
that the sulfur of cysteine was involved in a het-
ero cyclic ring between lysine and glutamic acid. An
unidentified ninhydrin-negative compound is attached
to lysine.
Lockhart, Newton and Abraham performed acid
hydrolysis at 37OCl9. They found the amino acid
sequences:
isoleucine-cysteine-leucine-glutamic acid and
ornithine-phenylalanine-isoleucine.
The latter peptide appeared to be an N-termi-
nal peptide.
Lockhart and Abraham postulated the following
partial structure for bacitracin A 2 0.
Aspartate
4
Aspartated-a Histidine
.) \
Lysine Phenylalanine
tLOrnithine-Isoleucine 9 -.Isoleucine
%
t
lsoleucine
+\ Glutamate-
+
Cys ine
Leucine
6 GLENN A. BREWER
They also indicated that the sequence lysine-orni-

thine-valine-phenylalanine occurs in bacitracin B.
Craig and co-workers confirmed the presence of
three isoleucine residues in bacitracin A and, on
this basiq postulated the emperical formula
C66H168 014N 17S for the antibiotic2l.
The same workers proposed the following struc-
ture for bacitracin A based on the products obtained
after partial hydrolysis22,23,24,
Isoleucine-cysteine-leucine-glutamic acid-isoleu-
cine-lysine
Aspartic acid-aspartic acid-histidine-phenylalanine-
isoleucine-ornithine
It should be noted that this structure differs sig-
nificantly from the one proposed by Abraham's
group20, and does not explain their earlier find-
ingsl4.
Craig and co-workers proposed that bacitracin
A contains a thiazole ring formed by the condensa-
tion of cysteine and isoleucine25. They began a
study of the chemistry of bacitracin F.
Further studies by Abraham and co-workers con-
firmed the fact that there were three isoleucine
residues in bacitracin A26,27. This had been previ-
ously indicated by Craig and co-workers21.
Lockhart and Abraham concluded that the lysine
residue in bacitracin A is linked to isoleucine
through the a-amino group and to aspartic acid
through the €-amino group28. This aspartic acid
residue has the L-configuration while the other as-
partic acid in bacitracin A has the D-configuration.
Wrinch proposed a structure for bacitracin A
based on the published information29.
Several reviews of the chemistry of bacitra-
cin A have been published30131132133,34.
Craig and Konigsberg established that bacitra-
cin F was a degradation product of bacitracin A35.
The conversion was accompanied by the loss of
BACITRACIN I
ammonia.
Swallow and Abraham found that the glutamic
acid residue was connected via the a-carboxyl group
and that the y-carboxyl group is free36. One of the
aspartic acid residues was present as an amide.
Stoffel and Craig synthesized a number of cys-
teine peptides modeled on the N-terminal portion of
bacitracin A37. They hoped to establish the substi-
tution that would give stable thiazoline rings.
Craig and co-workers studied the acid isomer-
ization of bacitracin A38. The transformation in-
volves the epimerization of the N-terminal isoleu-
cine residue.
Theodoropoulos established that both lysine
residues in bacitracin A are a-isoleucyl-(E-aspar-
tyl)-1ysine39.
Kaneko and co-workers published a series of
papers on the synthesis of peptide intermediates to
be used in the total synthesis of bacitracin A40r41,
42,43,44,45,46.
Ratti and co-workers established the optical
configuration of the aspartyl and asparaginyl resi-
dues of bacitracin A as D and L r e ~ p e c t i v e l y ~ ~ .
Cornell and Guiney established that the coor-
dination sites for zinc in bacitracin were the thia-
zolene ring and histidine residue48.
Manning developed a method to establish the
amount of racemization that occurred during acid hy-
drolysis 4 9 I50.
On the basis of NMR studies, a space-filling
model of bacitracin A was proposed51.
The presently accepted structures for the bac-
itracins can be found in Section 3.
2.2 Biosynthesis
The cell-free enzymatic synthesis of bac-
itracin A has been extensively studied by a number
of workers.
8 GLENN A. BREWER
Bernlohr and Sievert noted the similarity

of the amino acid composition of bacitracin and
Bacillus licheniformis spore coats52. This suggest-
ed that the antibiotic was a precursor of a struc-
tural entity of the bacterial cell.
Bernlohr and Novelli indicated that baci-
tracin was produced by postlogarithmic cells of
Bacillus lichenifnrmis which are in the process of
producing spores53. The amino acids were not incor-
porated into bacitracin by a normal mechanism.
Shimura and co-workers found that the
amount of bacitracin produced by B. licheniformis
was governed by the amount of cysteine present in
the medium54.
Cornell published a thesis OR the bio-
synthesis of b a ~ i t r a c i n ~ ~ .
The cell-free synthesis of bacitracin was
first achieved by Shimura and c o - ~ o r k e r s ~ ~They
.
utilized lysed protoplasts of B. licheniformis. The
incorporation of L-histidine was inhibited when var-
ious D-amino acids were added. The biosynthesis was
not inhibited by ribonuclease, chloramphenicol or
puromycin so it was concluded that the biosynthetic
pathway was different from that involved in protein
biosynthesis.
Pfaender also reported the bios nthesis
of bacitracin with a cell-free preparation5 7 . He
found that leaving out one of the required amino
acids or the substitution of a D-amino acid for an
L-amino acid stopped the synthesis.
Pfaender and co-workers fractionated the
enzyme system and found two fractions with molecular
weights of 200,000 and 350,000,which dissociated to
50,000 units on storage for one day in the cold5*.
Froyshov and Laland purified bacitracin
synthetase about ll-fold59. They showed that two
fractions were present,both of which were required
for the synthesis of bacitracin. The amino acids
required for the pyrophosphate-ATP exchange reac-
tions were determined for each fraction.
Froyshov reported that he had resolved
BACITRACIN 9
bacitracin synthetase nto three fractions by af-

finity chromatography66 .
Ishihara and Shimura purified bacitracin

synthetase 25-fold6I.
Froyshov continued his work and found

that fraction A was responsible for the chain
lengthening of bacitracin A62.
Ishihara published a review on the bio-
synthesis of bacitracin A with cell-free enzyme
preparations63.
.
Froyshov also reviewed rogress in cell-
free biosynthesis of bacitracin A 6%
Wang and c o - ~ o r k e r sand

~ ~ Umezawa and co-
workers66 have published reports on the practical
cell-free synthesis of bacitracin A.
3. DescriDtion
3.1 Composition, Formula, Molecular Weight
The bacitracin of commerce is a mixture
of components. The major component is bacitracin A.
The mixture of bacitracin components
[1405-87-41 will be referred to in this monograph as
bacitracin. Certain salts and derivatives of the
bacitracin complex have been utilized in feeds or
formulations
Zinc bacitracin 11405-89-61
Manganese bacitracin [1405-99-81
Sodium bacitracin 139436-06-11
Methylenebis [2-hydroxybenzoate]-[55852-84-11
3.11 Bacitracin A r22601-59-81
The structure of bacitracin A was
elucidated after almost twenty years of work by a
number of different groups (Section 2 ) . It is no
wonder then that there is disagreement in the liter-
ature on which group established the definitive
structure. Ressler and Kashelikar, using a dehydra-
tion-reduction technique established the final posi-
tion of the amino acids in the seven membered
10 GLENN A . BREWER
ring67. Craig and co-workers established the con-

formation of bacitracin A6*. The structure has been
confirmed by total chemical synthesis69.
CO+Leu+ Glu -Ile -P Lvs+

z
Orn+ Ile ---c Phe
Asn 4- Asp 4- His J
C66H103N170 16s
Molecular Weight 1422.73
3.12 Bacitracin B r1402-99-91
The structure of bacitracin B is
very similar to that for bacitracin A except that
valine replaces one of the isoleucine residues. The
exact residue is not certain but evidence suggests
the isoleucine in the seven membered ring is re-
placed by valine.
C65H101N17O 16S
3.13 Bacitracin F [22601-63-41
Bacitracin F is a degradation prod-
uct of bacitracin A (see section 6).
o=c- C k CH3
' 2HC' CH3
fC$
I
C- H
1 (L) (D) (L) (L) (D) (L) (D)
CO+Leu+Glu+Ile+L s+Orn+Ile+Phe
LYAsn+D-AspeL-HiX
BACITRACIN 11

3.14 Other Bacitracin Components
A number of other minor components
have been identified in the bacitracin complex. The
structures of these components are not known at the
present time.
Bacitracin B1 (57762-79-5)
Bacitracin B2 (57762-78-4)
Bacitracin C (1403-00-5)
Bacitracin D (1403-01-6)
Bacitracin E (1403-07-7)
Bacitracin F1 (1403-04-9)
Bacitracin F2 (1403-05-0)
Bacitracin G (1403-03-8)
Unless otherwise specified, in the
remainder of this profile when we use the name baci-
tracin we refer to the bacitracin complex.
4. Physical Properties
4.1 Spectra
4.11 Infrared SDectrum
The infrared spectrum of bacitra-
cin has been published by Hayden and co-workers70.
The infrared curves of bacitra-
cin and zinc bacitracin taken as mineral oil mulls
and as KBr pellets are shown in Figures 1-471.
4.12 Nuclear Magnetic Resonance
Spectrum
Chapman and Golden used NMR to
study deuterium exchange in bacitracin A51. This
work along with the tritium exchange studies con-
ducted by Craig and co-workers6* extablished the
conformation of bacitracin A in aqueous solution.
Coates and co-workers used 270
MHz NMR to measure pro9qn spin lattice relaxation
times for bacitracin A .
4J
a,
d
rl
a,
PC
k
a
.%
I
c
-ti
u
Id
k
4J
4
u
Id
a
u-4
0
k
4J
u
a,
a
zo
a
a
k
a
k
u-4
c
H
0
33NVBMOSBV
0 0 0
7
12
5
k
c1
u
al
a
rn
a
al
k
rd
k
w
c
H
33NVBIOSBV
13
c,
a,
rl
rl
al
PI
k
m
z
I
c
.ti
u
rd
k
c,
.ti
u
rd
m
u
c
.d
N
w
0
5
k
c,
u
a,
a
m
a
a,
k
rd
k
w
c
H
14
WAVELENGTH (MICRONS)
2.5 3 4 5 6 7 8 9 10 12 15 20 30 4050
Figure 4. Infrared Spectrum of Zinc Bacitracin-Mineral Oil Mull

16 GLENN A. BREWER
Reynolds and co-workers used C 1 3 mag-

netic resonance spectroscopy to establish the tau-
tomeric equilibrium of the histidine ring in
bacitracin A73.
The NMR spectrum of bacitracin in D20
is shown in Figure 5 7 4 .
4.13 Ultraviolet Absorlstion Slsectrum
The ultraviolet absorption spec-
trum of bacitracin was reported by Hayden, gt
The ultraviolet spectrum of baci-
tracin was determined in water, methanol, dilute ac-
id and dilute alkali75. In all solvents, a small
peak with an E ( 1 $ , 1 cm) of about 2 0 was exhibited
at about 2 5 0 nm. There was no significant shift in
wavelength or decrease in absorbance on standing in
dilute acid or alkali for periods up to 2 4 hours at
room temperature.
4.14 Fluorescence Spectrum
Bacitracin ex ibits a very weak
fluorescence in aqueous solution9 6 . In both acid
and alkaline solutions the excitation wavelength is
at about 2 9 2 nm and the emission occurs at about
3 2 5 nm.
4.15 Acoustic Absorption Spectrum

Slutsky, Madsen and White deter-
.
mined the acoustic bsorption spectrum of bacitracin
and other peptides 7 9
4.2 Crystal Properties

4.21 X-Ray Powder Diffraction
Samples of U.S.P. Reference stan-
dard of bacitracin and zinc bacitracin were exam-
ined by powder x-ray diffraction. Both substances
.
were found to be amorphous as indicated y the ab-
sence of any peaks in the x-ray pattern7B
4.22 Hygroscopicity
Hayashi and co-workers determined
Figure 5. N M R Spectrum of Bacitracin in D20
18 GLENN A. BREWER
the hygroscopicity of bacitracin at loo%, 93% and

63% relative humidity82.
Lannung also reported on the hy-

groscopicity of b a ~ i t r a c i n ~ ~ .
4.3 Solubility
4.31 Solubility in Pure Solvents
Weiss, Andrew and Wright publish-
ed data on the solubility of bacitracin and zinc
bacitracin in a number of solvents79.
Solvent Solubility in mg/ml
Bacitracin Zinc Bacitracin
water >20 5.1

acetone 0.75 1.0
1,4-dioxane 0.70 0.49
ethanol 9.1 2.0
ethylene glycol 220 7.95
formamide 19.9 >20
isopropyl alcohol 1.85 0.16
me thano1 >20 6.55
pyridine 9.15 4.05
benzene 0.025 0.065
benzyl alcohol >20 10.35
carbon disulfide 0.30 0.30
carbon tetrachloride 0.18 0.12
chloroform 0.0 0.01
cyclohexane 0.075 0.06
ethyl acetate 0.047 1.3
diethyl ether 0.065 0.02
ethylene chloride 0.025 1.1
isoamyl alcohol 1.65 2.6
isooctane 0.55 0.015
methyl ethyl ketone 0.20 0.85
petroleum ether 0.35 0.025
toluene 0.15 0.02
isoamyl acetate 0.09 0.45
Gross noted that bacitracin is more
sol ble in aqueous solution in the pH range 6.5 to
7.5 j 0
BACITRACIN 19
4.32 Distribution Coefficient

Carpenter and co-workers deter-
2-butanol-O.1N acid
-
An .
mined the distribut n coefficient of bacitracin in
4.33 Formulation Release

Nesbit and co-workers determined
the release of bacitracin from ointment bases84.
4.4 Physical Properties of Solutions
4.41 Metal Bindincr
Selzer noted that while mostanti-
biotics contain less than 30 p.p.m. of heavy met-
als, bacitracin, by virtue of salt formation,
maf;5
contain more than three times this concentration .
Garbutt, Morehouse and Harisen es-
tablished the following order for complex formation
of metal salts and bacitracin:
Cu>Ni>Co=Zn>Mn86
A l l the metals, except manganese,complexed the baci-
tracin group which titrates between 5.5 and 7.5.
Using titration data and the U.V. spectra of the
complexes, these workers postulated the involvement
of the imidazole group of histidine in the complex.
Using NMR and ORD measurements,
&.
Cornell and co-workers found that zinc comp xes be-
tween the thiazoline and histidine residues
Weinberg measured the stability
constants of the binding of copper, nickel, cobalt,
zinc and manganese to b a ~ i t r a c i n ~ ~ .
Storm and Strominger established
the association constants for bacitracins A and F
with magnesium88. Bacitracin F has a lower associa-
tion constant.
4-42 Optical Rotary Dis2ersion

Konigsberg and Craig reported
that bacitracin undergoes a change in rotation
20 GLENN A. BREWER
below p H 4 due tggthe epimerization of the terminal

isoleucine group .
Craig reported on O.R.D. studies
of bacitracin Ago.
Cornell and co-workers used
O.R.D. to study the attachment of zinc to bacitracin
A48.
Craig and co-workers studied the

conformation of bacitracin A in aqueous solutiong1.
4.43 Isoelectric Point
Messing patented a method to de-
termine the isoelectric point of proteinsg2. The
method was used to establish the isoelectric point
of bacitracin as 8.8. This value agrees well with
a determination of 8.5 using electrophoresis.
4.44 Dialysis
Craig and co-workers developed
the technique of thin-film dialysis to stud
conformation of large molecules in solutionjiOfhegac-
itracin A was one of the model compounds studied.
Klein and co-workers used bacitr-
acin as a model compound to establish the properties
of four cellulosic membranes93.
Craig and co-workers reported ad-
ditional studies on the dialysis o € bacitracin Ag4.
Krogerus used dialysis to study
the release rate of bacitracin from various ointment
bases95.
Several reviews have been pub-
lished on the ph sical and chemical properties of
the bacitracinsg: I 9 7 I 98.
5. Production
5.1 Microbiological
Meleney and co-workers described the
production of bacitracin on L-glutamic acid
BACITRACIN 21
synthetic and soybean digest media99.

Hendlin studied the formation of baci-
tracin by Bacillus subtilis and evaluated the ef-
fect of the addition of various ions, organic acids,
amino acids and carbohydrateslOO.
Inskeep and co-workers described a new

plant built for the production of bacitracinlOl I
Darker patented the addition of various

salts to so bean medium to stimulate production of
bacitracin14;2 .
Keko, Bennett and Arzberger patented a

soybean meal-starch medium for the production of
bacitracinlo3.
Su and Lu noted the increased production
of bacitracin in a peanut oil meal-starch medium
when calcium lactate and potassium phosphate were
addedlo4.
Cohen patented a soybean meal-dextrin
medium for the production of bacitracin105.
Wilk specified the pH ranges for the

growth and antibiotic production phases of a baci-
tracin-producing culture106 .
Freaney and Allen patented a fermenta-

187.
tion medium capable of su orting a yield of about
320 units/ml in 24 hours
Ziffer patented a soybean-sucrose medi-

um for bacitracin productionl08.
Ripoli published a report on the produc-
tion of bacitracin in five-liter flaskslog.
Siquiroff found that the production of
bacitracin was higher in surface culture than in
shaken flasks1l0
Zorn patented a fermentation medium con-
taining a water-soluble salt of cobaltlll- He pro-
posed that cobalt complexes of bacitracin were
formed which stabilized the bacitracin for use in
animal feed supplements.
22 GLENN A. BREWER
Aida and Ito describe the formation of

bacitracin A and bacitracin X com lex from bacterial
protoplasts (see Section 2.2) 112 ,P13 ,114,115 ,116.
Bacitracin X complex has a similar amino acid compo-
sition to bacitracins A and B but can be separated
by paper chromatography.
Cornell and Snoke showed by adding vari-
ous antibiotics and D-phenylalanine that the biosyn-
thesis of protein and bacitracin by Bacillus
licheniformis was accomplished by different metabol-
ic pathwaysll7. The same workers showed that
B. licheniformis is inhibited by bacitracin in the
early stages of growth118.
Brand1 and co-workers studied oxygen
transfer in the bacitracin fermentation119.
Weinberg and Tonnis showed that although
inhibitors of nucleic acid metabolism, messenger RNA
synthesis and protein synthesis inhibited the pro-
duction of bacitracin, the inhibition could be over-
come by the addition of a manganese saltl20.
Weinberg postulated the function of the
bacitracin peptide and other peptide antibiotics for
Bacillus species121.
Styczynska and co-workers noted that the

production of bacitracin by Bacillus subtilis was
stimulated when fermentation was conducted as a
mixed culture process with a Pseudomonas strain122.
Lubinski patented a process using a
strain of Bacillus subtilis ada ted to iron and
grown on a soy-fish meal medium538 .
Feuer and co-workers obtained a patent

on an antifoam composition which was useful in the
bacitracin fermentation123 .
Chigaleichik and co-workers defined a
synthetic medium for bacitracin production by
Bacillus polymyxa124 .
Simlot, Pfaender and Specht noted that

changes in the fermentation medium did not alter the
quantity of bacitracin synthesized but did change
the type produced125.
BACITRACIN 23
Haavik suggested that glucose inhibited

the formation of bacitracin primarily by lowering
the pH of the fermentation,and not by catabolite re-
pression controlla6 # 127. The same worker found that
phosphate only has an adverse effect when it alters
the optimum pH of the fermentation128.
Haavik postulated that bacitracin may
participate in manganese-ion transport throu h the
cell membrane of Bacillus l i c h e n i f ~ r m i s l, 1~9 ~
0
132.
.
Kurima and co-workers atented a process
for the production of bacitracin 133
Pass and Raczynska-Bojanowska found that

high bacitracin-producing strains of Bacillus
subtilis lack ornithine 6-transaminaselj4,ljb If .
ornithine is added to low producing strains, their
productivity is increased.
Vitkovic and Sadoff found that bacitra-
cin is a constituent of vegetative cell proteinl36.
Makukhina and co-workers described the
production of bacitracinl37.
Tyc and co-workers have patented a pro-
cess €or the production of bacitracin utilizing a
non-sporulating strainl38.
Lipavska and associates used acriflavine
to prevent infection of Bacillus licheniformis with
bacteriophage BLE139. They found that acriflavine
did not inhibit the production of bacitracin.
Tyc and Kadzikiewicz described their

method of producing ultraviolet mutants of Bacillus
licheniformis,and evaluating selected isolates for
bacitracin production140. Increases of 5 0 to 75%
were obtained with four isolates.
Haavik studied the metabolism of a high
yielding mutant strain of B. licheniformis and found
that the addition of L-leucine stimulated bacitracin
productionl41.
Raczynska-Bojanowska and co-workers pat-
ented a process for the simultaneous production of
24 GLENN A . BREWER
bacitracin and p r ~ t e a s e s l ~ ~ .
5.2 Isolation
Anker and co-workers used butanol ex-
traction to isolate the bacitracin from the fermen-
tation brothg9.
Gorley used ammonium sulfate salt frac-
tionation to purify crude bacitracinl43.
Johnson and Meleney patented a process
for the production and recovery of bacitracinl44.
There are four common ways in which bac-
itracin is isolated from fermentation broth. A num-
ber of patents and papers have been published on
these.
5.21 Precipitation From Broth
Various workers have used salts
to precipitate bacitracin from the fermentation
broth. After the bacitracin salt mixture is fil-
tered off,the pH is adjusted and the antibiotic is
extracted into a solvent145,146i147i148,149,150,
151,152,153,154,155,15611571158,1591160.
5.22 Ion Exchange of Bacitracin

A number of patents have been
issued for processes which involve the removal of
bacitracin from broth b means of an ion exchan e
~~~~~~161i162~163i164~1~5~166,167,168.169~17
172.
5.23 Solvent Extraction of Bacitracin
Solvent extraction has been used
less extensive1 than the first two methods based on
patents issued 5gi173,174i175. Apparently, the de-
velopment of this isolation procedure has been car-
ried out pri-marily by one company.
5.24 Metal Salts of Bacitracin
The metal salts of bacitracin are
used extensively as animal feed supplements (See
Section 1). These insoluble salts can be formed di-
rectly in the fermentation broth and isolated as a
BACITRACIN 25
crude concentrate for animal feed use177117811791

180,181,182,183.
5.25 Miscellaneous Methods
Namiki has published a report on
a method used to isolate high potency bacitracinl84.
Monroe and Ward have patented a
process to precipitate bacitracin on diatomaceous
earth185. The dried solid can be used as an animal
feed supplement.
Ores and Rauber have used the
non-ionic resin XAD-2 to isolate bacitracinl86.
Kindraka and Gallagher have used
ultrafiltration to remove bacitracin from fermenta-
tion broth187.
Malitskii and Mikhel'son have
noted that dry bacitracin has tendency to undergo
spontaneous combustionl88.
Brecka and co-workers inoculated
a bacitracin fermentor with Rhodotorula flava after
the antibiotic was produced189. The fermentor con-
tained both bacitracin and y-carotene at harvest.
The use of the second fermentation was to remove
fermentation by-products.
Stepanov and Rudenskaya have used
immobilized bacitracin to purify proteolytic enzymes
190.
6. Stability
6.1 Stabilitv of Solid
Bond, Himelick and MacDonald reported
that bacitracin was stable at temperatures up to
370C191. Craig and co-workers also indicated that
bacitracin is relatively stable as a solid192.
Gross studied the stability of bacitracin powder at
temperatures up to 60°C193. He indicated that after
a minor initial drop,the preparations were relative-
ly stable. There was no difference in stability be-
tween high and low potency preparations.
26 GLENN A. BREWER
Babin , Coustou and Brisou showed that

bacitracin in a mixture with papain enzyme powder
maintained its potency for a six month periodl94.
Gupta, Vyas and Sekhon showed that 15
Mrads of neutron and y-radiation did not change the
activity of bacitracin powderl95.
Tsuji and Robertson also showed that
6oCo radiation did not cause potency loss of baci-
tracin powder196. Ethylene oxide treatment caused
46% reduction in potency, but did not cause the for-
mation of bacitracin F. Dry heat sterilization
caused a 35% decrease in potency with a correspond-
ing increase in bacitracin F.
6.2 Stabilitv of Solutions
Anker and co-workers reported that so-
lutions of bacitracin were stable for 8 to 12 months
at 50Cg9. Hayashi and co-workers found that a so-
lution of bacitracin in pH 7 phosphate buffer lost
2 5 % of the initial potency after 6 days at room tem-
perature82.
Vasilescu and Molss found that solutions
of bacitracin were most stable at pH 4.498.
Craig and Konigsberg showed that baci-
tracin B was inactivated more rapidly than bacitra-
cin A35. In both cases,bacitracin F was a major de-
composition product.
The same workers showed that below pH
4.0 bacitracin undergoes an epimerization of the
terminal isoleucine residue89r38.
Pirila, Saukkonen and Santaoja sepa-
rated the degradation products of bacitracin in so-
lutionl97.
Herrmann, Woodward and Pulaski postu-
lated that the inactivation of bacitracin on pas-
sage through the gastrointestinal tract of rats is
due to degradationl98.
Pirila, Salo and Pirila found that the
complex of bacitracin with sodium dodecyl sulfate
was stable in solution, although the complex showed
BACITRACIN 27
diminished skin penetrationlgg.

Makinen found that bacitracin inhibits
the activity oz0gapain, subtilisin and leucine
aminopeptidase .
6.3 Light Stability
Wurtzen found that exposure to sunlight
and temperature variations between 2OoC and 35OC
caused 20-35% l o s s of activity in 6 days201.
6.4 Formulation Stability
Bond and co-workers reported that anhy-
drous grease based ointments were stable while water
miscible ointments were notlgl. A numb r f other
investigators agree with these findings82,902.
Hegarty and Verwey atented formulations

for bacitracin that were stable5 0 3 .
Plaxco and Husa established the stabili-

ty of bacitracin in a number of ointment bases204.
Other authors evaluated various other formulation
excipients205,206,207,208,2091239~
Gordon patented aerosol compositions of

bacitracin2I0.
Snyder patented a stable formulation of
bacitracin in animal feed211. The bacitracin was
coated with oil and the droplets absorbed on diato-
maceous earth to form a free-flowing powder.
Saito, Kawano and Ichijima patented a
bacitracin feed additive stabilized with 2-0x0-4-
methyl-6-ureidohexahydropyrimidine212.
6.5 Stability of Metal Salts
Gross, Johnson and Lafferty showed that
zinc bacitracin was more stable than bacitracin in
troches, ointments and tablets2l3. Other additives
have confirmed the increased stabilit
bacitracin with zinc and other metals
217,91.
3 Of
215,216,
Crisler and Weinberg indicated that

28 GLENN A. BREWER
while zinc salt of bacitracin was not more stable

than bacitracin to autoclaving, the salt enhances
the antibiotic activity of bacitracin ll-fold2l8 f
219.
Tanaka, Seki and Ito patented the use of

mineral salts of bacitracin as animal feed supple-
ments220, These salts were reported to have en-
hanced stability. Other patents have been issued on
the use of metal salts221t222.
7. Analytical Methods
7.1 Identitv Tests
7.11 Physical Methods
Landgren differentiated antibiot-
ics by measuring the refractive index of the crys-
tals using liquids of known refractive index223.
Zief and co-workers prepared the
tetraphenylboron derivatives of several antibiotics
224. The melting points of these derivatives were
used to identify them.
Matta and co-workers also utilized
the tetraphenyl borate derivative for antibiotic
i dentifi~at i o n 2 ~ ~ .
7.12 Colorimetric Tests
Fischbach and Levine utilized the
ninhydrin reaction as an identity test226.
Hayashi and co-workers reported
that bacitracin gave positive biuret, Adamkewitz,
Millon and Molisch reactions82. Wornick and Kuhn
indicated that bacitracin produces a violet color
with ninhydrin spray on paper227.
7.13 Chromatographic Methods
Almost any chromatographic system
for bacitracin could be used as an identity test for
the antibiotic. In this section we are listing
those systems specifically indicated as identity
tests, other chromatographic methods can be found in
Section 7.4.
c
O d N m - 3
m m m m m
N N N N N
c
0
.d
c,
u
a,
c,
a,
a
a,
c,
rd
c, X
a, 0
2 z
7
a
..
h I
N rl 0 c,
h -3
P c 51
v
c, z
z
w ow
I
..
h
0 rl Lrl I+
c, 7 0
.. ..
h N
c a N c I m
I k
.. ..
rd
$
rl
z
0
-3 a
0
a,
CI
rl z ! UJ
Lrl
0
m 2 -J
v
k
PI
a
3
In
v
c,
k
0
a
a
?
m
u u u
4 4 4
B B B
30 GLENN A . BREWER
7.14 ElectroDhoresis Methods

Peptides are commonly separated by
electrophoretic methods. A few methods specifically
designated as identity tests are listed here. Other
electrophoretic systems may be found in section
7.42.
Lightbrown and DeRossi utilized
agar gel e l e c t r o p h ~ r e s i s ~ ~Using
~. this basic meth-
od, Bozzi and Valdebouze developed a bioautogra hic
system for 14 antibiotics including bacitracin 276 .
Grynne developed a paper electrophoresis-bioauto-
graphic system for a number of antibiotics including
bacitracin237.
7.2 Microbiolosical Assavs
7.21 Tube Dilution Assay
Patrick, Craig and Bachman corre-
lated the results of serial dilution assa s with
those obtained by agar diffusion assays248 .
7.22 Turbidimetric Assay

Although the agar diffusion assay
technique is the primary method for bacitracin, a
number of turbidimetric methods have been reported.
Method Notes Authors Reference
Staphylococcus aureus Darker, g g. 241
Na resazurin indicator De Felip, et al. 242
Streptococcus faecalis Pain, Bose, 243
Dutta
Autoanalyzer nethod Platt, Gentile 244
and George
Na resazurin indicator Ruffo and Socci 245
Escherichia Coli Rappe , Mauquoy 246
and Bauer
Zinc bacitracin in Ragheb, Black 247
feeds and Graham
BACITRACIN 31
Kirschbaum, Arret and Harrison

published statistical procedures for determining the
dose-response curve €or turbidimetric assays248.
7.23 Agar Diffusion Assays

The majority of microbiological
assays €or bacitracin involve the use of agar diffu-
sion methods. Some highlights of these methods are
presented in tabular form.
Staphlococcus aureus, Darker,-
et -
al. 241
prediffusion
Development of diag- Patrick, Craig, 240
nostic discs Bachman
Effect of medium Neter , Murdock , 249
composition Kunz
Corynebacterium Porath 250
xerosis
Assay of bacitracin in Trolle-Lassen 251
Galenical products
Micrococcus flavus Pinzelik, Nisonger 252
Murrcly
Organisms resistant Friedman, 253
to other antibiotics Kirschbaum
Assay of emulsion Varma, Hall, 254
formulation Rising
Sarcina lutea Vuilleumier, Anker 255
Diagnostic discs Kirschbaum, 256

Kramer, Arret
Disc plate assay Rossi 257
Bacitracin in feed Craig 258
Disc plate assay Bauer 259

Sensitivity of method Pitton 260
32 GLENN A. BREWER

Antibiotic mixtures YonezawaI - _.
et al 261
Bacitracin in feed Craig 262
Bacitracin in tissue Freres, Valdebouze 263
Diffusion Cluzel, Cluzel, 264

characteristics Michel, Sirot
Bacitracin in milk Read, Bradshaw, 265
Swartzentruber
Bacillus Kabay 266
stearothermophilus
Bacitracin in feeds- Skodova, et al. 267
gel filtration
Sensitivity tablets Casals, Gylling, 268
Pedersen
Bacitracin in milk, Ryb inska 269
tissue
Bacitracin in Haavik 128
fermentation broth
Use of tetrazolium Picmanova,-
et -
al. 270
dyes
Bacitracin in tissue Smither 271
Bacitracin in tissue Kr st a-Skonieczna, 2 7 2

Rygrnzta
Bacitracin in feeds- Skodova, Skarka 273
molecular sieve
Interference Liskova , 274
Kohoutkova
Bacitracin in animal Pacini , 275
feeds Meneghini
Frozen inoculum Hadfield 276
Bacitracin in anti- DeCarneri 277

biotic mixtures
BACITRACIN 33

Vertical agar Lameris,et s. 278
diffusion
7.24 Feed Assays
Several of the microbiological
assays already mentioned can be used to assay baci-
tracin or zinc bacitracin in feeds. The following
papers detail extraction methods which can be used
to extract bacitracin from complex animal feeds.
Author Reference
Randall 279
Randall and Burton 280
Wright and Burton 281
Craig 282
Grynne and Hoff 283
Grynne 284
Grynne, Hoff, Silsand and Vaaje 285
Fassbender and Katz 286
7.25 Miscellaneous Assavs

The microbiological assay of baci-
tracin in antibiotic mixtures, soils and body fluids
has been discussed in some of the papers specified
in sections 7 . 2 1 through 7 . 2 4 . The following papers
are of special interest in the assay of these sam-
ples:
Assay Notes Authors Reference
Bacitracin and Lingnau and 287
neomycin Machek
Bacitracin and Balliu and 288
neomycin Boteanu
Bacitracin in soil Soulides 289
34 GLENN A . BREWER
Assay Notes Authors Reference

Blood level assay Eagle, et al. 290
Stool assay Wilson, Ing, 291
Metcalfe-Gibson
and Wrong
Animal tissue Kline and 292
Rathmacher
Bacitracin standard Kirschbaum, 293
Arret and Kramer
Review of methods Dennin 294
Temperature of Hinks, Daneo- 295
incubation Moore and
Braverman
Electrical Morris and 296
polarization Jennings
7.3 Chemical Methods
Although microbiological methods appear
to be preferred f o r bacitracin,several types of
chemical and biochemical assays have been proposed
for the antibiotic.
7.31 Gravimetric and Colorimetric
Maturana, Dannier and Brieva have
proposed a gravimetric phosphotungstic acid method
for b a ~ i t r a c i n ~ ~ ~ .
Doulakas has published a colori-
metric assay involving the reaction with phloro-
glucinol after the oxidation of the antibiotic with
hyp~bromite~~~.
7.32 Electrochemical Assays
Caplis, Ragheb and Schall have
proposed an alternating current polarographic assay
for bacitracin2 9.
Skarka and Sestakova have reported
a oscillopolarographic method as well as a
BACITRACIN 35
sensitive colorimetric method300.

Jacobsen, Pederstad and Oeystese
have utilized differential pulse polarography to
assay bacitracin and zinc bacitracin301. The deg-
radation product,bacitracin F, is reduced at a less
negative potential.
7.33 Determination of Zinc in Zinc
Bacitracin
Charles and Weiss utilized an
EDTA titration to measure the concentration of zinc
in zinc bacitracin302.
More recently, atomic absor tion
s ectroscopy has been utilized for this assay503 ,
394.
7.34 Biochemical Assays

As is the case with other anti-
biotics, investigators have established that certain
enzyme systems are inhibited by the presence of bac-
itracin. In general, these methods have not been
shown to be as useful as microbiological assays but
we have included a few references which may be of
general interest.
Enzyme System Author Reference
D-Amino acid oxidase Hayashi 305
Arginine diaminase Mikolajcik 306
Proteolytic enzymes Coppi and Bonardi 307
Pancreatic lipase Coppi and Bonardi 308
Human spermatozoa Schirren 309
7.4 Chromatographic Methods
7.41 Countercurrent Distribution
At the time when bacitracin was
discovered, countercurrent distribution was proba-
bly the most popular separation technique. Although
it has been supplanted by various types of
36 GLENN A. BREWER
chromatography on solid supports it is still useful

for the separation of large molecules such as the
bacitracins.
System Notes Authors Reference
Separation of Barry, Gregory 12
bacitracin in one major and Craig
and two minor fractions
Separation into more Craig 310
than one component
Amy1 alcohol-butanol- Newton and 6
pH 7.0 buffer Abraham
Isolated pure A, B Newton and 311
and C co-workers
Separation into 1 major Craig and 312
and 4 minor components co-workers
Separation into 10 Newton and 13
components Abraham
CHC13-methanol-water Konigsberg and 313
(2:2:l) Craig
PJ~~OH-H~O-C~H~-CHC
Ramachandrar?
~~ 314
(23:7:15: 15)
Separation of commercial Hausmann, 315
bacitracin into 10 Weisiger and
components Craig
A number of systems Craig arid 35
utilized Konigsberg
Countercurrent dist. of Craig, Hausmann 316
DNP derivative and Weisiger
Separation of bacitracin Craig, King and 317
A into two isomers Konigsberg
BuOH-C H N-AcOH-H~O Konigsberg, Hill 38
(20:5 :$ :30) separation and Craig
of degradation products
BACITRACIN 31
System Notes Authors Reference

30% Ethyl acetate-70% Craig and 91
1 butanol-pH 5.43 co-workers
buffer
7.42 Electrophoresis
Electrophoresis on a variety of
substances has been utilized frequently in the
separation of large molecular weight molecules pos-
essing an ionic charge.
Method Notes Authors Re€ erence
Starch column Flodin and 318
Porath
Cellulose column Porath 319
Paper electrophoresis Proenca da Cunha 320
and Baptista
Paper electrophoresis Paris and 321
Theallet
Paper electrophoresis Apreotesei and 322
Teodosiu
Paper electrophoresis Proenca Ca Cunha 323
and Gomes
Paper electrophoresis Pirila, Saukkonen 197
and Santaoja
Agar gel Swank and Munkres 325
Paper electrophoresis Maeda, Y a g i , 324

Naganawa, Kondo
and Umezawa
Polyacrylamide gel Swank and Munkres 325
Agar gel Dubost and Pascal 326
Polyacrylamide gel Coombe 327

38 GLENN A. BREWER

Low voltage Langner and 328
co-workers
Electrophoresis of Langner 329

feed and foods
Gelatin gel Bozzi and 236
Valdebouze
Identification test Grynne 237
Isoelectric focusing Froeyshov 330
in gel
7.43 Column Chromatography
Column chromatography utilizing a
variety of support materials has been used to per-
form crude separations of bacitracin fractions.
Charcoal-celite column Porath 250
Charcoal-celite (1:3)- Porath 319
0.1u acetic acid
Carboxymethylcellulose Konigsberg and 89

Craig
Carboxymethylcellulose Konigsberg, Hill 38
and Craig
Carboxymethylcellulose Storm and 331
Strominger
7.44 Gel Filtration
Gel filtration has been extensive-
ly used to separate macro molecules on the basis of
molecular size. Bacitracin has been utilized as a
standard in several systems since it is well charac-
terized.
BACITRACIN 39

Sephadex G-25 P.R. Carnegie 332
(Propanol-acetic acid-
water)
Sephadex G-10 (acetic Eaker and 333

acid-NaC1) Porath
Sephadex G-100 Reickert and 334
co-workers
Sephadex LH-20 Gregerman, 335
Weaver and
Kowatch
Agarose Bryce and 336
Crichton
Polyethyleneglycol Randau, Bayer 337
dimethacrylate gel and Schnell
Sephadex G-25, G - 5 0 Catsimpoolas 338
and Kenny
Polyacrylamide gel Stewart 339
Bio-Gel P-2 (tissues) Skarka, Skodova 340
and Skoda
7.45 Paper Chromatography
Paper chromatography is frequently
used for the separation of antibiotics because the
components can conveniently be located by bioautog-
raphy .
Bioautography of Snell , Ij ichi 228
various antibiotics and Lewis
Ninhydrin pyridine- Castel, Mus 341
acetic acid and Storck
Butanol-acetic acid- daCunha and 342
water (50:25:25) Baptista
40 GLENN A . BREWER

"Salting out" daCunha and 343
chromatography Baptista
Three solvent systems Paris and 321
Theallet
Dyes as detection Singh 344
reagents
Hydrophobic system Ritschel and 345
Lercher
Det. of Bacitracin in Louis 346
fodder
Separation of 42 Schmitt and 347
antibiotics Mathis
7.46 Thin Layer Chromatography
Thin layer chromatography is also
widely used for the chromatography of antibiotics
because of its rapidity.
Silica gel and Paris and 321
kieselgel Theallet
Silica gel ethanol, Umezawa and co- 348
NH40H-H20 (8:1:1) workers
Silica gel ethanol- Akita and Ikekawa 349
water (4:l)
Butanol-acetic acid- Umezawa and co- 350
water (3:l:l) workers
Separates Bacitracins Nussbaumer 351
A and F
Separates various Pitton 352
antibiotics
McGilveray and 353
Strickland
CuSO4 color reaction Guven and Ozsari 229
BACITRACIN 41

Identification of Wayland and 230
sensitivity discs Weiss
Bioautography Aszalos, Davis 354
and Frost
Fooks, McGilveray 355
and Strickland
Reimers 356
5 Solvent systems Stretton, Carr, 357
Watson-Walker
Butanol-H20-pyridine- Carr, Stretton 358
AcOH-ethanol and Watson-Walker
Dowex-50 plates Pauncz 359
Resin coated plates Pauncz 232
Detection of anti- Langner and Tuefel 231
biotics in meat
Bioautography Langner and Teufel 328
Cellulose plates Langner and Tuefel 329
Determination in Freres and 233
feed Va 1deb0uz e
Determination in Baldini and 360
tissue co-workers
Determination i n milk Bossuyt and 234
co-workers
7.47 High Pressure Liquid
Chromatography
High pressure liquid chromatogra-
phy is one of the newest chromatographic methods.
The technique combines a high resolution column
with a detector, so the method is generally not only
selective but precise.
Spechter has utilized a silica
42 G L E N N A. BREWER
column coated with Carbowax 2OM361.
Tsuji, Robertson and Bach used

Bondapak C18/Corasil with gradient elution to sepa-
rate the components of b a ~ i t r a c i n ~ ~ ~ .
Tsuji and Robertson improved on
the previous method by using a micro-Bondapak c18
columnl96.
Dr. Yeh adopted the general method
of Tsuji and Robertson196 for the examination of
some samples of commercial bacitracin obtained by
our laboratory407. (Samples of bacitracin and zinc
bacitracin were generously supplied by International
Minerals and Chemicals Corporation and by A/S Dumex
Ltd. In addition, the U.S.P. Standard of zinc baci-
tracin was chromatographed).
Although the column and solvent
system employed by Dr. Yeh were the same as those
reported by Tsuji and Robertson, he was unable to re-
produce the exact exponential gradient they utilized
because of equipment limitations. As a result, the
peaks were not as sharp and he was unable to obtain
separation of some of the components. The major
component in all the samples appeared to be bacitra-
cin A. A component eluting just before bacitracin
A was probably bacitracin B1 or B2. The other com-
ponents appeared to be present in much smaller con-
centrations. In all samples, eight to ten compo-
nents could be seen.
Dr. Yeh experienced some base line
drift because of the change in gradient composition.
We were gratified with the separation that Yeh was
able to achieve with the limited amount of time he
was able to devote to the project.
8. Mode of Action
Gale found that,like other antibiotics baci-
tracin interfered with protein biosynthesis 365.
Gale and Folkes studied the inhibition of incorpora-
tion of amino acids into proteins using a cell
homogenate364.
Schechter, Momose and Rudney found that
BACITRACIN 43
bacitracin interfered with biosynthetic athways

which involved polyprenylpyrophosphates 365.
Storm and Strominger found that bacitracin

interacted with C55 isoprenylpyrophosphate in the
cell membrane366. This altered the permeability of
the bacterial cell.
9. Derivatives of Bacitracin
A number of bacitracin derivatives have been
produced. Some of these have been suggested for
use in animal feeds.
Siminoff, Price and Bywater suggested that
the methylene disalicylic acid complex of bacitracin
was useful as a feed additive for swine and poultry
367. Radomski, Hagan, Nelson and Welch established
the toxicity and safety of this derivative368
This complex was approved as a feed a d d i t i ~ e ~ 6 ~ .
Man anese bacitracin is an approved feed
additive3 7 3 .
A Japanese patent was issued for the sodium

methanesulfate derivative of b a ~ i t r a c i n ~ ~ ~ .
A U.S. patent was issued to Lewis, Ninger and
Pattison for the synthesis of the sodium methane-
sulfonate derivative of bacitracin which they sug-
gested was suitable €or parenteral administration
37 2 Baldwin patented sodium, potassium, calcium,
zinc and manganese salts of bacitracin methane-
sulfonate373.
SPOFA United Pharmaceutical Works reacted
bacitracin with a number of aldehydes and then iso-
lated the corresponding zinc salts374. Vondracek,
Toscaniova and Hoffman have patented a furfural de-
rivative of b a ~ i t r a c i n ~ ~ ~ .
Kalina, Ulbert and Masita patented the diiso-
butylnaphthalenesulfonate derivative of bacitracin
376.
Atassi and Rosenthal reduced bacitracin with
dib~rane~~ Shipchandler
~. was issued a patent on
derivatives of bacitracin reduced with sodium boro-
h~dride~~*.
44 GLENN A. BREWER
Mancino, Tigelaar and Ovary compared the

antigenic properties of the three monodinitro-
phenyl derivatives of bacitracin with that of the
tri-dinitrophenyl derivative379.
A Japanese patent was issued in which baci-
tracin was reacted with polyamine ion exchange
resins by means of an aldehyde380. The resulting
product was insoluble. In the same way, a dimer of
bacitracin was produced by reacting the antibiotic
with g l y ~ x a l ~ ~ l .
10. Reviews
Two reviews have been published on the assay
of bacitracinlg3I 382.
A number of reviews have been ublished on
bacitracin383 I384 ,385,386 I 387 I 388 g8 I 389 390 I
391.
Many more reviews have included bacitracin

alon with other antibiotics 392 I 393 I 394 I 395 I 396 I
397,~98,399,400,401,402,403, 404,405,406,
BACITRACIN 45
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52 GLENN A. BREWER
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128. HaavikFH.1.; J. Gen. Microbiol. - 84, Part 1,
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129. HaavikFH.1.; Nature 254, 79-82 (1975).
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130. HaavikTH. I. ; Acta Pathol Microbiol Scand. ,
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BACITRACIN 53
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134. Pass, L. and Raczynska-Bojanowska, K.; Rocz.
Technol. Chem. Zywn. 24, 217-23 (1974).
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136. Vitkovic, L. and Sadoff, H.L.; Spores 6, 362-6
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54 GLENN A. BREWER
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155. KalinaTV.; Ulbert, S. and Masita, A.; Czech.
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BACITRACIN 55
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164. Eliott7A.R.: Chornock. F.W. and Zinn.. E . ;
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56 GLENN A. BREWER
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BACITRACIN 51
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263. Freres, D. and Valdebouze, P.; Bull. Acad. Vet.

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280. Randalc W.A. amd Burton, J.M.; J. Assoc.
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281. Wright, W.W. and Burton, J.M.; J. Assoc. Agr.
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296. M 0 r r i s F V . J . and J e n n i n g s , B . R . ; Biochim. B i o -

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298. D o u l a k z , J . ; J . P h a r m . S c i . - 64 307-10 ( 1 9 7 5 ) .
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302. C h a r l e s , J . L . a n d W e i s s , P . J . ; A n t i b i o t i c s and
C h e m o t h e r a p y 8 496-9 ( 1 9 5 8 ) .
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303. S a l v e s = , B . a n d 'Aaro, B. , Medd. N o r . F a r m .
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304. Anon.; F e d . R e g i s t . 4 0 ' 1 5 0 8 8 A p r i l 4 , 1 9 7 5 .
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305. H a y a s h c H. ; S e i k a g a k u - 32 45-52 ( 1 9 6 0 ) .
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3 0 6 . M i k o l a j c i k , E . M . ; J . D a i r y S c i . 48 1 4 4 5 - 9
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3 0 7 . C o p p i , G . and B o n a r d i , G . ; .-
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308. Coppi, G . a n d B o n a r d i , G . ; B i o c h i m . B i o l . S p e r .
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320. Proencada Cunha, A. and Baptista, M.L.D.M.;
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55 19136i (1961)).
321. Paris, R.R. and Theallet, J.P.; Ann. Pharm.
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322. ApreosGsei, C. and Teodosiu, M.; Farmacia 10
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323. Proencada Cunha, O.R. and Gomes, M.E.B.; Bol.
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329. L a n g n e 7 H . J . a n d T e u f e l , U . ; Chem., M i k r o b i o l . ,
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336. B r y c e , C.F.A. a n d C r i c h t o n , R . R . ;
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[C.A. 75 5869 1r(19%1) 1 .
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339. S t e w a r c J . A . ; Biochem. B i o p h y s . R e s . Commun.
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Ver. Apotheker - Ztg. 106 120-2 (1961).
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356. Reimerc F.; Arch. Pharm. Chemi. - 75 1064-68
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376. KalinaTV. ; Ulbert , S. and Masita, A. ; Czech.
Patent 118,498, May 15, 1966.
(C.A. 66 74945w (1967)).
377. Atassi7M.Z. and Rosenthal, A.F.; Biochem. J.
68 GLENN A . BREWER
111 593-601
- (1969).
(C.A. 70 1 0 6 8 6 7 ~( 1 9 6 9 ) ) .
378. Shipchandler, M.T.; U . S . Patent 3 , 9 6 6 , 6 9 9 ,
June 2 9 , 1 9 7 6 .
(C.A. 8 5 143521m ( 1 9 7 6 ) ) .
379. Mancinc D.; Tigelaar, R.E. and Ovary, 2 . ;
Immunology 1 8 739-47 ( 1 9 7 0 ) .
(C.A. - .
73 6 4 X l t ( 1 9 7 0 ) )
380. Sato, K.; Kurosawa, A.; Koyama, Y.; Nagai, Y.;
Abe, M.; Ouchi, M. and Shimizu, S. Japan
Kokai 7 3 6 7 , 2 5 1 , September 1 3 , 1973-
(C.A. - 80 6 9 6 5 s ( 1 9 7 4 ) ) .
381. Sato, H.; Kurosawa, A.; Koyama, Y.; Nagai, H.;
Ohuchi, M. and Shimizu, M.; Japan Kokai
77 4 1 2 0 1 , November 2 2 , 1 9 7 7 .
(C.A. 88 1 5 2 9 4 1 ( 1 9 7 8 ) ) .
382. Brewer, G.A. and Platt, T.B.; Encyclopedia of
Industrial Chemical Analysis Vol. 5 533-549
(1967).
383. Meleney, F.L. and Johnson , B.A. ; Conn. State
Med. J. 1 4 305-7 ( 1 9 5 0 ) .
4 4 1 0 7 8 5 a (1350) j .
(c.A. -
384. Anon.; Bacitracin 1 2 7 pages ( 1 9 5 2 ) S . B . Penick
co.
&
(C.A. -
47 39309 ( 1 9 5 3 ) ) .
385. Anon.; Arch. Pharm. Chemi.; 61 1-7 ( 1 9 5 4 ) .
(C.A. 48 4 1 8 1 i ( 1 9 5 4 ) ) .
386. Paylos, R.N. and Seijo, E.; Rev. farm. - 95 146-
56 ( 1 9 5 3 ) .
(C.A. 48 47749 ( 1 9 5 4 ) ) .
387. Aubertin, E . ; J. Med. Bordeaux 1 3 0 1257-63
(1953).
(C.A. 48 6652d ( 1 9 5 4 ) ) .
388. MeleneE F . L . and Johnson, B.A.; Semana med.
495-508 ( 1 9 5 4 ) .
(C.A. 49 1215d ( 1 9 5 5 ) ) .
389. JawetzTE.; Pediat. Clin. N. Am. 329-44 ( 1 9 5 6 ) .
(C.A. 5 1 6 8 7 4 a ( 1 9 5 7.) 1. .
390. HickeyTR. J . ; Progr. Ind. Microbiol. - 4 93-150
(1964).
(C.A. 62 584313 ( 1 9 6 5 ) 1 .
391. Weinberg, E.D. ; 'Antibiotics - 1 90-101 ( 1 9 6 7 ) .
(C.A. 68 479621 ( 1 9 6 8 ) ) .
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1 3 7 - 4 7 , 180-8 ( 1 9 4 9 ) .
(C.A. 4 3 388433 ( 1 9 4 9 ) ) .
3 9 3 . Brainerd, H.D.; Calif. Med. - 7 1 9-14 ( 1 9 4 9 ) .
(C.A. 4 3 8 5 5 0 c ( 1 9 5 9 ) ) .
394. Hooper,.R.; Science Counselor - 1 2 1 2 8 - 9 , 148-9
(1949).
BACITRACIN 69
(C.A. -44 1613f (1950)).

395. Anon.; Chem. Eng. News 29 1190-5 (1951).
(C.A. 45 4001f (1951)).
396. WernerTG.; Scientia Pharm. - 19 95-103 (1951).
(C.A. -
45 105101a (1951)).
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(C.A. 50 7404e (1956)).
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Med. Encyclopedia, N.Y. (1956).
(C.A. 50 14875c (1956)).
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Penau, H.; Hagemann; Romain, R.; Ziegle, M.;
Vignalou, J. and Quevauviller, A.; Therapie -11
961-1029 (1956).
(C.A. 52 20665e (1958)).
400. Brunnec R.; Osterr. Apotheker Ztg. - 11 455-60
477-9 (1957).
(C.A. 52 5748g (1958)).
401. JawetzTE.; Antibiotics Monographs - 5 1-95
(1956).
(C.A. -53 22520g (1959)).
402. Baker, W.B.; Drug ti Cosmetic Ind. -
87 172-3,
258-62, 264-7 (1960).
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350-60 (1962).
(C.A. 57 11307b (1962)).
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(C.A. 70 80793u (1969)).
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(C.A. -76 30462p (1972)).
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407. Yeh, P.; Personal Communication, 1980.
Literature search complete through 1978.

BRETYLIUM TOSYLATE
James E. Carter, Anton H . Amann, and

David M . Baaske
1. Description 72
I . 1 Chemical and Proprietary Names 72
1.2 Empirical Formula, Molecular Weight, and Structure 72
1.3 Appearance, Color, Odor, and Taste 72
2.1 Melting Range 72
2.2 Solubility Profile 73
2.3 Infrared Spectrum 73
2.4 Ultraviolet Spectrum 73
2.5 Proton Magnetic Resonance Spectrum 73
2.6 Mass Spectrum 78
2.7 Differential Scanning Colorimetry 78
3. Synthesis 80
4. Analysis 80
4.1 Elemental Analysis 80
4.2 Nonaqueous Titration 81
4 . 3 High Performance Liquid Chromatography (HPLC) 81
4.4 Gas- Liquid Chromatography (GLC) 82
4.5 Thin- Layer Chromatography (TLC) 84
5. Stability 84
6. Analysis of Biological Samples by Gas- Liquid Chromatography. 84
7. Absorption, Metabolism, and Excretion 85
8. Acknowledgment 85
9. References 86
Copyright 0 1980 by Academic Ress, Inc.

Analytical Profiles of Drug Substances, 9 71 All rights of reproductionin any form reserved.
ISBN: 0.12-260809-7
12 JAMES E. CARTER eta1
1. Description
1.1 Chemical and Proprietary Names
Bretylium tosylate is the non-proprietary name for

o-bromobenzylethyldimethylammonium p-toluenesulfonate. It
has been marketed as an antihypertensive agent but is no
longer used for this indication in the United States. Pro-
prietary names listed by the Merck Index are Bretylan, Brety-
late, Darenthin and Ornid.
The drug is now marketed as an antiarrhythmic agent with the

proprietary name Bretylol.
1.2 Empirical Formula
C18H24BrN03S
Molecular Weight 414.36 The Merck Index (1)

lists the molecular weight as 414.39. Based upon atomic
weights defined in 1973 by the International Union of Pure
and Applied Chemistry 414.36 is correct.
Structure
CH3
+ I
CH2-N-C2H,
so;
I
Br@ LH3
CH,
1.3 Appearance, Color, Odor and Taste
Bretylium tosylate is a white to off-white free

flowing, fine, odorless powder. It has an extremely bitter
taste.
2.1 Melting Range

96OC - 99OC
BRETYLIUM TOSYLATE 73
2.2 Solubility Profile
Bretylium tosylate is freely soluble in water,

methanol and ethanol. It is commonly recrystallized from hot
acetone. Chloroform and methylene chloride are the best
extraction solvents. Bretylium tosylate is essentially in-
soluble in ether, ethylacetate and hexane.
2.3 Infrared Spectrum
The KBr pellet infrared spectrum of 0.5% bretylium

tosylate obtained with a Perkin-Elmer 283 Infrared Spectro-
photometer is contained in Figure 1. Bretylium tosylate is
very hygroscopic. Unless the spectrum is obtained on dried
material the broad 0-H stretching band centered at 3460 cm-I
will be present. The aromatic (3100 - 3000 cm-l) and ali-
phatic (3000 - 2900 cm-l) C-H stretching bands are present
but not as strong as might be anticipated. The strong broad
peak centered at 1200 cm-l is the S - 0 stretching band. The
molecule contains both a para substituted aromatic (strong
C-H bending at 815 cm-l) and an ortho substituted aromatic
(strong C-H bending at 772 cm-l) ring.
For routine identification purposes a liquid infrared

spectrum is generally more reproducible. The spectrum of a
2% solution in dry chloroform is shown in Figure 2.
2.4 Ultraviolet Swectrum
Bretylium tosylate absorbs strongly in the ultra-

violet region of the spectrum with three distinct maxima be-
tween 230 nm and 300 nm. The spectrum (Figure 3) was ob-
tained with a Beckman Acta I11 double beam spectrophotometer.
The wavelength maxima and molar absorptivities are:
278 671
271 885
264 886
257 (shoulder) ---
2.5 Proton Magnetic Resonance Spectrum
The 60 MHz proton magnetic resonance spectrum was

obtained with a Varian Associates T-60A spectrometer. The
spectrum in CDC13 with tetramethylsilane (TMS) as internal
reference is contained in Figure 4. The integration and
Figure 1. KBr Infrared Spectrum of Bretylium Tosylate.
0
k
0
rl
c
-4
w
0
5k
4J
u
a,
a
Cn
a
a,
k
rd
k
u
c
H
N
a,
Ll
?
b
-4
h
16 JAMES E. CARTER et al.
Figure 3 . Ultraviolet Spectrum of Bretylium

Tosylate.
Figure 4. Proton Magnetic Resonance Spectrum of Bretylium Tosylate in CDC13.
78 JAMES E. CARTER er al.
multiplicities are consistent with the proton assignments.
Chemical shifts (6) in ppm relative to TMS are:
::
Br
ii CH *-+N
t d
7H3
-CH -2
J i b
i i
g
@ A 4 3
Proton # of Chemica1
Assignment Protons Shift ( 6 ) Multiplicity
3 1.35 triplet
3 2.27 singlet
6 3.07 singlet
2 3.65 quartet
2 4.73 singlet
4 7.17 mu1tiplet
4 7.67 multiplet
2.6 Mass Spectrum
The direct probe electron impact mass spectrum of

bretylium tosylate i s shown in Figure 5. The spectrum was
obtained with a Dupont Dimaspec GC/MS Model 321 (2). No
parent ion is seen because bretylium tosylate is a salt and
will not travel through the spectrometer intact. Principal
fragment ions in the spectrum are identifiable. The base
peak at m/z 91 is the tropylium ion (C7H7+) probably formed
by loss of SO3- from tosylate. The tropylium ion is also
possible from fragmentation of the bretylium ion. The m/z 58
is C3H8N' formed by loss of C2H5 (which is possible by a
number of different paths) from the bretylium quaternary
ammonium side chain. The two isotopes of bromine of mass 79
and 81 make the peaks at m/z 169, 171 and m/z 185, 187
readily identifiable as ~ 7 Br+~ and
6 C7HgN Br+ respectively.
2.7 Differential Scanning Calorimetry
Bretylium tosylate was heated at a rate of 20°/min

in a Perkin-Elmer Model DSC-2 differential scanning calori-
19
t
Figure 5. Electron Impact Mass Spectrum of Bretylium Tosylate.
80 JAMES E. CARTER c t a l .
meter. A single endotherm was observed with an onset temper-

ature of 97.5OC with the endotherm maximum at 102.5OC. The
onset temperature corresponds to the melting point. The heat
of transition ( H) calculated in relation to an indium
standard is 16.8 cal/g.
2.8 Crvstal Prowerties
Bretylium tosylate crystals examined with a polar-

izing microscope were found to be tetragonal prisms, elong-
ated parallel to the c crystallographic axis (3). X-ray
diffraction patterns (Table I) were also determined (3).
Table I. Powder x-ray diffraction pattern of

Bretylium Tosylate
Re1ative
20 Intensity d &
7.65 50 11.6
11.05 50 8.00
12.65 10 6.99
14.10 25 6.28
15.25 30 5.81
16.60 10 5.34
18.00 5 4.92
18.65 5 4.75
19.35 100 4.58
20.10 5 4.41
21.30 15 4.17
22.15 50 4.01
23.10 75 3.85
24.35 10 3.65
24.75 100 3.60
26.45 80 3.37
3. Synthesis
The Bretylium Unites States patent contains examples for

the synthesis of numerous bretylium salts (4).
4. Analysis
4.1 Elemental Analysis
Elemental analysis of a typical bretylium tosylate

BRETYLlUM TOSYLATE 81
sample is as follows:
Element % Theoretical* % Found**
C 52.18 52.40
H 5.84 5.76
Br 19.28 19.56
N 3.38 3.28
0 11.58 11.69
S 7.74 ----
*Calculated for C18H24BrN03S
**Determined on a dried sample
4.2 Non-aaueous Titration
Bretylium tosylate may be measured by non-aqueous

titration with 0.025 N perchloric acid in dioxane. The end
point is visually detected by a change from violet to blue-
green using crystal violet as the indicator.
4.3 High Performance Liquid Chromatography (HPLC)
Two reversed phase HPLC methods have been developed

for the quantitation of bretylium tosylate. In the first
method (5) bretylium and tosylate ions are determined simul-
taneously with benzenesulfonic acid as an internal standard.
Chromatography is carried out on a 10 um octadecylsilane
column with an isocratic mobile phase consisting of 30% meth-
anol in water (pH 5.0) containing a paired ion reagent,
tetrabutylammonium phosphate. Flow rate through the column
was 2.0 ml/min and the variable wavelength UV detector was
set at 220 nm. Standards containing from 0.1 to 0.5 mg
bretylium tosylate per ml of solution were employed. The
method is applicable for raw drug evaluations, analysis of
intravenous solutions and compatibility studies with other
drugs. It is not amenable to determination of bretylium or
tosylate in biological fluids. Total analysis time is less
than 12 minutes.
The second reversed phase HPLC method was employed for the
quantitation of bretylium ion (6). Bretylium tosylate
standard concentrations ranged from 10 to 400 ug/ml; the
internal standard was the 2,4-dichloro congener of bretylium
tosylate. The 30 cm by 3.9 mm column was packed with 10 um
alkylnitrile bonded silica. The compounds were eluted with
a mobile phase consisting of acetonitrile and 0.005 M sodium
phosphate monbasic in purified water (30:70) at a flow rate
of 2.0 ml/min. A fixed wavelength UV detector at 254 nm was
used to monitor the column effluent. As with the first HPLC

method this method is only applicable to evaluation of the
raw material and dosage forms. Total analysis time by this
method is approximately 1 5 minutes.
Both methods are specific, accurate, rapid and precise.
4.4 Gas-Liquid Chromatography (GLC)
A quantitative, stability indicating GLC assay for

bretylium tosylate is also applicable to dosage forms ( 7 ) .
The method is based upon a published assay for estimating

plasma and urine levels of the drug (8). p-Chlorobenzyl-
ethyldimethylammonium p-toluene sulfonate (bretylium is the
o-bromo congener) was synthesized and used as the internal
standard. The method has been used to evaluate the stability
of raw materia1,tablets and injections. The identity of the
peaks appearing in the chromatogram has been confirmed by
GLC-mass spectrometry. The method involves reaction of
bretylium and internal standard with sodium thiophenolate at
70° for 15 minutes. The resultant halogenated benzylthio-
ethers are quantitated by GLC. The reaction is specific for
quaternary amines.
Chromatography was performed with a suitable gas chromato-

graph equipped with a flame ionization detector. The 1.8 m
by 4 mm id glass column was packed with 3% OV 225 on 100-120
mesh Chromosorb W-HP. The column and inlet temperatures were
maintained at 210° while the detector temperature was 275O.
The carrier gas was helium at a flow rate of 5 0 ml/min.
The reaction scheme for bretylium and internal standard with

sodium thiophenolate is shown in Figure 6. Three peaks
appear in the chromatogram following the solvent front.
These were identified by GLC-mass spectrometry ( M S ) with a
DuPont DP-1 system in the electron impact ( E I ) mode ( 9 ) .
Diphenyldithiol appears at 3.0 min; EIMS, 218 ( M
'
)
. p-
Chlorobenzylphenylthioether appears at 4.0 min; EIMS, 234
(M+) while o-bromobenzylphenylthioether appears at 4.8 min;
EIMS 278 (M') and 280 (M').
All EIMS spectra showed the base peak at 110 corresponding to

the CgHgSH'ion.
The 15 minute reaction time and 6 minute analysis time is

much more practical than the 1 hour reaction time and 2 0
minute analysis time reported previously (8).
31
k
c,
a,
i?k
c,
u
*a
a, a,
cl a
R1
3 m
t
0
a,
s
04
0
I
+
4
a
C
0
k
pc
m
d
+ +
a,
Q d
.0
3
Jz
u
i
>.
u
a,
m
i
m
84 JAMES E. CARTER e t a l .
4.5 Thin-layer Chromatography (TLC)
Purity and stability of the raw drug have also been

assessed by thin-layer chromatography. Bretylium tosylate
has an Rf of 0.50 when chromatographed on Alumina-G with 1-
butanol saturated with water as solvent. o-Bromobenzyldim-
ethyl amine is the most likely contaminant and degradation
product. When the plate is sprayed with modified Dragen-
dorff's reagent it appears as a pink spot with an Rf of
0.85.
5. Stability
Bretylium tosylate is a very stable molecule. Solutions

of bretylium tosylate at 50 mg/ml in 1 hydrochloric acid,
1 N sodium hydroxide and 10% hydrogen peroxide were heated
for one hour at 90°C. The solutions were analyzed by the
stability indicating gas chromatographic (section 4.4) and
thin-layer chromatographic (section 4.5) methods. The ultra-
violet absorbance at 271 nm was also monitored.
The results (Table 11) indicate the potency of the solutions

did not change with this drastic treatment.
Table 11. Subjection of bretylium tosylatc to drastic

acid, alkaline and oxidative conditions.
Change
Solution Assay* Initial Assay Final Assay From Initial
1 HC1 GLC 100.0% 100.5% +O. 5%

TLC one spot** one spot no change
uv 0.824 0.832 +l.0%
N NaOH
1- GLC 101.5% 100.5% -1.0%
TLC one spot one spot no change
uv 0.825 0.838 +1.6%
10% H202 GLC 101.5% 102.9% +1.4%
TLC one spot one spot no change
uv 0.835 0.821 -1.7%
*GLC - gas-liquid chromatography

TLC - thin-layer chromatogrpahy
UV - ultraviolet absorbance at 271 nm.
**one spot indicates one spot with an Rf matching
bretylium tosylate standarii.
6. Analysis of Biological Samples by Gas-Liquid Chromato-

graphy
BRETYLIUM TOSYLATE 85
A quantitative method for the analysis of low concen-

trations of bretylium in plasma and urine has only recently
been developed (10). The method is based upon derivatization
as are the previously described GLC procedures ( 7 , 8 ) . To
enhance the sensitivity of the assay an electron capture
detector was employed and 2 , 4 , 5 trichloro sodium thiopheno-
late was substituted for sodium thiophenolate., Internal
standards employed were p-bromobenzylethyldimethylammonium
p-toluenesulfonate or o-methoxybenzylethyldimethylammonium p-
toluene sulfonate. Bretylium tosylate was quantitatively
extracted with methylene chloride after deproteinization with
acetonitrile.
The sensitivity of the method is 5 ng/ml.
Analysis was performed with a gas chromatograph and a 63Ni

electron capture detector. The 1.8 m by 4 mm id glass column
was packed with 3% OV 2 2 5 on 100/120 Supelcoport. Injection
port, column and detector temperatures were maintained iso-
thermally at 270°, 250° and 300°, respectively. Argon/
methane ( 9 5 / 5 ) was the carrier gas at a flow rate of 50
ml/min (30 ml/min through the column and 20 ml/min directly
t o the detector as a scavenger gas).
The retention times of the 2 , 4 , 5 trichlorophenylthioether

derivatives of the o-methoxybretylium congener, bretylium and
the p-bromobretylium congener were 6.1, 7.4 and 9.4 min
respectively.
7. Absorption, Metabolism and Excretion
Bretylium is not absorbed from the stomach and is poorly

absorbed from the gastrointestinal tract (11). Radioactive
tracer studies indicate that the drug is not metabolized and
is excreted primarily in the urine ( 8 , 1 2 ) . After an intra-
muscular administration of I 4 C bretylium 63% of the dose was
recovered in the urine and 31% was in the feces in the
following 4 days (8). Bretylium was found in high concentra-
tion in the bile which suggests bile as the source of bre-
tylium found in the feces.
The pharmacological and biochemical properties of bretylium

have been reviewed (13).
8. Acknowledgement
The manuscript was expertly typed by Ms. Deborah

Canfield.
References
1. The Merck Index, 9th Edition, 1376, Merck & Co. Inc.,
Rahway, NJ, 1976.
2. V. Diaz, Shilstone Engineering testing laboratory Inc.,
New Orleans, LA, 70112. Personal communication.
3. S. Palenik, Walter C. McCrone Associates, Inc., Chicago,
IL 60616. Personal communication.
4. -
Anon., Unites States Patent 3,038,004, June 5, 1962.
5. Y.C. Lee, D.M. Baaske, A.H. Amann and J.E. Carter,
Chromatography Newsletter, 8, 9 (1980).
6. C.M. Lair Z.M. Look, P.K. Lai and A. Yacobi, - J.
Liquid Chromatography, 3, 93 (1980).
7. J.E. Carter, H. Kesler, L.R. Klein, D.P. Carney, A.H.
Amann and L.A. Gardella, presented in part, American
Pharmaceutical Association 126 annual meeting, Anaheim,
CA, Apr., 1979.
8. R. Kuntzman, I. Tsai, R. Chang and A.H. Conney, - Clin.
Pharmacol. and Therap. , 11,829 (1970).
9. E. Chait, EI DuPont DeNemours and Co. Inc., Instrument
Products, Wilmington, DE 19898. Personal communication.
10. C.M. Lair B.L. Kamath, J.E. Carter, P. Erhardt, Z.M.
Look and A. Yacobi, J . Pharm. Sci., accepted for publi-
cation.
11. A.L.A. Boura and A. McCoubrey, 2. Pharm. Pharmacol., 14,
647 (1962).
12. W.G. Duncombe and A. McCoubrey, &. J . Pharmacol., 15,
260 (1960).
13. Pharmacological and Biochemical Properties of Drug
Substances, Vol 2, M.E. Goldberg, Ed., American Pharma-
ceutical Association, Academy of Pharmaceutical Sciences,
Washington, D.C., p. 148.
CARBAMAZEPINE
Hassan Y. Aboul-Enein and A. A . Al-Badr
I. Description 88
1 . 1 Nomenclature 88
1.2 Formulae 83
1.3 Molecular Weight 88
I . 4 Elemental Composition 88
1.5 Appearance 88
2.1 Melting Point 88
2.2 Solubility 89
2.3 Identification 89
2.4 Spectral Properties 89
3. Synthesis 94
4. Stability, Decomposition Products 96
5. Metabolism, Pharmacokinetics, and Absorption 96
6. Methods of Analysis 99
6.1 Spectrophotometric Methods 99
Acknowledgments 103
References 104
Copyright 0 1980 by Academic Ress. Inc.

Analytical h f i l e s of Drug Substances, 9 87 All rights of reproductionin any form ~ S C N C ~ .
ISBN: 0-12-260809-7
88 HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR
CARBAMAZEPINE
1. Description
1.1 Nomenclature
1.11 Chemical names
5H-Dibenz [b,f] azepine-5-carboxamide

5-Carbamoyl-5H-dibenz [ b , f] azepine
2,3 : 6,7-Dibenzazepine-l-carboxylic acid, amide
1.12 Generic Name
Carbamazepine
1.3 Trade names
Finlepsin, Tegretol, Tegretal
1.2 Formulae
1.21 Empirical C15H12N20
a?o
1.22 Structural
cow2
1.23 Wiswcsser Line Notation : TC 676 BNJ BVZ (1)
1.3 Molecular weight 236.26
1.4 Elemental composition
C 76.25%, H 5.12%, N 11.86%, 0 6.77%
1.5 Apearance
White to off-white powder.
2. Physical properties
2.1 Melting point
Melts within a range of 3O between 187 and 193' (2).

C ARB AMAZE PINE 89
2 . 2 Solubilig
Practically insoluble in water; soluble in alcohol,

acetone and propylene glycol ( 3 ) .
2 . 3 Identification
2 . 3 1 Infrared Spectroscopic test
USP XIX ( 4 ) cites the use of infrared absorption

spectrum of carbamazepine in methylene chloride
as a mean of identification comparing some
characteristic absorption bands of the drug. This
will be discussed in the infrared spectral proper-
ties of the drug.
2 . 3 2 Color test
Carbamazepine can be identified ( 5 ) by color test

with ammonium molybdate. A faint to blue color
is produced (sensitivity 1.0 1.18).
BP 1 9 7 3 ( 6 ) describes a color test in which 0.1 g

of the drug is treated with 2 ml nitric acid in a
water-bath for three minutes where an orange
color is produced.
2.33 Crystal test
Carbamazepine can be identified by forming crys-

tals with lead iodide solution where needles are
formed ( 5 ) .
2 . 4 Spectral properties
2 . 4 1 Ultraviolet spectrum
Carbamazepine in neutral methanol solution shows

maxima at 212 nm, an inflection at 2 3 6 nm,
283 nm; and a minimum at 256 nm. (Fig. 1).
Carbamazepine ( 5 ) in ethanol shows a maxima at

215 nm and at 285 nm, minimum at about 257 nm.
In 0 . 1 N sulphuric acid, the drug shows maxima
at 283 nm (E 1%,1 cm 1 4 7 ) and an inflection at
about 255 nm (E 1%,1 cm 2 7 4 ) .
90 HASSAN Y. ABOUL-ENEIN AND A. A. AL-BADR
- = .
400 380 360 340 320 300 280 260 240 220'200
Fig. 1 - Ultraviolet spectrum of carbamazepine

in methanol.
C ARB A MAZE PINE 91
The ultraviolet absorption spectrum of the drug

is used as a mean of identification of carbama-
zepine in BP 1973 (6). A 2 cm layer of 0.001 w/v
solution in alcohol (95%) exhibits a maximum only
at 285 nm; extinction at 285 nm, about 0.98.
The drug also exhibits an intense blue flores-

cence in the ultraviolet light at 366 nm.
2.42 Infrared spectrum
The infrared spectrum of carbamazepine is shown

(Fig. 2). The spectrum was obtained from nujol
mull. The structural assignments have been
correlated with the following band frequencies:-
Frequency (Cm-l) Assignments
3470
NH2
1680 c=o
1600 shoulder and 1590 Aromatic C = C
Clarke (5) cited the following bands as charac-

teristic principal peaks for carbamazepine when
determined in potassium bromide; 1678, 1388 and
1594 Cm-l.
2.43 Nuclear Magnetic Resonance Spectrum
A typical NMR spectrum of carbamazepine is shown

in (Fig. 3). The sample was dissolved in CDCl
3’
The Spectrum was determined on a Varian T-60A,
NMR spectrometer with TMS as the internal stan-
dard. The following structural assignments have
been made for (Fig. 3).
Chemical Shift (6) Assignments
Broad singlet at 4.83

NH2
Singlet at 6.87 CH = CH at CloCll
Multiplet centered at 7.33 Eight aromatic protons
on the two phenyl
groups.
2.44 Mass spectrum and fragmentometry
The mass spectrum of carbamazepine obtained by

' 0 0 0
C ? a W d
O N 0
C
d
I 1 I 1
I 8
m
m
p:
C
0
0
Lc
h
d
0
N
z 0
n u
l
c
f
0
N a
r
0
E 0
0
r
z 0
0
N
r
9
a
0
0
2 P
0
0
9
: w
0
W
8
5
L!
m c)
r U
a,
a
(0
z 0
0
8
a
01
h
(d
L!
w
C
H
: 0
0
N
Lo
0
0
m
0
4
m
0
!0
n
m
x 8
I
0
(D
I
0
d o
N
0
0
0
rt
92
W
c3
I n I
c 6 3 1 ' * 1 ' I!. ' ' * 1 ' * 1 . . . . 1 . . , , 1 . . . . 1 . . . .
8.0 7.0 6.0 5.0 PPM(6) 4.0 3 .O 2.0 1 .o (
Fig. 3 - NMR spectrum of carbamazepine in CDCl containing TMS

as internal standard. 3
e ectron impact ionization shows a molecular ion

M
$1 at m/e 236 (relative intensity 9.1%) Fig. 4 ,
and a base peak at m/e 193.
Frigerio et. a1 (7,8) had published the mass

spectrometric properties of carbamazepine and its
metabolites, carbamazepine - 1 0 , U epoxide and
10,11-dihydro-10,11-dihydroxy-5H-dibenz [b,f]
azepine-5-carboxamide. The fragmentation pattern
are shown in Scheme 1. Frigerio et.al., dis-
cussed the fragmentation pattern of carbamaze-
pine and its epoxide in details (7,8).
CONH 2 H
m/e 236 mie 193 m/e 192
3. Synthesis
a 1
H
Br
coc12
Toluene
@& I
COCl
Me
(PhC02)
Pressure -
COCl
coNH2
a) Carbamazepine can be synthesized as follows:-
Iminodibenzyl in toluene was treated with C0Cl2 to give

95% of 5-chlorocarbonyl iminodibenzyl which in turn was
dissolved in CC1 and was treated with 1,3-dibromo-5,
4
5-dimethyl hydantoin and (PhCO ) to give 90% 5-chloro-
carbonyl-10-bromoimino-dibenzyj. The latter was dis-
solved in xylene and heated at about 100' in an auto-
clave with gaseous NH to give 85% o f 5-carbamoyl-5H-
3
dibenzo [b,f] azepine (9).
11846 SCAN 50 S I G M A 4 RT-0 1 1 B ~ C K = 1 7 0 ~ X 1 0 100'/.=
0 444000
CQRBWAXPXNE
Fig. 4 - Mass spectrum of carbamazepine (EI) determined

by direct probe insertion.
96 HASSAN Y. ABOUL-ENEIN AND A. A . AL-BADR
cow2
b) 5H-Dibenzo [b,f] azepine, which may be prepared by

thermal decomposition of 2-(0-aminostyry1)-aniline
hydrochloride, is condensed with carbamoyl chloride by
refluxing in an inert solvent in the presence of
sodamide (10).
4 . Stability, Decomposition products
Carbamazepine is relatively stable drug at room tempera-

ture. However, it is recommended that it should be kept
and stored in a well closed container, protected from light
and in dry place.
BP 1973 (6) had described a test for identification of

foreign substances namely iminodibenzyl using tlc for this
purpose .
5. Metabolism, Pharmacokinetics and absorption
Meinardi (11) had published a review on carbamazepine in

1972 in which he discussed the determination, metabolism
and pharmacology of the drug.
Carbamazepine is readily absorbed from the gastrointestinal

tract. Peak concentration in serum have been reported at
about 2% h after a dose. It is believed to have a half-
life between 14-29 h. (12).
Studies on the plasma kinetics of carbamazepine suggested

that it induced its own metabolism (13).
Frigerio et. al., ( 7 ) had isolated carbamazepine-lO-11-

epoxide as a urinary metabolite from humans following oral
CA RB A M AZE PINE 91
administration. The epoxide formation was confirmed by the

in vitro studies of the activity of the liver microsomal
--
monooxygenases. SKF 525A inhibited the formation of car-
bamazepine oxide by 80X (14). Goenechea and Hecke-
Seibicke (15) had detected seven metabolites in human
urine in addition to unchanged drug by tlc. l0,ll-Dihydro-
10-11-dihydroxy-5H-dibenzo [b,f] azepine-5-carboxamide was
identified on the basis of UV, IR, mass and NMR Spectra.
Iminostilbene was also isolated as a minor urinary meta-
bolite from rats (16).
The N-glucuronide of carbamazepine was identified in the

bile of isolated perfused rat lever by the mean of per-
methylation GC/MS (17).
The pharmacokinetic of carbamazepine was studied in

several species :-
A) Humans
Gerardin et. al., (18) had discussed the pharmacoki-

netics of the drug in normal humans after single and
repeated doses. It was reported that the plasma
concentration of the drug following single dose (100,
200, 600 mg) to normal healthy humans were fitted by
a one-compartment open model. The elimination half-
life after a single dose was 37.7h; it decreased dur-
ing chronic treatment to a calculated value around 21h.
The steady-state plasma concentration, lowers than
expected from the single dose study, was adequately
predicted from the single-dose data when a correction
was made for the increased elimination rate constant.
These findings contrast with the apparantly unpredic-
table plasma levels reported during carbamazpine
therapy.
Palmer et. al., (19) reported that following oral

administration of the drug (200 mg) to two healthy
fasting subjects, peak plasma concentration occured
after 6-8 h . and remained constant for 24 h before
declining over the subsequent 6 days. The plasma half-
life was about 36 h.
B) Rhesus monkey
The pharmacokinetics of carbamazepine (20) after a

20 mg/kg dose was administered by I.V. (5. min) infu-
sion and orally. A l l semilogarethmic plasma concen-
&
Km 0.34nM
0.41 nmo1/
I minlmg protein
I
"I
I \
corn2
corn2 Glucuronide
(igb
OH OH OK I
I
I
CONJQ cowz OH
1
Glucuronide
I
Glucuronide
Oil
Identified Metabolites of Carbamazepine.

CARBAMAZEPINE 99
tration-time curve after I . V . administration exhibited

an irregular decay behavior in the first 3-hr period,
followed by a linear disappearance phase (T% =, 1.0-
2.4 hr). Urinary extraction measurements confirmed
the short elimination half-time and showed that < 1%
of the dose was excreted unchanged. Oral studies also
yielded a short elimination half-life (1.0-1.60 hr),
which was confirmed by urinary excretion measurements.
The fraction of the oral dose reaching the systemic
circulation ranged between 58 and 87%. Measurable
(but insignificant) amounts of drug were found in the
feces after I.V. and oral administrations.
C) Adult male, female and pregnant rats
After treatment with single and repeated doses of car-

bamazepine, male rats eliminated the drug faster than
females; the total body clearance (TBC) was 1 6 ml/min/
kg and 9.4 ml/min/kg. respectively. Two dose levels
(25 and 50 mg/kg) had the same pharmacokinetic pro-
perties in young rats. Pregnant rats cleared the drug
to a lesser extent than controls. Carbamazepine acce-
lerated its own elimination after repeated administra-
tion in both adult and young rats as revealed by the
shortening of its half-life and an increase of 50% in
clearance. Moreover the protection against electro-
sock was significantly reduced after repeated adminis-
tration, compared with a single-dose administration,
(21) 9
The mean amount of carbamazepine not bound in vitro to

plasma protein from 24 healthy subjects was 18.2%; the
mean amount not bound in plasma from 5 4 patients tak-
ing the drug was 26.9% (range 7 . 9 to 60%). There was
no significant difference in binding capacity between
plasma from patients with renal disease and that from
healthy subjects but the plasma from patients with
level disease bound a slightly lower percentage o f
carbamazepine than did normal plasma (22).
6. Methods of Analysis
6.1 Spectrophotometric methods
6 . 1 1 Ultraviolet spectrophotometric methods
a) Both BP 1973 and USP XIX ( 4 ) describe an ana-

lytical procedure for carbamazepine and its
tablet formulation depending on measuring
HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR
the absorbance of the solution prepared at 285

nm. The solvent used in BP 1973 is alcohol
95% while USP XIX uses dehydrated alcohol-
methanol (95:5) as a solvent system in the
ultraviolet determination of carbamazepine.
b) Fellenberg eta (23,24) reported a method for the

determination of carbamazepine in blood. The
method has a detection threshold of <O.l mg/
100 ml of blood. The method is based on the
catalytic rearrangement of carbamazepine to
9-methylacridine. After extraction of the
drug from blood with methylene chloride, which
is then washed with alkali and acid. An ali-
quot of the extract is evaporated to dry-
ness and the residue heated briefly with HC1 at
150'. Following removal of nonspecific inter-
ference with n-heptane, the absorbance of
9-methylacridine is determined at 258 nm. The
method is reported to be rapid, sensitive and
specific and is suitable for routine clinical
use.
6.12 Nuclear magnetic resonance spectrometry
Carbamazepine has been assayed (25) in its tablet

form by the application of PMR spectrometry. The
method involves comparing the integral of the
aromatic and olifinic protons of the drug, in the
range of 6.60-7.60 ppm, to that of the singlet of
known amount of hexamethylcyclotrisilazine (at
0.00 ppm) used as internal standard. The method
is simple, rapid and accurate. The average per-
cent recovery of carbamazepine from its tablets
is 96.88 5 0.93.
6.2 Chromatographic methods
6.21 High pressure liquid chromatography
Several methods have been published for the quan-

titative determination of carbamazepine and its
epoxide metabolite in biological fluids (plasma,
serum, urine) by HPLC. Kitazawa and Komuro (26)
had described a method for determination of car-
bamazepine and other anti-convulsant drugs in
human blood plasma. The method involved two step
extraction procedures with chloroform and use of
2x50 cm long stainless steel column packed with an
CARBAMAZEPINE 101
a n i o n exchange r e s i n . The m o b i l e p h a s e w a s 4mH

ammonium p h o s p h a t e b u f f e r s o l u t i o n of pH 6.2 a t
a f l o w r a t e o f 0 . 4 0 ml/min.
The r e s u l t s p r e s e n t e d showed l i n e a r c a l i b r a t i o n
c u r v e s and 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 as low a s
1 . 0 v g I 0 . 5 m l plasma. The method w a s e f f i c i e n t
t o d e t e c t t h e d r u g i n plasma a f t e r t h e r a p e u t i c
c l i n i c a l doses.
Eichelbaum and B e r t i l s s o n ( 2 7 ) d e s c r i b e d a method

u s i n g HPLC-mass s p e c t r o m e t r y f o r s i m u l t a n e o u s
d e t e r m i n a t i o n of carbamazepine and i t s a c t i v e 1 0 ,
11-epoxide m e t a b o l i t e i n plasma. The method
r e q u i r e d no d e r i v a t i z a t i o n and had a l o w e r l i m i t
of s i n s i t i v i t y of 4 ng f o r carbamazepine and 4 ng
f o r i t s m e t a b o l i t e . The method i s v e r y s p e c i f i c
and had a p r e c i s i o n s of 2.2% f o r t h e d r u g and
4.2% € o r i t s m e t a b o l i t e s .
Karba Marton (28) p u b l i s h e d a method f o r d e t e r -

m i n a t i o n of carbamazepine i n t h e whole blood by
HPLC. The d r u g w a s w e l l s e p a r a t e d from normal
blood c o n s t i t u e n t s i n less t h a n 8 m i n u t e s . The
s e n s i t i v i t y o f t h i s method i s 0.25 mg of t h e
d r u g 1 1 i n a 2 m l s a m p l e , and t h e l o w e r l i m i t of
d e t e c t i o n i s 100 ng.
Another method (29) w a s r e p o r t e d f o r d e t e r m i n a t i o n

of carbamazepine i n plasma i s d e s c r i b e d i n which
t h e drug w a s e x t r a c t e d with e t h e r , i s o l a t e d with
L i c h r o s o r b RP8 and d e t e c t e d by UV s p e c t r o s c o p y
a t 280 nm. The r e c o v e r y of t h e d r u g w a s a t c o n c e n -
t r a t i o n 1-2 ug/ml. T h e r e w a s no i n t e r f e r e n c e by
t h e d r u g m e t a b o l i t e o r endogenous plasma com-
ponent s .
6.22 P a p e r chromatography
C l a r k e ( 5 ) d e s c r i b e d s e v e r a l s o l v e n t s y s t e m s used
f o r p a p e r c h r o m a t o g r a p h i c d e t e c t i o n of carbamaze-
p i n e as shown i n T a b l e 1.
6 . 2 3 Thin Layer Chromatography
Carbamazepine WAS d e t e r m i n e d among o t h e r a n t i c o n -

v u l s a n t a g e n t s i n serum by t l c . The serum w a s
e x t r a c t e d w i t h toluene and t h e d r i e d e x t r a c t w a s
d i s s o l v e d i n c h l o r o f o r m and s p o t t e d on t o a t l c
Table 1
Solvent System Visualizing agent Rf

Citric acid : H 0 : n-butanol Ultraviolet blue 0.34
( 4 . 8 gm : 1 3 0 m12: 870 ml) fluorescence
Acetate Buffer Ultraviolet, blue 0.29

(pH 4 . 5 8 ) fluorescence
Phosphate Buffer Ultraviolet, blue 0.30

(PH 7 . 4 ) fluorescence.
plate. After development, the plate was scanned

at 215 nm without staining. Most of the inter-
fering substances that occur naturally in serum
were soluble in and eliminated by the liq. front.
(30).
Clarke (5) reported the use of a solvent system

consisting of strong ammonia solution :methanol
(1.5 : loo), the solvent system is recommended to
be changed after two runs. Carbamazepine gives an
R value o f 0 . 7 3 . The chromatogram (Silicagel G)
f
is visualized by potassium permangnate spray.
6.24 Gas-Liquid Chromatography
Gas-Liquid chromatography is considered to be the

main procedure for the quantitation and analysis
of carbamazepine specially in biological fluids
and tissues.
The drug has been determined by several authors

( 3 1 , 32, 3 3 , 3 4 ) through derivatization to its
methylated derivative. The methods reported show
lower limit of detection of about 0.5 mgflitre.
Carbamazepine had beer, determined by GLC without
derivatization on several stationary phases as
shown in Table 2.
Toseland et al., ( 3 8 ) described the determination

of carbamazepine among other anticonvulsants and
barbiturates in plasma and tissue using the nit-
rogen flame detector.
C ARB A M AZEPl NE 103
Table 2
Stationary phase Detector used Reference
3% OV - 17 Flame ionization. (35)
2% SP - 1000 Isothermal (36)
Cab-0-Sil deactivated
with benzyltriphenyl
phosphonium chloride
and OV - 225 Isothermal (37)
Marozzis. &., (39) had reported the gas chroma-

tographic retention indexes (I ) of 232 compounds
of toxicological interest whi% were determined
isothermally at 180' on SE 30, OV.1, OV-17, among
which was carbamazepine.
Clarke (5) reported the retention time of carbama-

zepine to be 0.81 relative to codeine and 3.76 re-
lative to diphenhydramine using 2.5% SE-30 on
80-100 chromosorb W AWHMDS and the conditions
specified in the monograph.
ACKNOWLEDGEMENTS
The authors would like to thank Mr. Dennis Charkowski,
Department o f Pharmacology, Unitersity of Iowa, Iowa City,
Iowa 5 2 2 4 2 , U.S.A., for determining the mass spectrum of
carbamamazepine, Mr. Said E. Ibrahim, for his help in the
Library search, Mr. Essam A. Lotfi and Mr. Khalid N.K. Lodhi,
for their technical assistance in the ultraviolet and nmr
determination, and Mr. Altaf Hussain Naqvi for typing the
manuscript.
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3. Merck Index, Ninth edition, Merck 6 Co., Inc., Rahaway,

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copeial Convention, Inc., Rockville, Md., 20852, page 69.
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Abstr., -
77, 101406 g (1972)
10. Schindler, U . S . Pat. 2, 948, 718 (1960, Geigy).
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Chem. -
12. Martindale, The Extra Pharmacopoeia, 27th edition, The

Pharmaceutical Press, London, edited by Ainley Wade,
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Pantarotto, A. Frigerio and G. Belvedere, Xanobiotica,
6,
- 593 (1976).
CARBAMAZEPINE 105
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Biochem. 10,1 1 2 ( 1 9 7 2 ) ; t h r o u g h Chem. A b s t r . , 77,
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J. Pharm. Pharmacal., 25, 340 ( 1 9 7 3 ) .
--
17. J . E . Bauer, N . G e r b e r , R.K. Lynn, R.G. Smith and R.M.
Thompson, E x p e r i e n t i a 32, 1032 (1976).
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Theobald, J. Pharmacokint, Biopharm, 4, 521 ( 1 9 7 6 ) .
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Martis, J . Pharm. S c i . , 64,302 (1975).
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423 (1976).
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Letters, 12, 951 (1979).
26. S. Kitazawa and T. Komuro,ClinChim A a , 73, 31 (1976).
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(1975).
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-
22, 1070 ( 1 9 7 6 ) .
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89, (1977).
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1 7 , 267 ( 1 9 7 6 ) ; t h r o u g h Chem. A b s t r . , 86, 65258 k (1977).
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36. P.A. T o s e l a n d , J. Grove and D . J . B e r r y , C l i n . Chim. Acta
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38, 321 (1972).
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and C.A. Meijers, C l i n . Chem. Acta, 73, 97 (1976).
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(Winston-Salem, N.C.) 21, 98 (1975).
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14793 c (1976).
CEFACLOR
Leslie J . Lorenz
1. Description 108
1.1 Name 108
1.2 Structure, Formula, and Molecular Weight 108
1.3 Appearance 108
2.2 Nuclear Magnetic Resonance Spectrum 109
2.5 Optical Rotation 112
2.6 Differential Thermal Analysis 112
2.7 Thermogravimetric Analysis 114
2.8 Dissociation Constants (pKa) 1 I4
2.9 Solubility Properties 114
2.10 Crystal Properties 1 I4
3. Chemical Synthesis 115
4. Stability 117
4.1 Bulk Stability 1 I7
4.2 Solution Stability 117
5. Drug Metabolism 117
6. Method of Analysis 118
6.1 Identification tests 118
6.2 Quantitative tests 118
6.3 Impurity Tests 119
7. Determination in Body Fluids 122
8. Acknowledgments 122
9. References 123
Copyright 0 1980 by Academic Ress. Inc.

Analytlcal Rofiles of Drug Substances, 9 107 All rights of reproduction in any form reserved.
ISBN: 0-12-260809-7
108 LESLIE J . LOREN2
1. Description
1.1. Name
C e f a c l o r i s 3-chloro-7-d- (2-phenylglycinamido) -
3-cephem-4-carboxylic a c i d , monohydrate.
1.2. S t r u c t u r e , formula and m o l e c u l a r weight
mC-
COOH
molecular weight 385.82
1.3. Appearance
C e f a c l o r is a w h i t e t o cream c o l o r e d c r y s t a l l i n e
powder. The m a t e r i a l i s o d o r l e s s going t o s l i g h t l y
sulphurous.
2.1. I n f r a r e d spectrum
The i n f r a r e d spectrum o f c e f a c l o r monohydrate
i n a potassium bromide p e l l e t i s p r e s e n t e d i n f i g u r e 1.
An i n t e r p r e t a t i o n o f t h e spectrum i s given i n t a b l e 1.
-Q
-8
(D
0
-0
O o + J
a,
d
z
0 d
-0 a,
a
k
0 a
a,
-0 k
3 rd
k
0
-I3
c9 a,
0
I I I I I -8
'p
110 LESLIE J. LOREN2
Table 1
Wave1ength (cm-' ) Ass ignment
3680-3000 OH from H20 and

( s e r i e s o f broad bands) amide NH s t r e t c h
2580 (broad) C0.j
1775 (strong) 8-lactam C=O s t r e t c h
1693 (strong) amide C=O s t r e t c h
1600 (strong)
RC - 0 carboxylate s t r e t c h i n g
1560 (weak) aromatic C=C
1500 (medium) aromatic C=C
1365 (strong) co2 (sym)

697 (sharp) c-c1
2.2. Nuclear magnetic resonance spectrum

Figure 2 shows t h e proton magnetic resonance
spectrum of cefaclor. The spectrum was recorded on a
60 MHz instrument. An i n t e r p r e t a t i o n of t h e spectrum i s
presented i n t a b l e 2 .
Table 2
Proton Magnetic Resonance Spectrum
COOD
I I I I I I
8.00 7.00 6.00 5.00 4.00 3.00 2.00 1.oo .oo
PPM
Figure 2 The NMR Spectrum of Cefaclor i n D20+DC1
112 LESLIE J . LORENZ
Peak Assignments
PP! Mu1t ip l i c it y Assignment
3.66 q u a r t e t AB J=19Hz CH2 (2)
5.17 doublet H (6)
5.35 singlet @-CH- CO-
I
ND3
5.78 d o u b l e t J=5HZ
7.60 singlet phenyl
2.3. Mass spectrum

C e f a c l o r i s amenable t o f i e l d d e s o r p t i o n t e c h n i q u e s
€or o b t a i n i n g a meaningful mass spectrum. Using t h i s
t e c h n i q u e , a small quasi-molecular i o n i s s e e n €or c e f a c l o r
a t m/e o f 369 which corresponds t o t h e molecular weight o f
c e f a c l o r a n h y d r a t e . The major ion i n t h e mass spectrum
appears a t m/e o f 331. T h i s i s caused by t h e l o s s o f HC1
from t h e molecule. The oiily o t h e r s i g n i f i c a n t i o n i n t h e
spectrum a p p e a r s a t m/e o f 287. T h i s i o n corresponds t o
a molecule which has l o s t HC1 and decarboxyolated.
2.4. U l t r a v i o l e t spectrum
F i g u r e 3 shows t h e u l t r a v i o l e t spectrum f o r c e f a -
c l o r . The chromophores i n c e f a c l o r a r e 3-cephem, phenyl
and amide. O f t h e s e , o n l y t h e 3-cephem group c o n t r i b u t e s
s i g n i f i c a n t l y about 2 2 0 nm. T h e w r * 3-cephem t r a n s i t i o n
has ~ ( 2 6 5nm) 2 8400. There i s a n + r * transition at
about 230 nm due t o t h e 3-cephem group. The phenyl group
has a weak a b s o r p t i o n a t 260 nm with E N 200.
2.5. Optical r o t a t i o n
The s p e c i f i c r o t a t i o n € o r c e f a c l o r determined on
a one perceng s o l u t i o n o f c e f a c l o r i n 0.1 M h y d r o c h l o r i c
a c i d a t Na2' i s +105.6'0n an anhydrous b a s i s .
D
2.6. D i f f e r e n t i a l thermal a n a l y s i s
The thermogram o f c e f a c l o r g e n e r a l l y shows a small
broad endotherm between 40OC and 12OoC corresponding t o t h e
l o s s o f water and o t h e r v o l a t i l e s from t h e sample. The
major endotherm i n t h e DTA curve f o r c e f a c l o r i s o b s e r v e d
around 22OoC where t h e m a t e r i a l decomposes.
h
cu
I14 LESLIE J . LORENZ
2.7. Thermogravimetric a n a l y s i s
Cefaclor gives a reasonable thermogravimetric
curve and shows a l o s s of w a t e r and o t h e r v o l a t i l e s from
about 4 0 O C t o 12OoC. A t about 18OoC c e f a c l o r samples
begin t o l o s e weight i n d i c a t i n g t h e b e g i n n i n g o f decomposi-
t i o n o f t h e sample.
2.8. D i s s o c i a t i o n c o n s t a n t pKa
The f o l l o w i n g d i s s o c i a t i o n c o n s t a n t s have been
determined f o r c e f a c l o r :
Solvent PKa
Carboxvl Amino
1.5*0.2 7.17
H2°
66% DMF 4.33 7.34
2.9. Solubility properties

The s o l u b i l i t y p r o p e r t i e s o f c e f a c l o r are
d e s c r i b e d i n t a b l e 3.
Table 3
So 1v e n t S o l u b i l i t y mg/ml
Water 10.0
pH 1 . 2 (USP XIX) >5 b u t <10
pH 4 . 5 (USP XIX) 4
pH 7.0 (USP XIX) >5 b u t <10
Met hano 1 co.5
Octanol <0.5
Is opropano 1 <0.5
Diethyl e t h e r <0.5
Ethyl a c e t a t e C0.5
Ch 1or0 form <0.5
Benzene <0.5
Cyclohexane <0.5
2.10. Crystal properties

Polymorphs o f c e f a c l o r are p o s s i b l e . Such
polymorphs are a f u n c t i o n o f t h e s o l v e n t from which
c e f a c l o r i s c r y s t a l l i z e d . The o n l y polymorph o f g e n e r a l
importance i s c e f a c l o r monohydrate. The X-ray powder
d i f f r a c t i o n d a t a f o r c e f a c l o r monohydrate are given i n
table 4.
CEFAC LO R
Table 4
X-ray Powder D i f f r a c t i o n Data

C e f a c l o r Monohydrate
x=l.5418
"d" Value (A) I n t ens i t i e s ( 1/ I, )
12.90 0.75
10.05 0.17
6.58 0.13
6.08 0.13
5.42 0.96
5.01 1.oo
4.75 0.04
4.06 0.54
3.86 0.04
3.69 0.29
3.53 0.58
3.41 0.04
3.29 0.17
3.23 0.13
3.13 0.04
2.99 0.21
2.81 0.25
2.67 0.08
2.52 0.08
2.48 0.04
2.35 0.17
2.26 0.17
2.15 0.04
2.07 0.08
1.99 0.21
1.94 0.08
3. Chemical s y n t h e s i s
F i g u r e 4 p r o v i d e s a flow s h e e t o f t h e chemical
s y n t h e s i s f o r c e f a c l o r . In t h i s procedure, p e n i c i l l i n V (1)
i s e s t e r i f i e d with p - n i t r o b e n z y l bromide (PNB-Br) and
oxidized with peracetic acid t o give p e n i c i l l i n sulfoxide
e s t e r ( 2 ) . Ring expansion and o z o n o l y s i s p r o v i d e t h e
3-hydroxy- 3- cephem s u l f o x i d e ( 4 ) . Sul f o x i d e r e d u c t i o n
and e n o l c h l o r i n a t i o n o c c u r s w i t h phosphorous t r i c h l o r i d e
i n N , N-dimethyl formamide. S i d e c h a i n c l e a v a g e i s
accomplished w i t h phosphorous p e n t a c h l o r i d e and p y r i d i n e
followed by a l c o h o l y s i s w i t h i s o b u t y l a l c o h o l . The
r e s u l t i n g n u c l e u s h y d r o c h l o r i d e (5) i s n e u t r a l i z e d w i t h
116 LESLIE J. LORENZ
? ? 0
1
,) PNB-Br ,
1) NCP, CaO, 8
\2)CH3CO3H ‘2) SnC14
Toluene, A
COzK COzPNB
1 2
0 0
II
? 1) PCl3, DMF
*
2) PC15, Py, CHpClz
OH 3)i-BuOH
COzPNB COzPNB
3 4
HzNu,
Hcl’HzNz/cl CH3CN:HzO
TEA
* D
0” 0” CI
COzPNB C02PNB
5 6
coz - coz -
7 a
Cefaclor Monhydrate
Figure 4 The Chemical Synthesis o f Cefaclor

CEFACLOR 117
t r i e t h y l a m i n e and a c y l a t e d w i t h a N-protected D-phenyl-

glycine t o give c e f a c l o r (7). This is then hydrated i n
w a t e r t o y i e l d t h e d e s i r e d monohydrate ( 8 ) .
4.1. Bulk s t a b i l i t y
C e f a c l o r i s a r e a s o n a b l y s t a b l e molecule i n t h e
dry s t a t e . When c e f a c l o r i s p r e s e n t i n t h e monohydrate
c r y s t a l l i n e form i n t h e dry powder, two y e a r s t a b i l i t y
can b e e a s i l y o b t a i n e d . The powder becomes l i g h t l y
yellow upon aging, however, l i t t l e d e c r e a s e i n t h e potency
of c e f a c l o r i s observed.
On d e g r a d a t i o n , c e f a c l o r appears t o l o s e HC1
q u i t e e a s i l y . F u r t h e r d e g r a d a t i o n s t e p s seem t o b e q u i t e
r a p i d and no o t h e r compounds have been i s o l a t e d . In
an a t t e m p t t o g e n e r a t e such compounds, some s t u d i e s have
been c a r r i e d o u t on t h e p - n i t r o b e n z y l e s t e r o f c e f a c l o r .
This s t u d y showed t h a t c e f a c l o r can undergo i n t r a m o l e c u l a r
n u c l e o p h i l i c a t t a c k by t h e s i d e c h a i n amine group t o
produce a d i k e t o p i p e r a z i n e with t h e f o l l o w i n g s t r u c t u r e (1) :
0
..
4.2. Solution s t a b i l i t y
C e f a c l o r i s s t a b l e i n s o l u t i o n s o f pH n o t h i g h e r
t h a n 4.5. S o l u t i o n s p r e p a r e d i n pH 2 . 5 and 4.5 b u f f e r s
c o n t a i n a t l e a s t 90 p e r c e n t o f t h e i r i n i t i a l a c t i v i t y
a f t e r 72 hours a t 4OC ( 2 ) . In n e u t r a l o r a l k a l i n e s o l u t i o n s ,
c e f a c l o r undergoes a r a p i d l o s s o f a c t i v i t y . When h e l d
i n Mueller-Hinton b r o t h a t 37OC o v e r n i g h t , 30 t o 60 p e r c e n t
o f t h e i n i t i a l a c t i v i t y o f t h e s o l u t i o n is l o s t ( 3 , 4 ) .
5. Drug metabolism
When c e f a c l o r was a d m i n i s t e r e d t o normal v o l u n t e e r s ,
peak serum c o n c e n t r a t i o n s o f c e f a c l o r o c c u r r e d about one hour
a f t e r a d m i n i s t r a t i o n . A 250 mg dose gave an approximate
peak l e v e l of 7 mcg/ml. A 500 mg dose gave an approximate
peak level o f 13 mcg/ml, and a 1 gram dose gave a n
approximate peak l e v e l o f 23 mcg/ml ( 5 , 6 ) . The mean serum
118 LESLIE J. LORENZ
h a l f l i f e o f c e f a c l o r i n normal a d u l t v o l u n t e e r s a s d e t e r -
mined by s e v e r a l i n v e s t i g a t o r s ranges from 29 t o 60
minutes (5-10).
C e f a c l o r i s r a p i d l y e x c r e t e d i n t h e u r i n e . In s e v e r a l
s t u d i e s , 38 t o 54 p e r c e n t o f t h e drug was d e t e c t e d i n t h e
u r i n e i n t h e f i r s t two hours a f t e r a d m i n i s t r a t i o n ( 7 ) .
After e i g h t hours 43 t o 79 p e r c e n t o f t h e drug was found i n
t h e urine (6,lO).
These s t u d i e s would t e n d t o i n d i c a t e t h a t about 85

p e r c e n t of t h e drug is e x c r e t e d i n t o t h e u r i n e a s t h e
unchanged drug. When c e f a c l o r i s metabolized i n t h e body
o r n a t u r a l d e g r a d a t i o n o c c u r s , t h e e f f e c t on t h e molecule
i s s e v e r e and no r e c o g n i z a b l e p r o d u c t s have been found.
6. Methods o f a n a l y s i s
6.1. Identification tests

6.1.1. Infrared
The i n f r a r e d spectrum o f a sample i n a
potassium bromide p e l l e t may be used f o r i d e n t i t y . In such
c a s e s , t h e i n f r a r e d spectrum compares f a v o r a b l y with t h e
c e f a c l o r r e f e r e n c e spectrum o v e r t h e range o f 2 . 5 t o 16
microns when recorded i n a s i m i l a r manner.
6.1.2. Nuclear magnetic resonance
The NMR spectrum of a c e f a c l o r sample
d i s s o l v e d i n deuterium oxide with deuterium c h l o r i d e present,
over t h e r a n g e of 0 t o 10 ppm has chemical s h i f t s and
i n t e g r a t i o n s which compare f a v o r a b l y t o a c e f a c l o r r e f e r -
ence m a t e r i a l handled i n l i k e manner.
6.2 Quantitative t e s t s
6.2.1. Microbiological
For b u l k and formulated p r o d u c t s , e i t h e r
an a g a r d i f f u s i o n a s s a y w i t h B a c i l l u s s u b t i l i s (ATCC 6633)
( 2 ) o r an automated t u r b i d i m e t r i c a s s a y (AUTOTURW ) with
Staphylococcus a u r e u s (ATCC 9144) (2) may b e used f o r a s s a y
of c e f a c l o r . Agar d i f f u s i o n a s s a y s w i t h e i t h e r B . s u b t i l i s
(2,8,10,11,12,13) o r S a r c i n a l u t e a (ATCC 9341) (2,13) are
used t o a s s a y t h e a n t i b i o t i c i n t i s s u e s and b i o l o g i c a l
f l u i d s . The g r e a t e s t s e n s i t i v i t y i s o b t a i n e d w i t h -
S. -l u t e a
a s c o n c e n t r a t i o n s o f c e f a c l o r a s low as 0.025 mcg p e r mg
may be determined ( 2 ) .
CEFAC LOR 119
6.2.2. High performance l i q u i d chromatography

HPLC i s t h e t e c h n i q u e o f c h o i c e f o r
determining t h e p u r i t y o f c e f a c l o r i n raw m a t e r i a l ,
f o r m u l a t e d p r o d u c t s and i n body f l u i d s . C e f a c l o r i s run i n
an a c i d i c medium on a r e v e r s e phase column. A Waters
Microbondapaks C18 o r o t h e r a l t e r n a t i v e c o l m w i t h similar
r e t e n t i o n c h a r a c t e r i s t i c s i s u s e d t o determine t h e p u r i t y .
The approximate s o l v e n t system c o n s i s t s o f 2 p a r t s g l a c i a l
a c e t i c a c i d , 1 2 p a r t s a c e t o n i t r i l e and 86 p a r t s w a t e r .
The s u b s t a n c e i s u s u a l l y monitored a t 254 nm; however,
s l i g h t l y improved s e n s i t i v i t y can be o b t a i n e d a t about
265 nm. The samples a r e d i s s o l v e d i n an a c i d i c media s u c h
a s a pH 4.6 b u f f e r o r i n d i l u t e f o r m i c o r a c e t i c a c i d .
6.2.3. Iodometric t i t r a t i o n
An i o d o m e t r i c t i t r a t i o n procedure similar
t o t h a t used f o r c e p h a l e x i n (14) has been adapted f o r
c e f a c l o r . In t h i s procedure a s w i t h o t h e r c e p h a l o s p o r i n s ,
t h e i n t a c t a n t i b i o t i c does n o t consume i o d i n e , w h i l e t h e
a l k a l i - h y d r o l y s i s product o f c e f a c l o r does. The a l k a l i n e
h y d r o l y s i s o f c e f a c l o r r e s u l t s i n t h e cleavage o f t h e
6-lactam r i n g . This product t h e n reacts w i t h i o d i n e t o
give a q u a n t i t a t i v e t i t r a t i o n procedure f o r cefaclor.
This t e s t can be run i n a manual mode a s well a s i n an
automated mode with b e h a v i o r s i m i l a r t o t h a t o f c e p h a l e x i n
(15). T h i s t e s t i s n o t n e c e s s a r i l y a s t a b i l i t y i n d i c a t i n g
t e s t f o r c e f a c l o r s i n c e any molecule w i t h an i n t a c t 6-
lactam moiety w i l l g i v e a t e s t i n t h i s procedure.
6.2.4. C o l o r i m e t r i c d e t e r m i n a t i o n with
hydroxylamine
The r e a c t i o n o f hydroxylamine w i t h c e f a c l o r
has been used t o determine t h e drug (16). The method i s
based on t h e f a c t t h a t hydroxylamine c l e a v e s t h e P-
lactam r i n g (pH 7 . 0 ) t o form a hydroxamic a c i d . T h i s
hydroxamic a c i d forms a c o l o r e d complex w i t h f e r r i c i o n .
Again any e n t i t y having t h e B-lactam r i n g i n t a c t w i l l g i v e
a t e s t w i t h t h i s p r o c e d u r e . Thus, t h i s t e s t may n o t b e
a s t a b i l i t y indicating t e s t f o r cefaclor.
6.3. I m p u r i t i e s
6.3.1. Colorimetric determination o f 3-chloro
nuc 1e u s
120 LESLIE J . LORENZ
The 3-chloro n u c l e u s o f c e f a c l o r
H
COOH
has a f r e e a-amino group a d j a c e n t t o a 6-lactam. T h i s
t y p e o f moiety i s s e n s i t i v e t o a t e s t w i t h n i n h y d r i n t o
form a c o l o r e d r e a c t i o n p r o d u c t . The r e a c t i o n i s c a r r i e d
out i n a c i t r a t e b u f f e r and a pH o f about 3.0. A f t e r
30 minutes f o r c o l o r development, t h e absorbance i s r e a d
a t 560 nm.
6.3.2. Phenyl g l y c i n e
Phenylglycine c o n t e n t o f c e f a c l o r can b e
determined by a high performance l i q u i d chromatographic
procedure. I n t h i s procedure, p h e n y l g l y c i n e i s determined
u s i n g a Waters Microbondapaks C18 column o r o t h e r s i m i l a r l y
s u i t a b l e r e v e r s e phase column. The e l u t i n g s o l v e n t c o n s i s t s
o f 0.01 M potassium dihydrogen phosphate t i t r a t e d t o a
pH o f 2.7 with phosphoric a c i d and 1 p e r c e n t by volume
o f a c e t o n i t r i l e . The column e l u e n t i s monitored a t 2 2 0 nm
f o r d e t e r m i n a t i o n o f p h e n y l g l y c i n e . Phenylglycine i s t h u s
determined v e r s u s t h e r e s p o n s e o f a p h e n y l g l y c i n e s t a n d a r d
handled i n l i k e manner.
A s might be expected, c e f a c l o r e l u t e s
very l a t e i n such a system and may be removed from t h e column
i n a quick g r a d i e n t o r s t e p g r a d i e n t procedure where t h e
a c e t o n i t r i l e composition o f t h e mobile s o l v e n t i s i n c r e a s e d
u n t i l t h e e l u t i o n o f t h e c e f a c l o r has been completed.
6.3.3. Cephalexin
Cephalexin might be a p o t e n t i a l i m p u r i t y
a r i s i n g from t h e rearrangement o f t h e 3-exomethylene
i n t e r m e d i a t e i n t h e s y n t h e s i s o f c e f a c l o r . Trace l e v e l s
o f c e p h a l e x i n may b e determined by high performance l i q u i d
chromatograph t e c h n i q u e s . To determine t h e c e p h a l e x i n
c o n t e n t o f c e f a c l o r , a r e v e r s e phase system i s employed
u s i n g a Waters Microbondapaks C18 o r o t h e r s u i t a b l y s i m i l a r
HPLC column. The e l u t i n g s o l v e n t c o n s i s t s o f 9 1 p a r t s
water, 4 p a r t s a c e t o n i t r i l e and 5 p a r t s g l a c i a l a c e t i c
a c i d . The column e l u e n t i s monitored a t 265 nm f o r
d e t e r m i n a t i o n o f c e p h a l e x i n . The r e s p o n s e o b t a i n e d a t t h e
CEFACLOR 121
e l u t i o n volume o f c e p h a l e x i n i s compared t o t h e r e s p o n s e
of a sample c o n t a i n i n g c e p h a l e x i n a t a known composition t o
d e t e r m i n e t h e c o n t e n t of c e p h a l e x i n i n t h e c e f a c l o r sample.
6.3.4. 3-Exomethylene a n a l o g o f c e f a c l o r
The 3 -e xomethylene a n a l o g o f c e f a c l o r
COOH
i s a p o t e n t i a l i m p u r i t y which may c a r r y through t h e s y n t h e -
sis i f t h e ozonolysis o f t h e corresponding intermediate
s h o u l d be incomplete. T h i s compound i s determined by h i g h
performance l i q u i d chromatography. To determine t h i s
compound, a Waters Microbondapaks C 1 8 o r o t h e r s u i t a b l y
s i m i l a r r e v e r s e phase column i s employed. The e l u t i n g
solvent f o r t h i s determination i s 1 p a r t g l a c i a l a c e t i c
a c i d , 7 . 5 p a r t s methanol, and 91.5 p a r t s water. The column
e l u e n t i s monitored a t 225 nm. The 3-exomethylene a n a l o g
i s determined by measuring t h e r e s p o n s e o f t h e 3-exo-
methylene peak i n t h e sample chromatogram and comparing
i t t o t h e r e s p o n s e o f a sample with a known c o n t e n t of t h e
3-exomethylene a n a l o g .
6.3.5. Other i m p u r i t i e s
G r a d i e n t HPLC p r o c e d u r e s can be u t i l i z e d
f o r t h e determination o f o t h e r unidentified impurities i n
c e f a c l o r . I n t h i s p r o c e d u r e , a Waters Microbondapap
C18 column, o r a Dupont Zorbaxs TMS column o r o t h e r
s u i t a b l y s i m i l a r HPLC r e v e r s e phase column i s employed.
A l i n e a r g r a d i e n t i s run from 2 p e r c e n t g l a c i a l a c e t i c
a c i d i n water t o 2 percent g l a c i a l a c e t i c a c i d i n aceto-
n i t r i l e . Most compounds o f i n t e r e s t e l u t e e a r l y i n t h e
system s o a 2 p e r c e n t change p e r minute i s used f o r 25
minutes f o l l o w e d by a f a s t e r s l o p e such as 5 p e r c e n t change
p e r minute f o r t h e remainder o f t h e chromatogram. Samples
a r e p r e p a r e d a t about 25 mg p e r m l i n f o r m i c a c i d and a b o u t
2 0 u l o f such a s o l u t i o n i s c h r o m a t o g r a p h i c a l l y examined.
The column e l u e n t i s monitored a t 254 nm. The peak a r e a s
of a l l u n i d e n t i f i e d peaks a r e i n t e g r a t e d and compared t o
t h e r e s p o n s e o f a c e f a c l o r s t a n d a r d a t about one p e r c e n t
o f t h e c o n c e n t r a t e d s o l u t i o n . The assumption i s made t h a t
122 LESLIE J . LOREN2
a l l o t h e r i m p u r i t i e s have s i m i l a r s p e c t r a l p r o p e r t i e s a s
c e f a c l o r and an approximation of t h e i r l e v e l s i n t h e sample
can t h e n be made.
7. Determination i n body f l u i d s
M i c r o b i o l o g i c a l and h i g h performance l i q u i d chromato-
g r a p h i c procedures have been employed f o r t h e d e t e r m i n a t i o n
of c e f a c l o r i n b i o l o g i c a l f l u i d s . Generally, p r o t e i n
has been p r e c i p i t a t e d from t h e samples by c l a s s i c a l means
and t h e f l u i d s are t h e n examined by one o f t h e s e t e c h n i q u e s .
When h a n d l i n g b i o l o g i c a l f l u i d s which c o n t a i n c e f a c l o r ,
care must b e t a k e n so t h a t t h e s o l u t i o n s a r e kept c o l d i n
an i c e b a t h o r f r o z e n from t h e time of sampling t o t h e t i m e
o f a s s a y . Also, i f p o s s i b l e , i t i s a d v i s a b l e t o a c i d i f y
t h e samples t o p r e v e n t l o s s of c e f a c l o r due t o i t s
i n s t a b i l i t y a t h i g h e r pH's.
8. Acknowledgements
The a u t h o r wishes t o e x p r e s s h i s s i n c e r e t h a n k s
t o t h e f o l l o w i n g people who have provided t h e
necessary information f o r s p e c i f i c portions of
t h i s chapter:
D. E. Dorman €or t h e NMR i n t e r p r e t a t i o n .
L. D . H a t f i e l d f o r t h e s y n t h e t i c p r e p a r a t i o n o f
ce f a c l o r.
J . L . Occolowitz f o r t h e mass s p e c t r a l i n t e r -
p r e t a t ion.
H. W. Smith f o r t h e X-ray c r y s t a l i n t e r p r e t a t i o n .
L. G. Tensmeyer f o r t h e i n f r a r e d assignments.
T. C. T r o x e l l f o r t h e u l t r a v i o l e t s p e c t r a l
interpretation.
P. G . Wassel f o r t h e c e p h a l e x i n t e s t and t h e
3-chloronucleus t e s t .
C. L . Winely f o r t h e m i c r o b i o l o g i c a l a s s a y
port ions.
CEFACLOR 123
9. References
1. J . M. I n d e l i c a t o , A. Dinner, L . R. P e t e r s , and
W. L. Wilham, J . Med. Chem., 20, 961 (1977).
2. M. A. Fogelsong, J . W. Lamb, a n d J . V. D i e t z ,
Antimicrob. Agents Chemother. , 13, 49 (1978).
3. C . C. Sanders, Antimicrob. Agents Chemother.,
-
1 2 , 490 (1977).
4. D. A . P r e s t o n , Postgrad. Med. J . , - 55, (Supplement
No. 4 ) , 2 2 (1979).
5. G . R. Hodges, C. Liu, D . R. Hinthorn, J . L. Harms,
and D. L . Dworzack, Antimicrob. Agents Chemother.,
14, 454 (1978).
-
6. B. R. Meyers, S . Z . Hirschman, G . Wormser, G .
Gartenberg, and E. S r u l e v i t c h , J . C l i n . Pharmacol.,
-
18, 274 (1978).
7. 0. M. Korzeniowski, W. M. Scheld, M. A. Sande,
Antimicrobl. Agents Chemother. , 1 2 , 157 (1977).
8. J . Santoro, B. N. Agarwal, R. M a z i n e l l i ,
N . Wenger, and M. E. Levison, Antimicrob. Agents
Chemother., 13, 951 (1978).
9. D. A. SpykerTB. L. Thomas, M. A. Sande, and
W. K. Bolton, Antimicrobl. Agents Chemother.,
14, 1 7 2 (1978).
-
10. R. Bloch, J . J . Szwed, R. S. Sloan, and F. C . L u f t ,
Antimicrob. Agents Chemother. , 1 2 , 730 (1977).
11. S. J . Berman, W. H. Broughton, G. Sugihara,
E. G . C . Wong, M. M. Sato, and A. W . Siemsen,
Antimicrob. Agents Chemother., 1 4 , 281 (1978).
12. C. Simon, and U. Gatzemeier, Postgrad. Med. J . ,
55 (Supplement No. 4) , 30 (1979).
-
13. G . H . McCracken J r . , C. M. Ginsburg, J . C . Clahsen,
and M. L. Thomas, J . Antimicrob. Chemother.,
-
4 , 515 (1978).
14. Federal R e g i s t e r , 21CFR 141, 506.
15. C . E. Stevenson, and L . D. Bechtol, Private
Communication.
16. .
Federal R e g i s t e r , 21CFR 442, 40(b) (1) ( i i )
CEFAMANDOLE NAFATE
Rafik H . Bishara and Eugene C . Rickard
Introduction i26
1. Description 126
1. I Nomenclature 126
1.2 Formula 127
1.4 Appearance, Color, Odor, and Taste 127
2.2 Simple Solubility Profile 127
2.3 Specific Rotation 128
2.4 pH Range 128
2.5 Dissociation Constant (pKJ 128
2.6 Thermal Analysis 128
2.7 Crystallinity 128
2.9 Circular Dichroism Spectrum 131
2. I 0 Infrared Spectrum 132
2. 11 Nuclear Magnetic Resonance
Spectrum 134
3. Synthesis 136
4. Stability-Degradation 138
5. Pharmacology, Bacteriology, Pharmacokinetics, and Metabolism 140
5.1 Pharmacological Action 140
5.2 Antibacterial Activity 140
5.3 Protein Binding 142
5.4 Pharmacokinetics 142
5.5 Metabolism 143
6. Method of Analysis 144
6 . 2 Microbiological Assay 144
6.3 Iodometric Assay 145
6.4 Hydroxylamine Assay 145
6.5 Electrochemical Assay 146
6.6 Chromatography 147
6.7 Analysis of Related Materials 148
7. Analysis of Biological Samples 148
7.1 Microbiological Assay 148
7.2 Liquid Scintillation Assay 148
7.3 Chromatographic Assay 149
8. Analysis of Pharmaceutical Formulations 149
10. References 151
Copyright 0 1980 by Academic Press. Inc.
Analytical Profiles of Drug Substances. 9 125 All rights of reproduction in any form resewed.
ISBN: 0-12-260809-7
126 RAFIK H. BISHARA AND EUGENE C. RICKARD
Introduction
Cefamandole nafate is a semisynthetic broad-spectrum
cephalosporin antibiotic for parenteral administration. The
dosage form of cefamandole nafate also contains 6 3 mg of
sodium carbonate per gram of cefamandole free acid activity
(0.275 moles of sodium carbonate per mole of cefamandole free
acid activity). After addition of diluent, cefamandole
nafate rapidly hydrolyzes to cefamandole, and both compounds
have microbiologic activity in vivo.
1. Description
1.1 Nomenclature
1.1.1 Chemical Name
7-D-Mandelamido-3-<<(l-methyl-lH-tetrazo1-5-
yl)thio>methyl>-3-cephem-4-carboxylic acid, formate (ester),
sodium salt
7 4 D - <(Formyloxy)phenylacetyl>amino>-3-< < ( 1-
methyl-1H-tetrazol-5-yl)thio~methyl~-3-cephem-4-carboxylic
acid, sodium salt
7-D-Mandelamido-3- [ [ ( 1-methy1-lH-tetrazol-5-
yl)thio] methyl] -8-0x0-5-thia-1-azabicyclo [4.2.0] -oct-2-ene-
2-carboxylate formate(ester)
1.1.2 Nonproprietary Name
Cefamandole nafate
1.1.3 Proprietary Name
Mandol @, Mandokef 8
CEFAMANDOLE NAFATE 127
1.2 Formula
1.2.1 Empirical
C19H17N606S2-Na Salt
1.2.2 Structural
H
1.3 Molecular Weiaht
512.49
1.4 Appearance, Color, Odor, and Taste
White t o off-white, o d o r l e s s powder with a s l i g h t l y
bitter taste.
2 . Physical P r o p e r t i e s
2.1 Meltina Ranae
Cefamandole n a f a t e s t a r t s t o d i s c o l o r with e v o l u t i o n
of gas a t about 190°C under USP c o n d i t i o n s f o r C l a s s I
substances (1).
2.2 Simple S o l u b i l i t y P r o f i l e
The sample i s s o n i c a t e d f o r one minute a t ambient
temperature.
Solvent mg/ml
Water ) 3 3 3 - <lo00
pH 1 . 2 (USP X I X ) c0.5
pH 4.5 (USP X I X ) 3 3 3 3 - <lo00
pH 7 . 0 (USP X I X ) >,333-<1000
Met hano 1 310-<33.3
Octanol <O. 5
I sopropanol < 0.5
D i e thy l e t h e r <0.5
Ethylacetate < 0.5
Chloroform <0.5
Benzene <0.5
Cyclohexane < 0.5
128 RAFIK H. BISHARA A N D EUGENE C. RICKARD
2.3 Specific Rotation

R o t a t i o n measured a t sodium D l i n e (approximately
589nm) o f a 10% s o l u t i o n of cefamandole n a f a t e i n pH 5.0
a c e t a t e b u f f e r (1.04M) is -38 f 5 c a l c u l a t e d on an
anhydrous b a s i s . I t i s t o be noted t h a t t h e s p e c i f i c
r o t a t i o n i s a f u n c t i o n of c o n c e n t r a t i o n . I n unbuffered
s o l u t i o n s o r s o l u t i o n s b u f f e r e d a t p H 6.0-7.5, t h e conversion
o f cefamandole n a f a t e t o cefamandole c a u s e s a d r i f t , w i t h
t i m e , of t h e measured r o t a t i o n . There i s minimal d r i f t and
pH dependence i n t h e range o f pH 4.5-5.5.
2.4 pH Range
The pH o f a 10% aqueous s o l u t i o n i s between 3.5 and
7.0.
2.5 D i s s o c i a t i o n Constant
The c a r b o x y l a t e PKa o f cefamandale n a f a t e i s about
2.6-2.9 as determined by aqueous t i t r a t i o n o r 3.0 a s
determined by spectrophotometry ( 2 ) .
2.6 Thermal Analysis
2.6.1 D i f f e r e n t i a l Thermal A n a l y s i s
A DTA thermogram of cefamandole n a f a t e , a t a
h e a t i n g r a t e o f 5OC p e r minute i n a n i t r o g e n atmosphere o f
40cc p e r minute, shows ( f i g u r e 1) an exotherm a t 207'C
i n d i c a t i n g decomposition.
2.6.2 Thermogravimetric A n a l y s i s
A TGA thermogram of cefamandole n a f a t e , run
simultaneously with t h e above DTA, shows ( f i g u r e 1) a weight
l o s s beginning a t 63OC r e s u l t i n g i n a 0 . 2 % loss a t 137OC. A t
163'C a n o t h e r loss b e g i n s r e s u l t i n g i n a continuous loss
through decomposition.
2.7 Crystallinity
2.7.1 C r y s t a l l i n e Habit
The anhydrate form ( y ) o f cefamandole n a f a t e
g e n e r a l l y c r y s t a l l i z e s a s small n e e d l e s .
2.7.2 X-Ray Powder D i f f r a c t i o n
The following d a t a d e s c r i b e t h e p a t t e r n f o r
t h e anhydrate form ( y ) o f cefamandole n a f a t e , where d i s
equal t o t h e i n t e r p l a n a r s p a c i n g measured i n terms of
Angstroms (x). The r a t i o 1/11 i s t h e i n t e n s i t y of t h e X-ray
maxima based upon a v a l u e o f 100 f o r t h e s t r o n g e s t l i n e .
Cu-Ni-A 1.5405
d I/I 1 d
- - -
17.80 30 3.72 100
11.76 30 3.51 5
9.39 10 3.32 2
7.49 70 3.06 10
7.18 20 2.91 15
6.20 15 2.83 15
5.52 40 2.75 10
5.00 40 2.56 5
4.74 20 2.36 10
4.54 80 2.17 10
4.20 50 2.11 10
3.98 10
TGA 63OC 137OC 163OC 100%
X D T A
90%
0
i
I
K
80%
c
a
70%
60 '10
I 1 I I I I I I I I I
Figure 1. Thermogravimetric Analysis and Differential Thermal

Analysis Thermograms of Cefamandole Nafate
PH UNBUFFERED CMPD CEFAMANDOLE NAFATE
CONDITIONS 0.5 NM RESOLUTION
1 CM PATHLENGTH LOT# REFERENCE STANDARD
CONC - 7 MCG./ML IN WATER
MTE 2-eW
Figure 2. Ultraviolet Spectrum of Cefamandole Nafate
2.8 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t spectrum o f cefamandole n a f a t e i n
w a t e r i s g i v e n i n f i g u r e 2 . The spectrum e x h i b i t s a maximum
a t 269nm w i t h a molar a b s o r p t i v i t y of 10,800 ( E 1 - c m / l % a b o u t
211). The chromophores i n cefamandole n a f a t e a r e 3-cephem,
t h i o t e t r a z o l e , p h e n y l , amide, and e s t e r . Of t h e s e , o n l y t h e
3-cephem and t h i o t e t r a z o l e make s i g n i f i c a n t c o n t r i b u t i o n s
above a b o u t 225nm. For t h e 3-cephem group i n H 2 0 , ~( 2 6 1 nm) =
9200 i s e x p e c t e d from a TI + IT* t r a n s i t i o n ; t h e r e i s p r o b a b l y
a TI -f TI* t r a n s i t i o n a t a b o u t 230 nm. a l s o . Thiotetrazole
h a s a 245 nm peak w i t h E a b o u t 1 2 , 8 0 0 . On s u b s t i t u t i o n i n t o
t h e a n t i b i o t i c , t h e peak a p p a r e n t l y r e d s h i f t s t o around
275 nm w i t h a d r a m a t i c i n t e n s i t y d e c r e a s e t o E a b o u t 4000.
T h i s can be s e e n by comparison o f -OH v e r s u s t h i o t e t r a z o l e
substitution.
2.9 C i r c u l a r Dichroism Spectrum
The c i r c u l a r d i c h r o i s m (CD) spectrum o f cefamandole
n a f a t e i n w a t e r i s g i v e n i n f i g u r e 3. The A E maxima and z e r o s
are:
A ,nm AE
264 7.11
246.7 0
228.5 -19.81
209 -12.70
195 0
lo 1
AE
- 10
- 20
195 220 245 270 295
Wavelength, nm
F i g u r e 3. C i r c u l a r Dichroism Spectrum o f Cefamandole N a f a t e

132 RAFIK H. BISHARA A N D EUGENE C . RICKARD
The CD of cefamandole above 220 nm appears t o be

t h a t t y p i c a l t o t h e 3-cephem group. The 228.5 nm negative
CD comes from t h e n -+ T* 3-cephem t r a n s i t i o n . The p o s i t i v e
CD peak a t 264 nm probably l o c a t e s t h e p o s i t i o n of t h e
T 7 ~ * 3-cephem t r a n s i t i o n .
-f The f a c t t h a t t h e absorption
peak i s a t 269 nm r a t h e r than 264 i s probably due t o t h e
t h i o t e t r a z o l e a b s o r p t i o n , from which CD i s e i t h e r weak o r
absent. S i m i l a r l y , t h e t h i o t e t r a z o l e group i s responsible
f o r t h e absorption above 295 nm, where t h e CD i s zero. The
lack of o p t i c a l a c t i v i t y (CD) i n t h e t h i o t e t r a z o l e 275 nm
t r a n s i t i o n may r e s u l t from i t s conformational m o b i l i t y , a s
implied by molecular models ( 3 ) .
2.10 I n f r a r e d Spectrum
The i n f r a r e d spectrum of cefamandole n a f a t e i n a
potassium bromide p e l l e t i s given i n f i g u r e 4. Major band
assignments a r e a s follows:
I n f r a r e d Absorption, cm- 1 Group Responsible
3 5 0 0 , very broad H-bonded H20

3260, sharp H-bonded t r a n s N-H in
secondary amide
3040-3020 CH i n mono-substituted
phenyl r i n g
2940 CH s t r e t c h i n N-CH3 and
2880 S-CH2
1755 carbonyl i n @-lactam

1712 carbonyl i n e s t e r
1668 carbonyl i n amide
\
(termed Amide I )
t
1620 .carboxylate s a l t
1610 C=C, conjugated t o a c i d
group
1600 C=C i n phenyl r i n g
1530 Amide I1
1490 aromatic C=C
1450 N-CH3
1385 o r 1357 carboxylate s a l t

1174 C=O i n e s t e r
I
1100 t e r t i a r y n i t r o g e n , -N-
745 CH p a t t e r n f o r mono-
692 s u b s t i t u t e d phenyl r i n g
PATH PELLET CMPD CEFAMANDOLE NAFATE
ISM# 16 MINUTE SCAN TIME
CONDITIONS 3 WAVENUWBER RESOLUTION LOT# REFERENCE STANDARD
CONC 0.87 MG. IN KBr
DATE 4-20-79
Figure 4. Infrared Spectrum of Cefamandole Nafate
134 RAHK H. BISHARA AND EUGENE C. RICKARD

2.11.1 Proton NMR
The 60 MHz proton NMR spectrum of cefaman-
dole nafate in deuterated dimethylsulforide is given in
figure 5. Assignment of the resonances are as follows:
I I
o=c 4’
C02Na
H a
Description of Resonance:- Assignment:
d/9.38 p.p.m. (J = 8.5) NH
-
s/8.37 p.p.m. CHO
-
m/7.45 p.p.m. (5H) aromatic protons
s/6.13 p.p.m. -CH-CO
I--
/O
dd/5.53 p.p.m. (J = 8.5, 5) H-7

d/4.87 p.p.m. (J = 5) H-6
AB-system/4.32 p.p.m. (2H) (JAB = 12) CH2(3’)
s/3.90 p.p.m. (3H) NCH3
b
m/E. 3.4 (2H)- (J 2 18) CH2 (2)
AB
m/2.53 solvent
a Unless otherwise specified, each resonance represents a

single proton. Coupling constants (J) are in Hz.
This resonance is superimposed by that due to moisture in

the solid sample.
2.11.2 3C-NMR
The fully decoupled 13C-NMR spectrum of
cefamandole nafate in deuterium oxide is given in Figure 6.
The spectrum was obtained on a Varian FT80-A instrument at
I
SWEEP TIME 250

SWEEP WIDTH 1000
SPIN RATE 45
CMPD
LOT#
l
3. 75
n .
CEFAMANDOLE NAFATE
REFERENCE STANDARD
. . l
2.50
, . . . I .
1.25
. . ,
t
CONC 108.2 MG./ML IN DMSB-DB
DATE 2/12/80
F i g u r e 5. P r o t o n NMR Spectrum o f Cefamandole N a f a t e

ambient temperature and using a 5mm sample tube. The data

consist of 15,000 acquisitions of 16,384 data points over a
5000 Hz spectral width. The reference line is at 1348 Hz
(67.46). Assignment of the resonances are as follows:
a
Description Assignment
27.4 CH2 ( 2 )
34.8 -
N-CH3
37.1 CH2 (3’)
58.3 C (6)
59.5 c (7)
75.6 cgH5 -cH
119.0 c (3)
128.4 C (c),C (e)-aromatic
129.9 C (b),C (f)-aromatic
130.6 C (d)-aromatic
131.7 c (4)
134.5 C (a)-aromatic
154.6 C (1)-tetrazole
162.8 CHO
-
164.6 C (8)
168.3 c (4’)
171.8 CO-NH
-
a
Chemical shift, 6 , in ppm from TMS (0.0 ppm). Each
resonance represents a single carbon unless otherwise
stated.
3. Synthesis
D ( - ) Mandelic acid (I) is formylated to produce
0-formylmandelic acid (II), which is then treated with excess
thionylchloride to form D (-1 0-formylmandeloyl chloride
(111). Formylation of 7-aminocephalosporanic acid (IV)
produces 7-formamidocephalosporanic acid (V), which is then
treated with l-methyl-1H-tetrazole-5-thi01, sodium salt (VI)
to afford 7-formamido-3-(l-methyl)-1H-tetrazol-5-ylthiomethyl)
-3-cephem-4-carboxylic acid (VII). Deformylation of (VII)
yields 7-amino-3-(l-methyl-lH-tetrazol-5-ylthiomethyl)-3-
.
cephem-4-carboxylic acid (VIII) The nucleus (VIII) is
silylated with monosilylacetamide (MSA) and is then acylated
i
I
zw
I
,80
I
150 isu 110
1
170 110
I
iw 'IU
I
an 70
I
1x1 50
1
40 10
I
20 to o
cwmlhl
Figure 6. I3C-NMR Spectrum of Cefamandole Nafate

138 RAFIK H . BISHARA AND EUGENE C. IUCKARD
with 0-formylmandeloyl chloride (111) to provide 7- (D-2-

fonnyloxy-2-phenylacetamido)-3-(l-methyl-lH-tetrazol-5-
ylthiomethyl)-3-cephem-4-carboxylic acid (IX). Alternatively,
(IV) is acylated with (111) to produce 7- (D-2-formyloxy-2-
phenylacetamido)-3-cephem-4-carboxylic acid (X). Addition of
1-methyl-5-thio-1, 2, 3, 4-tetrazole (XI) to (X) produces
(IX). The sodium salt (XII) is produced by treating (1x1
with sodium 2-ethylhexanoate in acetone. The flow diagram of
the synthesis presented above (4-7) is shown in figure 7.
4. Stability-Degradation
The ester function of cefamandole nafate is quite labile
to nuclcophilic attack by water or hydroxide ion in slightly
acidic to slightly alkaline aqueous solutions in vitro (81,
giving cefamandole (11) as the product (figure 8). Indelicato
et.al..found that the formyl moiety of cefamandole nafate
hydrolyzes with a half-life at 37OC which ranges from about
290 minutes at pH 5.5 to about 7 minutes at p H 8. In un-
buffered solutions, the addition of bases such as sodium
carbonate, ethanolamine and tromethainine produce rapid
hydrolysis. For sodium carbonate, the fraction of
cefamandole nafate which hydrolyzes is approximately equal to
the number of equivalents of carbonate added per mole of
cefamandole nafate and the hydrolysis reaches steady state in
about 30 minutes or less for 0.28, 0.60 and 0.90 mole equi-
valents of carbonate. Ester hydrolysis is essentially com-
plete within a few minutes when one mole of amine is added
per mole of cefamandole nafate. Retention of chirality in
the 7-D-mandelamido sidechain is observed for carbonate
hydrolysis, which indicates cleavage of the acyl-oxygen bond.
The hydrolysis of cefamandole nafate also occurs very rapidly
in vivo with half-lives of 6-7 minutes and 10-17 minutes for
dogs and humans respectively (9).
In artificially accelerated degradation studies (2, 10,

ll), ester hydrolysis is observed in unbuffered solutions
stored at 25, 37 or 6OoC for 24 hours, in 0.1N hydrochloric
acid at 25OC for 24 hours, in aqueous solutions (46OC) ex-
posed to a high intensity UV light source for 64 hours, and
in the reconstituted formulation. Hydrolysis of the solid
sample varies with water content (11); no hydrolysis is
observed for a dry (<0.1% water) sample after 2 months at
5OoC (11), 24 hours at 100°C or 22 days at 6OoC (10), but
hydrolysis is observed in a wet sample heated for 3 weeks at
5OoC (11). The 1-methyl-5-thio-1, 2, 3, 4-tetrazole (111)
product is formed under the same conditions which
produce ester hydrolysis. In addition to these
a,
a,
V
O="
reactions, other products can be formed. In neutral or un-

buffered solutions, the nucleus which remains after loss of
I11 may form the hydroxymethyl compound (IV) or, in slightly
acidic solutions, dehydrate to the a,@-unsaturated lactone
(V). In extremely acidic conditions, the mandelic acid
moiety (VI) is cleaved and the 8-lactam system is destroyed.
Strongly basic conditions produce mandelic acid and destruc-
tion of the @-lactam. This information is summarized in
figure 8 ; the protonation of compounds I-IV and VI will
depend upon pH.
5. Pharmacology, Bacteriology, Pharmacokinetics and

Metabolism
5.1 Pharmacological Action
Cefamandole nafate is a parenteral cephalosporin
antibiotic. In vitro, it is rapidly converted to cefamandole
by hydrolysis of the formyl ester after dissolution ( 8 ) .
Because of rapid in vivo conversion of cefamandole nafate to
cefamandole, the latter is the predominant circulating moiety
after administration of cefamandole nafate to laboratory
animals and humans (9). The calculated rate constant for
hydrolysis of cefamandole nafate is higher in dogs than in
humans, yielding t+ values of 6-7 minutes and 10-17 minutes
respectively. Disappearance of cefamandole nafate from the
plasma of dogs (due to hydrolysis and elimination) is
slightly faster than from humans with a half-life (t+) of
4-6 minutes and 6-9 minutes, respectively.
5.2 Antibacterial Activity
5.2.1 In Vitro
In many conventional laboratory evaluation
procedures, the in vitro antibacterial activities of
cefamandole and cefamandole nafate appear virtually identical
due to the hydrolysis of cefamandole nafate (2). Cefamandole
is active in vitro against a variety of gram-pos,itive and
gram-negative microorganisms. In addition, it is active
against a number of gram-negative aerobic organisms and both
gram-positive and gram-negative anaerobes that have not
traditionally been in the spectrum of the cephalosporins.
Extensive in vitro studies have documented this expanded
spectrum for cefamandole ( 1 3 - 2 6 ) .
5.2.2 In Vivo
The efficacy of cefamandole is identical to
that of cefamandole nafate in treating experimental animal
infections, indicating that rapid conversion of cefamandole
nafate to cefamandole occurs in vivo ( 1 2 ) .
HO -
H003- t k l ( 0 ) IA A
@NO03
A1
CH3 CH3
I eN003 I eN003 H
I1 " I S - ' H 3 f z 0 3=0
io
I1 N- N HN-03-H3 1
5.3 Protein Binding

The protein binding of cefamandole is 74 percent
when determined by an ultrafiltration method (27) and 67
percent with a range of 56-78 percent when measured by
equilibrium dialysis (28). However, this data is obtained
from an initial antibiotic concentration of 25mcg/ml and
20mcg/ml, respectively. Cefamandole appears to be rapidly
dissociated from the serum proteins as indicated by its
relatively short half-life and its rapid appearance in the
urine (18).
5.4 Pharmacokinetics
5.4.1 Serum Concentrations
After intramuscular administration of a 500mg
dose of cefamandole to normal volunteers, the mean peak serum
concentration is 13mcg/ml (18, 26, 29, 30). After lg doses,
the mean peak level is 25 mcg/ml (18, 26, 27, 30, 31). These
peaks occur at 30-120 minutes. The decline of antibiotic
concentration in the serum is biphasic with a rapid fall in
the first two hours. Thereafter, levels decrease more slowly.
Detectable concentrations are present for six to eight hours
(18). Multiple-dose studies in patients given cefamandole
intramuscularly show no evidence of accumulation (30, 32).
Following intravenous doses of 1, 2 and 39, serum concen-
trations are 139, 240 and 533mcg/ml at 10 minutes, respec-
tively (18, 27, 29, 33). These concentrations decline to
0.8, 2.2 and 2.9mcg/ml at four hours. Intravenous adminis-
tration of 49 doses every 6 hours produce no evidence of
accumulation in the serum.
5.4.2 Half-Life
The half-life after an intravenous dose is
32 minutes: after intramuscular administration, the half-life
is 60 minutes (27, 28, 33, 34).
5.4.3 Serum Clearance and Apparent Volume of
Distribution (AVD)
A mean serum clearance of 230 ? 99/ml/min./
1.73m2 is found (27, 28) following intravenous administration
of cefamandole. The AVD ranges from 10-67 percent of body
weight. Following intramuscular administration of
cefamandole the mean AVD is 17.11 liters, or 24 percent of
the body weight (29), which is similar to the findings in the
intravenous studies described above.
5.4.4 Urine Concentrations, Excretion, and Renal

Clearance
Cefamandole, in addition to being excreted by
glomerular filtration, is also secreted by the renal tubules
(32). Sixty-five to eighty-five percent of cefamandole is
excreted by the kidneys after intramuscular injection of the
drug, in the first 8 hours (18). The mean eight-hour urine
concentrations are 254mcg/ml after 500mg doses and 1357
mcg/ml after lg doses. Similar results are obtained by other
investigators (27, 30). After intravenous administration of
cefamandole, 75 to 100 percent is excreted in the urine in
the first six to eight hours, and concentrations exceed 1000
mcg/ml with 500mg doses (27, 29). Probenecid slows tubular
excretion and doubles the peak serum level and the duration
of measurable serum concentrations. The renal clearance of
cefamandole before and after administration of probenecid is
302 f 60 and 80 ? 14ml/min./l. 73m2 , respectively (32, 34) .
In the presence of renal impairment, urinary excretion of
cefamandole is slowed (32).
5.4.5. Body Fluid and Tissue Concentrations
Distribution of cefamandole in body fluids
and tissues following therapeutic doses of the antibiotic has
been determined in bones and joints (351, gallbladder (361,
interstitial fluid (37) and uterine tissue (38, 39). Tissue
analysis gives primarily qualitative rather than meaningful
quantitative data as to the presence or absence of an anti-
biotic in a particular body fluid or tissue. Therapeutic
efficacy cannot be predicted by the level attained in a
specific body fluid or tissue.
5.5 Metabolism
A study of the metabolic fate of 14C-cefamandole in
rats and dogs shows that after rapid in vivo hydrolysis of
cefamandole nafate to cefamandole, the antibiotic is very
resistant to metabolic degradation in both species (40). In
dogs, cefamandole escapes metabolism and is eliminated as
unaltered antibiotic almost exclusively by renal excretion.
In rats, cefamandole is somewhat labile to metabolism.
However , a major portion of the administered antibiotic is
eliminated unchanged principally by renal excretion.
Essentially all of the administered radiocarbonnoteliminated
by renal excretion is eliminated via biliary excretion. The
fraction of radiocarbon dose remaining in the body of the
rats after 24 hours amounts to less than 2%. The half-life
of cefamandole in the blood of both species ranges from 30
to 42 minutes. Tissue level studies reveal no abnormal
deposition of the antibiotic or metabolite in any tissue,
although all tissues examined contained concentrations of the

antibiotic. The only tissues possessing significantly higher
levels than that found in the blood are the kidney in both
species and liver in dogs.
6. Method of Analysis
Theory ( % )
_____
Element Sodium Salt Free Acid
~
C 44.53 46.53
H 3.34 3.70
N 16.40 17.13
0 18.73 19.57
S 12.51 13.07
Na 4.49
6.2 Microbiological Assay
Cefamandole nafate is rapidly hydrolyzed to
cefamandole in vivo ( 9 ) or in aqueous solutions of pH 5.5-8
(8). The in vitro activity of cefamandole nafate relative to
that of cefamandole is different for some organisms when the
assay conditions do not produce hydrolysis of cefamandole
nafate (12, 4 1 ) . Thus, a chemical hydrolysis is required
prior to the microbiological assay in order to obtain results
which are valid measurements of the bioactivity, and to avoid
experimental difficulties due to partial hydrolysis during
the assay. The microbiological assay is described for
turbidimetric and agar diffusion methods. These assays are
not specific for cefamandole nafate in the presence of
impurities and/or degradation products. However, most con-
taminants will tend to have lower specific activity than
cefamandole nafate so that a certain degree of selectivity is
achieved.
6.2.1 Turbidimetric Method
The turbidimetric assay is performed after
hydrolysis to cefamandole, e.g., 1 5 minutes at room temper-
ature with 0.87 moles of sodium carbonate per mole of
cefamandole nafate followed by dilution with 0.1M phosphate
buffer, pH6. The sample is further diluted to the reference
concentration with O.lM, pH6 phosphate buffer and added to
medium # 3 ( 4 2 ) inoculated with Staphylococcus aureus
(ATCC 9 1 4 4 ) . Dose response concentrations are 0.02 to l.0mcg
cefamandole per ml of inoculated medium. The precision of
the assay is about 2.5% as measured by the relative standard
d e v i a t i o n ( R S D ) of t h e assay ( 4 3 ) .
6.2.2 Agar Diffusion Method
For p e n i c y c l i n d e r agar d i f f u s i o n a s s a y s of r a w
m a t e r i a l s o r f i n a l dosage forms of cefamandole n a f a t e , e i t h e r
S. -
- aureus (ATCC 6538P) o r B a c i l l u s s u b t i l i s (ATCC 6633) may
be used. With an a g a r p l a t e system c o n s i s t i n g of 1 0 m l of
agar medium N o . 2 ( 4 2 ) as base l a y e r and 5 m l of a g a r medium
No. 1 ( 4 2 ) a s seed l a y e r , dose response c o n c e n t r a t i o n s of
0.5 t o 2 . 0 mcg of cefamandole n a f a t e p e r m l are a p p r o p r i a t e
f o r both organisms. Sample p r e p a r a t i o n , h y d r o l y s i s followed
by d i l u t i o n i n 0 . 1 M phosphate b u f f e r (pH 6.0), i s c a r r i e d
o u t i n t h e same manner a s f o r t h e t u r b i d i m e t r i c assay. For
assay of b i o l o g i c a l f l u i d s , t h e B. s u b t i l i s assay i s used.
When s e n s i t i v i t y g r e a t e r than 0.s mcg p e r m l i s r e q u i r e d , a
5 m l s i n g l e l a y e r p l a t e , medium No. 1, with dose response
concentrations of 0 . 1 t o 1 . 0 rncg p e r m l may be employed.
Samples a r e n o t hydrolyzed s i n c e cefamandole n a f a t e i s con-
v e r t e d t o cefamandole i n vivo ( 9 , 1 2 ) , b u t standard m a t e r i a l
m u s t be hydrolyzed t o cefamandole f o r p r e p a r a t i o n of s t a n d a r d
curves.
6.3 I o d a n e t r i c Assay
Cefamandole n a f a t e can be determined b y a n i o d o m e t r i c
t i t r a t i o n procedure s i m i l a r t o t h a t a p p l i e d t o o t h e r
cephalosporins ( 4 5 ) . The 6-lactam r i n g i s hydrolyzed f o r
about 1 0 minutes with a l k a l i (0.2N N a O H ) , a c i d i f i e d
(0.5N HC1) and allowed t o r e a c t with i o d i n e f o r about 5
minutes. A l l r e a c t i o n s are thermostated t o 37'C. The
d i f f e r e n c e i n i o d i n e uptake i s measured between a blank (no
sodium hydroxide added) and t h e sample. The RSD of t h i s
assay i n an automated mode i s about 1-2% ( 2 ) . The measure-
ment of i o d i n e uptake i s not s p e c i f i c f o r cefamandole n a f a t e ,
e s p e c i a l l y i n t h e presence of i m p u r i t i e s and/or degradation
products which contain t h e i n t a c t 6-lactam system.
6.4 Hydroxylamine Assay
The hydroxylamine assay i s an a l t e r n a t e chemical
assay procedure f o r cefamandole n a f a t e ( 4 4 ) . The method i s
based upon cleavage of t h e 6-lactam by hydroxylamine t o form
a hydroxamic a c i d which i s then r e a c t e d with a c i d i f i e d f e r r i c
ion t o give a colored complex t h a t can be monitored a t 480nm.
A blank c o r r e c t i o n €or i n t e r f e r i n g non-6-lactam chemical
s p e c i e s which r e a c t with hydroxylamine i s incorporated by
adding t h e hydroxylamine t o an a c i d i c s o l u t i o n of t h e sample
( t h e a c i d d e s t r o y s a l l 6-lactam e n t i t i e s ) . However, i t i s
not p o s s i b l e t o c o r r e c t f o r i n t e r f e r e n c e s due t o i m p u r i t i e s
and/or degradation products which contain an i n t a c t 6-lactam.
146 RAFlK H. BISHARA AND EUGENE C. RICKARD
6.5 Electrochemical Assay

The electrochemical assays r e l y on t h e r e d u c t i v e
cleavage of t h e t h i o e t h e r linkage a t t h e 3' p o s i t i o n of t h e
cephalosporin ( 2 ) . This reduction occurs a t t h e dropping
mercury e l e c t r o d e (DME) with a half-wave p o t e n t i a l of about
-0.75V VS. SCE ( s a t u r a t e d calomel e l e c t r o d e ) f o r a 0 . 4 m
sample concentration (0.2mg/ml) i n a pH 2 . 4 McIlvaine b u f f e r ,
and v a r i e s with concentration and pH ( 2 ) . The electrochemi-
c a l methods include c o n t r o l l e d p o t e n t i a l coulometry, DC o r
sampled DC polarography and d i f f e r e n t i a l p u l s e polarography.
6.5.1 Controlled P o t e n t i a l Coulometry
The c o n t r o l l e d p o t e n t i a l coulometric d e t e r -
mination has been p r e v i o u s l y described ( 2 ) . Coulometry i s an
absolute method i n which t h e t o t a l charge consumed i s
measured during an exhaustive e l e c t r o l y s i s . That i s , no
comparison t o a standard i s r e q u i r e d ( 4 6 ) . Thus, it i s an
important a n a l y t i c a l t o o l f o r e v a l u a t i o n of p u r i t y o f s t a n d a r d
m a t e r i a l s , b u t i t i s not normally used f o r r o u t i n e a s s a y s .
For cefamandole n a f a t e , a p o t e n t i a l of -0.875V vs. SCE i s
used and t h e r e d u c t i o n i s performed a t a s t i r r e d mercury pool
e l e c t r o d e . The p r e c i s i o n of t h i s method applied t o cefaman-
dole n a f a t e i s approximately 0.95% as measured by t h e RSD
( 4 6 ) . This assay measures a l l compounds which have function-
a l groups t h a t a r e reduced a s e a s i l y o r more e a s i l y than
cefamandole n a f a t e . However, t h e most common i m p u r i t i e s and
degradation products do not i n t e r f e r e ( 2 ) .
6.5.2 DC and Sampled DC Polarography
DC and sampled DC ( T a s t ) polarography can be
applied t o cefamandole n a f a t e ( 2 ) . These methods e x h i b i t a
p r e c i s i o n of about 1.4-1.5% f o r t h e measurement of a sample
versus a standard ( 2 , 4 7 ) . Their s e l e c t i v i t y depends upon
t h e d i f f e r e n c e between half-wave p o t e n t i a l s of t h e v a r i o u s
s p e c i e s . Since t h e s e methods can d i s c r i m i n a t e a g a i n s t more
e a s i l y r e d u c i b l e a s w e l l as l e s s e a s i l y r e d u c i b l e s p e c i e s ,
both a r e more s e l e c t i v e than c o n t r o l l e d p o t e n t i a l coulometry
( 2 , 48). These methods a r e more s e l e c t i v e than micro-
b i o l o g i c a l , iodometric o r hydroxylamine a s s a y s , b u t n o t as
s e l e c t i v e a s t h e high performance l i q u i d chromotographic
assay. An automated, microprocessor c o n t r o l l e d polarographic
system has r e c e n t l y been described f o r t h i s assay (49) and
t h e l i n e a r i t y range i n v e s t i g a t e d ( 4 7 ) .
6.5.3 D i f f e r e n t i a l P u l s e 'Polarography
The d i f f e r e n t i a l p u l s e polarographic assay i s
t h e o f f i c i a l chemical assay i n t h e Code of Federal Regula-
t i o n s ( 4 5 ) . I t r e l i e s upon t h e same electrochemical
CEFAMANDOLE NAFATE I47
reduction process and achieves s i m i l a r ( o r s l i g h t l y g r e a t e r )

s e l e c t i v i t y than DC polaroqraphy. The p r e c i s i o n f o r t h i s
assay i s about 1 . 2 % f o r a sample measured versus a s t a n d a r d
using t h e automated, microprocessor c o n t r o l l e d system. The
l i n e a r i t y and o t h e r c h a r a c t e r i s t i c s of t h i s assay have been
r e c e n t l y described ( 4 7 ) .
6.6 Chromatography
6.6.1 Paper Chromatography
Cefamandole n a f a t e may be chromatographed on
u n t r e a t e d Whatman N o . 4 paper using a methyethyl ketone/water
(92:8 v/v) developing s o l v e n t ( 4 0 , 5 0 ) . When about 30 ml of
developing s o l v e n t i s used and development t i m e i s 6-7 h o u r s ,
t h e s o l v e n t f r o n t w i l l run o f f t h e paper and t h e cefamandole
n a f a t e zone w i l l move about 0.75 of t h e d i s t a n c e t o t h e end
of t h e paper. g . s u b t i l u s innoculated i n an agar medium
(6g peptone, 39 y e a s t e s t r a c t , 1.59 beef e x t r a c t , 209 a g a r
d i s s o l v e d i n 1 l i t e r of water and pH a d j u s t e d t o 7 . 2 ) can be
used f o r d e t e c t i o n when approximately 1 mcg of sample i s
applied.
6.6.2 Thin Layer Chromatography
The R value i s about 0.52 f o r cefamandole
f
n a f a t e when chromatographed on a s i l i c a g e l 60 F254 t h i n
l a y e r p l a t e developed by ethylacetate/acetone/glacial a c e t i c
acid/water (5:2:1:1 v/v/v/v) i n a s a t u r a t e d chamber ( 2 ) . The
sample may be dissolved i n water o r i n t h e developing s o l -
vent. The sample i s v i s u a l i z e d under s h o r t wavelength UV
l i g h t (254nm) o r under white l i g h t a f t e r exposure t o i o d i n e
vapors. The Code of Federal Regulations 45 describes a
continuous flow t h i n l a y e r chromatographic (TLC) system f o r
i d e n t i f i c a t i o n which employs a s i l i c a g e l G t h i n l a y e r p l a t e
developed by n-butanol/glacial a c e t i c acid/water ( 4 : l : l v / v / v ) .
This t e s t u s e s a s t a r c h i o d i d e / g l a c i a l a c e t i c acid/O.lN
i o d i n e spray reagent f o r v i s u a l i z a t i o n . Other TLC systems
and v i s u a l i z a t i o n procedures f o r cephalosporins are d e s c r i b e d
by M a r r e l l i ( 4 4 ) .
6.6.3 High Performance Liquid Chromatography
A reverse-phase high performance l i q u i d
chromatographic (HPLC) assay may be performed using a C8
column with an i s o c r a t i c e l u t i n g s o l v e n t ( 2 % g l a c i a l a c e t i c
a c i d ) , 20% a c e t o n i t r i l e and 78% water, by volume) a t a f l o w
r a t e of 2 m l / m i n u t e ( 5 1 ) . Samples are prepared i n g l a c i a l
a c e t i c a c i d , d e t e c t i o n i s by W absorbance a t 254 nm. and t h e
r e t e n t i o n t i m e f o r cefamandole n a f a t e i s about 9.5 minutes.
This method e x h i b i t s a p r e c i s i o n of about 2-3% (RSD).
Cefamandole n a f a t e may a l s o be determined by ion-pair
chromatography using the C8 column with an eluting solvent of

25% acetonitrile, 2% acetic acid and 73% water containing 2%
glacial acetic acid and 500 mg/ml of tetrabutylammonium
dihydrogenphosphate (52). With a flow rate of 2.5 ml/minute,
the retention time is about 15 minutes. Sample preparation
and detection are identical to that described above.
6.7 Analysis of Related Materials
Water may be determined by the KarlFischertitration
procedure. Other solvents, such as methanol, may be
determined by a gas chromatographic (GC) assay. In the GC
assay, the sample is dissolved into water and an internal
standard solution such as ethanol is added. A 6 foot glass
column packed with Porapak Q (60/80 mesh) and operated at
15OoC with a helium carrier gas flow rate of 80 ml/minute
gives retention times of approximately 1.1 and 2.4 minutes
for methanol and ethanol, respectively. A Chromosorb 104
column may be used also. The flame ionization detector is
used.
The hydrolysis product cefamandole and a potential
impurity, 7-(D-2-formyloxy-2-phenylacetamido)-3-cephem-4-
cephalosporanic acid (Compound X, figure 7 ) may be observed
by TLC (R values of 0.46 and 0.58 respectively) (2) or
determine5 using either of the HPLC methods described in
section 6.5.3 (retention times of about 4.5 and 8.75 minutes
for the first method and 6 . 2 5 and 13.5 minutes for the second
method, respectively). Another possible impurity and de-
gradation product 1-methyl-5-mercepto-1, 2, 3, 4-tetrazoleI
can be measured polarographically (2). It is oxidized at
the DME in a pH 5.8 acetate buffer. The concentration of
this species is proportional to the sum of the currents for
the absorption prewave and the main wave (half-wave potentials
about -0.20V and -0.05V vs. SCE, respectively).
7. Analysis of Biological Samples
7.1 Microbiological Assay
Serum and urine are assayed microbiologically using
-
B. subtilis as the microorganism (18, 30, 40, 53-59). The
microbiological assay is also used for determining the
cefamandole levels in aqueous humor (591, occular tissues
(60-611, interstitial fluids, bile (55), pulmonary and sub-
cutaneous tissue (58). A klebsiella strain is used as the
indicator organism for assaying urine samples by an
Autoturb (18).
7.2 Liquid Scintillation Assay
14C-cefamandole is assayed by liquid scintillation
procedure in metabolic and tissue distribution studies (40,
60, 61).
7.3 Chromatographic Assay
7.3.1 Paper Chromatography
Cefamandole is assayed in biological samples
on Whatman No. 4 paper developed with methylethylketone/water
(92:8) for 7 hours to give a mobility of about 15cm from the
point of application (40).
7.3.2 Thin Layer Chromatography
Separation of cefamandole, cefamandole nafate
and two metabolites is accomplished on silica Gel F plates
developed with ethylacetate/acetone/glacial acetic acid/water
(5:2:2:1) solvent. The R values are 0 . 5 5 , 0.63 and 0.81,
E
respectively (40).
7.3.3 High Performance Liquid Chromatography
In studying the hydrolysis of cefamandole
nafate to cefamandole, the biological samples are
chromatographed on a column packed with Vydac reverse phase
(30/44um) and eluted with 20-25% acetonitrile in 0.1%
aqueous acetic acid. At a flow rate of 2.0 ml/minute and
monitoring the absorption at 254 nm, the retention time for
cefamandole nafate and cefamandole are 4.4 and 7.2 minutes,
respectively (9). Other HPLC work is performed on VBondapak
C18 column eluted with 30% methanol-0.01M sodium acetate,
pH 5.2 at a flow rate of 2 ml/minute and monitoring at
270nm. The accuracy of the latter system is ? 3% and the
reproducibility measurements yield a coefficient of variation
of 4.6% (62).
8. Analysis of Pharmaceutical Formulations
The pharmaceutical formulation contains a buffering agent
such that the reconstituted material has a pH between 6.0 and
8.0. Thus, when the material is reconstituted in water, it
will be a mixture of cefamandole nafate and cefamandole (the
hydrolysis product). The hydrolysis can be minimized by
dissolving the sample in acetic acid or other acidic solvents.
Somewhat different chromatographic, spectroscopic and physical
characteristics will be observed on partially hydrolyzed
samples compared to the corresponding data for the raw
material.
The microbiological, iodometric, hydroxylamine, and
polarographic (63) assays described in section 6 for the raw
material are applicable to the formulation. The chromato-
graphic methods will generally yield two zones (or peaks)
corresponding to cefamandole nafate and cefamandole. Speci-
fically, cefamandole gives a zone which is about 0.50 of the
d i s t a n c e t o t h e end o f t h e paper i n t h e paper chromatographic

system, a R v a l u e o f 0.45 i n t h e TLC system ( 2 ) and i s
f
observed i n t h e HPLC systems as d e s c r i b e d i n s e c t i o n 6.6.
9. Acknowledgements
The a u t h o r s a r e t h a n k f u l t o M r . H. W . Smith, D r . T .
T r o x e l l , D r . L . G . Tensmeyer, and D r . D. Dorman f o r t h e i r
h e l p w i t h t h e d a t a and i n t e r p r e t a t i o n f o r c r y s t a l l i n i t y ; UV
and CD; I R ; and NMR s p e c t r o s c o p y , r e s p e c t i v e l y .
10. References
1. The United States Pharmacopeia, XX, The National
Formulary XV, p. 961.
2. E. C. Rickard and G. G. Cooke, J. Pharm. Sci., 66,
379 (1977).
3. R. Nagarajan and D. 0. Spry, J. Amer. Chem. SOC., 93,
2310 (1971).
4. L. G. Tensmeyer, U.S. Patent No. 3,947,414 (1976).
5. L. G. Tensmeyer, U.S. Patent No. 3,947,415 (1976).
6. J. M. Greene and J. M. Indelicato, U.S. Patent
No. 3,928,592 (1975).
7. L. D. Hatfield, Proc. R. SOC. London, Ser. €3, in
press (1980).
8. J. M. Indelicato, W. L. Wilham & B. J. Cerimele,
J. Pharm. Sci., 65, 1175 (1976).
9. J. S. Wold, R. R. Joost, H.R. Black and R.S.Griffith,
J. Infect. Dis., 137 (Suppl.), S17 (1978).
10. A. Dinner, R. J. Templeton and A. D. KOSSOY, Eli Lilly
and Co., Indianapolis, IN 46206,personal communication.
11. M. J. Pikal, A. L. Lukes and J. E. Lang, J. Pharm.
Sci., 66, 1312 (1977).
12. J. R. Turner, D. A. Preston and J. S. Wold,
Antimicrob.Agents, Chemother. , 12, 67 (1977).
13. G. P. Bodey and S. Weaver, Antimicrob. Agents
Chemother., 9, 452 (1976).
14. G. Darland and J. Birnbaum, Antimicrob. Agents
Chemother., 11,725 (1977).
15. E. C. Ernst, S. Berger, M. Barza, N. V. Jacobus and
F. P. Tally, Antimicrob. Agents Chemother., 9, 852
(1976).
16. S. Eykyn, C. Jenkins, A. King and I. Phillips,
Antimicrob. Agents Chemother., 3, 657 (1973).
17. S. Eykyn, C. Jenkins, A. King and I. Phillips,
18. R. S. Griffith, H. R. Black, G. L. Brier and J. D.
Wolny, Antimicrob. Agents Chemother., 10,814 (1976).
19. B. R. Meyers, B. Leng and S. Z. Hirschman, Antimicrob.
Agents Chemother., S,737 (1975).
20. B. R. Meyers and S. 2 . Hirschman, J. Infect. Dis.,
137 (Suppl.), S25 (1978).
21. T C . Neu, Antimicrob. Agents Chemother., 6, 177
(1974).
22. V. L. Sutter and S. M. Finegold, Antimicrob. Agents
Chemother. , 10, 736 (1976).
23. J. A. Washington, Mayo Clin. Proc., 51, 237 (1976).
24. A. E. Weinrich and V. E. Del Bene, Antimicrob. Agents
Chemother., 10,106 (1976).
25. W. E. Wick and D. A. Preston, Antimicrob. Agents

Chemother. , I,
221 (1972).
26. Data on file, Lilly Research Laboratories, Indiana-
polis, IN 46206.
27. I. W. Fong, E. D. Ralph, E. R. Engelking and W. M. M.
Kirby, Antimicrob. Agents Chemother., 2, 65 ( 1 9 7 6 ) .
28. M. Barza, S. Melethil, S. Berger and E. C. Ernst,
29. B. R. Meyers, B. Ribner, S. Yancovitz and S. Z.
Hirschman, Antimicrob. Agents Chemother., 9, 1 4 0
(1976).
30. N. K. Shemonsky, J. Carrizosa and M. E. Levison,
Antimicrob. Agents Chemother. , 8, 679 (1975).
31. H. D. Short, L. 0 Gentry and S. Sessoms, J. Anti-
microb. Chemother., 2, 345 ( 1 9 7 6 ) .
32. H. E. Mellin, P. G. Welling and P. 0. Madsen,
Antimicrob. Agents Chemother., 11, 262 ( 1 9 7 7 ) .
33. W. E. Grose, G. P. Bodey and D. Stewart, Clin.
Pharmacol. Ther., 20, 579 ( 1 9 7 6 ) .
34. R. S. Griffith, H. R. Black, G. L. Brier and J. D.
Wolny, Antimicrob. Agents Chemother., 11, 809 (1977).
35. D. J. Schurman, data on file, Lilly Research
Laboratories, Indianapolis, IN 46206.
36. E. L. Quinn, T. Madhaven, R. Wixson, E. Guise,
N. Levin, M. Block, K. Burch, E. Fisher, A. Suarez
and R. del Busto, proceedings of the 10th Inter-
national Congress of Chemotherapy, Zurich, September
18-23, 1977, p. 803. Washington, D.C.: American
Society for Microbiology, 1978.
37. J. S. Tan and S. J. Salstrom, Antimicrob. Agents
Chemother. , 11,698 ( 1 9 7 7 ) .
38. R. Carter, data on file, Lilly Research Laboratories,
Indianapolis IN 46206.
39. S. Gall, data on file, Lilly Research Laboratories,
Indianapolis, IN 46206.
40. H. R. Sullivan, S. L. Due, D. L. Kau, J. F. Quay and
W. M. Miller, Antimicrob. Agents Chemother., 12,
73 ( 1 9 7 7 ) .
41. C. L. Winely, J. C. Spears and J. K. Scott, Anti-
microb. Agents Chemother., 16,424 ( 1 9 7 9 ) .
42. U.S. Code of Federal Regulations (1974, admended
March 15, 19771, Food and Drugs, Title 21, part
436.102 (a), Washington, D.C. , U . S . Government
printing office.
43. C. L. Winely, Lilly Research Laboratories, Indiana-
polis, IN 46206, personal communication.
44. L. P . Marrelli, "Analytical Procedures for Cephalos-
porins," in Cephalosporins and Penicillins, E. H.
Flynn, ed., Academic Press, New York NY, 1972,
chapter 14.
45. U . S . Code of Federal Regulations (1974, admended

April 6, 1979), Food and Drugs, Title 21, part 442.8,
Washington, D.C., U.S. Government printing office.
.
46. E. C Rickard , "Coulometry," in Modern Methods of
Pharmaceutical Analysis, R. E. Schirmer,ed., CRC
Press, West Palm Beach, FL, in press (1980),
Chapter V.
47. T. Getek (Food and Drug Administration, Washington,
D.C. 20204) and E. C. Rickard (Lilly Research
Laboratories, Indianapolis, IN 46206), manuscript in
preparation.
.
48. E. C Rickard , "Polarography," in Modern Methods of
Pharmaceutical Analysis, R. E. Schirmer, ed., CRC
Press, West Palm Beach, FL, in press (19801,
Chapter VI.
49. R. E. Cooley, C. E. Stevenson and E. C. Rickard,
J. Automated Chem., in press (1980).
50. N. E. Davis, Lilly Research Laboratories, Indiana-
polis, IN 46206, personal communication.
51. L. J. Lorenz, Eli Lilly and Co., Indianapolis, IN
46206, personal communication.
52. J. Kennedy, Eli Lilly and Co., Indianapolis, IN
46206, personal communication.
53. H. C. Neu, J. Infect. Dis., 137 (Suppl.), S80
(1978).
54. G. B. Appel, H. C. Neu, M. F. Parry, M. J. Gold-
berger and G. B. Jacob, Antimicrob. Agents Chemother.,
10, 623 (1976).
-
55. N. G. Waterman, H. U. Eickenberg and L. Scharfen-
berger, Antimicrob. Agents Chemother., 10,733 (1976).
56. M. M. Agbayani, A. J. Khan, P. Kemawikasit, W.
Rosenfeld, D. Salazar, K. Kumar, L. Glass and H. E.
Evans, Antimicrob. Agents Chemother., 15,674 (1979).
57. P.H. Azimi, Antimicrob. Agents Chemother., 2, 955
(1978).
58. F. Daschner, E. Blume, H. Langmaack and W. Wolfart,
J. Antimicrob. Chemother., 2, 474 (1979).
59. J. L. Axelrod and R. S. Kochman, Amer. J. Ophthalmol.,
85, 342 (1978).
60. Barza, A. Kane and J. Baum, Amer. J. Ophthal., 86,
121 (1978).
61. P. Young, M. Barza, A. Kane and J. Baum, Arch.
Ophthalmol., 97, 717 (1979).
62. N. S. Aziz, J. G. Gambertoglio, E. T. Lin, H.
Grausz and L. 2 . Benet, J. Pharmacokinet. Biopharm.,
6, 153 (1978).
-
63. U . S . Code of Federal Regulations (1974, Admended

April 6, 1 9 7 9 ) , Food and Drugs, Title 21, part
442.208, Washington, D.C., U . S . Government printing
office.
The literature is surveyed through January 1980.

CYPROHEPTADINE
Hassan Y. Aboul-Enein and A . A. Al-Badr
1. Description 156
1.1 Nomenclature 156
1.2 Formulae 156
1 .3 Molecular Weight 157
1.4 Elemental Composition 157
1.5 Appearance, Odor, Color, and Stability 157
2.2 Solubility 157
3. Synthesis 166
4. Metabolism 167
5.1 Spectrophotornetric Methods 170
5.2 Titrimetric Method 171
Acknowledgments 177
6. References 178
Copyright @ 1980 by Academic Rcss. Inc.

Analytical Pmfiks of Drug Substances, 9 155 All rights of reproduction in any form reserved.
ISBN: 0-12-2608097
CYPROHEPTADINE
1. Description
1.1 -Nomenclature
1.11 Chemical names

4-(5H-Dibenzo [a,d] cyclohepten-5-ylidene)-1-methyl
piperidine; l-methyl-4-( 5H-dibenzo- [a,d] cyclohep-
tenylidene ) piperidine; 5-( 1-methyl piperidyli-
dene-4)-5H-dibenzo [a,d] cycloheptene; l-methyl-
4-(5-dibenzo [a,e] cycloheptatrienylidene) pipe-
ridine; 4-(1,2: 5,6-dibenzocycloheptatrienylidene)
-1-methyl piperidine.
1.12 Generic name

Cyproheptadine.
1.13 Trade names
Anarexol, Antegan, Nuran, Periactin, Vimicon,

Cipractin, Peritol, Dronactine, Periactinol.
1.2 Formulae
1.21 Empirical
c21 H21
1.22 Structural
-1
1.23 Wiswesser line notation
L C676 BYJ BUDT6N DYTJ A (1)

CY PRO HE PTADINE 157
1.3 Molecular weight

Anhydr0u.s base 287.39, Anhydrous HC1 323.86
Sesquihydrate HC1 350.89.
1.4 Elemental composition

C 87.76%, H 7.37%, N 4.87%.
1.5 Appearance, color, odor and stability
White to slightly yellow, crystalline powder that is

odorless or practically odorless and has a slightly
bitter taste; relatively stable in light, stable at
room temperature and nonhygroscopic; the sequihydrate
is stable in air (2 ).
2.1 Melting point
The anhydrous form melts at agout 250'. The sesqui-

hydrate melts about about 162 (2). Crystals from
absolute ethanol + ether, dec. 252.6 - 253.606 Hydro-
chloride monohydrate crystal melts at 214-216 ( 3 ).
Crystals frgm dilute ethanol (the base ) melts at
112.3-113.3 ( 3 ) .
2.2 Solubility
1 Gm in about 1.5 m l methanol, about 16 m l chloroform,

about 35 m l alcohol and about 275 ml H20; practically
insoluble in ether (2).
2.3 Identification
The following tests are cited from the BP 1973 (4).
a) The infrared absorption spectrum exhibits maxima

which are compared to the authentic cy-prohepta-
dine hydrochloride.
b) The light absorption, ir. the range of 230-350 nm,

of a 2 cm layer of 0.0016% w/v solution in etha-
nol 95%, exhibits a maximum only at 286 nm, and ex-
tinction about one.
c) A saturated solution yield the characteristic test

for chloride.
The following are certain color tests ( 5 ) which are

useful in the identification of cyproheptadine in
micro amount :-
Reagent. Color Sensitivity
Sulphuric acid/formal- Gray green 0.5 Pg

dehyde.
Ammonium molybdate. Blue green to 0.1 Pg
green.
Ammonium vanadate Purple-brown. 0.5 Pg
Furthermore, crystal tests can be used for the iden-

tification of the drug ( 5 ) , f o r example, with ammonium
thiocyanate solution, it gives branching needles
(sensitivity 1 : 1000). With potassium iodide solu-
tion, dense roset and fans of rods (sensitivity
1 : 1000).
Yalcindag and Onur ( 6 ) had published a report which

described the identification for some drug containing
basic nitrogen including cyproheptadine through the
microscopic appearance of crystals formed with a num-
ber of reagents and by some color tests.
2 . 4 Spectral properties
2 . 4 1 Infrared spectrum
The infrared spectrum of cyproheptadine base

(Nujol mull) is given in Fig. 1, major brand assi-
gnments are as follows :-
-1
Frequency, cm Assignment.
3380, 2 4 0 N-CH3
1590 Aromatic phenyl
stretch.
1640 C=C at Cl0 - cll
Other finger print bands has been assigned by

Clarke ( 5), these peaks are :-
749, 797, 765 and 776cm-’, all of which are seen
in the spectrum which is shown in Fig. 1. Further
information with regard to the infrared spectrum
of cyproheptadine is given in reference (1).
Fig. 1 - Infrared spectrum of cyproheptadine in N u j o l mull.
HASSAN Y . ABOUL-ENEIN AND A. A. AL-BADR
2.42 Ultraviolet spectrum
Cyproheptadine in methanol solution exhibits maxi-

ma at 224 nm, an inflextion at 240 m and at 283
nm as shown in Fig. 2 .
Clarke ( 5 ) reported the "ultraviolet absorption
spectrum for cyproheptadine in 0.1N H Sc! to show
maxima at 224 nm (El% 1 ern 1656) and 2 at4 285 nm
(El% 1 em 3 5 5 ) , and an inflextion at 240 nm.
2.43 Nuclear Magnetic Resonance Spectrum:
A typical NMR spectrum of cyproheptadine is pre-

sented in Fig. 3 . It was obtained on a Varian
T-60A NMR Spectrometer with TMS as the internal
standard. The sample was dissolved in CDCL
3'
The following structural assignments have been
made for Fig. 3 :
Chemical shift (8) Assignment .
- Singlet at 2.1 N-CH
3
- Broad complex multiplets 8 protons of the
between 2.27 and 3.47 piperidine ring
system.
- Singlet at 6.87. CH = CH bridge at
Cl0 and C .
- Multiplet centered 8 protons'$or the
at 7 . 2 3 . aromatic phenyl.
rings.
These data are in agreement with the data publish-
ed by Englehardt -
et -
al. ( 7 ) .
2.44 Mass spectrum and Fragmentometry
The low resolution mass spectrum of cyproheptadine

is shown in Fig. 4 . It was obtained on a Finni-
gan 1015 L quadrupole mass spectrometer at an
ionization potential of 70eV. The spectrum shown
is obtained by direct insertion of cyproheptadine
base. It shows a molecular ion Id at m/e 287
(Relative intensity 5 . 2 % ) , a promenant peak at m/e
96. The most important prominant ions are shown
in Table 1.
CYPROHEPTADINE 161
Fig. 2 - U l t r a v i o l e t spectrum of cyprohep-

t a d i n e i n methanol.
I . I . . . i . . . . i .1 . . . i . . . . i I . . . . . ' i . . . . I i .
8.0 7.0 6.0 S.O(PPM)s 4.0 3.0 2.0 1.0
Fig. 3 - NMR spectrum of cyproheptadine i n C D C l 3 with TMs

as i n t e r n a l standard.
96
cn
W
215
50 100 150 200 250 308
Fig. 4- Mass spectrum of cyproheptadine ( E I ) determined

by d i r e c t probe i n s e r t i o n .
T+*
Fragment Table 1 m/e Relative dntensity
287 5.1
&+'
CH3
M+ - CH 272 1.3
215 17.4
202 4.5
'
0 I
CH3
+- 96 80.2
l+
CI, 4 H3
70 39.1
Frigerio -
et -
a1 ( 8 , 9 ) had discussed the fragmenta-
tion of some of the metabolites of cy-proheptadine
mainly cyprohepladine-lO,ll-epoxide, desmethyl
cyproheptadine, desmethyl cyproheptadine-10, 11-
epoxide. Frigerio - et - a1 (8)suggested a frag-
mentation pathway of these epoxide metabolites
as shown on Scheme 1.
CY PROHEPTADINE 165
I I
R H
m / e 289 ( R = H) I
m / e 303 ( R = CH3)
- CHO'
c
II
cH2
I
m / e 203 R
m / e 260 (R = H)
S c h e m e 1.
CH
3. Synthesis
a) Phthalic anhydride ( I ) is reacted with phenylacetic

acid (11) to form 3-benzylidenephthalide (111) which on
isomerization and hydrogenation, gives 2-phenylbenzoic
acid (IV). This is converted to its acid chloride
which then undergoes condensation to close the 7-nem-
bered ring and give 10, 11-dehydro-5H-dibenzo [a,b]
cyclohepten-5-one (V). Bromination at the 1- pesition
followed by dehydrobromination introduces the 10, 11-
double-bond. Grignardization of this ketone with 4-
chloro-1-methyl piperidine followed by dehydration of
the resulting carbinol yields cy-proheptadine (base)
which on reaction with equimolar quantity of hydrogen
chloride, forms the hydrochloride salt ( 10) .
I I1 I11 IV
@ -HBr ~
1 ) N-methgl-4-
piperidyl
magnesium
chloride
2 ) Hydrolysis.
*
"4i"
9- I 3
CH H/ \CH3
CY PROHEPTADINE I67
b) Converting 5-cyanodibenzo [a,dl cycloheptadine ( I ) to

its corresponding pipertdine derivative I1 which on
treatment with sodium hydride, anhydrous dimethylsul-
phoxide at 170-180° then refluxed with 10% HC1, cypro-
heptadine was obtained in 87% yield (11).
I
I CH
I 11 CH3 3
c) Dibenzo [a,d] cycloheptene I11 was treated with phenyl

lithuim in tetrahydrofuran and ether, the anion genera-
ted was reacted with l-methyl-4-piperidone to give IV
which was dehydrated by refluxing with acetic acid in
concentrated HC1 ( 11).
I11 IV dH3 CH3
4. Metabolism
The metabolism of cyproheptadine has been extensively stu-
died in several species including humans.
Hucker -
et -
a1 (12) had published a report on the physiologi-
cal disposition and urinary metabolite in the dog, rat and
CH3
9 0
Rat
Dog, c a t
Dog,cat I
I --
H
CH3
I I
CH3 H
Scheme 2.
Urinary metabolites of cy-proheptadine identified

i n dog, r a t and c a t . Underlining i n d i c a t e s t h a t t h e
s t r u c t u r e was a major metabolite i n t h a t species.
CY PROHEPTADINE 169
cat. It was found that, cyproheptadine was well absorbed

and excreted almost equally in the urine and feces of
these species. However, the plasma levels of the radio-
activity were considerably higher in the dog than in the
rat. About 17% or the dose was excreted in the dog bile in
6 hours. The urinary metabolites identified in the dog,
rat and cat as, reported by Hucker --
et a1 (12), are shown
in Scheme 2.
Rats excreted the drug almost entirely as the desmethyl

cyproheptadine-10, 11-epoxide. This statement was sub-
stantiated by the study reported by Hintze -
et -
a1 (13).
Hintze --
et a1 (13) reported that after intzoducing a dose of
14c labelled cyproheptadine hydrochloride, the major meta-
bolite in the rat urine was unconjugated, but the majority
of the radioactive materials found in mouse and human urine
were conjugated with glucuronic acid. The rat metabolite
(the desmethyl cy-proheptadine-10, 11-epoxide) accounted for
25% of 45 mg dose of the drug per kg. None of this epoxide
was found in human.
The dihydrcdiclswhich could arise from the 10, 11 epoxy me-

tabolite were not found in the urine of the rat, mice and
humans. The epoxide found in the rat urine reported to be
unusually stable in the in vivo hydrolysis. Hintze - et -
a1
( 1 3 ) suggested possible implications of these results in
the species-selected pancreatotoxicity of cyproheptadine
in the rat. A detailed study on the B-sell toxicity of
cyproheptadine is published by Rickert (14) and by Wold and
Fischer (15). Frigerio - et -
a1 (8)had identified cyprohep-
tadine-10, 11-epoxide, desmethylcyproheptadine-10, 11-
epoxide, and desmethylcyproheptadine in rat urine after
administration of 40 mg/kg I.P. of the drug by mass spec-
trometry and confirmed their structure Furthermore,
Frigerio - et -
a1 (9)in another report had identified the
presence of the desmethylcyproheptadine-10, 11-epoxide in
the urine of human volunteers. Porter - a1 (15) had
et -
reported a full study on the metabolism of cyproheptadine
in humans. The metabolites identified are shown in
scheme 3 .
Aromatic ring hydroxylation followed by glucuronide conju-

gation, N-demethylation and heterocyclic ring oxidation
were shown to occur in man. The principal metabolite,
however, was identifed as a quarternary ammonium glucuro-
nide-like conjugate of cyproheptadine. They reported (16)
no evidence for any metabolic changes at C
10’
Cll bridge
N Glucuronide
I
HO
O
#
= \CH3 H
Scheme 3.
of the drug in humans. All metabolites seen in scheme 3,

were identified by GLC, Ms, NMR and IR spectrometric tech-
niques.
5. Methods of Analysis%
5.1 Spectrophotometric methods
5.11 Colorimetric methods

Cyproheptadine had been determined in pharmaceuti-
cal formulation colorimetrically by Adamski (18).
The ground tablets were extzacted with chloroform,
the extract was shaken with phosphate buffer and
bromocresol green. The chloroform was re-extrac-
ted with 0.N NaOH measuring the extinction at 615
nm refering the results to a calibrating graph.
~ ~~ ~ ~~
X Shapoval -
et -
a1 (17)published a report which include
the physical, chemical and biological properties of
the drug in tablets and the metho&of its evaluation.
CY PROHEPTADINE 171
The error was reported to be > 1.2%.
Beltagy - a1 (19)reported a method for the de-

et -
termination of 18 drugs in the free form and in
various formulations colorimatrically using tro;
peolin 000. Cyproheptadine was included in that
method of assay in which the drug was treated
with tropeolin 000 in a pH 1.09 buffer. The com-
plex formed was extracted with methylene chloride,
the dye liberated by the acid addition was measu-
red at 485 nm. The method gave results comparable
to those obtained by the method of the B.P.
1973 ( 4 ) .
5.12 Ultraviolet Spectrophotometric method
This method has been adopted by the B.P. 1973 ( 4 )

for the assay of cy-proheptadine tablets. The
method cited in the B.P. 1973 depends on the
measurement of the extinction of 1-cm layer of the
alcoholic (95%) solution at a maximum at about
286 nm. The content of cyproheptadine hydrochlo-
ride is calculated taking 355 as value of E 1%
1-cm.
Demir and h a 1 (20) had published a similar pro-

cedure.
5.2 Titrimetric methods
5.21 Nonaqueous titration
The B.P. 1973 (4) analyses cy-proheptadine hydro-

chloride, free drug, by the non-aqueous titration
using 0.1 N perchloric acid as a titration after
the addition of mercuric acetate solution using
crystal violet solution as indicator.
5.3 Chromatographic methods
5.31 Counter-Current Distribution
Hintze -
et -
a1 (13) had isolated cyproheptadine and
its epoxide metabolite from rat urine by the coun-
ter current distribution method. The pooled urine
was adjusted to pH 8 and extracted several times
with methylene chloride, the organic layer was
in vaccu to 2 m l . After addition of
concentrated --
172 HASSAN Y . ABOUL-ENEIN AND A. A . AL-BADR
10 ml of 0.05M of phosphate b u f f e r (pH 7 . 5 ) , t h e

remainder of t h e organic phase was evaporated. The
buffer s o l u t i o n was placed i n a 100 tube-counter
current d i s t r i b u t i o n apparatus and d i s t r i b u t e d b e t -
ween 0.05M phosphate buffer (pH 7.5 ) and benzene.
After a 100 cycles, the solvents were decanted i n t o
glass-receiving tubes an? a l i q u o t s of t h e benzene
l a y e r were removed f o r determination of radio-
a c t i v i t y . The benzene l a g e r s of tubes 75-90
were combined and dried out over sodium s u l f a t e .
Mass spectrometry, and TLC were u t i l i z e d t o d e t e r -
mine t h e p u r i t y of t h e metabolite and t h e unchanged
drug t h a t was i s o l a t e d from r a t urine.
Another method reported by P o r t e r -- e t a1 (16) for

t h e counter current d i s t r i b u t i o n . A gum i s o l a t e d
from human u r i n e iiigesting 1% cy-proheptadine ( 5 ,
10,11-11, 4mg, 16 JJ C i per s u b j e c t ) a f t e r passing
C
t h e urine through XAD-2 r e s i n columns. The gum was
subjected t o f r a c t i o n between water and butanol/
benzene (1:l v/v). Cyproheptadine and o t h e r meta-
b o l i t e s were separated by t h i s system and i d e n t i -
f i e d by TLC and GC.
5.32 Paper chromatography

Clarke ( 5 ) described a s e v e r a l solvent systems which
a r e used f o r t h e i d e n t i f i c a t i o n of cy-proheptadine
a s shown i n Table 2.
Table 2
Solvent system I Visualizing agent 1 Rf

C i t r i c acid : water : U l t r a v i o l e t , Iodo- 0.77
n-but anol platinate.
4.8g : 103 ml : (Weak reaction),Bro-

870 ml mocresol green (weak
reaction).
Acetate Buffer U l t r a v i o l e t Iodo- 0.22
(PH 4 . 5 8 ) platinate.
Phosphate Buffer U l t r a v i o l e t Iodo- 0.00
(PH 7 . 4 ) platinate
CYPROHEPTADINE 173
5.33 Thin k y e r Chromatography

Several reports had appeared in the literature
concerning the tlc of cyproheptadine and its me-
tabolites describing the separation and identifi-
cation of cyproheptadine and its metabolites ( 5 , 8 ,
12, 1 3 , 21, 22). The systems are given in Table
3. Ultraviolet light at 254 nm was used to detect
the drug and its metabolites unless otherwise
stated.
Hucker - a1 (12), Hintze -

et - a1 ( 1 3 ) an$ Porter -
et - et
-
a1 (16)had published the Rf values of cyprohep-
tadine metabolites in several solvent systems
which can be useful i~nclinical identification of
the drug and its metabolites in biological fluids.
Furthermore,Virgnoli --
et -
a1 (21) published a report
on the identification of cyproheptadine among
other drugs using Silica-gel as an absorbsnt in
the following solvent systems :-
A) Diethyl ether : acetone : diethylamine

90 19 1
B) Benzene : dioxane : diethylamine

400 95 6
The chromatograms were sprayed by iodoplatinate

followed by dilute H2S0 or 1% potassium permang-
nate in 5% H2S04 and iohoplatinate reagent.
5.34 Gas Liquid Chromatography

During the metabolic study of cyproheptadine in
humans and other species, several gas chromatogra-
phic analyses were reported f o r the determination,
identification and quantitation o f cyproheptadine
and its metabolites. The drug was chromatographed
without derivatization. The gas chromatographic
conditions are given in Table 4 .
t
. 0 m m
r:
ni
0 0 D
W
0 0 0 0 d
2
w
.. .- .. ..
M
a,
d
k-
\
w k-
In Ln 0 In
d
..
0 Ln 0 Ln
d M 03 r(
..
Ln 0 0 In
W a d 0
I74
c\i m M m
ri ri r- r-
..
0
0
r-
.. ..
I75
176 HASSAN Y. ABOUL-ENEIN AND A. A . AL-BADR
-Table 4
Carrier Column lefer-

Column. Temp. C
Gas. :nces.
6 f e e t column packed w i t h h-ogramned from 16

3% OV-17 on acid-washed L50-250' a t a
and s i l a n i z e d Gas-Chrom F :ate of 5% per
ninute.
Glass tubing ( 1m long a n d 250 8

4 nm i . d . )packed with
100-120 mesh Gas-Chrom
Q and coated with OV-17.
6 f e e t x &-inch g l a s s 238 12
column packed w i t h 1.5%
OV-17/Gas-Chrom Q.
5 f e e t x &-inch 0 . d . 225 13
g l a s s column, 3% OV-225
on Supelcoport ( 80-100
mesh).
5 f e e t x 4 mm i . d . g l a s s 225 5
column, packed w i t h 2.5%
SE 30 on 80-100 mesh
Chromosob W AWHMDS.
5.35 P a r t i t i o n Column Chromatography
Porter --
e t a 1 ( 1 6 ) have separated cy-proheptadine
metabolites by f r a c t i o n a t i o n on columns packed
with Cellex SE (H') and on Bio-Rex 63 (H') resin
column.
CYPROHEPTADINE 177
ACKNOWLEDGEMENTS
The authors would like to thank M r . Dennis Charkowski,

Dept. of Pharmacology, the University of Iowa, Iowa City,
Iowa 52242, U.S.A., for determining the mass spectrum of
cyproheptadine; M r . Said E. Ibrahim, for his help in the
library search, M r . Essam A. Lotfi and M r . Khalid N.K.Lodhi
for determining the ultraviolet and nmr spectra, and M r . Altaf
Hussain Naqvi for typing the manuscript.
A sample of cyproheptadine HC1 was kindly donated by
D r . E.L. Engelhardt of Merck Sharp and Dohme Research labo-
ratories, West Point, Pa. 19486, U.S.A.
REFERENCES
1. IfAtlasof Spectral data and physical constants of Organic

compounds", edited by J.G. Grasselli and W.M. Ritchey.
Vol 3, CRC Press 1975, p. 160.
2. Remington's Pharmaceutical Sciences, 15th edition, Mack

Publishing Co., Easton, Pa 18042, 1975, Page 1066.
3 , Merck Index, Ninth edition, Merck & Co. Inc., Rahaway,
N.J., U.S.A., 1976, Page 2765.
4. British Pharmacopoeia 1973, London Her Majesty's Station-
ary Office 1973, Page 139.
5. E.C.G. Clarke, "Isolation and Identification of Drugs",
The Pharmaceutical Press, London, 1969, Page 278.
6. O.N. Yalcindag and E. Onur, Turk. Hij terc. -
Biyol.-
Derg.,
33 25 (1971)Through - -Abst.
Anal. - 22, Abstract No.2649
(1972 ) .
7. E.L. Engelhardt, E.C. Zell, W.S. Saari, M.E. Christy and
C.D. Colton, J. --
Med. Chem., -8, 829 (1965), and referen-
ces were cited therein.
8. A. Frigerio, N. Sossi, G. Belvedere, C. Pantarotto and
S. Garattini, J. Pharm. Sci, 63, 1536 (1974).
9. A . Frigerio, N. Sossi, G. Belvedere, C. Pantarotto and S.
Garattini, Adv. Mass Spectrum. Biochem. Med., -1, 109
( 1976) .
10. L.M. Atherden, "Bentley and Driver's Textbook of Pharma-

ceutical Chemistry", Eight edition, "London, Oxford Uni-
versity Press, 1969, Page 591.
11. L. Vargha, E. Kasztreiner, E. Meszaros and G. Szidagyi,
Ger. Offen. 1, 921, 934; through Chem. Abstr. -
72, 31625~
(1970r
12. H.B. Hucker, A.J. Balleto, S.C. Stauffer, A.G. Zacchei
and B.H., Arison, Drug.Metab. Dispos. -
2, 406 (1974).
13. K.L. Hintze, J.S. Wold and L.J. Fisher, Drug Metab.
Dispos., 2, 1, (1975).
CY PROHEPTADINE 179
1 4 . D.E. Rickert, -
Diss. -
Abstr. -
I n t . B, -
35, 4079 (1975).
183,
1 5 . J . S . Wold and L . J . Fischer, J. Pharmacol. E X ~ .Ther., -
188 (1972 ).
16. C . C . P o r t e r , B.H. b i s o n , V.F. Gruber, D.C. T i t u s and
W.J.A. Vandenheuvel, Drug Metab. Dispos., - 3, 189 (1975).
17. E.E.S. Schapoval, M.M. M a r t i n e l l i , L.C. Chiaaini and
E . J . C . De Castro, Rev. B r a z i l . Farm., 53, 1% (1972)
through - Chem.
-A- bstr.79mOam73).
18. S. Adamski, Acta Pol. Pharm., 311 (1965 ) through -

Anal.
- 13, Abstract No. 6503 (1966).
Abstr.
-
31, 484,
19. Y.A. Beltagy, A. I s s a and S.M. R i d a , Pharmazie, -
( 1976 ) .
20. S. Demir and H. Amal, I s t a n b u l Univ. E c z a c i l i k Fak. Mecm.,
-
6, 1 4 , ( 1970) ; through Chem. Abstr. 73, 1 1 3 0 2 4 - 9 v
21. L. Virgnoli, B. C r i s t a n , F. Gouezo and J.M. Vassalo, Bull.
Tran. SOC. Pharm. u o n . , 9, 277 (1965); through Anal.-
14, Abstract No. 3731. (1.5176).
Abstr. -
22. F. Schmidt, Dtsch. Apoth. Ztg., 114, 1593 (1974); through

Chem. Abstr. -
-- 82, 64614d (1975).
DIBENZEPIN HYDROCHLORIDE
Alfred Egli and Werner R. Michaelis
I. Introduction 182
1.1 History 182
1.2 Name, Formula, Molecular Weight 182
1.3 Appearance, Colour, Odour 182
2. Physicochemical Properties 182
2.2 Spectra 183
2.4 Solubility 191
2.5 Dissociation Constant 193
2.6 Partition Coefficients 193
3. Synthesis 193
4. Stability 194
4.1 Stability in Bulk 194
4 . 2 Stability in Solution 195
4.3 Stability in Dosage Forms 195
5. Biophannaceutical Aspects 195
5 . 2 Metabolism 196
6. Acute Toxicities 197
7. Analytical Methods 198
7.1 Titration 198
7.2 Spectroscopic Methods 198
7.4 Analysis of the Dosage Forms 20 1
7.5 Determination in Body Fluids 204
8. References 205
Copyright @ 1980 by Academic R s s . Inc.

Analytical Profiles of Drug Substances. 9 181 All rights of reproductionin any form reserved.
ISBN: 0-12-260809-7
182 ALFRED EGLI A N D WERNER R. MICHAELIS
1. Introduction
1.1 History
In 1959 and 1962, patent applications were filed for
dibenzepin hydrochloride [l]. The drug substance shows
remarkable histaminolytic and anti-anaphylactic effects [21.
According to clinical trials this antidepressant can be
classified among the thymoleptic drugs between Imipramine and
Amitryptiline [ 3 , 41.
Dibenzepin hydrochloride is the active ingredient of the
NOVERIL@ dosage forms.
1.2 Name, Formula, Molecular Weiqht

Dibenzepin hydrochloride is 10-[2-(dimethylamino)ethyl]-
-5,10-dihydro-5-methyl-11H-dibenzo[b,e][1,4]diazepin-11-one,
monohydrochloride
CH34jCH3
N
- HCI
TI@
Molecular Formula:
7 &iD 5a
0
II
La
1
3
C18H22C1N30
Molecular
331.85 Weight:
6 I L
CH3
Chemical Abstracts Registry Number: 315-80-0
1.3 Appearance, Colour, Odour

Finely crystalline to crystalline, white or buff white
powder; odourless o r of weak, characteristic odour.
2. Physicochemical Properties

Element % Calculated % Found
C 65.2 65.3
H 6.7 6.5
c1 10.7 10.6
N 12.7 12.6
0 4.8 5.0
DIBENZEPIN HYDROCHLORIDE 183
2.2 Spectra
2.21 I n f r a r e d
The I R spectrum i n a KBr p e l l e t as obtained on a
PERKIN-ELMER 283 i n f r a r e d spectrophotometer i s presented i n
f i g . 1.
The main c h a r a c t e r i s t i c bands a r e t h e f o l l o w i n g :
Wave number (cm-') Assignment
3100 - 2800 C-H stretching vibrations

2400 - 2560 N-H+ stretching vibrations
1630 C=O stretching vibration
1600 C=O i n - p l a n e deformation v i b r a t i o n
775 C-H out-of-plane deformation
vibration (1,2 disubstitution)
2.22 U l t r a v i o l e t
The UV spectrum i n 0.1 N h y d r o c h l o r i c a c i d as o b t a i n e d
on a Z E I S S DM4 spectrophotometer i s presented i n f i g . 2.
A maximum occurs a t about 204 nm w i t h a l o g molar a b s o r p t i v i t y
o f 4.530, another maximum a t about 220 nm w i t h a l o g molar
a b s o r p t i v i t y o f 4.458 and a shoulder a t about 285 nm w i t h a
l o g molar a b s o r p t i v i t y o f 3.421.
2.23 Fluorescence
I n 0.1 N h y d r o c h l o r i c a c i d t h e drug substance shows no
fluorescence ( e x c i t a t i o n from 220 t o 400 nm).
2.24 P r o t o n Nuclear Magnetic Resonance

The PMR spectrum i n d e u t e r a t e d d i m e t h y l sulphoxide as
o b t a i n e d on a BRUKER HX-90-E spectrometer i s presented i n
f i g . 3. TMS served as i n t e r n a l standard. The c h a r a c t e r i s t i c s
o f t h e spectrum a r e g i v e n i n t h e f o l l o w i n g t a b l e :
4
a,
rl
4
a,
a
k
m
Y
m
c
.rl
a,
-0
.rl
k
0
rl
r
o
0
k
-0
x
I
c
.rl
a .
a,M
NOJ
C N
a,
ncc
w
.rl
F i q u r e 2 : U l t r a v i o l e t Spectrum o f D i b e n z e p i n H y d r o c h l o r i d e
i n 0.1 N H y d r o c h l o r i c Acid.
CA = 0.0505 m g / m l ; CB = 0.0101 m g / m l .
I n s t r u m e n t : Z E I S S DM4.
[ PPm 1
F i g u r e 3: P r o t o n Nuclear Magnetic Resonance Spectrum o f Dibenzepin H y d r o c h l o r i d e i n (CD3) SO.
Instrument: BRUKER HX-90-E.
Chemical S h i f t
Intensity Multiplicity Assignment
[PPml
11.5 1 H singlet 3'-H+

(broad)
7.6 1 H doublet o f H-Cl
doublet
7.4-7.5 2 H multiplet H-C3, H-C9
7.3 1 H doublet o f H-C6
doublet
7.15-7.3 3 H mu1t i p l e t ti-C4, H-C7, H-C8

7.1 1 H triplet H-C2
4.6 1 H mu1t i p l e t
4.2 1 H mu1t i p l e t
H-C1 '
3.35 2 H triplet H-C2 '

3.3 3 H singlet 5-CH3
2.8 6 H singlet 3 ' -CH3
2.25 Carbon-13 N u c l e a r Maqnetic Resonance

The C-13 NMR spectrum i n d e u t e r a t e d d i m e t h y l s u l p h o x i d e
as o b t a i n e d on a BRUKER HX-90-E s p e c t r o m e t e r i s p r e s e n t e d i n
f i g . 4. TMS s e r v e d a s i n t e r n a l standard. The assignment o f
the i n d i v i d u a l signals i s given i n the following table:
Chemical S h i f t Carbon Chemical S h i f t
Carbon
PPm 1 [PPml
c- 1 131.2 C-8 124.6

c- 2 122.6 c-9 123.5
c- 3 132.4 C-9a 134.8
c-4 116.4 c-11 168.0
C- 4a 153.6 C-lla 126.5
5-CH3 36.6 c-1' 44.6
C-5a 148.3 c-2' 53.5
C-6 119.1 3 ' -CH3 42.0
c- 7 126.3
.tlz low0 8 WO 6wO 4wo 2wo
tiz 5000 4wo 3O W 2wo ,000
HZ 25M 22.53 MHz-C" 2 ow 1500 1 000 5w
m lW
1W 50
50 25
w w
2b0 1'50 1'00 5'0
[ PPm I
F i g u r e 4: C-13 Nuclear Magnetic Resonance Spectrum o f Dibenzepin H y d r o c h l o r i d e i n (CD3)gS0.
Instrument: BRUKER HX-90-E.
2.26 Mass
The low r e s o l u t i o n e l e c t r o n impact mass spectrum ( 7 0 eV)
as o b t a i n e d on a AEI MS 30 mass spectrometer u s i n g d i r e c t
i n s e r t i o n probes a t 80 O C i s presented i n f i g . 5. The
f r a g m e n t a t i o n pathways a r e as f o l l o w s :
CH3
- CH3, N- CH = CH2
CH3'
HI 0
II .q
r3
- C H O -29
-+
&ib
CH3
-cHi-14
CHI 0
m/e=237
CH3
100- F i q u r e 5: Low R e s o l u t i o n E l e c t r o n Impact Mass Spectrum
o f Dibenzepin H y d r o c h l o r i d e .
90 - I n s t r u m e n t : AEI MS 30 (Energy: 70 eV,
Ion S o u r c e Temperature: 80 OC).
80-
70 -
60.
50-
40-
30-
20 -
10-
0 I I
11
I I
I,
,
,1
2
: , , , , , , , , ,
100 150 200 250 300 350 400
2.31 M e l t i n g P o i n t
238 O C ; t h e d e t e r m i n a t i o n was c a r r i e d o u t on a METTLER
FP 1 ( s t a r t i n g t e m p e r a t u r e 230 O C , h e a t i n g r a t e 2 O C / m i n > .
2.32 Polymorphism
So f a r no polymorphism has been observed by I R s p e c t r o s -
copy and d i f f e r e n t i a l scanning c a l o r i m e t r y .
2.33 D i f f e r e n t i a l Scanninq C a l o r i m e t r y
The DSC thermogram, o b t a i n e d w i t h a PERKIN-ELMER DSC-2
i n s t r u m e n t a t a h e a t i n g r a t e o f 10 O C / m i n and i n a n i t r o g e n
atmosphere, i s shown i n f i g . 6.
The DSC c u r v e shows o n l y a s h a r p m e l t i n g endotherm accompanied
by decomposition o r s u b l i m a t i o n .
2.34 Thermoqravimetry
The t h e r m o g r a v i m e t r i c curve, c a r r i e d o u t on a PERKIN-
ELMER TGS-1 thermobalance, i s g i v e n i n f i g . 6. The sample
t e m p e r a t u r e was r a i s e d a t a r a t e o f 1 0 O C / m i n m a i n t a i n i n g a
n i t r o g e n atmosphere.
No l o s s o f w e i g h t i s observed u n t i l m e l t i n g . A s t r o n g l o s s o f
w e i g h t i s observed d u r i n g t h e m e l t i n g process.
2.4 Solubility
The s o l u b i l i t y was determined i n a v a r i e t y o f s o l v e n t s
e q u i l i b r a t e d by v i b r a t i o n d u r i n g 24 h o u r s a t 25 O C .
Solubility Solubility
Solvent
i n mg/g i n g/1OO m l
water more t h a n 200 more t h a n 20

methanol more t h a n 200 more t h a n 20
ethanol 86 7.0
2-propanol 9.2 0.69
acetonitrile 14.4 1.1
acetone 2.2 0.17
e t h y l acetate 0.5 0.04
chloroform 13.1 19.3
benzene 1.7 0.15
hexane 0.7 0.04
192 ALFRED EGLI AND WERNER R. MICHAELIS
50 100 150 200 "C
F i q u r e 6: D i f f e r e n t i a l Scanning C a l o r i m e t r y and Thermo-

g r a v i m e t r y Curves o f Dibenzepin Hydrochloride.
Instruments: PERKIN-ELMER DSC-2
PERKIN-ELMER TGS-1
(Heating r a t e s 10 T/min>.
A t 22 * 2 O C dibenzepin h y d r o c h l o r i d e d i s s o l v e s more t h a n
2 % (w/v) i n propylene g l y c o l and e t h a n o l 95 per cent, and
more t h a n 20 % (w/v) i n e t h a n o l 50 per cent; i t i s p o o r l y
s o l u b l e (0.056 76 (w/v>) i n n-octanol.
2.5 D i s s o c i a t i o n Constant
T i t r a t i o n o f a 0.003 M s o l u t i o n i n water a t 20 - 22 OC
y i e l d e d as pKa 5.25 * 0.05 f o r t h e 3'-Nitrogen.
2.6 Partition Coefficients

The p a r t i t i o n c o e f f i c i e n t s between s i m u l a t e d g a s t r i c
f l u i d pH 1.2 ( w i t h o u t enzyme) and n-octanol on one hand, and
s i m u l a t e d i n t e s t i n a l f l u i d pH 6.8 ( w i t h o u t enzyme) and
n - o c t a n o l on t h e o t h e r , have been determined a t 37.0 * 0.5 O C
g a s t r i c f l u i d pH 1.2/n-octanol: 1 : 0.27
i n t e s t i n a l f l u i d pH 6.8/n-octanol: 1 : 18.3
3. Synthesis
C a t a l y t i c hydrogenation o f 2-[methyl(2-nitrophenyl) -
aminolbenzoic a c i d m e t h y l e s t e r l e a d s t o t h e corresponding
aminoester, 2-[(2-arninophenyl)methylamino]benzoic a c i d m e t h y l
e s t e r , which i s t h e n converted by c y c l i z a t i o n w i t h a s t r o n g
base (e.g. sodium amide) t o t h e lactam 5,10-dihydro-5-methyl-
11H-dibenzo[b,e][l,4]diazepin-ll-one. Alkylation with
2-chloro-N,N-dimethylethanamine y i e l d s dibenzepin base, whose
h y d r o c h l o r i d e i s formed by r e a c t i o n w i t h gaseous h y d r o c h l o r i c
a c i d i n e t h a n o l i c s o l u t i o n . F i n a l l y t h e product i s r e c r y s -
t a l l i z e d f r o m e t h a n o l [2l.
The s y n t h e s i s o f C-14-labelled drug substance i s d e s c r i b e d
i n [51.
0 0
NaNHz
H
1 O
I
CH3
CH3, ,CH3
CI-CHz -CHz - N ( C H 3 4
CY, ,CH3
1
* K;D
N H CI N
{ o 5:
K;% I I
CH3 CH3
4. Stability
Dibenzepin h y d r o c h l o r i d e i s a v e r y s t a b l e s u b s t a n c e ; a
d e g r a d a t i o n c o u l d o n l y b e o b s e r v e d i n a c i d s o l u t i o n under
d r a s t i c conditions.
4.1 S t a b i l i t y i n Bulk
Samples s t o r e d i n g l a s s b o t t l e s f o r 1 5 y e a r s a t 2 1 O C
and f o r 8 y e a r s a t 35 O C were i n v e s t i g a t e d by TLC ( 3 s y s t e m s ) :
no d e g r a d a t i o n p r o d u c t c o u l d b e d e t e c t e d ( d e t e c t i o n l i m i t
0.05 ?A).
4.2 S t a b i l i t y i n Solution
Dibenzepin h y d r o c h l o r i d e i s a l s o very s t a b l e i n s o l u t i o n :
a f t e r r e f l u x i n g a 1 0 p e r c e n t aqueous s o l u t i o n (pH 3.6) f o r
10 days o n l y t h e a c t i v e i n g r e d i e n t and no d e g r a d a t i o n p r o d u c t
c o u l d be d e t e c t e d by TLC ( 3 systems, d e t e c t i o n l i m i t 0.05 %).
To degrade t h e a c t i v e i n g r e d i e n t v e r y d r a s t i c c o n d i t i o n s a r e
necessary: a f t e r r e f l u x i n g a 10 p e r c e n t aqueous s o l u t i o n o f
pH 1 f o r 15 days about 10 E (w/w) o f t h e f o l l o w i n g degradation
p r o d u c t c o u l d be i s o l a t e d and i d e n t i f i e d :
CH3, 7 3
N
i HC' N-(Z-(dimethylamino)ethyl)-N'-
-methyl-N'-phenyl-1,Z-benzene-
KNB I
(3-43
diamine h y d r o c h l o r i d e
No o t h e r d e g r a d a t i o n p r o d u c t c o u l d be detected.
4.3 S t a b i l i t y i n Dosaqe Forms

Dibenzepin h y d r o c h l o r i d e i s marketed as NOVERIL@ t a b l e t s ,
sugar-coated t a b l e t s , i n j e c t i o n and c o n c e n t r a t e i n t e n d e d f o r
i n j e c t i o n by i n t r a v e n o u s i n f u s i o n .
Since t h e a c t i v e i n g r e d i e n t i s s t a b l e i n these dosage forms
too, t h e s h e l f - l i v e s i n a temperate c l i m a t e and i n a h o t
c l i m a t e o f a l l these p r e p a r a t i o n s a r e a t l e a s t 5 years [61.
5. Biopharmaceutical Aspects
The a b s o r p t i o n , d i s t r i b u t i o n and e x c r e t i o n o f t h e C-14-
l a b e l l e d drug substance was i n v e s t i g a t e d i n t h e mouse a f t e r
o r a l and i . v . a d m i n i s t r a t i o n o f s i n g l e doses and a l s o a f t e r
S.C. a d m i n i s t r a t i o n t o t h e r a b b i t . I n a d d i t i o n , radiochromato-
g r a p h i c examinations o f t h e b r a i n e x t r a c t s o f mice, r a t s and
r a b b i t s were made.
196 ALFRED EGLI A N D WERNER R . MICHAELIS
I n t h e mouse, o r a l l y a d m i n i s t e r e d dibenzepin h y d r o c h l o r i d e
was promptly and completely absorbed. A f t e r i.v. a p p l i c a t i o n ,
t h e r a d i o a c t i v i t y disappeared r a p i d l y from t h e b l o o d because
t h e substance i s taken up r a p i d l y by t h e organs. The d i s t r i -
b u t i o n o f t h e a c t i v i t y i n t h e v a r i o u s organs i s independent o f
t h e mode o f a d m i n i s t r a t i o n . The l a r g e s t c o n c e n t r a t i o n s were
found i n t h e l i v e r , kidneys, g a l l bladder, and t h e lungs.
The drug substance i s r a p i d l y excreted. H a l f o f t h e adminis-
t e r e d a c t i v i t y had a l r e a d y been e x c r e t e d 5 h a f t e r o r a l admi-
n i s t r a t i o n and 100 min a f t e r i . v . a p p l i c a t i o n . A f t e r e i t h e r
o r a l o r i . v . a d m i n i s t r a t i o n 80 76 were e x c r e t e d i n t h e u r i n e
and 20 76 i n t h e feces.
The a c t i v i t y p a t t e r n i n t h e r a b b i t was s i m i l a r t o t h a t i n t h e
mouse: r a p i d and complete a b s o r p t i o n and a c t i v i t y concen-
t r a t i o n i n l i v e r , kidneys, g a l l bladder, and lungs.
5 min a f t e r i . v . a p p l i c a t i o n , 2.6 76 o f t h e dose were found i n
t h e b r a i n o f t h e mouse and 1.6 % i n t h e b r a i n o f t h e r a t .
30 min a f t e r S.C. a d m i n i s t r a t i o n t o t h e r a b b i t , 0.3 76 were
found i n t h e b r a i n . Between 1/2 and 4 h a f t e r a d m i n i s t r a t i o n
t o t h e r a b b i t t h e s p e c i f i c a c t i v i t y found i n t h e b u l b i o l f a c t .
was lower and t h a t i n t h e caudate nucleus was somewhat h i g h e r
than i n the r e s t o f the brain.
Radiochromatographic examination showed t h a t t h e a c t i v i t y
found i n t h e b r a i n o f mice, r a t s , and r a b b i t s c o n s i s t e d m o s t l y
o f unchanged dibenzepin. Besides t h i s t h e r e were found t h e
m e t a b o l i t e I11 ( c f . 5.2) and two minor b a s i c components o f
unknown s t r u c t u r e , which t o g e t h e r amounted t o no more t h a n
10 x.
5.2 Metabolism [El
Compound
R1 R2 R3
R1, 3 2
I CH3 CH3 CH3
N
I I1 H Y 3 CH3
111 CH3 CH3 H
IV H CH3 H
V H
H CH3
R3
VI H H H
The m e t a b o l i t e s o f o r a l l y a d m i n i s t e r e d d i b e n z e p i n h y d r o -
c h l o r i d e e x c r e t e d i n t h e u r i n e o f man, dog and r a b b i t have
been s t u d i e d .
The compound i s n o t r e t a i n e d i n t h e body, b u t i s r a p i d l y meta-
b o l i z e d and e x c r e t e d i n t h e u r i n e . I n a l l o f t h e s p e c i e s , none
o f t h e m e t a b o l i t e s was more t o x i c t h a n t h e p a r e n t compound.
Man and dog e x c r e t e d t h e unchanged compound and 5 d e m e t h y l a t e d
derivatives (11-VI) . R a b b i t s e x c r e t e d t h e unchanged compound
and t h e compounds I1 and 111. I n a l l t h r e e s p e c i e s , meta-
b o l i t e s c o n t a i n i n g p h e n o l i c h y d r o x y g roups were e x c r e t e d . F o r
t h e most p a r t , t h e s e appeared i n t h e u r i n e a s g l u c u r o n i d e s .
The dog e x c r e t e d a b o u t 1 6 76 o f t h e a d m i n i s t e r e d doses as f r e e
b a s i c m e t a b o l i t e s . About 8 76 were c o n j u g a t e d w i t h g l u c u r o n i c
a c i d . 48 h a f t e r t h e l a s t dose, n o e x c r e t o r y p r o d u c t s r e l a t e d
t o t h e d r u g s ub s ta n c e were f o u n d i n t h e u r i n e .
Man e x c r e t e d 20 - 30 76 o f t h e a d m i n i s t e r e d dose a s f r e e b a s i c
m e t a b o l i t e s . The amounts p r e s e n t a s t h e g l u c u r o n i d e s were
r e l a t e d t o t h e dose. The f o r m a t i o n o f g l u c u r o n i d e s was depen-
d e n t o n t h e dosage s c h e d u l e ; d i v i d e d doses gave l a r g e r amounts
t h a n a s i n g l e dose.
The r a b b i t e x c r e t e d t h e d r u g p r i n c i p a l l y a s c o n j u g a t e s o f t h e
metabolites.
6. A c ut e t o x i c i t i e s
The a c u t e t o x i c i t i e s ( LD50) o f d i b e n z e p i n h y d r o c h l o r i d e
were f ound t o be: i n t h e mouse, 22 mg/kg i . v . and 225 mg/kg
p.0.; i n t h e r a t , 22.2 mg/kg i . v . and 220 mg/kg p.0.; and i n
t h e g u i n e a - p i g , 110 mg/kg p.0. [61.
7. A n a l y t i c a l Methods
7.1 Titration
Dibenzepin h y d r o c h l o r i d e may be assayed i n g l a c i a l a c e t i c
a c i d / a c e t i c anhydride 1:l ( v / v ) by t i t r a t i o n w i t h 0.1 N per-
c h l o r i c acid. The end p o i n t i s determined p o t e n t i o m e t r i c a l l y
u s i n g a glass/calomel e l e c t r o d e system.
The h y d r o c h l o r i c a c i d c o n t e n t o f t h e dibenzepin h y d r o c h l o r i d e
i s u s u a l l y determined by t i t r a t i o n w i t h 0.1 N s i l v e r n i t r a t e .
The end p o i n t i s detected p o t e n t i o m e t r i c a l l y u s i n g a s i l v e r /
potassium s u l f a t e e l e c t r o d e system.
7.2 Spectroscopic Methods
7.21 I n f r a r e d
I n f r a r e d spectroscopy i s u t i l i z e d f o r i d e n t i f i c a t i o n
purposes d u r i n g t h e a n a l y s i s o f t h e drug substance (see 2.21).
7.22 U l t r a v i o l e t
The drug substance can be assayed d i r e c t l y by measurement
o f t h e e x t i n c t i o n a t about 221 nm (maximum) or a t about 280 nm
(shoulder) i n 0.1 N h y d r o c h l o r i c acid. The method i s n o t
s p e c i f i c , because by-products w i t h t h e same chromophore a r e
determined simultaneously. F o r t h e s p e c i f i c assay o f t h e
a c t i v e i n g r e d i e n t i t i s necessary f i r s t t o separate t h e by-
products by t h i n l a y e r chromatography and t h e n t o i s o l a t e t h e
substance by e l u t i o n from t h e s i l i c a g e l o f t h e p l a t e w i t h
0.1 N h y d r o c h l o r i c acid. The a c t i v e i n g r e d i e n t i s determined
i n t h e f i l t e r e d 0.1 N h y d r o c h l o r i c acid.
7.23 C o l o r i m e t r y
I n moderately a c i d i c s o l u t i o n s dibenzepin h y d r o c h l o r i d e
r e a d i l y forms i o n p a i r s with m e t h y l orange, which a r e e x t r a c -
t a b l e w i t h chloroform. A procedure has been developed f o r
assay w i t h AUTO ANALYZER. Therein dibenzepin h y d r o c h l o r i d e i s
allowed t o r e a c t w i t h m e t h y l orange a t pH 4.0. The r e s u l t i n g
i o n p a i r i s e x t r a c t e d w i t h c h l o r o f o r m and i t s c o n c e n t r a t i o n
determined a t 425 nm.
7.24 P r o t o n Magnetic Resonance

PMR spectroscopy may be used f o r i d e n t i f i c a t i o n o f t h e
drug substance (see. 2.24).
7.3 Chromatoqraphy
7.31 T h i n Layer Chromatoqraphy

The f o l l o w i n g systems can be used f o r t h e s e p a r a t i o n o f
by-products, degradation products, m e t a b o l i t e s and e x c i p i e n t s :
System S t a t i o n a r y Phase Mobile Phase
1 s i l i c a g e l 60 F 254 chloroform/methanol
(MERCK t l c p l a t e s , no 5 7 1 5 ) 1:l (v/v)
2 s i l i c a g e l 60 F 254 e t h y l acetate/
(MERCK t l c p l a t e s , no 5 7 1 5 ) g l a c i a l acetic acid/
water 5 : 2 : 2 (v/v/v)
3 s i l i c a g e l 60 F 254 chloroform/cyclo-
(MERCK t l c p l a t e s , no 5 7 1 5 ) hexane/diethy lamine
5 : 4 : 1 (v/v/v)
4 s i l i c a g e l 60 F 254 c h l o r o f o rm/cy c l o -
(MERCK t l c p l a t e s , no 5 7 1 5 ) hexane/diethylamine
1 : E : l (v/v/v),
t w i c e developed
5 aluminium o x i d e F 254 n-heptane/chloroform/
(MERCK t l c p l a t e s , no 5 7 1 3 ) e t h a n o l ( 9 5 per c e n t )
Y : 9 : 2 (v/v/v)
V i s u a l i s a t i o n i s accomplished under UV l i g h t 254 nm and by

s p r a y i n g w i t h D r a g e n d o r f f ' s reagent. The RSt values are:
RSt Value
Substance System 1 System 2 System 3 System 4 System 5
dibenzepin 1.0 1.0 1.0 1.0 1.0

hydro- ( R f 0 . 4 0 ) ( R f 0 . 4 5 ) ( R f 0.50) ( R f 0.42) ( R f 0.64)
chloride
degradation 0. Y O 1.15 1.20 1.75 1.15
product"
* N- ( 2 - (dimethylamino) e t h y l ) - N ' -methyl-" -phenyl-1, 2-benzene-

diamine h y d r o c h l o r i d e ( f o r f o r m u l a see 4 . 2 ) .
System 4 i s most s u i t a b l e f o r t h e d e t e c t i o n and semiquantita-
t i v e d e t e r m i n a t i o n o f t h e d e g r a d a t i o n product.
The f o l l o w i n g reagents can be used f o r t h e v i s u a l i s a t i o n o f

dibenzepin h y d r o c h l o r i d e :
Systems 1 - 4 System 5
Reagent
Detection Colour Detection
Colour
Limit [pgl L i m i t [pgl
Dragendorff I s brown 0.02-0.05 brown 0.05

reagent(1)
i o d i n e vapor brown 0.2 brown 0.5
2,6-dichloro-p- grey t o 1.2 grey t o 0.2
-benzoquinone-4- g r ey b r own green
-chlorimine-
(modified
Gibbs r e a g e n t ) ( 2 )
Folin-Ciocalteus blue 0.1 blue 0.05
reagent ( 3
sodium n i t r o - white t o 0.5 pink 0.5
prusside/ light
acetaldehyde violet
potassium j o d i d e / pinkbrown 0.5 pink 1
hexachloro- to violet
p l a t i n i c acid
potassium blue 0.2-0.5 brown 1
d i c hr omat e/
sulfuric acid
(40 per c e n t )
(1) Dragendorff s reagent with consecutive s p r a y i n g with

a m i x t u r e o f 20 r n l hydrogen peroxide ( 3 0 per c e n t )
and 10 m l o f e t h a n o l (95 per c e n t ) .
(2) 90 - 110 mg 2,6-dichloro-p-benzoquinone-4-chlorimine a r e
d i s s o l v e d i n a m i x t u r e o f 25 m l chloroform, 25 m l e t h a n o l
95 per c e n t and 3 m l dimethylforrnamide.
( 3 ) Sprayed w i t h F o l i n - C i o c a l t e u s reagent, MERCK no 9001
d i l u t e d with water 1:3 ( v / v > and a f t e r w a r d s t r e a t e d w i t h
ammonia gas.
The d e t e c t i o n l i m i t s under UV l i g h t 254 nm a r e 0.1 - 0.2 pg.
DIBENZEPIN HYDROCHLORIDE 20 1
7.32 Gas L i q u i d Chromatoqraphy

The f r e e base o f dibenzepin h y d r o c h l o r i d e can be d e t e r -
mined b y GC due t o i t s v o l a t i l i t y and i t s t h e r m a l s t a b i l i t y .
The c o n d i t i o n s a r e t h e f o l l o w i n g :
Column: glass; l e n g t h 2 m; i n t e r n a l diameter 2 mm
S t a t i o n a r y phase: D e x s i l @ 300, 1 X on Chromosorb@ W, AW-DMCS
( 8 0 - 100 mesh)
M o b i l e phase: n i t r o g e n , flow r a t e 35 m l / m i n
Temperatures: i n j e c t o r : 250 O C
d e t e c t o r : 300 O C
column: 200 O C f o r 2 min; temperature
g r a d i e n t : 8 OC/min; f i n a l temperature: 300 OC.
F i g . 7 shows a gas chromatogram o f a dichloromethane s o l u t i o n

o f dibenzepin s p i k e d w i t h t h e degradation product and
octacosane as an i n t e r n a l standard.
7.33 H i g h Performance L i q u i d Chromatoqraphy

A HPLC system has been developed on reversed phase
( o c t y l s i l a n i s e d s i l i c a g e l column) f o r assay and p u r i t y t e s t i n g
o f dibenzepin h y d r o c h l o r i d e .
HPLC-conditions
S t a t i o n a r y phase: LiChrosorb@ RP-8 (MERCK), 1 0 pm i n s t a i n l e s s
s t e e l , 25 cm x 4.6 mm i.d.
Mobile phase: i s o c r a t i c : m e t h a n o l / l p e r c e n t ammonium
carbonate s o l u t i o n 65:35 ( v / v )
UV d e t e c t i o n : a t 221 nm
F i g . 8 shows a chromatogram o f t h e drug substance s p i k e d w i t h
t h e d e g r a d a t i o n product. The f l o w was s e t a t 2.0 m l / m i n .
7.4 A n a l y s i s o f t h e Dosaqe Forms
7.41 I d e n t i f i c a t i o n
The i d e n t i f i c a t i o n o f dibenzepin h y d r o c h l o r i d e i n t h e
dosage forms can be c a r r i e d o u t b y t h i n l a y e r chromatography
u s i n g s i l i c a g e l p l a t e s w i t h chloroform/cyclohexane/diethyl-
amine 1:8:1 (v/v/v) and subsequent UV v i s u a l i s a t i o n a t 254 nm.
The most advantageous s p r a y i n g reagents i s D r a g e n d o r f f ' s
reagent w i t h consecutive s p r a y i n g by a m i x t u r e o f 20 m l hydro-
gen p e r o x i d e 30 p e r c e n t and 1 0 m l o f e t h a n o l ( c f . 7.31).
' -
-i- ! '-
! !I
_-
c
. -
min 18 16 14 12 10 8. 6 4 2 0
F i q u r e 7: Gaschromatogram o f Dibenzepin s p i k e d w i t h t h e
degradation product and octacosane ( i n t e r n a l
standard).
Instrument: PERKIN-ELMER 900.
Key:
1 =dichlormethane ( s o l v e n t )
2 = N-( 2-(dimethylamino)ethyl)-N' -methyl-N' -phenyl-
-1,2-benzenediarnine h y d r o c h l o r i d e ( d e g r a d a t i o n
product 1
3 = dibenzepin
4 = octacosane ( i n t e r n a l standard)
min 15 12 9 6 3 0
Fiqure 8: High Performance Liquid Chromatogram

o f Dibenzepin Hydrochloride spiked
with the degradation product.
Reversed-phase Mode, isocratic,
UV detection at 2 2 1 nrn.
Key:
1 dibenzepin hydrochloride
2 N-( 2-(dimethylamino)ethyl)-N'-methyl-
-N'-phenyl-1,2-benzenediamine hydro-
chloride (degradation product)
Dibenzepin can a l s o be i d e n t i f i e d by I R spectroscopy a f t e r

e x t r a c t i o n from t h e dosage form with chloroform.
7.42 Assay
Dibenzepin h y d r o c h l o r i d e i n N o v e r i l @ coated t a b l e t s and
t a b l e t s may by assayed i n a n o n - s p e c i f i c way by d i r e c t
UV spectrophotometry a f t e r e x t r a c t i o n with 0.1 N h y d r o c h l o r i c
a c i d o r , i n case o f s o l u t i o n s ( i n j e c t i o n o r concentrate
intended f o r i n j e c t i o n by i n t r a v e n o u s i n f u s i o n ) a f t e r d i l u t i o n
w i t h 0.1 N h y d r o c h l o r i c acid.
A s p e c i f i c assay o f dibenzepin h y d r o c h l o r i d e i n t h e dosage
form may be c a r r i e d o u t by t l c f o l l o w e d b y UV spectrophoto-
metry ( t h e system can a l s o be used f o r i d e n t i f i c a t i o n pur-
poses). The a c t i v e i n g r e d i e n t i s e x t r a c t e d w i t h methanol. The
chromatographic c o n d i t i o n s are: s i l i c a g e l , mobile phase:
chloroform/cyclohexane/diethylamin 1:8: 1 (v/v/v) .
The spot
corresponding t o dibenzepin i s e x t r a c t e d with 0.1 N hydrochlo-
r i c acid, and t h e c o n c e n t r a t i o n i s determined a t about 285 nm
(shoulder) by spectrophotometry.
A f u r t h e r s p e c i f i c assay i s t h e HPLC d e t e r m i n a t i o n o f dibenze-
p i n h y d r o c h l o r i d e a f t e r e x t r a c t i o n with methanol/water 8:2
(v/v) from t h e dosage form u s i n g LiChrosorb@ RP-8 as s t a t i o -
nary phase and a c e t o n i t r i l e / l per c e n t ammonium carbonate
s o l u t i o n 65:35 (v/v) as t h e m o b i l e phase. UV d e t e c t i o n wave-
l e n g t h i s s e t a t 221 nm.
7.5 Determination i n Body F l u i d s

The i s o l a t i o n , s e p a r a t i o n and i d e n t i f i c a t i o n o f dibenze-
p i n h y d r o c h l o r i d e and i t s m e t a b o l i t e s from u r i n e o f man,
r a b b i t s and dogs i s described i n [81. The methods used a r e
e x t r a c t i o n , t h i n l a y e r and gas chromatography; 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 s were made by UV spectroscopy. Gas chromato-
g r a p h i c procedures f o r t h e d e t e r m i n a t i o n o f t h e drug and i t s
b a s i c m e t a b o l i t e s v i a a c e t y l a t i o n o f t h e demethylated
compounds a r e described i n [ 9 - 131.
The i s o l a t i o n o f dibenzepin h y d r o c h l o r i d e from plasma and
o t h e r body f l u i d s by e x t r a c t i o n or column chromatography and
i t s i d e n t i f i c a t i o n by gas chromatography a r e g i v e n i n [14,151.
A gas chromatographic d e t e r m i n a t i o n o f t h e a c t i v e i n g r e d i e n t
and i t s b a s i c m e t a b o l i t e s i n b i o l o g i c a l m a t e r i a l a f t e r tri-
f l u o r a c e t y l a t i o n i s described i n C161. I n t h a t paper a s i m p l e
procedure i s g i v e n f o r t h e s e p a r a t i o n o f dibenzepin hydrochlo-
r i d e and i t s demethylated m e t a b o l i t e s by i o n - p a i r e x t r a c t i o n .
HPLC may a l s o be used f o r t h e s e p a r a t i o n and d e t e r m i n a t i o n o f

t h e a c t i v e i n g r e d i e n t [17, 181.
Acknowledqements
The a u t h o r s a r e i n d e b t e d t o many c o l l e a g u e s f o r t h e i r
most v a l u a b l e h e l p , i n p a r t i c u l a r t o Mrs. D.A. Giron-Forest
and Messrs. H.-R. L o o s l i , Ch. Quiquerez and W.D. Schoenleber
o f SANDOZ L t d .
Furthermore, t h e a u t h o r s wish t o express s p e c i a l thanks t o
Miss I. Andre f o r h e r s e c r e t a r i a l a s s i s t a n c e i n p r e p a r i n g
t h i s manuscript.
8. References
1. F. Hunziker and J. Schmutz ( t o WANDER L t d . ) , Chem. Abstr.

61, 13331 (1964) and -
- 67, 64455 (1967)
2. F. Hunziker, H. Lauener and J. Schmutz, Arzneim.-Forsch.
(Drug Res.) 2, 324 (1963)
3. D. Bente, M.P. Engelmeier, K. H e i n r i c h , H. H i p p i u s and
W. Schrnitt, Arzneim.-Forsch. (Drug Res.) 14,538 (1964)
4. G. S t i l l e , H. Lauener and E. Eichenberger, Schweiz. med.
Wschr. - 95, 366 (1965)
5. F. Hunziker and 0. S c h i n d l e r , Helv.chirn.acta 48, 1590
(1965)
6. WANDER L t d . unpublished r e s u l t s
7. W. M i c h a e l i s , Arzneim.-Forsch. (Drug Res.) 17,181 (1967)
8. H. Lehner, R. Gauch and W. M i c h a e l i s , Arzneim.-Forsch.
(Drug Res.) 17,
185 (1967)
9. R. Brochon, H. Lehner, R. Gauch and 0. Rudin,
Arch. T o x i c o l . -
24, 249 (1969)
10 a R. Bonnichsen and B. Schubert, Z. Rechtsmed. 68, 253
(1971)
11. E. Klug, Z. Rechtsmed. 71, 27 (1972)
12. W. K i s s e r , Wien Med. Wochenschr. 123, 747 (1973)
13. A. De Leenher and A. Heyndrickx, J. Pharm. Sci. 62, 31
(1973)
14. H.P. Gelbke, T.H. G r e l l and G. Schmidt, Arch. T o x i c o l .
-
39, 2 1 1 (1978)
15. R. Pentz and A. Schutt, Arch. Toxicol. 2,225 ( 1 9 7 8 )

16. H.J. S c h l i c h t and H.P. Gelbke, J. Chromatogr. 166,599
(1978)
17. D.R.A. Uges and P. Bouma, Pharm. Weekbl., ,.I
Sci. Ed 417
(1979)
18. J. Husser and C. Hesse, 5 t h E u r . Symp. Basic Res.
Gerontol. [Lect.] 1976 (Pub. 19771, 739
DIGOXIN
Penelope R . B. Foss and Steven A . Benezra
1. Description 208
1.1 Names 208
1.2 Formula, Structure, Molecular Weight 208
1.3 Appearance, Color, Odor 208
2.7 Solubility 217
3. Synthesis 217
4. Stability 217
5 . Pharmacokinetics, Metabolism, and Protein Binding 217
5.1 Pharmacokinetics and Metabolism 217
6.1 Elemental Analyses 220
6.2 Identification Tests 220
6.3 Fluorometric Analysis 220
6.4 Chromatography 22 1
6.5 Polarography 225
6.6 Colorimetry 230
7. Methods of Analysis-Biochemical Applications 230
7.2 Polarography 239
7.3 Radioimmunoassay 239
8. References 240
Copyright 0 1980 by Academic Press, Inc.

Analytical Profiles of Drug Substances, 9 207 All rights of reproduction in any form reserved.
ISBN: 0-12-260809-7
208 PENELOPE R. B . FOSS A N D STEVEN A . BENEZRA
1. Description
Digoxin is a cardiotonic glycoside obtained from the

leaves of Digitalis lanata Ehrhart (Fam. Scrophulariaceae).
1.1 Names
3~-[(O-2,6-Dideoxy-~-D-~-hexopyranosyl-(l~4)-0-2,6-
dideoxy-~-D-~-hexopyranosyl-(l~4)-2,6-dideoxy-~-D-ribo-
hexopyranosyl)oxy] - 1 2 ~ , 1 4 - d i h y d r o x y - 5 ~ - c a r d - 2 0 ( 2 2 )-enolide2
Cordioxil, Davoxin, Digacin, Dilanacin, Dixina,

Lanocardin, Lanicor , Lanoxin, Rougoxin, Vanoxin2
1.2 Formula, Structure, Molecular Weight
780.96
‘41H64’14
HO
1.3 Appearance, Color, Odor
Digoxiii is an odorless, white crystalline powder.

DIGOXIN 209
2. P h y s i c a l P r o p e r t i e s
The i n f r a r e d spectrum of d i g o x i n i s shown i n F i g u r e 1.3

I t was t a k e n a s a 0.2% d i s p e r s i o n of d i g o x i n i n KBr w i t h a
N i c o l e t Model 7199 FT-IR. Table I g i v e s t h e i n f r a r e d
assignments c o n s i s t e n t w i t h t h e s t r u c t u r e of d i g o x i n .
Table I
I n f r a r e d S p e c t r a l Assignments f o r Digoxin
Band (cm-l) Assignment

3445 0-H s t r e t c h
2930 C-H s t r e t c h of CH3-,
-CH -
1725 C=O s g r e t c h c h a r a c t e r -
i s t i c of 01, $ u n s a t -
urated y lactone
1625 C=C s t r e t c h
1445, 1405, 1375, 1320, 1270 C-H bending v i b r a t i o n s
of -CH , and -CH2-
1163, 1150, 1080, 1020 C-0 s t r e ? c h f o r a l c o h o l s
and e t h e r s
865 C-H bend of t r i s u b s t i -
t u t e d C=C
2.2 Nuclear Magnetic Resonance (NMR) S p e c t r a
The 'H and 13C NMR of d i g o x i n a r e shown i n F i g u r e s 2

and 3.5 T e t r a m e t h y l s i l a n e i s t h e i n t e r n a l s t a n d a r d i n t h e
s o l v e n t s used f o r t h e p r o t o n and carbon NMR.
The 'H NMR was o b t a i n e d w i t h a Varian XL-100A a t

100 MHz w i t h d e u t e r a t e d chloroform a s t h e s o l v e n t . The lH
NMR i s v e r y complex and n o t a l l p r o t o n s can be a s s i g n e d .
P r o t o n s 18-CH3 and 19-CH , p l u s t h e HOD s i g n a l a r e between
0.82-0.95 ppm. The 4',%', and 4"' p r o t o n s a r e from
3.16-3.30 ppm. P r o t o n s 5', 5", 5"' appear between
3.70-3.98 ppm, and p r o t o n s 3', 3", 3"' and 3 a r e between
3.98-4.34 ppm. P r o t o n s l', 1" and 1"' a r e between
4.80-5.02 ppm.6
The 13C NMR was o b t a i n e d w i t h a Varian CFT-20 i n s t r u -

ment a t 20 MHz. 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 was t h e
s o l v e n t . Table I1 g i v e s t h e carbon assignments f o r t h e 13C
NMR.6
0
5:
0
0
0
7
0
0
m
7
u
U
aJ
a
Cn
z
V
0
0
0
cu
0
0
m
N
0
0
0
@Y
0
0
m
Cr)
0
0
0
-r
0 0 0 0 0 0 0 0 0 0 1
~ Q ) c o b c D m - r c 3 c u -
33NVlll WSNVtll
210
21 1
F i g u r e 2 - 'H Nuclear Magnetic Resonance Spectrum of Digoxin

Figure 3 - 13C Nuclear Magnetic Resonance Spectrum of Digoxin
DIGOXIN 213
Table I1
13C NMR Assignments for Digoxin
Carbon No. Chemical Shift (ppm)

7 21.31
10 34.59
13 55.64
14 84.30
17 45.16
18 9.34
19 23.58
20 176.69
22 115.84
23 173.82
1’ 98.91
1” 98.91
1’ ’ ’ 95.30
HO
214 PENELOPE R . B. FOSS AND STEVEN A. BENEZRA
2.3 Ultraviolet (W)Spectrum
The W spectrum of digoxin in ethanol was taken with a

Beckman ACTA CIII W spectrophotometer and is shown in
Figure 4.4 Digoxin has o2e maximum in the W spectrum at
220 nm with E = 1.28 x 10 .
2.4 Mass Spectrum
The mass spectrum of digoxin as shown in Figure 5 was

obtained with a Varian MAT CH5-DF mass spectrometer.7 The
direct probe temperature was 290', and the electron energy
was 70 eV. The major fragmentation pattern characteristic
of the aglycone portion of digoxin is outlined below.a
HO - - C23H3304
-H20
Ci?3H3103
M/E 390' M/E 373 M/E 355

C23H3406
2.5 Optical Rotation

The optical rotation of di.goxin has been determined
under different conditions.
25 + 13.6' to 14.2' (C=lO in pyridine)'

['I Hg
[a]:' + 18.9' (C=l in pyridine)2
+ 30.4' (C=1.2 in alcohol)2

2.6 Melting point
Digoxin melts and decomposes between 23Oo-265'C.

0
0
m
G
.I4
X
0
M
0 .I4
Lo n
cv W
0
I I I 1 I 1 I I I 1 1
0 0 0 0 0 0
0
7
co CD w- cv
AlISN31NI 3A11Vl3tl
DIGOXIN 217
2.7 Solubility
Digoxin is freely soluble in pyridine, slightly soluble
in 1 : l ethanol:water, chloroform, and practically insoluble
in water and in ether.1'2
3. Synthesis
No successful synthesis of digoxin has been reported.

Digoxin is obtained commercially from the ethanolic extrac-
tion of Digitalis lanata leaves followed by chromatographic
purification.
4. Stabilityg
Digoxin is stable indefinitely when kept in the dark in

well closed containers. No degradation is noted in tablets
after five years when stored in tightly closed containers.
Solutions of digoxin hydrolyze in the presence of acids

yielding digoxigenin bis-digitoxoside, digoxigenin mono-
digitoxoside and digoxigenin. The latter degrades further
to anhydrodigoxigenin under anhydrous acid conditions.
Neutral solutions of digoxin in ethanol and propylene

glycol are stable up to five years.
Digoxin solutions are relatively stable to light except

when stored under intense light for long periods o f time.
Degradation is by apparent opening of the lactone ring and
can be detected by a lowering of the ultraviolet absorbance
and by HPLC assays.
Only chromatographic procedures can be used to

determine digoxin in the presence of all its breakdown
products.
5. Pharmacokinetics, Metabolism, and Protein Binding
5.1 Pharmacokinetics and Metabolism
In man digoxin is 60-80% absorbed and has a biologic

half life of 1.5 to 2.0 days.1° In the anuric patient the
half-life is prolonged to four to six days. To determine
which dosage form has the best bioavailability digoxin was
given by intravenous infusion, intramuscular injection, oral
elixir, and tablet to human subjects." The fate of the
glycoside is similar regardless of the dosage method used. l2
The bioavailability of the dosage forms was compared by
218 PENELOPE R . B. FOSS AND STEVEN A. BENEZRA
serum concentration levels and cumulative urinary excretion.l1

The dosage forms, in order of highest to lowest resulting
serum concentration levels, and excretion, were intravenous
infusion, intramuscular injection, oral elixir, and tablet.
An improved digoxin tablet with a more rapid dissolution
rate, showed twice the absorption rate of the previous
tablet and a forty percent increase in urinary excretion.l3
A new encapsulated liquid digoxin (a soft gelatin capsule
containing the glycoside in a dissolved form) was superior
in bioavailability to the rapid dissolution tablet and the
solution.14''' The capsule's absorption approaches that
of the intravenous dosage forms.
In postmortem examinations of patients with normal

renal function, the highest concentration of digoxin was in
the kidney, followed by the heart and liver.16 The lowest
concentration of digoxin was in the brain. Studies in
anephric patients and those with renal failure show the
highest concentration of digoxin to be in the heart followed
by the liver, and the kidney. When digoxin content was
measured in samples of left ventricular papillary muscle,l7
skeletal muscle, and plasma of human patients during heart
surgery, the papillary muscle digoxin concentration averaged
77 ng/g, the skeletal muscle, 1 1 . 3 ng/g and plasma, 1-2 ng/mL.
A significant amount of total body digoxin is stored in the
skeletal muscle since skeletal muscle represents 43% of the
body weight. A relatively wide range of digoxin concen-
tration in atrial heart tissue is commensurate with satis-
factory digitalization. Myocardial tissue samples taken two
hours after the intravenous administration of tritiated
digoxin revealed a significant variation in digoxin concen-
tration in and around the infarcted zone. The infarcted
tissue'' demonstrated a tissue to serum ratio of 12:l. The
therapeutic activity of digitalis is likely to depend on the
concentration at the active sites in the tissues rather than
in the plasma.
The quantity of digoxin excreted each day is a function

of the amount present in the body. Excretion during the
first twenty-four hours has been determined to be between
20-50% of the dose.l g y 2 O Digoxin undergoes appreciable
biliary excretion after intravenous dosing in man, however,
total fecal recovery is low, with figures ranging from 6-20%
of the dose of digoxin.lg''O'l' Doherty -
et -
a1.21 determined
that only 6-8% of the given dose of digoxin is recycled
through the bowel. Digoxin is excreted predominantly through
the kidney.
In dogs, it was found that approximately sixty percent2*

DIGOXIN 219
of the metabolism of digoxin takes place in sites other than

the liver. The heart muscle was found to have a negligible
role in digoxin metabolism. A significant amount of digoxin
is excreted unmetabolized. The following digoxin metabo-
lites are present in the lipid-extractable fraction of urine
or plasma : dihydrodigoxin, digoxigenin bis-digitoxoside,
digoxigenin mono-digitoxoside, dihydrodigoxigenin monodigi-
toxide, and digoxigenin. 23 Dihydrodigoxin is the major
metabolite. In various animals the activity of dihydrodig-
oxin has been measured to be 1 / 7 to 1/20 the activity of
digoxin.24 All glycolytic reduced and nonreduced metabo-
lites were found except for dihydr~digoxigenin~~ which is
often detectable only in patients using very high doses of
digoxin. Metabolic conversion of digoxin includes the
stepwise hydrolysis of the sugar units, conjugation to form
water soluble metabolites, epimerization at C-3, and reduc-
tion of the lactone ring which destroys the activity of
digoxin.23
5.2 Protein Binding
Digitalis protein binding is important because tissue

uptake is related to free drug and not to total drug concen-
tration. The variations reported in protein binding of
digoxin may result from differences in methodology and may
a l s o occur when using the same method, but in different
laboratories.
Significant species differences in binding have been

reported for digoxin. B a g g ~ treported
~~ the range to be
17-40% binding. Storstein26 reported the range to be 5-60%.
Most investigators found digoxin binding to be about 20%.
Storstein26 used equilibrium dialysis and ultrafil-

tration for measuring the protein binding of digoxin. With
equilibrium dialysis 21-24% of digoxin was found to be
protein bound. The glycoside concentration was within
therapeutic range, and the dialysis was performed at room
temperature. Serum or human albumin was used for the equi-
librium dialysis. Storstein reported that the ultrafiltra-
tion results were not accurate.
Doherty and Hall27 reported that the lack of affinity

for serum protein binding for digoxin appears to be a func-
tion of its polarity. The polar structure of digoxin tends
to render chemical protein binding of the drug less likely
to occur.
Protein binding of digoxin was found to be normal in

220 PENELOPE R. B. FOSS AND STEVEN A. BENEZRA
uremic patients, but decreases during hernodialysis.26
6. Methods of Analvsis
Elemental analysis2 of digoxin as C41H64014

C 63.06%
H 8.26%
0 28.69%
6.2 Identification Tests'
Digoxin is dissolved and diluted with hot ethanol and

an aliquot is evaporated to dryness. Acid-ferric chloride
TS is added to the residue. A green color develops that
slowly changes to a deep green-blue.
Digoxin is dissolved and diluted with hot ethanol. An

aliquot of the solution is evaporated to dryness then dis-
solved in a solution of methanol and chloroform (1:Z). The
sample is spotted onto Whatman No. 1 filter paper that has
been impregnated with a solution of formamide and acetone
( 3 : 7 ) . The sample is developed with chloroform saturated
with formamide. After development the paper is heated for
fifteen minutes at 90°C then sprayed with trichloroacetic
acid in chloroform and hydrogen peroxide and reheated to
90°C for ten minutes. The sample is viewed under W light
and compared to the standard.
6.3 Fluorometric Analvsis
Fluorometry has been used to simultaneously determine

digitoxin and digoxin in leaves, tincture, tablets, and
drug.28 An Aminco Bowman spectrofluorometer was used for
the determination of the excitation and emission spectra,
and a Turner model 110 was the fluorometer used for the
analysis. The reagent was a mixture of acetic anhydride,
acetyl chloride, and trifluoroacetic acid. Digoxin has two
excitation peaks, one at 470 nm, the same as digitoxin, and
a second at 350 nm. The fluorescence peaks for both digi-
toxin and digoxin occur at 500 nm. With a 47B + 2A-12
filter combination the reading was found to be a sum o f the
fluorescence of digoxin and digitoxin. To correct for
digoxin fluorescence the 7-60 + 2A-2ND filter combination
was used because it allows the determination o f the emission
of digoxin alone. The results were linear over the concen-
tration range of 0.5 to 6 pg/mL. The accuracy, based on
2 pg/mL was 99.2% of theory.
DIGOXIN 22 1
Fluorinietric analysis was also used for the determi-

nation of digoxin in tablets.29 A Technicon automatic
analyzer was used for the analysis. The reagents and solu-
tions were, 70% SD3A alcohol in water, hydrochloric acid,
ascorbic acid, hydrogen peroxide, and standard digoxin.
Three standards of appropriate levels and samples of the
intact or powdered tablets were used. Excitation and
emission wavelength maxima for digoxin were 350 nm and
490 nm respectively. Spectral measurements were made on a
Farrand Spectrofluorometer. The procedure was stability
indicating, and a linear relationship existed between
fluorescent intensity and digoxin concentration. The
relative standard deviation of a 0 . 2 5 mg digoxin sample was
21.2%. None of the tablet excipients interfered with the
procedure.
The following fluorimetric assay procedure has also

been used for the analysis of digoxin in tablets.30 Ten mL
of 80% alcohol was added to a tablet, in a volumetric flask,
warmed on a steam bath until the tablet was dispersed, and
the alcohol boiled. The mixture was cooled, swirled, and
diluted to 20 mL with 80% alcohol. After standing for
15 minutes 5 mL of the supernatant was pipetted into a 20 mL
volumetric flask and diluted to volume with 80% alcohol.
Three mL of 0.1% solution of ascorbic acid in methanol,
0 . 2 mL of .009 M hydrogen peroxide in water were added to a
1 mL aliquot of the sample solution. The solution was
diluted to ten mL with hydrochloric acid and allowed to
stand for two hours in the dark. The standard was prepared
in a similar manner. For the fluorescence measurement the
excitation maximum was at 355 m and the emission maximum
was at 490 nm.
6.4 Chromatography
Paper c h r ~ m a t o g r a p h yhas
~ ~ been used to separate the
components of a digitalis tincture. Whatman 3MM paper
impregnated with formamide and developed in chloroform gave
an R f = 0 . 3 3 for digoxin. a variety of spray reagents were
used to detect digoxin.
Table I11 gives various thin layer chromatography

systems which have been used for the separation of digoxin.
Table I11
Thin Layer Chromatography for Digoxin
Comment, R f or
Relative Order of
Adsorbent Mobile Phase Spray Reagent Elution Ref.
-
Silica Gel G Cyclohexane-acetone- 50% aq sulfuric acid Digoxin appears as 32
acetic acid ( 4 9 : 4 9 : 2 ) a blue spot under
385 nm W light
or or
Ethyl acetate-water- 30% aq soln chloramine
methanol ( 8 0 : 5 : 5 ) and 25% alcoholic soln
trichloracetic acid
(1:4)
Silica Gel F Ethyl acetate-methanol- 6 g of trichloroacetic Digoxin 0 . 3 33
water ( 8 0 : 5 : 5 ) acid in 25 mL chloro-
form and 0.5 mL 30%
w/v hydrogen peroxide
Silica Gel Chloroform-acetone 50% methanolic sulfuric Digoxin, digoxigenin 34
(non-fluorescent) (1:l) acid bis-digitoxoside,
digoxigenin mono-
digitoxoside,
a-anhydrodigoxigenin,
8-anhydrodigoxigenin
Comment: 2-25 hrs
continuous development
Dichloromethane- Digoxin, gitoxigenin,
methanol ( 9 :1 ) digitoxigenin
Table I11 (continued)
Comment, R or
f
Relative Order of
Adso rbent Mobile Phase Spray Reagent Elution Ref.
-
Silica Gel 60 F -
Ethylacetate-dichloro- 20% v/v orthophosphoric Gitoxigenin, 35
254 methane-methanol-water acid digoxigenin, f3-acetyl
(60:36:3.5:2) digoxin, digoxigenin
mono-digitoxoside,
or-acetyl digoxin,
digoxigenin bis-digi-
toxoside, gitoxin,
digoxin, digitoxin,
digitoxigenin
Chloroform-pyridine Digitoxin, digoxin
N
N
W (60: 1 0 )
Dichloromethane- Digitoxin, digoxin
methanol (9O:lO)
Silica Gel F Chloroform-acetone 20% v/v orthophosphoric Digoxigenin, dig- 36
254 (1:l) acid oxigenin mono-
digitoxoside,
digoxigenin-
bis-digitoxoside,
digoxin
Kieselgel 60 Ethylacetate-dichloro- All cardenolides are 37
DC-Fertigplatten methane-methanol- referenced relative
water ( 1 2 0 : 7 2 : 7 : 4 ) t o digoxin.
Table I11 [continued)
Comment, R or
f
Relative Order of
Ad so rben t Mobile Phase Spray Reagent Elution -
Ref.
and Dichloromethane- Comment :

methanol (9 :1) Mobile phase 1: con-
tinuous development
3 hrs, mobile phase 2:
continuous development
2 hrs. The plate is
turned 90° after
development by mobile
phase 1.
DIGOXIN 225
6.43 Gas Chromatography
Digoxin, as a tablet or as the powdered drug,32 was

converted to digoxigenin for analysis by gas chromatography.
The separation was achieved by using three columns, (A) a
two meter glass U tube packed with 2.5% OV-1 on 80-100 mesh
Chromosorb A , (B) a 0.5 meter copper U tube with 2.5% OV-1
on 80-100 mesh Chromosorb A , and (C) 3% OV-17 on 80-100 mesh
Chromosorb A . Cholesterol was used as the internal standard.
The oven temperature was 285OC. The injection port and the
flame ionization detector temperature were 330OC. All
injections were made with a 5 pL syringe. The detection
range was 0.05-0.2 mg. There was little difference in the
retention times of the silylated drug and standard versus
those of the unsilylated drug and standard.
Retention times (min)
Column A B
- C
-
Cholesterol unsilylated 2.0 0.67 0.5
s ilylated 2.0 0.67 0.5
Digoxigenin unsilylated 15.0 5.33 3.67

silylated 15.0 5.0 3.67
6.44 High Performance Liquid Chromatography
Table IV gives various HPLC systems used for digoxin.
6.5 Polarography
Polarography has been used for the assay of digoxin

tablets.4 7 The working electrode was a dropping mercury
electrode with a one second drop time, and the reference
electrode was a saturated calomel electrode (SCE) with a
platinum wire as an auxillary electrode. The linear poten-
tial sweeps were constant at 5 mV/sec, and the pulse modu-
lation was 25 mV. A 2-mL aliquot of the extraction of the
ground tablets plus 0.2 mL of 0.2 M TBAI, tetrabutylammonium
iodate, or of 0 . 2 M TBAH, tetrabutylammonium hydroxide, the
supporting electrolytes, was added to 2 mL of isopropanol.
Before each experiment the solution was deaerated with
isopropanol saturated nitrogen, which was also passed over
the solution during the assay. The potential was scanned
cathodically from -1.8 volt. The peak potential of digoxin
was -2.285 xolts. The usef -8 1 analytical range of the assay
was 5 x 10- M to 2.5 x 10 M of digoxin.
Table IV
HPLC Systems for Digoxin
Flow (mL/min)
or
Column Mobile Phase Pressure Retention Time (min) Detection Ref.
Li Chrosorb SI60 n-Butanol- 1.3 3.5 225 nm 38

(25 cm x 3 mm id) acetonitrile-heptane-
water (230:100:700:10)
t-Butanol-acetonitrile- 2.2 10
heptane-water
(220:70:800:10)
(204:93:712:10.4) 3.6
n-Pentanol-aceto- 1.3 3.8

nitrile-iso-octane-
water (270:93:660:9.3)
(230:100:700:10) 1.4 5.2
(170:60:620:10) 1.3 10.4
(175:60:620:6) 8.2
Table IV continued
Flow (mL/min)
or
Column Mobile Phase Pressure Retention Time (min) Detection __
-~ Ref.
Merckosorb S160 Comment: The digitalis glycosides are derivatized 254 nm 39

5 I.rm with 4-nitrobenzoyl chloride (4N BC1) or
(15 cm x 3 mm id) 260 nm
n-Hexane-methylene 1.5 5.6

chloride-acetonitrile
(10:3 :3 )
n-Hexane-chloroform- 1.5 5.9

acetonitrile (30:10:9)
Li Chrosorb S160 8% Methanol in methy- 2.0 1.3 230 40

5 I.rm lene chloride saturated
(15 cm x 3 mm id) with water
Nucleosil CI8 37% Acetonitrile 1.4 4 220 40

(30 cm x 3.5 mm id) in water
40% Solution of 1.3 5.4

1:l acetonitrile-
dioxane in water
Table IV continued
Flow (mL/min)
or
Column Mobile Phase Pressure Retention Time (min) Detection Ref.
Whatman ODs-1 540 mL of acetonitrile 3.0 5.6 220 nm 41
(25 cm x 4.2 mm id) diluted to two liters (tablets)
with water 7.0
(injection)
5.4
(pediatric
injection)
450 mL of acetonitrile 3.0 12.4 (elixir)

diluted to two liters
with water
Li Chrosorb S160 Cyclohexane-absolute 2 8 265,234 nm 42

(25 cm x 4 mm id) ethanol-acetic
acid (60:9: 1)
Whatman ODs-1 780 mL Acetonitrile 2.25 9 220 nm 43

(30 cm x 4.2 mm id) diluted to three liters
with water
Perisorb RP Water with 25% aceto- 0.75 4.5 254 nm 44

( 1 x 2 mm id) nitrile
Table IV continued
Flow (mL/min)
or
Column Mobile Phase Pressure - Retention Time (min) Detection Ref.
Zo rbax-S IL 6% Methanol + 0.15% 1500 p s i 4.5 254 nm 45
(25 cm x 2 . 1 mm id) acetic acid in
methylene chloride
3% Methanol + 0.1% 0.5 10 254 nm, 46

acetic acid in or 235 nm
methylene chloride 1200 p s i
N
W
N
6.6 Colorimetry
An alkaline d i n i t r ~ b e n z e n ereagent
~~ has been used for
a colorimetric assay of crystalline powder, tablets, injec-
tions, and elixirs containing digoxin. The dinitrobenzene
reagent was added to standard and sample preparations that
have been evaporated to dryness. The mixture was allowed to
stand for five minutes, with frequent stirring, at a room
temperature not exceeding 3OOC. The absorbance o f the
resulting blue color was measured at 620 nm versus the
reagent blank and the USP digoxin standard.
The following method of assaying dosage samples used a

color reagent of glacial acetic acid49 containing ferric
chloride and sulfuric acid. After an initial extraction
procedure tailored to the sample type, the sample was dis-
solved or diluted in a chloroform-methanol solution (65:35)
then diluted with glacial acetic acid. An aliquot of
digoxin solution was diluted with color reagent and allowed
to stand for two hours. The absorbance of the sample was
measured at 590 nm versus that of a digoxin standard.
The following two colorimetric procedures have been

used for the assay of tablet samples. The first employed an
alkaline sodium picrate reagent. A crushed digoxin tablet
was placed in a 10-mL volumetric flask and diluted with 6-mL
of absolute alcohol. The flask was heated to 4OoC and
shaken for two hours. The solution was diluted to volume
with alcohol, then centrifuged. A 3-mL aliquot of the
reagent was added to a 5-mL aliquot of the sample. The
solution was stored in darkness for 30 min. The absorbance
was measured at 490 nm versus a reagent blank.
The Xanthydrolso method was another tablet assay pro-

cedure. In a 50-mL volumetric flask a tablet was crushed in
a solution of three mL of hot chloroform/methanol (65:35)
and two mL of glacial acetic acid. Twenty mL of xanthydrol
reagent was added to the mixture. The flask was heated for
five min in a 7 5 O C water bath then cooled for five min in an
ice bath. The standard and blank were prepared in a similar
manner. The absorbance was measured at 540 nm.
7. Methods of Analysis - Biochemical Applications
7.1 Chromatography
Table V gives various paper chromatography systems

Table V
Paper Chromatography for Digoxin and Metabolites
Comment, Rf,
or Relative Order
Adsorbent Mobile Phase Spray Reagent of Elution Ref.
-
Whatman No. 1 Chloroform saturated 25% trichloroacetic 51
filter paper with formamide acid solution in dihydrodigoxin,
impregnated chloroform with four
with formamide drops of hydrogen
(30% in acetone) peroxide/50 mL
Whatman No. 3 Chlorofo rm-methanol m-dinitrobenzene digoxin 0.50 52

filter paper (1:l)
soaked with
formamide-acetone
(1:3)
which have been used for the separation of digoxin and its
metabolites.
Digoxin and its metabolites have been separated and

assayed by reverse phase high performance liquid chromato-
graphy.53 The column was a pBondapak C 1 8 (30 cm x 4 mm
id). The sample solvent was 95% ethanol, and the injection
size was 50-75 pL. The detector was set to 220 nm. Listed
below are four mobile phases that achieved the desired
separation.
Isocratic systems:
The flow rate was 3 mL/min
1. 25% acetonitrile in water R of digoxin was 13 min.
t
2. 33% acetonitrile in water Rt of digoxin was 23 min.
Gradient systems:
The flow rate was 2.2 mL/min
1. 25% acetonitrile in water to 40% acetonitrile in
water at 5%/min. Rt of digoxin was 10 min.
2. 100% water to 30% acetonitrile in water at 6.67%/
min. R of digoxin was 23 min.
t
7.13 Column Chromatography
Column chromatographys4 has been used for the separa-

tion of digoxin and dihydrodigoxin extracted from urine
samples. The adsorbent was diethylaminoethoxypropylated
Sephadex LH-20 (DEAE-Sephadex LH-20). The mobile phase was
chloroform-methanol (85:15). Samples were applied in
0.2-0.5 mL volumes of eluting solvent. The flow rate for a
40 x 1.0 cm column was 0.25 mL/min.
Dihydrodigoxin Ve/Vt = 0.25
Digoxin Ve/Vt = 0.34
The flow rate for a 36 x 2.5 cm column was 0.20 mL/min.
Dihydrodigoxin Ve/Vt = 0.43
Digoxin Ve/Vt = 0.48
DIGOXIN 233
Table VI gives various thin layer chromatography sys-

tems which have been used for the separation of digoxin and
its metabolites extracted from biological samples.
A single column gas chromatographic determination62 of

digoxin and its metabolites has been achieved with either
isothermal or temperature programming. Digoxin and its
metabolites were converted to trimethylsilyl (TMS) deriva-
tives prior to analysis. The column (U shaped, 1 ft x 4 mm
id) was packed with 1.6% SE 30 on 80-100 mesh Gas Chrom Q.
The sample injection volume was 10 pL. The instrument used
was a Barber-Colman 5000 gas chromatograph equipped with a
hydrogen flame ionization detector. Under isothermal condi-
tions the column temperature was set to 3OOOC and the detector
temperature was set to 320OC. The injection block temperature
was maintained at column temperature. The nitrogen flow was
125 mL/min. The retention time o f digoxin was approximately
eighteen minutes. With temperature programming from 23OOC
to 33OOC at boC/rnin, one minute initial delay, the retention
time of digoxin was approximately twenty-four minutes. The
detector temperature was 3 4 O O C and the nitrogen flow was
60 mL/min.
Digoxin and its metabolites, derivatized with hepta-

fluorobutyric anhydride,6 0 ’ 5 6 have been resolved on a gas
chromatographic column packed with 3% OV-1 on Gas Chrom Q.
Digoxin and its metabolites were extracted from urine,
plasma, biological tissue, and fecal samples. The compounds
were initially separated by paper and/or thin layer chroma-
tography. Before the extraction 3H-digoxin can be added as
an internal standard. The gas chromatograph used was a
Tracer MT-220 with a 63Ni electron capture detector. With a
U-shaped column ( 4 ft x 2 mm id) at 25OOC and a detector at
35OoC the retention time for digoxigenin HFB was nine minutes.
With a U-shaped column (6 ft x 2 mm id) at 250°C and detector
at 325OC the digoxigenin HFB retention time was eight minutes.
A Varian CH-7 GC-MS combinations6 was used for an

determination of digoxin and its metabolites. For the GC a
column packed with 3% OV-1 on Chromosorb W AW DMCS 100/120
(6 ft x 2 mm id) was used. The column temperature was
25OoC, injector temperature, 26OoC, and the molecular separa-
tor, 260OC. For the mass spectrometer, electron energy was
20 eV, the ion source temperature, 25OoC, and the trap
current, 300 PA. The accelerating voltage was 3KV and the
Table VI
Thin Layer Chromatography for Digoxin
Comment, Rf or
Relative Order
-
Silica Gel G Cyclohexane-acetone- Lieberman-Burchard Digoxin: 0.21 55

acetic acid (65:33:2) (acetic anhydride- Comment: Plates are
sulfuric acid-ethanol developed six times
(5:5 :50) to a height of 15 cm.
Chloroform-ethanol Digoxin: 0.77

(2: 1) Comment: Plates
E
P are developed
once to 10 cm
Silica Gel GF Chloroform-acetone Digoxin: 0.32 56
(13:7)
Silica Gel H Cyclohexane-acetone- 20% sulfuric acid soln Digoxin: 0.09 57

acetic acid (65:33:2) or Comment: plates
Anisaldehyde reagent developed four
(0.5 mL anisaldehyde, times
1.0 mL sulfuric acid,
50 mL acetic acid)
Table VI (continued)
Comment, Rf o r
Relative Order
Adsorb en t Mobile Phase Spray Reagent of Elution Ref.
~
Silica Gel F Solution of conc sulfuric 58

254
acid in ethanol ( 1 : 4 )
Cyclohexane-acetone-
acetic acid Digoxin: 0 . 1 6 (lined tank)
( 4 9 :49 :2) 0.33
( 4 9 :49 :2 ) 0 . 3 4 (lined tank)
(45:45:10) 0.59
(16:80:4 ) 0.13
Chloroform-pyridine
(64:6)
% formamide in
acetone for
impregnation
10% (64:6) 0.38
2-Butanone-xylene-formamide 0.12
10 ( 5 0 : 5 0 :0)
10 (50:50:4 ) 0.09
15 (50:50:4) 0.10
20 (50:50:4 ) 0.09
10 ( 7 0 :3 0 : 0) 0.36
2-Butanone-xylene
15 (50:50) 0.16
Table VI (continuedl
Comment, Rf or
Relative Order
-
Silica Gel G Ethyl acetate-chloro- Digoxin 0.15 one 59

Or G254
form-acetic acid development
( 9 0 : 5 :5 ) 0 . 2 8 two
developments
Cyclohexane-acetone- Digoxin: 0.29

acetic acid ( 4 9 : 4 9 : 2 )
acetic acid ( 6 5 : 3 3 : 2 0.36 one development
and in each mobile phase
acetic acid ( 4 9 : 4 9 : 2 )
Cyclohexane-acetone-acetic acid
( 6 5 : 3 3 : 2 ) and 0.12 one development
Ethyl acetate-chloroform in each mobile phase
(9:1 )
Chloroform-isopropanol-acetone 0 . 1 8 two developments
(80:5: 15)
Table VI (con tinued)
Comment, Rf or
Relative Order
-
Silica Gel Chloroform-methanol Chose one of following: 0.33 two developments 51

F254
(saturated with ( 1 ) 25% trichoroacetic
AgN03)-ammonia ( 9 : l : l ) acid soln in chloroform
with four drops of
hydrogen peroxide per
50 mL.
( 2 ) acetic anhydride-
sulfuric acid-abs ethanol
( 5 : 5 : 100)
( 3 ) 0 . 0 5 mL p-anisalde-
hyde, 0.2 mL conc
sulfuric acid, 10 mL
acetic acid
( 4 ) 20 mg ascorbic
acid, 19 mL methanol,
30 mL conc hydro-
chloric acid, 2.1 pL
30% hydrogen-peroxide.
( 5 ) 10 mL of 3% aq soln
chloramine T, 40 mL 25%
trichloroacetic acid in
ethanol
Comment, Rf or
Relative Order
Cellulose Chloroform saturated Reagent 5 above 0.33 51
(MN-300) with formamide
predipped
with formamide
in acetone
Mallinckrodt Isopropyl ether- 0.09 developed five
Chromar 7GF methanol (9:l) times
Isopropyl ether- 0.26 developed four
methanol (9:1) times in mobile
and phase 1, developed
2-Butanone-chloroform (3:l) one time in mobile
phase 2
Kieselgel 60 Chloroform-methanol- Digoxin: 0 . 2 4 61
DC acetone-water
Fertigplatten (64:6:28:2)
Any of the following
techniques can be used for
enhanced detection:
(1) Chloramine-trichloro-
acetic acid spray
(2) HC1 vapor
( 3 ) Coating the plate with
a thin film of parafin
DIGOXIN 239
spectrum was scanned every six seconds.
7.2 Polarography
The polarographic analysis63 of digoxin has been used

for assaying the drug as well as blood samples containing
the drug. The study of the polarographic characteristics of
digoxin in a 50% alcohol solution containing tetraethyl
ammonium hydroxide showed a half wave potential of -1.965
volts for an alcoholic solution of the drug and -1.958 volts
for the drug extracted from blood samples. At concentrations
of 0.1-0.4 pg of digoxin in the blood, the error o f the
method was 20.02 pg.
7.3 Radioimmunoassay
Employing 3H digoxin tracer and antiserum solutions

available in a commercial kit, optimum conditions were
determined for the radioimmunoassay of digoxin in plasma,
serum, and urine.64 A summary of the procedure is given
below.
Phosphate buffered saline solution, plasma, and 30%

ethanol water were added to each tube and vortexed. The
antiserum was added, vortexed, and preincubated, then the
tracer solution was added, vortexed, and incubated. The
charcoal suspension was added, vortexed, and centrifuged.
The supernatant was decanted into 15 mL of liquid scintil-
lation fluid and counted. The range of the assay was
0.05 pg/mL to 5 ng/mL of digoxin.
Tritiated digoxin has also been used €or the determi-

nation of digoxin in liver tissue.65 A liquid-liquid
extraction was used to obtain the glycoside. The radio-
activity was determined with a liquid scintillation counter.
The solvent was 5 mL of 95% ethanol plus 15 mL of toluene.
The total counting volume contained 4 g/L of 2,5 diphenyl-
oxazole (PPO) and 50 mg/L of 1,4-bis-2(5-phenyloxazole)-
benzene (POPOP). The average recovery rate for the
procedure was 95.6% and the sample size was 1 mg.
The amount of digoxin in human plasma has been assayed

by radioimmunoassay with an iodinated tracer.66 The reagents
for the assay were digoxin, labelled digoxin (3-0-succinyl
digoxigenin [1251]tyrosine derivative), a dilute phosphate
buffer containing sodium chloride, bovine albumin powder,
sodium azide, anti-digoxin serum, dextran coated charcoal,
and normal digoxin free human serum. All standards and
specimens were set up in assay tubes and had cold dextran-
coated charcoal suspension added. The tubes were centri-

fuged and the supernatant placed in a separate assay tube.
The supernatant fluid and charcoal were both counted for one
minute. Digoxin values of pg/L of plasma were calculated
from a standard curve of the percent tracer bound versus pg
of digoxin per liter. The useful working range of the assay
is 0.2 to 8 pg/L of plasma. Fifty pL of plasma was used.
The amount of 1251-labelled digoxin has been determined

in a 10-pL sample of serum with a modification of a Curtis
Digoxin RIA kit assay procedure. The following is a descrip-
tion of the micro-radioimmunoassay procedure. Each polymer
tablet was dissolved in the sodium chloride solution, and
100-pL aliquots of the solution were pipetted into the test
tubes. Ten pL aliquots of each standard (0-4.8 pL of
digoxinlliter) and patients sera were pipetted into a
polymer slurry, mixed, and let stand for ten minutes. Ten
pL of 1251-labelled digoxin was pipetted into each test
tube, mixed and let stand for 30 min. Twice, saline was
added to each test tube, centrifuged, and decanted. The
contents of the test tubes were counted for ten minutes for
radioactivity.
8. References
1. The United States Pharmacopeia, XIX, (1975).

2. Merck Index, Ninth Edition, (1976).
3 . W. Martin, Burroughs Wellcome Co., private communi-
cations, (1979).
4. P.R. Booze FOSS, Burroughs Wellcome Co., unpub-
lished data.
5. R. Crouch, Burroughs Wellcome Co., private communi-
cation, (1979).
6. S . Hurlbert, Burroughs Wellcome Co., private
communication, (1979).
7. R.L. Johnson, Burroughs Wellcome Co., private
8 . P. Chandrasurin, Burroughs Wellcome Co., private
9. J . E . Murphy, Burroughs Wellcome Co., private
10. A.F. Lyon, C.C. DeGraff, American Heart Journal,
7216, 838 (1966).
11. D.J. Greenblatt, D.W. Duhme, J. Koch-Weser,
T.W. Smith, The New England Journal of Medicine,
-
289, 651 (1973).
12. J.E. Doherty, W.H. Perkins, The American Journal
of Cardiology, - 15, 170 (1965).
D1GO XI N 24 1
13. D. Falch, A. Teien, C.J. Bjerklund, British Medical

Journal, 1, 695 (1973).
14. J. LindenEaum, Clinical Pharmacology and
Therapeutics, 21, 278 (1977).
15. G.I. Mallis, D X . Schmidt, J. Lindenbaum, Clinical
Pharmacology and Therapeutics, 18, 761 (1975).
16. J.E. Doherty, W.H. Perkins, W.J. Flanigan, Annals
o f Internal Medicine, 66, 116 (1967).
17. J. Coltart, M. Howard,x. Chamberlain, British
Medical Journal, 2, 318 (1972).
18. A.J. Thompson, J. Hargis, M.L. Murphy, J.E. Doherty,
American Heart Journal, 88, 319 (1974).
19. J.E. Doherty, W.H. Perkins, American Heart Journal,
63, 528 (1962).
20. EI.Marcus, L. Burkhalter, C. Cuccia, J. Pavlovich,
G.G. Hapedia, Circulation, 34, 864 (1966).
21. J.E. Doherty, W.H. Hall, M.L. Murphy, O.W. Beard,
- 59, 433 (1971).
Chest,
-
22. R.M. Abel, R.J. Luchi, G.W. Perkin, H.L. Corn, Jr.,
L.D. Miller, The Journal of Pharmacology and
Experimental Therapeutics.,L O , 463 (1965).
23. U. Peters, L.C. Falk, S.M. Kalman, Archives o f
Internal Medicine, 138, 1074 (1978).
24. D.R. Cork, S.M. K a l G , Drug Metabolism and
2, 148 (1974).
Disposition, -
25. J.D. Baggot, L.E. Davis, Res. Vet. Sci., 15, 8 1
(1973).
26. L. Storstein, Clinical Pharmacology and
Therapeutics, 20, 6 (1976).
27. J.E. Doherty, C H . Hall, J. Sherwood, D. Gerkin,
J. Gammill, The American Journal o f Cardiology,
28, 326 (1971).
-
28. I.M. Jakovljavic, Analytical Chemistry, 35,
1513 (1963).
29. L.F. Cullen, D.L. Packman, G.J. Papariello, Journal
o f Pharmaceutics Sciences, 3,697 (1970).
30. A. Waghorn, J.A. McCrerie, The Wellcome Foundation
Ltd., private communication.
31. H. Brindle, G . Rigby, S.N. Sharma, Journal of
Pharmacy and Pharmacology, 1,942 (1955).
32. A.H. Kibbe, O.E. Aracys, Journal of Pharmaceutical
Sciences, 62, 1703 (1973).
33. L. Hicks, The Wellcome Foundation Ltd., private
communication.
34. J.D. Mills, The Wellcome Foundation Ltd., private
communication.
35. C.J. Clarke, P.H. Cobb, The Wellcome Foundation,
private communication.
36. C.J. Clarke, The Wellcome Foundation; private

communication.
37. C.J. Clarke, P.H. Cobb, Journal of Chromatography,
1 6 8 , 541 ( 1 9 7 9 ) .
38. K L i n d e r , R.W. Frei, Journal of Chromatography,
117, 81 ( 1 9 7 6 ) .
39. TNachtmann, H. Spitzy, R.W. Frei, Journal of
Chromatography, 122, 293 ( 1 9 7 6 ) .
40. F. Erni, R.W. Frei, Journal of Chromatography,
1 3 0 , 169 ( 1 9 7 7 ) .
41. C.H. Powell, Jr., Burroughs Wellcome Co., private
communication.
42. P.H. Cobb, Analyst, 1 0 1 , 768 ( 1 9 7 6 ) .
43. J.L. Ebron, Burroughsellcome Co., private communi-
cation.
44. Hewlett Packard, Application Sheet E-6, ACS Short
Course: "Solving Problems with Liquid
Chromatography.
45. D.R. Baker, Dupont Instruments, private communi-
cation.
46. D.R. Baker, Dupont Instruments, Application Sheet
E - 7 , 7A-C, ACS Short Course: "Solving Problems
with Liquid Chromatography."
47. K.M. Kadish, V.R. Spiehlor, Analytical Chemistry,
4 7 , 1714 ( 1 9 7 5 ) .
-
48. A.E.H. Hoak, T.G. Alexander, D. Banes, Journal of
the American Pharmaceutical ASSOC., 48, 217 ( 1 9 5 9 ) .
49. A.C. Caws, The Wellcome foundation Ltd., private
communication.
50. A Waghorn, J.A. McCrerie, The Wellcome Foundation
Ltd., private communication.
51. J.J. Sabatka, D.A. Brent, J. Murphy, J. Charles,
J. Vance, M.H. Gault, Journal of Chromatography,
1 2 5 , 523 ( 1 9 7 6 ) .
52. T W a t s o n , P. Tramell, S.M. Kalman, Journal of
Chromatography, 69, 157 ( 1 9 7 2 ) .
53. M.C. Castle, Journal of Chromatography, 115,437
(1975).
54. D. Sugden, M. Ahmed, M.H. Gault, Journal of
Chromatography, 1 2 1 , 401 ( 1 9 7 0 ) .
55. W.E. Wilson, S .AxJohnson, W.A. Perkins,
J.E. Ripley, Analytical Chemistry, 2 , 40 ( 1 9 6 7 ) .
56. E. Watson, D.R. Clark, S.M. Kalman, Journal of
Pharmacology and Experimental Therapeutics, 184,
424 ( 1 9 7 3 ) .
57. W.H. Bulger, R.E. Talcott, S.J. Stohs, Journal o f
Chromatography, 7 0 , 187 ( 1 9 7 2 ) .
58. L. Storstein, Journal of Chromatography, 117,
87 ( 1 9 7 6 ) .
DIGOXIN 243
59. M.L. Carvalhas, M.A. Figueira, Journal of

Chromatography, 86, 254 (1973).
60. E. Watson, P. Tramell, S.M. Kalman, Journal o f
Chromatography, 69, 157 (1972).
61. D.B. Faber, A . d z o k , U.A.T. Brinkmann, Journal
o f Chromatography, 143, 95 (1977).
62. W.E. Wilson, S . A . Johnson, W.H. Perkins,
J.E. Ripley, Analytical Chemistry, 39, 40
(1967).
63. J.H. Hilton, Science, 110, 526 (1949).
64. J.G. Wagner, M.R. Hallmark, E. Sakmar, J.W. Cryres,
29, 787 (1977).
Steroids, -
65. K.C. Wang, L.J. Spratt, Biochemical Pharmacology,
12, 577 (1963).
66. K D . Horgan, W.J. Riley, Clinical Chemistry, 19,
187 (1973).
67. B . Calesnick, A. Dinan, Clinical Chemistry, 22,
903 (1976).
DOXORUBICIN
Aristide Vigevani and Martin J . Williamson
I. Description 246
1 . 1 History 246
1.3 Appearance, Color 247
2.2 Nuclear Magnetic Resonance Spectra 247
2.3 Mass Spectra 25 1
2.4 Ultraviolet and Visible Spectrum 253
2.5 Fluorescence Spectra 255
2.6 Circular Dichroism 255
2.9 X-ray Diffraction 255
2.10 Differential Scanning Calorimetry 260
2.11 Solubility 260
2.12 Ionization Constant 260
2. I3 Polarography 260
3. Synthesis 260
3.2 Chemical 263
4. Stability 263
5. Metabolism 265
6.2 Spectrophotometric Analysis 267
6.3 Electrochemical Analysis 267
6.4 Paper Chromatography 267
6.5 Thin Layer Chromatography 268
6.6 Liquid Chromatography 268
7. Determination of Doxorubicin in Biological Fluids 270
8. Analysis of Pharmaceutical Formulations 270
9. Miscellaneous 270
1 1. References 270
CopytighI @ 1980 by Academic Ress, Inc.

Analytical h f i l c s of Drug Substances, 9 245 AIIrighrs Of n - ~ u c t i o nin any form resewed.
ISBN: &12-260809-7
246 ARISTIDE VIGEVANI A N D MARTIN J . WILLIAMSON
1. Description
1.1 History
Doxorubicin is an antineoplastic antibiotic isolated
from a culture of Streptomyces peucetius var. caesius or by
chemical synthesis from daunorubicin. The injectable dosage
form is supplied as the hydrochloride salt in combination
with lactose as a freeze-dried powder.
1.2 Name, Formula, Molecular Weight

Doxorubicin is chemically named (8S-lOS)-lO-
(3-amino-2,3,6-trideoxy-a. -L-e-hexopyranosy1)oxy-
methoxy-5,12-naphthacenedione. (CAS-23214-92-8) .
7,8,9 ,l0-tetrahydro-6,8,1l-trihydroxy-8-hydroxyacetyl-l-
Originally
named (7S:9S)-9-hydroxyacetyl-4-methoxy-7~8,9,lO-tetrahydro-
6,7,9,11-tetrahydroxy-7-~-(2,3,6-trideoxy-3-amino-cl - L - w -
hexopyranosyl)-5,12-naphthacenedione.
NHq H
27H29N01 1 Mw 543.5
Hydrochloride salt (CAS-25316-40-9)

C27H2gNOll.HC1 Mw 580.0
DOXORUBICIN 247
1.3 A p p e a r a n c e , Color
The h y d r o c h l o r i d e s a l t i s a r e d , f r e e - f l o w i n g
c r y s t a l l i n e powder, a n d t h e f r e e z e - d r i e d f o r m u l a t i o n
c o n t a i n i n g lactose i s a r e d cake.
2 Physical Properties
A review of the c a r b o n y l a b s o r p t i o n s o f
a n t i n e o p l a s t i c ( a n t i tumor) a n t h r a c y c l i n e s h a s b e e n
published1. The i n f r a r e d s p e c t r u m o f d o x o r u b i c i n
h y d r o c h l o r i d e recorded from a KBr p e l l e t ( 0 . 4 ) % o n a
Perkin-Elmer model 457 g r a t i n g s p e c t r o p h o t o m e t e r is shown i n
F i g u r e 1. The i n t e r p r e t a t i o n o f t h e m a i n a b s o r p t i o n b a n d s i s
g i v e n i n T a b l e 1.
TABLE 1
I n f r a r e d spectrum o f doxorubicin hydrochloride

~
I R A b s o r p t i o n Band, c m - l A s s i g n men ts
3560-3160 0-H s t r e t c h ( h y d r o g e n bonded)

3160-2300 NH3+ s t r e t c h and OH
s t r e t c h (hydrogen bonded)
1724 C=O (ketone)
1 6 1 3 and 1580 C=O s t r e t c h ( i n t r a hydrogen
bonded q u i n o n e )
1282 C-0-C s t r e t c h ( e t h e r )
1115 C-0 ( t e r t i a r y alcohol)
1071 C-0 (secondary a l c o h o l )
1008 C-0 (primary a l c o h o l )
2.2 N u c l e a r M a g n e t i c Resonance Spectra

Proton magnetic resonance spectrometry h a s been
e x t e n s i v e l y u s e d a s a f u n d a m e n t a l tool for t h e d e t e r m i n a t i o n
o f t h e s t r u c t u r e o f daunorubicin and r e l a t e d
compounds2r3r4. The lH-NMR s p e c t r u m o f a d r i a m y c i n o n e
p e n t a a c e t a t e i n CDC13 h a s b e e n d e s c r i b e d and t e n t a t i v e l y
assigned5. The lH-NMR s p e c t r u m o f d o x o r u b i c i n
h y d r o c h l o r i d e i n DMSO-d6 s o l u t i o n r e c o r d e d a t 1 0 0 MHz on a
V a r i a n HA-100 s p e c t r o m e t e r a t 8 O o C ( f o r b e t t e r r e s o l u t i o n ) i s
shown i n F i g u r e 2. The i n t e r p r e t a t i o n o f t h e s p e c t r u m is
g i v e n i n T a b l e 2.
I I I
0
L
n
(v
-s
0
0
-0
W
0
C
.d
<-0
0
z-
3 V
-s
0-
bl
0 Icl
-0 C
0
N U
DO XO R UBICIN 249
TABLE 2
lH-NMR d a t a of d o x o r u b i c i n h y d r o c h l o r i d e i n D M s 0 - d ~
s o l u t i o n a t 80°C (TMS as i n t e r n a l r e f e r e n c e ) .
Proton Mu 1t i p l i c i t y a J or WH (Hz)
CH3-5 d 1.15 6.5

H2-2 m 1.77 -
H2-9 m 2.15 -
H2-7 s 2.92 -
H-3 I m 3.31 -
H-4 I bs 3.62 6.0
CH3O S 3.94 -
H-5 dq 4.14 and
H2-14 S 4.57 -
OH-4 bs 4.46 -
H-10 bs 4.90 10.0
H-1 bs 5.25 7.0
H-3 t 7.53 7.0
H-2
H-4
+
3 d 7.79 7.0
-
NH3 bs 7.96
13. 08b
OH-11 two s and
13. 85b
a) s - S i n g l e t ; d = Doublet; t = Triplet;
m = M u l t i p l e t ; b s = Broad s i g n a l ; dq = Double
quartet.
b) A t room t e m p e r a t u r e .
C
.d
u
0
DOXORUBICIN 25 I
The 13C NMR spectra of doxorubicin hydrochloride,

daunorubicin hydrochloride, the corresponding aglycones and
of a-methyl daunosaminide in DMSO-d6 solutions and the
interpretation has been reported6. Figure 3 shows the FT
13C NMR proton noise-decoupled spectrum of doxorubicin
hydrochloride in D20 solution, recorded at 80 MHz on a
Varian CFT-20 NMR spectrometer. Dioxane, which is not shown,
was used as the internal standard. The interpretation of the
spectrum is given in Table 3 .
TABLE 3
13C-NMR data of doxorubicin hydrochloride in D20 solution

values (ppm) are referred to 734s.
Carbon 6 Carbon 6
1 161.0 4a (134.5)
2 (119.2) 5a 111.0
3 137.3 6a (134.0)
4 (120.2) 10a (134.5)
5 (185.9) lla 111.0
6 154.6 12a (120.0)
7 32.8 CH3O 57.2
8 76.6 1' 99.4
9 36.0 2' 28.5
10 69.0 3' 47.7
11 156.2 4' (67.9)
12 (186.1) 5' (67.0)
13 213.9 CH3-5 ' 16.6
14 65.3
Assignments with similar shift values

given in parentheses may be interchanged.
2.3 Mass Spectra

The mass spectrum of doxorubicin hydrochloride
itself cannot be obtained by electron-impact ionization, but
this technique can be used to obtain the spectra of
adriamycinone5 and daunosamine'. A study of
-
N-acetyldaunosamine derivatives has been published8. The
mass spectra of some E-acylated daunorubicin derivatives have
been published9 and also the GC-MS of persilylated aglycone
derivatives of doxorubic in and daunorubic inlo.
E
a
P
-m
0 C
.r(
w
0
0
-0
m
r-l
u
0
-r m
aJ
U
3
cn
-4
E
-8
N
DOXORUBICIN 253
The i n t a c t molecule can be examined by f i e l d

desorption i o n i z a t i o n mass s p e c t r o m e t r y l l . Figure 4 shows
t h e spectrum obtained on a Varian MAT31lA spectrometer,
equipped w i t h a combined FD/FI/EI source ( e m i t t e r heating
c u r r e n t 2 O m A ) l 2 . Table 4 g i v e s t h e assignments of t h e
major fragmentation peaks.
TABLE 4
F i e l d desorption mass spectrum of doxorubicin hydrochloride
m/e assignment
544 M+1
543 Molecular ion
414 a d r iamycinone
l+
CH30 0 bH
7, -b isanhyLi0a-L iamycinone
0 OH
336
2.4 U l t r a v i o l e t and V i s i b l e Spectrum

The u l t r a v i o l e t and v i s i b l e spectrum of doxorubicin
hydrochloride i n methanol ( c = l % )is shown i n Figure 513.
The molecular a b s o r p t i v i t i e s a r e given i n Table 5.
m
=.t
o m
= II
4
U
u
aJ
a
co
m
I 0
V I
a,
U
3
(D m
m 4
m h
DOXORUBICIN 255
TABLE 5
U l t r a v i o l e t a n d V i s i b l e Molecular Absorptivities of
Doxorubicin Hydrochloride i n Methanol
Wavelength E
233 38150
253 255 00
290 8400
471 13050
495 13000
530 7200
2.5 Fluorescence Spectra

T h e f l u o r e s c e n c e spectra of d o x o r u b i c i n
h y d r o c h l o r i d e i n water a n d e t h a n o l a t a p p r o x i m a t e l y 5 ppm,
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 MPF-2A s p e c t r o f l u o r i m e t e r ,
a r e shown i n F i g u r e 6.13
2.6 C i r c u l a r Dichroism
T h e c h i r a l c e n t e r s a t C-8 a n d C-10 are r e s p o n s i b l e
f o r t h e C o t t o n e f f e c t s a t 3 4 5 a n d 2 8 5 nm. T h e c i r c u l a r
d i c h r o i s m c u r v e s of m e t h a n o l s o l u t i o n s of d o x o r u b i c i n a n d
d a u n o r u b i c i n h y d r o c h l o r i d e s a n d of a d r i a m y c i n o n e a n d
daunomycinone i n d i o x a n e , d e t e r m i n e d u s i n g a Roussel-Jouan
D i c h r o g r a p h 11, a r e shown i n F i g u r e s 7 a n d 8. T h i s t e c h n i q u e
h a s been used i n t h e deduction o f stereochemical
r e l a t i o n s h i p s i n t h e f i e l d of a n t h r a c y ~ l i n o n e s ~ ~ .
2.7 Optical R o t a t i o n
The o p t i c a l r o t a t i o n C C ] ~ ~of d o x o r u b i c i n
h y d r o c h l o r i d e i n m e t h a n o l ( 0 . 1 ) was d e t e r m i n e d a t 5 8 9 nm
u s i n g a P e r k i n - E l m e r Model 2 4 1 MC polarimeter t o b e +255O.
2.8
Melting Point
D o x o r u b i c i n h y d r o c h l o r i d e melts a t 2 0 5 T w i t h
decomposition.
2.9 X-ray D i f f r a c t i o n
A t t h i s t i m e n o X-ray d i f f r a c t i o n s t u d i e s have been
r e p o r t e d o n d o x o r u b i c i n h y d r o c h l o r i d e or i t s d e r i v a t i v e s .
S i n g l e c r y s t a l X-ray d i f f r a c t i o n of
-
N-bromoacetyldaunorubicin s o l v a t e with acetone15 confirmed
t h e s t r u c t u r e and a b s o l u t e c o n f i g u r a t i o n o f daunorubicin,
which h a d p r e v i o u s l y been d e t e r m i n e d b y c h e m i c a l
s t u d i e s 2 , 3,4. More r e c e n t l y t h e s e r e s u l t s were c o n f i r m e d
b y a n X-ray a n a l y s i s of d a u n o r u b i c i n , a s t h e h y d r o c h l o r i d e
0
0
ln 4
0 2m
aD c
m
s
4J
0
ln
In c
.r(
0
d aJ
d 5
.d
o c
nl '-
I n k
0
0
In
0
a,
d
0
ln
d
0
0
d
0 w
(u 0
P
0
0
d
0
a,
m
0
ln
m
0
d
c)
5
c
0
(u
m
0 4J
Q)
0 rl
0 0
0 .r(
3
0 m
U
a, 4J
(u rl
3
0
ln
(u
In
0
d
nl
256
70 70
> 60 60
h
ul
5 50 50
I-
z
z 40 40
0
2 30 30
5
L
g 20 20
-
!z
-I 10 10
W
&
0 0
r 1 1 1 1 1 1 1 1 ~ 1
480 520 560 600 640 680 480 520 560 600 640 680
WAVELENGTH (nm)
F i g u r e 6. F l u o r e s c e n c e S p e c t r a of D o x o r u b i c i n H y d r o c h l o r i d e i n Water ( l e f t )
and E t h a n o l ( r i g h t ) . C o n c e n t r a t i o n s a p p r o x i m a t e l y 5 mg/l.
258 ARISTIDE VIGEVANI AND MARTIN J . WILLIAMSON
At2
3t
D AU NORUB ICI N
At2 h(nrn1
3r
I
2
--)--c.---c.
DOXORUBICI N
-1
-2
-3
-4 4
260 2i O 360 3;O 3iO 3 i O 3iO 460
400
A(nmi
Figure 7. Circular Dichroism Curves of Doxorubicin and

Daunorubicin Hydrochlorides in Methanol
DOXORUBICIN 259
A&
27
-2 1
260 O
2; 360 3;O 3iO 3kO 360 460
?dnm 1
A€
-2
260
I 260 360 3iO 3 i O 3kO 360 460
A (nml
Figure 8. Circular Dichroism Curves of Adriamycinone

and Daunomycinone in Dioxane Solution
monohydrate p y r i d i n e s a l t 1 6 . Some c o n f o r m a t i o n a l
d i f f e r e n c e s were o b s e r v e d w i t h respect t o t h e N-bromoacetyl
derivative.
2.10 D i f f e r e n t i a l Scanning Calorimetry

The h e a t i n g c u r v e of d o x o r u b i c i n h y d r o c h l o r i d e
o b t a i n e d w i t h a Perkin-Elmer Model DSC-1B s c a n n i n g
calorimeter a t a t e m p e r a t u r e g r a d i e n t o f 8OC/min. i s shown i n
F i g u r e 917. I t shows a n endotherm, c o r r e s p o n d i n g t o t h e
s o l i d - l i q u i d t r a n s i t i o n a t 202-2O5OCI p a r t i a l l y superimposed
by a n endotherm due t o d e c o m p o s i t i o n which i s a t a maximum a t
26OOC and c o n t i n u e s t o h i g h e r t e m p e r a t u r e s .
2.11 S o l u b i l i t y
Doxorubicin h y d r o c h l o r i d e i s r e a d i l y s o l u b l e i n
water, normal s a l i n e , methanol, a c e t o n i t r i l e and
t e t r a h y d r o f u r a n , b u t o n l y s l i g h t l y s o l u b l e or i n s o l u b l e i n
less polar o r g a n i c s o l v e n t s . The a p p a r e n t p a r t i t i o n
c o e f f i c i e n t (Papp) between 1 - o c t a n o l and T r i s b u f f e r a t pH
7.0 w i t h c o n s t a n t i o n i c s t r e n g t h ( I = 0.1) is 0.52 a t room
t e m p e r a t u r e (22-24OC) a f t e r s h a k i n g f o r 1 5 hours18.
2.12 I o n i z a t i o n C o n s t a n t
A pKa o f 8.22 was d e t e r m i n e d f o r t h e h y d r o c h l o r i d e
w i t h N/20 sodium hydroxide. Solutions of doxorubicin
h y d r o c h l o r i d e show i n d i c a t o r - 1 i k e p r o p e r t i e s I t u r n i n g from
orange-red t o b l u e - v i o l e t a b o u t pH = 913. V a l u e s o f -5.9,
8.2, 10.2, and 13.2 f o r pK1, pK2, pK3 and pK4, d e t e r m i n e d
by s p e c t r o p h o t o m e t r i c methods, have been r e p o r t e d 1 9 .
2.13 m l a r o g r a p h y
Due t o i t s q u i n o i d a l system, d o x o r u b i c i n g i v e s
c h a r a c t e r i s t i c p o l a r o g r a m s a t d i f f e r e n t pH v a l u e s . These
c u r v e s , d e t e r m i n e d u s i n g a Leeds-Northrup Electro-Chemograf
t y p e E p o l a r o g r a p h , are shown i n F i g u r e
3. Synthesis
3.1 M i c r o b i o l o g i c a l
Doxorubicin c a n be o b t a i n e d by a e r o b i c f e r m e n t a t i o n
o f S t r e p t o m y c e s peucetius v a r . c a e s i u s f o l l o w e d by e x t r a c t i o n
w i t h a c i d i c a c e t o n e and p u r i f i c a t i o n by p a r t i t i o n
chromatography o n a column o f cellulose b u f f e r e d a t pH 5.4.
The a n t i b i o t i c is r e c o v e r e d from t h e e l u a t e s i n 1 - b u t a n o l
s a t u r a t e d w i t h pH 5.4 p h o s p h a t e b u f f e r by back e x t r a c t i o n
w i t h d i l u t e a c i d pH 3 , f o l l o w e d by r e - e x t r a c t i o n i n t o
c h l o r o f o r m a t pH 8.6. The c h l o r o f o r m s o l u t i o n i s
c o n c e n t r a t e d and d o x o r u b i c i n c r y s t a l l i z e d a s t h e
h y d r o c h l o r i d e on a d d i t i o n o f a n e q u i v a l e n t o f m e t h a n o l i c
F C
.rl
0
LD .rl
w
0 0
8 c
m
V
vl
u
:
+J
.rl
0
0
.co
U
m
c
..
.rl
C
C
m
0 V
0 vl
(D r(
U m
.rl
+J
c
a,
u
a,
w
w
0 4
0 c1
U
U
a,
u
3
0 cn
2
U
-4
E
4
0.3 0.4 0.5 0.6 0,7 0.8 0.9
1 1.1 1.2 1.3Volt
Figure 10. Polarograms of Doxorubicin i n S o l u t i o n s

of D i f f e r e n t pH Values
DOXORUBICIN 263
hydrogen chloride. Final purification is performed by

crystallization from ethanol or from a methanol/l-propanol
mixture20.
3.2 Chemical
Doxorubicin can be obtained2l by reacting
daunorubicin hydrochloride in a methanol/dioxane solvent
mixture with a chloroform solution of bromine, forming
14-bromodaunorubicin. This is then hydrolyzed with an
aqueous methanolic solution of sodium hydroxide under a
nitrogen atmosphere. After dilution with water, the solution
is extracted with chloroform and the organic extracts dried
over anhydrous sodium sulfate, concentrated, treated with
hydrogen chloride in anhydrous methanol, and then diluted
with ethyl ether. The precipitate formed is doxorubicin
hydrochloride, which is purified by crystallization from a
mixture of methanol and 1-propanol. The above reaction
pathway can be summarized as shown in Figure ll, in which the
anthraquinone moiety is not shown.
4. Stability
Doxorubicin hydrochloride is very stable in the solid
state. It has been stored for years at room temperature
without any loss in potency or indications of degradation.
The lyophilized powder of doxorubicin hydrochloride with
lactose is also stable, if dry and stored in well closed
containers at room temperature13. The active drug
substance has also been found to be stable for three months
at 60°C, and for three months in light of 500 ft. candles of
illumination at room temperature. The lyophilized
formulation is stable mder similar lighting conditions, and
at 6OoC if the moisture content in the sealed vial is less
than 1.0%.
The effect of pH values and buffer concentrations on the

stability of aqueous solutions of doxorubicin hydrochloride
has been determined by spectrophotometric and chromatographic
methods. Doxorubicin is stable in acidic solutions in the pH
range 3.0 to 6.5, but decomposes at increasing rates as the
pH is increased from 6.5 to 12. Decomposition in aqueous
solution gives complex mixtures of pigmented compounds with a
wide range of chromatographic polarities. Apart from the
isolation of adriamycinone from dilute acid solutions13 the
identification of the components of these mixtures has not
been accomplished.
F i g u r e 11. S y n t h e t i c Pathway for Doxorubicin
DOXORUBICIN 265
5. Metabolism
The two major metabolic transformations of doxorubicin in
laboratory animals and in man are:
a) The reduction of the side chain carbonyl group to a

secondary alcohol, giving 13-dihydrodoxorubicin
(adriamycinol) .
b) The reductive cleavage of the daunosamine moiety with
the formation of 10-deoxyadriamycinone.
The first reaction is catalyzed by an enzyme named

"daunorubicin reductase," an aldo-keto reductase of a very
ubiquitous nature. The reductive splitting of the benzylic
glycosidic bond is, on the contrary, rather unique as no
other examples of enzyme catalysis of this otherwise
chemically very facile reaction have been described1. The
aglycone-like compounds thus formed are then further
metabolized by other typical reactions such as 2-methylation
and conjugat ion22* 23.
Doxorubicin and its metabolites extracted from the urine

of patients treated with the drug were separated by
chromatography on columns of silicic acid. The following
compounds were isolated (in order of increasing polarity)
(see Figure 1 2 ) : 13-dihydroadriamycinone (3), lO-deoxy-13-
dihydroadriamycinone(4), l-demethyl-lO-deoxy-13-
dihydroadriamycinone(5), doxorubicin(1) ,
13-dihydrodoxorubicin(2), l-demethyl-lO-deoxy-13-
d ihydr oadr iamyc inone-1-2-s u1fate( 6 ) , 1-demethy1-10-deoxy-13 -
dihydroadriamycinone-l-~-R-D-glucuronide(7). A total of 60%
of the fluorescence in the urine was due to metabolites and
the remainder was unchanged drug. As the recovery of
doxorubicin fluorescence in bile and urine from a patient was
about 60% of the administered dose, the authors pointed out
the possibility of the presence of non-fluorescent
metabolite^^^. The above mentioned metabolites were also
detected in the plasma of patients under doxorubicin
treatment25.
Protein binding studies using the ultracentrifugation

method suggested that doxorubicin is bound to rabbit and
human plasma proteins to an extent of 50%26, but a
re-examination of the original Scatchard plot data changed
this value to 90%27. Other studies using equilibrium
dialysis have suggested complex binding relationships that
need further investigation2*.
&
I
0
I I
0
o\
I =
0
I
kt
= I
0
0 0-
0
I I N
0
I
0-
0
X I I 0
X .rl
u
3
It
\
8-&-
0\ I
0
0- p
0 -0
IOU2
0
0-Ul
I
0
I
0 0
I 0
0
0,
I
DOXORUBICIN 261
The main biochemical effects of doxorubicin are concerned

with nucleic acid synthesis. The binding of this drug to DNA
is considered responsible for the interference with template
DNA function29. The DNA-doxorubicin binding constant has
been determined to be approximately 2 x lo6 M-l (30).
The elemental analysis of doxorubicin hydrochloride
(Farmitalia reference standard batch GDA 1) is as follows:
% Theory % Found
C 55.91 56.08
H 5.22 5.33
N 2.41 2.16
c1 6.11 5.85
6.2 Spectrophotometric Analysis

The visible absorption maximum at 495 nm (El% = 223)
lcm
can be used for the quantitation of doxorubicin in dosage
forms.
The fluorescence properties of doxorubicin can be used

for the determination of total anthracycline at low
concentrations31.
6.3 Electrochemical Analysis

ChronopotentiometricJ1, cyclic voltammetr ic32
and p~larographic~~r33 assays of doxorubicin hydrochloride
have been reported. These techniques determine the total
anthracycline content.

Doxorubicin can be separated from the aglycone,
adriamycinone, by paper chromatography using either of the
following two systemsl3.
A) 1-butanol saturated with pH 5.4 M/15 phosphate buffer.

B) 1-propanol/ethyl acetate/water, 7/1/2 by volume.
The Rf values for doxorubicin and adriamycinone are

0.1 and 0.3, and 0.25 and 0.65 for systems A and B
respectively .
268 ARISTIDE VIGEVANI AND MARTIN J. WILLIAMSON

Thin l a y e r c h r o m a t o g r a p h i c s y s t e m s f o r d o x o r u b i c i n
are g i v e n i n T a b l e 6.
TABLE 6
Thin l a y e r c h r o m a t o g r a p h i c s y s t e m s f o r d o x o r u b i c i n .
Adsorbent S o l v e n t System -
Rf Reference
Silica gel Methylene c h l o r i d e /

me t h a n o l / w a t e r (100/20/2) 0.17 13
Silica g e l 1-butanol/acetic acid/water

(4/1/5) 0.33 13
Silica gel Chloroform/95% e t h a n o l /

trifluoroacetic acid
(75/20/5) 0.23 34
Silica gel Chloroform/me t h a n o l / a c e t i c

acid (93/5/2, p l a t e d r i e d ,
t h e n 76/20/4) 0.2 35
Silica gel Ch loroform/me thanol/wa ter

sprayed with (140/60/10)
phosphate
buffer
(pH = 7.0) 0.3 36
Polyamide/ l-Butanol/2-propanol/isopropyl
cellulose e t h e r / a c e t i c acid/wa ter
(35/6/6/9/44) 0.3 37
6.6 L i q u i d Chromatography
L i q u i d c h r o m a t o g r a p h i c s y s t e m s for d o x o r u b i c i n
h y d r o c h l o r i d e a r e g i v e n i n T a b l e 7.
DOXORUBICIN 269
TABLE 7
Liquid chromatographic systems for doxorubicin
Stationary Mobile Approximate Doxorubicin Ref.

Phase Phase Capacity Factor (k’)
Silica 2-pr opanol/i sopropyl

(5 micron) e ther/0 .125 M acetate
buffer pH 4.5 (65/30/5) 10 38
Silica 2-propanol/0.5 sodium

(5 micron) acetate buffer pH 4.5
(96.2/3.8) 4 39
Silica Methylene chloride/

(5 micron) methanol/25% ammonia/
water (90/9/0.1/0.8) 4 40
Cyanopropylsilica Chloroform/methanol/
(10 micron) acetic acid/water
(79.8/14.1/4.7/14 ) 3 41
Cyanopropylsilica Ch lor oform/me thanol/

(10 micron) water (96/5/1) - 42
Corasi1-pheny 1 Linear gradient, 16%

(37-75 micron) acetonitrile/in water
to 20% acetonitrile/
80% pH 4 formate buffer 10 43
Coras i1-pheny1 Linear gradient 0 to 40%

(37-50 micron) acetonitrile in pH 4.0
ammonium formate buffer 10 44
Octadecyl-silica Me thanol/aqueous solution

(10 micron) of PIC B-7 (heptane-
sulfonic acid) (50/50) 14 45
Oc tadecyl-s ilica Acetonitrile/aqueous

(10 micron) phosphoric acid pH 2,
(31/69) 3 46
Octadecyl-silica Me thanol/water/acetic
(10 micron) acid (66/33.2/0.8) 1.4 47
Octyl-silica Acetonitrile/10’2 M.
(10 micron) aq. phosphoric acid
(40/60) 2 48
7. Determination of Doxorubicin in Biological Fluids

Total anthracycline compounds in biological fluids can be
determined by fluorimetric method^^^,^^. Radioimmunoassay
procedures have also been reported50. The emphasis is now
on the separate determination of metabolites and intact drug
in biological fluids. One such method coupled liquid
chromatography followed by RIA^^ but it was rather
time-consuming. TLC followed by fluorescence scanning has
been reported35 and used for disposition prediction^^^.
The most recently published methods have used

rever sed-phase 1 iquid ~ h r o m a t o g r a p h y 5s2
~ or normal phase
liquid ~ h r o m a t o g r a p h y ~with
~ , ~ ~fluorescence detection.
These methods have been applied to tissue distribution
studies5l. Similar methodss4 used for daunorubicin and
metabolites should also be applicable.
8. Analysis of Pharmaceutical Formulations

The identification and/or determination of doxorubicin
hydrochloride in Adriamycin involves the use of visible
spectrophotometry, thin layer chromatography followed by
spectrophotometry or microbiological agar diffusion55.
However, the recently published liquid chromatographic
procedure46 is replacing the above physical methods.
9. Miscellaneous
Ph a rmace u t ica 1 preparations of doxor u b i c in hy dr och lor ide ,
trade-marked Adriamycin, have been patented20.
10. Acknowledgments
Acknowledgment is made to Drs. F. Arcamone and S. Penco
of Farmitalia-Carlo Erba SPA. and Drs. G. Davis, W. Hausmann
and J. Short of Adria Inc., for their useful advise during
the preparation of the manuscript.
11. References - Literature covered until March, 1979.

F. Arcamone, in "Topics in Antibiotics Chemistry,"
-
P. Sammes Ed., Vol. 2, Ellis Horwood Publ.,
Chichester, 1978, pp. 102-239, and references therein.
F. Arcamone, G. Franceschi, P. Orezzi, S. Penco, and

R. Mondelli, Tetrahedron Letters, 3349 (1968).
F. Arcamone, G. Cassinelli, G. Franceschi, P. Orezzi,

and R. Mndelli, Tetrahedron Letters, 3353 (1968).
F. Arcamone, G. Cassinelli, G. Franceschi,

R. Mondelli, P. Orezzi, and S. Penco, Gazz. Chim.
Ital., 100, 949 (1970), and references therein.
DOXORUBICIN 27 1
F. Arcamone, G. F r a n c e s c h i , S. Penco, and A. S e l v a ,

T e t r a h e d r o n Letters, 1007 (1969).
A. Arnone, G. Fronza, R. M o n d e l l i , and A. V i g e v a n i ,

T e t r a h e d r o n Letters, 3349 (1976).
B. G i o i a , F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e
Communication (1972).
A V i g e v a n i , B. Gioia, and G. C a s s i n e l l i , C a r b o h y d r a t e
as., 32, 321 (1974).
P. P. Roller, M. S u t p h i n , and A. A. A s z a l o s , Biomed.

Mass Spectrom., 3, 166 (1976).
K. K. Chan, and E. Watson, J. Pharm. Sci., 67,1748

(1978).
K. H. Maurer, U. Rapp, K. Chan, and W. Sadee,

Communication p r e s e n t e d a t t h e "Mario Negri 2nd
I n t e r n a t i o n a l Symposium on Mass S p e c t r o m e t r y i n
B i o c h e m i s t r y and Medicine", M i l a n , 24-26 J u n e , 1974.
B. Gioia, F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e
F. Arcamone, G. C a s s i n e l l i , G. F r a n c e s c h i , S. Penco,
C. P o l , S. R e d a e l l i , a n d A. S e l v a , " I n t e r n a t i o n a l
Symposium o n Adriamycin," S. K . C a r t e r , A. D i Marco,
M. Ghione, I. H. K r a k o f f , and G. Mathe Eds.,
S p r i n g e r - V e r l a g , B e r l i n , 1972, pp. 1-22.
H. Brockmann, H. Brockmann Jr., and J. Niemeyer,

T e t r a h e d r o n Letters, 4719 ( 1 9 6 8 ) .
R. A n g i u l i , E. F o r e s t i , L. Riva d i S a n s e r v e r i n o ,
N. W. Isaacs, 0. Kennard, W. D. S. Motherwell,
D. L. Wampler, and F. Arcamone, Nature. New Biology,
-234, 78 (1978)
S. N e i d l e and G. T a y l o r , Biochim. Biophys. Acta.,

-
479, 450 (1977).
E. P e l l a , Carlo Erba Research Laboratories, P r i v a t e

G. G o n f a l o n i e r i and G. Vasconi, Carlo Erba R e s e a r c h

L a b o r a t o r i e s , P r i v a t e Communication, (1975).
19) R. J. S t u r g e o n , and S. S. Schulman, J. Pharm. Sci.,

-
66, 958 (1977).
20) F. Arcamone, G. C a s s i n e l l i , G. F a n t i n i , A. G r e i n ,
P. O r e z z i , C. p o l , and C. Spalla, B i o t e c h n o l .
Bioeng., &, 1 1 0 1 (1969).
21) F. Arcamone, G. F r a n c e s c h i , and S. Penco, U.S. Patent

3,803,124 (Apr. 9, 1974).
22) M. A. A s b e l l , R. Schwartzbach, F. J. B u l l o c k , and

D. W. Yesair, J. Pharmacol. Exp. Ther., 182, 63
(1972).
23) F. J. B u l l o c k , R. J. B r u n i , M. A. A s b e l l , J.
Pharmacol. Exp. Ther., 182, 70 (1972).
24) S. Takanashi and N. R. Bachur, Drug Metabolism and

D i s p o s i t i o n , Q, 79 (1976).
25) R. S. Benjamin, C. E. Riggs, Jr., and N. R. Bachur,

Cancer Res., 37, 1416 (1977).
26) P. A. H a r r i s and J. F. Gross, Cancer Chemother, Rep.,

-
59, 819 (1975).
27) K. K. Chan, J. L. Cohen, J. F. Gross, K.

J. H i m e l s t e i n , J. R. Bateman, Y. Tsu-Lee, and
A. S. Marlis, Cancer Treatment Reports, 62, 1161
(1978).
28) M.Menozzi, F a r m i t a l i a Research L a b o r a t o r i e s , P r i v a t e

29) A. DiMarco and F. Arcamone, Arzneim.-Fbrsch. (Drug

R e s e a r c h ) , 25, 368 (1975) and r e f e r e n c e s c i t e d
t h e r ein.
30) S. R. Byrn and G. D. Dolch, J. Pharm. S c i . , 67, 688

(1978) and r e f e r e n c e s c i t e d .
31) L. Dusonchet, N. Gebbia, and F. G e r b a s s i , Pharm. Res.

Commun., 2, 55 (1971).
32) G. M. Rao, J. W. LOwn, and J. A. Plambeck, J.

Electrochem. Soc., 125, 534 (1978).
33) L. A. S t e r n s o n and G. Thomas, A n a l . Letters, 10, 99

(1977).
DO XO RUBICIN 273
G. W. C l a r k , Adria Laboratories , P r i v a t e
Communication, (1979).
E. Watson and K. K. Chan, Cancer Treatment R e p o r t s ,

-
60, 1611 (1976)
Federal Register 41, 14184, A p r i l 2, 1976 S e c t i o n

436.315.
Federal Register 41, 14184, A p r i l 2, 1976 S e c t i o n

436.314.
F a r m i t a l i a SpA., J u n e 1975, P r i v a t e Communication.
H . G. B a r t h and A. Z. COnner, J. Chromatogr., 131,

375 (1977).
R. Hulhoven and J. P. Desager, J. Chromatogr., 125,

369 (1976).
P. A. Harris, Proc. Amer. A s s o c . Cancer R e s e a r c h , 2,

131 (1976).
V. P. M a r s h a l l , E. A. R e i s e n d e r , and P. F. W i l l e y .
J. A n t i b i o t i c s , 29, 966 (1976).
J. J. Langone, H v a n Vunakis, and N. Bachur, Biochem.

Med., 2,283 (1975).
M. I s r a e l , W. J. Pegg, P. M. W i l k i n s o n and
M. B. G a r n i c k , " B i o c h e m i c a l / B i o l o g i c a l A p p l i c a t i o n s
of L i q u i d Chromatography," G. L. Hawk, e d i t o r , Marcel
D e k k e r Inc., N.Y. 1978, Chap. 22.
Waters P h a r m a c e u t i c a l A p p l i c a t i o n Note #312.
Federal Register 43, 44836, September 29, 1978,

S e c t i o n 436.322.
L. M a l s p e i s , Ohio S t a t e U n i v e r s i t y , P e r s o n a l
Communication, (1978).
S. Eksborg, J. Chromatogr., 149, 225 (1978).

N. R. Bachur, A. L. Moore, J. G. B e r n s t e i n a n d A.
L i u , Cancer Chemotherap. Rep., 54, 89 (1970).
H.van Vunakis, J. J. Langone, L. J. R i c e b e r g and L.

Levine, Cancer R e s e a r c h , 34, 2546 (1976).
51) M. Israel, W. J. Pegg., P. M. Wilkinson and M. B.

Garnick, J. Liquid Chromatogr., 1,795 (1978).
52) R. Baurain, D. Deprez-DeCampeneere and A. Trouet,

Analytical Biochemistry, 99, 112 (1979).
53) J. H. Peters and J. F. Murray Jr., J. Liquid

Chromatogr., 2, 45 (1979)
54) S. Eksborg, H. Ehrsson, B. Anderson and M. Beran, J.

Chromatogr., 153, 211 (1978).
55) Federal Register 41, 14184, April 2, 1976, Section

450.24.
FLUPHENAZINE DECANOATE.
Geofiey Clarke
1. Description
I . 1 Name, Formula, Molecular Weight 276
2.1 lnfrared Spectrum 216
2.4 Fluorescence Spectrum 219
2.5 Mass Spectrum 28 1
2.6 Melting Range 28 1
2.7 Refractive Index 28 1
2.8 Solubility 28 1
2.9 pKa 284
2.10 Differential Thermal Analysis 284
3. Synthesis 284
4. Stability 284
6.2 Non-aqueous Titration 286
6.4 Colorimetric Analysis 286
6.5 Fluorometric Analysis 287
6.6 Chromatographic Analysis 287
7. Body Fluid and Tissue Analysis 29 1
8. References 293
Copyright 01980 by Academic Press. Inc

Analytical Profiles of Drug Substances. 9 215 All rights ofreproduction in any form reserved.
ISBN: 012-260809-7
216 GEOFFREY CLARKE
1. Description
1.1. Name, Formula, Molecular Weight.
Fluphenazine decanoate is 4-[3-[2-(trifluoromethyl)phenothia-

z i n -1O-ylI propyl] -1 piperazine ethanol decanoate ester;
P r o l i x i n decanoate; S Q 10,733
(CH2)3 N
nN (CH2)2 0
9
c (CH2)8 CH3
I W
Molecular weight 591.7
1.2. Appearance, Odor, Color.
Fluphenazine decanoate is a pale yellow to yellow orange

viscous liquid w i t h a characteristic odor. A t r o o m tempera-
ture the l i q u i d w i l l slowly crystallise.
2.1 Infrared spectrum
The infrared spectrum o f fluphenazine decanoate(1ot 117102,

p u r i t y 99%) in the liquid phase (as a thin f i l m ) is given in
.
figure 1. The following W j g n m e n t s have been made f o r the
most characteristic bands
Frequency(cm-') Ass ignment
2 940 A r o m a t i c C-H stretching vibra-

tions
1740 Ester carbonyl stretching vibra-

tions
1605 aromatic r i n g skeletal vibrations

1575
930,870 C-H out o f plane bending vibra-

820,750 tions
"t I I II
F i g u r e 1. I n f r a r e d s p e c t r u m o f F l u p h e n a z i n e d e c a n o a t e as a t h i n f i l m .
I n s t r u m e n t : Unicam SP 1000.
U l t r a v i o i f t s p e c t r u m of F l u p h e n a z i n e d e c a n o a t e i n m e t h a n o l
d
Figure 2.
0
SLH
ffl
a,d
(15ug m l ) . I n s t r u m e n t : P e r k i n E l m e r 137.
FLUPHENAZINE DECANOATE 279
The four bands between 750 and 930cm-1 are reported t o be
t w o bands a t 1605 d 1575cm

-!
characteristic o f 2, 1 0 disubstitu ed phenothiazines and t h e
c h a r a c t e r i s t i c f o r pheno-
thiazines in general (397 .
2.2. U l t r a v i o l e t spectrum
The u l t r a v i o l e t spectrum o f fluphenazine decanoate(1ot

117102, p u r i t y 9%) in methanol is given in figure 2. Similar
spectra are obtained i n ethanol and c h l o r o f o r m although t h e
shoulder a t 240nm i s obscured in c h l o r o f o r m due t o absorption
o f t h e solvent. The spectrum given in figure 2 is
characteristic o f a phenothiazine and t h e location o f the m o s t
intense peak a t i s consistent w i t h a halogen substituent
in t h e 2-position .
1Yo
4cm
240nm 261nm 315nm
Chlor0 f o r m - 551 74.3

Methanol 220 562 65.9
Ethanol(95%) 221 586 68.4
2.3 Fluorescence spectrum
Fluphenazine decanoate does n o t e x h i b i t any s i g n i f i c a n t l y

measurable fluorescence in ethanolic solution. Fluorescence
measurement can be made however a f t e r p r i o r oxidation to
t h e sulphoxide. (See section 6.5).
2.4 Nuclear magnetic resonance spectrum.
The 100 M H z spectrum o f fluphenazine decanoate in DMSO-

d&internal reference TMS) i s given i n figure 3.
F i g u r e 3 . N u c l e a r m a g n e t i c resonance s p e c t r u m of F l u p h e n a z i n e d e c a n o a t e
i n DMSO-d6. I n s t r u m e n t : Thompson P a c k a r d .
FLUPHENAZINE D E C A N O A T E 281
The c h y g i c a l s h i f t s produced can be assigned to the following

protons . d e
a b c n f g h i k
proton chemical shi fts(dppm).
a 3.97 triplet(6.5 Hz)

b t I 1.77 multiplet
c,d,e,d Ye ,f 2.22 multiplet
9 4.07 triplet(6.5 Hz)
h 2.22 multiplet
j 1.20 singlet
k 0.82 triplet(7.0 Hz)
Aromatic 6.97
7.11
7.22
2.5 Mass Spectrum
The low resolution mass spectrum i s given in figure 4. The

molecular ion a t rn/e 591 and t h e fragmentation p a t t e r n depicted
i n figure 5 are consistent w i t h the structure given f o r fluphen-
azine decanoate. The m / e 4ffi may be due t o either fragmen-
t a t i o n or t o fluphenazine base .
2.6 M e l t i n g range
The m e l t i n g range o f crystallised fluphenazine decanoate has

been determined as 30-3Z°C.(Lot 117102,purity 99%).
2.7 R e f r a c t i v e index
The r e f r a c t i v e index has been determined as 1.5395 a t 25OC on a

sample o f m a t e r i a l o f 99% purity.(Lot 117102).
2.8 Solubility
-1
Fluphenazine decanoate is inso uble in water ( d O u g mlml) b u t
extremely soluble(>lOOOrng m l ) in chloroform, d i e t h y l ether,
cyclohexane, methanol an$ythanol. It is also extremely soluble
i n coconut and sesame oil. .
282
RELATIVE INTENSITY
a
; 8 g : % 8 8 S f 3 $ E
2a
40
60
80
100
120
140
160
180
200
220
240
260
2 280
'? 300
rtm 7,
' 0
w 5 320
m U
340
..
I Y 360
% 380
400
420
440
460
480
500
520
540
560
580
600
620 -
0 h) W P Ul
PERCENT TOTAL IONIZATION Z

39
a,
c,
hl I rd
S
E SI 0
c
I-----
tO - -rll rd
u
I
el
110
a"
f
I I
I1s 0
a,
c
-4
N
rd
r:
a,
.c
a
7
r--- d
b4
4-1
0
I
I
I
1
I
I
I
3
N +
283
284 GEOFFREY CLARKE
2.9 pKa
The p K a and p K a values f o r fluphenazine decanoate have n o t

been reported. dowever they would be expected t o be very
similar t o those reported f o r fluphenazine enanthate o f about 3.4
and 8.0.(see analytical profiles of drug substances, volume 2).
2.10 D i f f e r e n t i a l thermal analysis
Beoween 15OC and 200°C only the endotherm due t o m e l t i n g a t

30 C is observed f o r the crystalline material. Thg c(iidro-
chloride salt gives an endotherm due t o m e l t i n g a t 180 C. .
3. Synthesis
Fluphenazine decanoate(1) can be prepared from fluphenazine(I1)

by refluxing a chloroform solution o f (11) w i t h decanoyl chloride.
(See Figure 6). The fluphenazine decanoate is e x t r a c t e d as t h e
hydrochloride salt and recrystallised f r o m a m i x t u r e o f anhy-
drous acetone and ether. A f t e r reconversion t o the base w i t h
aqueous sodium carbonate t h e fluphenazine decanoa
ed into ether, dried and concentrated b y evaporation t 8 , h.
extract
4. Stability
Fluphenazine dpjanoate(1) w i l l hydrolyse i n alkaline medium t o

fluphenazine(I1) .In the presence o f peroxides, oxidation o f t h e
piperazine nitrogen(506afj N -oxide(III) occurs probably by a f r e e
radical mechanism. ’ ’ Fluphen ine decanoate w i l l undergo
photolysis t o f o r m a sulphoxide(IV{?(See Figure 6).
5. Drug metabolism
Studies w i t h 14C labelled fluphenazine decanoate i n t h e dog have

been reported. The fluphenazine decanoate i s hydrolysed t o
fluphenazine by plasma esterases and excreted in the urine. The
metabolism o f fluphenazine decanoate, in the dog, is therefore
similar to(@at o f fluphenazine dihydrochloride and fluphenazine
enanthate .(See analytical profiles o f drug substances, volume
2). Traces o f residual fluphenazine decanoate and/or i t s meta-
bolites were found in t h e l ~ liver,
, kidney, skin and h e a r t of
the dog b u t none i n the brain .
n
/
light
n
(CH2)3.N-N.(
I
c H2)2 0
Figure 6. Chemistry of Fluphenazine decanoate.

286 GEOFFREY CLARKE
6. Methods o f analysis
6.1 Elemental analysis
The following analysis has been made(10)
Ca1c u1a t ed Found
C 64.94 65.19
H 7.42 7.68
N 7.09 7.2Y
6.2 Non-aqueous t i t r a t i o n
Fluphenazine decanoate can be t i t r a t e d w i t h perchloric a c i d in

glacial acetic acid using c r y s t a l violet as the indicator. The
neutralisation equivalent is 295.75. End point detection either
visually v f h malachite green or potentiometrically has also been
reported .
6.3 Spectrophotometric analysis
The U V absorbance a t 261nm o f fluphenazine decanoate in

methanol can be used as a quantitative assay. UV spectrophoto-
m e t r y however w i l l only d i f f e r e n t i a t e between fluphenazine
decanoate and its sulphoxide, therefore a chromatographic sepa
r a t i o n o f fluphenazine decanoate f r o m other related substances
usually precedes UV measurement(See section 6.6).
6.4 C o l o r i m e t r i c analysis
Fluphenazine decanoate can be extracted as an ion-pair w i t h

bromothymol blue into toluene f r o m a p H 2.55 g l y c i n e / q I
buffer. The absorbance o f the solution i s measured a t 400nm. (7
A solution of fluphenazine decanoate in e t h y l acetate when
shaken w i t h a pH2 b u f f e r e d solution o f palladium chloride w i l l
f o r m a complex. This complex is s o h@e,jtj, the e t h y l acetate
phase and can be measured a t 440nm. . Palladium com-
plexes o f any other esters o f fluphenazine present as trace
impurities, would also be formed i n the e t h y l acetate phase.
However, the major hydrolysis product fluphenazine forms an
aqueous soluble complex and would r e m a i n in the b u f f e r phase.
Therefore, spectrophotometric measurement o f the aqueous b u f
f e r phase coulq& used as an assay f o r fluphenazine i n fluphena-
zine decanoate .
FLU PH EN A ZI N E DEC A N 0ATE 287
6.5 F l u o r i m e t r i c analysis
A spectrofluorimetric procedure has been reported i n which a

solution of fluphenazine decanoate in methanol/sulphuric acid
(80:20),after oxidation w i t h c e r i c ioTy@ t h e sulphoxide, fluoresces
a t 400nm when activated a t 343nm. .
6.6 Chromatographic analysis
6.6.1 Column chromatography
Fluphenazine decanoate and r e l a t e d substances can be adsorbed

onto a column o f silica gel f r o m a chloroform solution. The
fluphenazine decanoate can be selectively eluted f r o m t h e
column w i t h a solvent m i x t u r e o f cyclohexane/methanol/methyl-
acetate (67.2:35.6:97.2). A f t e r removing t h e solvent by evapor-
ation t h e fluphenazine decanoate can be quantified b y f i y o l v i n g
i n methanol and measuring the U V absorbance a t 261nm The .
major degradation products, fluphenazine and fluphenazine de-
canoate N-oxide are n o t eluted f r o m t h e silica gel column.
6.6.2 Paper chromatography
The following systems have been reported, although no R values

are quoted. Benzene/acetic/water(Z:Z:l) descending o n Wfiatman
No.1 paper and sodium formate(1 molar) ascending on Whatman
3 M M paper for the separation o f fluphenazine decanoate, flu-
phenazine and fluphenazine sulphoxide. Methanol/water(85:15)
descending on Whatman No.1 paper impregnated w i t h castor
oi1(2% in ether) f o r t h e separation o f fluphenazine decanoate,
fluphenyfae, fluphenazine octanoate and fluphenazine dode-
canoate .
Location i s by U V l i g h t and quantitation by elution
w i t h 95% ethanol and measuring the absorbance a t 261nm.
6.6.3 Thin layer chromatography
A summary o f t h e solvent systems and separations reported is

given in table 1. The adsorbent used in a l l systems is silica gel G
w i t h a fluorescent indicator. Location o f separated compounds
i s made b y fluorescence quenching of UV light(366 or 254nm) or
c o l o r i m e t r i c a l l y b y spraying w i t h 50% sulphuric a c i d t o produce
r e d zones. The solvent systems r e f e r r e d t o in table 1 a r e as
follows
Rf values
Solvent Fluphenazine Fluphenazine Fluphenazine Fluphenazine Fluphenazine

System sulphoxide dec anoat e dec anoat e dec anoate
N-oxides sulphoxide
I 0.60 0.25 0.00, 0.16 0. ao 0.73
II 0.73 - 0.00, 0.20" 0.84 0.80

0.08, 0.25
III 0.10 0.0 0.0 0.50 0.48
IV 0.1 0 0.0 0.0 0. ao 0.75
V 0. ao - - 0.90 -
*All 4 zones have n o t been positively identified as N-oxides.
Table 1. Thin layer chromatography.

I Cyclohexane/acetone/ammonia(30:80:5) (17)
II Chloroform(saturated w i t h ammonia)/methanol(80:2) (18)
111 Cyclohexane/acetone/ammonia(36:60:0.6) (17)

IV Methanol/ethyl acetate/cyclohexane/chloroform (19)
(9:25 :17 :3 8)
V Chloroform/methanol/ammonia(9:10:0.5) (20)
Solvent system (111) has been used as t h e basis f o r a quantitative

assay, the separated zones being
UV absorbance measured a t 261nm @if
ed w i t h methanol and the
6.6.4 Gas l i q u i d chromatography
Fluphenazine decanoate has been separated f r o m fluphenazine by

chromatographing the silylated omixture on a 5' x 4'' column o f
3% JXR* on Gas C h r o m Q a t 280 C. The carrier gas was nitrogen
and detection was b y f l a m e ionisation. Perphenazine was used as
an i n t T S y 1 standard and t h e following retention times were
report. .
Fluphenazine 4 minutes
Perphenazine 7 minutes
Fluphenazi ne decanoa t e 24 minutes
The silylation procedure was necessary in t h e above method t o
satisfactorily chromatograph the fluphenazine. However, flu-
phenazine esters do n o t require s i l y l a t i n g and t w o other pro-
cedures have been reported f o r the separation o f fluphenazine
decanoate f r o m o t h e r fluphenazine esters. W i t h the exception o f
t h e temperature and the absence o f silylation the conditions
were as above. The following separations were reported.
305°C(23) 330°C(24)
Fluphenazine octanoate 5.2 minutes
Fluphenazine decanoate 7.4 minutes 1m i n u t e
Fluphenazine 11.2 minutes
dodecanoate
Fluphenazine stearate/ 3.4 minutes
oleate/linoleate
*JXR-Applied Science Laboratories 1nc.State College P.A.U.S.A.
290 GEOFFREY CLARKE
6.6.5 High performance liquid chromatography
A reversed phase HPLC system f o r t h e separation o f fluphena-

zine f r o m fluphenafB7 decanoate and other fluphenazine esters
has been reported. .The e f f e c t s o f t h e p H o f t h e mobile
phase and the chain length o f the stationary phase were studied
and the following separation reported.
Column : Partisil-TMS*(trimethylsilane)
200 x 4.6mm ID
Mobile phase : Methanol/acetonitrile/
1%ammonium carbonate
(1:l:O. 3)
-1
Flow r a t e : 2ml min
D e t e c t ion : U V a t 260nm
Retention times : Fluphenazine 2.6 minutes
Fluphenazine decanoate 3.3 minutes
Fluphenazine m y r i s t a t e 4.2 minutes
Fluphenazine palmi t a t e 4.8 minutes
Fluphenazine stearate 5.8 minutes
Slight variation in the ammonium carbonate concentration had

l i t t l e e f f e c t over the range 0.1-1%. However, changes in the
r a t i o o f t o t a l organic p m t o aqueous phase has a marked
.
e f f e c t on r e t v $ p n times Hence t h e following separation has
been reported .
Column : Partisil-TMS*
250 x 4.6mm ID.
0.45% ammonium carbonate
(1:l:l) -1
Flow r a t e : 2 m l min
Detection : U V a t 260nm.
Retention times : Fluphenazine 4.5minutes
Fluphenazine decanoate a) 9 minutes
N-oxi des b) 11 minutes
Fluphenazine decanoate 14.5 minutes
FLUPHENAZINE DECANOATE 29 1
Using a similar mobile phase t o the above a separation o f the

octanoate and d o q y y n o a t e impurities i n fluphenazine decanoate
has been reported .
Column : Bondapak C18**
300 x 3.9mm ID
0.4% ammonium carbonate
(1:1:0.5).
Flow rate : 2ml min-l
Detection : 254nm
Retention times : Fluphenazine 2 minutes
Fluphenazine octanoate 3 minutes
Fluphena z i ne de canoa t e 4.5 minutes
Fluphenazine dodecanoate 6.5 minutes
*Partisil, Reeve Ange1,Clifton NJ. US.A.

**Bond a pa k, Waters Ass oci a t es .
7. Body f l u i d and tissue analysis
The metabolism o f fluphenazine decanoate is m a i n l y t h a t o f

i t s hydrolysis product fluphenazine. Whilst traces o f the
y8 hy dr o I ys edc2 f $ ~hpena z ine d e c ano a t e ha v e been d e t ec t ed by
C tracing, most o f the reported work has been directed
towards the detection and estimation o f fluphenazine and i t s
metabolites. In man, p p o d plasm 1 o f fluphenazine have
been determined by C tracing "fi', a f t e r i n i t i a l extrac-
t i o n of the alkaline plasma w i t h n-heptane. Further parti-
tioninq34yeparated various metabolites in the urine and
faeces U r i n e and plasma e x t r a c t s have also been analysed
b y GLC(34) using an alkali-bead nitrogen sensitive detector
and a column o f 3% OV-17" o n Chromosorb W a t 215OC. A
similar p r o c e q y e using a f l a m e ionisation detector has also
been reported .
Another G L C procedure has been repo
fW.
f o r determining fluphenazine and i t s metabolites in urine
The urine was extracted by adsorption onto a n A m b e r l i t e
X A D - 2 column, washing w i t h pH8.5 ammonium chloride b u f f e r
and elution w i t h methanol. The extracted metabolites were
chromatographed as t h e i r t r i r n e t h y l s i l y l derivatives on 2% SE-
30 on Gas Chrom Q a t 225OC. D e t e c t i o n was b y f l a m e
ion is at ion.
292 GEOFFREY CLARKE
A f l u o r i m e t r i c procedure f o r blood plasma has been reported

in which the plasma is e x t r a c t e d w i t h hepiary&yamyl
alcoho1(98.3:1.7) a f t e r alkaline hydrolysis a t 100 C The
metabolites are back e x t r a c t e d i n t o 0.1M phosphate b u f f e r and
oxidised w i t h hydrogen peroxide. Fluorescence measurement
was a t 405nm, e x c i t i n g a t 350nm.
A n HPLC procedure has been reported f o r hexane e x t r a c t e d

serum UTW a glassy carbon electrode as an electrochemical
detector . The mobile phase was methanol/O.O5M
phosphate buffer, p H 6.9(53:47) and t h e separation was
achieved on a Lichrosorb S1(60)column(Merck, Darmstadt,
GFR), t r e a t e d w i t h dichlorodirnethylsilane.
A TLC r a t i o n of metabolites i n animal tissues has been

reportedy5? The tissues were extracted w i t h dichloromethane
and chromatographed on silica gel i n chloroform/isopropyl
alcohol(l0:l) and isopropyl alcohol/chloroform/arnmonia/water
( 3 2 16:Z:l).
*OV-17, Applied Science Laboratories Inc.,

State College P A U.S.A.
Acknowledgement
The author wishes t o thank Mrs. M. Watson f o r her invaluable

secretarial help.
8. References
M.S.Puar and P.T.Funke; Squibb Institute, p r i v a t e commun-

ication.
P.R.Wood; Squibb Institute, p r i v a t e communicaton.
P.Dondzila; Squibb Institute,private communication.
H.L.Yale, A.1.Cohen and F.Sowinski; J.Med.Chem.6, - 347,
(1963).
H.K ad in; Squ ibb Inst it u t e ,private communication.
R.D.G.Woolfenden;Squibb Institute,private communication.
B.J.Millard;School of Pharmacy, London, p r i v a t e communi-
cation.
J.Dreyfuss, J.J.Ross and E.C.Schreiber; J.Pharm.Sci., 60,
829,( 1971).
U.Timm and S.Pfeifer;Pharmazie a,11,(1973).
H.L.Ya1e and R.C.Merril1; U.S.Patent 1 194,733(1965).
J.A.Hill;Squibb Institute,private communication.
H.Kadin; Squibb Institute,private communication.
L.Cavatorta;J.Pharm.Pharmacol,c 49(1959).
G.A.Brewer jnr;Squibb Institute,private communication.
M.Parr y; Squibb Inst i t u t e ,pr ivat e com municat ion.
H.R.Roberts;Squibb Inst itute,private communication.
S. Shand;Squi bb Inst itute,pri vat e communication.
M.Parry and 1.M.Jackson;Squibb Inst itute,private communi-
cation.
C .G .Hug hes ;Squ ibb Inst itut e ,pr iv a t e communication.
C.L.Kroll;Squibb Inst itute,private communication.
W .F. H eyes;Squi bb Inst itut e,private communication.
W.F.Heyes;Squibb Institute,private communication.
T.Cowen;Squibb Inst itute,private communication.
M. Parr y; Squ i bb Inst itute,pr iv a t e com municat ion.
W.F.Heyes and J.R.Sa1mon;J.Chromatog.E 309(1978)
W.F.Heyes;Squibb Institute,private communication.
P.Y eh ;Squ ibb Inst itute,pr iv a t e communication.
E.C. Schre ibe r and M.L.Gro z ier;T hera p i e 3441( 1973).
294 GEOFFREY CLARKE
M.I.Kelsey,A.Keskiner and E.A.Moscatelli; J.Chromatog.

75 294(1973).
-
E.C.Dinovo,L.A.Gotlschalk,B.Naudi and P.G.Geddes;
J. Phar m. Sci .65
- 66 7(197 6).
A.B.Smulevitch,E.l.Minsker, N.A.Mazayyena, R.P. Volkora
and S.K.Lukanina; Comprehensive Psych. 14
227(1973).
C.P.Chien, T.L.Chan, D.Daniano and K.Chung; Abstracts,
10th congress C.I.N.P., Quebec(1976).
(33) F.Qui t ken, A. R it k i n and D.F.Klei n., A r c h .Gen .Psychat.

-
32(10)1276(1973).
(34) R.Whelpton and S.H.Curry;J.Pharm.Pharmacol. 28 869(1976).
-
(35) U.R.Tjaden, J.Laukelma, H.Poppe and R.G. Muusze;

-275(1976).
J.Chromatog.125
(36) N.J.Gaertner, U.Breyer and G.Liornin;

Biochem.Pharm.23 303.
L
(37) R. J. W arren, I. B. Eisdorf er, W .E. Thompson and J. E. Zarern bo;

. .
J. Phar m Sci -
55( 2) 144( 1966).
GENTAMICIN SULFATE
Bernard E. Rosenkruntz, Joseph R. Greco,
John G. Hoogerheide, and Edwin M . Oden
1. Description 296
1 . 1 Drug Properties 296
1.2 Chemical Properties and Structure 296
1.4 The USP Standard 298
2.3 NMR Spectrum 300
2.5 Thermal Properties (DSC, TGA) 302
2.6 Electrometric Titration-pK Value 310
2.8 X-Ray Diffraction 310
2.9 Solubility 313
2.10 Countercurrent Distribution 314
3. Biosynthesis 314
4. Isolation and Purification Processes 314
5. Drug Metabolism and Pharmacokinetics 315
6. Stability 315
7.2 Determination of Sulfate 316
7.3 Loss on Drying and Moisture Content 317
7.4 Determination of Component Ratios 317
8. Chromatographic Analysis 320
8.1 Paper Chromatography 320
8.2 Thin Layer Chromatography 32 1
8.3 Ion Exchange Chromatography 326
8.4 Gas- Liquid Chromatography 326
8.5 High Pressure Liquid Chromatography 326
9. Electrophoresis 327
10. Determination in Body Fluids 330
10.2 Fluoroimmunoassay 330
10.3 Radioimmunoassay 330
10.4 Radioenzyme Assay 331
10.5 High Pressure Liquid Chromatography 33 1
12. References 334
Copynght @ 1980 by Academic Ress. Inc.
Analytical Rofiles of Drug Substances, 9 295 AU rights of reproduction in any form IXSSNC~.
ISBN: 0-12-260809-7
296 BERNARD E. ROSENKRANTZ et al.
Gentamicin S u l f a t e
1. Description
1.1 Drug P r o p e r t i e s
Gentamicin i s an important member of t h e aminogly-

c o s i d e class of a n t i b i o t i c s u b s t a n c e s t h a t w a s f i r s t
i s o l a t e d i n 1963 by W e i n s t e i n e t a l . 1 from two p r e v i o u s l y
undescribed s p e c i e s of Micromonospora. I s o l a t i o n and pre-
l i m i n a r y chemical s t u d i e s 2 demonstrated t h a t i t is a
m i x t u r e of b a s i c , water s o l u b l e a n t i b i o t i c s c o n t a i n i n g t h e
a m i n o c y c l i t o l 2-deoxystreptamine and 2 a d d i t i o n a l amino
sugars. Chromatographic s e p a r a t i o n of t h e g e n t a m i c i n
complex showed i t t o c o n s i s t of 3 major components d e s i g n a t e d
as C1, C2 and C l a . 3 9 4 The g e n t a m i c i n complex is used a s
t h e s u l f a t e s a l t i n v a r i o u s dosage forms i n c l u d i n g i n j e c t a b l e
and t o p i c a l p r e p a r a t i o n s , and is e f f e c t i v e a g a i n s t a wide
v a r i e t y of gram-negative and gram-positive organisms.
1.2 Chemical P r o p e r t i e s and S t r u c t u r e
Each of t h e t h r e e major components of t h e gentami-

c i n complex c o n t a i n s f i v e b a s i c amino f u n c t i o n s . A s i s
t y p i c a l of t h i s c l a s s of a n t i b i o t i c s , 6 g e n t a m i c i n s u l f a t e i s
o b t a i n e d a s a h y d r a t e d amorphous s o l i d w i t h o u t c h a r a c t e r i s t i c
m e l t i n g p o i n t , o r UV a b s o r p t i o n .
The e l u c i d a t i o n of t h e s t r u c t u r e and s t e r e o c h e m i s -
t r y of t h e components of t h e g e n t a m i c i n complex a r e d e s c r i b e d
i n p u b l i c a t i o n s by Cooper e t al.7-11 and Daniels.12 The
s t r u c t u r a l formulae, m o l e c u l a r w e i g h t s and t h e nomenclature
of t h e amino s u g a r u n i t s comprising t h e g e n t a m i c i n complex
are g i v e n i n F i g u r e 1; t h e common s u g a r u n i t has been named
garosamine and t h e d i s s i m i l a r 2,6-diamino s u g a r s have been
named purpurosamine A , B and C , corresponding t o g e n t a m i c i n s
C1, C 2 , and Cia, r e s p e c t i v e l y .
A number of i n v e s t i g a t o r s have r e p o r t e d on minor

components t h a t are coproduced w i t h gentamicin. In addition
t o g e n t a m i c i n A and g e n t a m i c i n B which were noted i n t h e
.
o r i g i n a l p a p e r by W e i n s t e i n e t al.1, t h e s e minor components
i n c l u d e gentamicins B1, X, C2a, A summary of t h e
methods used t o i s o l a t e and s e p aand c2k
r a t e t ese is g i v e n i n a
r e c e n t review.13
PURPUROSAMINE
/
/
0
/
/
/
/
2-DEOXYSTREPTAMINE
GENTAMICIN C1 R = R1 = CH3 C21 H 4 3 N 5 0 7 (M.W. 477.6)

GENTAMICIN C2 R = CH3; R, = H C20H41 N507 (M.W. 4 6 3 . 6 )
GENTAMICIN Cia R = R) = H C19 H 3 9 N 5 0 7 (M.W. 4 4 9 . 5 )
Figure 1: Structural Formula of Gentamicin Complex.

298 BERNARD E. ROSENKRANTZ el al.
1.3 Appearance, C o l o r , Odor
Gentamicin s u l f a t e is a w h i t e t o b u f f c o l o r e d ,
o d o r l e s s , h y g r o s c o p i c powder.
1.4 The USP S t a n d a r d
The b i o l o g i c a l a c t i v i t y of b u l k g e n t a m i c i n s u l f a t e
i s e x p r e s s e d i n mcg g e n t a m i c i n p e r mg g e n t a m i c i n s u l f a t e
based on a p o t e n c y of 1000 mcg p e r mg ( d r i e d b a s i s ) o r i g i n a l l y
a s s i g n e d t o t h e master s t a n d a r d b a s e . The c u r r e n t USP
S t a n d a r d of g e n t a m i c i n s u l f a t e h a s a p o t e n c y of 650 mcg/mg
on t h e d r i e d b a s i s and t h e minimum a c c e p t a n c e l i m i t on
potency f o r g e n t a m i c i n s u l f a t e b u l k s u b s t a n c e i s 590 mcg/mg
( d r i e d b a s i s ) . FDA c e r t i f i c a t i o n a l s o r e q u i r e s compliance
w i t h s p e c i f i c a t i o n s f o r i d e n t i t y , pH, l o s s on d r y i n g ,
o p t i c a l r o t a t i o n and g e n t a m i c i n C component r a t i o s . 1 4
The i n f r a r e d s p e c t r u m of a p o t a s s i u m bromide
(KBr) p e l l e t of Gentamicin S u l f a t e USP R e f e r e n c e S t a n d a r d
is g i v e n i n F i g u r e 2. I t was o b t a i n e d u s i n g a P e r k i n E l m e r
180 g r a t i n g s p e c t r o p h o t o m e t e r . The i n f r a r e d band a s s i g n -
ments a r e g i v e n below.l5 I t s h o u l d b e n o t e d t h a t bands are
n o t p r e s e n t which would p e r m i t d i f f e r e n t i a t i o n from s i m i l a r
aminoglycoside a n t i b i o t i c s .
-1
Wavenumber (cm ) Assignment
3500-2500 ( s , v b r ) OH, NH3

+, NH2 + stretch
1620 (m) NH3

+, NH2 + symmetric bend
1525 (m) NH3

+, NH2 + symmetric bend
1150-1000 ( v s , b r ) C-0, HS04

- stretch
610 ( s ) SO2 bend
Notation: w = weak, m = medium, s = s t r o n g , vs = v e r y

s t r o n g , b r = b r o a d , v b r = v e r y broad.
id
u
m
W
oa,
U
n C
ua,
ar-r
-la,
-lw
ala,
a&
..
cv
300 BERNARD E. ROSENKRANTZ ef (11.
The g e n t a m i c i n complex does n o t p o s s e s s u l t r a v i o l e t

l i g h t a b s o r b i n g p r o p e r t i e s ; b o t h t h e f r e e b a s e and s u l f a t e
show end a b s o r p t i o n only.
2.3 Nuclear Magnetic Resonance S p e c t r a
2.3.1 Proton Magnetic Resonance Spectrum
An 80 MHz p r o t o n NMR spectrum of a s o l u t i o n

of Gentamicin S u l f a t e USP Reference S t a n d a r d 15% w/v i n D20
i s g i v e n i n F i g u r e 3. It was o b t a i n e d u s i n g a Varian CFT-20
s p e c t r o m e t e r a t ambient t e m p e r a t u r e and sodium 2,2-dimethylY
2-silapentane-5-sulfonate (DSS) as t h e i n t e r n a l r e f e r e n c e .
The s p e c t r a l assignments g i v e n below a r e i n ppm ( 6 ) downfield
from DSS.I5
Chemical
Protons S h i f t s ( 6) Multiplicity Oripin
5‘-CH (a3) 1.30 doublets components of

(J=7.5 Hz) C1 and C
2
4”-CH3 1.35 singlet
2, 3‘, 4’CH2 1.75-2.5 broad
5’-CH ( CH3)NHa3 2.7 5 singlet
3”-NHCH3 2.95 singlet

‘1’ ‘2’ ‘la
3”-H 3.48 doublet
“1’ ‘2’ ‘la
(J=11.0 H z )
5”-CH20 eq 4.0 mu1t i p l e t
2”-H 4.25 d o u b l e t of d o u b l e t
C1, C 2 , Cla
(J=11.0, 4.0 Hz)
1”-H 5.16 doublet

(5-4.0 Hz)
5’c2’
1-H 5.88 ove r l a p p i n g
‘1’ ‘2’ ‘la
doublets
A d d i t i o n a l d i s c u s s i o n of NMR s p e c t r a l a s s i g n -
ments f o r g e n t a m i c i n i s g i v e n by Cooper e t a1.8,10,11
GENTAMICIN SULFATE
/i
I
I ' " ' I ~ ' " I I " ' I " I " ' I
~
I "
7 6 5 .I 4 3 2 1 0
F i g u r e 3: PMR Spectrum of G e n t a m i c i n S u l f a t e USP R e f e r e n c e S t a n d a r d .
30 1
302 BERNARD E. ROSENKRANTZ el al.
2.3.2 Carbon-13 Magnetic Resonance Spectrum
A carbon-13 NMR s p e c t r u m of a s o l u t i o n of
Gentamicin S u l f a t e USP R e f e r e n c e S t a n d a r d (80 mg/0.50 m l i n
D20) i s g i v e n i n F i g u r e 4. It was o b t a i n e d 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 a t ambient t e m p e r a t u r e and d i o x a n e as t h e
i n t e r n a l r e f e r e n c e . The chemical s h i f t a s s i g n m e n t s g i v e n
i n T a b l e 1 a r e i n ppm ( 6 ) w i t h r e f e r e n c e t o i n t e r n a l d i o x a n e
t a k e n as 67.40 ppm down from e x t e r n a l t e t r a m e t h y l ~ i l a n e . ' ~
A d i s c u s s i o n of C-13 NMR s p e c t r a l d a t a of t h e
g e n t a m i c i n C components C C 2 , and C1 i s g i v e n by
la'
Morton e t a1.I6
2.4 Mass Spectrum
The mass spectrum of g e n t a m i c i n f r e e b a s e , p r e p a r e d

by n e u t r a l i z a t i o n of Gentamicin S u l f a t e USP R e f e r e n c e
S t a n d a r d i s g i v e n i n F i g u r e s 5 and 5a. It w a s o b t a i n e d u s i n g
a V a r i a n MAT CH-5 medium r e s o l u t i o n s i n g l e f o c u s i n g s p e c t r o -
0
meter a t a probe t e m p e r a t u r e of 170 C and a s o u r c e t e m e r a t u r e
of 250°C. The m R s s a s s i g n m e n t s are g i v e n i n T a b l e 2. 11;
A d d i t i o n a l d i s c u s s i o n r e l a t i n g t o t h e mass s p e c t r o -
metry
--
et al.
~5,fegntamicin i s g i v e n by Cooper
and P a r f i t t e t a l . 1 9
e t a1.8,11, D a n i e l s
2.5 Thermal P r o p e r t i e s (TGA, DSC)
2.5.1 Thermogravimetric A n a l y s i s (TGA)
A t h e r m o g r a v i m e t r i c a n a l y s i s c u r v e w a s ob-
t a i n e d f o r Gentamicin S u l f a t e USP R e f e r e n c e S t a n d a r d ( s e e
F i g u r e 6 ) u s i n g a DuPont Nodel 950 Thermogravimetric A n a l y z e r
equipped w i t h a Model 900 Programmer-Recorder. The a n a l y s i s
w a s performed a t a h e a t i n g rate of 10°C/minute, u n d e r a
n i t r o g e n atmosphere.
The t h e r m o g r a v i m e t r i c a n a l y s i s of t h e USP
R e f e r e n c e S t a n d a r d i n d i c a t e s loss of a p p r o x i m a t e l y 12% water
from ambient t o 125OC. Decomposition s t a r t s a t 22OoC and
p r o c e e d s s t e p w i s e u n t i l 33OoC; above 330° a d d i t i o n a l de-
c o m p o s i t i o n o c c u r s , y i e l d i n g a f i n a l r e s i d u e of a b o u t 30%
which is a t t r i b u t a b l e t o t h e s u l f a t e s a l t . 1 5
- !- .-2
'3800 3500 3000 2500 2000 CJ 1800 1600 1400 1200 lo00 800 625:0
Wavelength urn
Figure 4: Carbon-13 NMR Spectrum of

Gentamicin Sulfate USP Reference Standard.
Carbon-13 chemical s h i f t assignments of Gentamicin S u l f a t e

USP Reference Standard i n ppm (6) w i t h r e f e r e n c e t o i n t e r n a l
dioxane t a k e n as 67.40 ppm down from e x t e r n a l t e t r a m e t h y l -
s i l a n e (see Figure 4).
1
Carbon Chemical S h i f t s
1 50.6
2 28.5
3 49.5
4 76.7
5 75.3
6 84.4
1' 95.4, 95.3, 95.0*
2' 49.5
3' 21.4
4' 24*0(C2,C1) , 26*3(CIa)
5' 70.0, 69.6*
5'-CH2NH2 43.5
5'-CH ( CH3) NH2 50.4 (13.1)
c2
5'-CH(CH3)NH(CH3) 58.3 (10.1) (32.0) C1
1" 102.0
2" 67.1
3" 64.3
4" 70.8
5" 68.7
3"-NHCH3 35.4
4"-CH3 21.8
'The o p e r a t i n g frequency of t h e s p e c t r o m e t e r was 25.2 MHz

( 1 3 C ) ; 8 K d a t a p o i n t s were a c q u i r e d w i t h a s p e c t r a l w i d t h of
5500 Hz a p u l s e w i d t h of 15.0 p s e c , y i e l d i n g a f l i p a n g l e
b
of 60 and a r e p e t i t i o n r a t e of 0.8 s e c .
* M u l t i p l i c i t y i s due t o t h e m i x t u r e of C components.
100
90
> 80
5z 70
5 60
W
J 50
2
40
J
W
E 30
0 50 100 D
MASSICHARGE
Figure 5: Mass Spectrum of Gentamicin Base

(mass range 0 to 600).
300 350 400 450 500 550
MASS/CHARGE
F i g u r e 5a: Mass Spectrum of Gentamicin Base

(mass r a n g e 300 t o 5 5 0 ) -
CENTAMICIN SULFATE 307
TABLE 2
Mass Spectral Assignments of

Gentamicin Base
Masses (amu)
Ions
'la c2 c1
(M+l)+ 450 464 47 8
(M-l7)+ (NH3) 432 446 46 0
@- 0 -@ - 0' = CHOH 350 35 0 35 0
@- 0 - @ - 0+ = CHOH 319 333 347
@- 0 -@- 0+H2 322 322 322
[@- 0 - B]+ 30 4 304 30 4
HO @- O+ = CHOH 191 19 1 191
160 160 160

@+
15 7 143 129
I @ OH]+ 145 145 145
See the following page for the definition of A ,B and C.

308 BERNARD E. ROSENKRANTZ ei al.
T a b l e 2 (Continued)
R
I
0
,
-51 1 I
I
I I 1 I 1
I
I I I I I
I
".
I I
,
1 1 .
I
. . I , , . . I , I , . . , I .
I " "
.I , . .
"
I
I , , , , 1 .
I "
" ' ' 7
' I '
+,,
I
' I , ,
" I '
h i
I I I 1 I I I I I I I I I ' ' I I 1 I " I ' " V '
Figure 6: Thermogravimetric Analysis (TGA) Curve of

2.5.2 D i f f e r e n t i a l Scanning C o l o r i m e t r y (DSC)
A d i f f e r e n t i a l scanning calorimetry curve

( s e e F i g u r e 7) w a s o b t a i n e d f o r Gentamicin S u l f a t e USP R e f e r -
ence S t a n d a r d u s i n g a DuPont Model 990 Thermal A n a l y z e r
equipped w i t h a Model 910 C e l l Base. The s c a n was performed
0
a t a t e m p e r a t u r e program r a t e of 10 C h i n u t e , u n d e r a n i t r o -
g e n atmosphere a g a i n s t an empty aluminum sample pan.
The 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 c u r v e
of t h e USP R e f e r e n c e S t a n d a r d h a s a b r o a d e n d o t h e r m i c peak
around 75OC due t o l o s s of water and a l a r g e endotherm a t
25OoC c o r r e s p o n d i n g t o m e l t i n g decomposition. ' 5
2.6 E l e c t r o m e t r i c T i t r a t i o n CurveIApparent pKa Value
Each of t h e t h r e e m a j o r g e n t a m i c i n C components con-

t a i n s 5 b a s i c amino groups. Because of t h e i r s i m i l a r b a s i c
s t r e n g t h , t h e e l e c t r o m e t r i c t i t r a t i o n c u r v e P o ( F i g u r e 8) g i v e s
one t i t r a t i o n "break" correspondilng t o f i v e e q u i v a l e n t s of
a c i d consumed. An a p p a r e n t pKa v a l u e ( h a l f n e u t r a l i z a t i o n ) of
7.9 i s d e r i v e d from F i g u r e 8. T h i s c u r v e w a s o b t a i n e d w i t h a
Mettler a u t o m a t i c t i t r a t i o n s y s t e m ( c o n s i s t i n g of modules
D V 1 1 , D K l O and DV103) and a Corning semimicro combination pH
e l e c t r o d e . About 180 mg g e n t a m i c i n b a s e w a s d i s s o l v e d i n
water and t i t r a t e d w i t h 0.5N h y d r o c h l o r i c a c i d . A pKa v a l u e
of 8.2 f o r g e n t a m i c i n w a s r e p o r t e d by Done2' and a l s o by
Newton and K l u z a . 2 2
2.7 Optical Rotation
Allowable l i m i t s f o r t h e s p e c i f i c r o t a t i o n of g e n t a -
m i c i n s u l f a t e are +107O t o +121° as g i v e n i n t h e Code of
F e d e r a l R g u l a t i o n s (CFR)23 as w e l l as i n t h e B r i t i s h Pharma-
copoeia.2' The CFR s t a t e s t h a t t h e measurement s h o u l d be
performed on a 1%aqueous s o l u t i o n a t 25OC, w h i l e t h e
B r i t i s h Pharmacopoeia s t a t e s t h a t a 10% aqueous s o l u t i o n
s h o u l d b e measured a t 2OoC. The s p e c i f i c r o t a t i o n of t h e
USP Gentamicin S u l f a t e R e f e r e n c e S t a n d a r d w a s found t o b e
+115.9' when measured as a 0.3% aqueous s o l u t i o n i n a Bendix
Series 1100 P o l a r i m e t e r a t 26OC. 25
2.8 X-Ray Diffraction
X-ray powder d i f f r a c t i o n s t u d i e s 2 6 show t h a t g e n t a -

m i c i n s u l f a t e i s e s s e n t i a l l y a n amorphous s u b s t a n c e ; no s p e c -
t r a l bands were observed when t h e USP R e f e r e n c e S t a n d a r d w a s
r u n on a P h i l i p s APD-3500 u t i l i z i n g Cu Ka r a d i a t i o n (1.54182).
Figure 7: Differential Scanning Calorimetry (DSC) Curve of
312 BERNARD E. ROSENKRANTZ er (11.
9-
0-
7-
PH
6-
5-
4-
3-
2-
F i g u r e 8: E l e c t r o m e t r i c T i t r a t i o n Curve of
Gentarnicin Base.
GENTAMICIN SULFATE 313
2.9 Solubility
Gentamicin s u l f a t e i s f r e e l y s o l u b l e i n w a t e r ,
0.1N h y d r o c h l o r i c a c i d , 0.1N sodium h y d r o x i d e (>1 g/ml i n
e a c h of t h e s e aqueous media). It is i n s o l u b l e i n a l c o h o l
and most o t h e r o r g a n i c s o l v e n t s . A s p a r t of a comprehensive
s t u d y of 5 1 a n t i b i o t i c compounds Marsh e t a1.W r e p o r t e d t h e
s o l u b i l i t y of g e n t a m i c i n s u l f a t e i n 26 s o l v e n t s a t room
temperature. Some of t h e s e d a t a are p r e s e n t e d below:
S o l u b i l i t y a t 28+4'C
Gentamicin S u l f a t e
Solvent (mdml)
Ethylene Glycol >20

Formamide >20
Propylene Glycol 6.332
Chloroform 0.678
Methanol 0.200
Dimethyl S u l f o x i d e 0.072
Is0p r opano 1 0.045
Acetone 0.042
Carbon D i s u l f i d e 0.028
Pyr i d i n e 0.028
Ethyl Acetate 0.025
Benzene 0.0
Carbon T e t r a c h l o r i d e 0.0
Is0 oct a n e 0.0
Diethyl Ether 0.0
Gentamicin b a s e complex was found v e r y s o l u b l e i n

water (>1 /ml) and i s more s o l u b l e t h a n t h e s u l f a t e s a l t i n
a number 2 8 of o r g a n i c s o l v e n t s . Some of t h e s e d a t a are
t a b u l a t e d below:
S o l u b i l i t y a t 25+loC
Gentamicin Base
Solvent (mnlml)
Methanol >25
n-But a n o l >25
Ethanol >25
Chloroform >25
Acetone >25
2-Butanone 2.6
Toluene 2.4
Ethyl Acetate 2.1
Cyc l o h exane 0.2
2-10 Countercurrent Distribution
I n 1977, Byrne e t a1.28 r e p o r t e d on t h e s e p a r a t i o n

of t h e g e n t a m i c i n C complex i n t o f i v e components by C r a i g
d i s t r i b u t i o n . I n a d d i t i o n t o t h e t h r e e m a j o r components
C1, C 2 , and C l a y t h e s e w o r k e r s s e p a r a t e d two a d d i t i o n a l com-
ponents, C and CZb. Gentamicin CZa w a s i d e n t i f i e d as t h e
2a
of g e n t a m i c i n C , w h i l e g e n t a m i c i n C2b w a s iden-
6'-C-epimer
t i f i e d a s 6'-N-methylgentam$cin C1 . The s e p a r a t i o n s were
c a r r i e d o u t i n a 1 0 2 0 - c e l l automat% C r a i g d i s t r i b u t i o n a p p a r -
a t u s of 10 m l f i x e d lower p h a s e volume, u s i n g a c h l o r o f o r m :
methanol:17% ammonia ( 2 : l : l ) s o l v e n t system.
3. Biosynthesis
The b i o s y n t h e s i s of a m i n o c y c l i t o l a n t i b i o t i c s , i n c l u d i n g
g e n t a m i c i n , i s d i s c u s s e d i n a r e c e n t comprehensive review.29
Glucose h a s been shown t o p r o v i d e t h e s k e l e t o n s of a l l sub-
u n i t s of t h e a n t i b i o t i c s s o f a r s t u d i e d ; however, d e t a i l s o f
t h e s t e p s i n v o l v e d a r e s t i l l unknown i n a l m o s t a l l cases.
Of t h e deoxystreptamine-containing a n t i b i o t i c s , t h e b u l k of
t h e e f f o r t h a s b e e n d i r e c t e d toward t h e b i o s y n t h e s i s of
neomycins.
Gentamicins d i f f e r from t h e neomycins, kanamycins, and

paromomycins i n t h a t t h e y c o n t a i n b o t h C-methyl and N-methyl
s u b s t i t u e n t s ; most s t u d i e s on g e n t a m i c i n s have been aimed a t
d e t e r m i n i n g t h e s o u r c e of t h e methyl g r o u p s . Studies carried
o u t by Lee e t a1.30 i n d i c a t e a h i g h e f f i c i e n c y of L-methionine
i n c o r p o r a t i o n i n t o gentami i n s . L a b e l l i n g experiments u s i n g
5
13C-methyl m e t h i o n i n e and H-methyl m e t h i o n i n e have shown
t h a t a l l of t h e m e t h y l g r o u p s i n g e n t a m i c i n are d e r i v e d from
m e t h i 0 n i n e . 3 ~ A d d i t i o n a l work by L e e e t a1.32 shows t h a t when
13 C-methyl-methionine w a s added a t t h e o n s e t of b i o s y n t h e s i s
of t h e g e n t a m i c i n components, i n c o r p o r a t i o n of l a b e l i n t o t h e
minor components preceded i n c o r p o r f k i o n i n t o t h e m a j o r compo-
n e n t s . D e g r a d a t i o n o c c u r r e d when C-methyl g e n t a m i c i n
major components w e r e added t o t h e g e n t a m i c i n - p r o d u c i n g c u l -
t u r e medium and shaken.
4. I s o l a t i o n and P u r i f i c a t i o n P r o c e s s e s
I n 1963 R o s s e l e t 3 3 and co-workers f i r s t r e p o r t e d o n t h e

i s o l a t i o n of t h e g e n t a m i c i n complex u s i n g ion-exchange chroma-
t o g r a p h y . V a r i o u s ion-exchange p r o c e d u r e s c o n t i n u e t o be u s e d
e x t e n s i v e l y f o r t h e s e p a r a t i o n and p u r i f i c a t i o n of g e n t a m i c i n
on a p r e p a r a t i v e s c a l e . A commonly used p r o c e d u r e is t o ad-
j u s t t h e whole b r o t h t o pH 2 w i t h s u l f u r i c a c i d , f o l l o w e d by
filtration. After a d j u s t m e n t t o pH 7 , t h e n e u t r a l i z e d f i l -
t r a t e i s p a s s e d t h r o u g h a n IRC-50 r e s i n column i n t h e
ammonium c y c l e , and t h e a n t i b i o t i c i s t h e n e l u t e d w i t h 2 N
aqueous ammonia. The g e n t a m i c i n C complex may be i s o l a t e d
from co-produced minor components u s i n g a Dowex 1x2 column
(OH-f o rm) - 5
5. Drug Metabolism and P h a r m a c o k i n e t i c s
Gentamicin s h a r e s w i t h many o t h e r a m i n o g l y c o s i d e
a n t i b i o t i c s t h e i m p o r t a n t p r o p e r t y of b e i n g s t a b l e i n
b i o l o g i c a l s y s t e m s . When a d m i n i s t e r e d t o man o r a n i m a l s ,
t h e m a j o r p o r t i o n i s e x c r e t e d i n t h e u r i n e by g l o m e r u l a r
f i l t r a t i o n . 34. Gentamicin i s n o t absorbed i n a p p r e c i a b l e
amounts from t h e i n t a c t g a s t r o i n t e s t i n a l t r a c t .
A f t e r i n t r a m u s c u l a r a d m i n i s t r a t i o n , p e a k serum concen-
t r a t i o n s u s u a l l y o c c u r between 30 and 60 m i n u t e s and serum
levels a r e m e a s u r a b l e f o r s i x t o e i g h t hours. When g e n t a m i c i n
i s a d m i n i s t e r e d by i n t r a v e n o u s i n f u s i o n o v e r a two-hour
p e r i o d , t h e serum c o n c e n t r a t i o n s are s i m i l a r t o t h o s e o b t a i n e d
by i n t r a m u s c u l a r a d m i n i s t r a t i o n . P r o t e i n b i n d i n g s t u d i e s have
i n d i c a t e d t h a t t h e d e g r e e of g e n t a m i c i n b i n d i n g i s low,
between 0 and 3O%.35
6. Stability
Gentamicin s u l f a t e powder is v e r y s t a b l e when s t o r e d

i n t i g h t l y c l o s e d c o n t a i n e r s a t room t e m p e r a t u r e . Gentamicin
s u l f a t e is s t a b l e f o r a t least f i v e years with r e s p e c t t o
p o t e n c y , s p e c i f i c r o t a t i o n and pH.
Gentamicin w a s a l s o s t a b l e i n b o i l i n g aqueous b u f f e r s
of pH 2 t o 14.36 It is p a r t i c u l a r l y r e s i s t a n t t o a t t a c k by
a l k a l i , and h a s b e e n r e f l u x e d i n 2 N sodium h y d r o x i d e f o r 2
h o u r s w i t h no a p p a r e n t l o s s i n a c t i v i t y . 3 7 More r e c e n t
s t u d i e s on g e n t a m i c i n c o n f i r m i t s e x c e l l e n t s t a b i l i t y i n
m o d e r a t e l y a c i d t o s t r o n g l y b a s i c aqueous media. Under h gh-
l y s t r e s s e d c o n d i t i o n s ( h e a t i n g i n 1N s u l f u r i c a c i d f o r 5
0
d a y s a t 60 C ) , a p p r o x i m a t e l y a 30% l o s s i n p o t e n c y w a s
f 0 u n d . 3 ~ Gentamicin s u l f a t e w a s a l s o shown t o be s t a b l e n
i n f u s i o n s o l u t i o n s 3 9 and i n a r t i f i c i a l t e a r s o l u t i o n s . 4 0
Gentamicin s u l f a t e e x h i b i t s e x c e l l e n t s t a b i l i t y i n v a r i -
ous p h a r m a c e u t i c a l dosage forms. I n p a r e n t e r a l s o l u t i o n s and
t o p i c a l o i n t m e n t s i t h a s b e e n shown t o be s t a b l e f o r a t l e a s t
0
f i v e y e a r s u n d e r normal s t o r a g e c o n d i t i o n s ( 2 t o 30°C).
316 BERNARD E. ROSENKRANTZ et a1
7. Methods of A n a l y s i s
7.1 Identification
Gentamicin is c o n v e n i e n t l y i d e n t i f i e d by t h i n - l a y e r
chromatography (TLC). Gentamicin is r e s o l v e d i n t o i t s 3 com-
ponents and a l s o can b e s e p a r a t e d from most o t h e r r e l a t e d
a n t i b i o t i c s u s i n g TLC. R e f e r t o t h e d i s c u s s i o n i n s e c t i o n
8.2 and e s p e c i a l l y Wilson e t a l . 4 l a n d Pauncz42 f o r r e l a t e d
discussion.
I n a t y p i c a l TLC method about 50 t o 100 ug of

gentamicin s u l f a t e is a p p l i e d t o a s i l i c a g e l TLC p l a t e and
developed u s i n g t h e lower phase of a m i x t u r e of e q u a l volumes
of chloroform, methanol and c o n c e n t r a t e d aqueous ammonia. 41
The s p o t s a r e t y p i c a l l y v i s u a l i z e d w i t h n i n h y d r i n r e a g e n t
o r w i t h i o d i n e vapors. R e s u l t s a r e compared w i t h t h o s e
o b t a i n e d from a s i m i l a r l y chromatographed r e f e r e n c e s o l u t i o n .
Paper chromatography i s a l s o u s e f u l f o r i d e n t i f i -
c a t i o n (see s e c t i o n 8.1). The B r i t i s h Pharmacopoeia 1973, p.
216 d e s c r i b e s a method where t h e s o l v e n t system chloroform:
methano1:concentrated aqueous ammonia:water (10:5:3:2) i s
used a l o n g w i t h n i n h y d r i n s p r a y d e t e c t i o n .
The BP a l s o d e s c r i b e s a method where a UV s p e c t r u m

is o b t a i n e d a f t e r t r e a t m e n t w i t h s u l f u r i c a c i d . No maximum
is o b t a i n e d f o r g e n t a m i c i n , which d i s t i n g u i s h e s i t from kana-
mycin, neomycin and paromomycin.
The Code of F e d e r a l R e g u l a t i o n s (444.20) d e s c r i b e s

a n i n f r a r e d s p e c t r o p h o t o m e t r i c t e c h n i q u e u s i n g a KBr d i s c .
The i n f r a r e d spectrum of g e n t a m i c i n s u l f a t e , however, is
v e r y similar t o t h a t of o t h e r aminoglycoside a n t i b i o t i c s and
i s t h e r e f o r e of l i m i t e d v a l u e as a n i d e n t i f i c a t i o n test.
7.2 Determination of S u l f a t e Content
A s d e s c r i b e d i n s e c t i o n 1 . 2 , g e n t a m i c i n s u l f a t e is
composed of t h r e e major components- S i n c e each component h a s
5 b a s i c n i t r o g e n s , 5 e q u i v a l e n t s of s u l f u r i c a c i d are re-
q u i r e d p e r mole of g e n t a m i c i n base. The l i m i t s f o r s u l f a t e
c o n t e n t g i v e n i n t h e B r i t i s h Pharmacopoeia 1973 are 31.0 t o
34.0% (anhydrous b a s i s ) .
The g r a v i m e t r i c p r o c e d u r e d e s c r i b e d i n t h e B€&3
i n v o l v e s p r e c i p i t a t i o n of barium s u l f a t e by t h e a d d i t i o n of
h y d r o c h l o r i c a c i d and barium c h l o r i d e t o a n aqueous s o l u t i o n
of t h e a n t i b i o t i c , f o l l o w e d by washing, i g n i t i n g and weighing

the residue. Each gram of r e s i d u e is e q u i v a l e n t t o 0.4116
gram of s u l f a t e .
7.3 Loss on Drying and M o i s t u r e Content
Gentamicin s u l f a t e is an amorphous, h y g r o s c o p i c
powder which t y p i c a l l y c o n t a i n s 10 t o 15% water. The U . S .
Government Code of F e d e r a l R e g u l a t i o n s (CFR) a l l o w s a
maximum of 18% l o s s on drying.44 I n t h e CFR method45 t h e
g e n t a m i c i n s u l f a t e sample i s h e a t e d a t a t e m p e r a t u r e of 110 C
f o r 3 h r i n a vacuum (5 5mm mercury). The B r i t i s h Pharma-
copoeia s p e c i f i c a t i o n f o r water c o n t e n t is 15%*4
F i s c h e r t i t r a t i o n and e l e c t r o n i c e n d p o i n t d e t e c t i o n .
It has b e e n shown t h a t l o s s on d r y i n g r e s u l t s and

Karl F i s c h e r t i t r a t i o n r e s u l t s are i n good agreement.47
7.4 D e t e r m i n a t i o n of Component R a t i o s
A number of methods have been used f o r t h e determi-

n a t i o n of g e n t a m i c i n C component r a t i o s . Brief d e s c r i p t i o n s
of n i n e of t h e s e methods are p r e s e n t e d i n t h i s s e c t i o n .
7.4.1 For U.S. c e r t i f i c a t i o n , a l l b a t c h e s of genta-

m i c i n s u l f a t e must conform t o t h e f o l l o w i n g r e q u i r e m e n t s f o r
component r a t i o s d e s c r i b e d i n t h e Code of F e d e r a l Regula-
t ions :
Not l e s s t h a n 25% n o r more t h a n 50%

C1:
C : Not l e s s t h a n 15% n o r more t h a n 40%
Not l e s s t h a n 20% n o r more t h a n 50%
C?:
The o f f i c i a l method g i v e n i n 2 1 CFR 444.20 a ( b ) ( 8 )
i s based on a p a p e r chromatographic p r o c e d u r e r e p o r t e d by
Kantor and S e l z e r . 4 8 I n t h i s method, two i d e n t i c a l sample
chromatograms are developed i n t h e lower p h a s e of chloroform-
methanol-17% ammonium hydroxide ( 2 : l : l ) . One chromatogram
i s sprayed w i t h n i n h y d r i n r e a g e n t t o l o c a t e t h e p o s i t i o n s of
t h e C components Cl , C 2 and C , which have approximate
R v a l u e s of 0.35, 8.50 and 0.$5 r e s p e c t i v e l y . The s p o t s i n
t h s s t r i p are used t o l o c a t e t h e c o r r e s p o n d i n g zones i n t h e
second s t r i p . The zones are c u t o u t , e l u t e d w i t h pH 8.0
0.1M phosphate b u f f e r , and a s s a y e d u s i n g t h e CFR microbiolog-
i c a l a g a r d i f f u s i o n assay.
7.4.2 Wagman e t a1.49 r e p o r t e d a d i f f e r e n t i a l chro-

matographic b i o a s s a y f o r t h e g e n t a m i c i n complex. The t h r e e
gentamicins are s e p a r a t e d u s i n g t h e same p a p e r chromatography
318 B E R N A R D E. ROSENKRANTZ er a1
system d e s c r i b e d i n s e c t i o n 7.4.1. A f t e r chromatographic de-

velopment, t h e p a p e r s t r i p s are d r i e d and p l a t e d a g a i n s t
Staphylococcus a u r e u s ATCC 6538P and t h e zones of i n h i b i t i o n
are q u a n t i t a t e d u s i n g s t a n d a r d zone r e s p o n s e c u r v e s .
7.4.3 I n a n o t h e r m o d i f i c a t i o n u s i n g t h e same p a p e r
chromatography s y s t e m d e s c r i b e d i n S e c t i o n 7 . 4 . 1 , t h e d e v e l -
oped p a p e r s are s p r a y e d l i g h t l y w i t h d i l u t e 2 , 4 , 6 - t r i n i t r o -
b e n z e n e s u l f o n i c a c i d (TNBSA) t o d e t e c t t h e g e n t a m i c i n C
components, which a p p e a r as y e l l o w zones. The zones a r e c u t
o u t , a d d i t i o n a l TNBSA i n a pH 9.4 b u f f e g is added and t h e
chromophore i s allowed t o develop a t 30 C f o r one h o u r .
The amount of each g e n t a m i c i n C component is q u a n t i t a t e d by
comparison of t h e a b s o r b a n c e s o b t a i n e d a t 420 nm w i t h t h o s e
o b t a i n e d from a s i m i l a r l y t r e a t e d chromatogram of t h e
r e f e r e n c e s t a n d a r d . 25
7.4.4 Wagman e t a1.50 r e p o r t e d a d i f f e r e n t i a l

n i n h y d r i n p a p e r chromatographic a s s a y f o r t h e g e n t a m i c i n
complex. After development, t h e p a p e r s t r i p s a r e d r i e d and
sprayed w i t h ninhydrin reagent. The c o l o r i s developed w i t h
h e a t and t h e c o l o r i n t e n s i t i e s are r e a d on a n i n t e g r a t i n g
s c a n n e r . The p r o p o r t i o n s of t h e t h r e e g e n t a m i c i n C compo-
n e n t s i n t h e s a m p l e are c a l c u l a t e d by comparison t o s t a n d a r d s
of t h e t h r e e i n d i v i d u a l C components s i m i l a r l y t r e a t e d .
7.4.5 Anhalt e t a1.51 r e p o r t e d a h i g h p r e s s u r e

l i q u i d chromatographic method f o r g e n t a m i c i n C component
determination. I n t h i s method a r e v e r s e d p h a s e LC column
s e p a r a t e s t h e t h r e e g e n t a m i c i n C components by p a i r e d - i o n
chromatography. The s e p a r a t e d components are d e r i v a t i z e d
w i t h o-phthalaldehyde t o g i v e f l u o r e s c e n t p r o d u c t s . Results
from t h i s p r o c e d u r e compare f a v o r a b l y w i t h a s s a y r e s u l t s by
t h e CFR m i c r o b i o l o g i c a l a g a r d i f f u s i o n method. S e e S e c t i o n
8.5 f o r a n expanded d i s c u s s i o n of t h i s HPLC t e c h n i q u e .
7.4.6 K a b a s a k a l i a n e t a1.52 r e p o r t e d a method f o r

t h e d e t e r m i n a t i o n of t h e g e n t a m i c i n components i n f ermenta-
t i o n b r o t h by i n - s i t u f l u o r i m e t r i c measurements of 4-chloro-
7-nitrobenzo-2-oxa-l,3-diazole (NBD) d e r i v a t i v e s . I n t h i s
method, f e r m e n t a t i o n b r o t h samples are a c i d i f i e d , c e n t r i f u g e d ,
a d j u s t e d t o pH 12, s p o t t e d on TLC p l a t e s and developed. The
d r i e d p l a t e s are dipped i n m e t h a n o l i c NBD c h l o r i d e , h e a t e d ,
c o o l e d and rechromatographed i n methanol. The f l u o r e s c e n t
s p o t s are scanned and i n t e g r a t e d u s i n g a d e n s i t o m e t e r . This
method p r o v i d e s a r a p i d means of f o l l o w i n g changes i n
component r a t i o s d u r i n g t h e c o u r s e of t h e f e r m e n t a t i o n .
7 . 4 . 7 Calam e t a1.53 r e p o r t e d a method f o r c o n t r o l

and m o n i t o r i n g t h e p r o p e r t i e s of t h e t h r e e g e n t a m i c i n C
components by lH 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 o s c o p y .
T h i s method i n v o l v e s measurement of t h e peak h e i g h t s of
s i g n a l s f o r N-methyl and C-methyl g r o u p s p r e s e n t i n a l l
t h r e e components and of t h o s e p r e s e n t i n C1 and C2 o n l y .
The peak h e i g h t r a t i o s a r e c a l c u l a t e d . The r e s u l t s a r e used
t o c o n t r o l and m o n i t o r c o m p o s i t i o n w i t h i n c e r t a i n l i m i t s and
n o t t o d e t e r m i n e t h e a c t u a l % c o m p o s i t i o n of each component.
The l i m i t s and t h e method a p p e a r i n t h e g e n t a m i c i n s u l p h a t e
monograph of t h e B r i t i s h Pharmacopoeia 1973, Addendum 1975.
. The g e n t a m i c i n C components have been mo i t o r e d

using l 3 C NMR by 2 4 hour a c c u m u l a t i o n of ~ p e c t r a . 5 ~
7 . 4 . 8 Thomas and Tappin55 r e p o r t e d a n ion-exchange

method w i t h d i r e c t o p t i c a l r o t a t i o n measurement t h a t i s
u s e f u l f o r examining C component d i s t r i b u t i o n i n g e n t a m i c i n
sulfate. I n t h i s method, 80 mg s a m p l e s of g e n t a m i c i n s u l f a t e
are d i s s o l v e d i n 0 . 5 m l 2 M sodium c h l o r i d e and added t o t h e
t o p of a column (0.9 x 15 cm) f i l l e d w i t h c e l l u l o s e p h o s p h a t e
P-11 ion-exchange material. A g r a d i e n t mixer d e l i v e r s sodium
c h l o r i d e s o l u t i o n i n i n c r e a s i n g m o l a r i t y t o t h e column. The
column e l u a t e i s monitored by a p o l a r i m e t e r w i t h a flow-
through microc e l l . The o u t p u t i s p l o t t e d on a p o t e n t i o m e t e r
r e c o r d e r . The peak areas are d e t e r m i n e d w i t h a p l a n i m e t e r
and e x p r e s s e d as p e r c e n t of t h e t o t a l area.
See Thomas56 f o r a comparison of r e s u l t s ob-

t a i n e d by t h e CFR m i c r o b i o l o g i c a l method, t h e lH NMR
method, and t h e above ion-exchange method.
7 . 4 . 9 Wilson e t a1.57 r e p o r t e d a g e n t a m i c i n C
component a s s a y method u s i n g t h i n - l a y e r chromatography
f o l l o w e d by d i r e c t d e n s i t o m e t r y . Gentamicin s u l f a t e is
s p o t t e d on s i l i c a g e l TLC p l a t e s f o l l o w e d by development
i n t h e lower p h a s e of chloroform-methanol-concentrated
ammonium h y d r o x i d e (1:l:l). A f t e r d r y i n g , t h e p l a t e s are
s p r a y e d w i t h n i n h y d r i n r e a g e n t y i e l d i n g magenta s p o t s o n a
w h i t e background. The s p o t s a r e examined by d i r e c t d e n s i t o -
metry and q u a n t i t a t e d w i t h a d i g i t a l i n t e g r a t o r . The a u t h o r s
c l a i m t h a t t h i s method i s f a s t e r and o f f e r s t h e s a m e p r e c i -
s i o n as m i c r o b i o l o g i c a l methods.
320 BERNARD E. ROSENKRANTZ er al.
7.5 M i c r o b i o l o g i c a l Assay
In 1963 Oden e t al.5 8 d e s c r i b e d a s t a n d a r d c u r v e

d i s c - p l a t e a g a r d i f f u s i o n assay using Staphylococcus aureus
ATCC 6538P as t h e t e s t o r g a n i s m f o r t h e a n a l y s i s of gentami-
c i n r a w materials. In t h i s p a p e r a s t a n d a r d c u r v e c y l i n d e r -
p l a t e a s s a y u t i l i z i n g B a c i l l u s s u b t i l i s ATCC 6633 w a s a l s o
r e p o r t e d f o r t h e d e t e r m i n a t i o n of g e n t a m i c i n i n s e r u m
samples. ( r e f e r t o s e c t i o n 10.1) F a c t o r s a f f e c t i n g t h e
a s s a y r e s u l t s u s i n g t h e s e a s s a y p r o c e d u r e s , s u c h as t h e e f -
f e c t of s a l t s in t h e a g a r media, a r e d e s c r i b e d .
The c u r r e n t o f f i c i a l m i c r o b i o l o g i c a l a s s a y proce-
d u r e d e s c r i b e d i n t h e U.S. Code of F e d e r a l R e g u l a t i o n s
(CFR)59 f o r t h e s u b s t a n c e and dosage f o r m s i s a c y l i n d e r
p l a t e a s s a y u s i n g S t a p h y l o c o c c u s e p i d e r m i d i s ATCC 2228
as t h e t e s t organism. The B r i t i s h Pharmacopoeia u t i l i z e s 6 0
a c y l i n d e r p l a t e a s s a y and B a c i l l u s p u m i l u s NCTC 8241 as
t h e t e s t organism. D e t a i l e d p r o c e d u r e s f o r c a r r y i n g o u t
t h e a s s a y s are g i v e n i n t h e compendia.
The minimum p o t e n c y r e q u i r e d by b o t h t h e CFR and

BP f o r a c c e p t a n c e of b u l k commercial g e n t a m i c i n s u l f a t e i s
590 mcg p e r mg o n t h e d r i e d (anhydrous) b a s i s .
8. Chromatographic Analysis
8.1 P a p e r Chromatography
.'
Gentamicin c a n be s e p a r a t e d i n t o i t s t h r e e
components (Cla,C2,C1) by d e s c e n d i n g p a p e r chromatography
u s i n g t h e s o l v e n t s y s t e m c h l o oform:methanol:17% aqueous
ammonia (2: 1: 1, lower p h a s e ) T h i s is a m o d i f i c a t i o n of
t h e s y s t e m g i v e n by Ikekawa e t a1.61 The a p p r o x i m a t e R
v a l u e s r e p o r t e d f o r t h e t h r e e g e n t a m i c i n C components62fare :
Component
Rf
0.21
0.40
c2
0.67
Other paper chromatography systems have been

r e p o r t e d b u t p r o v i d e l i t t l e o r no s e p a r a t i o n of t h e t h r e e
gentamicin c components.62-65
Gentami i n can be d e t e c t e d by s p r a y i n g w i t h
n i n h y d r i n reagent' (0.25% n i n h y d r i n i n p y r i d i n e - a c e t o n e
1 : l ) followed by h e a t i n g a t 105O f o r s e v e r a l minutes.
The s p o t s produced a r e purple-blue i n c o l o r a g a i n s t a w h i t e
background. Ninhydrin r e a g e n t p r e p a r e d by d i s s o l v i n g 1
gram of n i n h y d r i n and 0.1 gram of cadmium a c e t a t e i n a
s o l u t i o n of 3 m l water, 1.5 m l g l a c i a l a c e t i c a c i d and 100
m l of g-propanol has a l s o been used.48,66 A bioautography
method may a l s o be used where t h e paper s t r i p is p l a c e d on
a g a r seeded w i t h Staphylococcus a u r e u s ATCC 6538P. The Rf
v a l u e s of t h e r e s u l t i n g zones of i n h i b i t i o n a r e t h e same a s
t h e s p o t s produced with n i n h y d r i n d e t e c t i o n .
Table 3 i s a summary of paper chromatography

systems f o r gentamicin and c o n t a i n s r e f e r e n c e s t o q u a l i t a t i v e
and q u a n t i t a t i v e methods. An expanded d i s c u s s i o n of q u a n t i -
t a t i v e methods I s g i v e n i n S e c t i o n 7.4.
8.2 Thin-Layer Chromatography
Thin-layer chromatography i s an e f f e c t i v e means of

i d e n t i f y i n g and s e p a r a t i n g t h e components of t h e g e n t a m i c i n
complex. Table 5 g i v e s a l i s t of TLC systems t h a t have been
used f o r gentamicin. R e v i e w s concerning TLC of g e n t a m i c i n
and r e l a t e d a n t i b i o t i c s are availabIe.62,65,67,70
TLC i s v e r y u s e f u l f o r s e p a r a t i n g gentamicin from

o t h e r r e l a t e d aminoglycoside a n t i b i o t i c s . I t o e t a l e 6 8
l i s t s Rf v a l u e s f o r gentamicin and 14 o t h e r b a s i c water
s o l u b l e a n t i b i o t i c s u s i n g s o l v e n t system C i n Table 5.
Pauncz42 s e p a r a t e d gentamicin from s e v e r a l o t h e r deoxy-
s t r e p t a m i n e c o n t a i n i n g a n t i b i o t i c s and t h e i r decomposition
p r o d u c t s u s i n g s o l v e n t system E. This system d i d n o t , how-
e v e r , s e p a r a t e g e n t a m i c i n i n t o i t s t h r e e components.
Kabasakalian e t al.52 r e p o r t e d a q u a n t i t a t i v e TLC

method f o r t h e g e n t a m i c i n complex u s i n g f l u o r i m e t r i c d e t e c -
.
See S e c t i o n 7.4 f o r an expanded d i s c u s s i o n of t h i s
tion.
method
Table 3
Paper Chromatography Systems €or Gentamicin
Method Type Paper Solvent Detection Reference

See Table 4 See Table 4
Qualitative Whatman No. 1 A 1 0.59* 1
Qualitative Whatman No. 1 B 1 0.26* 1
Qualitative Whatman No. 1 C 1 0.10* 1

N
W
N Qualitative Whatman No. 1 D 1 0.30* 1
Qualitative Whatman No. 1 E 1’2 4
Quantitative Whatman No. 1 E 1 49
Quantitative Whatman No. 4 E 3 48
Quantitative S & S NO. 5 8 9 E 2 50
*Gentamicins C C2’ and C 1 are not separated.

la’
Table 4
Paper Chromatography Solvent Systems for Gentamicin
A. Methano1:water (4:l) + 3% sodium chloride vs. paper buffered with 0.95 Iy

sodium sulfate + 0.05 M sodium bisulfate.
B. Propano1:pyridine:acetic acid:water (15:10:3:12).
C. Propano1:water:acetic acid (50:40:5).
D. Aqueous phenol, 80%.

W
w
W E. Lower phase of chloroform:methanol:l7% ammonium hydroxide ( 2 : l : l )
Paper Chromatography Detection Methods for Gentamicin
1. Bioautography vs. Staphylococcus aureus ATCC 6538P.
2. Spray with 0.25% ninhydrin in pyridine:acetone (1:l). Heat at 105OC for

several minutes giving purple to blue spots.
3. Spray with Modified Barrollier reagent. Add 3 ml water and 1.5 ml glacial
acetic acid to 1 g ninhydrin and 0.1 g cadmium acetate and shake. Add to
100 ml n-propanol and shake until solution is complete.
Table 5
Thin-Layer Chromatography Systems for Gentamicin
Bf Values
Plate Medium Solvent Detection Cla, C2, C1 Reference
(see below) (see below) (see below)
A 1 C1a<C2<C 1 41
B 1,2,3 1' a<' 2" 1 4
C 1 0.20, 0.28, 0.35 68
W
b D 132 0.69, 0.76, 0.71 69
P
h)
d E 1 0.05* 42
*Gentamicins Cla, C2, and C1 are not separated.
Plate Medium
a. Silica Gel 60, 0.25 mm thickness, E.M. Laboratories

b. Silica Gel G
c. MN cellulose powder 300, 250 micron thickness
d. Dowex 50 x 8 ion-exchange resin-coated plate
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8.3 Ion-Exchange Chromatography
Ion-exchange chromatography h a s b e e n u s e d e x t e n s i v e -
l y f o r t h e p r e p a r a t i v e s e p a r a t i o n of g e n t a m i c i n components.
Gentamicins may be i s o l a t e d from f e r m e n t a t i o n media by p a s s a g e
o v e r ion-exchange r e s i n s ; 20-50 mesh A m b e r l i t e IRC-50, sodium
form33, A m b e r l i t e IRC-50, ammonium form69, and m - 2 - 7 ~ ~ ~
r e s i n s have been used. The g e n t a m i c i n C components may be
s e p a r a t e d from t h e minor A, B y and X components on a s t r o n g l y
b a s i c r e s i n , Dowex 1x2 w i t h d e i o n i z e d water as e l ~ e n t ~ 9 , 7 ~ .
B a s i c i o n exchange r e s i n s such as A m b e r l i t e IRA-400 (OH-) a r e
used t o c o n v e r t g e n t a m i c i n s u l f a t e s t o t h e c o r r e s p o n d i n g f r e e
b a s e s .33
An ion-exchange s e p a r a t i o n h a s b e e n s u g g e s t e d as
a q u a n t i t a t i v e measure of C component r a t i o s and c o n t e n t
i n g e n t a m i c i n samples.56 See S e c t i o n 7.4 f o r a d d i t i o n a l
d i s c u s s i o n of t h i s paper.
S i n c e g e n t a m i c i n h a s r e l a t i v e l y low v o l a t i l i t y ,
a l l g a s chromatographic a n a l y s e s i n v o l v e d e r i v a t i z a t i o n .
Cunningham and Matsen73 h y d r o l y z e d serum g e n t a m i c i n w i t h
6 HCL and a n a l y z e d t h e r e s u l t i n g 2-deoxystreptamine as i t s
t r i f l u o r o a c e t y l d e r i v a t i v e on a 3% OV-101 column a t 150°C
w i t h f l a m e i o n i z a t i o n d e t e c t i o n . Mayhew and G ~ r b a c h75 ~~,
determined serum g e n t a m i c i n by a two s t e p d e r i v a t i z a t i o n w i t h
N-trimethylsilylimidazole and N-heptafluorobutyrylimidazole;
q u a n t i t a t i v e measurements w e r e made by means of a n e l e c t r o n
c a p t u r e d e t e c t o r f o l l o w i n g chromatography on 3% OV-101 sup-
p o r t e d on Chromosorb W AW DMCS.
8.5 High P r e s s u r e L i q u i d Chromatography
High p r e s s u r e l i q u i d chromatography (HPLC) h a s been

used a s a n a s s a y method b o t h f o r t o t a l g e n t a m i c i n s and f o r
i n d i v i d u a l components. Samples which have been a n a l y z e d in-
c l u d e serum and u r i n e specimens a s w e l l as t h e b u l k d r u g
material.
A major o b s t a c l e t o g e n t a m i c i n HPLC a s s a y s h a s b e e n
t h e problem of d e t e c t i o n . Gentarnicin e x h i b i t s no s i g n i f i c a n t
UV bands above 190 nm and h a s no n a t i v e f l u o r e s c e n c e . In ad-
d i t i o n , a s s a y l e v e l s a r e g e n e r a l l y t o o low f o r t h e u s e of t h e
r e f r a c t i v e i n d e x d e t e c t o r . Hence, t h e several methods which
have been developed i n v o l v e d e r i v a t i z a t i o n of t h e drug.
Peng, e t a d 6 d e t e c t e d g e n t a m i c i n as i t s d a n s y l
d e r i v a t i v e . Following d e p r o t e i n i z a t i o n of serum, g e n t a m i c i n
w a s d e r i v a t i z e d w i t h d a n s y l c h l o r i d e and e x t r a c t e d i n t o e t h y l
a c e t a t e . The sample w a s t h e n chromatographed on a m i c r o p a r -
t i c u l a t e r e v e r s e d phase column by u s i n g a n aqueous a c e t o n i -
t r i l e e l u e n t and f l u o r e s c e n c e d e t e c t i o n .
Maitra e t a1.77,78 s e p a r a t e d g e n t a m i c i n from

serum by means of a s i l i c i c a c i d column. The d r u g w a s t h e n
r e a c t e d w i t h o - p h t h a l a l d e h y d e , chromatographed on a WBondapak
C18 column w i t h a methano1:water (79:21) m o b i l e p h a s e con-
t a i n i n g t r i p o t a s s i u m EDTA and d e t e c t e d by f l u o r e s c e n c e r e a d o u t .
Continuous-flow post-column d e r i v a t i z a t i o n of e n t a -
m i c i n w i t h o - p h t h a l a l d e h y d e w a s performed by Anhalt. 79,88 The
d r u g w a s d e t e r m i n e d by f l u o r e s c e n c e r e a d o u t a f t e r s e p a r a t i o n
on a pBondapak C column w i t h a m o b i l e p h a s e of methanol:
water (3:97) ~ i t k ~ 0 g . 2 N a SO 0.02 g sodium p e n t a n e s u l f o -
2 4’
n a t e and 0.1% ( v / v ) a c e t i c a c i d . A n h a l t , e t al.51 compared
t h i s method t o a normal p h a s e s e p a r a t i o n on a P a r t i s i l (30
cm x 3.9 mm i . d . ) column w i t h a diethy1amine:methanol:water
( 0 . 5 : 4 0 : 6 0 ) m o b i l e p h a s e and r e f r a c t i v e i n d e x d e t e c t i o n .
An e l a b o r a t i o n on t h e method of Anhalt i n c l u d e s t h e
u s e of n e t i l m i c i n as i n t e r n a l s t a n d a r d and r e s o l v e s minor
i m p u r i t i e s i n t h e bulk drug substance.81 S e p a r a t i o n s are ob-
t a i n e d on a n E.M. Merck RP-8 column, mobile p h a s e of 0.2 g
sodium s u l f a t e , 0.02 sodium p e n t a n e s u l f o n a t e , 0.1% ( v / v )
a c e t i c a c i d , and p o s t column d e r i v a t i z a t i o n w i t h o - p h t h a l a l d e -
hyde. T h i s method g i v e s a l i n e a r r e s p o n s e t o g e n t a m i c i n o v e r
a wide c o n c e n t r a t i o n r a n g e , up t o 0.15 mg/ml f o r e a c h compo-
nent. I t is s p e c i f i c , a c c u r a t e and p r e c i s e , and h a s b e e n u s e d
e f f e c t i v e l y t o d e t e r m i n e t h e g e n t a m i c i n c o n t e n t of b u l k s a m -
p l e s as w e l l as f i n i s h e d p r o d u c t s .
9. Electrophoresis
E l e c t r o p h o r e s i s h a s been u s e d t o s e p a r a t e g e n t a m i c i n from
o t h e r a n t i b i o t i c d r u g s i n serum and i n u r i n e . V a r i o u s s y s -
t e m s which have b e e n u s e d f o r t h e s e s e p a r a t i o n s are g i v e n i n
T a b l e 6. W h i t e l e y and co-workersg2 have u s e d a n electro-
p h o r e t i c s e p a r a t i o n t o demonstrate an in-vitro i n t e r a c t i o n
between g e n t a m i c i n and c e p h a l e x i n .
Table 6
Conditions for Electrophoresis of Gentamicin
Gentamicin
Detection Mobility Relative
Medium Buffer Method to Neomycin Reference
Filter Paper 1 g formic acid

(Whatman No. 4 ) 0.005 g p-toluenesulfonic acid
in n-PrOH:H20 (5:95) pH 1.8 Microbiological 0.95 83
Filter Paper 0.8 g formic acid

in n-PrOH:H20 (5:95) pH 1.9 Microbiologica1 83
W
N
W
Filter Paper 0.8 formic acid

in H20 pH 1.9 Microbiological 83
Filter Paper 0.2 NH OH

(Whatman No. 4 ) 0.005 3 AaOH
0.01 g p-toluenesulfonic acid
in n-PrOH:H20 (1:9) pH 10.8 Microbiological 1.74 83
Filter Paper 0.2 NH OH, 0.0025 g NaOH,

(Whatman No. 4 ) in H20 pA 11.5 Microbiological 1.43 83
Filter Paper 0.05 g NaOH in H20

(Whatman No. 4 ) pH 12.2 Microbiological 1.02 83
Table 6 (Continued)
C o n d i t i o n s f o r E l e c t r o p h o r e s i s of Gentamicin
Gentamicin
Detection Mobility Relative
Medium Buffer Method t o Neomycin Reference
F i l t e r Paper formic a c i d : a c e t i c a c i d : 0.25% n i n h y d r i n ,

(Whatman 3 b@f) water ( 6 : 2 4 : 170) 0.01% i s a t i n ,
1%l u t i d i n e i n
acetone 1 84
0.9% a g a r o s e T r i s - (hydr oxyme t h y 1)-

i n pH 5.6 aminomethane, m a l e i c a c i d i n Bio-autographical 0.85 85
buffer H20 pH 5.6
F i l t e r Paper 0.05 NH40Ac,

(Whatman No. 1) 0.05 NH40H i n H20
pH 9.4 Ninhy d r in n.a. 82
10. D e t e r m i n a t i o n i n Body F l u i d
Assays f o r g e n t a m i c i n i n body f l u i d s have been perform-

ed u s i n g a number of methods; t h e s e i n c l u d e m i c r o b i o l o g i c a l
a s s a y s , immunoassays, radioenzyme a s s a y s and h i g h p r e s s u r e
l i q u i d chromatographic a s s a y s .
10.1 M i c r o b i o l o g i c a l Assay
The m i c r o b i o l o g i c a l a s s a y of g e n t a m i c i n is an
a g a r d i f f u s i o n a s s a y b a s e d upon a comparison between t h e
growth i n h i b i t i o n zones produced by t h e t e s t sample and t h o s e
produced by g e n t a m i c i n s t a n d a r d of known p o t e n c i e s d i l u t e d i n
a p p r o p r i a t e body f l u i d . Oden e t al.58 d e s c r i b e d a s t a n d a r d
c u r v e c y l i n d e r - p l a t e a s s a y u t i l i z i n g B a c i l l u s s u b t i l i s ATCC
6633 as t h e t e s t o r anism w i t h a s e n s i t i v i t y of 0.05 mcg/ml.
A l c i d and Seligmangg r e p o r t e d t h e u s e of a m u l t i p l e a n t i b i o t i c -
r e s i s t a n t s t r a i n of S t a p h y l o c o c c u s e p i d e r m i d i s ATCC 27626 as
t h e t e s t organism. Gentamicin c o n c e n t r a t i o n s are e s t i m a t e d
u s i n g t h i s t e s t organism i n t h e p r e s e n c e of o t h e r a n t i b i o t i c s
w i t h o u t t h e u s e of enzymes, r a d i o a c t i v e material, o r e l a b -
o r a t e equipment and t e c h n i q u e s .
10.2 Fluoroimmunoassay
A f l u o r o m e t r i c irnmunoassay f o r g e n t a m i c i n i n
serum w a s developed by Watson and co-workers87, who h a v e
p r e p a r e d a f l u o r e s c e i n i s o t h i o c y a n a t e d e r i v a t i v e of t h e d r u g .
The p r i n c i p l e upon which t h e a s s a y i s b a s e d i s t h a t a complex
of t h e d e r i v a t i z e d g e n t a m i c i n w i t h a n t i b o d y would s c a t t e r i n -
c i d e n t p o l a r i z e d l i g h t more t h a n t h e smaller uncomplexed
d e r i v a t i v e molecule. The d e g r e e of a n t i b o d y b i n d i n g t h u s c a n
b e d e t e r m i n e d from changes i n t h e f l u o r e s c e n c e i n t e n s i t y .
10.3 Radioimmunoassay
Radioimmunoassay f o r g e n t a m i c i n i s b a s e d on t h e
b i n d i n g of t h e a n t i b i o t i c by a n a p p r o p r i a t e a n t i b o d y . By
adding b o t h a n t i b o d y and r a d i o - l a b e l l e d d r u g t o a sample con-
t a i n i n g g e n t a m i c i n , one e s t a b l i s h e s a n e q u i l i b r i u m between
t h e bound and f r e e l a b e l l e d and u n l a b e l l e d g e n t a m i c i n mole-
c u l e s . Following s e p a r a t i o n of t h e bound and f r e e f r a c t i o n s ,
c o u n t i n g of t h e bound g e n t a m i c i n p r o v i d e s a measure of t h e
p r o p o r t i o n of bound d r u g which i s r a d i o a c t i v e l y l a b e l l e d .
T h i s measurement l e a d s t o t h e c a l c u l a t i o n of t h e amount of
g e n t a m i c i n i n t h e o r i g i n a l sample.
S e v e r a l of t h e methods which have been developed

f o r g e n t a m i c i n radioimmunoassay a r e o u t l i n e d i n T a b l e 7.
G r i f f i t h s and co-workers88 have compared t h e 3H-

g e n t a m i c i n , 1251-gentamiciny radioenzyme, and m i c r o b i o l o g i c a l
a s s a y methods. Jonsson89 s t u d i e d t h e s p e c i f i c i t y of t h e
1251-gentamicin radioimmunoassay and compared t h e r e l a t i v e
r e s p o n s e s of t h e i n d i v i d u a l g e n t a m i c i n components t o t h e
r e s p o n s e s of several a m i n o g l y c o s i d e a n t i b i o t i c s . Similar
comparisons have b e e n made f o r t h e 3H-gentamicin r a d i o -
i m u n o a s s a y .go, 91992
10.4 Radioenzyme Assay
D e t e r m i n a t i o n of g e n t a m i c i n i n serum by r a d i o e n -
zyme a s s a y i s b a s e d on t h e enzyme-catalyzed d e r i v a t i z a t i o n of
t h e d r u g w i t h a l a b e l l e d s u b s t i t u e n t group. D e r i v a t i z e d
g e n t a m i c i n is a d s o r b e d o n t o some s t a t i o n a r y medium t o s e p a r -
a t e i t from u n r e a c t e d components. The r a d i o a c t i v i t y of t h e
a d s o r b e n t p l u s g e n t a m i c i n t h u s p r o v i d e s a measure of t h e
amount of d r u g p r e s e n t i n t h e sample.
Smith e t a1.97yg8 employed a n R-factor-mediated

enzyme t o a d e n y l y l a t e g e n t a m i c i n w i t h I 4 C - l a b e l l e d ATP
s e r v i n g as t h e s o u r c e of t h e a d e n y l group. A d e n y l y l a t e d
d r u g , b u t n o t ATP, w a s adsorbed o n t o Whatman P-81 phospho-
c e l l u l o s e paper. F u r g e r e t a1.99 used a s i m i l a r t e c h n i q u e
where t h e l a b e l l e d material w a s 3H-ATP. Smith and co-
workers100 a l s o r e p o r t e d t h e i s o l a t i o n , p a r t i a l p u r i f ica-
t i o n , and c h a r a c t e r i z a t i o n of t h e g e n t a m i c i n a d e n i n e mono-
n u c l e o t i d e t r a n s f e r a s e , a s w e l l as com a r i n g methods i n which
t h e s o u r c e s of t h e a d e n y l group were IEC-ATP and c~J~P-ATP.
The 1 4 C - l a b e l l e d enzyme a s s a y h a s a l s o been compared t o t h e
m i c r o b i o l o g i c a l assay.100,101,102
O ' N e i l l e t a l e 1 0 3 as w e l l a s B r o u g h a l l and Reeves104

have compared enzyme a s s a y s i n v o l v i n g a d e n y l y l a t i o n t o t h o s e
employing a c e t y l a t i o n . Two a d d i t i o n a l p a p e r s l o 5 , l o 6 have
d e s c r i b e d radioenzyme a s s a y s i n which t h e s u b s t i t u e n t g r o u p
was a c e t y l d e r i v e d from 1 4 C - a c e t y l coenzyme A.
10.5 High P r e s s u r e L i q u i d Chromatography.
Anhalt e t a 1 . 7 9 ~ 8 0 d e s c r i b e d a h i g h p r e s s u r e
l i q u i d chromatography (HPLC) p r o c e d u r e f o r t h e a s s a y of
g e n t a m i c i n i n serum. The t e c h n i q u e i n v o l v e s t h e s e p a r a t i o n
of g e n t a m i c i n from i n t e r f e r i n g compounds i n serum o n a CM-
Table 7
Radioimmunoassay Methods f o r Gentamicin
Source of Separation
Carrier Protein Antibody Method Isotope Sensitivity Reference
Human serum Rabbit dextran:charcoal 3H, 1251 --- 88

albumin adsorption
Human serum albumin Rabbit second antibody 3H 2 ng 93,94,95

bovine serum albumin precipitation
porcine thyroglobulin
keyhole limpet hemo-
cyanin
Human serum albumin killed protein-A con- 1251 89

Rabbit
taining Staphylococcus
aureus
Bovine serum albumin Rabbit dextran:charcoal 3H 80 Pg 90,96
Bovine thyroglobulin Rabbit dextran:charcoal 3H 10 ng 91

adsorption
Human serum albumin Rabbit dextran:charcoal 3H 0.01 ug/ml 92

adsorption
Sephadex column followed by a n a l y s i s u s i n g reverse p h a s e

i o n - p a i r chromatography. 1-N-acetylgentamicin C may be
used a s i n t e r n a l s t a n d a r d f o r t h e serum a s s a y . bontinuous-
flow, post-column d e r i v a t i z a t i o n w i t h 2-phthalaldehyde i s
used t o form f l u o r e s c e n t p r o d u c t s f o r d e t e c t i o n . The HPLC
t e c h n i q u e , when compared t o m i c r o b i o l o g i c a l a s s a y s , o f f e r s
advantages w i t h r e g a r d t o r a p i d i t y , s p e c i f i c i t y and p r e c i s i o n .
11. Acknowledgements
The a u t h o r s wish t o thank t h e Schering C o r p o r a t i o n

Research L i b r a r y , Microbiology and P h y s i c a l and A n a l y t i c a l
Chemistry s t a f f s , i n p a r t i c u l a r M r . Stephen Gruber,
D r . Mohindar S. Puar, M r s . J e a n e t t e S a b a t i n o , and
D r . M.D. Yudis f o r t h e i r a s s i s t a n c e i n t h e p r e p a r a t i o n of
this analytical profile.
334 BERNARD E. ROSENKRANTZ ef af.
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Corporation, unpublished data.
82. J.E. W h i t e l e y , J.R. Brown and D.N. E l l i o t , J. Pharm.

Pharmacol., 3 , 2 0 1 ( 1 9 7 8 ) .
83 S. Ochab, P o l . J. Pharmacol. P h a r . , 25, 1 0 5 ( 1 9 7 3 ) .
84 J.L. P o t t e r , J. P e d i a t r . , 84, 2 5 0 ( 1 9 7 4 ) .
85. D.S. Reeves and H.A. H o l t , J. C l i n . P a t h o l . , 28, 435

(1975)
86. D.V. A l c i d and S.J. Seligman Antimicrob. Agents and

Chemother. , 2, 559 ( 1 9 7 3 ) .
87. R.A.A. Watson, J. Landon, E . J . Shaw and D.S. Smith,

C l i n . Chim. Acta., 73, 5 1 ( 1 9 7 6 ) .
88. W.C. G r i f f i t h s , P. D e x t r a z e and I. Diamond, Ann. C l i n .

Lab. S c i . , 2, 1 4 1 ( 1 9 7 7 ) .
89. S. J o n s s o n , Proc. 9 t h I n t e r n a t . Cong. Chemother.,

London, J u l y 1 9 7 5 , i n Chemotherapy 2, 165 ( 1 9 7 5 ) .
90. A. Broughton and J . E . S t r o n g , C l i n . Chim. Acta., 66,

125 ( 1 9 7 6 ) .
91. B.H. Minshew, R.K. H o l m e s and C.R. B a x t e r , Antimicrob.

Agents Chemother., 2, 107 ( 1 9 7 5 ) .
92. W.A. Mahon, J . Ezer and T.W. Wilson, Antimicrob. Agents

Chemother., 2, 585 ( 1 9 7 3 ) .
93. J.E. L e w i s , J.C. Nelson and H.A. Elder, Nature, 239,

214 ( 1 9 7 2 ) .
94. L.S. Berk, J . L . L e w i s and J . E . Nelson, C l i n . Chem., 3,

1159 ( 1 9 7 4 ) .
95. J.E. L e w i s , J . C . Nelson and H.A. E l d e r , "The A p p l i c a t i o n

of Radioimmunoassay Techniques t o t h e Measurement of
A n t i b i o t i c s , " i n Automation i n M i c r o b i o l o p y and Immuno-
loKy, pp. 335-352 ( 1 9 7 5 ) .
340 BERNARD E. ROSENKRANTZ ef af
96. A. Broughton and J.E. S t r o n g , C l i n . Chem., 22, 726

(1976)
97. D.H. Smith, B. Van O t t o and A.L. Smith, N. Engl. J.

, .Med -
286, 583 (1972)
98. I. P h i l l i p s , C. Warren and S.E. Smith, J. C l i n . P a t h . ,

-
27, 447 (1974).
99. F. F u r g e r , A. R u s s i and F.H. Kayser, Schweiz. Med.

Wschr., 103, 779 (1973).
100. A.L. Smith and D.H. Smith, J. I n f e c t . D i s . , 3,391

(1974)
101. A.L. Smith, J.A. Waitz, D.H. Smith, E.M. Oden and
B.B. Emerson, Antimicrob. Agents Chemother. , 5, 316
(1974).
102. E. Ten Krooden and J.H. Darrell, J. C l i n . Path., 27,

452 (1974).
103. M.A. O ' N e i l l , B.M. Maxwell, A.D. B l a i r , A.W. F o r r e y ,

and R.E. C u t l e r , C l i n . Chem., 20, 897 (1974).
104. J.M. B r o u g h a l l and D.S. Reeves, J.Clin. P a t h . , 28,

140 (1975).
105. J . M . B r o u g h a l l and D.S. Reeves, Proc. 9 t h I n t e r n a t .

Cong. Chemother., London, J u l y 1975, i n Chemotherapy 2,
159 (1975).
106. P. Berman and P. Botha, S . Afr. Med. J., 51, 930

(1977) , A b s t r a c t .
HALOPERIDOL
Casirnir A . Janicki and Chan Yan KO
1. Description 342
1 . 1 Name, Formula, Molecular Weight 342
I .2 Appearance, Color, Odor 342
'2.4 Mass Spectrum 346
2.5 Melting Range 35 1
2.6 Differential Scanning Calorimetry 35 1
2.7 Solubility 35 1
2.8 pKa 353
2.9 X-ray Diffraction 353
3. Synthesis 353
5. Drug Metabolic Products 356
6.2 Non-aqueous Titrimetric Analysis 357
6.5 Thin- Layer Chromatographic Analysis 359
6.6 Gas Chromatographic Analysis 36 1
6.7 High- Performance Liquid Chromatographic Analysis 361
6.8 Differential Scanning Calorimetric Analysis 363
6.9 Polarographic Analysis 363
6.10 Fluorescence Analysis 364
7. Determination in Biological Fluids 364
8. Determination in Pharmaceuticals 365
Copyright0 1980 by Academic Ress. Inc.

Analytical F'rofiles of Drug Substances, 9 34 1 All rights of reproduclionin any form reserved.
ISBN: 012-260809-7
342 CASlMlR A . JANICKI AND CHAN YAN KO
1. Description
1.1 Name, Formula, Molecular Weight
Haloperidol is 4-[4-(4-chlorophenyl)-4-
hydroxy-l-piperidinyl]-l'-(4-fluorophenyl)-
1-butanone. The trademark of the manu-
factured dosage form is HALDOLa.
OH
The molecular weight is 3 7 5 . 8 7

C21H23C1FN02
White to faintly yellowish amorphous or
microcrystalline odorless powder.
The infrared spectrum is presented in
Figure 1. The spectrum was obtained from a
potassium bromide dispersion using a Perkin-
Elmer Model 283 grating infrared spectro-
photometer. A list of the assignments made
for some of the characteristic bands is given
in Table I (1,2).
Table I
IR Spectral Assignment for Haloperidol
3125 -OH
2953 -CH2
2918
2839
2822
0
- 0
*
-
0
- 0
m
-
0
- 0
(Y
r
--
- I
0
I
-8 ;
K
$ I
3
-
z
w
>
a
0 3
0
- 0
N
-
0
0
-0
..
P)
d
-
E
[%I 33NVlllWSNVHl
344 CASIMIR A. JANICKI A N D CHAN YAN KO
Table I (cont'd)
1681 -c=o
1597 Substituted
Aromatic Ring
1500
1454
1482 -CH2
1410
1221 -OH
1156 -CH deformation of
F substituted
aromatic ring
995 -C1 substltuted
aromatic ,ring
827 -CH deformation of
p-substituted
aromatic ring
541 -c=o
2.2 Nuclear
-____ Magnetic Resonance
-- ___
Spectrum
The 9 0 MHz spectrum of haloperidol pre-
sented in Figure 2 was obtained in de-
uterated dimethylsulfoxide (d6) at a con-
centration of 8 2 mg/ml with tetranethyl-
silane as the internal standard. Spectral
assignments are listed in Table I1 ( 2 ) .
c
10 9 8 7 6 5
PPM 161
4 3 2 1 0
F I G U R E 2: The NMR of Haloperidol in Deutcrated

Dimethylsulfoxide (d ) with TMS as the
Internal Standard. Pnstrument : Perkin-
Elmer R-32.
346 CASIMIR A. JANICKI A N D CHAN Y A N KO
Table I1
NMR Spectral Assignments for Haloperidol
Chemical Shift Multiplicity Characteristic of
( ppm 1 proton
8.05 mu 1tip1et a,b
7.32 singlet 1 ,m,n,p
7.31 mu 1tip1et c,d
4.77 singlet
2.98 triplet g
2.20-2.70 mu 1 tip1et e , h,i
1.38-1.97 mu 1tip 1et f,j,k
2.3 Ultraviolet Spectrum
The ultraviolet absorption spectrum of
haloperidol obtained from a 9:l 0.1 M hydro-
chloric acid: methanol solution is &own in
Figure 3. Two absorption maxima, were ob-
served at 245 nm and 221 nm, with molar ab-
sorptivities of about 13,300 and 15,000
respectively.
2.4 Mass Spectrum
The EI mass spectrum, is given in
Figure 4, and the fragmentation pattern is
presented in Table 111. The fragmentation
patterns were discussed in further detail in
papers by Blessington ( 3 ) , Leferink and Moes
(4), and Diding and Co-workers ( 5 ) .
The chemical ionization mass spectrum

obtained on a Finnigan model 3300 CI mass
spectrometer with a model 6100 data system
using methane as reagent gas is presented in
Figure 5 ( 6 ) . A strong hydrogenated
molecular ion was observed. The frag-
mentation pattern is presented in Table IV.
H A LOPE R1 DO L 347
OX
w 0.6
u
z
a
m
a
0
v)
m
a
0.4
0.2
0
210 260 310 360
WAVELENGTH I n m I
FIGURE 3: The U V Absorption Spectrum of Halo-

p e r i d o l in 3:l 0 . 1 M Hydrochloric a c i d :
Methanol.
..
U
j4 rLJ
k
..
=3
w
fx
3
Erc
348
HALOPERIDOL Direct I n l e t 1 CI methane 1
60 100 150 200 250 300 350 400 450 500
ml e
F I G U R E 5: The Mass Spectrum of Haloperidol,

Chemical Ionization with Methane
Instrument: Finnigan Model 3300 D.
350 CASIMIR A. JANICKI A N D CHAN YAN KO
T a b l e I11
Mass F r _ a q m e n t a t i o n P a t t e r n of H a l o p e r i d o l o n EI-MS
b e
I
1 I I
I I
4 4 4 1
a c d
375 M+
237 C-H
224 d
206 d-H2 0
165 e
123 b
95 a
Table I V
Major I o n s and F r a g m e n t a t i o n of t h e CI-MS of

H a l o p e r i d o l Methane as t h e R e a c t a n t G a s .
AC
/
I
F ~ ~ - C H ~ TII C HII ~ T C H ~ - N
lo I I /
HALOPERIDOL 35 1
m/e -
Ion
418 M*C H
+
7+
404
376
z5
M*C H
M a H
358 M. H + - Ho~
280 f
264 9
237 C - H
224 d
The isotopic ratio of ~ 1 ~ ~ : ~ 1 ~ ~was

, 3observed.
: 1 ,
2.5 Melting Range
The melting range of a haloperidol
sample, determined after drying in vacuum at
60'C for 3 hours, is between 147'-152OC
according to the USP X I X class I procedure.
No polymorphs of haloperidol have been re-
ported to date.
2.6 Differential Scanning Calorimetry (DSC)
The DSC of haloperidol is shown in
Figure 6. A melting endotherm is observed
around 423OK using a temperature program of
5' /minute ( 7 1 .
2.7 Solubility
The approximate solubilities obtained at
room temperature are listed in Table V (2, 7,
11).
426' 423" 420' 417' 414" 411"
QUALITATIVE DSC THERMOGRAMS OF MIXTURES

OF HALOPERIDOL WITH AN IMPURITY, 5"PER
MINUTE
0
z
0
2
v)
a Reference standard
b Mixture, purity = 98.75 Mole %
\ c I# ,, = 97.33 Mole %
d = 94.84 Mole %
wK
v) II
e ,, = 92.73 Mole %
sa f I, ,, = 89.93 Mole %
2
A
rl
f
1 TEMPERATURE, O K
FIGURE 6: DSC Thermogram of Haloperidol. Instrument: Perkin-Elmer DSC-2.

HALOPERIDOL 353
Table V
Solubility Data of Haloperidol at Room Temperature
Approximate
S o Ivent Solubility (g/lOO ml)
Acetone 2.0
Benzene 1.1
Chloroform 6.6
Citric Acid (0.1 M ) 1.3
Ethanol 1.7
Ether 0.5
Ethyl Acetate 1.8
n-Hexane 0.5
Lactic Acid 100
Methanol 1.8
n-Octano 1 <0.1
2-Propanol 0.5
Tartaric Acid (0.1 M ) 1.2
Water (pH 5.9) <0.01
2.8 pKa
The pKa of haloperidol is 8.3 calculated
by linear extrapolation using potentiometric
titration in 158, 258, 358, 45% methanol-
water (v/v) with 0.005 NaOH as titrant (1).
2.9 X-ray Diffraction
Reed and Schaefer (8) determined the
crystal and molecular structure of halo-
peridol by single crystal X-ray diffraction
techniques.
3. Synthesis
Haloperidol is synthesized by heating a
mixture of 4-chloro-l-(4-fluorophenyl)-l-butanone,
potassium iodide, and 4-(4-chlorophenyl)-4-
piperidinol in a toluene solvent to about 1 0 0 ° C ,
in a closed vessel, (9,lO).
OH
Foi-CH2CH2CH2-CI +
0 I
The 4 - c h l o r o - l - ( 4 - f l u o r o p h c n y l ) - l - b u t a n o n e was
obtained by a Friedel Crafts reaction using
fluorobenzene whereas the 4-(4-chlorophenyl)-4-
piperidinol was obtained in three steps from ct-
methylstyrene.
A major impurity that has been isolated and
identified is 4-(4-(4-chlorophenyl)-4-hydroxy-l-
piperidinyl)-l-(4-(4-(4-chlorophenyl)-4-hydroxy-
4-piperidinyl)phenyl)-l-butanone The structure is
given as:
OH OH
/
HALO PERIDO L 355
Diding and co-workers identified deschlorohalo-

peridol as an impurity in a sample of haloperidol
hydrochloride by GC-MS ( 5 ) . The structure is
given as:
OH
4. Stability Degradation
Haloperidol is a relatively stable compound.
Samples have been found to be stable for up to
five years stored at room temperature in amber
glass containers. Storage for up to one year at
4 5 ° C in amber glass containers did not adversely
affect the drug substance. Haloperidol discolors
and de-grades when exposed to natural sunlight for
long periods. However, no degradation was noted
when the drug substance was exposed to 2 0 0 0 foot
candles for two weeks. The drug is only very
slightly hygroscopic ( 2 , 7 ) .
Haloperidol suspensions (1 mg/ml) were re-
fluxed for 2 4 hours in water, 0.1 N sodium
hydroxide, and 1 sodium hydroxide. No de-
gradation was observed. Under the same reflux
condition but in 0.1 N hydrochloric acid solution
and 1 N hydrochloric acid solution approximately
108 and 5 0 % degradation was ob-served respectively
(7). The hydrolysis products have not been
identified.
A pH stability profile has been obtained from
pH 2 to pH 8 using citrate-phosphate buffers.
Good stability was observed at room temperature,
4 0 ° C and 6OoC for up to 2 weeks (11).
Pharmaceutical solutions in lactic acid with

a pH of about 3 have been found to be stable for
up to 5 years at room temperature, 2 years at 4 0 ° C
356 CASIMIR A . JANICKI A N D CHAN YAN KO
and 6 months at 6OOC. However, when the

solutions were exposed to natural sunlight they
became cloudy and discolored. A drop in halo-
peridol content was observed by assay and TLC.
Pharmaceutical tablets have been found to be
stable for up to 5 years at room temperature, 2
years at 4OoC and even 6 months at 6 O o C (7). The
stability depends on the particular tablet for-
mulation. An example was reported where halo-
peridol was found to be incompatible with 5-
(hydroxynethyl)-2-furfuraldehyde, an impurity in
anhydrous lactose (12). The adduct of haloperidol
with the furfural is shown below:
OH
5. Drug Metabolic Products

The excretion and metabolism of haloperidol
have been studied in rats after the administration
of tritium labeled haloperidol (13,14). Oxidative
N-dealkylation represents the major metabolic
pathway (14). The major urinary metabolite of
haloperidol is N-(2-(4-fluorophenyl)acetyl)glycine
(d) (13,141. Other metabolites found were 4-
fluoro-v-oxobenzenebutanoic acid (a) (13,14) and
4-fluorobenzeneacetic acid ( c ) (14). Only traces
of unmetabolized haloperidol were found in the
urine. Since the tritium label was located on the
fluorophenyl ring, the metabolism of the
piperidine part of the molecule was not followed.
HALOPERIDOL 357
In man, haloperidol is metabolized to 4-

fluoro-y-oxobenzenebutanoic acid and N-(2-(4-
fluoropheny1)acetyl)glycine ( 1 5 ) . This demon-
strates that the metabolism in man follows the
same general pattern as that in the rat. No
measurable amounts of any glutamine or glucuronic
acid derivatives could be demonstrated. A trace
of unmetabolized haloperidol was found in the
urine.
Recently a "reduced" form of haloperidol ( e )
has been detected and identified in serum and
urine of patients on high doses of haloperidol
(16). Hydroxylation was also proposed as another
pathway ( 1 7 ) . The proposed metabolic pathway in
man for haloperidol is summarized in Figure 7,
with oxidative N-dealkylation representing the
major metabolic path.
Element 8 Theory
Carbon 67.10
Hydrogen 6.17
Chlorine 9.43
Fluorine 5.05
Nitrogen 3.73
Oxygen 8.51
6.2 Non-aqueous Titrimetric Analysis

The non-aqueous titration procedure is
the official method listed in the United
States Pharmacopeia XIX ( 1 8 ) for the drug
substance. An accurately weighed sample of
haloperidol is dissolved in glacial acetic
acid. After the addition of 3 drops of
p-naphtholhenzcin TS, the solution is
titrated with standardized 0.05 perchloric
acid to the end point.
I
0
"\
n
I E
0, 0
0
0=0
Of+
Y U
H
4
0
m
0
0
I
0=0
U
!i i.
..
P
HA LOPERl DOL 359
6.3 Colorimetric Analysis

A general procedure for the quantitative
determination of butyrophenones was described
by Haemers and Van Den Bossche (19). Their
procedure involves the reaction of butyro-
phenones with 3,5-dinitrobenzoic acid in an
alkaline medium resulting in the formation of
a red colored complex. Using this procedure,
haloperidol was determined in pharmaceutical
solutions and tablets. Haloperidol also
forms a chloroform soluble methyl orange
complex at a pH of 5, which is suitable for
quantitative work (1,12). Another colori-
metric procedure involves the use of po-
tassium iodoplatinate (20). None of the
colorimetric procedures have been found to be
suitable for stability work since they lack
the necessary specificity.
6.4 __-
Spectrophotometric Analysis
The official USP XIX analysis of halo-
peridol tablets is a spectrophotometric
analysis. A portion of powdered tablets is
weighed and extracted with chloroform and 0.1
-
N sodium hydroxide. A portion of the
chloroform layer is extracted with 0.1 N
sulfuric acid and the U V spectrum recorzed.
The sample is compared against a reference
standard diluted to the same final concen-
tration in 0.1 N sulfuric acid, at the
maximum about 2 7 5 nm.
For solutions containing parabens, the
same assay described above can be used,
except that the final acid layer is extracted
twice with diethyl ether. This removes any
UV interference in the sulfuric a c i d solution
due to the parabens ( 7 ) .
6.5 Thin-Layer Chromatoqraphic Analysis
Some TLC methods for haloperidol are
given in Table VI, along with the various
detection methods. Braun and co-workers
published a method to follow the metabolites
of haloperidol in urine (13).
Table VI
TLC Methods
___-____ for Haloperidol
Solvent
Ad sor ben t System Rf Ref.
-
-
1; Silica GF Ethyl Acetate: 0.64 (2)
Ch1oroform:Methanol:
Sodium Acetate Buffer
( p H 4.7) (54:23:18:5)
2. Silica GF Ch1oroform:Methanol 0.40 (2)

(92:8)
3. Alumina GF Ch1oroform:Ethanol 0.50 (2)

(99:l)
4. Silica GF Ch1oroform:Methanol 0.61 (7)

Ammonium Hydroxide
(91:8:l)
5. Silica G Ethano1:lN hydro- 0.57 (7)

chloric acid (95:5)
6. Silica G Acetone 0.60 (21)

7. Silica G Benzene:Acetone: 0.90 (21)
Petroleum ether:
Ammonium hydroxide
(10:10:10:2)
8. Silica G Acet0ne:Petroleum 0.32 (21)

ether (7:3)
9. Silica G n-butano1:isopropanol: 0.86 (21)

Acetic Acid:Water
(3:3:2:4)
10. Silica GF
with Na2C03 Methano1:Acetone 0.50 (22)
(12:88)
HALOPERIDOL
11. Silica GF
with Na2C03 Ethano1:Chloro- 0.74
form (16:84)
12. Silica GF Ch1oroform:Methanol:
Formic Acid (85:10:5) 0.51
13. Silica GF Ch1oroform:Methanol:
25% ammonium hydro-
xide (85:14:1) 0.87
14. Silica GF Ethy1acetate:isopro-
pano1:Ammonium hydro-
xide (70:25:4) 0.81
15. Silica GF Cyc1ohexane:diethyl-
amine:benzene (80:15:5) 0.18
6.6 Gas Chromatographic Analysis
Haloperidol has been assayed using a
glass column packed with the following: 2%
OV-1 on Chromosorb W HP with a column tem-
perature of Z l O O C (25); a mixture of 0.3%
Versamid and 0.6% OV-17 on Gas Chrom Q with a
column temperature of 23OOC (26); 3% OV-17 on
Gas Chrom Q with a column temperature of
28OOC (27); and 3% OV-1 on Gas Chrom Q with a
column temperature of 24OOC (28). All of
these reported methods u s e an electron
capture detector to have the necessary sen-
sitivity for determining haloperidol in body
fluids. Bianchetti and Morselli used a 3 %
OV-17 on Chromosorb W at a column temperature
of 285OC along with a Nitrogen-Phosphorous
detector (29).
6.7 High-Performance Liquid Chromatographic
Analysis
A HPLC method was described in the
literature (30). A reverse phase column was
used with a solvent mixture of 44% tetra-
hydrofuran and 0.75% phosphoric acid in
362 CASIMIR A. JANICKI AND CHAN YAN KO
water. An UV detector at 254 nm was used.

A retention time of 5.5 minutes was re-
ported, but no specificity data were given.
A HPLC method has been developed, which
gives the necessary specificity, that can be
used to follow the stability of haloperidol
in tablets and oral and injectable solutions
(31). A 10 mg equivalent of haloperidol is
transferred to a 120 ml bottle. A 50 m l
aliquot of chloroform and 25 m l of 0.1 N
sodium hydroxide are added. The bottle-is
shaken for 30 minutes, centrifuged, and the
aqueous layer discarded. A 15 ml aliquot of
the chloroform layer is evaporated to dryness
in a stream of nitrogen. The residue is
dissolved in 4 ml of the intefnal standard
solution (0.6 mg/ml oxatomide in methanol).
A 5 1.11 sample is injected into the coluru?.
The chromatographic conditions a c e as
follows:
Column: LiChrosorb RP-18; 25 cm x 2.1 mm id
stainless steel column
Mobile Phase: 30% 0.1 -
M ammonium carbonate: 70%
methanol
Flow Rate: 1 ml per min
Wavelength: 270 nm
Retention times: Haloperidol - 3 . 3 min
Oxatomide - 10.0 min
The specificity of the method is demon-
strated by the data given in Table V I I .
-
~ ~~
1- 3- [ 4 - (diphenylmethyll-l-piperazinyllpropyl
1,3-dihydro-2H-bcnzinidazol-2-one.
HALOPERIDOL 363
Table VII
Separation of impurities, degradation products
and preservatives from haloperidol by HPLC.
Compound Retention Time
(minutes)
4-Fluorobenzoic acid 1.0
4-fluoro-y-oxobenzenebutanoic
acid 1.1
Methylparaben 1.3
Propylparaben 1.9
4-(4-Chlorophenyl)-4-piperidinol 2.0
1-(4-Fluorophenyl)-4-(4-hydroxy-
4-phenyl-l-piperidinyl)-l-butanone 2.5
4-chloro-l-(4-fluorophenyl)-l-
butanone 2.9
Haloperidol 3.9
Oxatomide 10.0
4-(4-(4-chlorophenyl)-4-hydroxy-
1-piperidiny1)-l-(4-
(4-chlorophenyl)-4-
hydroxy-4-piperidinyl).
pheny1)-1-butanone 11.3
4-[4-(4-Chlorophenyl)-3,6-di-
hydro-1(2H)-pyridyl]-4'-fluoro-
butyrophenone 22.0
6.8 Differential Scanning Calorimetric
Analysis
A quantitative analysis of the purity of
haloperidol can be obtained by DSC ( 7 ) , using
the method by Plato and Glasgow (32). Quali-
tative DSC thermograms of mixtures of halo-
peridol with an impurity are given in Figure
6 to show the specificity of the technique.
6.3 Polarographic Analysis
A polarographic analysis of haloperidol
was described by Volke et a1 using a dropping
mercury electrode (33). A half-wave po-
tential of -1.28 volts was obtained in a
364 CASIMIR A. JANICKI AND CHAN YAN KO
solvent of 1:l v/v acetate buffer, pH 5.3.;

DMF, and -1.64 volts using 1:l v/v 0 . 2 5
potassium hydroxide: ethanol as the solvent.
6.10 Fluorescence Analysis
Haloperidol exhibits weak fluorescence
in methanol, ethanol, and 2-propanol with an
excitation wavelength at 310 nm and emission
wavelengths at 410 nm, 3 7 5 nm and 390 nm
respectively ( 3 4 ) . Haloperidol is converted
into a strongly fluorescent derivative by the
action of potassium permanganate on its
alcoholic solution in an acid medium. An
excitation wavelength of 3 0 5 nm produces
emission at 3 8 3 nm ( 3 5 ) .
7. Determination in Biological Fluids
Several gas chromatographic assay methods
have been published for the determination of
haloperidol in biological fluids ( 2 5 , 2 6 , 2 7 , 2 8 , 2 9 )
and they were given in Section 6 . 6 .
Problems have been reported with the various
gas chromatographic assays, for example, as the
column temperature increased, the haloperidol
dehydrated. As the temperature reached about
285OC, the haloperidol peak decreased in size
while the dehydrated product peak greatly in-
creased. At 2 8 5 O , the large dehydrated-halo-
peridol peak was lost in the solvent peak ( 3 6 ) .
It is important that the temperature of the
column stay about 240OC.
The GC assay methods often lack the required
sensitivity or specificity to obtain reproducible
plasma data from clinical studies. So a sensitive
and specific GC/CI-MS method was developed for the
determination of haloperidol in plasma ( 3 7 ) . The
procedure involves the addition of the internal
standard (the chloro substituted analog of halo-
peridol), alkalinization of the sample with sodium
hydroxide and extraction with heptane containing
1 . 5 % isoamyl alcohol. Prior to injection, the
HALOPERlDOL 365
organic layer is evaporated to dryness and recon-

stituted in methanol containing 5 % triethylamine.
The GC conditions are as follows:
Instrument: A gas chromatograph equipped
with all glass-lined transfer
lines
Column: 10% OV-1 on Chromosorb WHP-0.3 m x
2 mm id Column Temperature: 260°
Carrier: Methane at 25 ml/min
Detector: Quadrupole mass spectroveter set to
monitor ion m/$ 376 (MH of halo-
peridol and MH -H20 of the internal
standard )
Haloperidol has a retention time of 0 . 7 minutes
and the internal standard of 1.1 minutes. The
response curve was linear between 0.2 ng and 6.0
ng
Radioimmunoassays have been published (17,
38,391. A radioimmunoassay kit is also available
(17) which is specific for haloperidol in the
presence of the metabolites a,b,c,d, and e given
in Figure 7 . The limit of detection of the assay
is reported to be 0.02 ng contained in 0.5 ml of
plasma.
8. Determination in Pharmaceuticals
Demoen (1) proposed a spectrophotometric
assay. However, no specificity data were given.
A method that has been found to be suitable for
stability work is that given in the U.S.P. XIX for
tablets (18). The method has been described in
Section 6.4. Janicki and Almond (12) used that
method to determine the haloperidol content in
direct compression tablets that contained an
unexpected degradation product of haloperidol.
The method can also separate and quantitate
haloperidol in the presence of the following
compounds considered model degradation products:
Institut National Des Radioelements,
6220 Fleurus, Belgium
366 CASIMIR A . JANICKI A N D CHAN YAN KO
4-chloro-l-(4-fluorophenyl)-l-butanone; 4-fluoro-
-0xobenzene butanoic acid; 4-fluorobenzoic acid;
and 4-(4-chlorphenyl)-4-piperidinol.
The method cannot separate the dehydrated
product of haloperidol from haloperidol. The
structure of the dehydrated product is given as:
The dehydrated product of haloperidol is a

theoretical degradation product and has not been
found to be an actual degradation product in
dosage forms to date (7). However, TLC solvent
systems 2 and 4 in Section 6.5 can separate the
dehydrated product from haloperidol, which runs
ahead of haloperidol in both systems.
The HPLC assay may be used for stability
analysis. The method and specificity were de-
scribed in Section 6.7.
The methyl orange procedure described by
Janicki and Almond (12), the colorimetric pro-
cedure using 3,5-dinitrobenzoic acid described by
Haemers and Van Den Bossche (191, and the colori-
metric determination using potassium iodoplatinate
described by Pawelezyk and Plostkowiak (20) cannot
be used for stability work since they all lack the
necessary documentation of specificity. The
fluorescence method of Baeyens and De Moerloose
(35) has been applied to dosage forms but no
specificity data were presented.
HALOPERIDOL 361
References
1. P. J . A. W . Demoen, J . Pharm. S c i . , 50, 350

(1961).
2. P. J . A. W . Demoen, J a n s s e n P h a r m a c e u t i c a ,
Beerse, B e l g i u m , u n p u b l i s h e d d a t a .
3. B. B l e s s i n g t o n , Org. Mass S p e c t r o m . , 2, 1113

(1971).
4. J. G . L e f e r i n k , a n d R . A . A . Moes, J . ASSOC.
O f f . Anal. Chem., 60, 2 1 ( 1 9 7 7 ) .
5. E. D i d i n g , H. S a n d s t r o m , J . O s t e l i u s , a n d B .
K a r l e n , A c t a Pharm. S u e c . , 13, 55 ( 1 9 7 6 ) .
6. C . Y. KO, M c N e i l L a b o r a t o r i e s , F o r t
W a s h i n g t o n , PA, u n p u b l i s h e d d a t a .
7. C. A. J a n i c k i , M c N e i l L a b o r a t o r i e s , F o r t
a. L . L. Reed, a n d J . P. S c h a e f e r , A c t a
C r y s t a l l o g r . , S e c t B . , 2, 1 8 8 6 ( 1 9 7 3 ) .
9. P. A. J . J a n s s e n , U . S. P a t e n t 3 , 4 3 8 , 9 9 1
(1969).
10. P. A . J. J a n s s e n , C . Van D e W e s t e r i n g h , A. H.
M. J a g e n e a u , P. J . A. Demoen, B. K . E.
Hermans, G . H. P. Van Daele, K . H. L.
S c h e l l e k e n s , C. A . M. Van D e r E y c k e n , a n d C .
J. E. Niemegeers, J. Med. Pharm. C h e m . , A,
281 (1959).
11. W. D . W a l k l i n g , M c N e i l Laboratories, F o r t
12. C . A. J a n i c k i a n d H. R. Almond J r . , J . Pharm.

Sci., 2,4 1 (1974).
13. G. A . B r a u n , G. I. P o o s , a n d W. S o u d i j n , Eur.
J. P h a r m a c o l . , L, 58 ( 1 9 6 7 ) .
368 CASIMIR A . JANICKI AND CHAN YAN KO
14. W. Soudijn, I. Van Wijngaarden, and F.

Allcwijn, Eur. J. Pharmacol., I,4 7 ( 1 3 6 7 ) .
15. A. Forsman, G. Folsch, M. Larsson, and R.
Ohman, Curr. Ther. Res., Clin. Exp., 21, 6 0 6
(1977).
16. A. Forsman and M. Larsson, Curr. Ther. Res.,

Clin. Exp., 24, 5 6 7 ( 1 9 7 8 ) .
17. J. Heykants, Symposium on Modern Trends in
Psychopharmacology and Psychiatry,
Kollekolle, Denmark, Sept. 1 9 7 8 .
18. United States Pharmacopeia, XIX Revision,

Mack Publishing Co., Easton, PA, 2 2 7 ( 1 3 7 5 ) .
19. A. Hacmers, and W. Van Den Bossche., J.
Pharm. Pharmacol., 2 , 5 3 1 ( 1 9 6 9 ) .
20. E. Pawelczyk, and Z. Plotkowiak, Chem. Anal.
(Warsaw), 17, 1 3 3 3 ( 1 9 7 2 ) .
21. M. P. Quaglio, G. S. Cavicchi, and M.
Muraglia, Boll. Chim. Farm., 110, 3 8 5 ( 1 9 7 1 ) .
22. E. Roder, E. Mutschler, and H. Rochelmeyer,
J. Chromatogr., 42, 1 3 1 ( 1 9 6 9 ) .
23. R. A. Egli, 2. Anal. Chem., -
259, 277 ( 1 9 7 2 ) .
24. Von S. Lauffer, E. Schmid, and F. Weist,

Arzncim. - Forsch., 3,1 3 6 5 ( 1 9 6 9 ) .
25. I. A . Zingales, J. Chromatogr., 54, 15
(1971).
26. A. Forsman, E. Martensson, G. Nyberg, and R.

Ohman, Naunyn - Schmiedeberg's Arch.
Pharmacol., 386, 1 1 3 ( 1 9 7 4 ) .
27. F. Marcucci, L. Airoldi, E. Mussini, and S.
Garattini, J. Chromatogr., 53, 1 7 4 ( 1 9 7 1 ) .
HALOPERIDOL 369
28. J. N. S. Tam, and W. A. Cressman, McNeil

Laboratories, Fort Washington, PA, un-
published data.
29. G. Bianchetti, and P. L. Morselli, J.
Chromatogr., 153, 2 0 3 ( 1 9 7 8 ) .
30. D. S. Greene, Drug Dev. and Ind. Pharm., 2
127 (1979).
31. L. T. Olszewski, C. M. Moral, and J. S.

Ranweiler, McNeil Laboratories, Fort
Washington, PA, unpublished data.
32. C. Plato, and A. P. Glasgow, Anal. Chem., -
41,
330 ( 1 9 6 9 ) .
33. J. Volke, L. Wasilewska, and A. Ryvolova -

Kejharova, Pharmazie, 26, 3 9 9 ( 1 9 7 1 ) .
34. W. Raeyens, Analyst, 1 0 2 , 5 2 5 ( 1 9 7 7 ) .
35. W. Baeyens, and P. DeMoerloose, Pharmazie,

-
32, 764 ( 1 9 7 7 ) .
36. H. R. Almond Jr., and J. A. Meschino, McNeil

Laboratories, Fort Washington, PA, un-
published data.
37. K. T. Ng, and J. J. Kalbron, 25th National

Meeting of the Academy of Pharmaceutical
Sciences, Hollywood, FL, Nov. 1 9 7 8 .
38. B. R. Clark, B. B. Tower, and R. T. Rubin,
Life Sci., 20, 3 1 9 ( 1 9 7 7 ) .
39. M. Shostak and J. M. Perel, Fed. Proc., 35
531 (1976).
KHELLIN
Muhmoud A . Hassan and Muhammad Uppal Zubair
1. Description 372
1.1 Nomenclature 372
1.2 Formulae 372
I .3 Molecular Weight 373
I .5 Appearance, Color, Taste, Odour 373
2.2 Solubility 374
2.4 Spot Tests 374
2.5 Microcrystal Tests 375
3. Isolation 380
4. Biosynthesis 382
5. Synthesis 382
6.1 Modified Zeisel-Viebock Method 386
6.2 Colorimetry 386
6.3 UV Spectrophotometry 387
6.5 Two Dimensional Thin Layer Chromatography 388
6.6 PMR Spectrometry 388
References 393
Analytical Rofiles of Drug Subslancss, 9 Copyright @ 1980 by Academic Press, Inc.

37 1 All rights of reproduction in any form E S ~ N ~ .
ISBN 0-12-260809-7
372 MAHMOUD A. HASSAN A N D MUHAMMAD UPPAL ZUBAIR
1. Description
1.1 Nomenclature
1.1 1 Chemical Names
a. 4,9-Dimethoxy-7-methyl-5 H-furo[3,2-g]
-[1] benzopyran-5-one.
h. 5,8-Dimethoxy-2-methyl-4,5-furo-6,7-
chromone.
c. 5,8-Dimethoxy-2-methyl-6,7-furano-
chromone.
d. 4,9-Dimethoxy-7-methyl-5-oxofuro
[3,2-g] 1,2-chremone
e. 4,9-Dirnethoxy-7-methyl-5-ox0furo
[3,2-g] [l] benzopyran.
f. 4,9-Dimethoxy-7-methyl-5-0~0-1,8-
dioxabenz [ f] indene.
The CAS Registry No. is [82-02-01.
1..1 2 Generic Name
Khellin
1.1 3 Trade Names
Ammincardine, Amicardine, Ammipuran, Ammivin

Ammivisnagin, Benecardin, Corafurone, Cardio-
khellin, Ceronin, Eskel, Kelamine, Kelicorin,
Kelicor, Keloid, Khellin, Gynokhellin, Khel-
fren, Lynamine, Methafrone, Norkel, Simes
Kellina, Visaminin, Visnagin, Visnagalin,
Vasokellina, Viscardin.
1.2 Formulae
1.2 1 Empirical
H O
‘14 12 5
KHELLIN 373
1.2 2 Structural
OCH3
The structure of Khellin has been elucidated

by degradative methods as well as by its
partial synthesis by reconstruction of the
chromone ring starting from Khellinone (1)
and also by its total synthesis.
1.2 3 Wiswesser Cine Notation
* T C 566 DO JV MOJ B01 HO 1L
1.3 Molecular Weight
260.24
1.4 Elemental ComDosition
C,64.61%; H, 4.65%; 0, 30.74%.
1.5 Appearance, Color, Taste, Odour
White odourless crystals sometimes with slight

yellowish tinge, and with a bitter taste.
2.1 Crystal Properties:
2.1 1 X-Ray Diffraction:
Khellin forms monoclinic-Prismatic crystals

with forms C 0 0 1 ~ { 1 0 0 ~ { 2 0 1 ) ( 0 1 0 ) { 1 0 1 ~Fro?
.
measurements a:b:c = 2.462:1:2.681; B=102 53-.
Cleavage parallel (100) good, (010) less
perfect. Optical constants: a= 1.478; B=1.741;
y =1.785. Orientation a=b; Optical axes
observed on (OlO), dispersion 6)8.25 ( f o r
3 14 MAHMOUD A. HASSAN A N D MUHAMMAD UPPAL ZUBAIR
yellow light2 48' 52 , angle c: y=6O(red),7'

(yellow) , 84 (green) ,lO$O(blue); 12'(violet).
Unit, cell for orientation a :bo:Co=2,007:1:
1.608; B=93' 39:a 14.49, b 7f22, C 11.61 A;
Z=4, space group C 5h - PZl/n (derived from
Polany rotation an$ Weissenberg diagram) ( 2 ) .
2.1 2 Melting Range
Khellin melts at 154-155'. Boils at 180-200'

at 0.05 mm Hg.
2.2 Solubility
25 mg/100 ml of water, 2.6 g/lOO ml of methanol,

1.25 g/100 ml of isopropanol, 0.5 g/100 ml of ether,
0.15 g/100 ml of skellysolve B, and much more solu-
ble in hot water and hot ethanol ( 3 ) .
2.3 Identification
1. Khellin gives a red-violet color with NaOH o r

KOH or m-dinitrobenzene in alkaline solution(4-6).
2. A solution containing khellin 0.5-1 ml is heated

to boiling with 0.3 ml of 2,4-dinitrophenyl-
hydrazine (0.5% solution in 1.5 N hydrochloric
acid) f o r 30 minutes, after cooling, 0.5 ml 30%
potassium hydroxide and 1-2 ml o f ethanol are
added. The resulting red-violet stable color is
proportional to the amount of khellin present.
The 2,4-dinitrophenyl hydrazone of khellin was
prepared and it5 m.p. is, 284-28S0(7).
3 . Add 2 ml o f phosphoric acid to 0.1 g of khellin,
orange-red crystals are formed, which dissolve on
heating. To the viscous orange solution of 0.5 g
of khellin in 1 ml phosphoric acid, gradually add
dry ethylacetate with trituration to obtain
yellow crystalline precipitate of the oxonium
phosphate; m.p. 126' -
with decomposition (8).
2.4 Spot Tests
1. To a few crystals of khellin on a white porcelain

plate add 2 drops of phosphoric acid.(sp.gr. .
1.75), an orange-colored crystals are formed (8).
KHELLIN 375
2. To a few crystals of khellin add few crystals

of alloxan, 5 drops of sulphuric acid and tritu-
rate with a thin glass rod till the solids
dissolve, an initial orange color is formed
which changes to violet color with an orange
edge after 20 minutes (8).
3 . This isa more specific test for khellin and is

not given by compounds devoid of 5-OH and 8-OCH3
substituents. The reaction is carried out by
dissolving a few crystals of khellin in HN03, and
then destroying the oxonium salt by diluting with
alkali to yield a color, which i s due to the
quinone formed by oxidation with HNC3 (9,lO).
2.5 Microcrystal Tests
Khellin gives oxonium salt compounds when the

following reagents are added to the dry substance
(11) :
1. I -KI reagent, gives trimorphic crystallisation.

2
In the outer zone of the precipitation there are
rather coarse needles of good bire fringence,
those broad enough showing a yellow to dark red
dichroisy; then a zone of small grains, some
dichroic, yellow to dark, mostly nondichroic, red
after the test has stood a little while, at the
central concentrations fine needles in purple
masses, some of these needles in outer tufts
showing dark blue to light brown dichroism. This
is a sensitive test and probably specific.
2. HC104, gives yellow rods and needles.
3 . HAuBr4 in dil HC104-H9AC gives fine needles,

and at central concentration large tablets, chips
and bars and fans of fairly large needles, with
yellow to orange dichroism.
2.6 Spectral Properties
2 . 6 1 Infrared Spectrum
The infrared spectrum of khellin is recorded as

a nujol mull on a Unicam SP 1025 spectrophoto-
meter and is shown in Fig. 1 . The assignments
Y
5
i
c
n
5
0
al
c:
A
Lk
0
P E:
3
h
U
r'
-..
M
.A
LL
KHELLIN 311
for the characteristic bands in the infrared

spectrum are listed in table 1.
Table 1
Frequency cm-1 Assignment
1690 c = o
1650,1640 c = o
1600 C = C (aromatic)
1580 ethylenic linkage
1250,1230
1190,1160 c-0-c
1090,1070
870,820 -CH out of plane
790,740 deformation.
720.
Other fingre print bands characteristic of

khellin are 13913,1370, 1050, 1040, 1000,
960, 920 and 910.
2.6 2 Ultraviolet Spectrum (UV)
The UV spectrum of khellin in ethanol was

scanned using Pye Unicam SP 800; from 400-
200 my, three maxima and three minima were
observed. The maxima are located at 220, 244
and 328 nm. The minima occur at 232, 272
and 300 nm. The spectrum is shown in Fig.2.
The UV spectral data of khellin and analogues
have also been reported (12).
2.6 3 Nuclear Magnetic Resonance Spectrum (NMR)

Proton Spectrum
The proton NMR spectrum of khellin in deute-

rated chloroform i s shown in Fig.3. It was
recorded on a Varian T-60AY60MHz N M R Spectro-
meter, using tetramethylsilane as an internal
reference. It is to be noted that both natural
and synthetic khellin are currently used in
pharmaceutical formulations. Natural khellin
might contain variable amounts of visnagin due
to incomplete purification.
378
a 3UEqxo s qv
2
ra,
Id4
350
300
250
200
!
Fig. 2: Ultraviolet spectrum of khellin in ethanol.
d
I
f
I 4.0 1'
PARTS PLH ?lILLIOE;.
rig. 3: YFIK spectrum of khellin and TMS in CDCl

3'
Therefore, the PMR spectrum of visnagin in

deuterated chloroform using tetramethylsilane
is shown in Fig. 4.
The PMR spectral assignments of khellin and

visnagin are given in Table 2 (13).
Table 2: PMR Comparison of Khellin and Visnagin
Chemical shifts(6)
2-CH 3-H 5-OCHs 8-OCH3 8-H
-
2-H
- 3-H
(s) (s) (5) (SJ (s) (d) (d)
Khe11in 2.40 6.05 4.20 4.05 - 7.63 7.00
Vi snagin 2.33 6.03 4.20 - 7.18 7.63 7.00
( s ) = singlet, (d) = doublet.

2.6 4 Mass Spectrum
The mass spectrum of khellin obtained by con-
ventional elect-fonimpact ionization shows+a
molecular ion M at m/e 260.07 (14). The M ion
peak is the base peak and is shown in Fig. 5.
The fragmentation of khellin and other furano-
chromones have been reported (15).
Fig. 5: Mass Spectrum of Khellin
3. Isolation
Khellin [5,8-Dimethoxy-2-methyl-4,5-furo-6,7-chromone] is
obtained as the main chromone constituent from the
fruits of Ammi visnaga (Fam. Umbelliferae) (16-19).
I I 3 I 1 I. 1 . I , I I
L - . l . 1 . ~ ~ . . . . ~ . . . . I . . . . I I . . . . I . . . 1 . f . . . 1. . . * . 1 1
u I a. 14 '43 LD 1.0 t.b
Fig. 4: PMR Spectrum o f visnagin and TYS i n C D C l

3
4. Biosynthesis
Geissman (20) on the basis of the striking similarity

between the furocoumarin isopimpinellin and the furano-
chromone khellin, suggests that the two heterocyclic
ring systems have a common origin, namely cinnamic acid
and they are formed by the shikimic acid - phenylalanine
pathway. Extension of the-cinnamic acid side-chain,
possibly at the orthohydroxylated intermediate as gluco-
side, by the addition of two carbon fragment, as
shown+
However, Chen et al(21) has reported that blpnthesis
of radio-active khellin and visnagin from C -acetate
by Ammi visnaga plants. Their results support the hypo-
thesis that furanochromones are biosynthesised via an
acetate condensation pathway rather than by the phenyl-
alanine-shikimic acid route as is the case for the very
closely related furocoumarins.
5. Synthesis
Several synthetic routes to khellin and derivatives

have been reported (22-35). Two of them are illustrated.
Route - I : Involves the condensation of 5,7-Dihydroxy-2

methylchromone ( I ) with bromoethyl acetate, followed
by nuclear oxidation of the product with alkaline persul-
fate to give quinol (111) (36). The partially methylated
quinol ( I V ) is condensed with hexamine to give the
aldehyde (V) which on complete methylation and hydrolysis
with dilute alkali gives aldehydic acid ( V I ) , which on
boiling with sod. acetate and acetic anhydride afforded
khellin ( V I I I ) .
Route - 11: Describes total synthesis of khellin starting

with 2,5-dimethoxyresorcinol ( I ) , (37,38). ( I ) is con-
verted into the coumaranone (11) by means of Hoesch
Reaction, using chloroacetanilide. Acetylation of (11)
and reduction yielded ( I V ) . After removal of the acetyl
group of ( I V ) it was subjected to a Hoesch Raction with
acetonitrite to yield dihydrokhellinone ( V I ) . Acetylation
of V I and reaction with N-Bromosuccinimide in carbon
tetrachloride and purification yielded pure khellinone
( V I I I ) . Khellinone was condensed with ethyl-acetate in
presence of sodium hydride to give a diketone ( I X ) which
is then cyclised to give k I l e l l i n
KHELLIN 383
2-methyleehromone
Biosynthesis of a 2-methylchromone.
384 MAHMOUD A . HASSAN A N D MUHAMMAD UPPAL ZUBAIR
I II
Ill IV
V VI
KHELLIN 385
ROUTE I 1
X
6.1 Modified Zeisel-Viebock Method (39)
Malysz et a1 (40) have applied this method for

determining methoxy groups by using a special
apparatus. The sample containing equivalent of
2-5 mg of methoxy group was mixed with 0.5 g of
phenol, 2.4 g of potassium iodide and 4 ml of
phosphoricoacid , then heated in an atmosphere of
C02 at 150 for 1.5 hours. The iodomethane produced
was distilled off in a stream of CO and absorbed
in 10 ml of bromine solution (dissolve 10 g of
potassium acetate in 100 ml of anhydrous acetic acid
and add 4 ml of Br2). The absorbent solution was
then mixed with 10 rnl of 2.5% aqueous sodium acetate
and diluted with 100 ml of water and the excess of
bromine was destroyed with 3 drops of formic acid.
The colorless solution was acidified with 10 ml of
2 N sulfuric acid, 1 g of potassium iodide was added
and after 5 minutes the liberated iodine was tit-
rated with 0.05N sodium thiosulfate solution and
starch as indicator. This method has been used to
determine khellin in the pure form and in pharma-
ceutical preparations. The results were within 20.1-
-
+1% of those obtained by various pharmacopoeia1
methods.
6.2 Colorirnetry
Different colorimetric methods have been used for
the determination of khellin, based on color
reaction with sulphuric acid (41,42), phosphoric
acid (43,45) and m-dinitrobenzene and potassium
hydroxide (45,47). A colorimetric method based
on the color developed by treating khellin with
nitric acid followed by sodium hydroxide is
officially adopted by E.P. 1972 (48). The method
is based on the oxidation of khellin with nitric
acid to produce quinone derivative which gives
violet color with sodium hydroxide solution.
The reaction was favourably carried out at room
temperature (20-300) , lower or higher temperature,
either slow the reaction or enhance it, respec-
tively. The absorbance of the resulting violet
color is measured at Xmax 540 nm within 15 minutes
KHELLIN 387
CH3
0
OCH3.
Khellin Khellin-Quinone
after the addition of Na0I-I solution. The formed

color obeys Beer's Law in a concentration range
of 200-1200 1.18 of khellin. This method has been used
to determine khellin in fruits and extracts of Ammi
visnaga and in pharmaceutical formulations (49) .
6.3 U\I Spectrophotometric Method
Khellin can be analyzed spectrophotometrically. The

sample is dissolved in ethanol (95%) to give a
concentration of about 12 Ug/ml, and the absorbance
of the solution so produced is measured at 244 nm.
The l o g E values are given in table 3 (SO).
Table 3
Xmax (nm) Log & Ash (nm) Log E
220 4.453 228 4.400
244 4.513 280 3.678
328 3.614
6.4 Two Dimensional Thin Layer Chromatography
Different solvent systems have been used for the

separation of khellin from other natural chromones.
388 MAHMOUD A. HASSAN AND MUHAMMAD UPPAL ZUBAIR
Table 4
Solvent System Rf Value Reference
Xylene-Acetone(4:1) 0.25 51
Xylene-Ethyl Acetate- 0.31 51

Acetic Acid (15:s: 1)
Xylene-Ethyl Acetate- 0.62 51

Pyridine (6:18:1)
Xylene-Pyridine-Formic 0.63 51
Acid (23:5:2).
Ether-Formamide- 0.86 52
ethanol (93: 2: 5).
Chloroform-Tetrahydro- 0.67 52
furan-formamide (50: 50:
6.5).
Ethylacetate-Benzene- 0.50 52
Water (50:50:50).
Ethylacetate-Benzene- 0.31 52
water (50:75:50)
(Fig.6).
6.5 Two Dimensional Thin Layer Chromatography
Karawya et a1 (53), reported two dimensional TLC

technique on silica gel G plates using ethylacetate
as developing solvent (Fig.7). This technique has
offered a better separation of khellin from visnagin
than the unidimensional multiple-run technique and
was used in the quantitative recovery of the two
constituents and their subsequent colorimetric
estimation. It is also used for the determination
of khellin in pharmaceutical formulations.
6.6 PMR Spectrometry
Hassan and Aboutabl (13) has published a rapid,

accurate and specific PMR method for the determi-
nation of khellin in bulk drug and pharmaceutical
KHELLIN 389
Ethyl acebfe Benzene Water 50/75/50
.-,
.__.
I gr ;;::
b r g <.-..‘*
-
,,-1
..:
, %.
____._--
\.
-
A B
Fig. 6: A:0.1 cc alcoholic extract
of Ammi visnaga f r u i t s .
I’ROV l U 1 U
‘’
EXttUCT
6.conA
- +
run
1
I
Fig. 7: Two dimensional T I L u s i n g e t h y l -

acetate as the developing system.
formulations. It also furnishes a specific means

of identification of khellin as well as simultaneous
detection and determination of the less potent 8-
demethoxy analogue, visnagin. Acetanilide exhibiting
three proton singlet at 2.30 ppm in CDC13 assigned
to its methyl group, is employed as an internal
standard. The two singlets at 4.2 and 4.05 ppm (in
CDCl ) assigned to the 5-and 8-methoxy protons of
khelqin respectively, were chosen for its quanti-
tative analysis (Fig.8). However, the presence of
other ingredients in injectables interfere with the
precise integration of the 5- and 8-methoxy signals.
For this reason the 2-methylprotons singlet appear-
ing at 2.4 ppm (in CDCl ) was used for assay of
khellin in injectables. zthanol-free chloroform
was used as a solvent, as its proton singlet of
7.25 ppm does not interfere with upfield protons
of both compounds. The method is rapid, accurate
and precise, with standard deviations of 2 0.76%
in synthetic mixtures and 0.94% in tahlets and
injectables respectively. No interference from
tablet excipients could be observed. Visnagin shows
in deuterated chloroform a very similar PMR spectrum
to that of khellin, except f o r the presence of an
aromatic proton singlet at 7.18 ppm and three proton
singlet at 2.33 ppm assigned to its 8-H and 2-CH
3
group. This has allowed facile detection of visnagin
in khellin in bulk drug and formulations. Moreover,
the ratio determination of visnagin to khellin is
achieved by integration measurements of the 2-methyl
protons singlets at 2.33 and 2.40 pprn respectively
(Fig.:)).
Ii
*r > .0.
rlu
**
TMS
@-coNHI
-a
..
l
I
. . . . l .
.,
. .
Fig. 8:
. I . .
..
. . l
so **a. <.I
PMR spectrum of khellin, acetanilide

and TMS in ethanol-free chloroform.
,
i
.W
I P "I
wrt
TMS
I H
I . . . . I . . . . I . . . .
I 0 I* ,4
Fig. 9: PMR spectrum of khellin, v i s n a g i n and TMS in CDC13.

KHELLIN 393
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Argentina, 40, 203 (1952).
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(1969).
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1 2 ( 3 ) , 307 (1964).
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12
(5) , 351-363 (1979).
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" R e g i s t r y of Mass S p e c t r a l Data," John Wiley and Sons,
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2, 54 (1952).
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52, 13801f (1958).
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24. R.Aneja, S.K. Mukerjee and T.R. S e s h a d r i , J . S c i . Ind.
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Research ( I n d i a ) , 17B, 382 (1958).
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93, 297 (1960).
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KHELLIN 395
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19 (41,
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p.547.
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Fayez, P l a n t a Mod., 15,
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J. Pharm. S c i , 59, 1025 (1970).
LORAZEPAM
Jay G. Rutgers and Charles M . Shearer
1. Description 398
1 . 1 Name, Formula, Molecular Weight 398
2.3 Ultraviolet Spectra 400
2.4 Mass Spectra 404
2.6 Differential Scanning Calorimetry 407
2.7 Solubility 407
2.9 Dissociation Constants 407
3. Synthesis 409
4. Stability and Degradation 411
5 . Metabolism and Pharmacokinetics 412
5.1 Metabolism 412
6. Identity 414
7. I Elemental Analysis 415
7.2 Phase Solubility Analysis 415
7.3 Direct Spectrophotometric Analysis 415
7.5 Electrochemical Analysis 416
7.6 Titrimetric Analysis 417
7.7 Chromatographic Analysis 417
8. References 424
Copyright 0 1980 by Academic Ress, Inc.

Analytical Rofiks of Drug Substances, 9 397 All rights of nproduction in any form ~ ~ e ~ e d .
ISBN: 0-12-260809-7
398 JAY G . RUTGERS A N D CHARLES M. SHEARER
1. Description
1.1
Name, Formula, Molecular Weight
The name used by Chemical Abstracts for lorazepam
is 7-chloro-5-(2-chlorophenyl)-1,3-d~hydro-3-hydroxy-2H-l,
4-benzodiazepin-2-one.
The Chemical Abstracts Registry Number is 846-49-1.
15H10C12N202 Mol. Wt. = 321.2

Lorazepam is a white or nearly white, practically
odorless, crystalline powder.
2.1
Infrared Spectrum
An infrared absorption spectrum of a potassium bro-
mide dispersion of lorazepam (Wyeth Reference Standard Lot
C-10684) is presented in Figure 1. The spectral band
assignments (1) are listed in Table I.
Table I
Infrared Spectral Assignments of Lorazepam
Wave number (cm-l) Vibration Mode

3500 to 2700 OH,NH stretch
1690 C=O stretch
1610 C=N stretch
1565 and 1475 Aromatic C=C stretch
825 Out of plane CH deformation
of 1,2,4 substituted aromatic
730 Out of plane CH deformation
of ortho disubstituted aromatic
750 and 695 1,2,4 substituted aromatic and
ortho disubstituted aromatic.
Unequivocal assignment cannot
be made.
WAVELENGTH (MICRONS)
2.5 3 4 5 6 7 8 9 10 12 14 16 18 20 25 30 40 50
01 I
4ooo 3500 Moo 2500 m 1800 1m 1400 1200 loo0 800 600 400 2GfJ
FREQUENCY (CM-ll
Figure 1 - I n f r a r e d Spectrum of Lorazepam (Wyeth Reference Standard,

L o t C-10684) KBr p e l l e t
JAY G. RUTGERS AND CHARLES M. SHEARER

The nuclear magnetic resonance spectrum sample
(Wyeth Reference Standard, Lot C-10684) was prepared by dis-
solving 75 mg of it in 0.5 ml of deutero-dimethylsulfoxide
containing tetramethylsilane as internal reference. The
spectrum was obtained on a 100 MHz Varian XL-100 spectro-
meter and is presented as Figure 2. Deuteration or irradi-
ation of the OH reduces the methine doublet to a sharp
singlet. The spectral assignments ( 2 ) are listed in Table
11.
Table I1
NMR Spectral Assignments of Lorazepam

Proton Chemica1 J
(No. Shift (ppm) ZYE (in Hz)
A1 iphatic 4.88 Doublet 7.5
C-H(1)
0-H( 1 ) 6.38 Doublet
Aromatic
C-H(4) 7.4 to 7.7 Mu1t ip let
Aromat ic
C-H( 1 ) 6.97 Doublet 2
Aromatic
C-H( 1) 7.6 Doublet of Doublet
Aromatic
C-H( 1 7.30 Doublet 9
N-H(~) 10.96 Broad singlet
2.3Ultraviolet Spectra
The ultraviolet spectrum of lorazepam in methanol
is presented in Figure 3. The spectra of lorazepam in 1N
NaOH and in 1N HC1 are presented in Figure 4. The absorpti-
vities and maximum wavelengths are given in Table 111.
These values agree with published data (3,4,5).
Levillain (6), has studied the relationship of
structure and the UV absorption characteristics of a series
of 1,4-benzodiazepines, including lorazepam, considering the
electronic distribution of the various substitutes and the
stereochemistry. The spectrum of lorazepam is consistent
with that of other benzodiazepines with similar structure.
1 I I I I I I I
-1
I,
II
I
I
1 I I I I I 1 I I I
9 8 7 6 5 4 3 2 1 0
ppm
Figure 2 - NMR Spectrum of Lorazepam (Wyeth Reference Standard,

Lot C-10684) i n deutero dimethylsulfoxide
402 JAY G. RUTGERS AND CHARLES M. SHEARER
0. t
0.5
0.4
z
U
m
cr
0
m
2 0.3
0.2
0.1
0.0
WAVELENGTH (nm)
Figure 3 - U l t r a v i o l e t Spectrum of Lorazepam (Wyeth Reference

Standard Lot C-10684) S o l v e n t - methanol
LORAZEPAM 403
0.1 -
0.0 '
220 240 260 280 300 320 340 360 380 400
WAVELENGTH (nm)
Figure 4 - U l t r a v i o l e t S p e c t r a of Lorazepam (Wyeth Reference

S t a n d a r d , L o t C-10684) S o l v e n t A , 1N N d H ;
Solvent B , 1 N HC1
404 JAY G. RUTGERS A N D CHARLES M . SHEARER
Table 111
Ultraviolet Spectral Characteristics
Solvent A M a x (nm) Absorptivity
methanol 320 6.1
229 116
1N NaOH 347 8.4
233 92
1N HCI 368 8.0
287 25
237 94
2.4 Mass Spectra

The mass spectrum o f lorazepam (Wyeth Reference
Standard, Lot C-10684) was obtained with Kratos DS-50-S Data
System coupled with a MS-902 double focusing, high resolution
mass spectrometer (7). The ionizing electron beam energy was
at 70 eV. Figure 5 is a bar graph of the mass spectrum with
the molecular ion at m/e 320. Identification of the pertinent
masses is presented in Table IV. A chemical ionization
spectrum showed the parent peak at M+1, 321.
Table IV
Mass Spectrum Fragmentation
Pattern of Lorazepam
mle
__ Species
320 M+
302 M+ - H20
291 M+ - CHO
274 M+ - H20 - CO
263 M+ - HCO - CO
239 M+ - H20 - CO - C1
2.5 Melting Range

The following melting range temperatures have been
reported.
OC Reference
167 170 (d) 4
166 - 168 (d) 8
Trace amounts of certain acidic impurities,including
6-chloro-4-(~-chlorophenyl)-2-qu~nazoline~rboxyl~c acid (a
possible degradation product), benzoic acid and salicylic
acid, will markedly depress the decomposition temperature of
lorazepam ( 9 ) .
I
U
3
a
$4
(d
a
2
cl
(A
4J
U
QJ
a
(A
0
n
z
W
I
0
X
w
50 100 150 200

O C
Figure 6 - Differential Thermal Analysis Spectrum of Lorazepam

(Wyeth Reference Standard, Lot C-10684)
LOR A ZE PA M 407
2.6 Differential Scanning Calorimetry

The DSC thermogram (10) of lorazepam (Wyeth Refer-
ence Standard, Lot C-10684) is shown in Figure 6 . The ther-
mogram was obtained at a heating rate of 10°C/min in a
nitrogen atmosphere using a Perkin-Elmer DSC-2. The thermo-
gram exhibits no endotherms or exotherms other than that
associated with the decomposition melt.
2.7 Solubility
The following solubilities at room temperature have
been reported.
- -
Solvent Solubility (mg/ml) Reference
Alcohol 14 11
Water 0.08 11
Propylene glycol 16 12
Chloroform 3 13
Ethyl Acetate 30 13
The X-ray powder diffraction pattern of lorazepam
(Wyeth Reference Standard, Lot C-10684) ,obtained (10) with a
Phillips diffractometer using CuKd radiation is presented in
Figure 7. The calculated "d" spacings are presented in
Table V. It is possible for lorazepam to form solvates and
other crystal forms (9).
The crystal and molecular structure of the ethanol
adduct of lorazepam have been characterized by X-ray analysis
(14). The asymmetric unit consists of one ethanol and two
lorazepam molecules linked together by hydrogen bonds. The
crystalsoare monoclini; with cell dimensions of a=13.446A,
b=19.259A and c=13.789A. The 0 angle i s 116.80O. The hete-
rocyclic seven-membered ring adopts a boat configuration.
The two phenyl rings are planar and the obtuse angles
between them are 106.6O and 99.1°.
2.9 Dissociation Constants
Two pKa's are observed for lorazepam (15,5). The
pKa values, determined spectrophotometrically in aqueous
buffers, are 1.3 and 11.5. Polarographically, the first pKa
was found ( 1 6 ) to be 1.8. Barret et a1 (5) have proposed
that three species, a protonated, a neutral, and a deproton-
ated form, are involved i n the equilibria. Protonation at
low pH occurs at the nitrogen in the 4 position. Deproton-
ation occurs at high pH with the loss of the hydrogen atom
from the 3-hydroxyl group.
5a
al
N
td
3
li-4
0
LORAZEPAM 409
Hagel et a1 (17) have studied the nonaqueous titra-

tion of lorazepam with tetrabutylammonium hydroxide and
perchloric acid. Their findings indicate that protonation
does occur at the N-4 position. However, they propose that
the deprotonation occurs at the N-1 position rather than at
the 3-substituent.
Table V
X-Ray Powder Diffraction Pattern
d I/I, d
- __ -
13.5 .46 3.65 .31
7.31 .26 3.54 1 .oo
6.71 .20 3.44 .18
5.68 .10 3.38 .13
5.47 .29 3.23 .40
4.96 .79 3.12 .20
4.86 .60 3.02 .23
4.56 .53 2.97 .29
4.46 .36 2.88 .07
4.40 .45 2.82 .15
4.10 .19 2.77 .17
3.99 .33 2.66 .10
3.87 .42 2.36 .10
3.83 .12 2.33 .ll
3.72 .07
2.10 Protein Binding

The protein binding of lorazepam and other benzo-
diazepines have been studied extensively by Mueller and
Wollert (18-24)using circular dichroism and gel filtration
techniques. The binding to albumin is decreased by the addi-
tion o f chlorine in the ring 2' position as evidenced by the
fact that oxazepam binding is greater than that o f lorazepam.
The binding is relatively independent of pH (pH 6.60 to 8.20).
3. Synthesis
One synthetic route for lorazepam is shown in Figure 8
beginning with 2-amino-2',5-dichlorobenzophenone (I). The
benzophenone is first converted to its oxime (11) with
CI
a
'c=q
N H 2 NHZOH HCI-..
R
CI
n
'
N H Z
C=NOH
\R R'
2-AMINO-Z', 5-DICHLORO- 2-AMINO-Z', 5-D ICHLORO- 6-CHCORO-Z-CHLOROMETHYL-4-

BENZOPHENONE BENZOPHENONE OX IME (Z-CHLOROPHENYL)-QUINAZOLlNE 3-OX IDE
I n m
3-ACETOXY -7-CHLORO-S-(Z-CHLOROPHENYL) 7-CHLORO-5-(2-CHLOROPHENYL)

1, 3-DIHYDRO-2H-1, 4-BENZODIAZEPIN-PONE 1,3-DlHYDRO-ZH-l,
P 4-BENZOD IAZEPIN-2-
ONE 4-OXIDE
Is!
R
R= bC1
LORAZEPAM
!l
l
Figure 8 - Synthesis of Lorazepam

LORAZEPAM 41 1
hydroxylamine. Reaction of the oxime with chloroacetyl

chloride produces (111) the quinazoline 3-oxide ( 2 5 ) . Ring
enlargement to the benzodiazepin-2-one &-oxide (IV) is
accomplished by treatment with sodium hydroxide (26).
Reaction with acetic anhydride and subsequent hydrolysis of
the ester (V) with base produces (VI) lorazepam (8,27). A
variation of this procedure is to react (IV) with isopropenyl
acetate to form (V) which is hydrolyzed with base to produce
lorazepam (28).
In another synthetic procedure the benzophenone anti-

oxime is reacted with 2,2-diacetoxyacetyl chloride to pro-
duce a dihydroxyacetanilide derivative. This intermediate is
cyclized with base and then hydrogenated to yield lorazepam
(29).
4. Stability and Degradation

Lorazepam can lose a molecule of water and rearrange to
form 6-chloro-4-(~-chlorophenyl)-2-quinazolinecarboxaldehyde
(30).
This quinazolinecarboxaldehyde can disproportionate and

be oxidized orreducedto form the corresponding quinazoline-
carboxylic acid or quinazoline alcohol respectively.
412 JAY G . RUTGERS A N D CHARLES M. SHEARER
cl%cl *YCozH I "',
\ \
Acid hydrolysis of lorazepam ultimately produces

2-amino-2',5-dichlorobenzophenone which is the basis for
numerous GLC, TLC and colorimetric analyses of lorazepam.
In base lorazepam rearranges ( 3 1 ) to 7-chloro-5-(2-chloro-
-
phenyl~-4,5-d~hydro-2H-l,4-benzod~azepin-Z,3~lH)-dione.
-
5. Metabolism and Pharmacokinetics

5.1 Metabolism
The metabolites of lorazepam which have been char-
acterized in human and animal studies are shown in Figure 9.
In man the major metabolite is the glucuronide ( 3 2 , 3 3 , 3 4 ) .
I4C labeled studies have shown that 88% of the administered
radioactivity was recovered in the urine and 7% in the stool.
The glucuronide comprised 86% of the urinary activity.
Minor metabolites are II,V,VI and VII. Characterization of
the metabolites was made by mass spectrometry and by thin-
CI @
/ JfcooH CI @yCOOH
LORAZEPAM GLUCURONIDE Ill HYDROXYLORAZEPAM Ill1
~-CHLORO-~-(O-CHLOROPHENY
Ll- (VII
2-QUINAZOLINECARBOXYLIC ACID IV)
HYDROXYMETHOXYLORAZEPAM I111I LORAZEPAM DIHYDRODIOL f I V I

CI &O CI mNH2 c=o
,HCOH
6-CHLORO-4-lo-CHLOROPHENYLl-2~lHl- 2-AMINO-2'. 5 - 0 ICHLOROBENZOPHENONE IVIIII
CI
OR QU INAZOLINE IVII)
LORAZEPAM D IHY DROD IOL [ IVI LORAZEPAM DIHYDRODIOL I I V I
Figure 9- M t a b o l i t e s of Lorazepam
layer chromatographic comparison to authentic samples. The

glucuronide of lorazepam, in particular, has been character-
ized extensively by chemical analysis and infrared and mass
spectroscopy of the trimethylsilyl derivative (35).
The glucuronide of lorazepam is also the major meta-
bolite in miniature swine, dogs and cats (32,33,36,37).
Minor metabolites in these species are II,III,V,VII and VIII.
The metabolic transformation in rats is quite different from
that in other animals investigated. Significant amounts of
metabolite are found in plasma, bile and tissue. Compounds
II,III,IV,V and VII have been identified as metabolic pro-
ducts (37).
A review of the metabolism of lorazepam has been
written by Elliot (38).
5.2 Pharmacokinetics
The pharmacokinetics of lorazepam has been studied
by Greenblatt et a l . (33). A 2 mg oral dose of I 4 C lorazepam
was administered to eight male adults. Blood samples were
collected for a period of 96 hours and urine and feces sam-
ples 120 hours after administration. The various fractions
were examined by means of a liquid scintillation spectro-
meter and gas chromatography. Data obtained on pooled plasma
samples indicate that there is a lag time of about 35 minutes
before the beginning of absorption. The apparent half-life
of the absorption process is about 15 minutes for free lor-
azepam and 39 minutes for the glucuronide conjugate. Maxi-
mum plasma levels observed were 16.9 mg/ml for free lorazepam
at 2 hours and 29.9 mg/ml for the conjugate at 4 hours. The
apparent elimination half-lives are approximately 12 and 16
hours respectively. 88% of the total radioactivity admin-
istered was eventually recovered in the urine predominantly
in the form of the conjugate. AR additional 7% was recovered
in the stool.
6. Identity
Kuhrent-Brandstaetter (4 ) has described several qualita-
tive tests based on melting point or formation of color which
can be used to identify lorazepam. A sample warmed in a
phenylhydrazine solution forms crystals slowly when cooled.
The crystals melt at 88-92OC. Upon continued heating the
melt recrystallizes to orange-yellow crystals which remelt at
LOR A ZE PA M 415
205-207OC. Heating a mixture of lorazepam and benzidine to

the melting point produces an orange-brown melt.
A method has been developed for the detection of loraze-
pam in urine (39). A urine sample is extracted with ether
and the ether extract examined under longwave UV light. A
blue fluorescence due to the quinazolinone metabolite is
indicative of lorazepam. The residue from the ether extract
is then heated in 6N hydrochloric acid to produce the benze-
phenone derivative. A blue color developed with Bratton-
Marshall reagent is a l s o indicative of lorazepam. However,
the test is not specific for lorazepam. Tetrazepam is
reported to give the same positive tests.
Infrared spectroscopy can be used directly on the drug
substance for its identification.
7.1Elemental Analysis
The elemental analysis of lorazepam (Wyeth Reference
Standard, Lot C-10684) is presented below.
Elemen t % Calculated % Reported (7)
C 56.10 56.05
H 3.14 2.99
N 8.72 8.65
c1 22.08 21.77
7.2 Phase Solubility Analysis
Phase solubility analysis (9 ) on lorazepam (Wyeth
Reference Standard, Lot C-10684) using isopropanol as the
solvent gave a purity of 99.8 f 0.2%.
7.3Direct Spectrophotometric Analysis
Seitzinger ( 3 ) has described an ultraviolet spectro-
photometric method for the analysis of lorazepam in tablets.
A sample equivalent to 5 mg of lorazepam is weighed into a
100-ml volumetric flask, 50 ml of alcohol is added and warmed
in a steam bath. After cooling, the sample is diluted to
volume with alcohol. The sample is filtered and a 10.0 ml
aliquot of the filtrate diluted to 100 ml with alcohol. The
absorbance is determined at 228 nm using alcohol as a blank
and compared with the absorbance of a standard solution of
lorazepam.
The adaption of the spectrophotometric method to

automated analysis has been reported (40).
7.4 Colorimetric Analysis
Lorazepam can be hydrolyzed with hydrochloric acid
to form 2-amino-2',5-dichlorobenzophenone. The aromatic
amine is diazotized with nitrous acid and the diazonium salt
coupled to N-(1-naphthy1)ethylenediamine. The absorbance of
the resulting colored solution i s determined at 5 5 5 nm. A
standard lorazepam solution is subjected to the same react-
ions for comparison. The procedure was applied to several
tablet dosage forms of lorazepam. The tablets were extrac-
ted initially with chloroform and a portion of the chloro-
form extract evaporated for color development. Results
obtained by the colorimetric procedure were in good agree-
ment with those obtained by the spectrophotometric methods
(3).
7.5 Polarographic Analysis
Lorazepam is reducible at the dropping mercury elec
trode over a wide pH range. In the pH range of 0 to 6 a
well defined wave is obtained. Above pH 6 the wave becomes
strongly affected by absorption of the reducible species on
the mercury electrode resulting in a distorted wave (41).
The optimum pH range for analytical applications is consid-
ered to be 3 to 6 . The diffusion current is linear with
concentration in the range of to 10-4M (16).
Several analytical procedures for lorazepam tablet
dosage forms employing methanolic acetate buffer (pH5)
have been reported (16,421. The procedure can be adapted
to differential pulse polarography(43,44,45). The polaro-
graphic technique has also been adapted to automated analy-
sis by interfacing a polarographic analyzer with a contin-
uous flow system (46,471. Polarographic analysis is sta-
bility indicating for the major route of degradation ( 3 0 ) .
Oelschlager (48) has investigated the reduction o f
lorazepam and found that it consumes four electrons in three
steps to form 7-chloro-5-(q-chlorophenyl)-l,3,4,5-tetra-
hydro-2H-1,4-benzodiazepin-2-one. The first step in the
postulated mechanism is the reduction of the 4,f-N=C double
bond with the consumption of two electrons. Water is eli-
minated with the formation of the aldimine. The aldimine is
subsequently reduced with the consumption of two additional
electrons.
LORAZEPAM 417
7.6 .-Titrimetric Analysis

The tetrabutylammonium hydroxide titration proce-
dure for oxazepam (NF-XIV, 1975) was shown to be applicable
to the titration of lorazepam. The titration is considered
to proceed through the deprotonation at the N-1 position.
Titration of lorazepam with perchloric acid in glacial ace-
tic acid resulted in poorly defined potential breaks (17).
7.7 Chromatographic Analysis
Lorazepam may be evaluated on a thin-layer
plate as the intact drug or, frequently, as the acid hydro-
lysis product, 2-chloro-2',5-dichlorobenzophenone. There
are certain cases where it is advantageous to develop lor-
azepam as its hydrolysis product. Conversion to the benzo-
phenone may be achieved by hydrolyzing in solution before
spotting (49) or hydrolyzing directly on the plate after
spotting (50). One method of detection is also based on
conversion of lorazepam to the benzophenone after develop-
ment ( 3 2 ) .
The various solvent systems used for thin-
layer chromatography of lorazepam on silica gel plates are
given in Table VI. The table indicates those cases where
the material was developed as the benzophenone. Table VII
lists the methods of detection used for lorazepam on thin-
layer chromatograms.
Gas chromatography has been used extensively in
metabolic and pharmacological studies of lorazepam. This
technique can provide the sensitivity which is required for
the low doses usually administered.
Lorazepam is not thermally stable. A number of
investigations (55-58) have shown that under gas chromato-
graphic conditions lorazepam can lose a water molecule and
rearrange to form 6-chloro-4-( 2'-chlorophenylquinazoline)
-2-carboxaldehyde. Consequently, in any gas chromatogra-
phic procedure where lorazepam is injected directly this
rearrangement must be considered. Another consideration is
that in metabolic studies the major metabolite is excreted
as the glucuronide and a preliminary enzymatic incubation
i s usually employed. However, Marucci (59) was able to
chromatograph the glucuronide directly by preparation of a
derivative. The conjugate was first reacted with diazo-
methane to methylate the uronic acid carboxyl group and also
the N position. The methyl derivative was then silylated
with hexamethyldisilazane. The mass spectrum was consistent
with a dimethyltrisilyl derivative. The procedure was
Table VI
Thin Layer Chromatopraphy for Lorazepam
Solvent Rf x 100 Application Reference
Benzene 46(as benzophenone) Separation of oxazepam and 49
lorazepam
Chloroform-Acetone- Rm=80 Identification of drugs in 52
Diethylamine (vs. meprobamate) biological media
( 5 0 : 50: 10)
Hexane - 25% diethyl- Rm=5 9 11 52
amine in ethanol (vs. nitrazepam)
(75: 2 5 )
To1uene-acetone Rm=5 II 52
e
m (80:20)(Tank contains (vs. thioridazine)
ammonia vapor)
Chloroform-ethanol- 33 Netabolic studies 32
acetone (8:1: 1)
E thy1 acetate-ethano1- 63 I1 11 32
conc. ammonia ( 5 : 5: 1)
Chloroform-methanol 36 Identity in tablets 3
(lo: 1)
Benzene 41 Separation of 1,4-benzodiazepines 53
Heptane-chloroform- 11 Separation of 1-4-benzodiazepines 51
ethanol ( 5 0 : 5 0 : 5 )
Solvent Rf x 100 Application Reference
Ethyl acetate- 1,2- 25 Separation of 1-4-benzodiazepines 51
dichloroethane - 25%
amnonium hydroxide
(80:20:5)
Ethyl acetate-ethanol- 61 11 51
25% ammonium hydroxide
(50: 50: 10)
Heptane-chloroform-ethanol 24 II 51
(50:50: 10)
Ethyl acetate- 1,2-dichloro- 20 II 51
,P
a
ethane - 25% ammonium
hydroxide (80:20:10)
Cyclohexane-ethyl acetate- 42 (as Identification of 1,4-benzodiazepines 54
ethanol - 25% ammonia benzophenone) in urine
(20:20: 7:O.l)
420 J A Y G. RUTGERS A N D CHARLES M . SHEARER
Table V I I
TLC D e t e c t i o n Methods f o r Lorazepam
Method -
Color Detect ion Reference
L i m i t (pg)
Quenching of Dark s p o t 0.1 3

Phosphorescence a g a i n s t a green
on a phosphor- background
escent p l a t e
under shortwave
W light
Conc. HCl s p r a y , B 1ue - v i o le t 0.02 3

h e a t , followed by
Bratton-Marshall
spray
Conc. I.r, SO,, Green 0.01 51

s p r a y , observe
under longwave
W light
Mercuric c h l o r i d e - Blue 52
diphenylamine
spray
* Data n o t a v a i l a b l e
LORAZEPAM 42 1
applied to glucuronide levels in urine from humans and ani-

mals.
An alternate technique is conversion of lorazepam
to its benzophenone derivative prior to injection. An
example of this is the procedure developed by Knowles et al.
( 6 0 ) for determination of free and conjugated lorazepam in
serum,urine and feces. The biological sample is adjusted
to pH 7 and extracted with ether to remove free lorazepam.
The aqueous phase is adjusted to pH 4 . 5 and incubated over-
night with$ -glucuronidase to cleave the conjugate. The
aqueous phase is again adjusted to pH 7 and extracted with
ether. Lorazepam is re-extracted into 12N sulfuric acid
and then heated at looo to form the benzophenone. The sam-
ples are dissolved in toluene prior to analysis. The limit
of detection was 0.01 ug/ml.
The conditions for various methods are given in
Table V I I I .
Gonnet ( 6 4 ) has developed an isocratic elution
technique for separating lorazepam from a series of other
benzodiazepines (medazepam, diazepam, nitrazapam, chloaze-
pate, oxazepam and chlordiazepoxide). Separations were
achieved on a 20 cm x 4 . 6 mm column packed with Lichrosorb
SI 6 0 , 5 micron, at a pressure of 1090 psi and a flow rate
of 2 . 6 ml/min. The mobile phase was dichloromethane-iso-
propanol ( 9 6 : 4 ) saturated with water.
de Silva (65) discusses the high performance liquid
chromatography of lorazepam and other benzodiazepines. A
reverse phase column (a Bondapak C - 1 8 ) was used with an
eluant consisting of methanol (500 ml); isopropanol (50 ml);
pH 3.25 1 M potassium phosphate buffer (1 ml): diluted to
1000 ml with distilled, deionized water. A normal phase
system ( 1 0 Partisil
~ silica gel) had an eluant of methylene
chloride (95):methanol (5). The reverse phase column was
the better.
Lorazepam can be separated (12) from its degrada-
tion products (see Section 4 ) by an eluant consisting of
acidic aqueous acetonitrile on a reverse phase column.
High performance liquid chromatography has been
used to analyze for benzodiazepines including lorazepam in
tissue ( 6 6 ) . An eluant of 60% methanol (v/v) in phosphate
buffer (pH 7.8) eluted lorazepam in 4 . 2 ml from a column of
Spherosorb-5-ODS (150 mm x 4 . 6 mm id).
Table V I I I
Gas Chromatographic Systems for Lorazepam
Injected Column Col- Carrier Column Detec- -

Ref.
As Packing -
umn Gas Temp. -
ort
Lor azepam 3% O V 1 7 on l m x 2mm Nitrogen a t 255°C Electron 55

Gas Chrom Q glass 42 ml/min capture
Lor a zepam 3% O V 1 7 on 4 ft x 4mm Argon-methane 240°C Electron 61
Gas Chrom Q boros i li- (90: 10) capture
( 60/80) cate glass 120 ml/min
e
N
Lor a z e pam 3% O V 1 7 on
Gas Chrom Q
2m x Pmm Helium
30 ml/min
280°C Mass Spectro-
meter
56
(100-120)
Lor a ze Pam 3% O V 1 7 on 4 f t x 4mm Argon-me t hane 230, Electron 62
Gas Chrom Q borosili- (90: 10) 210% capture
( 60/80) cate glass 75 ml/min
Trimethyl- 3% OV17 on 4 f t x 4rnm A r gon-me thane 2 10"C Electron 62
s i l y l deri- Gas Chrom Q boros i li- (90: 10) capture
v a t i v e of (100-120) cate glass 75 ml/min
Lor a zepam
Table V I I L ( c o n t i n u e d )
Injected Column Col- Carrier Column Detec- -

Ref.
As Packinq -umn Gas Temp. tor
M e t h y 1t r i - 3% OV17 on l m glass Nitrogen 320°C Flame 59

methyl- G a s Chrom Q 30 ml/min ionization
s i l y l deri- (100-120)
v a t i v e of
Lorazepam
glucuronide
Benzo- 3% O V 1 7 on 10 f t x Helium 2sooc E l e c t r on 60
phenone Chromosorb 2im s t a i n - 20 ml/min capture
w
N W (80/100) less s t e e l
Benzo- SE 30 2m g l a s s Nitrogen 240" C F 1a m e 53
phenone 40 ml/min ionization
Benxo- 6% QF-1 on 6 ft x H e 1ium 24OOC Electron 34
phenone Anakrom ABS 1/8" 40 ml/min capture
(80/90) stainless
steel
Lorazepam 3% OV-7 on 2m x 4mm Argon 25OOC Electron 63
Varaport 30 glass 80 ml/min capture
80/100
424 JAY G. RUTGERS A N D CHARLES M. SHEARER
8. References
1. Bellmy, L. J., "The Infra-Red Spectra of Complex

Molecules'1,2nd Ed., John Wiley & Sons, Inc., New
York 1964.
2. Hoffman, B., Wyeth Laboratories, Inc., personal
communication.
3. Seitzinger, R.W.T., Pharm. Weekbl., 110,1109 (1975).
4. Kuhnert-Brandstaetter, M., Kofler, A., and
Friednich-Sander, G., Sci. Pharm., 42, 234 (1974).
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6. Levillain, P., Bertucat, M . , and Perrot, B., Eur. J.
Med. Chem.-Chem. Therapeutica, 10,433 (1975).
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communic ation.
8. Bell, S.C., McCaully, R.J., Gochman, C., Childress,
S.J., and Gluckman, M.I., J. Med. Chem., 2, 457
(1.968).
9. DeAngelis, N.J., Wyeth Laboratories, Inc., personal
communic ation.
10. Sivieri, L., Wyeth Laboratories, Inc., personal
communication.
11. Kyriakopoulos, A.A., in "Pharmacokinetics of Psycho-
active Drugs", Edited by Gottschalk, L.A. and Merlis,
S., John Wiley and Sons, New York 1976.
12. Snodgrass-Pilla, C., Wyeth Laboratories, Inc., per-
sonal communication.
13. Bissinger, J.M., Wyeth Laboratories, Inc., personal
communication.
14. Bandoli, G. and Clemente, D.A., J. Chem. SOC.,
Perkin Trans., 11,413 (1976).
15. Davidson, I.E. and Smyth, W.F., Proc. SOC. Anal.
Chem., 9, 209 (1972).
16. Goldsmith, J.A., Jenkins, H.A., Grant, J., and Smyth,
W.F., Anal. Chem. Acta, 66, 427 (1973).
17. Hagel, R.B. and DebesisTE.M., ibid., 78,439
(1975).
18. Mueller, W. and Wollert, U., Arch. Pharmacol., 278,
301 (1973).
19. Mueller, W. and Wollert, U., ibid.,=, 229 (1973).
20. Mueller, W. and Wollert, U., ibid.,=, R68 (1974).
21. -
Mueller, W. and Wollert, U., ibid.,283, 6 7 (1974).
22. Mueller, W. and Wollert, U., ibid.,=, 17 (1975).
23. Mueller, W. and Wollert, U., Biochem. Pharmacol., 25,
141 (1976).
24. Mueller, W. and Wollert, U., ibid.,%, 147 (1976).
LORAZEPAM 425
25. S t e r n b a c h , L.H., K a i s e r S., and Reeder, E., J. Am.

Chem. SOC . , 82, 475 (1960).
26. B e l l , S.C., Sulkowski, T.S., Gochman, C., and
C h i l d r e s s , S.J., J. Org. Chem., 27, 562 (1962).
27. B e l l , S.C. and C h i l d r e s s , S.J., ibid., - 27, 1691
( 1962).
28. B e l l , S.C., U.S. P a t e n t 3,296,249, Jan. 3, 1967.
29. McCaully, R.J., U.S. P a t e n t 3,926,952, Dec. 16, 1975.
30. R u t g e r s , J.G., Wyeth L a b o r a t o r i e s , I n c . , unpublished
results.
31. Nudelman, A . , McCaully, R.J., and B e l l , S.C., J.
Pharm. S c i . , 2, 1880 (1974).
32. S c h i l l i n g s , R.T., S h r a d e r , S.R., and R u e l i u s , H.M.,
Arzneim.-Forsch., 21, 1059 (1971).
33. Greenblatt, D.J., S c h i l l i n g s , R.T., Kyriakopoulos,
A.A., Shader, R . I . , Sisenwine, S.F., Knowles, J.A.,
and R u e l i u s , H.W., Clin. Pharmacol. Ther., 20,
329 (1976).
34. E l l i o t , H.W., Nomof, N., Navarro, G., R u e l i u s , H.W.,
Knowles, J.A., and Comer, H.W., ibid., - 12, 468
(1971).
35. Chang, T.T.L., Kuhlman, C.F., S c h i l l i n g , R.T.,
Sisenwine, S.F., T i o , C.P., and R u e l i u s , H.W.,
E x p e r i e n t i a , 2, 653 (1973).
36. S c h i l l i n g s , R.T., Sisenwine, S.F., Schwartz, M.H.,
and R u e l i u s , H.W., Drug Metabolism and D i s p o s i t i o n ,
-
3 , 85 (1975).
37. S c h i l l i n g s , R.T., Sisenwine, S.F., and R u e l i u s , H.W.,
ibid., 5, 425 (1977).
38
39.
- E l l i o t , H.W., B r . J. Anesth., -
Lafarque, P., J. Eur. Toxicol., 6, 136 (1973).
48, 1017 (1976).
40. C u l l e n , L.F., S c h l e i f e r , A . , B r i n d l e , M.P., and

P a p a r i e l l o , G.J., Anal. Chem., - 47, 1936 (1975).
41. C l i f f o r d , J.M. and Smyth, W.F., Z. Anal. Chem., &,
149 ( 1 9 7 3 ) .
42. O e l s c h l a g e r , H. and Sengun, F.I., Pharmazie, 2,
770 (1974).
43. d e S i l v a , J.A.F., Bekersky, I., and Brooks, M.A., J.
Pharm. S c i . , 2, 1943 (1974).
44. Van Doorne, P . , Pharm. Weekbl., 110, 149 (1975).
45. Smyth, W.F., Smyth, M.R., Groves, J.A., and Tan, S.B.,
A n a l y s t , log, 4 9 7 ( 1 9 7 8 ) .
46. C u l l e n , L.F., B r i n d l e , M.P., and P a p a r i e l l o , G.J.,
J. Pharm. S c i . , 62, 1708 (1973).
47. C u l l e n , L.F., B r i n d l e , M.P., and P a p a r i e l l o , G . J . ,
Adv. Autom. Anal., Technicon I n t . Congress, 2,
9 (1972).
426 J A Y G. RUTGERS A N D CHARLES M. SHEARER
48. O e l s c h l a g e r , H. and Sengun, F.I., Chem. Ber., 108,

49.
3303 (1975).
S c h u l t z , C. and S c h u l t z , H., Arch. Tox.,
(1973).
z, 183
50. S c h u l t z , C. and S c h u l t z , H., Z. K l i n . Chem. Klin.

Biochem. , l0, 528 (1972).
51. Meeles, M.T.H.A., Dreijer-Van Der Glas, S.M., and
Kho, G.L., Pharm. Weekbl., 110, 492 (1975).
52. F l o u v a t , B., Roux, A., I r o n d e l l e , B., and Sanchez A . ,
Eur. J. T o x i c o l . , 8, 305 (1975).
53. Maier, R.D. and Wehr, K.H., Arch. T o x i c o l . , 32, 341
(1974).
54. Krugers Dagneaux, P.G.L.C. , Pharm. Weekbl. , 108,
1025 (1973).
55. Marucci, F., M u s s i n i , E . , A i r o l d i , L., G u a t a n i , A.,
G a r a t t i n i , S., J. Pharm. Pharmacol., &, 6 3 (1972).
56. F r i g e r i o , A . , Baker, K.M., and B e l v e d e r e , G., Anal.
Chem. , 45, 1846 (1973).
57. Forgione, A., F r i g e r i o , A., M a r t e l l i , P., Proc. I n t .
Symp. Gas Chromatogr. Mass Spectrom., 213 (1972).
58. F r i g e r i o , A., Boll. Chim. Farm., 111, 709 (1972).
59. Marucci, F. , B i a n c h i , R. , A i r o l d i , L. , Salmona, M.,
F a n e l l i , R., Chiabrando, C., F r i g e r i o , A . , M u s s i n i ,
E. , and G a r a t t i n i , S. , J. Chromatogr. , 1 0 7 , 2 8 5 (1975).
60. Knowles, J.A., Comer, W.H., and R u e l i u s , H.W.,
Arzneim.-Forsch. , 2 l , 1055 (1971).
61. d e S i l v a , J.A.F., Bekersky, I . , and P u g l i s i , C.V.,
J. Chromatogr. S c i . , 11, 547 (1973).
62. d e S i l v a , J.A.F., Bekersky, I . , P u g l i s i , C.V., Brooks,
M.A., and Weinfeld, R.E., Anal. Chem., 48, 10 (1976).
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65. d e S i l v a , J.A.F., S t r o j n g , N . , and P u g l i s i , C.V.,
Anal. L e t t e r s , g, 135 (1978).
66. O s s e l t e n , M.D., Hammond, M.D., and T w i t c h e t t , P.J.,
J. Pharm. Pharmacol., 29, 460 (1977).
METHOXSALEN
Mohammed A . Lou& and Mahmoud A . Hassan
1. Description 428
1.1. Nomenclature 428
1.2 Formulae 428
I . 4 Elemental Composition 429
1.5 Appearance, Color, Taste, Odor 429
2.2 Solubility 429
3. Isolation 435
4. Biosynthesis 437
5. Synthesis 437
6. Metabolism 440
7.1 Colorimetry 440
7.2 Spectrophotometry 441
7.3 Fluorimetry 441
7.4 TLC-Fluonmetry 441
7.5 Time- resolved Phosphorimetry 442
7.6 Electrophoresis 442
8. References 450
Copyright @ 1980 by Academic Rcss, Inc.

AU rights of reproduction in any form rexrved.
AnaIyTicfd Rofiles of Drug Substances, 9 427 ISBN: 0-12-260809-7
428 MOHAMMED A . LOUTFY A N D MAHMOUD A . HASSAN
1. Description
1.1 Nomenclature
1.11 Chemical Names
9-Methoxy-7H-furo 13 ,3-gl
111 benzopyran-?-one; 6 -Lactone
of 6-hydroxy-7-methoxy-5-benzo-
furanacryJi: acid; 8-Methoxy
[ furano-3,2, : 6,7 - coumarin];
8-Methoxy-4, 5 :
' 6,7-furano-
coumar in.
1.12 Generic Names
Ammoidin,Xanthotoxin, 8-Methoxy-
psoralen, Methoxsalen.
1.13 Trade Names
Meladinin, Meloxine, 8-MOPr 8-MP,

Methoxa-Dome, New-Meladinin,
Oxsoralen.
1.2 Formulae
1.21 Empirical
C12H804
1.22 Structural
METHOXSALEN 429
1.23 Wiswesser Line Notation

T C566 DO LV OJ BOI
1.3 Molecular Weight

216.18
1.4 Elemental Composition
C, 66.67%; H, 3.73%; 0, 29.60%.
1.5 Appearance, Color, Taste, odor
Silky fluffy needles or long rhombic
prisms, white to cream-colored, bitter
taste followed by tingling sensation,
odorless.
2.11 X-Ray Diffraction
The crystallographic properties of
methoxsalen has been reported(1).
2.12 Melting Point
Methoxsalen melts between 143 and
148O(2).
2.2 Solubilitv
Methoxsalen is practically insoluble in
cold water, sparingly soluble in boiling
water and freely soluble in chloroform.
It is soluble in boiling alcohol, ace-
tone, and acetic acid. It is also
soluble in aqueous alkalis with ring
cleavage, reconstitution occurs upon
neutralisation.
430 MOHAMMED A. LOUTFY AND MAHMOUD A . HASSAN
OCH3 OCH3
2.3 Identification
a- An alcoholic solution of methoxsalen
gives a deep-violet color with 8-
amino-5-hydroxy-2-methyl-furo-4; 5
6, 7 - chromone, in the presence of
alkali (3). The test is sensitive
to 0.02 mg of the drug.
b- The UV absorption spectrum of a 1 in
125,000 solution in alcohol exhibits
maxima and minima at the same wave-
lengths as that of a similar solution
of USP Methoxsalen Reference Standard,
concomitantly measured (4) .
c- Dissolve, by heating, about 10 mg in
5 ml of diluted nitric acid, the
solution turns yellow. Render the
solution alkaline with sodium hydro-
xide, the solution turns brown (4).
2.4 SDectral ProDerties
Methoxsalen exhibits a character-
istic UV Spectrum (Fig.l), in 95%
ethanol, with the following
electronic absorption bands ( 5 ) :
1Max Log E A min Log E

219 4.48 232 4.23
245 4.44s. 262 4.23s.
249 4.46 276 3.90
262 4 -23s.
301 4.16
s.= Shoulder
METHOXSALEN 43 1
Other UV spectral data for metho-

xsalen and other psoralens have
been reported (6-9).
The IR spectrum of methoxsalen has
been determined in nujol on a
Unicam SP - 200 (Fig.2). The
structural assignments have been
correlated with the following
band frequencies:
Frequency Cm-1 Assignment
3110, 3080, 3040 CH
1705 C=O ( a-pyrone)
1620 C=C (aromatic and
a-pyrone)
1580 , 1540 C=C (aromatic)
1150 c-0-c
875 Furan ring
800 Isolated H
(penta substi-
tuted aromatic)
These findings are in agreement
with reported data (5,9). Other
finger print bands characteristic
of methoxsalen are: 1400, 1380,
1340, 1300, 1220, 1180, 1120,
1100, 1020, 1000,820 and 760 cm-l
2.43 Nuclear Elagnetic Resonance
Spectrum
The proton magnetic resonance
spectra of methoxsalen and other
furocoumarins have been investi-
gated (5,9,10,11). A typical PMR
spectrum of methoxsalen is shown
in Fig.3. The sample was dissolv-
ed in CDC13 and the spectrum was
recorded on a T60A NMR spectro-
meter, using tetramethylsilane as
a reference standard.
0
0
. w
0
0
0
, r l
L
I . I
I . . . . I . , . , I . . . . ( . _ , I , I , . L . I , , . . I . , , ,
8.0 7.0 6.0 5.0 4.0 3.0 2.0
PARTS PEIt M I L L I O N , 6
Fig. 3 - PMR swtm of Methoxsalen and TMS, tetrartgthylsilane

in deutrated chlorofom.
METHOXSALEN 435
The PMR d a t a of m e t h o x s a l e n a n d
i t s b i o l o g i c a l l y i n a c t i v e isomer,
b e r g a p t e n ( 5 - m e t h o x y p s o r a l e n ) are
i l l u s t r a t e d i n t a b l e I.
T a b l e I: PMR c h a r a c t e r i s t i c o f M e t h o x s a l e n
and Bergapten.
Chemical s h i f t s ( 6 )
5-ocH 8 a 3 3-H 4-H 5-H 8-H 4'-H 5'-H
sa3 s dk d s d d d
Metho- - 4.35 6.38 7.75 7.33 - 6.83 7.66

xsalen
Bergap
ten 4.25 - 6.23 8.10 - 7.25 7.03 7.53
A l s o o t h e r PMR s t u d i e s o n metho-
x s a l e n and a n a l o g u e s have been
published (5,9,10).
2.44 Mass S p e c t r u m
The mass s p e c t r u m of m e t h o x s a l e n ,
o b t a i n e d by c o n v e n t i o n a l e l e c t r o n -
i m p a c t i o n i z a t i o n , shows a mole-
c u l a r i o n M+ a t m / e 216.04 (12,131.
The M+ i o n p e a k i s t h e b a s e p e a k
(Fig.4). The f r a g m e n t a t i o n
p a t t e r n s of m e t h o x s a l e n a n d o t h e r
p s o r a l e n d e r i v a t i v e s have been
reported (5,13,14 1 .
3. Isolation
Fahmy and Abu-Shady ( 1 5 - 1 7 ) h a v e r e p o r t e d t h e

i s o l a t i o n of m e t h o x s a l e n f r o m t h e f r u i t s o f
E g y p t i a n , u m b e l l i f e r o u s Ammi r n a j u s ( L )
plant. The d r i e d powdered Ammi m a j u s ( L ) (Fam.
L
..
'0
-W
.N
~0
-r+
.N
---! 0
N
0
03
c
0
W
4
0
V
d d
I
0
W
m
I
2
i;
L
0
i"
u)
N
N
d
'in L l
0
u)
4
N
0 0 0 0 0 ' 5 -
o r n w w ~
d
METHOXSALEN 431
umbelliferae) fruits are exhaustively extracbd

with petroleum ether (60-80°). The deep green
extract is concentrated and kept for overnight
and the greenish resinous crystalline deposit
is filtered and crystallised from ethanol. The
yield is 0.25%. The isolation of methoxsalen
and other furocoumarins have been also des-
cribed by other co-workers (18-26).
4. Biosynthesis
The exact biogenetic pathway leading to the
formation of methoxsalen is uncertain. One of
the problems still to be resolved concerns the
mechanism by which ?near furocoumarins are
formed from their 2 , 3’-dihydro-2’- isopropyl
counterparts (27). It seems certain that
marmesin is directly involved and tracer ex-
periments indicate that (+) - ( S ) - form, rather
than the ( - 1 - ( R ) - (nodakenetin), is pre-
ferentially incorporated (28,29). The sequence
of further substitution of furanocoumarins has
been studied by Caporale, et -- a1 (30, 31). The
most recent suggestion is that further sub-
stitution,hydroxylation followed by methylation,
occurs after furan ring formation. Scheme I
illustrates the formation of methoxsalen start-
ing from trans-cinnamic acid.
5. Synthesis
The synthesis of methoxsalen has been achieved
by two main routes.
Route I: Benzofuraneroute (32-34), which
involves the hydrogenation of 6,7-dihydroxy-
benzofuran (I) or 6,7-dihydroxycoumaran-3-one
(11) to afford 6,7-dihydroxycoumaran (111) .
I11 was then converted, by Pechman? reaction
with malic acid, to 8-hydroxy-4’,5 -dihydro-
6,7,3’,2’-f~ran0~0~rnarin (IV). Methylation of
IV followed by dehydrogenation gives metho-
xsalen ( V ) .
Route 11: Umbelliferore route (35), involves
the use of a coumarin ring instead of a benzo-
furan derivative. Although it is an improve-
ment on the previous route, it gives a low
438 MOHAMMED A. LOUTFY A N D MAHMOUD A. HASSAN
-
Scheme I.
Biosynthesis of methoxsalen from trans-cinnamic
acid.
HO glucose
METHOXSALEN 439
Route I (Benzofuran route):
OH OH
I I1 I11
R o u t e I1 ( U m b e l l i f e r o n e r o u t e ) :
HO
OH
I
OCH3
I1
0
-
BrCH2COOEt
RH2C
nCH
VI
v
R = COOEt
QCH3
440 MOHAMMED A. LOUTFY A N D MAHMOUD A . HASSAN
yield (33%).In this route, 7-hydroxy-8-methoxy-

coumarin (111) is the key intermediate for the
synthesis of methoxsalen. 111 is converted
into the 6-formyl derivative which in turn is
cyclised, using ethyl bromoacetate to effect
ring closure, to VII. This approach is attrac-
tive in that an umbelliferone is an intermedi-
ate rather than 6-hydroxycoumaran. Umbellifero-
nes can be more easily synthesised and are more
readily available from natural sources.
Other methods for the synthesis of methoxsalen
and analogues are also reported (19,36-39).
6. Metabolism
Very little is known about the metabolism of
photosensitising furocoumarins (40). By
contrast no data are so far available about the
metabolismof methoxsalen. However studies have
been performed in mice and human volunteers on
the absorption, metabolism and excretion of
psoralen and trimethylpsoralen (41,42).
7.1. Colorimetry
Colorimetric determination of methoxsalen
in the Ammi majus fruits and tablet
formulation has been described (43,44).
The method involves the addition of 0.5M
potassiumhydroxide solution to the drug,
after 30 minutes, diazotized sulfanilic
acid solution (a 1:l - mixture of 0.64%
sulfanilic acid solution in dilute
hydrochloric acid and 0.4% aqueous sodium
nitrite) is added. The extinction of
the solution is then measured and referr-
ed to acalbbration curve prepared by
a standard methoxsalen solution.
Schonberg, -
et -a1 (3) have described
another colorimetric procedure for the
determination of methoxsalen and other
psoralens. The method involves the
addition of 1 ml of a 0.1% solution of
8-amino-5-hydroxy-2-methylfuro (4’,5’,
6,7) chromone to 2 ml of an alcoholic
solution of the psoralen. The reaction
METHOXSALEN 441
mixture is made alkaline with 10 ml of a

buffer solution (pH 11.61, a violet color
is gradually developed and can be measured.
7.2 Spectrophotometry
Methoxsalen and bergapten have been
determined in the plant material, by
measuring the absorbance of the chloro-
form extract at 300 and 311 nm, respec-
tively (45). Fedorin and Georgrievskii
(46) have described a spectrophotometric
procedure for the estimation of metho-
xsalen and bergapten in Beroxan prepa-
rations. Yeargers and Auqenstein (47)
have described the absorption and emission
spectra of methoxsalen and psoralen in
powders and in solutions.
_ -a1 (48 ) have developed
Chakrabarti , et
a spectrophotometric method for the
estimation of methoxsalen in the plasma.
The method is based on extraction of the
plasma, acidified with hydrochloric acid,
with benzene-ethyl acetate mixture (9:l)
and the solvent is evaporated, to dryness.
The residue is dissolved in xylene and
the absorbance is measured at 300 nm.
The drug extracted from plasma is charac-
terised by TLC, UV absorption spectrum
and GC/MS fragmentation pattern. The
U.S.P. method for the assay of metho-
xsalen is spectrophotometric (4) one.
7.3 Fluorimetry
The assay of methoxsalen and bergapten
in Beroxan preparations has been achieved
fluorimetrically (46).
7.4 TLC - Fluorimetry
Chakrabarti, _
et -
a1 (49) have described
a rapid and sensitive method for the
determination of methoxsalen in the
plasma. The plasma samples are acidified
with hydrochloric acid and heated in a
boiling water-bath to release the plasma-
442 MOHAMMED A. LOUTFY AND MAHMOUD A. HASSAN
bound drug. The drug is extracted by

a solvent system (benzene-ethylacetate,
9:l). The extract is evaporated to dry-
ness and the residue is dissolved in
methylene chloride and is spotted on
thin layer plates and developed with the
same solvent system. The plates are
visualized under UV light (320-400 nm)
and scanned. The method is sensitive up
to 2 0 ng of methoxsalen. The overall
recovery of the drug from the plasma is
84%. The identity of the recovered drug
was confirmed by GC/MS (Fig.5).
7.5 Time-resolved phosphorimetry
Phosphorescence spectra for methoxsalen
and psgralen have been recorded for 300°K
and 77 K. The peaks for 300° fluoresceme
excitation spectra obtained from a "front
face" cell agreed with peaks in the ab-
sorption spectra, when correlations have
been made for the output of the exciting
lamp. At 770K the phosphorescence life-
times vary from 0.4 to 1.1 seconds (47).
7.6 ElectroDhoresis
Berlingozzi and Parrini ( 5 0 ) have described
a method for separation of methoxsalen
from other coumarin derivatives by cir-
cular paper electrophoresis.The compounds
are first subjected to circular paper
chromatography in water-acetic acid-
butylene glycol (86:10:6). The Rf values
found in strips, complete circle, and a
9 0 0 sector of circle, and the color of
fluorescence in Wood's hight are, res-
pectively 0.602, 0.779 and 0.786, light
green colour for methoxsalen. The
electrophoretic experiments are conduc-
ted in a buffer (pH9) of sodium barbital,
sodium acetate, potassium oxalate,O.l N
hydrochloric acid, and water. The
travelling distances for methoxsalen and
other coumarins are reported. A better
separation has been obtained by means of
electrophoresis than chromagography.
METHOXSALEN 443
7.7. Chromatography
7.71 Paper chromatography
Methoxsalen and other psoralens
have been separated by paper
chromatography. The following
solvent systems have been used,
in a unidimensional ascendinq
method (51): water; water-methy-
ethylketone (17:3 ) ; water-ethanol-
methylethylketone ( 1 5 : 3 : 2 ) i water-
formamide-methylethyl ketone
(9:3:2)); formarnide-ethylacetate-
water ( 8 : 5 : 3 ) and butanol-acetic-
acid-water ( 4 :1 :1) .
Grujie-Vasie- ( 5 2 ) has described
a paper chromatographic separation
of methoxsalen and some psoralens,
using the following solvent
systems: water-saturated ammonium
hydroxide; butanol-ethanol-
concentrated ammonium hydroxide-
water ( 4 : 4 : 1 : 1 ) : and propanol-
water ( 9 0 : 1 0 , 8 0 : 2 0 , 7 0 : 3 0 , and
20:80).
Beyrich ( 4 5 ) has reported a method

for the quantitative determination
of methoxsalen, bergapten, and
imperatorin in the dried plant
material. The powdered plant is
extracted with chloroform in a
Soxhlet, the extract is evaporated
to dryness and the residue is
dissolved in toluene. An aliquot
(10-100 1-11)is applied to a paper
imprignated with .dimethylformamide
and developed with heptane-
benzene mixture ( 4 : 2 ) . The sepa-
rated methoxsalen is eluted with
chloroform and determined spectro-
photometrically. Heptane-benzene
mixture ( 4 : l and 7 : 3 ) has been
also used ( 5 3 , 5 4 ) on paper impriq-
nated with formamide, for the
detection of methoxsalen and other
psoralens. The detection has been

achieved by their fluorescence
alone, and after treatment with
0.5N ethanolic potassium hydroxide,
by the diazoreaction, and by the
use of Emerson phenazone-potassium
ferricyanide reagent.
Lutomski, -
et -
a1 ( 4 3 ) have des-
cribed a paper chromatographic
method for quantitation of metho-
xsalen in Ammi majus fruits and
__.____
their preparations. The powdered

sample is extracted with methanol
in a Soxhlet and the extract is
applied to Whatman No.1 paper,
previously imprignated with
dimethylformamide-ethanol mixture
(2:3 ) . The chromatogram is then
developed with heptane-benzene
(7:3 ) by the descending-solvent
technique. The spot of metho-
xsalen is eluted with ethanol and
then determined colorimetrically.
The isolation and detection of
methoxsalen from other coumarins,
by TLC methods, have been reported
(55,56). The chromatoplates are
prepared in the usual manner ( 5 7 )
using silica gel G as the adsorbant.
Development has been carried out
in different solvent systems
(Table 11). The developed plates
are dried and then observed under
UV light (yellowish green). The
plates are finally sprayed with a
0.5% Iodine-potassium iodide solu-
tion, the colors are observed in
daylight (reddish brick-red) and
under UV light, and the Rf values
are determined (Table 11).
METHOXSALEN 445
Table I1
Solvent system Rf
a- Toluene-ethylformate- 0.65
formic acid
( 5 : 4 : 1)
b- Benzene-ethylacetate 0.39
( 9 : 1 )
c- Benzene-acetone 0.71
( 9 : 1 )
A better resolution has been
effected by two-dimensional chro-
matography on silica gel thin
layers (Fig.61, and a l s o by the
use of wedge-shaped ( 5 6 ) chromato-
gram (Fig.7).
7.73 Gas Liquid Chromatography
Stewart, and Shyluck ( 5 8 ) have
developed a GLC method for the
separation of methoxsalen and
certain coumarins. The relative
retention time of methoxsalen,
relative to herniarin, is 3 . 6
under the following conditions:
SEG column of copper tubing ( 0 . 6 1
m X 5mm 0.d.) packed with succin-
ate-ethyleneglycol polyester on a
support of 6 0 - 8 0 mesh chromosorb
W; column temperature, 208O;
helium flow rate, 100 ml/min;
injector temperature, 2 4 5 0 ; and
recorder sensitivity, 1 mv.
7.74 High Performance Liquid
Chromatoqraphy
A HPLC method has been reported
( 5 9 ) for the estimation of metho-
xsalen in the blood.
Another method for its determin-
ation in tablet dosage form has
been also described ( 6 0 ) . It
Fig. 5
No. 114
Cursory Nos m/e

4 (a1 4 Ib)
-
-Im! n " '
c
A - - 1
Methoxsalen (a and b ) .
Fig. 7
Fig. 6
M*sional TLC Wedge-shaped TLC

shminq a-Methoxsalen showing, a-*tho-
and b-&rgapten. xsalen and b-Bergapten.
METHOXSALEN 447
i n v o l v e s t h e u s e of a l o w volume
p o s i t i v e d i s p l a c e m e n t pump, u n i -
v e r s a l i n j e c t o r , and a s i n g l e
w a v e l e n g t h d e t e c t o r (254 nm) A .
uBondapak C18 s t a i n l e s s s t e e l
column ( 3 . 8 mm x 3 0 c m ) i s u s e d .
The column t e m p e r a t u r e i s a m b i e n t ,
t h e o p t i c a l d e n s i t y i s set a t
0.05 a . u . f . s . , t h e recorderis set
a t 1 0 mv f u l l s c a l e , and t h e
c h a r t s p e e d i s 0.25 c m p e r m i n u t e .
The s o l v e n t ( m o b i l e p h a s e ) i s
methanol-water m i x t u r e ( 6 0 : 4 0 ) ,
and t h e f l o w r a t e i s c o n t r o l l e d
a t 2 m l p e r minute. A t y p i c a l
chromatogram o f m e t h o x s a l e n , u s i n g
Khellin a s an i n t e r n a l s t a n d a r d ,
i s shown i n F i g . 8 . The r e c o v e r y
o f m e t h o x s a l e n h a s been f o u n d t o
b e 86-95% of t h e s t a t e d amount.
7.8 PMR S D e c t r o m e t r v
L o u t f y and Hassan (11) h a v e re-

p o r t e d a r a p i d and s i m p l e PMR
p r o c e d u r e f o r t h e e s t i m a t i o n of
m e t h o x s a l e n i n b u l k d r u g and 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
method i s b a s e d on t h e integration
of t h e t h r e e methyl p r o t o n s
s i n g l e t of t h e 8-methoxy g r o u p o f
m e t h o x s a l e n a p p e a r i n g a t 4.35 ppn.
Acetanilide, exihibiting three
methyl p r o t o n s s i n g l e t a t 2.16
ppm, h a s been employed as a n
.
i n t e r n a l s t a n d a r d ( F i g . 9 ) Etha-
n o l - f r e e c h l o r o f o r m h a s been u s e d
a s a s o l v e n t i n t h e a s s a y . The
method i s a c c u r a t e , w i t h a s t a n d -
a r d d e v i a t i o n of 2 2 . 4 , and a n
a v e r a g e r e c o v e r y of 9 8 . 3 % , i n
t h e t a b l e t d o s a g e form. Themethod
proved t o be r e l i a b l e f o r t h e
d e t e c t i o n of t h e b i o l o g i c a l l y
i n a c t i v e isomer, b e r g a p t e n . T h i s
i s a t t r i b u t e d t o t h e presence of
4 - H p r o t o n d o u b l e t of b e r g a p t e n
a p p e a r i n g a t 8 . 1 ppm. ( F i g . 1 0 )
TMS
L8.C
Fig. 9
7.0 6.0
I
5.0 PPM 1 4.0
PARTS PER MILLION,
- PMR Spectrum of Wthmsalen,

6
I
3.0
.
I2.0
I
l . . I . . I . . . . I . . . . I . . . . 1 . . . . 1 . . . . 1 , . . .
Acetanilide and TMS in CL?C13.

$ 0
449
This finding has contributed

greatly to the specificity of the
met hod.
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--
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METHOXSALEN 45 1
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c
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METHOXSALEN 453
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54. T. Beyrich and H. Poser, -
J. Chromat., 32,
87 (1968).
55. E.A. Abu-Mustafa, B.A.H. El-Tawil, and M.B.
E. Fayez, Phytochemistry, 3, 701 (1964).
56. F.M. Abdel-Hav, E.A. Abu-Mustafa, B.A.H.
El-Tawil , and-M. B. E. Fayez , Planta Medica ,
-
13, 91 (1965).
57. E. Stahl , "Thin Layer Chromatography",

G. Allen and Unwin Ltd., (London), 1969.
58. A.B. Stewart and J.P. Shyluck, -
Anal. -
Chem.,
-
3 4 , 1058 (1962).
59. C.V. Puglisi, A.F. de Silva, and J.C. Yeyer;

- -
Anal. 10, 39 (1977).
Lett., -
60. A.H. Hikal, A.R.M. Morad, and S. P1-Houfy,

Chromatoqraphia (in press).
NADOLOL
Lidia Slusarek and Klaus Florey
1. Introduction 456
1 . 1 History 456
2. Synthesis 456
3.2 Solid State Properties 470
3.3 Racemate Composition 47 5
3.4 Solution Data 416
4. Analytical Tests and Methods 477
4.2 Identification Tests 477
4.4 Titrimetric Methods 478
4.5 Gas Chromatography/Mass Spectrometry 479
5.1 Solid State Stability 480
5.2 Solution Stability 48 1
6. Analysis of Body Fluids 48 1
8. References 483
Copynght Q 1980 by Acackmic F’ress. Inc.

Analytical Profiles of Drug Substances, 9 455 All nghts of reproduction m any form reserved.
ISBN: 0-12-260809-7
456 LIDIA SLUSAREK AND KLAUS FLOREY
1. Introduction
1.1 History
Nadolol, a "B-blocking" antiarrhythmic
agent, was synthesized,' tested2 and developed in
the laboratories of the Squibb Institute for
Medical Research.
1.2Name, Formula, Molecular Weight
Nadolol (SQ 11,725) is 2,3-cis-1,2,3,4-
tetrahydro-5-(2-hydroxy-3-(tert-butylam~no~propoxy)-
2,3-naphthalenediol; CAS:42200-33-9.
CH3
I
OCH -CH-CH2-NH-C-CH3
I
bH CH 3
17H27N04 Molecular Weight 309.41
1.3Appearance, Color, Odor

Nadolol is a white to off-white
crystalline, odorless powder.
2. Synthesis
The multistep synthesis of nadolol is presented
in Figure 1: 5,8-Dihydronaphthol (1) via its
acetyl derivative (2) is converted to cis-5,6,7,8-
tetrahydro-l16,7-naphthalene trio1 (3). This in
turn is converted with or without intermediate
formation of the acetonide (4) to 2,3-cisI 1,2,3,4-
tetrahydro-5-(2,3-epoxypropoxy)-2,3-naphthalene
diol ( 5 ) which forms nadolol ( 6 ) on the addition of
tertiary butylamine. For details, see reference 1.
It can also be prepared by attaching t-butylamine
to 5,8 dihydro-l-(2,3-epoxypropoxy) naphthalene (7)
to form (8) and subsequent oxidation of the double
bond and resolution to obtain nadolol ( 6 v f 4
NADOLOL 451
Figure 1
Synthetic Pathways to Nadolol
(1)
OH
[wj OCOCH3
J
r 1
L OH OH
(4) (3)
HO
- HO I
CH
l 3
I OCH -CH-CH2-NH-C-CH3
2 1 I
OH
CH3
(5) (6)
I
OCH2 -CH-CH2
\/
0
I 2 3 5 6 7 6 9 D II 12 13 I5
WAVELENGTH IMICRONSI
Sq 11,725
Curve 70207
Mineral Oil Mu13
Figure 2. IR Spectrum of Nadolol, Mineral Oil Mull.

Instrument: Perkin Elmer Model 21
NADOLOL 459
The infrared spectrum of nadolol
taken as a mineral oil mull is shown on Figure 2.
The following assignments have been made for
structurally significant bands:'
Frequency (cm-l) Assignment
3510 sharp 0 - H stretch
3280 broad OH stretch
1570 aromatic ring C = C stretch
1260 =c-0 stretch of
1240) aromatic ether
1090) C - 0 stretch of
1060 hyd roxy1s
An infrared assay has been developed5
for the determination of the racemates of nadolol
(Section 3.3).
3.12 Nuclear Magnetic Resonance Spectra
Figures 3 and 4 show the 100 MHz
nuclear magnetic resonance spectra of nadolol and
its corresponding D20 exchange in dimethyl
sulfoxide - d ~ .The
~ proton assignments are listed
in Table I.
Table I
Figure 3. NMR Spectrum of Nadolol in DMSO-d6. Instrument: Varian XL-100-15
Internal Standard: Tetramethylsilane
NADOLOL 46 1
Table I (continued)
T Value*
Proton P o s i t i o n ( C o u p l i n g C o n s t a n t , J , i n Hz)
1 3.3211 ( 8 . 0 Hz)
2 3 . 0 0 t (8.0 Hz)
3 3.24d ( 8 . 0 Hz)
4 (3 protons) 5.53
5 6.75
6 7.28m
6'; 6'' 7.28m
7 6.15b
7' 6.15b
8 8.97
*d-doublet; t-triplet; m-multiplet, b-broad
An i n s p e c t i o n o f t h e D20 e x c h a n g e
s p e c t r u m ( F i g u r e 4 ) shows d i s a p p e a r a n c e of t w o
r e s o n a n c e s a t ~ 5 . 5 3a n d ~ 6 . 7 5 . They c o r r e s p o n d t o
t h e four interchangeable protons: t h r e e hydroxyls
a n d o n e NH p r o t o n .
The C-13 NMR d a t a of n a d o l o l i n
d i m e t h y l s u l f o x i d e -dg ( F i g u r e 5 ) and i t s t e t r a -
b e n z o a t e d e r i v a t i v e i n CDC13 ( F i g u r e 6 ) a r e
compared i n Table II.6
T a b l e I1
C-13 NMR P a t t e r n of Nadolol a n d i t s

Tetrabenzoate Derivative
RO
OCH2-CH-CH2-N-C ( CH3)
I I
OR R
t",
Figure 4. NMR Spectrum of Nadolol in DMSO-d6, D20 exchange.

Instrument: Varian XL-100-15
Internal Stardard: Tetramethylsilane
i I/ L
40
Figure 5. C-13 NMR Spectrum of Nadolol in DMSO-d6.

Instrument: Varian XL-100-15, operated at 25.2 MHz
I
1""""'d"'
Figure 6. C-13 NMR Spectrum of Tetrabenzoate Derivative of Nadolol in CDC13.

Instrument: Varian XL-100-15, operated at 25.2 MHz.
NADOLOL 465
Table I1 (continued)
Carbon Nadolol Tetrabenzoate

R=H R=COC 6H5
Chemical Shifts, ppm.
c-1 34.38 31.04, 31.27

c-2 67.82 69.86**
c-3 67.82 71.19**
c-4 28.81 26.60, 26.77
C-4a 122.94 121.12
c-5 156.13 155.37
C- 6 107.91 107.91
c-7 125.92** n.a.
C- 8 120.71"" 121.46
C- 8a 135.49 138.70
OCH2 70.33 65.82, 66.23
CHO 68.98 69.10, 69.21
CH2N 45.25 47.47, 47.76
*C (CH3) 49.39 56.82
C (*CH333 28.81 28.76
* - indicates to which carbon the shift is

ascribed.
** - these carbons may be interchanged.
n.a. - not assigned.
The chemical shift assignments are
made on the basis of non-decoupled spectra and
known substituent effects. The data of the tetra-
benzoate derivative show double resonances for side
chain carbons (OCH2CH(OR)CH2N) as well as the tetra-
hydro carbons (>CH2). This can be ascribed to the
presence of two racemates A and B in nadolol
(see Sec. 3.3).
3.13
Mass Spectra
The low resolution mass spectrum of
nadolol is shown on Figure 7. The high resolution
mass spectrum yields a molecular ion at m/e 309.1956
with the formula C1 H27NO4 consistent with the
assigned structure.7
Typical of compounds containing
-
t-butyl groups is the loss of 15 a.m.u. from the
t 19.25
1919s
1982
1992
19h2
1922 d
0
d
I3192 0
a
la
5g I z
u-4
1
991, 0
19h I k
4J
52 1
a19 1
1
98
1
99
5h
52
1 5
m m m m m m m m m m m
m r n r n ~ t ~ m ~ m m -
3
hlISN31NI 3 A I l V 1 3 t l
NADOLOL 467
molecular ion to yield a peak at m/e 294. The loss

of 44 a.m.u. to give the peak at m/e 265 (formula
C15H23N03) is interpreted as the elimination of
CH2 = C H - OH from the tetrahydronaphthalenediol
portion of the molecule (see schematic below).
m/e 309 m/e 265+CH2=CH-OH

Fragmentation of the aliphatic side
chain yields ions at m/e 252, 222 and 180, the
latter two accompanied by the proton transfer shown
below:
OCH -CH-CH2-NH-C (CH3)

2 1
OH
Nadolol
/ \
\
I'Ofq
HO
HO
+
OCH CHCH2NH OH
21
OH
m/e 252 m/e 180
HO
+
OCH CH
21
0
m/e 222
468 LIDlA SLUSAREK AND KLAUS FLOREY
A fragment ion at m/e 116, corres-

ponding to C6H14N0, can arise from the side chain
cleavage depicted below:
HO
I
m/e 116
OCH2-\- CHCH2NHC ( CH3)
I
OH

The ultraviolet spectrum of nadolol
in methanol' is shown on Figure '8. It depicts a
shoulder at 218 nm (concentration 0.017 mg/ml) and
two well-defined peaks at 270 and 278 nm (concen-
tration 0.171 mg/ml). The table below lists the
absorbances in methanol:
1%
-
X nm Elcm
219 (shoulder) %275
270 37.5
278 39.1
Inspection of this table reveals
that the UV absorbance of nadolol is very low. It
should also be noted that an appropriate blank
correction would be necessary for the true
value at 219 nm (see also 4.31).
lcm
3.15Fluorescence Spectroscopx
Nadolol has no native fluorescence
in 95% ethanol, aqueous 0.1N sodium hydroxide or
0.1N hydrochloric acid. It can, however, be
induced by heating samples at 100°C. in concen-
trated sulfuric acid (excitation maximum at 260 nm;
emission maximum at 400 nm).' Nevertheless, this
approach has not been utilized for analytical
purposes because of interferences and variations in
fluorescence.9
In
rl
w
0
3.2 Solid State Properties

3.21 Melting Range
Nadolol : 124-136; C.’
Racemate A: 134-1360 C. 1 0
Racemate B: 148-157 C.”
Differential Thermal Analysis (DTA)
3.22
Nadolol shows a single endotherm at
about 130° C. ’’
The twooracemic com ounds A and B
showed endotherms at 140 C. and 1468 C.,”
respectively, which correlate with their melting
ranges (see Section 3.21). The differential thermal
analysis curves were recorded on a DuPont 900
Thermoanalyzer with a temperature rise of 15O C. per
minute.
Differential Scanning Calorimetry
3.23
(DSC)
Attempts were made to determine the
purity of nadolol by differential scanning
calorimetry.” However, the results obtained were
difficult to interpret due to the complex melting
behavior exhibited by racemic mixtures.
Polymorphism
3.24
No polymorphism has been reported
for nadolol. However, it was observed” that an
amorphous form of nadolol can be obtained by lyo-
philizing an aqueous solution of the compound. The
amorphous nature of the lyophilate was ascertained
through x-ray and thermal analysis. The amorphous
form exhibited diffused melting behavior at 50° C.
and was at least ten times as soluble in water at
room temperature as the crystalline form.
X-Ray Powder Diffraction
3.25
The x-ray powder diffraction patterns
of nadolol (Table 111, Figure 9) and racemic-
compounds A (Figure 10) and B (Figure 11) are
presented. ’
The diffraction patterns of the A and
B racemates are quite different as can be seen from
Figures 10 and 11. Consequently an X-ray powder
diffraction method was developed‘ to measure the
percentages of racemate A and racemate B in samples
of nadolol. The range of concentrations measurable
N A DO LO L 47 1
by this technique is 30% to 70% with an accuracy

+ 5% (see Section 3.3).
of -
Table I11
X-Ray Powder Diffraction Pattern of Nadolol*
28 (Deg.) d (Ao) 1/10

6.32 13.98 1.000
7.34 12.04 0.433
10.57 8.37 0.123
13.12 6.75 0.303
15.07 5.88 0.293
15.33 5.78 0.316
15.67 5.66 0.346
17.03 5.21 0.349
18.64 4.76 0.437
19.41 4.57 0.170
19.83 4.48 0.140
21.36 4.16 0.358
21.79 4.08 0.584
22.38 3.97 0.434
22.72 3.91 0.508
23.32 3.81 0.436
24.08 3.70 0.222
24.93 3.57 0.153
26.12 3-41 0.125
27.06 3.30 0.117
28.76 3.10 0.200
30.29 2.95 0.133
30.80 2.90 0.107
37.85 2.38 0.094
-
Twice the angle of incidence or
"28
reflection.
d - Interplanar distance.
1/10 - Relative peak intensity based on
highest intensity as 1.000
3.26
Single Crystal X-Ray Diffraction
Single crystal x-ray diffraction
data of the hydrobromide-salt of racemate A have
been collected.'4 Crystals of the hydrobromide
salt were large, well formed rods of the triclini
system. The unit cell dimensions were a = 7.822 1,
b = 12.535 8, c = 9.712 8, a = 101.80,B = 93.10,
6 = 89.2O. There are two molecules in the unit
cell, centrosymmetrically related.
I0
5
N
Figure 9. X-Ray Powder Diffraction Pattern of Nadolol.

Instrument: Phillips 120-101-11
5
W
Figure 10. X-Ray Powder Diffraction Pattern of Racemate A of Nadolol.

Figure 11. X-Ray Powder Diffraction Pattern of Racemate B of Nadolol.
NADOLOL 475
3.3 Racemate Composition

Nadolol consists of two sets of enantio-
mers. They are present as two racemic compounds:
racemate A and racemate B.I4
The following table illustrates the
relative optical activity:
Table IV
Enantiomers of Nadolol
Optical Rotation -
Racemate
The composition of the racemates can be

determined by infrared spectroscopy of mineral oil
mulls, powder x-ray diffraction (Section 3 . 2 5 ) or
by NMR techniques. The infrared spectroscopic
method5 is based on the presence of specific
absorption bands for racemate A and for racemate B:
A: 1260 cm-l (7.9 p )
B: 1240 cm-l ( 8 . 0 5 p ) and 3 5 8 0 cm-’ ( 2 . 8 p )
These bands are recognizable in mixtures
of A and B in the range from 30%A - 70%B to 70%A -
308B. Either one or both racemates may be measured
independently with an accuracy of about -
+ 5%.
The NMR method of analysis is based upon
the chemical shift difference of t-butyl groups of
the tetrabenzoates of racemate A 7 6 1.60) and
racemate B (6 1.57).6 Quantitation of each racemate
was obtained with -
+ 2 % accuracy for samples contain-
ing 20 to 7 0 % of racemate B. Structural assign-

ments were made on the basis of europium shift
reagent studies. Kiralshift reagent allowed for
the separation of t-butyl group resonances of the
d,l isomers of theside chains. Use of this
reagent permits the determination of the optical
purity of the side chain of racemate A and B or of
the mixture of racemates.
An x-ray powder diffraction method for the
quantitation of racemates was also developed' (see
Section 3.25 and Figures 10 and 11).
An attempt was also made to separate race-
mates A and B by thin-layer chromatographic proced-
ures. I 5 Sufficient separation was not achieved,
however, even after 202 solvent systems and 14
chromatographic adsorbents were examined.
3.4 Solution Data
3.41 Solubility
Solubility data are summarized in
Table V . 1 2 ~ 1 6 t 1 7
Table V
Solubility of Nadolol
Solvent -
Temp.
CO
Solubility
mg/ml
0.1N HC1 37 42.5
pH 5.0, 0.2M citrate R.T. 40.1
pH 5.0 , 0.2M phosphate II
40.2
pH 7.0, 0.2M phosphate 30.4
Propylene glycol 37 97.5
50% Aq. PEG 400 II
46.0
Methylene Chloride R.T. 2.0
Methanol fl
>200.0
Isopropanol 5.0
l,l,l-Trichloroethane Inso lub1e
95% Ethanol Freely soluble
Chloroform Slightly soluble
Acetone Insoluble
Benzene Inso 1uble
Ethyl Ether Insolub1e
Hexane Insoluble
N ADOLOL 477
3.42 pKa
A pKa value of 9.67 was determined
potentiometricaIly.16
3.43
Partition Coefficient
The partition coefficient of nadolol
was determined in the octanol/Krebs buffer system
at room temperature.” The composition of Krebs
buffer is the following: KC1-5mM; KH2P04-lmM;
NaHC03 - 26mM and NaC1-122mM. The table below
shows the results obtained:
Partition Coefficient PH
0.25 8.1
1.3 8.7
4. Analytical Tests and Methods
The followincr results were obtained on a
Squibb Research Standird:
Element % Theory 8 Found
C 66.99 65.92
H 8.80 8.76
N 4.53 4.38
4.2 Identification Tests
Identification of nadolol in tablet formu-
lations is based on a color reaction of the oxidized
drug with phenylhydrazine and ferricyanide.” The
-
cis-hydroxy groups are first oxidized to aldehydes
with periodate and then reacted with phenylhydrazine
to form a hydrazone. In acid solution, the hydra-
zone gives a red color with potassium ferricyanide.
Thin-layer chr~rnatography’~(Section 4.61)
and infrared spectroscopy (Section 3.11) have also
been used to identify the drug.
4.3 Spectrophotometric Analysis
4.31 Ultraviolet Analysis
Nadolol displays three absorption
peaks in the ultraviolet region at about 218, 270
and 278 nm (Section 3.14). Although the molar
absorptivity of nadolol is quite low, it is adequate
for the study of dissolution rates of nadolol

tablets.20 Beer’s law is obeyed up to at least 4 mg
of nadolo1/100 ml, as measured in pH 1.2 hydro-
chloric acid at 277
Colorimetric Methods
4.32
Complexation of the amino group of
nadolol with bromophenol blue in chloroform yields
a yellow color with an absorption maximum at 414 nm.
This is of potential usefulness for a quantitative
assay of nadolol in formulation.”
A colorimetric assay for the deter-
mination of nadolol in tablet formulation is based
on a hydrazone absorption at 352 nm in chloroform?2
The two vicinal hydroxyl groups are oxidized to the
corresponding dialdehyde, which is condensed with
2,4-dinitrophenylhydrazine yielding the hydrazone.
Fluorescence Spectrophotometric
4.33
Analysis
Although nadolol does not exhibit
native fluorescence, it can be modified to yield a
strongly fluorescent derivative. A fluorometric
assay for the quantitation of nadolol in serum and
urine at nanogram and microgram levels has been
described.23
The drug is oxidized with periodic
acid to the corresponding dialdehyde and coupled
with o-phenylenediamine to produce a fluorescent
compound. Using a suitable filter, the emission
peaks of the reagents and nadolol derivative are
well separated ( A excitation = 305 nm and
X emission = 445 nm).
4.4Titrimetric Methods
4.41 Reaction with Chloramine-T*
Nadolol is oxidized with Chloramine-
T and the excess reagent is reacted with potassium
iodide. The liberated iodine is titrated with
sodium thiosulfate. The mechanism of the reaction
of the drug with Chloramine-T is not known. This
reaction can be used for the determination of
nadolol in tablet formulations. However, the more
readily controlled colorimetric method is preferable
(Section 4.32).
N ADO LO L 419
Nonaqueous Titrations
4.42
By virtue of the presence of an amino
group, titration with acetous perchloric acid can
serve to quantitate nadolol. 2 4 Quinaldine red or
crystal violet indicators are used to determine the
end-point. The amino group is titrated indirectly?'
First, an ammonium salt of nadolol is formed with
glacial acetic acid. Then, the released acetate
ion is titrated with perchloric acid to the end-
point monitored potentiometrically or with an
internal indicator. The method has good precision
and the results obtained using both indicators were
comparable. It was used to develop bulk, batching
and formulation assays.
4.5 Gas Chromatography/Mass Spectrometry
A method to determine the serum concentra-
tion of nadolol by selected ion monitoring (SIM)
and gas chromatography/mass spectrometry (GC/MS) of
the tri(trimethylsily1) ether derivative has been
described.26 The drug is extracted from serum and
a known amount of internal reference, N-methyl-
nadolol, is added. After lyophilization of the
acidic extract, the resulting solid is reacted with
N-trimethylsilylimidazole. The m/e 8 6 fragment ion
of nadolol and the m/e 100 ion of the internal
reference N-methyl-nadolol are monitored to
establish the relative concentration ratio.
The detection level of this method is 2 . 6
ng/ml. No interferences are detected from extracts
of fresh human serum at the relatively low mass ions
of m/e 8 6 and 100. However, significant interfer-
ences were observed with several commercial serum
samples at these masses. They probably result from
contamination by plastic or rubber components used
during the serum processing. Parallel measurements
by spectr~fluorometry~~ (Section 4 . 3 3 ) on duplicate
samples, demonstrate a correlation coefficient of
0 . 9.
4.6
Chromatographic Methods
A-thin-layer- chromatographic method
has been developed15 to measure quantitatively the
purity of nadolol samples. The TLC separation is
achieved on silica gel GF plates using the solvent
system acetone-chloroform-2N ammonium hydroxide
480 LIDIA SLUSAREK A N D KLAUS FLOREY
(80:lO:lO). The position of the nadolol zone is

located under short-wave ultraviolet light (maxi-
mum at %254 nm). The isolated zone is eluted with
95% ethanol and the absorbance of the eluate is
measured at 278 nm. This procedure provides an
excellent separation of ultraviolet absorbing
impurities and allows for the quantitative measure-
ment of the drug. This assay has been adapted for
measuring the stability of nadolol in tablet
formulations.
As mentioned in Section 3.3, attempts
to separate the two racemates of nadolol by TLC
were unsuccessfu1.l5
A gas chromatographic method has
been developed2’ for the quantitative measurement
of nadolol in solutions. The drug is extracted with
dichloromethane, filtered and evaporated together
with added brompheniramine maleate as an internal
standard. After evaporation to dryness, the tri-
methylsilyl derivative is formed. The GC parameters
are as follows: oven temperature is 210° C. and the
circular glass column is 1.7 m with 3 mm i.d.,
packed with 3% (w/w) OV-17 on 60-80 mesh Gas Chrom
Q (silanized). Retention times of a typical run
are: nadolol-8.5 min and brompheniramine standard -
4.5 min.
4.63 High Pressure Liquid Chromatography
An HPLC method for the quantitative
determination of nadolol has been developed. * a A
reverse phase ethylsilane column was used, operated
at pressures of 200 to 2,000 psi and equipped with
a precision loop injector and a fixed wavelength
(254 nm) or variable wavelength (220 nm) detector.
As mobile phase, a 35% methanol-65% aqueous 0.0005M-
hydrochloric acid-0.05M sodium chloride solution
was used.
5. Stability - Degradation
5.1 Solid State Stabilitv
_ ~ .-
_ _ ~
_... _ ~ _
Nadolol exhibits excillent stabilitv as a
solid. There was no apparent degradation of the
bulk samples which were held at high temperatures
for prolonged periods. The same TLC patternowas
obtained for samples held at 5O C. and at 50 C. for
NADOLOL 481
over two years." Results of a light stability

study" shows that nadolol and its racemic compo-
sition are stable under 9 0 0 foot candle light.
Visual examination of a sample exposed to liqht for
6 months showed slight discbloration.
5.2 Solution Stability

Lyophilized sterile solutions of nadolol
in 0.1M, pH 7.4 sodium phosphate buffer, showed no
evidence of decomposition when held at room temper-
ature for 51 days.-'' In unbuffered solutions, -
samples prepared at various H's were stable after
3 months' storage at 50° c. 38 A very slight dis-
coloration was noted in some samples after 3 months
at 50° C. Storage of nadolol solutions at 80° C.
for 2 months produces degradation and discoloration
at most pH's. Exposure to intense light results ir,
discoloration of solutions at pH 2, 2 . 9 2 and 9 . 8 ,
after 2 weeks'storage. Variation in the pH values
with temperature and time are below 1 pH unit for
most solutions with the exception of those stored
at 80' C.
6. Analysis of Body Fluids
A sensitive fluorometric method, capable of
measuring microgram or nanogram levels bf nadolol
in human urine and serum has been developedz3
(Section 4.33). There is no interference in this
assay from: dialyzing medium used during the
clinical study, the diuretics hydrochlorothiazide
and furosemide, and epinephrine and norepinephrine.3
This fluorometric method has been adapted for
nadolol determinations in human bile at levels from
0.005 to 5 ug/ml.'
Another technique, Selected Ion Monitoring Gas
Chromatography/Mass Spectrometry, is described in
Section 4.5, for application to nadolol quantitation
in serum.'6 Suitable detection levels are obtained
and no interferences from blood components or other
administered drugs are observed. The SIM-GC/MS
method shows lower detection limit and better sensi-
tivity than the spectrofluorometric assay. Both
SIM-GC/MS and fluorometric methods, in the absence
of fluorescing metabolites, yield equivalent
results. The fluorometric method is more adaptable
to processing a large number of samples while the
SIM-GC/MS method should be selected where specifi-
city is required or where the serum levels are

extremely low.
7. Drug Metabolism
Metabolic studies with n a d ~ l o l - ~were
~C
carried out in patients at a dose that could safely
be given both orally and intravenously. Maximum
concentrations of radioactivity were attained in
plasma 2 to 4 hours after drug administration.
When given intravenously, concentrations of radio-
activity decreased rapidly during the first hour
after drug administration, reflecting distribution
of radioactivity into tissues. Terminal plasma
half-times are an average 12.2 hours after oral and
9.8 hours after intravenous administration. After
oral doses, an average of 24.6% and 76.9% of the
dose is excreted in urine and feces, respectively,
whereas, after intravenous doses, an average of
72.9% and 23.3% of the dose was excreted by the
same route.
The radiolabeled drug is excreted unchanged in
the urine and feces after either oral or intra-
venous administration indicating no biotransform-
ation of the drug.
The metabolism of nadolol has also been studied
in rats, dogs and monkeys.32,33
N ADO LO L 483
8. References
1. M.E. Condon, C.M. Cimarusti, R. Fox,

V.L. Narayanan, J. Reid, J.E. Sundeen and
F.P. Hauck, J. Med. Chem., 21, 913 (1978).
2. D.B. Evans, M.T. Peschka, R.J. Lee and
R.J. Laffan, Eur. J. Pharmacol., 35, 17 (1976).
3. F.P. Hauck and C.M. Cimarusti, Gen. Pat.

2,421,549 (see also Drugs of the Future, Vol.1,
No. 9, 434 (1976)).
4. F.P. Hauck, C.M. Cimarusti and V.L. Narayanan,

U.S. Patent 3,935,267 (1976).
5. B. Toeplitz, Squibb Institute, personal

communication.
6. M.S. Puar, Squibb Institute, personal
communication.
7. P.T. Funke, Squibb Institute, personal

communication.
8. E. Ivashkiv, Squibb Institute, personal

communication.
9. K. Bush, Squibb Institute, personal
communication.
10. G. Brewer, Squibb Institute, personal

communication.
11. H. Jacobson, Squibb Institute, personal

communication.
12. D. Wadke, Squibb Institute, personal
communication.
13. Q. Ochs, Squibb Institute, personal
communication.
14. J.Z. Gougoutas, B. Toeplitz, Squibb Institute,
personal communication.
15. F.P. Targos, Squibb Institute, personal

communication.
484 LIDIA SLUSAREK A N D KLAUS FLOREY
16. V. Valenti, Squibb Institute, personal

communication.
17. A. Weiss, Squibb Institute, personal

communication.
18. P. Valatin, Squibb Institute, personal
communication.
19. H.R. Roberts, Squibb Institute, personal

communication.
20. M.D. Ward, Squibb Institute, personal
communication.
21. C. Papastephanou, Squibb Institute, personal
communication.
22. E. Ivashkiv, J. Pharm. Sci., -
67, 1024 (1978).
23. E. Ivashkiv, J. Pharm. Sci., 66, 1168 (1977).
24. J. Alicino, Squibb Institute, personal

communication.
25. D.B. Whigan, Squibb Institute, personal
communication.
26. P.T. Funke, M.F. Malley, E. Ivashkiv, A. Cohen,

J. Pharm. Sci., 67, 6 5 3 ( 1 9 7 8 ) .
27. J.R. Salmon, Squibb Institute, personal
communication.
28. B. Pate1 and J. Kirschbaum, Squibb Institute,
personal communication.
29. C.R. Bennett, Squibb Institute, personal
communication.
30. I.S. Gibbs, Squibb Institute, personal
communication.
31. J. Dreyfuss, L.J. Brannick, R.A. Vukovich,
J.M. Shaw, D.A. Willard, J. Clin. Pharmacol.,
-
17, 300 (1977).
N ADO LO L 485
32. K . K . Wong, J. Dreyfuss, J.M. Shaw, J.J. Ross

and E.C. Schreiber, Pharmacologist, - 15, 245
(1973).
33. J.M. -
Shaw and J. Dreyfuss, Fed. Proc., 35,
365 (1976).
NITRAZEPAM
Hussun Y. AbouE-Enein, Ahmud I . Judo, and

Mohummed A . L o u ~
I, Description 488
1.2 Formulae 488
I .3 Molecular Weight 488
1.5 Appearance, colour, odour 489
2.2 Solubility 489
3. Synthesis 496
4. Stability and Decomposition Products 497
5. Metabolism 498
6.1 Titrimetry 500
6.2 Spectrophotometry 500
6.4 Polarography 51 1
7. Acknowledgement 513
8. References 514
Copyright Q 1980 by Academic Press, Inc.

Analytical Profiles of Drug Substances, 9 487 All rights of nproduction in any fonn ~ e ~ e ~ e d .
ISBN: 0-12-260809-7
1. D e s c r i p t i o n
1.1. Nomenclature
1.11 Chemical Names
1,2-Dihydro-7-nitro-~-oxo-
benzodiazepine.
1,3-Dihydro-7-nitro-5-phenyl-2H-l, 4-benzo-
diazepin-2-one.
1.12 Generic N a m e
Nitrazepam
1.13 Trade N a m e s
B e n z a l i n , Calsmin, E u n o c t i n , Megadon,
Mogadon, Mogadan, Nelbon, N i t r e n p a x , Paxis-
yn, P e l s o n , Radedorm, R e l a c t , Sonebon,
Sonnolin.
1.2 Formu l a e
1.2 1 Empirical
1.22 Structural
1.3 Mo 1ec u 1a r weight
281.26
1.4 Elemental Composition
C,64.05%; H , 3.94%; N , 14.94%; 0, 17.07%
488
NITRAZEPAM 489
1.5 Appearance, c o l o r , odor
A y e l l o w , c r y s t a l l i n e powder, o d o r l e s s .
2.1 Crystal properties
2.11 Crystallinity
Parch and Lapysh (1) had d e s c r i b e d micro-

crystallographic reaction, for the detection
of n i t r a z e p a m ( d e t e c t i o n l i m i t 0.1 ug) and
o t h e r benzodiazepine d e r i v a t i v e s . T h i s i s
based on e v a p o r a t i n g a s o l u t i o n of t h e
sample, on a w a t c h - g l a s s , t h e r e s i d u e i s
k e p t f o r 5 t o 1 0 m i n u t e s a f t e r adding one
d r o p of 0 . 1 N-HC1, t h e n one drop of a s u i t -
a b l e r e a g e n t s o l u t i o n i s added and t h e
m i x t u r e i s s e t a s i d e i n a m o i s t atmosphere.
The v a r i o u s t y p e s of c r y s t a l s formed have
been d e s c r i b e d .
2.12 Melting P o i n t
224-226'C (2) ; 226-229'C (3)
2.2 Solubility
Nitrazepam is s o l u b l e i n a l c o h o l , a c e t o n e , c h l o r o -
form, and e t h y l a c e t a t e ; i n s o l u b l e i n water, e t h e r ,
benzene, and hexane ( 3 , 4 ) .
2.3 Identification
B.P. 1973 (3) s p e c i f i e s t h e f o l l o w i n g i d e n t i f i c a t i o n

tests f o r nitrazepam:
a ) The i n f r a r e d a b s o r p t i o n spectrum e x h i b i t s m a x i m a
which are o n l y a t t h e same wavelengths a s , and
have s i m i l a r r e l a t i v e i n t e n s i t i e s t o , t h o s e i n
t h e spectrum of n i t r a z e p a m a u t h e n t i c specimen.
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 230 t o 250 nm,

o f a 2-cm l a y e r of a 0.0005% w/v s o l u t i o n , i n a
m i x t u r e of 1 volume of N h y d r o c h l o r i c a c i d and 9
volumes of methyl a l c o h o l , e x h i b i t s a maximum
o n l y a t 280 nm; e x t i n c t i o n a t 280 nm, about 0.91.
490 HASSAN Y. ABOUL-ENEIN er al.
c ) To 1 0 mg add 5 m l of h y d r o c h l o r i c a c i d and 1 0 m l
of water, h e a t on a w a t e r - b a t h f o r 1 5 m i n u t e s ,
and f i l t e r . To t h e clear f i l t r a t e add 1 m l of a
0.1% w / v s o l u t i o n of sodium n i t r i t e , a l l o w t o
s t a n d f o r 3 m i n u t e s and add 1 m l of a 0.5% w / v
s o l u t i o n of s u l f a m i c a c i d . Allow t o c o o l f o r 3
m i n u t e s and add 0.1% w/v s o l u t i o n of N-(1-naphth-
y l ) ethylenediamine hydrochloride, a red colour
is produced.
2.4 Spectral properties
2.41 U l t r a v i o l e t Spectrum:
Nitrazepam, i n n e u t r a l methanol s o l u t i o n ,
shows maxima a t 218, 258 nm, and a n i n -
f l e c t i o n a t a b o u t 308 nm ( F i g . 1).
Nitrazepam, i n e t h a n o l , e x h i b i t s ( 4 ) maxima
a t 218, 260 nm; minimum a t a b o u t 242 nm.
I n 0.1N s u l p h u r i c a c i d , t h e d r u g shows a
maximum a t 2 7 7 . 5 nm E l % lcm = 1500 and a n i n -
f e c t i o n a t a b o u t 340 nm.
The UV a b s o r p t i o n s p e c t r u m of n i t r a z e p a m i s
used a s a mean of i d e n t i f i c a t i o n and a s s a y
of t h e d r u g i n t a b l e t f o r m u l a t i o n i n B.P.
1973 ( 3 ) .
2.42 I n f r a r e d spectrum
The I R spectrum of n i t r a z e p a m i s shown i n

F i g . 2 . The spectrum w a s o b t a i n e d from
N u j o l m u l l . 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
frequencies:
-1 Assignment
Frequency (cm )
1.680 c=o
1600 C=C a r o m a t i c
1370 NO2
Clarke (4) has c i t e d t h e following character-
i s t i c f i n g e r - p r i n t bands f o r n i t r a z e p a m
when d e t e r m i n e d i n p o t a s s i u m bromide d i s c :
1352, 1692, 702, and 1615 cm-’

NITRAZEPAM 49 1
Fig. 1 - Ultraviolet spectrum of Nitrazepam i n methanol

lOOL - a . !loo
5 . 80
-
LLI
80-
0
2
2 60- . 60
k -/
25 40- * 40
+
IT
20 - - 20
0
-
WAVENUMBER (CM-’)
Fig. 2 - Infrared spectrum of Nitrazepam in nujol mull.

NITRAZEPAM 493
2.43 N u c l e a r Magnetic Resonance Spectrum
A t y p i c a l NMR spectrum o f n i t r a z e p a m i s
shown i n F i g . 3 . The sample w a s d i s s o l v e d
i n d e u t e r a t e d c h l o r o f o r m (CDC1 >. The
spectrum w a s d e t e r m i n e d on a 3Varian T-60A
N M R s p e c t r o m e t e r w i t h TMS as t h e r e f e r e n c e
standard.
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 made f o r F i g . 3 :
Chemical S h i f t (6) Assignment
4.4 ( s i n g l e t ) CH2 a t C3
7.2 ( s i n g l e t ) C-H aromatic at C
9
7.4 ( d o u b l e t ) Five aromatic protons
of t h e phenyl group a t
c5.
8.2 ( s i n g l e t ) Two a r o m a t i c p r o t o n s a t
C6 and C
8
1 0 . 1 (broad N - H
singlet)
2.44 Mass s p e c t r u m and fragmentometry
The l o w r e s o l u t i o n m a s spectrum of n i t r a -
zepam i s shown i n F i g . 4. I t was o b t a i n e d
on a F i n n i g a n 1015 L q u a d r u p o l e m a s s
s p e c t r o m e t e r of a n i o n i s a t i o n p o t e n t i a l of
7 0 e V . The s p e c t r u m shown w a s o b t a i n e d by
d i r e c t i n s e r t i o n of n i t r a z e p a m . I t shows
a m o l e c u l a r i o n M+ a t m / e 281 ( r e l a t i v e
i n t e n s i t y 42.8%) and M+ + 1 a t m / e 282
( r e l a t i v e i n t e n s i t y 8.1%). Some of t h e
most prominent i o n s are g i v e n i n T a b l e I .
Table I
&
m Fragment
280 M-H
264 M-OH
254 M-HCN
253 M- (H,HCN)
252 M-(H,CO)
235 M-N02
. . . . . . . . . . . . . . . , . . . . . . . , . . . . . I
1 ' 8 1 1 I I ' I
¶a w 300 200 t 00 i *I
.U
P
P
W
Fig. 3 - NMR spectrum of Nitrazepam in CDCl

3
containing TMS
as an internal standard.
1:-' ?A
* 1 . 1
495
496 HASSAN Y. ABOUL-ENEIN el al.
m/e Fragment
234 M- (H ,NO2
207 M-(N02-CO)
206 M-(H-NO~-CO)
3. Synthesis
The two most f r e q u e n t l y used methods, w i t h good y i e l d s

( 5 , 6 ) , f o r t h e s y n t h e s i s of s i m p l e b e n z o d i a z e p i n o n e s a r e
shown i n Scheme 1.
Scheme 1
RI s
X-CO-CH-X
(X=halogen)
't x@NHco:"' p=0
ROCOCHR.HC1
I NH3
- a:-
Pyr i d i n e ,
heat NH2
_.
H
x?i?-oR
0
heat !
x c =O j H - R
NH2
y@
A s c a n b e s e e n , i n b o t h c a s e s , 2-aminobenzophenones a r e
used as s t a r t i n g m a t e r i a l s . Treatment of t h e a p p r o p r i a t e -
l y s u b s t i t u t e d aminobenzophenone w i t h a h a l o a c e t y l h a l i d e
y i e l d s a compound I1 which, on t r e a t m e n t w i t h ammonia,
g i v e s t h e b e n z o d i a z e p i n o n e I V v i a am amino d e r i v a t i v e 111.
T h i s method g e n e r a l l y g i v e s b e t t e r o v e r a l l y i e l d s of up t o
70-80%, a l t h o u g h i t i n v o l v e s more s t e p s . Another e x t e n -
s i v e l y used method i s t h e t r e a t m e n t of 2-aminobenzophenone
w i t h a n amino a c i d e s t e r h y d r o c h l o r i d e i n p y r i d i n e , l e a d -
NITRAZEPAM 491
i n g d i r e c t l y from 1 t o IV.
O t h e r r o u t e s f o r t h e c o n s t r u c t i o n of t h e 7-membered r i n g ,
which have been developed s u b s e q u e n t l y , i n v o l v e t h e u s e of
intermediates possessing a protected or potential glycine
moiety ( 7 , 8 ) .
Nitrazepam h a s been p r e p a r e d by t h e f o l l o w i n g method ( 9 ) :

Anhydrous h y d r o c h l o r i c a c i d i s p u t i n t o a s t i r r e d m i x t u r e
c o n t a i n i n g 2-amino-5-nitrobenzophenone, g l y c i n e , and
p y r i d i n e . The r e a c t i o n m i x t u r e i s r e f l u x e d f o r more t h a n
48 h o u r s , a t i n t e r v a l s , and t h e n c o n c e n t r a t e d undervacuum.
The r e s i d u e i s p a r t i t i o n e d between benzene and water. The
benzene l a y e r i s washed w i t h water and d r i e d o v e r an-
hydrousmagnesium s u l p h a t e and t h e n c o n c e n t r a t e d u n d e r
vacuum t o g i v e t h e d r i e d p r o d u c t .
Nitrazepam i s a l s o p r e p a r e d by t h e t r e a t m e n t of 2-amino-
5-nitrobenzophenone w i t h a f+acylaminoethyl h a l i d e ( 1 0 ) .
02N
C6H5
C6H5
4. S t a b i l i t y and Decomposition P r o d u c t s :
Beyer and Sadee (11) have p u b l i s h e d a monograph g i v i n g t h e

a n a l y t i c a l d a t a on l Y 4 - b e n z o d i a z e p i n e d e r i v a t i v e s , i n c l u d -
i n g n i t r a z e p a m , c o n c e r n i n g t h e s t a b i l i t y of t h e d r u g i n
s o l u t i o n . Nitrazepam i s a r e l a t i v e l y s t a b l e d r u g a t room
t e m p e r a t u r e . However, 2-amino-5-nitrobenzophenone i s con-
s i d e r e d a s a d e c o m p o s i t i o n p r o d u c t . The B.P. 1973 ( 3 )
d e s c r i b e s a method f o r t h e d e t e c t i o n of t h i s d e c o m p o s i t i o n
p r o d u c t , u s i n g TLC. Genton and K e s s e l r i n g ( 1 2 ) have
s t u d i e d t h e e f f e c t of t e m p e r a t u r e and r e l a t i v e h u m i d i t y
on t h e s t a b i l i t y of n i t r a z e p a m i n t h e s o l i d s t a t e . The
d r u g 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 have been d e t e r m i n e d
i n a 1%d i l u t i o n i n m i c r o c r y s t a l l i n e c e l l u l o s e . The
sample i s e x t r a c t e d by s h a k i n g w i t h methanol. The extract
i s chromatographed by TLC on K i e s e l g e l GF 254 u s i n g ben-
z e n e - e t h y l a c e t a t e - a c e t i c a c i d (15:9:1) a s a d e v e l o p i n g
498 HASSAN Y. ABOUL-ENEIN et al.
s o l v e n t . The d i f f u s e r e f l e c t a n c e of t h e s p o t s are measur-

ed a t 265, 365 and 295 nm f o r n i t r a z e p a m , 2-amino-5-nitro-
benzophenone and 3-amino-6-nitro-4-phenyl-4H-quinoline-2-
one, r e s p e c t i v e l y .
Meyer, e t a 1 (13) have p u b l i s h e d a r e p o r t on t h e s t a b i l i t y

and a n a l y s i s of t h e h y d r o l y t i c p r o d u c t s of n i t r a z e p a m .
T h e drug i s hydrolysed i n t o 5-nitro-2-aminobenzophenone
and 3-amino-6-nitro-4-phenyl-4H-quinoline-2-one ( r i n g
c o n t r a c t i o n ) . These h y d r o l y t i c p r o d u c t s can b e determined
s e p a r a t e l y by UV a b s o r p t i o n a f t e r f r a c t i o n a t i o n by TLC on
a K i e s e l g e l PF 254 u s i n g benzene-isopropanol (9:l) as a
s o l v e n t ; o r by means of t h e a b s o r p t i o n of t h e i r diazonium
salts. A l t e r n a t i v e l y , t h e hydrolytic products can a l s o
be determined t o g e t h e r by means of a (dead-stop) t i t r a t i o n
w i t h 0 . 0 1 N sodium n i t r i t e s o l u t i o n . Meyer e t a1 (14)
have a l s o s t u d i e d t h e e f f e c t of pH on t h e s t a b i l i t y of
1,4-benzodiazepine d e r i v a t i v e s i n i n j e c t i o n f o r m u l a t i o n s .
5. Metabolism
The m e t a b o l i t e s of n i t r a z e p a m i n man and rat i s shown i n

F i g . 5 . With t h e e x c e p t i o n of s u b s t a n c e IV, which w a s
d e s c r i b e d by Beyer and Sadee ( 1 5 ) , and s u b s t a n c e X , which
i s s t i l l h y p o t h e t i c a l , t h e o t h e r compounds l i s t e d have
been proved by Rieder and Wendt (16) t o b e b i o t r a n s f o r -
mation p r o d u c t s of t h e d r u g a p p e a r i n g i n t h e u r i n e . They
have been i s o l a t e d by v a r i o u s p r o c e d u r e s of e x t r a c t i o n ,
column chromatography, and t h i n - l a y e r chromatography, and
t h e i r chemical s t r u c t u r e s have been e l u c i d a t e d by chemical
r e a c t i o n s , comparison w i t h a u t h e n t i c samples, m a s s spec-
t r o m e t r y , n u c l e a r magnetic r e s o n a n c e s p e c t r o m e t r y , and,
i n t h e c a s e s of I1 and 111, also by u l t r a v i o l e t and i n -
f r a r e d s p e c t r o m e t r y . The main m e t a b o l i c pathway i n man
and rat i n d i c a t e s ( F i g . 5 ) t h e r e d u c t i o n of t h e n i t r o
group t o t h e c o r r e s p o n d i n g amine I1 and - by a c e t y l a t i o n
of I1 - t o t h e 7-acetamido d e r i v a t i v e 111, which i s t h e
major m e t a b o l i t e . A s m a l l p r o p o r t i o n of I1 and 111 i s
hydroxylated i n p o s i t i o n 3 , y i e l d i n g compounds IV and V.
Another m e t a b o l i c pathway c o n s i s t s of t h e c l e a v a g e of t h e
benzodiazepine r i n g , w i t h t h e f o r m a t i o n of t h e benzophen-
one d e r i v a t i v e s VI, VII, and VIII. There i s someevidence
t h a t t h e a c i d X is formed d u r i n g t h e g e n e r a t i o n of t h e
benzophenones, which can b e c a l l e d an opened lactam. The
end p r o d u c t of t h i s l i n e , i n man, t h e 2-amino-3-hydroxy-
5-nitro-benzophenone VII and, i n r a t , t h e 2-amino-5-
notro-4-hydroxy-benzophenone VIII. P a r t of compound VIII
w
0
ffl
I
h
rd
3
I
5
(d
O"
a
U
-d
4
0
e
rd
u
a,
E
a
al
may b e p o s s i b l y d e r i v e d from t h e 4-hydroxylated n i t r a z e -

Pam-IX, which h a s been found i n t h e u r i n e of r a t , b u t n o t
i n man ( 1 6 ) . The p h e n o l i c s u b s t a n c e s VII, V I I I , and I X
are e x c r e t e d a l m o s t e x c l u s i v e l y ; I1 and V I o n l y t o a
minor p a r t i n c o n j u g a t e d form.
The d i s t r i b u t i o n , e x c r e t i o n and p h a r m a c o k i n e t i c s of n i t r a -
zepam have been d i s c u s s e d by R i e d e r and Wendt ( 1 6 ) .
6. Methods of A n a l y s i s
6.1 Titrimetry
6.11 Aqueous
Blaszek-Bodo, e t a 1 ( 1 7 ) have d e s c r i b e d a
d i a z o m e t r i c method f o r t h e d e t e r m i n a t i o n of
n i t r a z e p a m i n p u r e form and 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 method i s based on d i a -
z o t i s a t i o n r e a c t i o n i n which t h e d r u g i s
f i r s t hydrolysed with h y d r o ch lo r ic a c i d i n
t h e p r e s e n c e of z i n c t o a f f o r d 2,5-dia-
minobenzophenone. T h i s p r o d u c t i s t i t r a t e d
a g a i n s t s t a n d a r d sodium n i t r i t e s o l u t i o n .
The method proved t o b e a c c u r a t e and t h e r e
i s no i n t e r f e r e n c e from t h e d r u g e x c e p i e n t s .
6.12 Non-aqueous
A non-aqueous t i t r a t i o n method h a s been

described ( 3 ) f o r the quantitative analysis
of n i t r a z e p a m a s t h e p u r e d r u g . The d r u g
i s t i t r a t e d by p e r c h l o r i c a c i d i n a c e t i c
a c i d and t h e e n d p o i n t i s d e t e r m i n e d p o t e n t i -
ometrically .
6.2 Spectrophotometry
6.21 Colorimetry
C o l o r i m e t r i c methods have been used f o r t h e

d e t e r m i n a t i o n of n i t r a z e p a m i n v a r i o u s p r e -
p a r a t i o n s . Wassel and Diab (18) have
developed t h e f o l l o w i n g p r o c e d u r e s f o r t h e
d e t e r m i n a t i o n of n i t r a z e p a m i n pharmaceuti-
c a l f o r m u l a t i o n s and u r i n e samples:
NITRAZEPAM 501
a ) F e r r o u s hydroxamate p r o c e d u r e :
To a n e t h a n o l i c s o l u t i o n (1 m l = 0.2 t o
5 mg of n i t r a z e p a m ) , add f i l t e r e d Goddu
r e a g e n t 112.5% m e t h a n o l i c hydroxylammoni-
urn c h l o r i d e - 12.5% m e t h a n l o i c sodium
h y d r o x i d e (1:1)] ( 3 m l ) . The s o l u t i o n
i s h e a t e d a t 45OC f o r 50 m i n u t e s
t h e n c o o l e d and t h e f e r r o u s r e a g e n t
[ (NH4) 2 S04Fe2 (S04) 3 . 24H20) ( 2 0 gm) d i s -
s o l v e d i n 70% aqueous p e r c h l o r i c a c i d
(10 ml) i s added. Shake t h e s o l u t i o n
and d i l u t e t o 25 m l w i t h a c e t a t e b u f f e r
s o l u t i o n ( 0 . 1 M sodium a c e t a t e a d j u s t e d
t o pH 1 . 5 w i t h 70% aqueous p e r c h l o r i c
a c i d ) . Measure t h e e x t i n c t i o n a t 550 nm
and o b t a i n t h e amount of n i t r a z e p a m by
r e f e r e n c e t o a c a l i b r a t i o n g r a p h , which
i s r e c t i l i n e a r from 0 . 1 t o 5 mg of n i t r a -
zepam.
Treat u r i n e samples (200 t o 400 ml) w i t h

ammonia ( t o pH 10) and e x t r a c t n i t r a z e p a m
and i t s m e t a b o l i t e s w i t h c h l o r o f o r m
(4x100 m l ) . Wash t h e combined e x t r a c t s
w i t h water, add anhydrous e t h a n o l (5 ml)
and e v a p o r a t e t o d r y n e s s i n vacuo a t 500.
D i s s o l v e t h e r e s i d u e i n anhydrous e t h a n o l
( 5 ml) , add t h e Goddu r e a g e n t ( 3 ml) and
c o n t i n u e a s above.
b) C i t r i c a c i d method
To t h e e t h a n o l i c s o l u t i o n ( l m l up t o
50 ug of n i t r a z e p a m ) , add 5 m l of c i t r i c
a c i d r e a g e n t [ c i t r i c a c i d ( 2 gm) d i s s o l -
ved i n e t h a n o l (10 ml) and anhydrous
a c e t i c a c i d (90 m l ) ] and h e a t t h e s o l u -
t i o n a t 7O-8O0C f o r 20 m i n u t e s . D i l u t e
t h e s o l u t i o n t o 25 m l w i t h e t h a n o l and
measure t h e e x t i n c t i o n a t 510 nm. T h i s
procedure is s u i t a b l e f o r r o u t i n e analy-
ses.
Diab (19) h a s developed a c o l o r i m e t r i c

method f o r t h e a n a l y s i s of t h e d r u g i n
f o r m u l a t i o n s and i t s m e t a b o l i t e s i n blood
and u r i n e . The method d e p e n d s on t h e
c o l o r r e a c t i o n of P o r t e r ( 2 0 ) f o r t h e
502 HASSAN Y . ABOUL-ENEIN er al.
a r o m a t i c n i t r o compounds. The method i s

is e s s e n t i a l l y as follows: Transfer 5 m l
of s t a n d a r d s o l u t i o n ( 2 mg of n i t r a z e p a m
i n 100 m l e i t h e r dimethylformamide o r
a c e t o n e ) i n t o s e p a r a t e t e s t - t u b e s . Add
0 . 1 m l of 10% tetraethylammonium hydro-
x i d e s o l u t i o n t o t h e d i m e t h y l formamide
s o l u t i o n o r 0 . 1 m l of 10% sodium hydro-
x i d e s o l u t i o n t o t h e acetone s o l u t i o n ,
s h a k e t h e m i x t u r e s and measure t h e
e x t i n c t i o n s a t 410 nm. The c o l o r formed
by e i t h e r r e a c t i o n is s t a b l e f o r more
t h a n two h o u r s . For t h e a s s a y o f t a b l e t s ,
a q u a n t i t y of powdered sample i s e x t r a c -
t e d w i t h e t h a n o l . The combined f i l t e r e d
e t h a n o l extracts are d i l u t e d t o a c e r t a i n
volume and 1 m l of t h e s o l u t i o n i s evapo-
r a t e d t o d r y n e s s on a steam b a t h . The
r e s i d u e is d i s s o l v e d i n e i t h e r dimethyl-
formamide o r a c e t o n e and proceed a s f o r
t h e s t a n d a r d s o l u t i o n . For samples of
blood and u r i n e , n i t r a z e p a m i s e x t r a c t e d
w i t h benzene and d e t e r m i n e d a s above.
M e t a b o l i t e s a r e d e t e r m i n e d w i t h 4-
dimethylaminobenzaldehyde r e a g e n t (0.125
gm i n 100 m l of 65% s u l p h u r i c a c i d p l u s
0 . 1 m l of 5% aqueous f e r r i c c h l o r i d e
s o l u t i o n ) and measurement of t h e e x t i n c -
t i o n a t 420 nm.
Raber and Gruber ( 2 1 ) have d e s c r i b e d a

p h o t o m e t r i c method f o r e s t i m a t i o n of
n i t r a z e p a m and o t h e r 1 , 4 - b e n z o d i a z e p i n e
d e r i v a t i v e s . Nitrazepam i s h y d r o l y s e d
w i t h h y d r o c h l o r i c a c i d and t h e r e s u l t i n g
2-aminobenzophenone d e r i v a t i v e i s
d i a z o t i s e d and c o u p l e d w i t h 1 - n a p h t h o l .
t h e e x t i n c t i o n of t h e s o l u t i o n i s t h e n
measured a t 607 nm. The method i s
a p p l i c a b l e f o r e s t i m a t i o n of a m i x t u r e
of n i t r a z e p a m and o t h e r d e r i v a t i v e s .
Also, t h e p r o c e d u r e i s a p p l i c a b l e f o r t h e
d e t e r m i n a t i o n of n i t r a z e p a m i n t a b l e t o r
capsule formulations
NITRAZEPAM 503
Beyer and Sadee (15) have e s s e n t i a l l y

a p p l i e d t h e same p r i n c i p l e used b e f o r e for
t h e d e t e r m i n a t i o n of n i t r a z e p a m (and
o t h e r 5-phenyl-1,4-benzodiazepines) and
f o r i n v e s t i g a t i o n s on t h e metabolism of
n i t r a z e p a m . The diazonium s a l t b e i n g
t r e a t e d w i t h 2% sulphamic a c i d s o l u t i o n
and t h e d i a z o compound is t h e n c o u p l e d
w i t h N-1-naphthylethylenediamine d i h y d r o -
c h l o r i d e . The e x t i n c t i o n of t h e r e s u l t -
i n g a z o d y e i s measured a t 533 t o 535 nm
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 .
R e c e n t l y , Blaszek-Bodo, e t a 1 (22) have

r e p o r t e d a method f o r e s t i m a t i o n of
n i t r a z e p a m . The drug i s h y d r o l y s e d and
s i m u l t a n e o u s l y reduced and t h e r e s u l t i n g
2,5-diaminobenzophenone i s d i a z o t i s e d
and coupled w i t h N-l-naphthylethyl-
ened i a m i n e .
Egg (23) h a s d e v i s e d a q u a l i t a t i v e method
f o r t h e d e t e c t i o n of n i t r a z e p a m and o t h e r
d e r i v a t i v e s by t h e c o l o u r r e a c t i o n of
S a w i c k i and Johnson. B e n z o d i a z e p i n e s
c a n b e d e t e c t e d a f t e r TLC s e p a r a t i o n by
s p r a y i n g t h e chromatogram w i t h 1%2 , 5 -
dimethoxytetrahydrofuran s o l u t i o n i n
a c e t i c a c i d and d r y i n g f o r 5 t o 1 0
m i n u t e s a t 1OOOC; a r e d d i s h - v i o l e t s p o t
i s produced on r e - s p r a y i n g w i t h 2% 4-
dimethylaminobenzaldehyde s o l u t i o n i n
a c e t i c a c i d - conc. h y d r o c h l o r i c a c i d
(17:3). The s e n s i t i v i t y of t h e c o l o u r
r e a c t i o n i s dependent on t h e s u b s t i t u -
e n t s i n t h e benzodiazepine molecule.
6.22 Spectrofluorimetry
T h i s method h a s been used f o r t h e i d e n t i -

f i c a t i o n i n urgent t o x i c o l o g i c a l a n a l y s i s
of 1 , 4 - b e n z o d i a z e p i n e s used i n t h e r a p e u t i c
t r e a t m e n t ( 2 4 ) . The g a s t r i c f l u i d i s made
n e u t r a l o r weakly a c i d and t h e d r u g i s
e x t r a c t e d by e t h e r . The 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
t h e n d i s s o l v e d i n HCl04, H3PO4 o r H2SO4.
504 HASSAN Y . ABOUL-ENEIN et al.
6.23 Ultraviolet
The determination of nitrazepam and other

benzodiazepines in solutions, injections,
tablets and syrups pharmaceutical formu-
lations by UV spectrophotometry has been
described (25). Nitrazepam is determined
at 259 nm in neutral 96% ethanol.
Another report (26) has been published for

the spectrophotometric determination of
nitrazepam in methanolic solution at 259 and
309 nm in concentration ranges 0.2 to 2 and
0.4 to 3 mg dl-’, respectively.
The B.P. 1973 ( 3 ) describes a method for the
assay of nitrazepam tablets depending on
acid hydrolysis and the resulting 2-amino-
5-nitrobenzophenone is measured spectro-
photometrically at a maximum of about 280nm
(Elcm 1% = 910).
6.3 Chromatography
A compilation of qualitative colour and

precipitation reactions, spectrophotometric
and TLC data (useful for identification
purposes and for quantitative assay methods)
related to nitrazepam has been reviewed by
Dobrecky, et a1 (27). Several Reports had
been published concerning the chromatograph-
ic identification and separation of nitra-
zepam and its metabolites as shown in
Table 2.
Table 2
Solvent System Absorbent Detection Reference
Toluene-acetone-conc. Kieselgel -UV at 254 or356 28

aq. ammonia GF254 nm
(50 : 50 : 1) -d iazotisation
and coupling
NITRAZEPAM 505
Solvent System Absorbent Detection :eierence
Ethyl acetate-propa- -reduction by

nol-diethyl-amine.
Na2S204
(70 : 30 : 1) to give coloured
d er ivat ive
Methanol-1,2-dichlo-
roethane-conc. aqu.
ammonia
(10 : 90: 1)
Toluene-diethylamine Lieselgel -UV at 254 nm 29

:F
(4 : 1) 254
Ethyl acetate-metha- jilica gel -UV at 254 nm 30
nol-acetic acid
(9 0 : 10 : 1)
Heptane-chloroform- jilica gel 31

ethano1 :F
254
(5 : 5 : 1)
Ethyl acetate-1,2-
dichloroethane-25% -UV at 254 nm
aq. ammonia -Spraying with
conc. H SO4,HC1
(8 : 2 : 1) H3P04, Hc104 ti
the color of the
fluorescene in
radiation at 254
and 366 nm is
noted.
Dioxane-benzene- Ciesel gel -fluorescence 32

hexane-onc. aq.ammonii ;F
254
-spray with Drag-
endorff reagent
( 9 : 10 : 14 : 1)
-1% 2-furaldehyde
chloroform-acetone- solution in acetone
tetrahydrofuran solution of 10 gm
H2SO4 in 90 ml
( 9 : 1 : 1)
acetone
506 HASSAN Y . ABOUL-ENEIN ei al.
Solvent system AbsorEent Detection .eference
Shellsol A -methanol- Silica gel -Dragendorff 33

25% aq. ammonia G reagent diluted
1:lO with 10%
(85 : 15 : 1) HC1
Chloroform-benzene- 34
ether-tetrahydrofur- (UV at 230-300
an-acetone-acetic
I
acid
(35: 15: 16: 10: 5:3)
Chloroform-ether Silica gel -UV at 254 nm and 35

(3:2) G sprayed with
ethanolic 0.01%
N-l-naphthyl-
ethylenediamine
Chloroform-toluene- Merck -UV at 254 nm

ethano1 Aluminium -Spraying with
(20 : 30: 1) K2PtI6
(type
Oxide F354
E
IChloroform-ethanol Whatman
SG 81
Benzene-chloroform Aluminium -UV at 254 nm 24

(3:l) oxide
Chloroform-ethanol -Fluorescent spot
f29:l) F254
at 366 nm
-diazot isation
followed by spray
ing with 0.1% aq
N-1-naphthyl-NN-
d iethylpropane-1
2-d iamine hydro-
chloride and hea
at 50OC.
Negritescu et a1 (37) have described a TLC

separation method of the reaction products
formed during synthesis of nitrazepam by
nitration of 2,3-dihydro-5-phenyl-lH-l,4-
NITRAZEPAM 507
benzodiazepine-2-one. Nitrazepam i s formed

a l o n g w i t h a d i n i t r o d e r i v a t i v e . The l a t t e r
can be s e p a r a t e d from t h e reac%ion m i x t u r e by

TLC on K i e s e l g e l H , u s i n g 6 s o l v e n t s y s t e m s ,
namely:
1) benzene-n-butanol-formic a c i d (50: 28:8)

2) d i b u t y l e t h e r - e t h y l a c e t a t e - f o r m i c a c i d
(25 :75 :8 )
3) benzene-ethyl a c e t a t e - formic a c i d
(25:75:5 ; 25:75:10; 25:75:15; 25:75:20)
A f t e r d r y i n g t h e chromatograms a r e sprayed
w i t h HN03 (0.15 m l of conc. HNO3 i n 1 0 m l of
e t h a n o l ) and t h e p l a t e s a r e t h e n examined
under UV r a d i a t i o n .
Schuetz (38) h a s developed a chromatographic

method f o r t h e d e t e c t i o n of n i t r a z e p a m and
i t s major m e t a b o l i t e s . The sample ( e . g .
u r i n e e x t r a c t ) i s s u b j e c t e d t o TLC, withben-
zene-isopropyl alcohol-25% aq. ammonia
(80:20:1) a s t h e s o l v e n t . The d r u g and i t s
m e t a b o l i t e s , are t h e n hydrolysed and reduced
by s p r a y i n g w i t h a c i d i c T i C 1 3 s o l u t i o n . The
p l a t e i s t r e a t e d w i t h gaseous ammonia t o
c o n v e r t any amine s a l t s i n t o t h e f r e e b a s e s .
A second development a t r i g h t a n g l e s w i t h
t h e same m o b i l e phase is t h e n c a r r i e d o u t .
D i a z o t i s a t i o n followed by c o u p l i n g w i t h N-l-
n a p h t h y l e t h y l e n e d i a m i n e makes i t p o s s i b l e t o
d e t e c t amounts a s low a s 0.02 ug p e r s p o t .
6.32 Column Chromarography
Golovenko, e t a l . (39) have r e p o r t e d a method

f o r t h e s e p a r a t i o n of n i t r a z e p a m and i t s
m e t a b o l i t e s from r a t u r i n e . P o r t i o n s (10 t o
100 ug each) of n i t r a z e p a m and i t s p o s s i b l e
m e t a b o l i t e s , d i s s o l v e d i n chloroform-hexane
(l:l), a r e a p p l i e d t o a column (10 cm x 0 . 5
cm)of KSK-1 S i l i c a g e l (76 mesh) and t h e
column i s washed w i t h 1 0 m l of hexane; c l e a n
s e p a r a t i o n a r e o b t a i n e d by s t e p w i s e change
of e l u e n t . The c o l l e c t e d 1 - m l f r a c t i o n s
a r e e v a p o r a t e d i n vacuum, e a c h r e s i d u e i s
d i s s o l v e d i n 4 m l of e t h a n o l , and t h e absorb-
a n c e s of t h e r e s u l t i n g s o l u t i o n s a r e measured
a t t h e a p p r o p r i a t e w a v e l e n g t h s . The o r d e r
of e l u t i o n of compounds i n v e s t i g a t e d i n model
m i x t u r e s and e l u e n t s used ( a s 10-ml p o r t i o n s )
are a s f o l l o w s :
Nothing e l u t e d i n C C 1 4 ; n i t r a z e p a m , hexane-
a c e t o n e (4:1); 7-amino-l,2-dihydro-5-phenyl-
3H-1,4-benzodiazepin-2-one, chloroform-
a c e t o n e ( 4 : l ) ; and 7-acetamido-1,2-dihydro-
5-phervl-3H-1, 4-benzodiazepin-2-one, c h l o r o -
form-acetone (4:l).
Sawada, e t a 1 (30) have d e s c r i b e d a method

f o r t h 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
n i t r a z e p a m and i t s m e t a b o l i t e s i n r a b b i t
u r i n e . The method i n v o l v e s t h e s o r p t i o n on
a column of XAD-2 r e s i n , which i s e l u t e d by
methanol and e t h y l a c e t a t e - m e t h a n o l - a c e t i c
a c i d (9O:lO:l). Conjugated m e t a b o l i t e s i n
t h e e l u a t e from t h e column a r e h y d r o l y s e d
e n z y m i c a l l y , and t h e l i b e r a t e d compounds a r e
extracted into ethyl acetate.
Missen (40) h a s r e p o r t e d a p r o c e d u r e f o r
e x t r a c t i n g n i t r a z e p a m from t h e b l o o d , by
column chromatography. The p r o c e d u r e i n v o l -
v e s t h e a b s o r p t i o n of t h e d r u g on a c t i v a t e d
c h a r c o a l , A m b e r l i t e XAD-2 r e s i n and C e l i t e
e l u t i n g with chloroform.
6.33 High Performance L i q u i d Chromatography

Moore, e t a1 ( 4 1 ) have r e p o r t e d a HPLC method
f o r t h e a n a l y s i s of n i t r a z e p a m and i t s m e t a -
NITRAZEPAM 509
b o l i t e s , i n u r i n e . The u r i n e sample i s
a d j u s t e d t o pH7 w i t h a c e t a t e b u f f e r s o l u t i o n
and e x t r a c t e d w i t h e t h y l acetate. The com-
b i n e d e x t r a c t s are 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 d i s s o l v e d i n e t h y l acetate.
P o r t i o n s of t h i s s o l u t i o n a r e s u b j e c t e d t o
HPLC on a s t a i n l e s s s t e e l column (50 cm x
2mm) packed w i t h Zipax SAX ( 3 0 um) and o p e r -
a t e d w i t h hexane-ethyl acetate (7:3) as
m o b i l e p h a s e , a t a r a t e of 1 m l p e r m i n u t e ,
and d e t e c t i o n a t 260 nm. T h i s method i s
s u i t a b l e f o r t h e d e t e r m i n a t i o n of t h e d r u g
and of i t s 7-amino-and 7-acetamido-metaboli-
tes up t o 700 ng of e a c h i n j e c t e d . D e t e c t i o n
l i m i t s r a n g e from 20-100 ng and t h e r e c o v e r y
of t h e added compounds i s 80%.
H a r z e r and B a r c h e t ( 4 2 ) have d e s c r i b e d a
method f o r t h e a n a l y s i s of n i t r a z e p a m and
o t h e r b e n z o d i a z e p i n e s and t h e i r h y d r o l y s i s
p r o d u c t s , namely; benzophenones, by r e v e r s e d -
p h a s e HPLC. The method i s a p p l i e d t o t h e
a n a l y s i s of e x t r a c t s from blood and u r i n e .
The method i s based on t h e s e p a r a t i o n , by
HPLC on a column (25 cm x 4 mm) of LiChrosorb
SI-100 ( g r a i n s i z e 10 um), o p e r a t e d a t room
t e m p e r a t u r e and 750 p . s . i . w i t h aqueous
methanol ( 6 0 t o 100% of methanol) a s t h e
m o b i l e p h a s e a t t h e r a t e of 0.75 m l p e r
m i n u t e . Another p r o c e d u r e h a s been r e p o r t e d
( 4 3 ) f o r t h e a n a l y s i s of b e n z o d i a z e p i n e s ,
i n c l u d i n g n i t r a z e p a m , and t h e i r m e t a b o l i t e s ,
by enzymic d i g e s t i o n and high-performance
l i q u i d chromatography. The p r o c e d u r e i n -
v o l v e s t h e l i b e r a t i o n and e x t r a c t i o n of t h e
d r u g s a n d / o r m e t a b o l i t e s , w i t h e t h e r . The
e t h e r e x t r a c t i s d r i e d and e v a p o r a t e d and t h e
r e s i d u e i s d i s s o l v e d i n anhydrous e t h a n o l .
Few m i c r o l i t r e s of t h e e t h a n o l i c s o l u t i o n
a r e s u b m i t t e d t o HPLC on a a column (150 mm
x 4.6 mm) packed w i t h Spherisorb-5-ODS and
o p e r a t e d w i t h 0.025 M Na2HP0 -methanol ( 2 : 3 ) ,
4
a d j u s t e d t o pH 7 . 8 , as m o b i l e p h a s e , a t a
r a t e of 1 m l p e r m i n u t e . The d e t e c t i o n i s
done by UV s p e c t r o s c o p y a t 254 nm.
6.34 Gas L i q u i d Chromatography
GLC h a s been e x t e n s i v e l y used a s a method

f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m i n phar-
maceutical preparation; a l s o f o r t h e deter-
m i n a t i o n of t h e d r u g and i t s m e t a b o l i t e s 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 . F u r t h e r m o r e ,
GLC i s one of t h e most c o n v e n i e n t methods
f o r t h e d e t e c t i o n and d e t e r m i n a t i o n of n i t r a -
zepam i n t o x i c o l o g i c a l s c r e e n i n g . The d r u g
is chromatographed w i t h o u t d e r i v a t i s a t i o n o r
a f t e r a c i d h y d r o l y s i s i n t o 2-amino-5-nitro-
benzophenone. The g a s l i q u i d chromatographic
c o n d i t i o n s are given i n Table 3 .
Stationary
phase
Detect o r
Table 3
: a r r i e r Zolumn
:as temper-
0
i t u r e ,C
--Jr
Remarks* Refer-
2% of OV-17 on Flame 260

N2
Chromosorb G-Hr i o n i s a t i o n
3% of OV-1 on Flame 245 - 45
N2
Chromosorb Q ionisat ion
(60 t o 8 0 mesh)
3% of OV-1 on Flame 250 For t h e d r u g 46

ionisat ion N2
Chrom Q ( l O 0 t o & i t s meta-
120 mesh) bolites
210 For t h e hy-
drolysis
~ ~ ~~~ ~ ~
products
3% of OV-17 on 6 3 ~elec- ,r-CH 245 After acid 47
4
Diatomate CQ(8( t r o n c a p t u r e 9:l) hydrolysis
t o 100 mesh)
3% of OV-17 o r 63Ni elec- 245 a s hydroly- 48

‘2
Sp-2250 on t r o n capt- t i c product
shromosorb W ur e
275
o r Supelcoport
(100 t o 120
mesh) -----L-
NlTRAZEPAM 511
'
Stationary Refer-
phase 5nce
3% of OV-17 on 6 3 N i e l e c t - A r 235 a f t e r acid 49

Gas-Chrom Q ( 6 0 r o n cap- h y d r o 1y s i s
t o 80 mesh) ture
,
3.8% o f SE-30 Flame He 240 a f t e r acid 50
ionisat ion hydrolysis
2% of OV-17 on 6 3 N i e l e c t - He 27 5 - 51
c hromosorb ron capture
W.H.P. (80 t o
1 0 0 mesh)
* U n l e s s o t h e r w i s e s t a t e d i n t h e r e m a r k s , t h e d r u g h a s been
determined underivatised.
Lafargue,eta1(24)have r e p o r t e d a gas c h r o m a t e
g r a p h i c metFod f o r t h e i d e n t i f i c a t i o n of
n i t r a z e p a m i n t h e g a s t r i c f l u i d . The l a t t e r
i s e x t r a c t e d a f t e r b e i n g made n e u t r a l o r
weakly a c i d i c , w i t h e t h e r . The extract i s
t h e n examined by GLC i n a 2-metre column
packed w i t h 3% of OV-17 on G a s Cbrom Q ( 1 g O
t o 120 mesh) and o p e r a t e d a t 250 ( o r 210
f o r the hydrolysis products).
6.4 Polarography-
S e v e r a l methods have been p u b l i s h e d f o r t h e d e t e r -

m i n a t i o n of n i t r a z e p a m and r e l a t e d d e r i v a t i v e s i n
pharmaceutical formulations as w e l l as i n biologi-
c a l f l u i d s , ( b l o o d , u r i n e , and serum).
O e l s c h l a e g e r , e t a 1 (52) had r e p o r t e d a p r o c e d u r e
f o r t h e d e t e r m i n a t i o n of n i t r a z e p a m a f t e r t h e d r u g
i s s e p a r a t e d by a TLC on s h e e t s h a v i n g s i l i c a g e l
o r a l u m i n a a s a d s o r b e n t on a polyethylenetetra-
p h t h a l a t e b a c k i n g w i t h o u t removal of t h e p l a s t i c f o i l
512 HASSAN Y . ABOUL-ENEIN el al.
'Ihe p l a s t i c f o i l is s t a b l e t o all s o l v e n t s used and tk bind-

er and t h e s o r b e n t d o n o t i n t e r e f e r e w i t h t h e develop-
ment of t h e c u r r e n t v e r s u s p o t e n t i a l c u r v e . Any
z i n c - c o n t a i n i n g i m p u r i t i e s d o , however, i n t e r f e r e
and must b e marked w i t h EDTA b e f o r e measurement. A
dropping-mercury e l e c t r o d e i s used i n t h e determin-
a t i o n i n which n i t r a z e p a m and i t s 7-amino r e d u c t i o n
product are determined. Dimethyl s u l f o x i d e and
dimethylformamide a r e used as s o l v e n t s . The r e c o v e r y
is more t h a n 95%. For t h e amino compound, good
r e c o v e r y i s achieved o n l y i f t h e s o r b e n t i s removed;
t h i s i s not necessary f o r nitrazepam.
E l l a i t h y , e t a 1 ( 5 3 ) , had r e p o r t e d t h e d e t e r m i n a t i o n
of some b e n z o d i a z e p i n e s , among which n i t r a z e p a m i s
i n c l u d e d , by d i f f e r e n t i a l p u l s e polarography w i t h
a dropping-mercury i n d i c a t o r e l e c t r o d e and a s a t u -
r a t e d mercuric sulphate r e f e r e n c e electrode. The
c a l i b r a t i o n g r a p h of peak c u r r e n t v e r s u s drug con-
c e n t r a t i o n i s r e c t i l i n e a r f o r c o n c e n t r a t i o n down t o
0.14 ug p e r m l . Nitrazepam i s d i s s o l v e d i n ace-
t o n i t r i l e and t h e s o l u t i o n is b u f f e r e d a t pH 4 . 8 .
The method i s a p p l i c a b l e f o r t h e d e t e r m i n a t i o n of
n i t r a z e p a m and some o t h e r b e n z o d i a z e p i n e s i n u r i n e
( 2 ml) w i t h o u t p r i o r e x t r a c t i o n .
Halvorsen, e t a1 (54) have r e p o r t e d t h e e l e c t r o -

r e d u c t i o n and p o l a r o g r a p h i c d e t e r m i n a t i o n of n i t r a -
zepam i n serum. The e l e c t r o - r e d u c t i o n of nitrazepam
h a s been s t u d i e d by p o l a r o g r a p h y , c y c l i c v o l t a -
mmetry, chromopotentiometry and c o n t r o l l e d - p o t e n t i a l
coulometry. I n a phosphate b u f f e r s o l u t i o n of pH
6.9 t h e r e a r e two r e d u c t i o n s t e p s ; t h e f i r s t g i v i n g
a well-defined p o l a r o g r a p h i c wave b e i n g a f o u r -
e l e c t r o n r e d u c t i o n of t h e n i t r o - g r o u p and t h e
second b e i n g a two-electron r e d u c t i o n . The o x i d i s e d
form of n i t r a z e p a m i s s t r o n g l y adsorbed on t h e
e l e c t r o d e s u r f a c e and t h u s i t i s p o s s i b l e t o d e t e r -
mine n i t r a z e p a m i n t h e p r e s e n c e of p r o t e i n s .
The p o l a r o g r a p h i c d e t e r m i n a t i o n of n i t r a z e p a m i n
whole blood, i n a c u t e p o i s o n i n g , h a s been r e p o r t e d
(55). The procedure i s based on a d m i n i s t r a t i o n of
n i t r a z e p a m t o r a t s and t h e homogenised blood samples
a r e d i l u t e d w i t h an e l e c t r o l y t e c o n s i s t i n g of 1:l
m i x t u r e of methanol w i t h Britton-Robinson b u f f e r
s o l u t i o n of pH 2 . 2 t o 3.3. The s o l u t i o n s a r e
examined p o l a r o g r a p h i c a l l y i n t h e r a n g e 0.0 to0.6V.
NITRAZEPAM 513
The p o l a r o g r a p h i c and s p e c t r a l b e h a v i o u r of 7-amino

and 7-acetamido n i t r a z e p a m m e t a b o l i t e s have been
u t i l i s e d t o e f f e c t s e p a r a t i o n s of m i x t u r e s ( 5 6 ) .
Changes of UV a b s o r p t i o n s p e c t r a w i t h pH in s o l u t i o n
a r e used t o d e t e r m i n e pKa values f o r n i t r a z e p a m
.
m e t a b o l i tes 7 -Ace t a m i d o -n it r a z epam gives t w o
pKa v a l u e s , c o r r e s p o n d i n g t o p r o t o n a t i o n i n a c i d
and d e p r o t o n a t i o n of t h e n e u t r a l m o l e c u l e i n a l k a l -
i n e media. 7 - h i n o n i t r a z e p a m g i v e s t h r e e pKa
v a l u e s , t h e t h i r d one being due t o a d d i t i o n a l
p r o t o n a t i o n i n a c i d media. The s p e c t r a a r e e x p l a i n -
ed by c o n s i d e r i n g them t o b e superimposed s p e c t r a of
t h e two benzene r i n g s , one m o n o s u b s t i t u t e d , and o n e
t r i s u b s t i t u t e d w i t h i n t h e molecule. D i f f e r e n c e s i n
t h e pK v a l u e s o r t h e p o l a r o g r a p h i c b e h a v i o u r b e t w e e n
n i t r a z g p a m and i t s m e t a b o l i t e s a r e used t o e f f e c t
n o v e l s e p a r a t i o n a f t e r s o l v e n t e x t r a c t i o n s from
aqueous b u f f e r e d s o l u t i o n s .
ACKNOWLEDGEMENT
The a u t h o r s w i s h t o t h a n k M r . Dennis Charkowski, o f t h e

Toxicology C e n t e r , The U n i v e r s i t y of Iowa, Iowa C i t y , Iowa
52242, U.S.A., f o r d e t e r m i n i n g t h e mass spectrum of n i t r a -
zepam, and M r . E s s a m A. L o t f i f o r h i s h e l p i n t h e l i b r a r y
research.
A sample of nitrazepam-R04-5360/000, w a s k i n d l y d o n a t e d
by D r . R. Amrein and D r . S. Kessler of F. Hoffmann - L a Roche
& Co. L i m i t e d , B a d e , S w i t z e r l a n d .
514 HASSAN Y. ABOUL-ENEIN er ai.
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NITRAZEPAM 515
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NITRAZEPAM 517
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NITROGLYCERIN
Edward F. McNiff, Peter S. K. Yap, and

Ho- Leung Fung
1. Description 520
2.5 Vapor Pressure and Boiling Point 523
2.6 Melting and Crystal Properties 523
2.7 Density 523
2.8 Viscosity 523
2.9 Solubility 524
3. Synthesis 524
4. Stability 525
4.1 Chemical Stability 525
4.2 Physical Stability 527
5. Metabolism 527
5.1 Biochemistry 527
5.2 Site of Metabolism 528
5.3 Metabolic Fate 529
6. Pharmacokinetics 53 1
6.1 Tissue Distribution 53 1
6.2 Intravenous Administration 532
6.3 Oral and Topical Administration 532
7.1 Official Methods 533
7.2 Spectrophotometnc 533
7.3 Thin Layer chromatography 534
7 . 4 Polarography 534
7.5 Gas Chromatography 535
7.6 High Performance Liquid Chromatography 535
8. References 531
Copyrighi1 0 1960 by Academic Ress, Inc.

Analytical Profiles of Drug Substances, 9 AU rights of reproduction in any form reserved.
519 ISBN: &12-260809-7
5 20 EDWARD F. McNIFF, et u1.
1 . Description
1 . 1 Name, Formula, Molecular Weight
Nitroglycerin (glyceryl trini t r a t e , t r i n i t r o g l y c e r o l )
i s 1,2,3-propanetriol t r i n i t r a t e .
C3H5N309 CH20N02
M.W. 227.09
I
~HONO~
I
CH20N02

Pale yellow, odorless, o i l y liquid w i t h a sweet,
burning t a s t e .
An NMR sDectrum of nitroalvcerin i s shown i n Fia. 1 .
The sample was isolated from a l a h o s e adsorbate by eth&
extraction. After solvent evaporation, the sample was p u r i -
fied by hexane elution from a neutral s i l i c a column, followed
by gentle hexane evaporation under a stream of nitrogen. A
CDC13 solution spectrum was r u n on a Varian T-60A spectrom-
eter using trimethylsilane as the internal reference. The
multiplet a t - 4.8 6 i s assigned t o the four protons a t the
1 and 3 carbon atoms, and t h a t a t - 5.5 6 i s assigned t o the
proton a t the C - 2 position. Small s i n g l e t s appearing a t - 1 . 5
6 and - 7 . 2 6 a r e apparently due t o t r a c e impurities of
hexane residue and non-deuterated chloroform, respectively.
The integrated areas c o r r e l a t e well w i t h the structural
assignments. No attempts were made t o i n t e r p r e t the s p l i t -
t i n g patterns.
2 . 2 Infrared Spectrum
The IR spectrum, F i g . 1 , was obtained on a Perkin-
Elmer model 272B infrared spectrophotometer. Nitroglycerin
was isolated and purified as described i n 2.1. Sample
m o u n t i n g was by formation of a c a p i l l a r y film of neat n i t r o -
glycerin between NaCl plates. The band a t 850 cm-l i s found
i n a l l organic and inorganic n i t r a t e s . Bands a t 1650 and
1280 cm-’ a r e attributed t o symmetrical stretching and de-
formation vibrations of the NO2 group, respectively. T h i s
spectrum compares well w i t h one t h a t was reported along w i t h
a large number of other n i t r a t e s by Pristera e t a l l .
I , . . . : . . . I . . 1 . . .
1 . . . . 1 . . . . 1 . . . . 1 . . . . 1 . , . . 1
I . . . 1 I . . . .. . .l
1 I . . ' .
. . . . I . . . . I ,
, I
80 70 LO 50 ppu 1 .O 10 20 I 0 0
F i g . 1: NMR Spectrum of n i t r o g l y c e r i n
ii
..
hl
NITROGLYCERIN 523
2.3 U 1 t r a v i o l e t Spectrum
Nitroglycerin, i n a solution of neutral pH, has no
appreciable absorbance i n the near u l t r a v i o l e t and v i s i b l e
region2.
2.4 Mass Spectrum

The mass spectra of 21 nitrate e s t e r s , including
nitroglycerin, were run on an A.E.I. MS2H single focusing
mass spectrometer, operating a t 70 ev3. The base peak o f M/e
46 (NO$) i s c h a r a c t e r i s t i c o f the lower n i t r a t e s . Other
major peaks, w i t h t h e i r corresponding structural assignments,
a r e shown i n Table I :
TABLE 13
Mass Spectral Characteristics o f Nitroglycerin

-
M/ e Relative Intensity S tructura 1 Assi gnment
28 6 cot
29 15 CHO'
30 24 NO'
43 5
46 100
76 9
2.5 Vapor Pressure and Bioling P o i n t
The vapor pressure of nitrogl cerin a t Z O O , 25O and
37O has been reported4y5 as 2.6 x lo-', 5.5 x and 2 . 2 x
Torr, respectively. The gravimetric Knudson effusion
technique has been used t o study the vapor pressure of nitro-
glycerin i n molded t a b l e t s 6 .
Pure nitroglycerin has an a parent boiling p o i n t of
1450 C ( w i t h violent decomposition) . 1:
2.6 Melting and Crystal Properties
A t low temperatures, nitroglycerin e x i s t s i n two
crystal forms. I t freezes t o form a s t a b l e dipyramidal poly-
morph which melts a t 13.20 C . Under some conditions, an
unstable t r i c l i n i c crystal (m.p. 2 . 2 0 C ) may form. This
l a b i l e polymorph will convert into the more s t a b l e form upon
standi n g l .
2.7 Density
The density of nitroglycerin i s 1.601 a t 15' C4.
2.8 Viscositv7
524 EDWARD F. McNIFF, et N/
Viscosity ( c P )
- Temperature (OC)
35.5 20
21 .o 30
9.4 50
6.8 60
2.9 Solubility
The following information i s available from r e f e r -
ence 7: nitroglycerin has an aqueous s o l u b i l i t y o f 1.73 and
2.46 mg/ml a t 200 and 600 C respecti'vely; ethanol dissolves
nitroglycerin t o the extent of 375 mg/gm a t Oo and 540 mg/gm
a t 200; h o t ethanol i s miscible w i t h nitroglycerin i n a l l
proportions; other solvents completely miscible w i t h n i t r o -
glycerin are: acetone, ether, glacial a c e t i c acid, ethyl-
acetate, benzene, toluene, phenol, ni trobenzene, chloroform,
ethylene chloride and n i t r i c e s t e r s . Additionally, i t has
been reported4 t h a t nitroglycerin i s miscible with pyridine
and ethylene bromide, b u t i s only sparingly soluble in
petroleum ether, liquid petrolatum and glycerol. The solu-
b i l i t y in methanol and carbon d i s u l f i d e i s 56 mg/gm and 8.3
mg/gm
- - respectively .
Using the- aqueous sol u b i 1 i ty o f nitroglycerin and
partitioning data, Horhota and Fung8 calculated n i t r o
glycerin s o l u b i l i t y i n d i f f e r e n t water-polyethylene g ycol
400 co-solvent systems. For instance, the calculated sol u-
b i l i t y of nitroglycerin i n a 90% (w/v) polyethylene g ycol
400-water mixture was estimated a t 135 mg/ml.
3. Synthesis
Organic n i t r a t e synthesis i s commonly accomplished by
e s t e r i f i c a t i o n of the corresponding a l c ~ h o l , ~l o, .~ In the
case of nitroglycerin, the n i t r a t i n g mixture consists of
equal volumes of n i t r i c and s u l f u r i c acids. A small amount
o f urea o r urea n i t r a t e i s added as a scavenger f o r any
excess nitrous acid present. Esterification i s carried out
by slow addition of glycerol t o the mixed acids.
NO; + R-OH -3 R-O-N02 + H

+
Careful control o f temperature and r a t e of addition reduces

or eliminates the side reaction of alcohol oxidation. The
e s t e r can be separated by p o u r i n g the reaction mixture i n t o
cold water o r by careful d i s t i l l a t i o n .
NITROGLYCERIN 525
4. Stability
4.1 Chemical S t a b i l i t y
4.11 Hydrolysis
The s tabi 1 i ty of nitroglycerin i n a1 coho1 i c so-
lutions as a function of pH has been studied by Arnshlerll.
The compound i s r e l a t i v e l y s t a b l e i n neutral and weakly
acidic solutions b u t degrades very rapidly i n the presence
o f a1 kal i’* 3 3 .
Alkaline hydrolysis o f n i t r a t e e s t e r s can pro-
ceed v i a three possible mechanisrnsl4:
( a ) Nucleophilic substitution ( S N ~ )
CH2R
0HniH2- 0.NO2-+R.CH2-CH2-OH + NO; (alcohol t

nitrate)
( b ) 6-hydrogen elimination ( E 2 )
I
RCH-CH2--U.N02+H20 + R C H : C H ~ + NO; ( o l e f i n +
nitrate)
-
( c ) a-hydrogen elimination (ECo2)
OH
nH
1
3
RCH2*CH-0-NO2+ H20 + RCH2 * C H O + NO;
(carbonyl t n i t r i t e )
The i n i t i a l step of alkaline hydrolysis of
nitroglycerin involves a-elimination a t the secondary
n i t r a t e g r o u p resulting in the formation o f n i t r i t e ion and
a carbonyl . T h i s electronegative carbonyl g r o u p causes
e i t h e r of the remaining primary n i t r a t e s t o be more suscep-
t i b l e t o nucleophilic attack. A slower reaction on the
primary n i t r a t e , producing the alcohol and n i t r a t e ion,
becomes more important w i t h increasing r a t i o s o f hydroxide
ion t o nitroglycerin2. Alkaline degradation of n i t r o -
glycerin i s accompanied by the appearance and subsequent
disappearance of an u l t r a v i o l e t absorption peak near 335 nm
due, presumably, t o the monocarbonyl intermediate. The
maximum absorbance and the peaking time of this chromophore
are dependent upon i n i t i a l concentrations o f nitroglycerin
and hydroxide ion. T h i s reaction i s the basis f o r a kinetic
526 EDWARD F. McNIFF, e r a / .
assay procedure f o r n i t r o g l y c e r i n l 5 - 1 7 which i s discussed

1a t e r.
Acid c a t a l y z e d h y d r o l y s i s o f n i t r o g l y c e r i n was
found t o occur a t a much slower r a t e than t h a t o f a l k a l i n e
h y d r o l y s i s l ' + , 1 * . I n c u b a t i o n o f n i t r o g l y c e r i n a t 370 f o r 15
minutes i n 4 N NaOH r e s u l t e d i n e s s e n t i a l l y complete d e n i t r a -
t i o n , w h i l e i n 4 N HC1, n i t r o g l y c e r i n was degraded o n l y 28%
a f t e r 6 h o u r s l s . Under a c i d c o n d i t i o n s , t w i c e as much
g l y c e r y l - 1 , 2 - d i n i t r a t e i s formed compared t o g l y c e r y l - l , 3 -
d i n i t r a t e l g , suggesting t h a t t h e i n i t i a l r e a c t i o n s i t e i s on
t h e p r i m a r y n i t r a t e . The k i n e t i c s o f n i t r o g l y c e r i n h y d r o l y -
s i s i n n i t r i c a c i d a t 200 t o 800 C has a l s o been studied20.
Klason and Carlson21 observed t h a t a1 k a l i n e
degradation o f n i t r o g l y c e r i n i n t h e presence o f phenylmer-
captan r e s u l t e d i n t h e f o r m a t i o n o f d i p h e n y l d i s u l f i d e and
g l y c e r o l . I t was l a t e r shown t h a t reduced g l u t h a t h i o n e (GSH)
r e a c t s w i t h n i t r o g l y c e r i n t o produce i n o r g a n i c n i t r i t e ions22.
Subsequent s t ~ d i e s c~h a ~ r a, c~t e~r i z e d t h e r e a c t i o n as:
C3H5(0N02)3 f 2GSH-+C3H5(0N02)2 OH + GSSG f HN02
T h i s r e d u c t i o n process was found t o be r e l a t i v e l y slow. With

equal and 10 molar e q u i v a l e n t s o f GSH, 0 and 22% o f n i t r o -
g l y c e r i n were degraded w i t h i n 1 hour a t 37O C, r e s p e c t i v e l y .
B i o t r a n s f o r m a t i o n o f n i t r o g l y c e r i n i n t h e body i s a p p a r e n t l y
c l o s e l y r e l a t e d t o t h e above r e a c t i o n . The - i n_v_
i v o process,
however, i s a much f a s t e r r e a c t i o n because i t i s enzymatic-
a l l y c a t a ysed.
4 12 P h o t o l y t i c and Thermal S t a b i l i t y
Although i t was suggested t h a t n i t r o g l y c e r i n i s
suscepti b e t o p h o t ~ l y s i s ~t h~e,r e i s no s u p p o r t i n g evidence
i n t h e li e r a t u r e . I n aqueous s o l u t i o n , exposure t o l i g h t
does n o t ead t o a c c e l e r a t e d disappearance o f n i t r o g l y c e r -
i$ 6 .
The thermal decomposition o f n i t r o g l y c e r i n i s
h i g h l y dependent on t h e r a t i o o f n i t r o g l y c e r i n mass t o t h e
volume o f t h e r e a c t i o n presumably due t o p r o d u c t
i n h i b i t i o n by NO2. W i t h i n t h e temperature range o f 140° t o
160' and a mass t o volume r a t i o o f 3.5 x gm vapor
phase degradation f o l l o w s f i r s t o r d e r k i n e t i c s and obeys t h e
Arrhenius r e l a t i o n s h i p w i t h an energy o f a c t i v a t i o n (Ea) o f
approximately 36 kcal/mole. D e v i a t i o n from f i r s t o r d e r
k i n e t i c s i s observed i n t h e l i q u i d phase, and i s p r o b a b l y
due t o a u t o c a t a l y t i c e f f e c t s 2 7 . Below 1400, t h e decomposi-
t i o n r e a c t i o n s a r e a l s o a f f e c t e d by a u t o c a t a l y s i s 2 * .
NITROGLYCERIN 527
4.2 P h y s i c a l S t a b i l i t y
I n s t a b i l i t y o f n i t r o g l y c e r i n i n pharmaceutical
dosage forms can g e n e r a l l y be a t t r i b u t e d t o two processes,
v i z : ( a ) v o l a t i z a t i o n l e a d i n g t o l o s s o f d r u g t o t h e atmos-
phere,and ( b ) s o r p t i o n o f d r u g t o p l a s t i c s . The a p p r e c i a b l e
v o l a t i l i t y o f n i t r o g l y c e r i n a t room temperatures has been
shown t o be a m a j o r cause o f l o s s o f potency and i n t e r -
t a b l e t m i g r a t i o n o f drug d u r i n g storage o f u n s t a b i l i z e d
sub1 i n g u a l tablet^^,^^. T h i s problem has been somewhat
a l l e v i a t e d by t h e a d d i t i o n o f p o l y e t h y l e n e g l y c o l 400 and
povidone as s t a b i 1 izers 3 0 - 3 4 . Drug 1oss due t o s o r p t i v e
phenomena has been imp1 i c a t e d when n i t r o g l y c e r i n t a b l e t s
a r e s t o r e d i n p l a s t i c c o n t a i n e r s and u n i t dose s t r i p pack-
a g e ~ ~ ~FDA ' ~r e ~ g u .l a t i o n s (promulgated i n 1972)36 r e q u i r e
t h a t n i t r o g l y c e r i n t a b l e t s be packaged i n t i g h t c o n t a i n e r s ,
p r e f e r a b l y o f g l a s s w i t h metal screw caps, and dispensed i n
t h e o r i g i n a l , unopened c o n t a i n e r w i t h a s p e c i a l warning
l a b e l . No more t h a n 100 t a b l e t s s h o u l d be dispensed i n each
container.
Problems o f s t a b i l i t y and potency r e l a t i n g t o
extemporaneously prepared n i t r o g l y c e r i n i n f u s i o n s have r e -
c e n t l y been p o i n t e d Extensive loss o f n i t r o g l y c e r i n
from i n t r a v e n o u s s o l u t i o n s s t o r e d i n p l a s t i c i . v . bags can
be a t t r i b u t e d t o s o r p t i ~ n ~ ~ ,s i~n c~e -i n~ t a~ c,t d r u g can be
recovered from t h e c o n t a i n e r 4 0 . P l a s t i c t u b i n g used f o r t h e
a d m i n i s t r a t i o n o f intravenous n i t r o l y c e r i n s o l u t i o n s a l s o
causes d r u g l o s s due t o s ~ r p t i o n ~ ~ High , ~ ~ d. e n s i t y p o l y e t h y -
l e n e t u b i n g , however, i s n o n - a d s o r p t i ~ e ~ ~ .
5. Metabolism
The metabol ism o f n i t r o g l y c e r i n and o t h e r o r g a n i c n i t r a t e s
has been e x t e n s i v e l y r e ~ i e w e d ~ ~ Only - ~ ~ a. summary o f t h e
m a j o r f i n d i n g s r e g a r d i n g t h e metabolism o f n i t r o g l y c e r i n i s
presented here.
5.1 B i o c h e m i s t r
Heppel and i i l m o e 2 2 showed t h a t t h e spontaneous r e a c -
t i o n between n i t r o g l y c e r i n and GSH t o be c a t a l y z e d by a hog
1 i v e r microsomal enzyme. I n i t i a l c h a r a c t e r i z a t i o n o f t h e
enzymatic process u s i n g p a r t i a l l y p u r i f i e d hog l i v e r acetone
powder demonstrated t h a t t h e system i s anaerobic, has an
o p t i m a l pH o f 7-8, i s i n h i b i t e d by c u p r i c s u l f a t e and s t i m u -
l a t e d by cyanide. Subsequent i n v e s t i g a t i o n s showed t h a t t h e
l i v e r enzymes, p u r i f i e d f r o m r a t and guinea p i g l i v e r and
named o r g a n i c n i t r a t e reductases (ONR), c o n s i s t e d o f 2
d i s t i n c t fragments w i t h d i f f e r e n t a c t i v i t y f o r n i t r o g l y c e r i n
and o t h e r o r g a n i c n i t r a t e s 4 7 ,4*. The two d i f f e r e n t enzymes
were e s t i m a t e d t o have a m o l e c u l a r w e i g h t o f 14,000 and
528 EDWARD F. McNIFF, er al.
43,700 r e s p e c t i v e l y .
Needleman and Hunter23 developed a r a p i d and s e n s i -
t i v e enzymatic assay t o q u a n t i f y t h e r e l a t i v e a c t i v i t i e s o f
r a t l i v e r ONR toward d i f f e r e n t o r g a n i c n i t r a t e s . T h i s assay
measures t h e disappearance o f reduced tri phosphopyridi ne
n u c l e o t i d e (TPNH), which i s consumed f o r t h e p r o d u c t i o n o f
GSH, which i n t u r n i s r e q u i r e d f o r t h e d e n i t r a t i o n o f t h e
organic n i t r a t e ( F i g . 3 ) .
ORGANIC
ORGANIC NITRATE GLUTATH IONE

REDUCTASE REDUCTME
F i g u r e 3. Biochemical r e a c t i o n s i n v o l v e d i n t h e d i n i t r a t i o n
o f organic n i t r a t e s .
The maximum v e l o c i t i e s o f t h e enzymatic r e a c t i o n o f

d i f f e r e n t organic n i t r a t e has been r e p o r t e d 2 3 . P o l y n i t r i c
e s t e r s a r e r a p i d l y metabolized by l i v e r ONR, i n t h e o r d e r o f
manni t o 1 hexani t r a t e >> e r y t h r i t o l t e t r a n i t r a t e >> n i t r o -
g l y c e r i n . Replacement o f a n i t r a t e group w i t h a hydrogen
atom o r a hydroxy group, o r i n t r o d u c t i o n o f an e t h e r l i n k a g e
i n t o a l i n e a r c h a i n n i t r a t e compound, decreases t h e r a t e o f
enzymatic t r a n s f o r m a t i o n . Branched c h a i n a l c o h o l n i t r a t e s
a r e also s i g n i f i c a n t l y l e s s s u s c e p t i b l e t o organic n i t r a t e
reductase degradation. I n v i t r o s t u d i e s have a1 so demon-
s t r a t e d t h a t t h e metabolism o f n i t r o g l y c e r i n i n l i v e r
homogenates can be enhanced o r depressed upon p r e t r e a t m e n t
o f t h e experimental animals w i t h b a r b i t u r a t e s and bromoben-
zene r e s p e c t i ~ e l y ~ ~ I, t~ was ~ . suggested51 t h a t pheno-
b a r b i t a l pretreatment caused increase i n t h e amount o f reduc
tase enzyme as w e l l as t h e a c t i v i t y o f GSH g e n e r a t i n g
capacity.
5.2 S i t e o f Metabolism
Needleman and H a r k e ~compared ~ ~ t h e r a t e o f degrada-
t i o n o f n i t r o g l y c e r i n i n i s o l a t e d perfused r a t l i v e r t o t h e
-i n_v_ i v o b i o t r a n s f o r m a t i o n r a t e s . The i n v i t r o h a l f t i m e o f
2‘ minutes was comparable t o t h a t observed i n i n t a c t e x p e r i -
mental animals. I n e v i s c e r a t e d r a t s , t h e b i o l o g i c a l h a l f -
NITROGLYCERIN 529
l i f e o f n i t r o g l y c e r i n was 7 t o 8 minutes as compared t o l e s s

than 2 minutes i n c o n t r o l s 5 2 . These experiments c l e a r l y
e s t a b l i s h t h e importance o f h e p a t i c metabolism o f n i t r o -
g l y c e r i n i n experimental animals. Recently, Maier e t a l S 3
showed t h a t a r e l a t i o n s h i p e x i s t s between i n v i v o n i t r o -
g l y c e r i n b i o a v a i l a b i l i t y (100 mg/kg o r a l l y 7 n x s ) and t h e
--
i n v i t r o l i v e r ONR a c t i v i t y i n i n d i v i d u a l animals.
Glutathione-dependent ONR a c t i v i t y was found o n l y i n t r a c e
q u a n t i t i e s i n t h e kidney and was absent i n t h e lung, small
i n t e s t i n e , h e a r t and s k i n . These d a t a suggested t h a t f i r s t -
pass metabolism of o r a l l y administered n i t r o g l y c e r i n occurred
primarily i n the l i v e r .
Under p h y s i o l o g i c a l c o n d i t i o n s , r a t serum a l s o
hydrolyzed n i t r o g l y c e r i n t o d i n i t r a t e s and mononitrates, b u t
a t a much slower r a t e . The h a l f - l i f e o f serum degradation
was found t o be 15 t o 20 minutes a t 37O C 2 4 9 5 4 . The e f f e c t s
of c o n c e n t r a t i o n , temperature, r e d blood c e l l hemolysi s and
s i l v e r n i t r a t e a d d i t i o n on n i t r o g l y c e r i n s t a b i l i t y i n human
and r a t plasma have a l s o been examineds5. W i t h i n t h e tempera-
t u r e range o f -200 t o 370 C, degradation was shown t o obey
t h e Arrhenius r e l a t i o n s h i p w i t h an apparent energy o f
a c t i v a t i o n (Ea) o f 24.1 and 19.0 kcal/mole f o r human and r a t
plasma, r e s p e c t i v e l y . Depending w o n t h e temperature,
n i t r o g l y c e r i n i s degraded 10-50 times f a s t e r i n r a t plasma
compared t o human plasma.
Hepatic and blood metabol ism o f n i t r o g l y c e r i n has
been demonstrated i n o t h e r animal speciess6. Human 1 i v e r
biopsy samples were shown t o c o n t a i n a g l u t a t h i o n e dependent
ONR capable o f r a p i d b i o t r a n s f o r m a t i o n o f n i t r o g l y c e r i n t o
i t s lower n i t r a t e s 5 7 . The s i t e and mechanism o f o x i d a t i o n
o f n i t r o g l y c e r i n t o carbon d i o x i d e has a l s o been i n v e s t i -
gated58. I n v i t r o experiments demonstrated t h a t homogenates
o f the liv%,kidney, b r a i n and s k e l e t a l muscle o x i d i z e d
g l y c e r o l , a m e t a b o l i t e o f n i t r o g l y c e r i n , t o C02 b u t c o u l d n o t
o x i d i z e n i t r o g l y c e r i n t o C02. E v i s c e r a t i o n o f r a t s i n h i b i t e d
C02 p r o d u c t i o n a f t e r t h e a d m i n i s t r a t i o n o f n i t r o g l y c e r i n b u t
n o t g l y c e r o l . Pretreatment o f t h e rodents w i t h p h e n o b a r b i t a l
o r SKF 525A had no e f f e c t on n i t r o g l y c e r i n o x i d a t i o n , nor
was t h e r e an enhancement o f C02 r e l e a s e i n n i t r o g l y c e r i n -
t o l e r a n t animals. Thus, C02 p r o d u c t i o n may have r e s u l t e d
from b i o t r a n s f o r m a t i o n a t e x t r a h e p a t i c s i t e s subsequent t o
hepatic d e n i t r a t i o n o f n i t r o g l y c e r i n .
5.3 Metabolic Fate

Upon o r a l a d m i n i s t r a t i o n o f 10 mg/kg o f 1,3-C14
n i t r o g l y c e r i n t o r a t s s 9 20% o f t h e l a b e l e d dose was e x p i r e d
as carbon d i o x i d e w i t h an equal amount o f t h e r a d i o a c t i v i t y
e x c r e t e d i n t h e u r i n e a t t h e end o f 4 hours. TLC-radio-
530 EDWARD F. McNIFF, er ol.
chromatographic analysis revealed t h a t the cumulative urinary

excretion consisted of 7% glycerol , 1 % glyceryl-1 , Z - d i n i t r a t e ,
0.5% glyceryl-1 , 3 - d i n i t r a t e Y 4% glyceryl mononitrates and 8%
of unidentified water soluble metabolites. Needleman e t
a1 5 * administered a smaller dose ( 5 mg/kg) of radioactive
nitroglycerin subcutaneously t o rats. They observed t h a t 17%
of the dose was eliminated as expired C O Z Y w i t h urinary
excretion accounting f o r another 50% of the r a d i o a c t i v i t y in
0-24 hours. The major urinary metabolites were the glyceryl
mononitrates (32% of dose). The sum of the mononitrates and
water soluble metabol i t e s (unidentified) accounted f o r 80%
of the excreted l a b e l . A small f r a c t i o n of the labelled dose
( 1 .3%) was excreted as unchanged nitroglycerin.
I n a more recent study, Hodgson and Lee60 adminis-
tered a very h i g h oral dose, 180 mg/kg ( L D lo%), of n i t r o -
glycerin t o r a t s . Radioactive C02 accounted f o r 26% of the
dose and 40% of the label was eliminated i n the urine w i t h i n
24 hours. These authors showed (Table 11) t h a t the major
urinary metabolites a r e glyceryl d i n i t r a t e gl ucuronide (14%
of dose) glyceryl mononi t r a t e (1 1 % ) and glycerol ( 7 % ) .
T h i s study was the f i r s t w h i c h showed t h a t conjugation plays
a major r o l e in the metabolism of nitroglycerin.
TABLE 1160
Metabol i t e s of Nitroglycerin i n Rat Urine 24 Hours
After Oral Administration of
IL4C1 Nitroglycerin (180 mg/kg)
Metabolite % of Administered Dose
Nitroglycerin 0.1
Glyceryl-l,3-dini t r a t e 0.4 + O.Za
Glyceryl-1 , Z - d i n i t r a t e 0.7 0.4
Glyceryl mononi t r a t e 10.6 1.3
Glyceryl-l,3-dinitrate glucuronide 3.5 0.4
Glyceryl-1 ,2-di n i t r a t e gl ucuronide 10.0 0.7
Glyceryl mononi t r a t e glucuronide 1 . 5 T 0.2
G1ycerol 6.9 - 0.8
Unidenti f ied =-6
a
+ SE of three r a t s
Mean -
The metabolic f a t e of nitroglycerin i n the r a t can,
therefore, be schematically summarized a s follows ( F i g . 4):
NITROGLYCERIN 53 1
Nitroglycerin
I
G l y c e r y l - l , 3 - D i n i t r a t e Glucuronide
Bile
G1 ycogen Polar
co2
Proteins Components
(Expired
Lipids
Air)
RNA & DNA
Fig. 4 M e t a b o l i c Fate o f N i t r o g l y c e r i n
The m e t a b o l i c f a t e o f n i t r o g l y c e r i n i n man has n o t

been s t u d i e d i n g r e a t d e t a i l . So f a r , o n l y g l y c e r y l mono-
n i t r a t e s have been i d e n t i f i e d a s t h e major u r i n a r y m e t a b o l i t e
o f n i t r o g l y c e r i n i n man61.
6. Pharmacokinet i c s
6.1 Tissue D i s t r i b u t i o n
N i t r o g j y c e r i n i s r a p i d l y and e x t e n s i v e l y d i s t r i b u t e d
i n t h e body. F o l l o w i n g intravenous a d m i n i s t r a t i o n o f r a d i o -
l a b e l e d n i t r o g l y c e r i n i n t h e r a t , Needleman and c o - ~ o r k e r s ~ ~
found t h a t t h e apparent d i s t r i b u t i o n phase o f unchanged
n i t r o g l y c e r i n from blood has a h a l f - l i f e o f l e s s than 20
seconds. T i ssue r a d i o a c t i v i t y was not, however, measured
i n t h e study. D i C a r l o e t a1.59, s t u d i e d t h e d i s t r i b u t i o n o f
CC141 a f t e r o r a l a d m i n i s t r a t i o n (10 mg/kg) o f I C 1 4 ) n i t r o -
g l y c e r i n i n t h e same species. They measured t h e dioxane
e x t r a c t a b l e and n o n - e x t r a c t a b l e r a d i o a c t i v i t y i n t h e t i s s u e s
as a f u n c t i o n o f time. The l i v e r and carcass appeared t o be
t h e major s i t e s o f d i s t r i b u t i o n o f absorbed r a d i o a c t i v i t y .
The h e a r t , lung, kidney and spleen took up o n l y small q u a n t i -
t i e s o f t h e r a d i o - l a b e l . S i g n i f i c a n t accumulation o f non-
e x t r a c t a b l e r a d i o a c t i v i t y was shown i n t h e carcass, l i v e r and
G I t r a c t , suggesting t h a t n i t r o g l y c e r i n and/or i t s b i o t r a n s -
f o r m a t i o n products m i g h t be i n c o r p o r a t e d i n t o t h e t i s s u e s .
532 EDWARD F. McNIFF, e r a / .
This o b s e r v a t i o n agrees w i t h t h a t o f a l a t e r study62 which

showed t h a t t h e r a d i o a c t i v i t y from { 1 4 C ) n i t r o g l y c e r i n c o u l d
be i n c o r p o r a t e d i n t o r a t l i v e r glycogen, l i p i d , p r o t e i n , RNA
and DNA. Hodgson and Lee60 a l s o s t u d i e d t h e d i s t r i b u t i o n o f
{ 1 4 C ) i n t h e r a t a f t e r o r a l a d m i n i s t r a t i o n o f 180 mg/kg o f
r a d i o a c t i v e n i t r o g l y c e r i n i n peanut o i l . They found
however, no accumulation o f absorbed r a d i o a c t i v i t y i n t h e
carcass a f t e r 4 hours p o s t dosing. A t t h e same time, about
60% o f t h e r a d i o a c t i v i t y was detected i n t h e GI t r a c t .
6.2 Intravenous A d m i n i s t r a t i o n
The pharmacokinetics o f n i t r o g l y c e r i n a r e c h a r a c t e r -
i z e d by an extremely r a p i d plasma c l e a r a n c e o f drug. Follow-
i n g intravenous a d m i n i s t r a t i o n i n r a t s (0.35-2.5 mg/k ) ,
plasma n i t r o g l y c e r i n clearance i s about 0.6 L/min/kg6 9. In
man, plasma n i t r o g l y c e r i n c l earance has been r e p o r t e d as
23.6 and 28 L/min f o l l o w i n g intravenous i n f u s i o n 6 4 and sub-
l i n g u a l a d m i n i s t r a t i o n , r e ~ p e c t i v e l y ~ Since
~. these values
a r e i n excess o f l i v e r blood flow, i t has been suggested65
t h a t t h e r e must be s u b s t a n t i a l e x t r a - h e p a t i c e l i m i n a t i o n .
Plasma drug clearance i s a f u n c t i o n o f t h e apparent
volume o f d i s t r i b u t i o n and t h e e l i m i n a t i o n r a t e constant,
both o f which a r e q u i t e h i g h f o r n i t r o g l y c e r i n . An apparent
volume o f d i s t r i b u t i o n o f about 3 L/kg has been c a l c u l a t e d
f o r n i t r o g l y c e r i n i n r a t s 6 6 , which i s c o n s i s t e n t w i t h t h e
e x t e n s i v e t i s s u e d i s t r i b u t i o n discussed e a r l i e r . F o l l o w i n g
doses o f 0.7 mg/kg i n r a t s 6 6 and t h e r a p e u t i c doses i n
man64, 6 5 , plasma e l i m i n a t i o n appears monoexponenti a1 w i t h an
e l i m i n a t i o n h a l f - 1 i f e o f approximately 3-4 minutes. Admin-
i s t r a t i o n o f h i g h e r doses (2.5 and 3.5 mg/kg) i n r a t s r e -
s u l t e d i n an apparent b i e x p o n e n t i a l decay w i t h a t + B
min.63.
-
I t i s p o s s i b l e t h a t a t t h e r a p e u t i c doses, m u l t i -
15
exponential d i s p o s i t i o n cannot be c h a r a c t e r i z e d because o f

a n a l y t i c a l u n c e r t a i n t i e s encountered w i t h t h e extremely low
plasma c o n c e n t r a t i o n s ( < 1 ng/ml) found.
6.3 Oral and Topical A d m i n i s t r a t i o n

The r a t i o n a l e f o r o r a l use o f o r g a n i c n i t r a t e s
has been a c o n t r o v e r s i a l s u b j e c t . F o l l o w i n g o r a l a d m i n i s t r a -
.
t i o n and p o r t a l v e i n i n f u s i o n o f n i t r o g l c e r i n t o r a t s , i n
doses up t o 0.5 mg/kg, Needleman e t a1 5 7 observed no blood
pressure response and n e g l i g i b l e blood c o n c e n t r a t i o n s o f
i n t a c t drug. These authors a l s o observed human l i v e r b i o p s y
samples t o have m e t a b o l i c c a p a c i t y f o r o r g a n i c n i t r a t e s
s i m i l a r t o t h a t found i n r a t s , and t h e y concluded t h a t t h e
systemic a v a i l a b i l i t y o f n i t r o g l y c e r i n f o l l o w i n g o r a l admin-
i s t r a t i o n i s n e g l i g i b l e . C l i n i c a l s t u d i e s demonstrating
e f f i c a c y o f o r a l l y administered n i t r o g l y c e r i n 6 7 imply,
NITROGLYCERIN 533
however, t h a t systemic a v a i l a b i l i t y o f n i t r o g l y c e r i n may

be s i g n i f i c a n t i n man, s i n c e t h e m e t a b o l i t e s o f n i t r o g l y c e r i n
are considerably less active.
The use o f t o p i c a l n i t r o g l y c e r i n has been shown t o
g i v e s u s t a i n e d hemodynamic e f f e c t s i n man68. F o l l o w i n g
t o p i c a l a d m i n i s t r a t i o n o f a 2% n i t r o g l y c e r i n ointment,
e q u i v a l e n t t o 16 mg o f n i t r o g l y c e r i n , plasma c o n c e n t r a t i o n s
were s i m i l a r t o t h o s e seen a f t e r o r a l a d m i n i s t r a t i o n o f a 6.5
mg s u s t a i n e d r e l e a s e capsule69. S i t e dependency i n t h e p e r -
cutaneous a b s o r p t i o n o f n i t r o g l y c e r i n has been observed i n
man70 and i n r a t s 8 b u t n o t i n t h e rhesus monkey71. The s u r -
f a c e area o f a p p l i c a t i o n has a l s o been shown t o be an impor-
t a n t f a c t o r i n t o p i c a l a b s o r p t i o n when assessed by hemo-
dynamic e f f e c t s 7 2 .
7. Methods o f A n a l v s i s
7.1 O f f i c i a l Methods
The " O f f i c i a l Methods o f A n a l y s i s " p u b l i s h e d by t h e
A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l Chemists73, d e s c r i b e s two
methods f o r t h e d e t e r m i n a t i o n o f n i t r o g l y c e r i n . The f i r s t
i n v o l v e s e t h e r e x t r a c t i o n f o l l o w e d by t h e r e d u c t i o n o f n i t r o -
gen t o ammonia and subsequent d e t e r m i n a t i o n by t i t r a t i o n
w i t h a c i d . A second method u t i l i z e s t h e i n f r a r e d a b s o r p t i o n
peak near 7.89 pm and r e q u i r e s a n i t r o g l y c e r i n r e f e r e n c e
standard f o r q u a n t i t a t i o n .
The assay f o r n i t r o g l y c e r i n developed by Hohman and
Levine7!+ i s t h e b a s i s f o r t h e o f f i c i a l USP75 procedure. T h i s
technique uses column chromatography t o separate n i t r o -
g l y c e r i n from i t s d e g r a d a t i o n p r o d u c t s f o l l o w e d by a c i d
h y d r o l y s i s t o n i t r a t e i o n and subsequent s p e c t r o p h o t o m e t r i c
d e t e r m i n a t i o n o f n i t r a t e d p h e n o l d i s u l f o n i c a c i d . Potassium
n i t r a t e i s used as a r e f e r e n c e standard. Both t h e AOAC reduc-
t i o n method and t h e USP procedure a r e u s e f u l as p r i m a r y
s t a n d a r d i z i n g procedures f o r up t o m i l l i g r a m q u a n t i t i e s o f
nitroglycerin.
7.2 Spectrophotometric
Q u a n t it a t i o n o f n i t r a t e and n i t r i t e i o n f o l l o w i n g
hydrolysis o f organic n i t r a t e s i s possible using colorimetric
methods. Spectrophotometric measurement o f n i t r o x y l e n o l
formed from t h e r e a c t i o n o f h y d r o l y z e d o r g a n i c n i t r a t e w i t h
e i t h e r 2,4-xylenol o r 2,6-xylenol i s t h e b a s i s of t h e x y l e n o l
procedure76y77. A p p l i c a t i o n o f t h e G r i e s s r e a c t i o n and v a r i -
ous m o d i f i c a t i o n s have been used i n b i o l o g i c a l work f o r
n i t r a t e rr~easurement~~-~O However,
. t h e s e methods do n o t
possess t h e r e q u i s i t e s e n s i t i v i t y f o r t h e a n a l y s i s o f n i t r o -
g l y c e r i n i n b i o l o g i c a l f l u i d s d u r i n g d r u g therapy.
534 EDWARD F. McNIFF, el al.
Several spectrophotometric methods a r e a v a i l a b l e f o r

qua1 i t y c o n t r o l d e t e r m i n a t i o n s o f n i t r o l y c e r i n i n dosage
forms. I n t h e assay described by Be1 1 89 3 8 2 , n i t r o g l y c e r i n
i s hydrolyzed w i t h s t r o n t i u m hydroxide t o form n i t r i t e i o n .
F o l l o w i n g d i a z o t i z a t i o n w i t h N-(1-naphthyl) e t h y l e n e diamine
d i hydrochloride, q u a n t i t a t i o n i s achieved b y c o l o r i m e t r i c
d e t e r m i n a t i o n o f t h e azo dye. The use o f s t r o n t i u m h y d r o x i d e
i s s a i d t o reduce t h e i n t e r f e r e n c e due t o l a c t o s e . Since t h e
conversion o f n i t r o g l y c e r i n t o n i t r i t e i s n o t s t o i c h i o m e t r i c ,
absolute quanti t a t i o n requires a n i t r o g l y c e r i n reference
standard. T h i s method has been a ~ t o m a t e d ~ ~ , @ Use~ .o f t e t r a -
methyl ammonium hydroxide t o hydrolyse n i t r o g l y c e r i n has been
r e p o r t e d 8 4 t o produce s t o i c h i o m e t r i c conversion o f 2 moles
o f n i t r i t e per mole o f n i t r o g l y c e r i n as p r e d i c t e d by Hay85. I t
should t h e r e f o r e be p o s s i b l e t o use potassium n i t r i t e as a
r e f e r e n c e standard f o r t h e B e l l assay w i t h t h i s m o d i f i c a t i o n .
A k i n e t i c method has been developed which i s s u i t a b l e
f o r t h e a n a l y s i s o f s i n g l e dosage u n i t s l 5 - I 7 . T h i s assay i s
based upon t h e stepwise degradation o f n i t r o g l y c e r i n i n
a l k a l i n e a l c o h o l i c s o l u t i o n s , w i t h t h e f o r m a t i o n o f a chromo-
p h o r i c i n t e r m e d i a t e . The absorbance maximum a t 328 nm was
shown t o be p r o p o r t i o n a l t o t h e i n i t i a l n i t r o g l y c e r i n concen-
t r a t i o n present i n t h e r e a c t i o n .
The s p e c i f i c i t y o f t h e USP, B e l l and k i n e t i c assays
was examined by Morrison and FungE6. They found t h e USP and
k i n e t i c assay procedures t o be s t a b i l i t y - i n d i c a t i n g whereas
t h e B e l l assay i s p r e d i c t a b l y i n t e r f e r e d w i t h by i n o r g a n i c
n i t r i t e . However, under t h e r e a c t i o n c o n d i t i o n s o f t h e B e l l
method, t h e d i n i t r a t e s , mononitrates and i n o r g a n i c n i t r a t e d i d
not i n t e r f e r e .
7.3 T h i n Layer Chromatography

T h i n l a y e r chromatoqraphy - * " has been used t o separate
t h e 14C-glyceryi n i t r a t e s ( n i t r o g l y c e r i n and i t s m e t a b o l i t e s )
p r i o r t o q u a n t i t a t i o n o f t h e r a d i o a c t i v i t y . The system r e -
p o r t e d by Crew and DiCarlo18 (Table 111) i s r e p r e s e n t a t i v e
o f o t h e r ~ t~h a~t ,have ~ ~ been r e p o r t e d .
7.4 Polargraphy
The p o l a r g r a p h i c behavior o f n i t r o g l y c e r i n , penta-
e r y t h r i t o 1 t e t r a n i t r a t e and e t h y l e n e g l y c o l d i n i t r a t e has been
s t u d i e d i n an ethanol-water system based on t h e r e d u c t i o n o f
n i t r a t e a t t h e dropping mercury e l e c t r o d e . Tetramethyl -
ammonium c h l o r i d e was used as t h e s u p p o r t i n g e l e c t r o l y t e .
The e f f e c t s of pH, number o f n i t r a t e groups, mercury column
h e i g h t , b u f f e r s and s o l v e n t on t h e half-wave p o t e n t i a l (meas-
ured a g a i n s t t h e s a t u r a t e d calomel e l e c t r o d e (El vs. S.C.E.)
and t h e d i f f u s i o n c u r r e n t ( i . d . ) was examinedE9?
NITROGLYCERIN 535
Using t h i s technique f o r the assay of s i n g l e sub-

lingual t a b l e t s , Flann88 reported a E% of -0.91 v o l t s (vs.
S.C.E.) and the i . d . t o be dependent on nitroglycerin con-
c e n t r a t i o n . A non-aqueous polargraphic method has a l s o been
described by Woodson and A1 berE9.
TABLE 11118
T h i n-Layer Chromatography of Nitroglycerin
TLC p l a t e s : 250 1-1 s i l i c a gel G bound w i t h calcium s u l f a t e
Sol vent: benzene:ethylacetate:acetic acid (16:4:1)
Rf values: nitroglycerin 0.60
glyceryl-1 , 3 - d i n i t r a t e 0.45
glyceryl-l,2-dinitrate 0.30
glyceryl-l-mononitrate 0.10
glyceryl-2-mononitrate 0.10
g 1ycerol 0.00
Several GC procedures have been described f o r the
analysis of organic n i t r a t e s . This technique i s e s p e c i a l l y
s u i t a b l e f o r determination of nitroglycerin i n biological
f l u i d s a f t e r d r u g administration. T h e use of the e l e c t r o n
capture d e t e c t o r gives the necessary s e n s i t i v i t y . Table IV
gives chromatographic conditions t h a t have been u t i l i z e d f o r
nitroglycerin determination.
7.6 High Performance L i q u i d Chromatography
Several HPLC methods have been reported f o r the assay
of nitroglycerin i n dosage forms. Two normal phase methods -
, ~ ~ i s lacking on t h e i r s p e c i f i c i t y .
a r e a ~ a i l a b l e b~u ~t data
Table V l i s t s the chromatographic conditions of two proced-
ures shown t o be s p e c i f i c f o r nitroglycerin i n the presence
o f degradation products.
TABLE I V
GC C o n d i t i o n s f o r N i t r o g l y c e r i n
Temperature (OC) Sensi -

( I = I n j e c t i o n port, C = Sample t i v ity
Reference Column Detector Column, D = D e t e c t o r ) Ana 1yzed (ng/ml)
90 3.5% QF - 1 on 60-80 ECD I= 160, C = 120, 5 m l human 0.5

Gas Chrom Q . D = 180 plasma
91 3% SP-2401 on 100-120 ECD I= 160, C = 140, 0.2 m l r a t / 0.1
Supel c o p o r t . D = 180 human plasma
92 3% SE-30 on 50-60 TCD C = 130, D = 192 Tab1 e t --
Anakrom AB15. extract
93 0.4% OV-17 on 60-80 EC D I = 150, C = 120, 2 m l human blood 0.1-2
g l a s s beads D = 150 or urine
94 3% SE-30 on 100-120 ECD I = 200, C = 150, 3 m l human ?
Gas Chrom Q. D = 175 blood
65 10% OV-101 on 100-120 ECD I = 150, C = 130, 4 m l human ?
Chromosorb W-HP D = 200 p l asma
95 30% SE-30 on 80-100 ECD I = 150, C = 130, 5 m l human - 0.5
Chromosorb W-HP D = 210 plasma
96 3.8% OV-101 on 80-100 FID I = 70, C = 70-220 @ \li t r o c e l l u l o s e --
Gas Chrom Q; 2.5% 60/min, D = 225 propellants
OV-210 on Chromosorb
W-HP; 1.1% OV-225 on
Gas Chrom Q.
97 3% XE-60, 3.5% QF-1 on FID I = 160, C = 150, solvent
60-80 Gas Chrom Q. D = 200 mixtures
I = 160 C = 120,
ECD D = 260
NITROGLYCERIN 531
TABLE V
HPLC C o n d i t i o n s f o r Assay o f N i t r o g l y c e r i n
_
R_e f_l o o Reflol
Column C18 m i c r o p a r t i c u l a t e A1 k y l phenyl bonded t o

s i l i c a gel
M o b i l e Phase 60% MeOH A c e t o n i t r i l e-Tetrahydro-
furan-Water (26:64:10)
Flow r a t e
(ml / m i n) 2.0 2.0
Detection u.v 200 nrn u . v 218 nm
Detection
1i m i t 30 ng on column 50 ng on column
R e t e n t i on
time 4 rnin 10 m i n
Acknowledgement
Supported i n p a r t by N I H g r a n t 22273. We thank D r .
Dinesh Gala f o r r u n n i n g t h e i n f r a r e d and nmr s p e c t r a .
8. References
1. F. P r i s t e r a , M. H a l i k , A. C a s t e l l i and W. F r e d e r i c k s ,
Anal.Chem. 32, 495 (1960).
2. W.M. Ayres, G.C. Whitnack and R.T. Merrow, NAVWEPS
r e p o r t 7608, NOTS T e c h n i c a l P u b l i c a t i o n 2604 (1961)
U.S.Nava1 Ordinance T e s t S t a t i o n , China Lake, C a l i f . ;
Chem. Abs t. 60, 5266f ( 1964) .
3. R.T.M. F r a s e r a n d N.C. Paul, J . Chem. SOC. (B), 659
(1968).
4. M. Windholz (ed.), The Merck Index, 9 t h ed., Merck &
Co., Rahway, N. J. , 1976.
5. M.J. P i k a l , A.L. Lukes and L.F. E l l i s , J. Pharm. S c i .
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-
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NITROGLYCERIN 5 39
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51. N . H . Lee and F.M. B e l p a i r e , Biochem. Pharmacol. - 21,
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540 EDWARD F. McNIFF. er al.
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C i r c u l a t i o n 3, 585 (1979).
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38, 1031 (1976).
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73. W. H o r w i t z (ed. ) " O f f i c i a l Methods o f A n a l y s i s " ,
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(1964).
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79. L.J. Cass, W.S. F r e d e r i k and H. Delucia, A n g i o l o g y
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80. O.J. L o r e n z e t t i , A. Tye and J.W. Nelson, J. Pharm.
S c i . 55, 105 (1966).
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84. C.E. Wells, H.M. M u l l e r a n d Y.H. Pfabe, J . A. 0. A. C.
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85. M. Hay, M o n i t . S c i . P a r i s 15, 424 (1885).
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89.
B.C. F l a n n , J . Pharm. S c i . z, 122 (1969).
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( 1 969).
90. M.T. Rosseel and M.G. Bogaert, J. Pharm. S c i . - 62,
754 (1973).
NITROGLYCERIN 541
91. S.K. Yap, E.F. M c N i f f and H.-L. Fung, J. Pharm.

S c i . 67, 682 (1978).
92. E.T. E s s e l , J . Gas Chromatog. 179 (1965).
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94. Y. G i v a n t and F.G. Sulman, E x p e r i e n t i a 3-
4 , 643
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364 (1972).
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Chromatogr. 100, 185 (1974).
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Sci. - 68, 481 (1979).
L i t e r a t u r e reviewed up t o 4/1/80.
TRIFLUOPERAZINE HYDROCHLORIDE
Alex Post, Richurd J . Wurren, und John E . Zarembo
Description 544
I . 1 Nomenclature 544
I .2 Formula, Molecular Weight, Structure 544
Physical Properties 545
2. I Spectral Properties 545
2.2 X-Ray Diffraction Pattern 55 1
2.3 Solubility 555
2.4 Apparent Partition Coefficients 556
2.5 pKa 556
2.6 Thermal Properties 556
3. Synthesis 558
4. Identification 559
4.1 Derivatives 559
4.2 Color Reactions 559
4.3 Microscopy 560
4.4 Miscellaneous Identification Tests 56 1
5. Stability and Degradation 561
6. Metabolism 562
6.1 Metabolic Products 562
6.2 Biological Half-Life 565
7.2 Titrimetric Analysis 566
7.3 Complexometric Analysis 567
7.4 Spectrophotometnc Analysis 567
7.5 Spectrofluorometric Analysis 568
7.6 Chromatographic Methods of Separation 568
8. Miscellaneous 578
8. I Adsorption Phenomena 578
8.2 Surface Activity 578
9. References 579
Copyri%I 0 1980 by Acadcrnic R s s . Inc.

Analytical RofiLs of Drug Subsfnnces. 9 543 All rights of reproduction in any form reserved
ISBN: 0-12-260809-7
544 ALEX POST et ul.
I. Description
I. I Nomenclature
1.11 Chemical Names
Several chemlcal names have been used t o denote

t r i f I uoperazi ne hydroch l o r i de:
(a) IOH-Phenothl azlne. IO-[3-(4-methyI-l-p i p e r a z i n y 1 )propy I]-
2- ( t rif I uoromethy I )-d ihydroch Io r id e l
( b ) lO-f3-(4-methyl-l -p p e r a z i n y l Ipropy 1 l - Z - ( t r I f Iuoromethy I )
phenothiazine d i h y d r o c h l o r i d e l
(c1 D l h y d r o c h i o r i d e o f 0-~3-(4-methyIplperazine-I-yI )propyl]-

2-trl f Iuoromethylphenothia~ine~
(d) 2-TrlfIuor~lethyI-I0-~3(I-methyI-4-piperazinyI)propyI]
phenothiazinej
I. 12 Trade Names
I a t r o n e u r a l , J a t r o n e u r a l , Eskazinyl, Eskazine, S t e l a z i n e@ 4 ,
Terfluzine
1.2 Formula, Molecular Weight, S t r u c t u r e
1.21 E m p i r i c a l Formula, Molecular Weiqht
C~~H~L,FSJN~S-~HCI 400.420
1-22 S t r u c t u r e
a s D C F s .2HCI
I
CH2CHzCH2-N
n
wN-CH3
1.3 b p e a r a n c e . Color, Odor

Both t h e National Formulary1 and t h e B r i t i s h Pharmacopoeia2 d e s c r i b e
T r i f l u a p e r a z i n e Hydrochloride.as f o l l o w s :
A w h l t e t o o f f - w h l t e (cream colored) c r y s t a l l i n e powder w i t h

l i t t l e o r no odor.
TRI FLUOPERAZINE HYDROCHLORIDE 545
2. Phys i ca I P r o p e r t i e s
2. I Spectra I P r o p e r t i e s
2. I I I n f r a r e d Spectra
F i g u r e 1 i s t h e i n f r a r e d spectrum o f t r i f l u o p e r a z i n e f r e e base
and F i g u r e 2 i s t h e i n f r a r e d spectrum o f t h e h y d r o c h l o r i d e s a l t o f t r i f l u o -
perazine taken i n mineral 011 d i s p e r s i o n from 4000-625 cm-l on a Perkin-Elmer
Model 457A. The s i g n i f i c a n t bands I n t h e spectra are assigned as f o l l o w s :
Free Base HCI S a l t

Wave I ength cm-l Assipnment Wavelength cm-l Assionmnt
1600, 1575, 1500 c=c, aromatic 2700-2 I00 NH+
1330, 1250, 1130 CF3 1600, 1570, 1470 C=C, a r o m a t i c

830 I ,2,4-trl sub- 1320, 1340, 1115 CF,
s t i t u t e d aromatic
a29 I ,2,4-trisub-
750 1,2-substltuted s t l t u t e d aromatic
aromat ic
760 1,2-substituted
aromat i c
The i d e n t i f i c a t i o n and d i f f e r e n t i a t i o n o f phenothiazine t y p e

t r a n q u i l i z e r s by t h e I R spectra o f s a l t s as d e r i v a t i v e s has been r e p ~ r t e d . ~ ~ ~
The u l t r a v i o l e t a b s o r p t i o n spectrum o f t r l f l u o p e r a z i n e i n 95%

ethanol 1s shown I n F i g u r e 3. Maxima a t 258 nm ( l o g E 4.50) anj3307.5 nm ( l o g c
3.50) a r e bands c h a r a c t e r i s t i c of a 2 - s u b s t i t u t e d phenothiazine .
The u l t r a v i o l e t spectrum o f t r i f l u o p e r a z i n e has been used i n
the analysis o f biological s p e ~ i m e n s ’ ’ ~ * ~ as~ w ’ ~e l~l as i n t h e a n a l y s i s Of
t h e drug i t s e l f and i t s d e r i v a t i v e s . 8 The importance of c a r e f u l c o n t r o l of
instrumental Parameters such as s l i t wtdth and t h e absorption o f UV and
v i s i b l e I i g h t by phenothiazines has a t s o been r e p o r t e d . l l
2.13 Nuclear Magnetic Resonance Srjectra
2.131 Proton Spectrum
The p r o t o n NMR spectrum (Figure 4 ) was obtalned on a

deuterochlorofonn s o l u t l o n c o n t a i n i n g approximately 100 mg/ml o f t r l f l u o p e r -
a z l n e and t e t r a m e t h y l s l l a n e as i n t e r n a l reference standard. The spectrum was
obtained on a Perkin-Elmer R32 NMR. The NMR s i g n a l s a r e assigned as follows:
546 ALEX POST c'f ti/.
Protons Chemical S h i f t , ppm Mu I t I c i p I b :3

a I .94 mu I t 1 p l e t
b 2.25 sing l e t
c, c '
d, d' 2.3 - 2.6 broad m u l t l p l e t , s i g n a l s
over I app i ng
e
f 3.96
-
t r i plet
g ( a l i aromatics) 6.75 7.30 broad m u l t i p l e t , s i g n a l s
overlapping
2.132 13C Spectrum

The NMR Spectrum ( F i g u r e 5 ) was obtalned on a
deuterochloroform s o l u t i o n o f t r i f l u o p e r a z i n e w i t h t e t r a m e t h y l s i l a n e as a
reference. The spectrum was obtained on a Varian Associates Model FT-80
spectrometer. The NMR s i g n a l s a r e assigned as follows:
eJe Y 8.8' d'
e,e' d
Figure 1 : I n f r a r e d Spectrum of T r i f l u o p e r a z i n e Free Base
I
or
548
Trifluoperazine
- - - - - - --- Sulfoxide
Sulfone
vt
P
W
--__
_''.......... , ...:.
..
I I I I I I I I I I
220 240 260 280 300 320 340 360 380 400
F i g u r e 3- U l t r a v i o l e t A b s o r p t i o n S p e c t r u m of T r i f l u o p e r a z i n e i n 95% E t h a n o l
I
P
TRI FLUOPERAZINE HYDROCHLORIDE 55 i
Table I ( c o n t ' d )
-
m/e
266
248
,141
127
I13
99
70
An a lys i s and i d e n t i f i c a t i o n o f drugs by mass spectroscopy

has been reported. l4
2.2 X-Ray D i f f r a c t i o n P a t t e r n
The X-Ray d i f f r a c t i o n p a t t e r n of t r l f luoperazine dihydr cchlor ide

i s presented I n T ab l e 2 .
I
I
TIU FLUOPERAZINE HYDROCHLORIDE 553
Carbon Chemical S h i f t , ppm
a 24.15
b 45.23
C 45.95
d, d' 53.26
e, e ' 55.16
f 111.80
9 115.88
h 118.83
i 122.96
i 123.84
k 127.54
127.39
127.30
129.56 ( d o u b l e t c e n t e r )
129.79
144.23
145.68
124.29 ( q u a r t e t c e n t e r )
2.14 Mass Spectrum
The mass spectrum o f t r l f l u o p e r a r i n e wss obtained by d i r e c t

i n s e r t l o n i n t o an H f t a c h i Perkin-Eimer R4U-6E low r e s o l u t i o n mass spectrometer.
The r e s u l t s a r e presented i n t a b u l a r form i n Table I and as a b a r graph I n
Figure 6
Table I
-
m/e - Ion
407 M +
392 (M - CH3)+
307
294
280
n
TRIFLUOPERAZINE HYDROCHLORIDE 555
Table 2
X-Rav D i f f r a c t i o n P a t t e r n o f T r i f I uoperaz i ne D I hydroch t o r i de
-
2 0 -
1/10 d(Ao 1
10.10 33 8.75
13.80 12 6.44
15.30 98 5.79
15.60 58 5.68
17-30 8 5.12
20.50 I00 4.33
21 -90 8 4.06
23.20 24 3.03
23.50 24 3.78
23.80 8 3.74
27.60 12 3.23
27.80 16 3.21
30.30 16 2.95
32 -50 12 2.75
d = I n t e r p l a n a r spacing ( d i s t a n c e ) .
1/10 = R e l a t i v e i n t e n s i t y based on h i g h e s t i n t e n s i t y o f 103.
2.3 Solubility
So I vent Grams/100 ml Reference
water 66 15
water 59 2
0.2E HCi >66 15
0.2g NaOH i nsol ub l e 15
pH 7.4 b u f f e r 0.0014 16
ethanol (95%) 14.5 1s
ethyl ether insoluble 1
chloroform I .9 15
benzene i nsol ub I e 1
water 0.0013 17
( a ) f r e e base
ALEX POST ('t trl
2.4 Ppparent P a r t i t i o n C o e f f i c i e n t s (K)

I n an attempt t o c o r r e l a t e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s , several
i n v e s t i g a t o r s determined t h e apparent p a r t i t i o n c o e f f i c i e n t s i n a v a r i e t y o f
hydrophobic-hydrophilic systems. R e s u l t s o f these s t u d i e s a r e l i s t e d i n
the following table.
OrganidAqueous Phase -
K Reference
dodecane/pH 7.0 b u f f e r (30'C) 97 18

chloroform/water (pH I .O) 0.37 19
chIoroform/pH 7.6 b u f f e r >5000 19
chIoroform/O.l! HCI 0.7 20
n-octanoI/pH 7 b u f f e r >to5 21
n - o c t a n o l / O . l 2 5 ~ KCI 49.3 18
2.5 Apparent pKa
The a a r e n t pKal and pKa2 have been determined using t i t r i m e t r i c

and solubi I i t y " measurements. As reported by many o f these i n v e s t i g a t o r s ,
t h e d e t e n i n a t i o n o f t h e pKa o f phenothiazines, i n general, a r e d i f f i c u l t t o
o b t a i n because of t h e i r poor water s o l u b i l i t y . Thus t h e use o f t h e term
apparent pKa. However, Green16, u s i n g s o l u b i I ity measurements, d i d indeed
c o n f i r m t h a t apparent pKa2 f o r t r i f l u o p e r a z i n e i s approximately 8.1 -
confirming t h e r e s u l t s obtained by t i t r i m e t r i c measurements.
Apparent pKa
pKal PKal * Procedure Reference
3.9 8.4 tl t r i m e t r l c 22
3.9 8. I ti trimtri c ia
4.10 8.36 ti trl mtri c 23
8. I s o l u b l I ity 16
8.3 TLC ( a ) 24
( a ) Thin l a y e r chromatography
2.6 Thermal P r o p e r t i e s
2.61 M e l t i n g Range
T r i f l u o p e r a z i n e d i h y d r o c h l o r i d e melts a t about 242OC w i t h de-

composition' using t h e c a p i l l a r y m e l t i n g tube method. A m e l t i n g range o f
243244' was r e p o r t e d by Anderson, e t a1 .25
2.62 D l f f e r e n t l a l Scannlno Calorlmetrv
The DSC thermgram f o r t r l f l u o p e r a z i n e d l h y d r o c h l o r l d e 15 shown

i n Fiqure 7 . I t i s e v l d e n t t h a t m e l t i n g does appear t o s t a r t a t approximately
I 80°C w i t h subsequent r a p i d deconposi t ion a t about 250°C.26 The thermogram was
obtained a t a h e a t i n g r a t e o f 20'C per minute.
TRIFLUOPERAZINE HYDROCHLORIDE 557
OC
Figure 7 - Differential Scanning Calorimetry Curve of Trifluoperazine

Hydrochloride (USPI
558 A L E X POST e t a l .
3. Synthesis
The detailed synthesis of trifluo erazine dihydrochloride i s described
by Craig, e t a I 2 ’ a n d Anoerson, et a l g 5 . The schematic i s illustrated below.
4. Identification
4.1 Derivatives
Several salts have been prepared thbt can be used for identification
purposes (Table 3). As the preparation of the sulfoxide of trifluoperazine can
be easily orepared, it has also been used for the identification of the parent
compound. (refer to Figure 3)
Table 3
Salts of Trifluoperazine
-
Salt Melting Range Reference
Dihydrochioride c *42'(0) 1
Pi crate 242.0 (decomp) 6
Re i neckate 183.5 - 186.0' (decomp) 6
D i ma I eate 193 - 194' 29
196 - 197' 38
Dimethylsulfonate 257 - 258" 38
Difumarate 215' 38
Disuccinate 130 - 132' 38
Pmoate 152 - 167' 38
tienzcphenonedicarboxylate 158 - :61' 38
Dipyromellitate >240' 38
Dimethiodide Salt 162 - 163" 40
Trif luoperazine (free base) 'L 80" 38
T r i fI uoperaz i ne Su I foxide 173 - 175'

Ibi 27
Di h y a r o c h lor i de
(a) Refer to Section 2.6

( 6 ) With 3 M I S H20
4.2 Color Tests
Reactions with color reagents has been the method of choice for
differentiating trifluoperazine, its degradation products, and metabolites
efter a prel imirary saparation bv thin layer and paper c h r o m a t ~ g r a p h y . ~ ~ * ~ ~ - ~
A listing of several of these color reagents are given in Table 4 .
3 6 j 3 7
5 60 A L E X POST ei a / .
Table 4
I d e n t i f i c a t i o n o f T r i f l u o p e r a z i n e w i t h Color Reagents
Reagent Res Donse Reference
Bromine water + H2S04 cher r y - r e d 28

Selenious Acid amber brown-qreen 28
Concentrated HNO3 p i n k y e 1 low 6.28
68% HNO3 p u r p l e y e 1 low 29
1 % Cobalt a c e t a t e + I i g h t b I ue 6
isopropylamine
10% aq. Chloramine-7 creamy b I ue 6
Pal ladium c h l o r i d e orange brown 6
Uran i urn n i t r a t e orange 6
A m n i um vanadate ye1 low brown 6
S i I ver n i t r a t e creamy w h i t e 6
K e l l e r T e s t (Fee13 pink-mrange 29
2% aq. F e C l j red-wiolet 31
Ceric s u l f a t e red+coiorless 30
40-50% H2SO4 orange 31,33,34
Folin-Ciocalteau cameo 32
FF" Reagent pink 32
kbnde I i n flesh 32
Cinnamylaldehyde flesh 32
Furfura I cameo 32
FeCl3 amber 1
4.3 Microscopy
was successful i n u s i n s m i c r o c r v s t a l I ine r e a c t i o n s t o

d i f f e r e n t i a t e between phenothiazine t y p e t r a n q u i I i z e t s . Table 5 c o n t a i n s
the r e s u l t s f o r t r i f l u o p e r a z i n e o n l y .
Table 5
Sensitivily o f
Reaaent Description of C v s t a I s Detection ( u g h I )
Ammonium reineckate amorphous --

Picrlc acid yellow, b i r e f r i n g e n t 0. I
rosettes
Stannous C h l o r i d e c o l o r I ess. weak I v 0. I
b l refringen? irregular
r o s e t t e s and long needles
P I a t i num b rom ide amorphous --
Gold c h l o r l d e reddish-brown. weakly 0. I
b i r e f r i n g e n t needles,
dense r o s e t t e s
TRI FLCIOPE R A Z l NE HYDROCHLORIDE S6 I
In a subsequent col laborative.study6, Andres recommended that

stannous chloride was the reagent of choice to identify trif luoperazlne.
F ~ l t o n ~in~ ,an extensive study o f phenothiazines, was able to characterize
and distinguish trifluoperazine from other phenothiazines via the color of
crystals formed with gold, platinum, and palladium reaaents (Tabte 6).
Table 6
Color of Trifluoperazine in Microcrystal Tests
-
Reaaent Color Obtained
HAuC14 in (I+I)H2S0, orange
H2PtCI6 aq(l0). to (2+1) acetic ourple and violet

acid solution, to dryness
H2PdCI4 in (I+35)H2SO4, to (2+1) light salmon

acetic acid solution; then evaporated deep purple
H2PtC16 in diluted HClO~+-aceticacid brigh sky blue
H2PdCI4 In diluted HCIOt,-acetic acid red (purple to pale orange)
4.4 Miscellaneous Identification Tests

Ultraviolet and infrared absorption have been used as identity
the Rf and Rt of thin layer chromatography and gas liquid and high performance
liquid chromatography, respectively. have also been used. These will be cited
in subsequent sections. Proton and C-13 nuclear magnetic resonance spectra
and mass spectrometry are currently in use. Coupled techniques. i.e. GLC/mass
spec, GLC/IR and HPLC/mass spec38, are now more comnplace in identifying com-
ponents from biological tissues.
5. Stabi I ity and Deqradation
Trifluoperazine I s subject to air and light induced oxldative degradation.

The r n e ~ h a n i s m ~can
~ > be
~ ~‘considered a two-step reaction involvlng the inter-
mediate formation of a semiquinone free radical which Is then oxidized to the
s u 1 fox Ide.
ALEX POST et al.
The formation o f t h e s u l f o x i d e can be r e a d i l y followed spectrophoto-

n-&ricaIIy. As t h e o x i d a t i o n proceeds,the wavelength maximum, 255 nm, f o r
t r i f l u o p e r a z i n e f a l l s w i t h t h e concomitant increase i n t h e wavelength
maximum f o r t h e s u l f o x i d e a t 278 nm. S i m i l a r l y , degradation can be r e a d i l y
followed by many o f t h e methods c i t e d i n Section 7.6 and/or q u a n t i f i e d by
methods l i s t e d i n Sectlon 7.63-7.65.
Aqueous a c i d s o l u t i o n s o f t r i f l u o p e r a z i n e , f l u s h e d w i t h n i t r o g e n , and
k e p t i n t h e dark, a r e s t a b l e f o r several days. However, i n t h e l i g h t and
e s p e c i a l l y UV l i g h t , degradation occurs r a p i d l y . W i t h i n 15 minutes and under
UV l i g h t , d i s c o l o r a t i o n o f t h e s o l u t i o n i s evldent. I n ethanol o r a c i d i f i e d
ethanol, no such degradation i s observed w i t h i n 48 hours3*.
T r i f luoroperazine d i h y d r o c h l o r i d e s t o r e d a t r w m temperature f o r up t o
two years d i d n o t show degradation3’.
Lever and Hague4’ observed t h a t on d i l u t i n g concentrated s o l u t i o n s o f t r i -

fluoperazine w i t h c o m n d i l u e n t s used under c l i n i c a l s i t u a t i o n s l i . e . c o l a ,
coffee, tea, grape and apple j u i c e ) t h e r e was a c o l o r change and t u r b i d i t y and
o r p r e c i p i t a t i o n w i t h i n two hours a t room temperature. They recommended d i l -
u t i o n s be made f r e s h l y and w i t h d i s t i l l e d water only.
6. Metabolism
6. I Metabolic Products
The i n v i v o and i n v i t r o metabolism of t r i f l u o p e r a z i n e have been

e x t e n s i v e l y s t u d i e d by man i n v e s t i g a t o r s . .The f o l l o w i n g schematic a b s t r a c t e d
from several pub1 i n d i c a t e s several pathways t o t h e m e t a b o l i c
products i d e n t i f i e d . A summary of t h e f i n d i n g s along w i t h t h e l n d e n t i f l c a t i o n
and/or q u a n t i t a t i v e techniques used t o e s t a b l i s h t h e amounts p r e s e n t a r e
l i s t e d i n Table 7 . The c l t e d references c o n t a i n Information r e g a r d i n g t h e
pharmacokinetics o f t h i s compound as it r e l a t e s t o t h e mode of a d m i n i s t r a t i o n ,
and the amounts of t h e m e t a b o l i t e s present i n t h e various t i s s u e s .
TRI FLUOPE RAZ IN E HYDROCHLORIDE 563
Table 7
Metabo I i t e Ana I y t i ca I
l d e n t i f i e d and/ Technlques
Animal T Issue o r Quantlf ied Used Re f e r en ce
Rat Liver I, IV, VII, VIII TLC', M S ~ 40.49

K I dney I, IV, VII, VIT
Braln I, VII
U r Ine X
Man Urine I, VII, x TLC, MS 40.49

Bra I n I, IV SFe
P I asma I, IV S Fe
Rat L l v e r Mlcrosomes I , 111, v, VI, VII, x G L C ~ , MS 50
Rat B r a In VI I TLC. UVc 51

Llver I, 11, IV, VII, VIII, XI1
Lungs I. 111, VII
K i dney I, 111, VII
Rat L i v e r Mlcrosornes I TLC, UV 52

One d e - a i k y l a t e d analogue
Two h y d r o x y l a t e d analogues
Rat Ur I ne I . I1 TLC, UV 53
Rat L I v e r M i crosomes VT I T LC 54
Rat Ur I ne 111, XI TLC, UV 12
( a ) T h i n l a y e r chromatography
( b ) Mass s p e c t r o m e t r y
(c) U l t r a v i o l e t a b s o r p t i o n
( d ) Gas l i q u i d chromatography
(el Spectrofiuorometry
TRIFLUOPE RAZl N E HYDROCHLORIDE 565
Using 3 5 S tagged trlfluoperazlne, Flanagan, et a155 showed that

only about 11-136 of the o r a l l y admlnlstered dose was detected In the urine
of nonfasted rats after 96 hours. Of this amunt, 80-855 was found in the
24 hour urine collectlon. Using fasted rats, only about 96 of the total
dose was detected with about 90% found In the 24 hour collectlon.
Using 24 hour urine collections, West, et aIs6 found that trlfluo-

perazine was extensively metabolized by man with unchanged trifluoperazine
accounting for tess then I6 of the dose; the sulfoxide was 1-65 of the dose;
and that the excretion of the trlfluoperazine and Its metabolite was dose
dependent.
6.2 Biological Hal f-Life
Schrnalzing and &eyer5' showed that when trlf luoperazine is admin-

istered intravenously tomale rats, the biological half-life for the
trifluoperazine in brain, lung, kidney, and plasma was approximately 2.5
hours, and much longer In the liver. After o r a l administration, the concen-
tration in the liver was the same as after the intravenous dosage.
6.3 Protein Eindlnq
Nambu and Nagar59~tudied the binding of trif luoperazine t o bovine

serum albumin using an equillbrium dialysis method and a gel flltration pro-
cedure. They showed that binding increased with pH, the order of increase
was dependent on the ion species with citrate,succinatsphosphate>acetate,
was correlated with surface activity, and increased with the partition co-
efficient in dadecane/water system. Binding to B.9 in 1/30 g phosphate at
pH 7.00 at 10°C was 82-87s. (The results suggested that a hydrophobic
inferaction takes part I n the binding.) Zla and Price60apparently reached
the same conclusion when they used 2-(4'-hydroxybenzeneazo)benzoic acid
as a spectrophotometric probe and measured the dlfference absorption spectra
with binding of trifluoperazine to bovine serum albumin.
Gabay61and Huanglostudied the binding of trif luoperazine t o human

serum albumln, as well as that from the dog, rat, rabblt, pig. horse, sheep,
goat, and chlcken. They also used a UV difference spectrophotometric method
and an intrinsic protein fluorescence quenching method. From the shapes of
the W difference spectra, whlch were essentially ldeptical, indicated that
the overall binding s i t e environment (hydropholicl of the ten species were
s i m l lar.
7.1 Elemental Analysls
Conventional procedures for the determination of C, H. N, S, CI,

and F yielded the following results on a sample which passed NF X I V
spec I f i cat i ons. 62
566 A L E X POST e t a /
E I ement -
Found Theory
C 52.26 52.50
H 5.31 5.46
N 8.72 8.75
S 6.12 6.67
CI 14.87 14.76
F I I .87 I I .86
7.2 T i t r i m e t r i c Analysis
Several t i t r i m e t r i c procedures have been r e p o r t e d f o r t h e assay o f

t r i f I uoperazine d i h y d r o c h l o r i de:
7.21 T i t r a t i o n w i t h p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d i s
apparently t h e most f r e q u e n t l y used.63
The sample i s d i s s o l v e d i n g l a c i a l a c e t i c acid, mercuric

acetate T.S. i s added and the t i t r a t i o n e f f e c t e d w i t h standardized 0.1:
p e r c h l o r i c a c i d i n g l a c i a l a c e t i c a c i d t o t h e blue-green end-point o f c r y s t a l
v i o l e t . Each ml of O.IN 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 24.02 mo o f t r i -
fluoperazine dihydrochloride. The end-point can a l s o be determined poten-
t i o m e t r i ca I I y us i ng g I ass-ca lome I e I ectrodes. 64
7.22 T i t r a n t : Ceric Sulfate

Detection: Photometric Endpoint
Agarwal and Blake30 employed a photometric t i t r a t i o n procedure.

They t i t r a t e d an a c i d s o l u t i o n of t h e sample w i t h 0.02N c e r i c s u l f a t e ,
d e t e c t i n g t h e endpoint p h o t o m e t r i c a l l y a t t h e wavelength o f maximum absorbance
of c e r i c s u l f a t e , 420 nm. An e x c e l l e n t c o r r e l q t i o n w i t h t h e method noted i n
Section 7.21 was obtained.
7.23 Titrant: Ceric Sulfate

Detection: C o l o r l e s s Endpoint
The sample, dissolved i n d i l u t e s u l f u r i c a c i d i s t i t r a t e d w i t h

c e r i c s u l f a t e t o a c o l o r l e s s endpoint. The equivalence p o i n t corresponds t o
t h e a d d i t i o n o f two moles o f c e r i c i o n per mole o f t r i f l u o p e r a z i n e dihydro-
chloride. The a c i d - s t a b i l l z e d c o l o r e d f r e e r a d i c a l i s discharged when t h e
o x i d a t i o n t o t h e ' s u l f o x i d e ' i s completed.65
TRIFLCIOPERAZINE tlYDROCHLORIDE
7.3 Comp I exometrtc Analysis
Preclpltation of the trifluoperazine as its mono-Reineckate salt

with an excess of the precipitating reagent and titratin the excess bromo-
metrically is the basis of the method proposed by Olech.;10 The method is
rapid, requiring only mi I iaram amounts o f samole and with s n error o f
f1.0 - ~ ~ an excess of lead, cadmium, copper, or zinc
1-59. G a j e ~ s k aused
picrate to precipitate trifluoperazine. The lead picrate forms an insoluble
complex, while the others form 5:3 complexes. Titration of the excess cation
is made with standardized EDTA.
7.4 SDectrophotometric Analysis

Trifluoperazine dihydrochloride can be assayed by ultraviolet spectro-
photometry in dilute hydrochloric acid at its maximum wavelength ( Q 5 5 nm) or
via a two point analysis (Abs,,, nm- Abs278 nm). 38
Several approaches to the assaying of trifluoperazine in Various CM)-

mercial preDarations have been reported. The British Pharma~opoeia3~and the
National methods involve an extraction followed by the UV readout
o f suitably diluted solutions; the former report a method for tablets, the
latter for tablets, injection, and syrup.
Alternate procedures were Droposed by Watson, et a l
:
8 for the analysis
of trifluoperazine in fablets. In their first procedure, they partitioned a
t r i f l u o p e r a z i n e - b r o m o c r e s o l purple complex between an aqueous pH 6 buffer and
benzene-isoamyl alcohol and measured the absorbance of the yellow colored
organic phase at 410 nm. In the second method, a I %hydrochloric acid extract
i through an a I ka I i ne di atomaceous earth col umn, and the tri f I bocler-
azine eluted with chloroform. The chlorofom extract is nixed with methanol-
hydrochloric acid and the solution measured at 259 nm. These procedures eiim-
~na+edpotential interferences not accomodated by the British Pharmacopoeia
procedure.
A di fferentiaI spectrophotometric method was developed by Davi dson6'
which precluded interferences from the photochemical decomposition product
(sulfoxide) and excipients including the conventional coloring and flavoring
agents. The sample is treated with peroxyacetic acid to rapidly and quant-
itatively convert the trifluoperazine to its sulfoxide. The difference ab-
sorption maximum at 353 nm is a measure of the trifluoperazine. This procedure
has been used with sustained-release capsules, as well as other conventional
dosage f orms.
A highly specific procedure for the shenothiazine nucleus in biological
tissues was reported by Wal lach and Biggs7. A characteristic oxidation product
is obtained when the alkaline-extracted phenothiazine is treated with cobalt
( I l l ) ion and is stable in the hexane-tertiary butyl alcohol used. The wave-
length maximum occurs at 272 nm and the assay is linear over a range of 0 . 5 -
50.0 mcg/ml.
Huang and Bhansal i 5 3 separated the trifluoperazine and its sulfoxide

in urine using thin layer chromatography and after a quantitative elution from
the plate determined the amount of each present spectrophotometricall y . Using
Oeproteinated human blood and liver (with 5tj HCI), followed by extraction of
alkal inzed solution, Stevens. et aI7' quantified the amunt of trifluoperazine
spectrophotonetrical l y . Recoveries of 60-76% were obtained.
5 68 A L E X POST cf trl
U s i n g Sephadex LH-20, Malcolm71 separated t h e t r i f l u o p e r a z i n e and

then determined t h e amount p r e s e n t i n t h e v a r i o u s f r a c t i o n s s p e c t r o p h o t o -
m e t r i c a l l y t o determine t h e c o n c e n t r a t i o n . Reference samples were s i m i l a r l y
treated.
7.5 Spectrof lucrometric Analysis
The f l u o r e s c e n c e spectrum of a p h e n o t h i a z i n e i s unique and t h u s can

be used t o q u a n t i f y t h i s s p e c i f i c compound i n b i o l o g i c a l t i s s u e s , s o l u t i o n s
and t a b l e t s .
Me1 I i n g e r and Keeler72 showed t h a t when t r i f l u o p e r a z i n e i s t r e a t e d

w i t h KMNO,, i s a c i d , t h e f l u o r e s c e n c e s h i f t s t o s h o r t e r wavelengths w i t h an
increase i n i n t e n s i t y and c o n c o m i t a n t l y , t h e e x c i t a t i o n spectrum changes t o
form a c h a r a c t e r i s t i c wavelength p a t t e r n o f f o u r d i s t i n c t peaks. These
a u t h o r s used t h i s procedure f o r t h e q u a l i t a t i v e i d e n t i f i c a t i o n o f pheno-
t h i a z i n e s showing t h a t 0.6 - 0.8 pg/ml of body f l u i d c o u l d be d e t e c t e d . T h i s
was about a f i v e - f o l d i n c r e a s e o v e r u l t r a v i o l e t a b s o r p t i o n procedures. A
subsequent r e p o r t by t h e s e same a u t h o r s 7 3 showed t h a t s p e c t r o f I u o r o m e t r i c
a n a l y s i s c o u l d be used t o q u a n t i t a t e t r i f l u o p e r a z i n e i n b i o l o g i c a l t i s s u e s ,
ampuls, and t a b l e t s a t t h e f i n a l c o n c e n t r a t i o n o f 2 t o 20 ng/ml.
Ragland and K e n r ~ s s - W r i g h t found ~~ t h a t i f t h e o x i d a t i o n was e f f e c t e d

w i t b hydrogen p e r o x i d e i n 50% a c e t i c a c i d , t h e f l u o r e s c e n c e spectrum was more
s t a b l e and more i n t e n s e . They subsequently used t h i s procedure t o q u a n t i f y
nanogram q u a n t i t i e s of t h e p h e n o t h i a z i n e i n b l o o d serum. b r a i n t i s s u e , and
l i v e r . 7 5 T ~ m p s e t tconfirmed
~~ t h e a o p i i c a b j l i t y and r e l i a b i l i t y of t h i s
method f o r t h e q u a n t i t a t i o n o f t r i f l u o p e r a z i n e i n b l o o d serum.
West, e t a156 used s p e c t r o f Iuorometry t o determine b o t h t r i f l u o p e r -

a z i n e and i t s s u l f o x l d e i n u r i n e . plasma, and b r a i n . Recoveries o f 68-80%
were o b t a i n e d on 10 pg/ml o f u r i n e s o l u t i o n s . T h e i r r e s u l t s were i n e x c e l l e n t
agreement of t h o s e o b t a i n e d by Spano, e t at7’ who used a s p e c i f i c r a d i o i s o t o p e
procedure.
7.6 Chromatographic Methods o f Seoaration
Chromatography on paper and m o d i f i e d papers u s i n g an a s s o r t -

ment o f m b i l e phases has been used t o separate t r i f l u o p e r a z i n e and i t s
metabolites. Several o f t h e m o b i l e phases and s t a t i o n a r y phases a r e l i s t e d
i n Table 8 and d e t e c t i o n methods i n Table 9. Paper chromatography has been
used i n ana I y z i ng b io l o g i c a i t i s s u e s . 3 3 s 81 p a 2
TRI FL UOPE RAZ I N E HY D ROCH LO IU DE 569
Table p
Paper Chromatoaraphv o f T r i f i u o p e r a z i n e
M o b i l e Phase S t a t i o n a r y Phase R-
f Reference
!I Sodi um Formate Whatman 3MM 0.27 33
IN Sodium Forrnate-
o-propanol (90:lO) Whatman 3 M M 0.25 33
IN Sodium F o n a t e -
I N ammonia (9O:lO) Whatman 3MM 0.25 33
I N Sodium Formate-
95%Formic a c i d (97:3) Whatman 3 1 M 0.60 33
IN Sodium A c e t a t e Whatman 3MM 0.17 33
I N Sodium A c e t a t e -
n-propanol ( 9 0 : 1 0 ) Whatman 3 I M 0.35 33
Sodium C h l o r i d e -
n-proDaqoI ( 9 2 : 8 ) Whatman 34M 0.55 33
autanoi-hater-Ci t r i c Acid
(870:130:4.8 g )
Whatman # I impregnated
w i t h 5% sodium
0.34 36 .
d i hydrogen c i i r a t e
pH 4.58 A c e t a t e B u f f e r , Whatman # I impregnated 0.06,0.09 36981
r u n a t 95'~ w i t h 10% t r i b u t y r i n
pH 7.4 Phosphate B u f f e r , Whatman # I impregnated 0.03 36

r u n a t 86OC w i t h 105 t r i b u t y r i n
n-Butan c I -HC I -Water Whatnlan # I impregnated 0.91 78

(6:I :7.5) w i t h c i t r i c acid-phos-
phate b u f f e r , pH 4.0
n - B u t a n o l - A c e t i c Acid- Whatman # I impregnated 0.88 78

Water (6:I :7.5) w i t h c i t r i c acid-phos-
phate b u f f e r , pH 4.0
lsobutyl alcohol-2ropionic Whatman # I impregnated 0.91 70

a c i d - w a t e r (lO:1:4.5) w i t h c i t r i c acid-phos-
p h a t e b u f f e r , pH 4.0
55 Amronium S u l f a t e s a t - S A S #57€, Whatman #I 0.23 79

urated w i t h Isobutanol or 4
Cyclohexane-Benzene (9:l) Several papers imDreg- 0.78 80

nated w i t h formamide-
5% a m n i u m f o n a t e
570 A L E X POST era1
Table 9
Spray Reagents f o r D e t e c t i o n o f T r i f I u o p e r a z i n e
(Paper Chromatography 1
-
Reaae nt -
Co Io r Reference
40% H2SO4 orange 33
D r a g e n d o r f f ' s potas- purple genera I I y used

sium l o d o p l a t i n a t e
Mod i f i ed' conc. H2S04 red 81
Modified' Marquis red 81
Modified' Mandelin orangered 81
Modi f ieda Frohde red 81
Mod i f i ed' Mec ke red+ row n 81

Palladium Chloride ( 1 % ) red-orange 81
Bromine w a t e r d a r k green 81
UV, 263 nm bluish yellow 33
UV, 254 nm p u r p l e yellow 81
fa) T r e a t e d w i t h sodium s u l f a t e t o reduce r a t e o f r e a c t i o n .
7.62 T h i n Layer Chromatography
A s i g n i f l c a n t number o f m o b i l e phases have been used t o

chromatograph t r i f l u o p e r a z i n e on s i l i c a g e l . m d i f i e d s i l i c a g e l , and
aiumina, and a r e l i s t e d i n T a b l e 10 a l o n g w i t h t h e r e s p e c t i v e R f s o b t a i n e d .
S i m i l a r l y , a l a r g e number o f d e t e c t i o n reagents, i n c l u d i n g spray reagents,
have been used t o d e t e c t t r i f luoperazine.
T h i n l a y e r chromatographic s e p a r a t i o n s have been used t o

separate t r i f l u o p e r a z i n e from i t s m e t a b o l i t e s and a l l subsequently i d e n t -
i f i e d u s i n g d i f f e r e n t i a l spray r e a g e n t s ( T a b l e 10). s p e c t r o p h o t o m e t r i c p r o -
cedures, and mass spectrometry. F u r t h e r , on i s o l a t i o n o f t h e s e m a t e r i a l s ,
t h e y were q u a n t i f i e d u s i n g s p e c t r o p h o t o m t r i c procedures. T a b l e I I l i s t s t h e
t i s s u e s s t u d i e d and t h e ' r e a d - o u t ' used for t h e q u a l i t a t i v e and/or q u a n t i t a t i v e
analysis.
TRIFLUOPERAZINE HYDROCHLORIDE 57 I
T a b l e 10
TLC Systems
M o b i l e Phase Adsorbent -
Rf Reference
Cyc I ohexane-Benzene- S i l i c a Gel- 0.45 83

D i e t h y l a m i n e (75:15:10) 0.13 KOH
Methano I S i l i c a Gel- 0.49 83

0. ltj KOH
Aceione S i l i c a Gel- 0.19 83

O.IM KOH
Methanol S i I i c a Gel- 0.10 83

0: KHsot,
Ethanol ( 9 5 % ) S i I i c a Gel- 0.02 83

0.13 KHSO,
Ethylacetate-MeThanoI- S i l i c a Gel 0.12 84

Ammnia (85:10:5)
Ammn i urn Acetate-Methanol S i I i c a Gel 0.63 85

(10 ml 15%:40)
Methanol-121 Ammonium S i I i c a Gel 0.57 32

Hydroxide (100: I .5)
Cyclohexane-Diethylamine- S i l i c a Gel 0.54 32

Benzene (75:20:15)
Ace tone S l I i c a Gel 0.12 32
Ch lorofonn-Methanol (90:10) S i l i c a Gel 0.52 32
Benzene-Ethanol-I2N Arnmnium S i I i c a Gel 0.56 32

Hvdroxide (95:15:5)
Ethylacetate-Acetone-1:l 0.44 86
S i l i c a Gel
Arnmon i urn Hyd r o x i de i n
Ethanol (90:45:4)
Ethanol-Water-Acetic Acid S i I i c a Gel 0.28 07

(20:20: I )
Chloroform-Methanol (100: 10) S i l i c a Gel 0.45 88
ethyl acetate-Ch1oroform- S i l i c a Gel 0.33 89

Methanol-O.IbJ Sodium Acetate
pH 4.7 b u f f e r (54:23:18:5)
A L E X POST ('I crl.
Table 10 (continued)
Mobi l e Phase Ad so rbent -

Rf Reference
Benzene-Dioxane-Ammonia S i l i c a Gel 0.69 31
( 60: 35 :5 )
Ethanol-Acetic acid-Water S i l i c a Gel 0.33 31

( 50: 30 :20 )
Methanol-Butanol (60:40) Si I i c a Gel 0.40 31
t-Buty i a I coho I -IF Ammon i a S i l i c a Gel 0.18 33

(90: 10)
n-Propanol-IN Ammonia (88:12) S i I ica Gel 0.33 33
Ether, saturated w i t h water Si I i c a Gel 0.09 33
70% Methanol S i I i c a Gel 0.34 33
85% n-propanol S i I i c a Gel 0.12 33
n-Butanol saturated w i t h S i l i c a Gel 0.5 I 33

IN Ammonia
I sopropano I -Ch I o r o f o r m I .3N S i l i c a Gel 0.61 51
Ammonia water (l6:E:I:l)
Acetone-lsopropanol-I! S i l i c a Gel 0.77/0.8 I 12/51

Ammonia (27:21:12)
1.2-Dicholoroethane- S i I i c a Gel 0.5 I /O .32 12/51

Ethylacetate-Ethanol-Acetic
acid-Water(l5:26:12:8:7.5)
I sopropanol-Ch loroform-25% S i l i c a Gel 0.83 12

A m n i a - W a t e r (32: l6:25: I )
Ch I o r o f o rm- Ethano I-Arnmon i a S i I i c a Gel 0.80 52

(80:20: 1 )
Benzene-Dioxane-Diethylamlne- S i l i c a Gel 0.85 37

Ethanol (50 :40: 5: 5 1
Acetone-Ethyl acetate-Ethanol S i I i c a Gel 0.10 90

(5:4:l) s a t u r a t e d w i t h
Amnonium l a c t a t e pH 3
Acetone-Ethyl acetate-Ethanol S i I i c a Gel 0.30 90

(5:4: I ) saturated w i t h
Amnonlum l a c t a t e pH 7
TRl FLVOPE RAZINE HYDROCHLORIDE 513
Table 10 (continued)
Mcbi le Phase Adsorbent -

Rf Reference
Acetone-Ethyl acetate-Ethanol Silica Gel 0.17 90
( 5 : 4 : I ) saturated with
A m m n i u m lactate pH 9
Benzene-Acetone 95:5) Alumina neutral 1 0.20 91
Benzene-Acetone 95:5) A 1 u m i na basic) 0.40 91
Benzene-Acetone 90: 10) A I umi na basic) 0.53 91
Water-Acetone (70:30) Cel lulose c.35 91
Toluene-Chloroform-Methanol- S i I ica Ge 0.44 92

Ammonium Hydroxide
(60:40: 1O:O.Z)
Ethyl acetate-n-Propan6i- S i I ica Ge 0.53 43

Ammnia (70:25:4)
Ethyl acetate-Dichloroethane- Si I ica Gel 0.47 93

Pmmon i a ( 8 0 : 20: 5)
Cyclohexane-Diethylamine- S i I ica Gel 0.43 93

Benzene (80:15:5)
-
N N N N
m m m m
N
x x x x X
x x x x x x x x x x
x x x
-
a
x x x x x x x
x x x x x x x x x x x x x x
V
0
I a l
m -c
>
7.621 D e t e c t i o n Methods
Numerous d e t e c t i o n methods, i n c l u d i n g s p r a y r e a g e n t s ,
have been used t o v i s u a l i z e t r l f l u o p e r a z i n e on t h i n l a y e r p l a t e s . A listing
o f them can be found i n T a b l e 12.
T a bl e 12
D e t e c t i o n Methods Used i n TLC o f T r i f l u o p e r a z i n e
Reagent Response ( c o l o r ) R eference
Phosphomolybdic a c i d yellow-brown (tan) 86

+ Ferric chloride
Folin-Ciocalteau cameo 32
F e r r i c c h l o r i d e i n HCIOb t HN03 p in k-.o range 32.97
A m n i um vanadate flesh 32
Cinnamylaldehyde cameo 32
p-Dimethylaminobenzaldehyde pink 32
Fu r f u r a I cameo 32
l o d o p t a t i n a t e s p ray violet 85
Bromine orange-p i n k 94
A n i l i n e vapor f o l l o w e d mauve-purp le 94
by bromine
Te t r a c y a n o e t h y le ne i n brown+yellow 95
a c e t o n i t r i le
2 , 4 , 7 - T ri n i t r0 - 9 -f l u o r e n o n e in grey 95
aceton i tri l e
1,3,5-Trinitrobenzene i n toluene brown 95
Vanadi um pentoxide-HzSOb orange 96

97
p-Dimethylaminobenzaldehyde orange
40% HzSO,, + h e a t orange 92
0. I%Bromocresol p u r p l e blue 98
5% HgSO,+-ethanol orange 31
HC104 brown 99
N i t r i c acid brown-yel low 99
5% A m n i u m p e r s u l f a t e orange 52
7.63 H igh Performance L i q u i d Chromatography (HPLC)
A d s o r p t i o n . l o 0 i o n - e x ~ h a n g e , r~e v~e r~s e

~ ~phase,
~ ~ l C 1 and i o n -
p a i r i n g r e v e r s e phase102 systems have been used t o e v a l u a t e t r i f l u o p e r a z i n e .
The p r o c e d u r e i s r a p i d , y i e l d i n g good r e s o l u t i o n of s e v e r a l p h e n o t h i e z i n e s .
Table 13
HPLC Parameters f o r T r i f I u o p e r a z l z
Flow Rate R t (mln)

Co Iumn t b b l l e Phase (ml / m l n) Detector (approx) Re fererice
SII-x-I Ch1orobutane:Iso-octane
(Perk In-E I mer 1 conta I n I n g I % d i e t h y lami no LOO
I UV (254 nm) 16
ION-X-SC 0.01M (NHI, )2ttPO4 I n methanol :

H20 (2:3), adJusted t o pH 9 . 0 I UV (254 nm) e 100
U I trasphere-IP 0.01M %POI, + 0.01M Nonylamlne,

(Altex) adJuTted t o pH 3 . 0 , + 35%
10 I
acetonltrlle 2.0 uv 5
)1Bondapak C-18 10%0.25M Camphorsulfonlc acid,

(Waters) 60% methanol, 30% water,
a d j u s t e d t o pH 3.0 UV (262 nm) 30 I02
Alkylsulfonic Methanol-O.5M Ammonlum N i t r a t e

Acid s t r o n g (pH 6.0) (4:T) UV (254 nm) 10 103
cation
exchanger
TRiFLUOPERAZlNE HYDROCHLORIDE 577
7.64 Gas L i q u i d Chromatography
Gas l i q u i d chromatography continues t o p l a y an i m p o r t a n t r o l e

i n t h e d e t e c t i o n and d e t e r m i n a t i o n of t r i f l u o p e r a z i n e and 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 o f t r i f l u o p e r a z i n e and i t s m e t a b o l i t e s i n b i o l o g i c a l m a t e r i a l s .
I n Table 14 a r e l i s t e d a number o f s u b s t r a t e s and o t h e r parameters used t o
chromatograph t r i f I uoperaz i ne.
Using p y r o l y s i s techniques, Fontan, e t a t T t 1 c o u l d r e a d i l y

i d e n t i f y t r i f l u o p e r a z i n e and d i f f e r e n t i a t e i t from its m e t a b o l i t e s and o t h e r
phenothiazines. De LeenheerllZ coupled p r e p a r a t i v e gas I i q u i d chromatography
w i t h m i c r o - i n f r a r e d spectroscopy f o r t h e i d e n t l f i c a t i o n of t r i f l u o p e r a z i n e
and o t h e r phenothiazines. T r i f l u o p e r a z i n e was separated on a 1% FFAP column
a t 23OoC.
Table 14
Gas L i q u i d Chromatography
Parameters f o r t h e GLC of T r i f I uoperazi ne
Co I umn Carrier Rt
Column Temperature Gas Detector -
(min) -
Ref.
FID 22 104
5% QF-l on Anakrom 210' f o r 18 N2
ABS, 100/110 min. program-
med t o 240'
235' He FID 9.0 104
3.5% XE-60 on Gas
Chrom Q, 100/120
235O He FID 22.5 I04
3% OV-17 on Gas
Chrom Q
FID 9
5% Ov-1 on Diato- 230' N2 6.9
p o r t S, 80-100 mesh
9
2% FFAP on Diato- 230' N2 FID 13.6
port S , 80-100 mesh
85
3% SE-30 on Gas Chrom 210 He FID 8.6
Q, 80-100 mesh
105
1% HI-EFF-8BP + 10% 2200 N2 FID % 120
SE-52 on Gas Chrom Q,
80-100 mesh
106
2% SE-30 on Gas Chrom Q, 205' Argon 90SR 42
80-100 mesh
107
3%OV-l on Gas Chrom Q, 245' N2 FID 3. I
80-100 mesh
108
5% SE-30 on D i a t o p o r t S, 270° N .FID 5.5
2
60-80 mesh
109
10% SE-30 and I % t r l - 245O N2 FID 6.9
s t e a r i n on Gas C h r m W
1 % HI-EFF-86 on 2208 N2 FID 10.9 110
S l l a n i z e d Gas Chrom P
1% HI-EFF-8B o n 250° N2 FID I .6 I10
S i l a n i z e d Gas C h r m P
ALEX POST ei ul.
7.65 E I e c t rophores i s
Paper e l e c t r o p h o r e s i s on b u f f e r e d Whatman 3MM paper (pH 3.3
t o 9.3) s e p a r a te d t r i f l u o p e r a z i n e and i t s s u l f ~ x i d e . ~l d~e n t i f i c a t i o n was
made from t h e r e s p e c t i v e m i g r a t i o n d i s t a n c e and by t h e response t o a s u l f u r i c
a c i d spray r e a g e n t and by i t s f l uo resce nce .
Migration ( i n cml
f!E T r i f I uoperaz i ne Su I f o x i de
3.3 7.0 7.2

4.7 4.4 6 -2
6.0 4.7 5.8
1.2 4.7 5.4
8.0 3.2 5.4
9.3 1.7 5.9
8. M is c e lla n e o u s
8. I A d s o r p t i o n I sot he rm
A d s o r p t i o n isotherms o f t r i f l u o p e r a z i n e by carbon b l a c k , g r a p h i t e ,
s i I i c a g e l , and p o l y e t h y l e n e determined by Nogami, e t a1 'I4 showed a r e l a t i o n -
s h i p t o i t s n e u r o l e p t i c and h a e m o l y t i c a c t i v i t y . The amount absorbed was
r e l a t e d t o t h e m l e c u l a r volume o f R a t t h e 1 0 - p o s i t i o n , t h e b u l k i n e s s of
t h e s u b s t i t u e n t a t t h e 2 - p o s i t i o n , t h e pH o f t h e b u f f e r s o l u t i o n , and t h e
p a r t i t i o n c o e f f i c i e n t i n CHCt3/0. I& HCI. Sorby, e t determined t h e
a d s o r p t i o n is o th e r m s by k a o l i n , t a l c , and a c t i v a t e d carbon. They showed
t h a t t h e a d s o r p t i o n by k a o l i n and t a l c was aependent upon t h e pH o f t h e
medium whereas t h i s WBS n o t t h e case w i t h a c t i v a t e d carbon.
8.2 S u r fa c e A c t i v i t y
S e v e r a l p h eno t h i azi n es have been e v a l u a t e d f o r t h e i r e f f e c t on

s u r f a c e a c t i v i t y as an e x p l a n a t i o n for t h e i r p h y s i o l o g i c a l a c t i v i t y . Zografi
and Munski116 showed t h a t t r i f l u o p e r a z i n e was many t i m e s more e f f e c t i v e than
c hl orpro m a z ln e i n l o w e r i n g t h e s u r f a c e t e n s i o n of a pH 5.0, i o n i c s t r e n g t h 0.1,
s o l u t i o n a t 25OC. Seernan and B i a l y l l ' a t t r i b u t e d t h e a c t i v i t y o f t r a n q u l I l z e r s
t o t h e lowering of t h e surface tension a t the e r y t h r o c y t e surface v i a t h e
a d s o r p t i o n o f a m n o m l e c u l a r l a y e r o f t h e p h e n o t h i a z i n e analog. Trifluoper-
a z i n e was shown t o be s i g n i f i c a n t l y m r e e f f e c t i v e i n l o w e r i n g t h e s u r f a c e
t e n s i o n t h a n c h lo r p r oma zi ne , and, t h e r e f o r e , a more p o t e n t t r a n q u i I i z e r .
TRI F L U OPE R A Z I N E HY D R O C H LORI DE 579
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6 0 Zia, H. and P r i c e , J.C., Anal. Chem., 64, 1177 (1975).
6 1 Gabay, S. and Huang, P.C., The P h e n o t h i a z i n e s and S t r u c t u r a l l y R e l a t e d
Drugs, a d l t e d by 1.5. F o r r e s t , e t a l . , Raven Press, N.Y. (1974).
6 2 E.R. Reich, personal communication, Smith K I i n e & French Labs.
6 3 N a t i o n a l Formulary XIV, p. 737.
6 L Eno p o i n t d e t e c t i o n used a t t h e Smith K I i n e 8 French L a b o r a t o r i e s .
6 5 Chatten, L.G. e t al., J. Pharm. Sci., 60, 588 (1971).
66 Olech, A., Acta. Polon. Pharm., 2, 64 (1972).
67 Chemia .Analit., Is, 651 (1973). Ana!. Abct. #465 (January 19741.
68 Watson, J.R. e t a l . , J. Pharm. Sci., 2, 391 (1970).
69 Davidson, A.G., J. Pharm. Pharmacol., 2, 795 (1976).
70 Stevens, H.M. e t a l . , J. F o r e n s i c Sci., 17, 169 (1977).
?I Palcolm, H.M., ibid., 1 1, 57 (19771,
a, 35,
72 M e l l i n g e r , T.J. and Keeler, C.E.. Anal. Chem., 554 (1963).
73 M e l l i n g e r , T.J. and Keeler, C.E., ibid., 1840 (19641.
7 4 Ragland, J.B. and Kenross-Wright, V.J., i b i d . , 2, 1356 (1964).
75 Ragland, J.B. e t al., Anal. Biochem.,
76 Tompsett, S.L., Acta. Pharmacol. e t . Toxicol.,
u, 2. 60 (1965).
298 (1968).
77 Spano, P.F. e t al.. J. Pharmacol. Exp. Therap., 174, 20 (1970).
78 Eagelson, D.A., J. C I l n . Pathol., 39, 648 ( 1963).
79 Nadeau, G. and Sobollewski, E., J.Thromatog., 2, 544 (1959).
80 Macek, K. e t al., Pharmazie, 20, 605 (1965).
8 1 S t r e e t , H.V., Acta. Pharmacol. e t . Toxicol., 3, 312 (19621.
82 Heyman. J.J. e t al., Am. J. Psychiat., 117, 1108 (19601.
83 F i ke, W.W., Anal. Chem., 2, 1697 ( 1 9 6 6 r
84 Neesby, T., C l i n . Chem., 19, 356 (1973).
85 Kofoed, J. e t al., J. C h r z a t o g . , 23, 410 (1966).
86 Korczak-Fabierkiewicz, C. and Cimbpa, G., ibid., 53 413 (1970).
8 7 Clarke, V. and Cole, E.R., ibid., 24, 259 (19661..-'
8 8 Kiger, J.L. and Kiger, J.G., A n n a l z Pharmaceutiques Francaises, 2.
489 (19651,
89 Pluym, A., J. P h a n . Sci., 68,
1050 (1979).
90 Margasinski, 2 . e t al., Acta. Polon. Pharm., 5 (1964). 2,
9 1 Eiden, F. and Stachel, H.D., Deut. Apoth. Ztg.. 121 (1963). 103,
92 P e t e r Begosh, personal connnunication, Smith K I i n e b French Labs.
TRI FLUOPE RAZ I N E HYDROCHLORIDE 58 I
9 3 L a u f e r , V.S. e t al., Arzneim-Forsch., 19, 1965 (1969).

9 4 C l a r k e , V. and Cole, E.R., 3. Chromatog,, 53, 413 ( 1 9 7 0 ) -
95 F o r r e s t , J.E. and Heacock. R.A., 75,
ibid., 156 (1973).
96 N o i r f a l i s e , h . , i b i d . , Z&, 61 (1965).
9 7 De Leenheer, A., 75,
ibid., 156 (1979).
.
J. Pharm. S c i , 50, 350 ( I961 ) .
q 8 Dernoen, P.J.,
99 Eberhardt, H. e t a l . , Arzneim-Ersch..
100 Rogers, D.H., J . Chrom. Sci., 12,
z.
742 (1974).
804 ( 1 9 6 3 ) .
I o 1 Cooke, N.H.C. and O l s e n , J., American Lab., Aug. 1979, p . 45.

10: E.R. White, p e r s o n a l communication, Smith K I i n e & French Labs.
l o 3 Wht:aIs, B.B., J, Chromatog., 177, 263 (1979).
lo4 Procisc, H.F. and Lohmann, H.J., C I in. Chem.,2. 222 (1971 ).
1°5 Rader, 6.R. and Aranda, E.S., J. Phann. Sci., 57, 847 (1968).
l o 6 Anoers, M.W. and Mannering, G.J., J. Chromatog., 7, 258 ( 1962).
Io7 Wells, J. e t al., J. F o r e n s i c Sci., 0, 382 (1975r,
M a r t i n , H.F. e t al., Anal. Chem.. 35,. 1901 (1963).
l o g McMartin, C. and S t r e e t , H.V., J . Chromatog., 22, 274 (1966).
110 Jain, N.C. and K i r k , P.L., Microchern. J., 12, z 9 ( 1 9 6 7 ) .
Fontan, C.R. e t a l . , Mikrochim. Acta, 1 9 6 $ . 3 6 4 .
11* De Leenheer, h . , J. Chromatog., 74, 35 (1972).
1 1 3 Gudzinow;cz, B.J., Gas Chromatographic A n a l y s i s o f Drugs and P e s t i c i d e s ,
Marcel Dekker, Inc., N.Y. (1967).
114 Nogami, H. e t a l . , Chem. Pharm. B u l l . .
2,
,s, 1643 ( 1 9 7 0 ) .
785 ( 1 9 6 6 ) .
1 1 5 Sorby, D.L. e t a l . , J. Pharm. Sci.,
116 Z o g r a f i , G. and Munski. M.V., ibid., 59, 819 (1970:.
117 Seeman, P.M. and B i a l y , H.S., BiochemFPharm?coI., 2, 1181 (15633.
GRISEOFULVIN
Mahrnoud A . Hassan and Elsayed A . Aboutabl
1. Description 584
I . 1 Nomenclature 584
1.2 Formulae 584
2.2 Dissolution 587
3. Synthesis 592
4.1 Time- Resolved Phosphorimetry 594
4.2 Liquid Chromatography 594
4.3 Isotope Dilution 594
References 599
Copyright @ 1980 by Academic Press, Inc.

Analytlcal Rofiles of Drug Substances, 9 583 All rights of reproduction in any form reserved.
ISBN:0-12-260809-7
584 MAHMOUD A. HASSAN A N D E. A. ABOUTABL
GRISEOFULVIN
1. Description
1.1 Nomenclature
1.1.1 Other names: Curling Factor.
1.2 Formulae
1.2.1 Structural
s
Various structural formulae have been
proposed €or griseofulvin, but the
currently accepted is that suggested by
Grove --
et a1 (1). According to this formu-
lae the molecule of the antibiotic con-
tains three rings: the aromatic benzene
ring (A), a 5-membered heterocyclic ring
with an atom of oxygen (B) and a hydro-
aromatic 6-membered ring (C). The A 6 B
rings are condensed forming a coumaronone
system. Carbon atom 2, which is common
rings B & C is an assymetrical carbon
atom giving griseofulvin its spiran struc-
ture and causing its optical activity, to

GRISEOFULVIN 5 85
which the assymetrical 6 carbon also contri

-butes. The C ring may be regarded as the
methyl ether of the enol form of 2,4 dike-
tone o r as the methyl ether of 6-methyl
dihydroresorcinol.
1.2.2 Wiswesser Line Notation
T56 BOXVJ F01 H01 1G

C - & D L 6 V DX
BUTJ CO1 E l
1.2.3 Conformation
The preferred conformation of griseofulvin

in solution is that shown in the stereo-
structure given before ( 2 ) . This is based
on the finding o f relatively strong coup-
ling (J = 13.5 H z ) between the 6ca and the
/
5-a-protons. This relies on the application
o f an NMR shift reagent [Tris - (Dipevalo-
methanato) Europium], to the spectrum o f a
partially deuterated sample of griseoful-
vin.
2. Physical Properties:
2.1 Crystal Properties:

2.1.1 Crystallinity
Griseofulvin crystallizes from benzene as
stout octahedra o r rhombs. The crystals
5 86 MAHMOUD A . HASSAN A N D E. A. ABOUTABL
are generally up to 5 nm in maximum dimen-
sion, although larger particles which may
occasionally exceed 30nm may be present.
Crystal size affects the absorption of
griseofulvin when administered orally.
Microsize griseofulvin may be administered
in significantly smaller doses than the
conventional size powder to obtain the same
effect. The U.S.P. specifies that the offi-
cial product is the "Microsize" powder ( 3 ) .
Brown and Sim (4) carried out a quantita-
tive X-ray study of 5-bromogriseofulvin in
order to define unambiguously the stereo-

chemical relationship of the 2- and the 6 /-
centre. Crystals of 5-bromogriseofulvin
belong to the monoclinic system, space

2
group P2 (C ), with two molecules of C17
1 2
H BrClO in a unit cell of dimensions a =
16 6
/
10.96, b = 8.61, c = 10.27 A', = 108'30.
Initial phase determination was based on
the bromine and the chlorine atom and
several three dimensional Fourier syntheses
were evaluated, followed by least squares
refinement of the atomic parameters. The

GRISEOFULVIN 587
final discrepancy R over the 1 1 2 9 observed

reflexions is 14%.
2.2 Dissolution
The dissolution rate of griseofulvin had been
significantly enhanced by solid dispersion in
succinic acid. This had been initially attributed
to the extensive formation of a solid solution

of griseofulvin in succinic acid (5). Later, it
was shown by X-ray diffraction and differential
thermal analysis methods that solid solubility
was negligible and such a binary system could be
classified more adequately as a simple eutectic
mixture ( 6 ) . The dissolution profile of the
griseofulvin-succinic acid eutectic mixture sys-
tem was evaluated using the powder and constant

surface area tablet methods ( 7 ) . Contrary to the
original proposal of Sekigushi --
et a1 (8), disso-
lution rates of griseofulvin from solid disper-
sions were found to be markedly affected by their
particle size.
2.3.1 Ultraviolet Spectrum
In ethanolic solution, griseofulvin exhi-
bits a characteristic UV spectrum (Fig.1)

588 MAHMOUD A. HASSAN A N D E. A. ABOUTABL
I 1
1 ” ” I
4
I
---
- CH; 0
CH; 0
CL
50,000 45,000
-
40,000
cm-‘
35,000 3(
10, 1 1 I 1 1 1 , 1 I 1 I , I 1 1 I I I I
Fig. 1 - UV Spectrum of Griseofulvin in Ethanol.

CRISEOFULVIN 589
with maxima at 325, 292 and 236 nm. The
spectrum of isogriseofulvin is similar to
that of griseofulvin differing only by the

presence of a fourth maximum at 263 nm.
E;!n, at 292 nm = 686. The W spectral data
of griseofulvin analoges have also been
reported (9-11).
2.3.2 Nuclear MagnetiL Resonance Spectra
2.3.2.1 -
PMR
The proton magnetic resonance spec-
tra of' griseofulvin and its deri-
vatives have been investigated(l2,
14).
A typical PMR spectrum of griseo-
fulvin is shown in Fig.2. The sam-

ple was dissolved in deuterated
chloroform (CDC1 ) . The spectrum

3
was recorded on a Varian T-60A NMR
spectrometer with TMS as the refer-
ence standard. The following struc-

tural assignments have been made
(15)
r
Fig. 2 - PMR Spectrum of Griseofulvin in CDC13 and TMS.

GRISEOFULVIN 591
Chemical Shift ( 6 ) Assignment

0.97 (doublet) 6/ - CH3
2.70 (multiplet) 5/-,6/- H
3.60 (singlet) 2 / - OCH3
3.97 (singlet) 4 - OCH3
4.00 (singlet) 6 - OCH3

5.50 (singlet) 3/ - H
6.13 (singlet) 5 - H
The PMR spectrum o f griseofulvin

-5’, 5/-d exhibits only one ali-
2
phztic proton appearing as a quar-
tet at 2.75 6 ascribable to the
6/ -a-proton ( 2 ) . On stirring with
neutral alumina in chloroform this
compound undergoes stereoselective

partial replacement of the 5/ -6-
deuterium substituent with hydrogen
to give griseofulvin 5’-a-d. The

PMR spectrum of a mixture of the
2 compounds (Fig. 3-A) shows no peaks
in the region of 2.3 (501-H) but
exhibits a complex band at 2.7 -

2.96 (1,4H) representing the coup-
led and closely spaced 5/ -B- and

592 MAHMOUD A. HASSAN AND E . A. ABOUTABL
/
6 -a proton signals. A
strikingly altered PMR spectrum
(Fig. 3) was obtained on
application of Eu (DPM)3. Proton
signals are shifted downfield
in general proportion to their
closeness to the C-4 carbonyl
oxygen. The signals due to
6/ -CH3 (1.48), 6/ C-H ( - 3 . 9 8 ) ,
and 5 / B-H (5.36) constitute a
first order (A3MX) system in

which the doublet at 5.36 gives
J 5 / B-6 / a-13.5 Hz. A vicinal
coupling of this magnitude

must be due to trans diaxial
hydrogen substituent.
The PMR spectrum of griseoful-

vin is DMSO D6 has been reported
(16).
3. Synthesis
Several synthetic routes to griseofulvin have been reported

(17-20).
GRISEOFULVIN 593
I I
.I11
' 1111 I
I
.I
Fig. 3 - B
Fig. 3-A : NMR (100) Spectrum of Griseofulvin -5 / , /

d2
and its stereoselective Hydrogen Exchange
Product in CDC13.
Fig. 3 - B : The same with 0.4 molar equivalent of Eu (DPM)3
in CDC13.
594 MAHMOUD A. HASSAN AND E. A. ABOUTABL
4. Methods of 4nalysis
4.1 Time-resolved Phosphorimetry

Phosphorescene life times of griseofulvin and
dechlorogriseofulvin are shown to be 0,11 sec,
and 1.16 sec. respectively ( 2 2 ) . This 10-fold
difference was shown t o enable the use of time-
resolved phosphorimetry for the determination of
griseofulvin in mixtures with dechlorogriseoful-
vin
4.2 Liquid Chromatography
4.2.1 Column Chromatography
A liquid solid chromatographic method was
reported (23) €or the direct analysis of

griseofulvin is complex fermenter brothes.
The method is tedious and time consuming.
4.3 Isotope Dilution

Ashton (24) described an isotope dilution method
€or the assay of griseofulvin based on the esti-
mation of the radioactivity employing griseofulvin
labelled with radioactive 36Cl. McNall (25,26)
reported, another method using tritium-labelled
griseofulvin
GRISEOFULVIN 595
5.4 PMR Spectrometry
A rapid, accurate and specific PMR method for the
determination of griseofulvin in bulk drug and
pharmaceutical formulations has been developed
in our laboratory (15). From Fig.2, it is evident
that griseofulvin exhibits, among other peaks, two
singlets at 3.97 and 4.00 ppm (in CDCl,) assigned
to the 4/ - and 6/ - methoxy protons respectively,
Since the integration of these two peaks gives
the largest region f o r measurement, they are
chosen for the quantitative analysis of griseoful-
vin LI
Acetanilide, exhibiting a three protons signlet at
2.30 ppm (in CDC13), assigned to its methyl groups
is employed as internal standard. The determina-
tion is based on the integration of the 4’- and

6 / -methoxyprotons of griseofulvin relative to
that of the methylprotons of acetanilide, Accu-
rate determination is achieved, since the sig-
nals chosen for griseofulvin are widely separated
from that of acetanilide. Ethanol-free chloroform
is used as the solvent, its proton siTglet at
7.25 ppm does not interfere with the upfield
protons of both compounds. Fig. 4,

I - I
500 400 300 200 100 0;-
Fig. 4 - PMR spectrum of Griseofulvin, acetanilide and TMS in

ethanol-Eree chloroform.
GRISEOFULVIN 597
Assay of a series of known standard mixtures of

griseofulvin and acetanilide by this PMR techni-
que established the accuracy and precision of the

method with an average recovery of 99.55%. The
results of estimation of griseofulvin in tablets
and drysuspension powders are in agreement with
pharmacoepial requirements, No interference from
excipients could be observed.
4,s Microbiological
Dittmer ( 2 7 ) reported on the determination o f
microbiological activity of griseofulvin in body

fluids by dilution methods in liquid or solid
media using Tricophyton mentagrophyte as the
test organism.
--
Mrtek et a1 (28) developed the microculture
slide technique of Elliott --

et a1 (29). The assay
system consists of a suspension of Microsporum

gypseum macroconidia in Sabouraud liquid medium
containing nanogram quantities of griseofulvin.
Antifungal activity is determinzd on specially
prepared microculture slides by measuring changes
in the rate of hyphal elongation. A liner rela-
tionship of log dose to hyphal growth rate is
observed in the range of 0.001 - 0.01 mcg/ml gri-
seofulvin. This technique exhibited precision at
least equivalent to that of the agar cup proce-
dure
GRlSEOFULVlN 599
REFERENCES
1. J.F. Grove, J. Macmillan, T.P.C. Mulholland and M.A.

Thorold Rogers, J. Chem. SOC., 3977 (1952).
2. S.G. Levine and R.E. Hicks, Tetrahedron, Lett., (4),311,

(1971).
3. C.O. Wilson, 0 . Gisvold, R.F. Doerge, "Textbook of
Organic Medicinal and Pharmaceutical Chemistry", 7th Ed.,
J.B. Lippincott Co., Philadelphia, p.343 (1977).
4. W.A. Brown, G.A. Sim, J. Chem. SOC., 1050 (1963).
5. A.H. Goldberg, M. Gibaldi and J . L . Kanig, J. Pharm., Sci.

55, 487 (1966).
-
6. W.L. Chiou and S. Niazi, J. Pharm. Sci., 62, 498 (19731.
7. W.L. Chiou and S. Niazi, J. Pharm. Sci., 65, 1212 (1976).
8. N. Obi., Sekiguchi and Y. Ueda, Chem. Pharm. Bull., 9,

866 (1961).
9. V. Arkley, J. Attenburrow, G.I. Gregory and T. Walker,

J. Chem. SOC., 1260, (1962).
10. G.I. Gregory, P.J. Holton, H. Robinson and T. Walker,

J. Chem. SOC., 1269 (1962).
11. V. Arkley, G.I. Gregory and T. Walker, J. Chem. SOC.,

1603 (1963).
12. G.F.H. Green, J.E. Page and S.E. Staniforth, J. Chem.

SOC., 144 (1964).
13. S.G. Levine E R.E. Hicks, Tetrahedron Lett., 5409 (1968).

14. S.G. Levine 6 R.E. Hicks, Tetrahedron Lett., 311 (1971).
15. E. Aboutabl and M.M.A. Hassan, Talanta, (in press).
16. Edward R. Townley, ftAnalyticalProfile of Griseofulvin"

a chapter in Analytical Profile of Drug Substances, Vol.
8, Edited by K . Florey, Academic Press, Newyork,
Newvork. 1979 n.224.
17. C.H. Kuo, R.D- Hoffsommer, H.L. Slates, D. Taub and N.L.
Wendler, Chem. Ind., 1627 (1960).
18. G. Stork, M. Tomasz, J. Am. Chem. SOC., 84, 310 (19621,

ibid., 86, 471 (1964).
19. T. Fields, H. Newman and R.B. Angier, J.Med. Chem., -

13,
1242 (1970).
20. T. Fields, H. Newman and R.B. Angier, J. Med. Chem., 14,

767 (1971).
21. Y. Sato, T. Oda and S. Urano, Tetrahedron Lett., (31),

2695 (1976).
22. J.R. Meduffie and W.C. Neely, Anal. Biochem. -

54, 507,
(1973).
23. A. Holbrook, F. Bailey and G.M. Bailey, J. Pharm. Pharma
-co~., 2, 274 T. (1963).
24. G.C. Ashton, Analyst, -

81, 288 (1956).
25. E.G.McNal1, Antibiotics Annal., -

60, 674 (1959).
26. E.G.McNaI1, Arch. D e n . Chicago, -

81, 657 (1960).
27. W. Dittmer, Intern. Congr. Chemotherapy, Proc., Stutgart

1963 (1) , 728-32 (1964).
28. M.B. Mrtek, L.J. Lebeau, F.P. Siege1 and R.G. Mrtek, J.
Pharm. Sci., -58, 1363 (1969).
29. H.E. Elliott, L.J. LeBeau and M. Novak, Bacteriol.

Proc., 56, 55 (1956).
METHADONE HYDROCHLORIDE
Muhmoud A . Hassun and Abdulluh A . Al-Budr
1. Description 602
1.2 Conformation 602
2.1 Optical Rotatory Dispersion Spectrum 602
2.2 Circular Dichroism Spectrum 605
2 . 3 Crystallographic Properties 606
3.1 Gravimetric Analysis 607
3.2 Ultraviolet Analysis 607
3.3 Ion-Exchange Chromatography 607
3.4 Radio-Immunoassay 609
3.6 Gas Chromatography 610
3.7 High Pressure Liquid Chromatography 61 I
References 614
Copyright @) 1980 by Academic Press. h c .

Analytical Rofiks of Drug Substances. 9 60 1 All rights of reproduction in any form reSerVed
ISBN: 012-260809-7
602 MAHMOUD A. HASSAN A N D ABDULLAH A . AL-BADR
METHADONE HYDROCHLORIDE
1. Description
1.1 Nomenclature
1.11 Chemical Name
N,N-Dimethyl-1, 1-diphenyl-1-propan-1-one-methyl
propylamine hydrochloride (1).
1.12 Generic Name
Methadone hydrochloride; Metadone hydrochloride.
1.13 Trade Name
Tussal.
1.14 Wiswesser Line Notation
2VXR&RhlY&N1&1 &GH DL
1.2 Conformation
A probable conformation of methadone hydrochloride,

based upon crystallographic (2) and spectroscopic evi-
dence ( 3 ) , is shown in Fig.1. This conformation is
stabilised by a hydrogen-bonding interaction as has
been suggested by Beckett and Casy (4). Further evi-
dence of such conformation was also obtained by the
work of Henkel - et -
a1 (5).
2.1 Optical Rotatory Despersion Spectrum
The ORD characteristics of (+) and (-)-methadone have

been reported (1) and given below. The ORD curves
are shown in Fig. 2.
(+) Methadone :
METHADONE HYDROCHLORIDE 603
Q Me
PROBABLE CONFORMATION OF
METHADONE
FIG, 1
604 MAHMOUD A. HASSAN AND ABDULLAH A. AL-BADR
16
12
8
\ 4 I
I
I
4
$+
-
a 0
m
e -
4
i
I
B I
I
I
I
,'7 !
I J
200 300 400 500 600
S(mp)
Fig, 2: ORD Curves of (+ 1-methadone (a)

and (-)-methadone(b)
(-> Methadone :
[a]*36" (Cyclohexane). RD (C,O,ll;Dioxanne) : [@]600-185';

[ @ 1 5 0 0 - 1 8 5 ~ [; @ ] 4 0 0 - 9 3 " ; [@]37s+O0; [ @ ] 3 1 6 + 4 066O;
[@]29e+0°; [@]274- 1 4 974"; [@]270-12 796'; [@]268-
13611'; [@]251+-11433";[@]228- 1 1 9 8 0 ' .
UV : X- = 250 nm (log E =3,01) (inflexion),
286 nm (log E = 2 , 6 2 ) , 296 nm (log E = 2,62).
I
'1
:I
-.Ll
'"1
F i g . 3 : CD spectra of (+) - (6s) -methadone :

0.1% solutions in CH30H ( - ) ,
(---)y 3'"
hexane(*-.), and CH3CN(-*-. .
2.2 Circular Dichroism Spectrum
The CD spectral characteristics f o r (+)-methadone and

(+)-methadone hydrochloride have been reported (5) and
are given below. The CD spectra are shown in Fig. 3,
and Fig. 4.
Fig. 4 : CD spectra of (+)-(6s)-methadone hydro-

chloride, 0.025% solutions in CH30H (-)
and CHCl ( - - - ) .
3
2.3 Crystallographic Properties
Hanson and Ahmed (2) have reported the crystal struc-

ture and absolute configuration of monoclinic form of
d-methadone hydrobromide. The crystal is monoclinic,
probably P2 , a = 10.69, b = 8.74, c = 10.74
1
A " , B = 9 4 . 6 " , 2 = 2. The structure determination,

which was essentially three-dimensional, was begun by
the heavy atom method, and completed by means of dif-
ferential syntheses. The absolute configuration of
the molecule was determined by measuring the effect
on two selected sets of reflexions of the imaginary
part of the dispersion of copper radiation by the
bromine atom. A projection of a single molecule along
a convenient direction is shown in Fig.5. The abso-
lute configuration is that of the (+)-isomer. The
bromine atom, which is not shown, lies near the apex
of the pyramid formed by the nitrogen atom and its
neighbours.
3. Methods of Analysis:
3.1 Gravimetric Analysis:
Loucas et a1 (6) have published a gravimetric method

for the determination of methadone hydrochloride in
flavoured syrup formulation, by mixing the sample
(equivalent to 10-20 mg of the drug) with lOml of 1%
Molypdophosphoric acid solution, collecting the preci-
pitate on a Millipore membrance-filter and drying it
at 60"; lmg of the precipitate = 0.4mg of the drug.
Nitrogenous bases, particularly alkaloids interfered,
being co-precipitated with the drug.
3.2 Ultraviolet Analysis:
Caddy et a1 (7) described an oxidative analytical pro-

cedure for the determination of certain drugs contairdng the
diphenylmethylene group in blood and urine. The
method is based on the oxidation of the drug with
alkaline potassium permanganate to form benzophenone.
For calibration, a standard solution of the drug salt
is heated with alkaline potassium permanganate solu-
tion and heptane, and the extinction of the organic
layer is measured vs heptane at (247 nm). Beers Law
is obeyed for up to 20 u g of benzophenone per ml of
heptane solution. Urine or blood samples (adjusted to
pH 10.5) are extracted with ethyl ether the extract is
washed with N H C 1 , and the concentrated acid solution
is treated as for standard solution.
3.3 =Exchange Chromatography:
Knox et a1 ( 8 ) have described a chromatographic method

for the separation of methadone from mixtures of other
608 MAHMOUD A . HASSAN A N D ABDULLAH A . AL-BADR
0 Carbon
Fig. 5 : The d-methadone molecule, a s i t occurs i n

the monoclinic form of the bromine deri-
vative. The orientation t r i p l e t i s
composed of 1 A.'vectors, i n the directions
of the principal axes.
drugs. The method was carried out on a stainless

steel column (lm x 2.lmm) which was packed with Zipax
SCX (37-44 um), and sample was injected through a
septum and was eluted with aqueous borate buffer under
pressures of 500-1500lb per square inch; the elute
was passed through an 8 p.1 flow-cell and its extinc-
tion was measured. Methadone was separated with
buffer solution of pH 9.8.
3.4 Radio-Immunoassay:
Cleeland et a1 (9) published a review dealing with the

analysis of urine, blood, saliva and tissues for metha-
done with other drugs of abuse using radio-immunoassay.
3.5 Thin Layer Chromatography (TLC):
Gupta et a1 (10) have described a TLC method for scre-

ening of the major methadone metabolites and methyl
amphetamine in urine. Urine lml is placed in a screw-
capped PTFE-lined culture tube and 0.25 M CuSO4 (lml),
saturated aqueous sodium bicarbonate (lml) and chloro-
form (5ml) and added. The aqueous layer after centri-
fugation is aspirated off and the organic layer is
decanted into a test-tube to which is added 4-chloro-
7-nitrobenzofurazan chloride in chloroform. The solu-
tion is evaporated to dryness and the residue is
dissolved in chloroform. The solution is subjected
to TLC on silica gel (0.25 mm thick) by development
with ethylether-benzene(1:l). The methadone metabo-
lite 2-ethylidene-1,5-dimethyl-3,3-diphenyl pyrroli-
dine produces a blue-green and purple spots (% 0.94
and 0.84, respectively) with the above reagent.
Jain --
et a1 (11) have reported another TLC method
far the separation of methadone and its primary
metabolite in the presence of other drugs in
urine specimens. The sample was treated with conc.
aqueous ammonia and extracted with chloroform-ethyl
acetate-methanol (3:l:l). The organic layer was fil-
tered through phase-separating paper and evaporated
at 70" under N . The residue was dissolved in metha-
nol and applie8 to a silica gel E or FG precoated
plates. The best solvent systems were ethyl acetate-
dichloromethane-conc. aqueous ammonia (90:10:0.9),
ethyl acetate-octanol-conc. aqueous ammonia (93:7:1)
and ethylacetate-isopropylether-water-conc. aqueous
ammonia (90:lO:l.l). Spots were detected with
iodoplatinate spray reagent. Methadone and its
primary metabolite 2-ethylidene-1, 5-dimethyl-3,
3-diphenyl pyrrolidine were well separated from each

other. The limit of detection was 0.25 pg/ml both
for methadone and for its metabolite.
Davis et a1 (12) have reported an improved thin layer

chromatographic system for methadone and its meta-
bolites in biological samples using the Gelman instant
thin layer chromatography (ITLC) system. The ITLC was
modified by applying a thicker layer of silica gel to
the base of the imprignated fiber-glass strip, so as
to reduce the tendency to over load when working with
biological extracts. The technique described is
illustrated by the application to the separation of
labelled methadone and metabolites (pyrrolidine and
the N-oxide) in a kidney extract by the following
solvent systems:
a) ethylacetate-methanol-aqueous ammonia
17 : 2 : 1
b) benzene-ethylacetate
19 : 1
c) benzene-ethylacetate-methanol-aqueous ammonia
800 : 2000 : 12 : 1
followed by radiometric coating
3.6 Gas Chromatography:
Gas liquid chromatography systems for determination of

methadone in sustained-release tablets (13). The
method involves the extraction of a tablet at 37" with
successive portions of dissolution medium (mixtures of
gastric fluids and intestinal fluids of pH increasing
from 1.2-7.5). Each extract is made alkaline to phe-
nolphthalein and 10 ml portions were extracted with
chloroform (50 ml). Each chloroform extract was
dried over sodium sulfate and a 10 .nl portion was
evaporated with a chloroform solution of atropine
(internal standard). The residue was dissolved in
chloroform (2 ml) and a 1-2 1.11 portion was subjected
to GLC on a spiral siliconized glass column (3 ft.
long x 2 mm packed 3% of SP 2250-DP on Supelcophrt
(100-120 mfsh) and operated at 235" with a Helium
35 ml min- as a carrier gas and flame ionization
detection. The amount of methadone was calculated
METHADONE HYDROCHLORIDE 61 1
from the peak height and molar response ratios rela-

tive to atropine.
Lynn et a1 ( 1 4 ) has reported a new gas-chromatogra-

phic assay for determination of methadone in man and
animals (6). The internal standard.,2-dimethyldno-4
4-diphenylnonane-5-one is added to the specimen con-
taining the drug and then extracted with chlorobutane
at pH 9.8. Then it is extracted into 0.5M H SO4 and
and after alkalinization is extracted into choroform.
The extract was analysed on a column (6ft x 2mm) of
1.5% OV-101 on Gas-Chrom Q (100-120 mesh). Thf - tem-
perature is programmed from 170-250' at 1 min ,
with N2 as carrier gas (30ml min-l) and a H-flame
ionization detector. The peak area ratio of the
standard and the drug was obtained by electronic
integration. Tracer studies with (+)-14C-methadone
showed that the recovery was 9 3 + 2 % for the extrac-
tion and > 99% in subsequent stages.
3.7 High Pressure Liquid Chromatography (HPLC):
Knox and Jurand (15) have applied a high-speed liquid

chromatography €or the determination of methadone and
other narcotics. The chromatographic behaviour of
the narcotics studied has been investigated on a glass
or stainless steel column (80-100 cm x 2mm) packed
with Zipax Pellicular resins (37-44 m) and operated
at room temperature, with UV detection. Conditions
are outlined for rapid determination of methadone on a
column of strong anion exchange resin. The eluted
compound was identified by its W absorption and mass
spectrum.
Trinler and Renland (16) have reported a rapid screen-

ing of methadone and other narcotics by reverse phase
HPLC. The column (2ft x 0.125 inch, 0.d.) packed with
Bondapak c18 - Corasil; detection is by W spectro-
metry (254 nm). The eleuent is acetonitrile-water
( 9 : l ) and the fractions are collected for analysis by
W or IR spectrometry.
Goodman et a1 (17) have tried a combination of HPLC

and tritium exchange for the determination of common
drugs of abuse and their metabolites including metha-
done. The HPLC effluent is passed through the tri-
tium exchange system, which consisted of a PTFE-lined
stainless-steel column packed with a trituim exchange
612 MAHMOUD A. HASSAN A N D ABDULLAH A. AL-BADR
polymer followed by an ionization chamber detector.

The method was partially successful.
Hsieh et a1 (18) have recently reported a high-per-

formance liquid chromatographic analysis of methadone
in sustained release formulations. HPLC separation
of methadone was carried out using a reversed-phase
1-1 Bondapak c18 column. The column temperature was
ambient. The electrometer was set at 0.01 a.u.f.s.
with a recorded chart speed of 2 in. per 10 min. The
volume of the samples introduced into the column was
10 1-11. The solvent (mobile phase) flow rate was con-
trolled at l.Om/min. A stock solution of 0.1 mg/ml
anthracene in methanol was used as an internal stan-
dard. The sodium salt of 1-pentanesulfonic acid was
used as an ion-pair agent. Fig. 6a represents a
typical chromatogram of methadone hydrochloride using
a mobile phase of methanol-water (75:25), while
Fig. 6b illustrates the response of the same solution
when the ion-pair agent is present in the mobile
phase. It is seen that the ion-pair agent increases
the absorption and the resolution of the methadone
peak. The high sensitivity and the low quantities
(us) of drug detected by this method indicates that
this method may be successfully used for the in vivo
determination of methadone (Table 1).
Recovery data of methadone from sus-

tained release tablets.
Weight of Methadone in Methadone Recovery

sample sample recovered f 5%
(mg> (mg) (mg)
5 1.1 1.020 93
10 2.2 2.050 93
15 3.3 3.100 94
20 4.4 4.090 93
I I I I
Fig. 6: (a) Typical chromatogram of methadone hydro-

chloride in a methanol-water (75 :25) solution.
(b) Chromatogram of methadone hydrochloride
in the presence of an ion-pair agent (sodium
salt of 1-pentanesulfonic acid). M = Methadone;
S = internal standard.
614 MAHMOUD A . HASSAN A N D ABDULLAH A . AL-BADR
REFERENCES
1. P. Crabbe, P. Demoen and P. J a n s s e n , B u l l . SOC.

Chim. (France) 10,2855 (Fr) 1 9 6 5 .
2. A.W. Hanson and F.R. Ahmed, Acta C r y s t . , 11,

724 (1958).
3. A.F. Casy, J. Chem. SOC. B , 1158 (1966).
4. A.H. B e c k e t t and A.F. Casy, J. Pharm. Pharmacol.,

-
6, 986 (1954).
5. J.G. Henkel, K.H. B e l l and P.S. P o r t o g h e s e ,

J. Wd. Chem. 17 (l), 1 2 4 (1974).
6. S.P. Loucas, R.L. F e i n b e r g , P.A. Gunning, F.F.

Hartmann and B. Mehl, Am. J . Hosp. Pharm., 2,
(12) , 1193-1197 (1974).
7. B. Caddy, F. F i s h , P.W. Mullen and J. T r a n t e r ,

J . Forens. S c i . SOC., 13
(2), 127-135 (1973).
8. John H. Knox and Jadtriga J u r a n d ; J . Chromat.,

-
82 (2) , 398-401(1973).
9. R. C l e e l a n d , J . C h r i s t e n s o n , M. Usategni-Gomez,
J . Heveran; R. Davis and E. Grunberg, C l i n . Chem,
-
22 (6), 712-725 (1976).
10. R.N. Gupta, B.G. C h i t t i m and P.M. Keane, J. Chromat.

Sci 12 (2), 67 (1974).
11. Naresh C. J a i n , Wai J. Lenng, Robert D. Budd and

Thomas C. S n e a t h , J. Chromat., 103 (l), 85 (1975).
1 2 . C.M. David and D.C. Ferimore, J. Chromat., 104 (I),

193 (1975).
13. N i c o l a s , H. C h o u l i s and Harry Papadopoulas,

J. Chromat. , 106 (1), 180-183) (1975).
1 4 . R.K.Lynn; R.M. Leger, W.P. Gordon, G.D. Olsen

and N. Gerber, J. Chromat. 131,329 (1977).
15. John H. Knox and Jadwiga J u r a n d , J. Chromat.,

-
87, 95 (1973).
16. W.A. T r i n l e r , D . J . Reuland, J. Forens.

Sci. Soc., 15 (2), 153 (1975).
1 7 . P . Goodman, A . R e n n e r t and J. Downs, Rep. Atom.

Energy Commn. U . S . , 100-2292-1, (1974).
18. J. H s i e h , J.K.H. Ma, J.P.O. Donne11 and N.H.

C h o u l i s , J. Chromat. 1 6 1 (ll), 366 (1978).
CUMULATIVE INDEX
Italic numerals refer to volume numbers
Acetaminophen, 3, I Cyclothiazide, I, 66
Acetohexamide, 1. 1; 2. 573 Cyproheptadine, 9, 155
Allopurinal, 7, 1 Dapsone, 5 , 87
Alpha-tocopheryl acetate, 3, 11 1 Dexamethasone, 2, 163; 4, 518
Amitriptyline hydrochloride, 3, 127 Diatrizoic acid, 4, 137; 5 , 556
Amoxicillin, 7, 19 Diazepam, I, 79;4, 517
Amphotericin B, 6, 1; 7, 502 Dibenzepin hydrochloride, 9, 181
Ampicillin, 2, 1; 4. 517 Digitoxin, 3, 149
Aspirin, 8, 1 Digoxin, 9, 207
Bacitracin, 9, 1 Dihydroergotoxine methane sulfonate, 7, 8 1
Bendroflumethiazide, 5 , 1; 6 . 597 Dioctyl sodium sulfosuccinate, 2, 199
Betamethasone dipropionate, 6, 43 Diperodon, 6, 99
Bretylium Tosylate, 9, 71 Diphenhydramine hydrochloride, 3, 173
Bromocriptine methanesulfonate, 8, 47 Diphenoxylate hydrochloride, 7, 149
Clacitriol, 8, 83 Disulfiram, 4, 168
Carbamazepine, 9. 87 Dobutamine hydrochloride, 8, 139
Cefaclor, 9, 107 Doxorubicin, 9, 245
Cefamandole Nafate, 9, 125 Dmperidol, 7, 171
Cefazolin*; 4, 1 Echothiophate iodide, 3, 233
Cephalexin. 4. 21 Epinephrine, 7, 193
Cephalothin sodium, I. 319 Ergotamine tartrate, 6, I 13
Cephradine*, 5 , 21 Erythromycin, 8, 139
Chloral hydrate, 2, 85 Erythromycin estolate, I, 101; 2, 573
Chloramphenicol, 4 , 47, 517 Estradiol valerate, 4, 192
Chlordiazepoxide, I, 15 Ethambutol hydrochloride, 7, 231
Chlordiazepoxide hydrochloride, I, 39; 4, 517 Ethynodiol diacetate, 3, 253
Chloroquine phosphate, 5, 61 Fenoprofen calcium*, 6, 161
Chlorpheniramine maleate, 7. 43 Flucytosine, 5, 115
Chloroprothixene, 2. 63 Fludrocortisone acetate, 3, 281
Chlortetracycline hydrochloride, 8, 101 Fluorouracil, 2, 221
Clidinium bromide, 2, 145 Fluoxymesterone, 7, 251
Clonazepam, 6, 61 Fluphenazine decanoate, 9, 275
Clorazepate dipotassium, 4 , 91 Fluphenazine enanthate, 2, 245; 4, 523
Cloxacillin sodium, 4 , 113 Fluphenazine hydrochloride, 2, 263; 4. 518
Cyclizine, 6, 83; 7, 502 Gentamicin Sulfate, 9, 295
Cycloserine, I, 53 Gluthethimide, 5, 139
*Monographs in “Pharmacological and Biochemical Properties of Drug Substances”

M. E. Goldberg, D. Sc., Editor American Pharmaceutical Association.
617
618 CUMULATIVE INDEX
Gramicidin, 8. 179 Phenformin hydrochloride, 4, 319; 5 , 429

Griseofulvin, 8, 219, 9, 583 Phenobarbital. 7, 359
Halcinonide, 8, 251 Phenoxymethyl penicillin potassium, I, 249
Haloperidol, 9, 341 Phenylephrine hydrochloride, 3, 483
Halothane, I , 119; 2, 573 Piperazine estrone sulfate, 5 , 375
Hexetidine, 7, 277 Primidone, 2, 409
Hydralazine hydrochloride, 8, 283 Procainamide hydrochloride, 4 , 333
Hydroflumethiazide, 7, 297 Rocarbazine hydrochloride, 5. 403
Hydroxyprogesterone caproate, 4, 209 Promethazine hydrochloride, 5. 429
Hydroxyzine dihydrochloride, 7, 3 19 Pmparacaine hydrochloride, 6, 423
Iodipamide, 3, 333 Ropiomazine hydrochloride, 2, 439
Isocarboxazid, 2, 295 Propoxyphene hydrochloride, I , 301 ; 4, 5 19;
Isoniazide, 6, 183 6, 598
Isopropamide, 2, 315 hpylthiouracil, 6, 457
Isosorbide dinitrate, 4, 225; 5 , 556 Pseudoephedrine hydrochloride, 8, 489
Kanamycin sulfate, 6, 259 Reserpine, 4 , 384; 5 , 557
Ketamine, 6, 297 Rifampin, 5 , 467
Khellin, 9, 371 Secobarbital sodium, I, 343
Leucovorin Calcium, 8, 315 Spironolactone, 4, 431
Levarterenol bitartrate, I, 49; 2, 573 Sodium nitroprusside, 6, 487
Levallorphan tartrate, 2, 339 Sulphamerazine, 6, 515
Levodopa, 5, 189 Sulfamethazine, 7, 401
Levothyroxine sodium, 5 , 225 Sulfamethoxazole, 2, 467; 4, 520
Lorazepam, 9, 397 Sulfasalazine, 5 , 515
Meperidine hydrochloride, I, 175 Sulfisoxazole, 2, 487
Meprobamate, I, 209; 4 , 519 Testolactone, 5 , 533
6-Mercaptopurine, 7, 343 Testosterone enanthate, 4 , 452
Methadone hydrochloride, 3, 365;4, 519.9, Theophylline, 4 , 466
60 1 Thiostrepton, 7, 423
Methaqualone, 4, 245, 519 Tolbutamide, 3, 513; 5 , 557
Methimazole, 8, 351 Triamcinolone, I, 367; 2, 571; 4. 520, 523
Methotrexate, 5 , 283 Triamcinolone acetonide, I, 397, 416; 2, 571;
Methoxsalen, 9, 427 4 , 520; 7
Methyclothiazide, 5 , 307 Triamcinolone diacetate, I. 423
Methyprylon, 2, 363 Triamcinolone hexacetonide, 6, 579
Metronidazole, 5 , 327 Triclobisonium chloride, 2, 507
Minocycline, 6, 323 Trifluoperazine hydrochloride, 9, 543
Nadolol, 9, 455 Triflupromazine hydrochloride, 2, 523; 4, 520;
Nalidixic Acid, 8, 371 5 , 557
Neomycin, 8. 399 Trimethaphan camsylate, 3, 545
Nitrazepam, 9, 487 Trimethobenzamide hydrochloride, 2, 551
Nitrofuraptoin, 5 , 345 Trimethoprim. 7, 445
Nitroglycerin, 9, 519 Triprolidine hydrochloride, 8. 509
Norethindrone, 4, 268 Tropicamide, 3, 565
Norgestrel, 4 , 294 Tubocurarine chloride, 7, 477
Nortriptyline hydrochloride, I , 233; 2, 573 Tybarnate, 4, 494
Nystatin, 6, 341 Valproate Sodium and valproic acid*, 8.
oxazepam, 3, 441 529
Phenazopyridine hydrochloride, 3, 465 Vinblastine sulfate, 1. 443
Phenelzine sulfate, 2, 383 Vincristine sulfate, 1. 463

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Analytical Profiles

ACADEMIC PRESS 1980

Norman W. Atwater Salvatore A. Fusari

Academic Press Rapid Manuscript Reproduction

ACADEMIC PRESS, INC.

United Kingdom Edition published by

Main entry under title: (Revised)

Analytical profiles of drug substances.

Compiled under the auspices of the Pharmaceutical

H . Y . Aboul-Enein, Riyadh University, Riyadh, Saudi Arabia

C . A . Janicki, McNeil Laboratories, Fort Washington, Pennsylvania

Copyright @ 1980 by Academic Press. Inc.

Although bacitracin was known to be active

of harmful effects on any organ or tissue of the

glutamic acid, aspartic acid, lysine, histidine,

bacitracin A contains a thiazoline ring.

They also indicated that the sequence lysine-orni-

Bernlohr and Sievert noted the similarity

bacitracin synthetase nto three fractions by af-

Ishihara and Shimura purified bacitracin

Froyshov continued his work and found

Wang and c o - ~ o r k e r sand

ring67. Craig and co-workers established the con-

CO+Leu+ Glu -Ile -P Lvs+

Molecular Weight 1406.66

Figure 4. Infrared Spectrum of Zinc Bacitracin-Mineral Oil Mull

Reynolds and co-workers used C 1 3 mag-

4.15 Acoustic Absorption Spectrum

4.2 Crystal Properties

the hygroscopicity of bacitracin at loo%, 93% and

Lannung also reported on the hy-

water >20 5.1

4.32 Distribution Coefficient

4.33 Formulation Release

4-42 Optical Rotary Dis2ersion

below p H 4 due tggthe epimerization of the terminal

Craig and co-workers studied the

synthetic and soybean digest media99.

Inskeep and co-workers described a new

Darker patented the addition of various

Keko, Bennett and Arzberger patented a

Wilk specified the pH ranges for the

Freaney and Allen patented a fermenta-

Ziffer patented a soybean-sucrose medi-

Aida and Ito describe the formation of

Styczynska and co-workers noted that the

Feuer and co-workers obtained a patent

Simlot, Pfaender and Specht noted that

Haavik suggested that glucose inhibited

Pass and Raczynska-Bojanowska found that

Tyc and Kadzikiewicz described their

5.22 Ion Exchange of Bacitracin

crude concentrate for animal feed use177117811791

Babin , Coustou and Brisou showed that

diminished skin penetrationlgg.

Hegarty and Verwey atented formulations

Plaxco and Husa established the stabili-

Gordon patented aerosol compositions of

Crisler and Weinberg indicated that

while zinc salt of bacitracin was not more stable

Tanaka, Seki and Ito patented the use of

7.14 ElectroDhoresis Methods

7.22 Turbidimetric Assay