04 Analytical Profiles of Drug Substances, Vol 04

Analytical Profiles
of
Drug Substances
Volume 4
Edited b y
Klaus Florey
The Squibb Institute for Medical Research
New Brunswick, New Jersey
Contributing Editors
Norman W.Atwater Salvatore A. Fusari

Glenn A. Brewer, Jr. Boen T. Kho
Jack P. Comer Gerald J. Papariello
Frederick Tishler
Compiled under the auspices of the

Pharmaceutical Analysis and Control Section
Academy of Pharmaceutical Sciences
Academic Press New York San Francisco London 1975

A Subsidiary of Harcourt Brace Jovanovich, Publishers
EDITORIAL BOARD
Norman W. Atwater David ElGuttman'

Olenn A. Brewer, Jr. Erik H. Jensen
Lester Chafetz Boen T. Kho
Edward M. Cohen Arthur F. Michaelis
Jack P. Comer Gerald J, Papariello
Klaus Florey Bernard 2. Senkowski
Salvatore A. Fusari Frederick Tishler
* Until his death in 1974.
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Main entry under title:
Analytical profiles of drug substances.
Includes bibliographical references.

Compiled under the auspices of the Pharmaceutical
Analysis and Control Section, Academy of Pharmaceutical
Sciences.
1. Drugs-Collected works. 2. Chemistry, Medical
and pharmaceutical-Collected works. I. Florey, Klaus,
ed. 11. Brewer, Glenn A. 111. Academy of Pharmaceu-
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AFFILIATIONS OF EDITORS,
CONTRIBUTORS, AND REVIEWERS
N . W. Afwuter, Searle and Company, Chicago, Illinois
J. I. Bodin, Carter-Wallace Inc., Cranbury, New Jersey
G. A . Brewer, Jr., The Squibb Institute for Medical Research, New

Brunswick. New Jersey
L. C h j e t z , Warner-Lambert Research Institute, Morris Plains, New

Jersey
E. M . Colien, Merck, Sharp and Dohme, West Point, Pennsylvania
J. L. Cohen, School of Pharmacy, University of Southern California,

Los Angeles, California
J. P. Comer, Eli Lilly and Company, Indianapolis, Indiana
L. F. Cullen, Wyeth Laboratories, Philadelphia, Pennsylvania
R . D. Daley, Ayerst Laboratories, Rouses Point, New York
N. J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania
F. Eng, Parke, Davis and Company, Detroit, Michigan
K. Florey, The Squibb Institute for Medical Research, New Brunswick,

New Jersey
vii
AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS
S. A. Fusari, Parke, Davis and Company, Detroit, Michigan
D.E. Guttman, School of Pharmacy, University of Kentucky,

Lexington, Kentucky
W. W.Holl, Smith, Kline and French Laboratories, Philadelphia,

Pennsylvania
E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan
H. Kadin, The Squibb Institute for Medical Research, New Brunswick,

New Jersey
B. T. Kho, Ayerst Laboratories, Rouses Point, New York
J. Kress, Carter-Wallace Inc., Cranbury, New Jersey
E. P. K.Lau, Searle and Company, Chicago, Illinois
H. H. Lerner, The Squibb Institute for Medical Research, New Brunswick,

New Jersey
L. P. Marrelli, Eli Lilly and Company, Indianapolis, Indiana
D. L. Mays, Bristol Laboratories, Syracuse, New York
A. F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey
J. E. Moody, USV Pharmaceutical Corporation,Tuckahoe, New York
E. S. Moyer, Ortho Research Foundation, Raritan, New Jersey
N. G. Nash, Ayerst Laboratories, Rouses Point, New York
G. J. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania
v iii
AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS
V. E. Papendick, Abbott Laboratories, North Chicago, Illinois
D. M. Patel, William H. Rorer Inc., Fort Washington, Pennsylvania
R. B. Poet, The Squibb Institute for Medical Research, New Brunswick,

New Jersey
A . Post, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania
J. A . Raihle, Abbott Laboratories, North Chicago, Illinois
N. H.Reavey-Cantwell, William H. Rorer Inc., Fort Washington, Pennsylvania
P. Reisberg, Carter-Wallace Inc., Cranbury, New Jersey
R. E. Schirmer, Eli Lilly and Company, Indianapolis, Indiana
A . P. Schroff. Ortho Research Foundation, Raritan, New Jersey
B. Z. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey
L. A . Silvieri, Wyeth Laboratories, Philadelphia, Pennsylvania
A . M. Sopirak, Wyeth Laboratories, Philadelphia, Pennsylvania
J. L. Sutter, Searle and Company, Chicago, Illinois
D. Szulczewski, Parke, Davis and Company, Detroit, Michigan
F. Tishler, Ciba-Geigy, Summit, New Jersey
A . J. Visalli, William H. Rorer Inc., Fort Washington, Pennsylvania
J. J. Zalipsky, William H. Rorer Inc., Fort Washington, Pennsylvania
A . F. Zappala, Smith, Kline and French Laboratories, Philadelphia,

Pennsylvania ix
PREFACE
Although the official compendia list tests and limits for drug substances
related to identity, purity, and strength, they normally d o not provide other
physical or chemical data, nor d o they list methods of synthesis o r pathways
of physical or biological degradation and metabolism. For drug substances
important enough t o 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 o f Pharmaceuti-
cal Sciences, has undertaken a cooperative venture t o compile and publish
Analytical Profiles of Drug Substances in a series of volumes of which this is
the fourth.
The concept of Analytical Profiles is taking hold not only for compend-
ial drugs but, increasingly, in the industrial research laboratories. Analytical
Profiles are being prepared and periodically updated to provide physico-chem-
ical 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 compendia] status.
The cooperative spirit of our contributors had made this venture pos-
sible. All those who have found the profiles useful are earnestly requested to
contribute a monograph of their own. The editors stand ready to receive such
contributions.
This volume of Analytical Profiles is dedicated to the memory of David
E. Guttman, an enthusiastic member of the Editorial Board until his tragic
and untimely death in 1974.
Klaus Florey
xi
CEFAZOLIN
Alfred F. Zappala, Walter W . Holl, and Alex Post

ALFRED F. ZAPPALA eta/.
Content s
1. D e s c r i p t i o n
2. Physical Properties
2.1 I n f r a r e d Spectrum
2.2 Nuclear Magnetic Resonance Spectrum
2.3 U l t r a v i o l e t Spectrum
2.4 Optical Rotation
2.5 Melting Range
2.6 D i f f e r e n t i a l Thermal A n a l y s i s
2.7 Solubility
2.8 pKa
2.9 Crystal Properties
3. Synthesis
4. Stability
5. Drug Metabolic P r o d u c t s
6. Methods of Analysis
6.1 Elemental A n a l y s i s
6.2 Non-Aqueous T i t r a t i o n of C e f a z o l i n
6.3 Non-Aqueous T i t r a t i o n of C e f a z o l i n Sodium
6.4 Thin-Layer Chromatography
6.5 Spectrophotometric - UV Hydroxylamine Method
6.6 High P r e s s u r e Liquid Chromatographic Procedure
6.7 F e d e r a l R e g i s t e r Methods
2
CEFAZOLIN
1.1 Name, Formula, Molecular Weight
C e f a z o l i n is 3-[ [(5-Methyl-l,3,4-thiadiazol-2-
y l ) thio]methyl]8-oxo-7- [2- (1H-tetrazol-1-yl) acetomido] -
5-thia-1-azabicyclo [4.2.O]oct-2-ene-2-carboxylic acid.
It a l s o e x i s t s as t h e sodium salt. P a r e n t e r a l p r o d u c t s
a r e known as Ancef and Kefzol.
Mol. w t . 454.512 ( a c i d )
C14H14NgOqS3 (Na, -HI 476.495 ( s a l t )
1.2 Appearance, Color, Odor
White t o s l i g h t l y o f f w h i t e , o d o r l e s s .
The i n f r a r e d spectrum of c e f a z o l i n is p r e s e n t e d
i n Figure 1. The spectrum t a k e n was t h a t of a m i n e r a l
oil d i s p e r s i o n of t h e s t a n d a r d u s i n g a Perkin-Elmer 4578
Grating I R Spectrophotometer. A l i s t of t h e assignments
made f o r some of t h e c h a r a c t e r i s t i c bands is given i n
Table I (1).
3
MICRONS
1 5 3 0 4 0 50 .O 7 0 S O VO 10 11 I4 Ib I I 10 15 303540
DO
01
0
Y 01
L
0 03
:: 04
P 05
10
4000 3500 3000 1500 1000 IS00 1600 1400 1100 1000 SO0 b00 400 150
WAVENUMBER 1CM-l)
Figure 1: Infrared Spectrum of Cefazolin

Reference Standard, mineral o i l dispersion.
Instrument: Perkin-Elmer 4578
CEFAZOLIN
Table I - I R S p e c t r a l Assignments f o r C e f a z o l i n
Frequency (cm-’> C h a r a c t e r i s t i c of
3280 -NH-
3140
3075 3 -N=N-
-C=N-
1 tetrazole ring
-OH, bonded, -COOH

2580
1770 >c=o lactam
1715 >CEO acid
1670 >c=o amide I
1555 >c=o amide I1

The 60 MHz NMR spectrum of c e f a z o l i n p r e s e n t e d
i n F i g u r e 2 was obtained i n t r i f l u o r o a c e t i c a c i d a t a
c o n c e n t r a t i o n o f about 100 mg/ml and t e t r a m e t h y l s i l a n e
as i n t e r n a l s t a n d a r d . The s p e c t r a l assignments are
l i s t e d i n T a b l e I1 (1).
5
I 1 1 I I I I 1 I 1
I I I I I I I I I J
9 8 7 6 5 4 3 2 1 0
PPM
Figure 2: NMR Spectrum of Cefazolip

Reference Standard, i n TFA with TMS as internal standard.
Instrument: JEOL Co., Model JNM-C-60H
Table I1 - N M R S p e c t r a l Assignments f o r Cefazolin
Chemical I n t e g r a t i o n of
S h i f t (ppm) Multiplicity C h a r a c t e r i s t i c of No. of Protons
3.11 s i n g 1e t protons a t 1 3
3.85 singlet protons a t 2 2
4.71 singlet protons a t 3 2
5.40 doublet protons a t 4 1
5.75 overlapping protons a t 5 3

s i n g l e t & doublet and 6
8.21 doublet protons a t 7 1
proton a t 8 i s beyond 9 ppm; however, i t is masked by t h e s o l v e n t

The u l t r a v i o l e t a b s o r p t i o n spectrum of c e f a z o l i n
i n 0 . B NaHC03 is shown i n F i g u r e 3. When scanned between
350 a n z 220 nm, c e f a z o l i n e x h i b i t s a s i n g l e band w i t h a n
a bs or pt i on maximum a t 270 - 272 nm ( E = 13,100).
2.4 Optical Ro tatio n

The s p e c i f i c r o t a t i o n o f a 5% s o l u t i o n of
c e f a z o l i n i n 0;1M_ NaHC03 when measured a t 25OC i n a
1 decimeter t u b e is -170 5 70.
2.5Melting Range
Ce f azo lin starts t o decompose a t about 190°C
under USP c o n d i t i o n s f o r Class I s u b s t a n c e s (2).
2.6 D i f f e r e n t i a l Thermal An aly s is

A d i f f e r e n t i a l th ermal a n a l y s i s w a s performed on
c e f a z o l i n and t h e thermogram is p resen te d i n F ig u re 4.
The t y p i c a l meltin g endotherm is a b s e n t and o n l y t h e
decomposition exotherm a t about 205OC is p r e s e n t .
2.7 Solubility
The approximate s o l u b i l i t i e s o b ta in e d f o r
c e f a z o l i n a t rbbm temperature (25OC 5 l 0 C ) are l i s t e d i n
Table 111.
Table I11 - Approximate S o l u b i l i t i e s of Cefazolin
Solvent mg Cef a z o l i n / m l
ac e t one 5.7
acetone:water ( 4 : l v:v) 21.2
chlorof orm 0.02
95% e t h a n o l 1.1
e t h y l acetate 0.24
isobutylacetate 0.05
isopropyl alcohol 0.21
methanol 1.7
methylene c h l o r i d e 0.02
m e t hyl i sobut y lk eto n e 0.25
sodium c h l o r i d e , h a l f -satu rated 0.83
sodium c h l o r i d e , s a t u r a t e d 0.44
water 1.1
8
CEFAZOLIN
2.71 pH Solubility Profile

The pH solubility profile of cefazolin is
presented in Figure 5.
2.8 pKa
The pKa is 2.15 determined spectrophotometrical-
ly. A pKa of 2.05 determined titrimetrically has been
reported (3).

Several crystal forms of sodium cefazolin have
been reported (4). The pathways of conversion of one
form to another are shown below.
gain of treat with methanol or

water ethanol (loss of water)
8-f o m n-f orm

312 moles of water.
9
ALFRED F . ZAPPALAetal.
0.9 U.V. ABSORPTION SPECTRUM
COMPOUND: CEFAZOLIN
CODE: REFERENCE STANDARD
0.8
CONCENTRATION: 0.0294 m g / m l
SOLVENT: 0.1M NaHCOj
0.7 CELL PATH: 1 cm
0.6
Y
U
0.5
4
m
w
0
2 0.4
4:
0.3
0.2
0.1
0.0
2 00 250 300 350
WAVELfNGTH - nm
Figure 3: W Absorption Spectrum of Cefazolin

Reference Standard
Instrument: Cary Model 14
10
h
SAMPLE: C E F A Z O L I N
O R I G I N : REFERENCE STD.
SIZE: M I C R O C A P I L L A R Y
REF: GLASS BEADS
P R O G R A M M O D E : HEAT
RATE: 1 0 ° C / M I N
ATM: NITROGEN
c
c
1 I 1 I 1 I 1
0 50 100 150 200 250 300 350 400 450 500
T,"C
Figure 4 : DTA of Cefazolin, Reference Standard

Instrument: DuPont 900
G
rl
rl
0
N
a
w
a
U
w
0
a
4
rl
w
0
Ll
c4
h
U
rl
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rl
a
1
I-l
0
UY
X
I I I I I I 1
0 0 0 0 0
0
0
0
In
0
0 o
n o
In
N 0
(Y 2 2 In
lU/ NllOZV333 s h
12
0=&
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o=u
I
I
I
N
5I
T m
fll 74
n z-1”
m
W cn
*&
1
..a
u
z
t-l
R
00
G
rl
rl
rf
V w o
0
rl I
0
rcI
o=v
X
91
5
h
P
a
91
t-l
(d
+ +
a
a
t-l
a
a
P !
o=vI
h
(d N
B X
V
G I H
rl H
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91
”9
II
2-2
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V
13
ALFRED F. ZAPPALA e t a / .
4. S t a b i l i t y
The s t a b i l i t y of Ancef v i a l s a f t e r r e c o n s t i t u t i o n
w i t h water f o r i n j e c t i o n ( a ) , b a c t e r i o s t a t i c s w a t e r (b) ,
and normal s a l i n e s o l u t i o n (c) was determined ( 6 ) . The
r e s u l t i n g s o l u t i o n s were analyzed by t h e UV hydroxylamine
method ( S e c t i o n 6.5) i n i t i a l l y and a g a i n a f t e r s t o r a g e a t
50C f o r 96 hours. No d e g r a d a t i o n w a s observed.
The p h y s i c a l and chemical s t a b i l i t y of c e f a z o l i n

sodium in s e v e r a l i n t r a v e n o u s f l u i d s (Table I V ) w a s
evaluated (7). The s o l u t i o n s were assayed by t h e UV
hydroxylamine method ( S e c t i o n 6.5) i n i t i a l l y and
p e r i o d i c a l l y up t o 72 hours. I n s p e c t i o n i n d i c a t e d no
apparent p h y s i c a l change. The chemical s t a b i l i t y of t h e
s o l u t i o n s can b e s e e n from t h e d a t a i n T a b l e I V .
Lyophilized c e f a z o l i n sodium is s t a b l e € o r a t least

two y e a r s i n t h e d r y s t a t e a t room temperature. A
comparison of t h e chemical (8) and m i c r o b i o l o g i c a l (9)
assay methods is i l l u s t r a t e d i n Table V.
14
CEFAZOLIN
Table I V - S t a b i l i t y of
Cefazolin Sodium i n S e l e c t e d
Intravenous S o l u t i o n s *
Time in hrs.
- - r-
0 24
- -
- 'C
- - 48
-
72 24 72
10% d e x t r o s e 100 101 98.2 95.8 104 99.1
i n H20
5% d e x t r o s e 100 98.3 98.2 94.5 99.4 99.7

i n H20
5% d e x t r o s e w i t h 100 98.3 96.6 94.4 98.6 97.8

lactated Ringer
5%d e x t r o s e i n 100 101 100 96.3 100 93.2

Ringer's i n j e c t i o r
5% d e x t r o s e 100 100 99.4 98.5 102 97.4

i n 0 . 9 % NaCl
5% dextrose 100 100 100 96.0 9 8 . 0 100

i n 0.45% NaCl
10%d e x t r o s e 100 100 97.7 97.7 99.4 99.7

i n 0.9% NaCl
0.9% NaCl 100 101 99.7 98.9 105 100

l a c t a t e d Ringer 100 103 98.9 36.9 LO1 39.4
lnje c t i o n
linger injection too 102 101 36.9 LOO 99.7

kExpressed as a percent of i n i t i a l concentration
15
ALFRED F. ZAPPALA er a/.
Table V - Comparison of Chemical and Microbiological

Met hod s *
Months 1 3 6 9 12 18 24
Chem. 100 101 97.3 99.0 96.0 99.0 100

assay
Micro. 99.4 103 97.5 98.2 97.0 101 98.0

assay
*Expressed as a p e r c e n t of i n i t i a l c o n c e n t r a t i o n .
5. Drug Metabolic Products

S t u d i e s conducted t h u s f a r i n d i c a t e t h e r e i s very
l i t t l e b i o t r a n s f o r m a t i o n of p a r e n t e r a l l y administered
c e f a z o l i n sodium i n t h e body. Between 94 - 98% i s
excreted i n t h e u r i n e unchanged. Only trace amounts of
m e t a b o l i t e s have been seen b u t n o t i d e n t i f i e d (10).
6.1 Elemental Analysis

The r e s u l t s from an elemental a n a l y s i s of
c e f a z o l i n r e f e r e n c e standard are l i s t e d i n Table V I .
Table V I
Element Theory % Found
C 37.00 36.75
H 3.10 3.29
N 24.65 24.44
S 21.16 21.31
0 14.09 14.23
(by d i f f e r e n c e )
16
CEFAZOLIN
6.2 Non-Aqueous T i t r a t i o n of C e f a z o l i n
Reagents
(1) Dimethylsulf oxide (DMSO)
(2) Tetrabutylamonium hydroxide (TBAH) -
0.05N -
i n 9 :1 benzene:methanol; t h i s s o l u t i o n is s t a n d a r d i z e d
a g a i n s t benzoic a c i d ( N a t i o n a l Bureau of S t a n d a r d s ) .
Procedure
An a c c u r a t e l y weighed sample of about 200 mgs of
c e f a z o l i n is d i s s o l v e d i n 70 - 80 m l of DMSO. The
r e s u l t i n g s o l u t i o n is t i t r a t e d p o t e n t i o m e t r i c a l l y w i t h
standard 0.05E TBAH u s i n g a glass-calomel e l e c t r o d e p a i r
o r combination e l e c t r o d e . Each m i l l i l i t e r of 0.05N -
TBAH is e q u i v a l e n t t o 0.02273 g of c e f a z o l i n .
6.3 Non-Aqueous T i t r a t i o n of C e f a z o l i n Sodium

An a c c u r a t e l y weighed sample of about 200 mgs of
c e f a z o l i n sodium is d i s s o l v e d i n 70 - 80 m l of dimethyl-
s u l f o x i d e (DMSO). The r e s u l t i n g s o l u t i o n is t i t r a t e d
p o t e n t i o m e t r i c a l l y w i t h s t a n d a r d 0.05E a c e t o u s p e r c h l o r i c
a c i d u s i n g a glass-calomel e l e c t r o d e p a i r o r combination
e l e c t r o d e . Each m i l l i l i t e r of 0.05g p e r c h l o r i c a c i d is
e q u i v a l e n t t o 0.02383 g of c e f a z o l i n sodium.

The following t h i n - l a y e r method may be used f o r
t h e q u a l i t a t i v e p u r i t y e v a l u a t i o n of c e f a z o l i n and i t s
sodium s a l t .
About 50 and 100 micrograms of c e f a z o l i n ,

d i s s o l v e d i n a 4 : l m i x t u r e of acetone:water, are s p o t t e d
two cm from t h e edge of a S i l i c a G e l GF p l a t e . The p l a t e
is placed i n a s u i t a b l e chromatographic chamber l i n e d w i t h
f i l t e r paper s a t u r a t e d w i t h t h e developing s o l v e n t ( e t h y l -
a c e t a t e :acetone :acetic a c i d :water, 5 :2 :1:1 ) and allowed ,
t o e q u i l i b r a t e f o r t e n minutes. The s o l v e n t is t h e n
allowed t o r i s e t o a l i n e drawn a c r o s s t h e p l a t e 1 0 cm
from t h e o r i g i n . The p l a t e is removed from t h e chamber
and allowed t o a i r d r y i n a fume hood u n t i l s o l v e n t vapors
a r e no longer d e t e c t a b l e . The developed chromatogram may
b e v i s u a l i z e d under u l t r a v i o l e t l i g h t (254 and 365 nm),
exposure t o i o d i n e vapors, and s p r a y i n g w i t h potassium
permanganate. Cefazolin h a s an Rf v a l u e of about 0.45.
17
ALFRED F . ZAPPALA e t a/.
6.5 Spectrophotometric-UV Hydroxylamine Method

Reagents
(1) 0.5 Molar sodium b i c a r b o n a t e
(2) Acetate B u f f e r - Equal volumes of 0 . g
a c e t i c a c i d and 0 . g sodium acetate are mixed t o g e t h e r and
t h e r e s u l t i n g s o l u t i o n is a d j u s t e d t o pH 4.0.
(3) A l k a l i n e Sodium Acetate - 86.5 g of sodium
hydroxide and 1 0 . 3 g o f sodium acetate are d i s s o l v e d i n
s u f f i c i e n t water t o make 1000 m l .
-
(4) Hydroxylamine S o l u t i o n One volume of 5M
hydroxylamine h y d r o c h l o r i d e i s mixed w i t h two volumes z f
a l k a l i n e sodium acetate and t h r e e volumes of water.
Procedure
An a c c u r a t e l y weighed sample of approximately
50 mg is d i s s o l v e d i n 5.0 m l of 0.5E sodium b i c a r b o n a t e
and d i l u t e d t o 1000 m l w i t h water. F i v e m l a l i q u o t s of
t h i s s o l u t i o n are t r a n s f e r r e d t o each of two 100 m l
v o l u m e t r i c f l a s k s . To one f l a s k is added 5.0 r n l of
hydroxylamine s o l u t i o n . The f l a s k I s s w i r l e d and allowed
t o s t a n d f o r 45 minutes, a f t e r which b o t h s o l u t i o n s are
d i l u t e d t o 100 m l w i t h acetate b u f f e r . Two a l i q u o t s of a
standard s o l u t i o n of c e f a z o l i n are t r e a t e d i n t h e same
manner. The u l t r a v i o l e t a b s o r p t i o n spectrum of t h e
unreacted s o l u t i o n is recorded v e r s u s t h a t of t h e r e a c t e d
s o l u t i o n i n t h e r e f e r e n c e c e l l from 350 t o 240 nm i n 1 cm
c e l l s . The c a l c u l a t i o n of t h e p u r i t y of t h e sample is
accomplished by comparison of t h e absorbance d i f f e r e n c e
between 270 nm and 340 nm f o r t h e sample t o t h a t of t h e
s t a n d a r d . T h i s procedure h a s a l s o been automated (8).
6.6 High P r e s s u r e Liquid Chromatographic Procedure

Reagents
( 1 ) Mobile Phase: 0.02 Molar monobasic sodium
phosphate a d j u s t e d t o pH 6.2 2 0 . 1 w i t h 1l sodium
hydroxide.
(2) Standard S o l u t i o n : Approximately 20 mg of
r e f e r e n c e s t a n d a r d is a c c u r a t e l y weighed i n t o a 50 m l
volumetric f l a s k and d i s s o l v e d i n and d i l u t e d t o volume
w i t h 0.05M - sodium b i c a r b o n a t e .
18
CEFAZOLIN
I n s t r u m e n t a l Conditions
Column Packing: Strong anion exchange r e s i n
Column Diameter: 2.1 m I . D .
Column Length: 1 m
Column Temperature: Ambient
Column P r e s s u r e : 1000 p s i g
Flow Rate: 0.5 m l p e r minute
Detector: U.V., 254 nm
Procedure
An a c c u r a t e l y weighed sample of approximately 20 mg
is d i s s o l v e d i n and d i l u t e d t o 50 m l w i t h 0.05g sodium
b i c a r b o n a t e . D u p l i c a t e 20 p 1 a l i q u o t s of t h e s t a n d a r d
and sample s o l u t i o n s are i n j e c t e d . The r e t e n t i o n time
f o r c e f a z o l i n is approximately 20 minutes. The c a l c u l a -
t i o n of t h e p u r i t y of t h e sample is accomplished by
comparison of t h e average peak h e i g h t of t h e sample
s o l u t i o n t o t h a t of t h e s t a n d a r d s o l u t i o n . I n s t r u m e n t a l
c o n d i t i o n s may r e q u i r e m o d i f i c a t i o n s w i t h o t h e r HPLC
u n i t s and d i f f e r e n t l o t s of column packing.
6.7 "Federal R e g i s t e r " 38:31505-31509, 1973

A d d i t i o n a l methods l i s t e d in t h e F e d e r a l
R e g i s t e r are (1) 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 a s s a y ,
(2) iodometric assay, and (3) hydroxylamine c o l o r i m e t r i c
assay.
19
7. References
1. R. J. Warren, SmithKline Corp., Personal

Communication
2. U.S.P. XVIII, p. 935
3. Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan,

Personal Communication
4. J. of Antibiotics, Vol. XXIII, NO. 3, 131 - 136
(1970)
5. J. Hill and H. Winicov, SmithKline Corp.,
6. L. Ravin and E. Rattie, SmithKline Corp.,
7. L. Ravin and E. Rattie, SmithKline Corp.,
Personal Comunication
8. W. W. Holl et al, SmithKline Corp., Personal
Comunication, to be published
9. Antimicrobial Agents & Chemotherapy, Vol. 5 ,
NO. 3, 223 - 227 (1974)
10, J. of Antibiotics, Vol. XXV, NO. 2, 86 - 93
(1972)
20
CEPHALEXIN
Louis P.MarreUi
LOUIS P. MARRELLI
TABLE OF CONTENTS
Page
1. Description 3
1.1 Name: Cephalexin 3
1.2 Formula and Molecular Weight 3
1.3 Isomers 4
1.4 Hydrates 4
1.5 Appearance 4
2. Physical Properties 4
2.1 Spectra 4
2.11 Infrared Spectrum 4
2.12 Nuclear Magnetic Resonance b
Spectrum
2.13 Ultraviolet Absorbance 7
2.2 Crystal Properties 7
2.21 X-Ray Powder Diffraction 7
2.22 Differential Thermal Analysis 7
2.3 Solubility 7
2.4 Dissociation Constant 10
2.5 Optical Rotatory Dispersion 10
3 . Cephalexin Stability 10
4. Synthesis 10
5. Methods of Analysis 11
5.1 Identification Tests 11
5.2 Quantitative Methods 11
5.21 Titration 11
5.22 Colorimetric Determination 12
5.23 Thin Layer Chromatography 14
5.24 Paper Chromatography 15
5.25 Column Chromatography 15
5.26 Electrophoresis 16
5.27 Microbiological Assays 16
5.3 Assay Methods for Intermediates and 16
Imp uritie8
5.31 7-Aminodesacetoxy- 16
cephalosporanic Acid (7-ADCA)
5.32 Phenylglycine 17
6. Protein Binding 18
7. Pharmacokinetics 18
8. References 20
22
CEPHALEXIN
1. Description
1.1 -
Name: Cephalexin
Chemical Abstracts designates cephalexin as 5-thia-
1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, 7-( 2-amino-
2-ph eny1-ac etamido) -3-methyl-8-ox0
Ce halexin monohydrate is also known as 5-thia-1-

azabicyclo f4.2.01 oct-2-ene-2-carboxylic acid, 7-[(aIUino-
phenylacetyl) amino]-3-methyl-8-oxo monohydrate' * 7-( D-2-
amino-2-phenylacetamido) -3-me thyl-3-cephem-4-carboxylic acid
monohydrat3 * and 7-( D-cr-aminophenylacetamido)-3-methyl-3-
cephem-4-carboxylic acid monohydrate' .
1.2 Formula and Molecular Weight
c16 17 N304S'H20 365.41
23
LOUIS P. MARRELLI
1.3 Isomers
The s y n t h e s i s of t h e L epimer o f cephalexin has
been r e p o r t e d . The D-isomer e x h i b i t s considerably more
b i o l o g i c a l a c t i v i t y than t h e L-isomer. P e n i c i l l i n s derived
from D-cr-amino a c i d s a l s o show more b i o l o g i c a l a c t i v i t y than
t h e i r L-epimerg ' 6 .
1.4 Hydrates
P f e i f f e r e t . a1.' provided x-ray powder d i f f r a c t i o n
d a t a f o r the monohFdrac and dihydrate of cephlexin. Cepha-
l e x i n w a s found t o c r y s t a l l i z e from aqueous s o l u t i o n s a t
room temperature as t h e dihydrate b u t converted t o t h e mono-
hydrate when t h e r e l a t i v e humidity was below 70%. Refer t o
Section 2.21.
1.5
Appearance
Cephalexin i s a white t o cream-colored c r y s t a l l i n e
powder, having a c h a r a c t e r i s t i c odor.
2. Physical P r o p e r t i e s
2.1 Spectra
The i n f r a r e d spectrum of cephalexin mono-
hydrate recorded as a potassium bromide d i & i s presented
i n Figure 1. I n t e r p r e t a t i o n of t h e spectrum is given i n
Table 18. Changes i n the B-lactam carbonyl s t r e t c h i n g
region (1760 cm-l) can i n d i c a t e opening of the 6-lactam ring.
Morin' and coworkers have shown a r e l a t i o n s h i p between t h e
B-lactam carbonyl s t r e t c h i n g frequency and b i o l o g i c a l
a c t i v i t y . The importance of t h i s s t r e t c h i n g frequency has
been discussed i n a recent reviewlo.

Figure 2 shows the proton magnetic resonance
spectrum of cephalexin monohydrate. The solvent used was
deuterium oxide containing a small amount of t r i f l u o r o a c e t i c
a c i d t o enhance s o l u b i l i t y . 3-( Trimethylsilyl) -propane-
s u l f o n i c acid, sodium s a l t w a s added as t h e i n t e r n a l r e -
ference. "he spectrum was recorded on a Varian "60-A i n s t r u -
ment. The assignment of t h e spectrum i s shown i n Table IIe
A most c h a r a c t e r i s t i c region of t h e NMR spectrum i s t h a t
.
o r i g i n a t i n g from the two 6-lactam r i n g protons, H(6) and
H(7)
24
4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400
WAVENUMBER CM-'
FIGURE 1. Infrared spectrum of cephalexin monohydrate (potassium bromide disc).

8.0 7.0 6.0 5 .O 4.0 3.0 2.0 1.0 0
PPM (a)
FIGURE 2. NMR spectrum of cephalexin monohydrate (020 + trifluoroacetic acid).
TABLE I
Infrared Spectrum of Cephalexin Monohydrate
-1 Assignment
Wavelength (cm )
3500 - 3000 (series of broad bands) OH from H 0 and amide

NH stretch
2
+
2600 (broad) Y3
1760 @-lactam c = o stretch
1690 h i d e c = o stretch
0
(1600 [very broad] C-0 - (carboxylate
(1400 stretching)
1550 (unresolved) h i d e I1 band
820 - 690 Mainly skelectal

vibrations including
out-of-plane aromatic
hydrogen bending,
characteristic of mono-
substituted aromatic ring
TABLE I1
Proton Magnetic Resonance Spectrum
Peak Assignments
Relative
p .p .m. (6) Intensity Mu1t ip 1icity Assignment
2.07 3 singlet CH3 (3)
3.30 2 quartet (AB) CH* (2)
4.85 - singlet HOD (solvent)
4.97 1 doublet (J=4Hz) H (6)

5.34 1 singlet H (benzyl)
5.67 1 doublet (J=4HZ) H (7)
7.60 5 sing let

‘SH5
CEPHALEXI N
2.13 U l t r a v i o l e t Absorbance
An aqueous s o l u t i o n of cephalexin e i b i t s a
W absorption maximum a t 262 nm (Figure 3 ) . The El% Pcm
reported f o r cephalexin (on an anhydrous b a s i s ) was 23611
The u l t r a v i o l e t absorbance of cephalexin a6 w e l l as o t h e r
.
cephalosporins has been a t t r i b u t e d t o t h e 0 = C-N-C = C-
chromophore of t h e ring1*. Chou” u t i l i z e d t h e W absorption
a t 262 nm t o determine t h e cephalexin content of s o l u t i o n
f r a c t i o n s i s o l a t e d from human urine.
2.2 C r y s t a l Properties
2.21 X-Ray Powder Diffraction

Cephalexin was found t o occur i n s e v e r a l sol-
vated c r y s t a l forms, and o f t e n i n widely varying mixtures of
these f o r m d 4 . Some of t h e solvated c r y s t a l forms prepared
were the dihydrate, monohydrate, d i a c e t o n i t r i l a t e , formami-
d a t e , methanolate, and a c e t o n i t r i l e hydrate. X-ray powder
d i f f r a c t i o n data f o r cephalexin monohydrate i s presented i n
Table 111.
2.22 D i f f e r e n t i a l Thermal Analysis

D i f f e r e n t i a l thermal analysis’ of cephalexin
monohydrate was conducted on a W o n t Model 950 thermal
analyzer i n a nitrogen atmosphere. A heating r a t e of 2OoC
p e r minute w a s u t i l i z e d . An endotherm w a s noted at 123OC
i n d i c a t i n g the loss of water, and an exotherm of 203OC i n d i -
c a t i n g decomposition.
2.3 Solubility
The s o l u b i l i t y of cephalexin monohydrate i n t h e
following solvents has been reported’ :
Mg. Cephalexin Monohydrate Per M l .

Solvent Solvent, 25OC.
Water 13-5
Methanol 3- 4
N-oc tan01 0.03
Chloroform 4.01
Ether 4.01
Table I V r e l a t e s t h e s o l u b i l i t y of cephalexin
monohydrate i n water as a function of pH.
29
LOUIS P. MARRELLI
2.0
1.5
1.0
O.!
0.0
I I I
I 250 300 350
monohydrate. 49 mcg per ml in H20
30
CEPHALEXIN
TABLE I11
X-Ray Powder Diffraction Pattern of Cephalexin Monohydrate
Radiation; Cu/Ni. Norelco De Bye-Scherrer Camera
Cephalexin Monohydrate
d 1/11
- -
15.15 0.40
11.85 1.00
11.00 0.30
9.36 0.20
8.55 0.50
7.86 0.50
6.89 0.20
5.98 0.40
5.39 1.00
4.97 0.50
4.76 0.40
4.57 0.40
4.39 0.60
4.22 0.60
4.00 0.70
3.86 0.70
3.60 0.80
3.46 0.30
3.24 0.60
3.10 0.60
2.98 0.40
2.90 0.60
2.81 0.40
2.73 0.20
2.68 0.40
2.63 0.10
2.47 0.30
2.41 0.15
2.31 0.30
2.25 0.30
2.12 0.10
2.09 0.05
2.01 0.02
1.93 0.05
1.87 0.05
1.85 0.05
1.82 0.10
1.72 0.05
1.66 0.02
1.62 0.02
31
TABLE IV
Solubility - pH Profile of Cephalexin in Water (37°C)
Cephalexin Monohydrate Cephalexin Monohydrate

PH . .
mg Iml .
mg I d .
~
2.3 13
2.5 16
W 3 .O .o 24
N
3.5 40
4.0 75
5.0 100
CEPHALEXIN
2.4 Dissociation Constant (pKa)

The following d i s s o c i a t i o n constants were reported:
Solvent Carboxyl Amino Reference
66% rn 5-2 7.3 4

66% DMF 5.3 7.3 17
h0 7.1 18
2.5 Optical Rotatory Dispersion

Optical r o t a t i o n has been used as an a u x i l i a r y
method f o r the q u a n t i t a t i o n of cephalexir?. The s p e c i f i c
r o t a t i o n [@ID reported f o r cephalexin, calculated on an an-
hydrous b a s i s , w a s +153O (C = 1.0 i n & 0)l 9 .
3.0 Cephalexin S t a b i l i t y
The s t a b i l i t y of cephalexin i n solution i s depen-
dent on pH, degrading r a p i d l y i n b a s i c media and remaining
s t a b l e under mild a c i d i c conditions. No loss i n cephalexin
a c t i v i t y occurred i n 72 hours a t 25OC i n the pH range from
3 t o 5. The r a t e of degradation found at pH 6 and pH 7
(25OC) was approximately 3% and 18%per day, r e s p e c t i v e l y 0 .
With r e f r i g e r a t i o n , no appreciable loss occurs between pH 3
and pH 7 a f t e r 72 hours. I n U.S.P. hydrochloric a c i d b u f f e r
(pH 1.21, cephalexin l o s t 5% a c t i v i t y i n 24 hours a t 37OC as
compared t o a 45% loss i n phosphate b u f f e r at pH 6 . 9 l .
a n t i b i o t i c r e t a i n s a c t i v i t y well i n serum and u r i n e as no
The
loss i n a c t i v i t y was noted a f t e r storage a t -2OOC f o r 14

dayg2 . Cephalexin i n serum w a s found t o l o s e lo%, 5% and
75% a c t i v i t y , respectively, a f t e r storage a t 5'C, 25OC, and
37OC f o r 48 h o u r d l ' z z . Some organisms have been found t o
produce a @-lact m a s e ( cephalosporinase) which can r a p i d l y
degrade cephalexid' . Degradation of cephalexin a l s o r e s u l t s
from heat, s t r o n g alkali, strong a c i d s and u l t r a v i o l e t
l i g h t (260 nm) .
4.0 Synthesis
Two synthetic r o u t e s of general a p p l i c a b i l i t y have
been proposed f o r cephalexin' .
The f i r s t method i s based on the cleavage of t h e
'
acetoxyl group from cephaloglycid (I) by hydrogenation o r ,
more s a t i s f a c t o r i l y , from N-t-butoxy-carbonylcephaloglycin
(11) t o profuce the corresponding desacetoxy analogs V
(cephalexin) and I11 as shown i n Figure 4, Scheme I. The t-
33
LOUIS P. MARRELLI
SCHEME I
&lfON::@
!- N / CHzOAc @:b.lii> N / CH3
C02H COzH
I, R-H 111. R-Boc
II. A=Boc V. R-H
SCHEME I1
CO2H COZH
V IV
FIGURE 4. Synthasis routes for eaphelexin.
34
CEPHALEXIN
butoxy-carbonyl (BOC) group was removed from I11 with tri-

fluoroacetic a c i d and the r e a c t i o n product was converted t o
desacetoxycephaloglycin ( cephalexin, V) by treatment with
Amberlite LA-1 r e s i n .
The second method w a s s i m i l a r t o t h a t previously

used t o obtain cephaloglycid' .
The nucleus, 7-aminodes-
acetoxycephalosporanic a c i d ( 7-ADCA)25 was prepared, acylated
with BOC-protected D-phenylglycine employing a mixed anhy-
dride synthesis, and then deblocked with t r i f l u o r o a c e t i c
a c i d as shown i n Figure 4, Scheme 11.
5.0 Methods of Analysis

5.1 I d e n t i f i c a t i o n Tests
Cephalexin may be i d e n t i f i e d by i n f r a r e d
spectroscopy. B r i t i s h Pharmacopoeia' u t i l i z e s two charac-
t e r i s t i c color r e a c t i o n s f o r i d e n t i t y . Thin l a y e r (Sec.
5.23), paper (Sec. 5.24), and column chromatography (Sec.
5.25) have been u t i l i z e d f o r i d e n t i t y purposes.
5.2 Q u a n t i t a t i v e Methods
5.21 --.T i t r a t i o n
The iodometric t i t r a t i o n procedure has
been used f o r the determination of c e p h a l e x i d 6 '*'.
The
method i s based on the f a c t t h a t t h e i n t a c t cephalexin
molecule does not consume iodine, whereas the alkali-hydroly-
sis product of cephalexin does. Alkaline hydrolysis of
cephalexin r e s u l t s i n cleavage of t h e @-lactam ring. V a r i a -
t i o n s i n hydrolysis time, temperature, pH of t h e iodine solu-
t i o n and concentration o f cephalexin present influence t h e
consumption of iodine by t h e test solution. The method
compares favorably t o t h e microbiological cylinder-plate
method (Sec. 5.27) i n accuracy, and is much more rapid.
Possible intermediates used i n the s y n t h e s i s such as 7-ADCA
w i l l also respond t o the test.
An automated iodometric assay has been

used r e c e n t l y f o r t h e assay of cephalexin and formulations
!A;:(
thereof4
step
The procedure incorporates a sample hydrolysis
N, at 37OC f o r 10 minutes followed by a 5-minute
iodine consumption s t e p (pH 5.3-5.5, 37°C). Concentration
of the sample i s r e l a t e d t o the decrease i n iodine color
measured a t 350 MI. A reference standard i s run concurrently
through the analyzer f o r comparative purposes. The auto-
mated system gives excellent l i n e a r i t y of response f o r t h e
35
LOUIS P. MARRELLI
recommended concentration range of cephalexin (0 1.5 mg. -

per m l . sample s o l u t i o n ) , with a l l standard curve p l o t s
passing through t h e origin. The r e p r o d u c i b i l i t y of t h e
method on t h e same sample o r standard s o l u t i o n on a given
day i s generally b e t t e r than +l% r e l a t i v e standard deviation
(RSD). Cephalexin can be titf;ated with p e r c h l o r i c a c i d in a
g l a c i a l acetic acid m e d i d 9 . Crystal v i o l e t indicator
(2% i n g l a c i a l a c e t i c acid) may be used t o determine t h e
endpoint.
Moll and D6keio have reported using a

formol t i t r a t i o n procedure f o r t h e determination of cepha-
l e x i n , a m p i c i l l i n and r e l a t e d compounds. I n t h i s procedure
4 m l . of d i l u t e formaldehyde s o l u t i o n ( n e u t r a l i z e d t o the
phenolphthalein end p o i n t ) is added t o 10 m l . of an aqueous
s o l u t i o n containing 15.0 mg. of cephalexin. After 2 minutes
t h e s o l u t i o n is t i t r a t e d with 0.02N sodium hydroxide. A
p r e c i s i o n of +O.% RSD could be achieved i n t h e t i t r a t i o n o f
cephalexin mozohydrate raw m a t e r i a l samples. Acidic com-
pounds as well as amino a c i d s must not b e present as i m -
p u r i t i e s i n t h e sample. The formol t i t r a t i o n takes advan-
tage of the r e a c t i o n between an amino a c i d and formaldehyde
as a means of suppressing t h e b a s i c i t y o f the amino group
and thus making p o s s i b l e t h e t i t r a t i o n of t h e acid.
5.22 Colorimetric Determination

Reaction with hydroxylamine has been
u t i l i z e d f o r t h e colorimetric determination of cepha-
lexid’ ’32 . The method i s based on t h e f a c t t h a t hydroxy-
lamine cleaves t h e 8-lactam r i n g (pH 7.0) t o form a hydrox-
amic a c i d which forms a colored complex with f e r r i c ion.
Degradation products o r intermediates having an i n t a c t 8-
lactam r i n g r e a c t as well.
Kirschbaud’ has described a procedure

f o r the colorimetric determination of t h e a n t i b i o t i c ceph-
radine’ and r e l a t e d cephalosporins. An aqueous s o l u t i o n of
t h e compound (1 t o 30 mcg. p e r ml.) i s r e a c t e d with sodium
hydroxide, p a r t i a l l y n e u t r a l i z e d , and then r e a c t e d with 5*5’-
dithiobis-( 2-nitrobenzoic acid) resulting i n t h e formation of
a y llow chromophore (412 nm.). The molar a b s o r p t i v i t y E x
lo-$ reported f o r cephalexin when c a r r i e d through t h i s pro-
cedure w a s 1.29. The formation of the yellow chromophore
w a s a t t r i b u t e d t o the presence of t h e R - C q -CO-cephalo-
’Cephradine i s t h e generic name f o r 7-[D-2-amino-2-(lq4-

c yclohexadienyl) ace tamido] desac e toxycephalosporanic acid
36
CEPHALEXIN
sporin nucleus, i n which R i s a mono-, di- or tri- enyl

cyclohexyl ring.
A s p e c i f i c colorimetric t e s t w a s de-
veloped f o r t h e determination of cephalosporin d e r i v a t i v e s
having the following i n t a c t s i d e chain i n t h e 7- p o s i t i o n :
R - (3%-CO-cephalosporin nucleus, R being a heterocyclic
o r aromatic r i n 8 ' . The D-phenylglycine d e r i v a t i v e s of both
7-ADCA (cephalexin) and 7-ACA ( cephaloglycin)2 respond well.
These compounds (0.5 - 1.0 mg. per m l . i n %O) react with
acetone and sodium hydroxide a t 1 0 0 ° C t o form c h a r a c t e r i s t i c
red chromophores (520 nm.). A t the 1 mg. per ml. l e v e l , t h i s
t e s t w i l l v i s u a l l y d i f f e r e n t i a t e cephalexin from cephradine.
Cephaloglycin is the generic name f o r 7-( D-a-aminophenyl-

acetamido) cephalosporanic a c i d
31
LOUIS P. MARRELLI
5.23
Thin Layer Chromatography
The following t h i n l a y e r chromatographic
systems have been reported:
TABLE V
Adsorbent Solvent System Ref.

-
Rf -
Silica G e l Ace t o n i t r i l e / W a ter 0.67 13
(3:l)
Silica Gel Ethyl Acetate/Ace- 0.22 35
tone/Acetic Acid/
Water (5:2:2:1)
Cellulose
Chromatogram
Butanol/Acetic Acid/
Water (3:l:l)
- 36
Sheet
Sheet Ethyl Acetate/Acetic - 36

A c i o a t e r (3:l:l)
Sheet Acetonitrile/Ethyl
Acet at e/Water ( 3 :1 :1)
- 36
Cellulose Acetonitrilefiater 0.50 37

(3:1)
Cellulose Butanol/Acetic Acid/ 0.70 37
Water (3:l:l)
Cellulose is t h e p r e f e r r e d sorbent s i n c e
i t i s i n e r t toward cephalexin. Additional t h i n l a y e r chroma-
tography systems used f o r cephalexin and o t h e r cephalosporins
have been t a b u l a t e d ? . Cephalexin may be detected by u l t r a -
v i o l e t absorbance and quenching, ninhydrin, i o d o p l a t i n a t e ,
a l k a l i n e permanganate, and phosphomolybdic a c i d sprays.
Iodine detection and vanillin-phosphoric a c i d spray have
a l s o been u t i l i z e d . O f t h e microorganisms used, Sarcina
-
l u t e a i s p r e f e r r e d over B a c i l l u s s u b t i l i s o r Staphylococcus
aureus.
38
CEPHALEXIN
5.24
Paper Chromatography
The following paper chromatographic
systems have been reported:
Solvent Systems Ref.

-
Rf -
Butanol/Ac e t i c AcidJWate r 0.60 13, 36, 38
(3:l:l)
Ethyl Acetate/Acetic Acid/

Water (3:l:l)
- 36
Whatman No. 1*,untreated, was used

with both solvent systems and the e q u i l i b r a t i n g solvent w a s
t h e same as t h e developing solvent. Additional paper chroma-
tographic systems f o r cephalexin and o t h e r cephalosporins
have been t a b u l a t e d 8 . The butanol/acetic aciuwater
(3 :l:l) system w i l l separate cephaloglycin from cephalexin,
cephaloglycin being l e s s mobile. An acetonitrile/water
(9:l) s y s t e d 9 using Whatman No. 3 paper buffered a t pH 5.0
(16 hour development) h a s been u t i l i z e d t o s e p a r a t e cephra-
dine from cephalexin, cephradine being l e s s mobile. The
developed chromatogram can be examined under u l t r a v i o l e t
l i g h t , dipped i n ninhydrin, or bioautographed, using
Sarcina lutea'o .
5.25 Column Chromatography
The Moore-Stein amino acid analyzer has
been used f o r the determination of cephalexin i n u r i n e
.
samplesb1 Beckman Custom Research Resin Type PA-35, packed
t o a height of 9.0 cm. i n a water-jacketed column (0.9-cm.
i.d. x 2'3-cm. length) w a s used f o r t h e separation. The
u r i n e sample was d i l u t e d with an equal volume of sodium c i -
t r a t e buffer (pH 2.2) and 100 h applied t o t h e column. The
e l u t i o n time for cephalexin was approximately 61 minutes.
Excellent agreement was found between the analyzer method
and the microbiological method (Section 5.27) on a s e r i e s
of u r i n e samples tested. I n a d d i t i o n , t h i s technique has
been u s e f u l i n determining low l e v e l s of 7-ADCA and phenyl-
glycine i n cephalexin'l (Section 5.3). Determination of t h e
l e s s a c t i v e ( b i o l o g i c a l l y ) L-isomer i n cephalexin by t h e
Moore-Stein amino a c i d analyzer has been r e p o r t e 8 . Chou"
-
used the anionic r e s i n , Bio-Rad AG2 x8 ( a c e t a t e form),
t o i s o l a t e cephalexin from human urine.
*Whatman chromatography' paper, Reeve Angel, 9 Bridewell

Place, C l i f t o n , New Jersey
39
LOUIS P. MARRELLI
5.26 Electrophoresis
Paper e l e c t r o p h o r e s i s has been u t i l i z e d
by the B r i t i s h Pharmacopoeia' as test f o r impurities
present i n cephalexin (Section 5.3).
5.27 Microbiological Assays

Microbiological assays f o r cephalexin
have been discussed by Marrelli'*, Wick" Mann'' and
Simmond' and are l i s t e d i n the Federal R e g i s t e r C 6 . B r i e f l y ,
the two p l a t e systems well s u i t e d f o r t h e determination of
cephalexin i n pharmaceutical formulations a r e t h e cylinder
p l a t e methods u t i l i z i n g S t a hylococcus aureus ( ATCC 6535)
and Bacillus s u b t i l i s (A--e-ac range f o r both
i s approximately 2.5 t o 20 mcg. of cephalexin p e r ml.'2 '"
The B. s u b t i l i s p l a t e test h a s an advantage over t h e S.
aureus p l a t e t e s t i n t h a t b e t t e r defined zones of inhTbition
a r e obtained, thereby increasing t h e assay precision4
Since degradation product6 of cephalexin possess p r a c t i c a l l y
.
no antimicrobial a c t i v i t y 2 r a p i d and p r e c i s e photometric
microbiological assays a r e p o s s i b l e with cephalexin. The
t e s t organism f o r the photometric assay i s Staphylococcus
aureus 9144, 3 t o 3.5 hours being required f o r incubation.
I n Antibiotic No. 3 Broth, t h e concentration range is 0.2
t o 2.0 mcg. p e r m l . of broth. I f t h e automated AUTOTU@
System is used, p r e c i s i o n i n the order o f 1 2 % i s possible".
Cephalexin i n b i o l o g i c a l f l u i d s may be assayed by a Sarcina
l u t e a p l a t e system. The concentration range f o r assay i s
0.2 t o 3.5 m~g./ml.~+
5.3 Methods f o r Intermediates and Impurities
5.31 7-Aminodesacetoxycephalosporanic Acid

( 7-ADCA)
Cole'' u t i l i z e d the Moore-Stein amino
a c i d analyzer f o r the determination of 7-ADCA i n cepha-
lexin. Column s p e c i f i c a t i o n s were o u t l i n e d i n Section 5.25.
Twenty-five milligrams of cephalexin sample were dissolved
w i t h sodium c i t r a t e b u f f e r (pH 2.2) t o a t o t a l volume of
10.0 m l . and 1.0 m l . applied t o t h e column. The e l u t i o n
time f o r 7-ADCA w a s approximately 45 minutes. 'The s e n s i -
t i v i t y of the assay w a s 0.B 7-AKA.
A colorimetric procedure was developed

by M ~ ~ r r e l l iwhich
'~ permitted t h e d i r e c t determination of
low l e v e l s of 7 - A K A i n cephalexin (0.4 -
1.5%). The pro-
cedure i s based on t h e i n t e r a c t i o n of 7-ADCA with ninhydrin
under c o n t r o l l e d conditions t o produce a s p e c i f i c chromophore
40
CEPHALEXIN
( A max. a t 480 nm.) . Compounds having an a-amino group

adjacent t o a B-lactam r i n g respond i n general t o t h e
t e s t . B r i t i s h Pharmacopoeia' u t i l i z e d a paper e l e c t r o -
phoresis technique which estimated t h e 7-ADCA content i n
cephalexin a t the l% l e v e l . The b u f f e r s o l u t i o n s p e c i f i e d
i n the t e s t consisted of a mixture of 5 m l . of formic acid,
25 m l . of g l a c i a l a c e t i c a c i d , 30 m l . of acetone and
water t o a t o t a l volume o f 1000 m l .
A 2.0 pl. a l i q u o t of the cephalexin

sample s o l u t i o n (5.0% w/v i n 0.5N HC1) along with the
s p e c i f i e d amounts of reference standards and markers were
applied t o the paper. The voltage was adjusted t o about
20 v o l t s per cm. of paper and e l e c t r o p h o r e s i s w a s allowed
t o proceed u n t i l the c r y s t a l v i o l e t spot moved 9 cm. from
the base l i n e . Both ninhydrin spray and W were used f o r
detection purposes.
5.32 Phenylglycine
The ChromatonraDhic Drocedures o u t l i n e d
I &
i n Section 5.31 f o r the determination o f 7 - m A i n cepha-

l e x i n have been concurrently used for the determination of
phenylglycine i n cephalexin. The e l u t i o n time f o r phenyl-
glycine i n the amino a c i d analyzer assay was approximately
'56 minutes. The s e n s i t i v i t y of the assay w a s 0.01%. The
paper electrophoresis technique permitted estimation of
phenylglycine a t the 1%l e v e l . Hussepe has u t i l i z e d a
chromatographic procedure f o r phenylglycine similar t o
t h a t of the amino a c i d analyzer assay but incorporating
a Fluramm detection system'.
t
Hoffman-LaRoche Inc., Nutley, New J e r s e y
41
LOUIS P. MARRELLI
6. P r o t e i n Binding
Various values have been reported f o r t h e percentage
of cephalexin bound t o serum protein. Wicp' concluded
t h a t serum i n a c t i v a t i o n o r p r o t e i n binding of cephalexin
i s low. Addition of serum t o broth medium did n o t a f f e c t
-.i n -v.- i t r o minimal i n h i b i t o r y concentration determinations.
Cephalexin assays i n pH 7 b u f f e r and human serum r e s u l t e d
i n i d e n t i c a l standard curves when 6-mm d i s c s were satu-
r a t e d with s o l u t i o n s and t e s t e d by a Sarcina l u t e a micro-
b i o l o g i c a l assay. U t i l i z i n g a similar method, G r i f f i t h
and Black!) found t h a t p r o t e i n binding of cephalexin i n
human berum was % a t concentrations above 1.0 bg./ml.
and 4196 a t 0.2 yg./ml. Naumann and Fedde9O a l s o found
t h a t the amount o f cephalexin bound t o serum p r o t e i n s
varied with t h e cephalexin concentration. Using an u l t r a -
f i l t r a t i o n method, Kind e t . al.51 estimated t h e serum
binding as being 15%. O v a l r q h a n and Muggletor?2
obtained a value of 43% by u t i l i z a t i o n of t h e u l t r a f i l t r a -
t i o n technique.
7. Pharmacokinetics
Oral doses of cephalexin are r a p i d l y absorbed by ani-
mals and man, r e s u l t i n r a t h e r high blood serum l e v e l s ,
and a r e excreted unchanged i n t h e urine. Wick?* found
t h a t a 20-mg./kg. o r a l dose of cephalexin i n mice gave
a blood serum l e v e l of 18 pg./ml.
reported a similar value (17 Fg./ml.) with a 10 mg./kg.
--
Wells e t . al.5'
o r a l dose i n dog. The r a p i d i t y of o r a l absorption was

demonstrated by t h e f a c t t h a t a n t i b a c t e r i a l a c t i v i t y
i n t h e serum was t h e same within 1.5 hours a f t e r o r a l
or intramuscular administration; t h e r e a f t e r , the l e v e l s
were higher f o r t h e o r a l dose. I n man, a f t e r a 500-mg.
dose t h e mean of t h e peak a n t i b i o t i c a c t i v i t i e s found
*
by s e v e r a l i n v e s t i g a t o r s w a s 15 pg./ml.' '5' 'J and
u s u a l l y occurred a t 1-1.2 hours a f t e r treatment. The
compound is almost completely absorbed from the upper
small i n t e s t i n e i n both m a n and animals6'J6. I n
addition, t h e a n t i b i o t i c i s excreted unchanged i n
"'
u r i n e with almost 100% r e c o ~ e r f ' ~ ' 5 9 . A meal j u s t
.
p r i o r t o treatment r e s u l t e d i n lower blood l e v e l s and
increased t h e time required for peak t i t e ? ' 6 0
42
CEPHALEXIN
Kirby - et. - a1.61 c a l c u l a t e d the serum h a l f - l i f e of

intravenously administered cephalexin as 36 minutes.
Kabins - et. - al.62 and Naumann and FeddeSO c a l c u l a t e d
the serum h a l f - l i f e a f t e r o r a l dosage a s 54 minutes.
Thornhill - et. - al.5 found t h a t probenecid increased the
peak serum concentration by 50%. but Meyers e t . al.55 --
-
found a l e s s e r e f f e c t . Linquist --e t . a1.6sexamined the
disappearance of cephalexin from the blood s e r a of aneph-
r i c p a t i e n t s . H a l f - l i f e values ranged from 23.5 - 41.0
hours with a mean of 31 hours, c l e a r l y demonstrating t h e
dependence upon the kidney f o r excretion.
43
LOUIS P. MARRELLI
8. References
1. The United States Pharmacopoeia, XIX, Proof p. 2122.

2. Federal Register, 21CFR148w.6.
3. British Pharmacopoeia, 1973, p. 87.
4. Ryan, C.W., Simon, R.L., and Van Heyningen, E.M.,
J. Med. Chem., l2, 310 (1969).
5. Doyle, F.P., Fosker, G.R., Naylor, J.H.C., and Smith,
H., J. Chem. SOC., 1440 (1962).
6. Analytical Profiles of Drug Substances, Vol. 2 (K.
Florey, ed.) p.4, Academic Press, New York and
London, 1973.
7. Pfeiffer, R.R., Yang, K . S . and Tucker, M.A., J.
Phann. Sci., 59, 1809 (1970).
8. Underbrink, C.D., Eli Lilly Analytical Development,
Unpublished Data.
9. Morin, R.B., Jackson, B.G., Mueller, R.A.,
Lavagnino, E.R., Scanlon, W.B., and Andrews,, S.L.,
J. her. Chem. SOC., 91, 1401 (1969).
10. Flynn, E.H., ed., Cephalosporins and Penicillins.
Chemistry and Biology, Academic Press, New York and
London (1972), p. 315.
11. Flynn, op. cit., p. 631.
12. Chawette, R.R., et. al., J. Am. Chem. SOC., 84,
3401 (1962).
13. Chou, T.S., J. Med. Chem., l2, 925 (1969).
14. Pfeiffer, R.R., Eli Lilly Analytical Development,
Personal Communication, (1969).
15. Cole, T.E. , Eli Lilly Analytical Development,
16. Pfeiffer, R.R., Eli Lilly Analytical Development,
17. Flynn, op. cit., p. 310.
18. Hargrove, W.W. , Eli Lilly and Company, Personal
Communication, (1967).
19. Flynn, op. cit., p . 633.
20. Winely, C.L., Eli Lilly Analytical Development
Laboratories, Unpublished Data.
21. Simmons, R. J. , Anal. Microbiol., 11. , 193 (1972).
22. Wick, W.E., Appl. Microbiol., l5, 765 (1967).
23. Ott, J.L., and Godzeski, C.W., Antimicrob. Ag.
Chemother. 1966, 75 (1967).
24. Spencer, J.L., Flynn, E.H., Roeske, R.W., Siu, F.Y.,
and Chauvette, R.R., J. Med. Chem., 2, 746 (1966).
25. Stedman, R.J., Swered, K., Hoover, J.R.E., J. Med.
* .Chem - 7, 117 (1964).
26. Federal Register, 21CFR141.506.
44
CEPHALEXIN
27. British Pharmacopoeia, p. 88 (1973).

28. Stevenson, C.E., and Bechtel, L.D. (1971) Publication
submitted for review in J. Pharm. Sci.
29. Marrelli, L.P., Eli Lilly and Company, Personal
Cmunication (1967).
, -~
30. Moll, F., and Dzker, H., Arch. Phann., Berl., 305
(7), 548 (1972).
31. Federal Register, 21CFR141.507.
32. Plynn, op. cit., p. 615.
33. Kirschbaum, J., J. Pharm. Sci., 63, 923 (1974).
34. Marrelli, L.P., J. Pharm. Sci., 6 l , 1647 (1972).
35. Thomas, P.N., Eli Lilly and Company, Personal
Cmunication (1972).
36. Sullivan, H.R., Billings, R.E., and McMahon, R.E.,
J. Antibio., 22, 195 (1969).
37. Flynn, op. cit., p. 621.
38. Flynn, op. cit., p. 620.
39. Marrelli, L.P., Eli Lilly and Company, Personal
Cmunication (1972).
40. Miller, R.P., Antibiot. and Chemother., 12, 689
(1962).
41. Flynn, op. cit., pp. 629, 680.
42. Flynn, op. cit., p. 610.
43. Flynn, op. cit., p. 497.
44. Mann, J.M. , Anal. Microbiol., a, 207 (1972).
45. Simmons, R.J., Anal. Microbiol., s, 193 (1972)
46. Federal Register, CFR148~6.
47. Kuzel, N.R. and Kavanagh, F.W., J. Phann. Sci., 60,
767 (1971).
48. Hussey, R.L., Eli Lilly Analytical Development,
Personal Communication (1974).
49. Griffith, R.S. and Black, H.R., Postgrad. Med. J.,
47, February Suppl. , 32 (1971).
50. Kumann, P. and Fedder, J., Int. J. Clin. Pharmacol.,
Suppl., 2, 6 (1970).
51. Kind, A.C., Kestle, D.G., Standiford, H.C. and Kirby,
W.M.M., Antimicrob. Ag. Chemother., 405 (1968).
52. Flynn, op. cit., p. 438.
53. Wells, J.S., Froman, R.O., Gibson, W.R., Owen, N.V.,
and Anderson, R.C., Antimicrob. Ag. Chemother., 489
(1968).
54. Kunin, C.M., and Finkelberg, Z . , Ann, Inst. Med.,
72, 349 (1970).
55. Eyers, B.R., Kaplan, K., and Weinstein, L., Clin.
Pharmacol. Ther., 10, 810 (1969).
56. Muggleton, P.W., O'Callaghan, C.H., Foord, R.O.,
Kirby, S.M., and Ryan, D.M., Antimicrob. Ag.
45
LOUIS P. MARRELLI
Chemother., 353 (1968).

57. Perkins, R.L., Apicella, M.A., Lee, I., Cuppage,
F.E., and Saslaw, S., J. Lab. Clin. Med., 7 l , 75
(1968).
58. Thornhill, T . S . , Levison, M.E., Johnson, W.D., and
Kaye, D., Appl. Microbiol., l7, 457 (1969).
59. Gower, P.E. and Dash, C.H., Br. J. Pharmac., 37,
738 (1969).
60. O'Callaghan, C.H., Footill, J.P.R., and Robinson,
W.D., J. Pharm. Pharmac., 23, 50 (1971).
61. Kirby, W.M.M., de Maine, J.B., and Serrill, W.S.,
Postgrad. Med. J., 47, February Suppl., 46 (1971).
62. Kabins, S.A., Kelner, B., Walton, E., and Goldstein,
E., her. J. Med. Sci., 259, 133 (1970).
63. Linquist, J.A., Siddiqui, J.Y., and Smith, I.M.,
New Engl. J. Med., 283, 720 (1970).
ACKNOWLEDGEMENTS
The author wishes to express his indebtedness to Dr. C. L.
Winely for his contribution of the sections on micro-
biological assays, protein binding and pharmacokinetics.
46
CHLORAMPHENICOL
Dale Szulczewski and Fred Eng

D A L E SZULCZEWSKI A N D FRED ENG
CONTENTS
1. Description
1.1 Nomenclature
1.11 Chemical Names
1.12 Generic Names
1.13 Trade Names
1.2 Formulae
1.21 Empirical
1.22 Structural and Stereochemical
1.3 Molecular Weight
1.4 Elemental Composition
1.5 General
2.11 Crystallinity
2.12 X-Ray Diffraction
2.13 Melting
2.131 Range
2.132 As criteria of acceptability
2.133 In relation to purity determination-
differential scanning calorimetry
2.2 Solubility
2.21 Single Solvents
2.22 In mixed solvents or as a result of complexa-
tion
2.23 pH Effect
2.3 Distribution
2.4 Spectral Properties
2.41 Ultraviolet
2.42 Infrared
2.43 Nuclear Magnetic Resonance
2.45 Mass Spectrum
3 . Synthesis
4. Stability and Decomposition Products
4.1 Crystalline solid and solid dosage forms
4.2 In solution
4.3 In presence of microorganisms
5. Metabolism
6.1 Colorimetric
6.11 For identification
6.12 Quantitative Analysis
48
CHLORAMPHENICOL
CONTENTS (Cont ' d)
6.2 Polarographic
6.3 Spectrophotometric
6.4 Titrimetric
6.5 Microbiological
6.6 Chromatographic
49
DALE SZULCZEWSKI AND FRED ENG
1. Description
1.1 Nomenclature
1.11 Chemical Names

a. Dg-(-)-threo-2 ,2-dich1oro-N- [B-hydroxy-
a- (hydroxymethyl)-p-nitrophenethyl]acetamide .
b. Dg-(-)-threo-l-(~-nitrophenyl)-2- (2,2-
dich1oroacetamido)-l,3 propanediol
1.12 Generic Name
Chloramphenicol
1.13 Trade Names
Chloromycetin (The Merck Index' lists 45
other trade names.)
1.2 Formulae
1.21 Empirical
C11H12C12N205
1.22 Structural and Stereochemical
CH20H
C12CHCONHCc
I r l ~D -(or Ls)-threo-l-(p
I3
I nitrophenyl)-2-(2,2-
)Lc *OH dichloroacetamid )-
bI or
-
-1,3 propanediol9
(lR, 2R) l-(E-nitro-

phenyl)-2-(2,2-
dich1oroacetamido)-
N02 1,3-propanediol
1.3 Molecular Weight 323.14
1.4 Elenental Composition

C-40.88; H-3.74; C1-21.95; N-8.67; 0-24.76
50
CH LORAMPHENICOL
1.5 General3
Fine, white to grayish white or yellowish white,
needle-like crystals or elongated plates. Its
solutions are practically neutral to litmus.
2.11 Crystallinity
Chloramvhenicol is a crystalline solid. A
typical photomicrograph4 of chloramphenicol is shown in
Figure 1.
2.12 X-ray Diffraction
Analytical X-ray diffraction powder data5
indexed using single crystal diffraction data6 for chloram-
phenicol follows:
Radiation. CuKct(A1.5418); Filter. Ni.

I/I, Diffractometer.
System. Orthorhombic. Space Gr0up.C222~(DX)

a, 17.6A: b, 7.35A; C, 22.38.
2. 8.
Ect. 1.519; nuB. 1.601; EY. 1.668.

Sign. Negative. 2V. 7 8 O .
Measured Density. 1.49.
51
Fig. 1 Photomicrograph of Chloramphenicol.
52
CHLORAMPHEN ICOL
2.12 X-ray Diffraction (continued)
Powder data for sample of chloramphenicol.

(Chloromycetin sample from Parke, Davis & Company, Detroit,
Lot I1 573326.)
dA 20, deg. C u m 111

-1 h k-&-
-
11.08 7.98' 12 002
8.76 10.10 1 200
8.15 10.85 32 201
6.87 12.89 64 202
5.64 15.72 57 203
4.98 17.81 32 113
4.68 18.98 61 204
4.47 19.85 57 311
4.37 20.30 79 400
4.28 20.74 100 401,114
3.95 22.50 11 205
3.88 22.91 11 313
3.70 24.05 18 115,006
3.66 24.31 64 020
3.61 24.63 4 021
3.44 25.93 54 404
3.38 26.37 7 220
3.34 26.68 7 221
3.28 27.17 7 023
3.23 27.61 18 222
3.16 28.25 4 br* 510,315
3.13 28.55 10 br* 511,405
3.05 29.25 7 024
2.97 30.05 7 207
2.92 30.64 79 600,513
2.89 30.90 50 601
2.87 31.19 14 316,117
2.82 31.69 89 406,602,025
plus other lines
* broad
53
2.12 X-ray D i f f r a c t i o n (continued)
Further information regarding confirmation

of t h e chemical s t r u c t u r e of chloramphenicol through analy-
k.
sis of x-ray d i f f r a c t i o n p a t t e r n s obtained on t e a n t i -
b i o t i c and i t s bromo analog are given by Dunitz
2.13 Melting
2.131
Range
Bartz7 determined the melting range of
chloramphenicol t o be 149.7 -
150.7 'C (corrected).
As a c r i t e r i a of a c c e p t a b i l i t y
2.132
The United S t a t e s Food and Drug
Administration8 and the U.S.P.3 both specify a melting range
of 151k2'C.
2.133I n r e l a t i o n t o p u r i t y determination
Information p e r t a i n i n g t o t h e p u r i t y
of chloramphenicol can be obtained through i n t e r p r e t a t i o n
of t h e thermograms obtained v i a D i f f e r e n t i a l Scanning
Colorimetry. A t y p i c a l thermogramg obtained on chloram-
phenicol follows as Figure 2.
2.2 Solubility
2.21 Single Solvents

The following d a t a are a b s t r a c t e d from the
Merck Index1:
S o l u b i l i t y a t 25' i n water = 2.5 mg./rnl.; i n propylene gly-

c o l = 150.8 mg./ml. Very s o l u b l e i n methanol, ethanol,
butanol, e t h y l a c e t a t e , acetone. F a i r l y s o l u b l e i n e t h e r ;
i n s o l u b l e i n benzene, p e t r . e t h e r , vegetable o i l s . Solu-
b i l i t y i n 50% acetamide soln. about 5%. S o l u b i l i t i e g de-
termined by Weiss e t a l . , A n t i b i o t i c s & Chemotherapy 7,374
(1957) i n mg./ml. a t about 28': Water 4.4; methanol 720;
ethanol >20; isopropanol >20; isoamyl alcohol 17.3; cyclo-
hexane 0.13; benzene 0.26; toluene 0.145; p e t r . e t h e r 0.085;
isooctane 0.022; carbon t e t r a c h l o r i d e 0.295; e t h y l a c e t a t e
>20; isoamyl a c e t a t e >20; acetone >20; methyl e t h y l ketone
>20; e t h e r >20; ethylene c h l o r i d e 2 . 3 ; dioxane >20; chloro-
form 1.95; carbon d i s u l f i d e 0.35; pyridine >20; formamide
>20; ethylene glycol >20; benzyl alcohol 14.6.
54
CHLORAMPHENICOL
PARKE D A V I S LOT H700171

4.421 mg
4 millicalories f u l l scale
1.25"/min
% P U R I T Y = 99.863
FIG. 2 - THERMOGRAM AND P U R I T Y DETERMINATION OF CHLORAMPHENICOL.

DALE SZULCZEWSKI A N D F R E D ENG
2.22 I n mixed s o l v e n t s o r as a r e s u l t of complex-

ation
The s o l u b i l i t y p r o f i l e s f o r chloramphenicol
i n s e v e r a l aqueous solvent mixtures were determined by
Negoro and Associates". H i s r e s u l t s are summarized i n
Figure 3.
Kostenbauderll determined the s o l u b i l i t y of

t h i s a n t i b i o t i c i n aqueous s o l u t i o n s of N, N , N ' , "-tetra-
methylphthalamides as p a r t of a study on t h e complexing
p r o p e r t i e s of these amides. Results obtained indicated a
moderate influence on s o l u b i l i t y as shown i n Figure 4.
Aqueous s o l v e n t s containing 5% Tween 20-80

i n c r e a s e t h e water s o l u b i l i t y of chloramphenicol approxi-
mately 3 fold12. The s o l u b i l i t y of chloramphenicol i n
serum and u r i n e i s approximately t h e same as i t is i n
wa t e r l 3 .
The s o l u b i l i t y of chloramphenicol i n water

is increased by a d d i t i o n of boraxl4. This s o l u b i l i t y in-
c r e a s e i s explained on t h e b a s i s of t h e formation of a
1:2 complex between the b o r a t e i o n and t h e a n t i b i o t i c . Re-
s u l t s are summarized i n Table 1.
Table 114
S o l u b i l i t y of Chloramphenicol i n Borax Solutions
Molar Borax Solubility PH

Concentration % of Solution
0 0.375 4.70
0.0001 0.391 7.15
0.001 0.438 8.65
0.005 0.614 8.65
0.01 0.732 8.65
0.02 1.23 8.65
0.05 2.14 8.70
0.11 3.46 8.90
0.125 3.67 8.90
0.15 3.87 9.00
56
CHLORAMPHENICOL
h
401
0,
\
v
F
>
I-
-Tm 301
3
.J
0
v)
20(
101
~~
50 1 10
CONCENTRATION OF PURE SOLVENT ( w t . %)
I. Dimethylacetamide, 3 1 ° C
II. Acetone, O°C
I I I. Methanol
IV. Ethanol
V. N-Propanol
VI. Propylene Glycol
10
FIG. 3 - SOLUBILITY CURVES OF CHLORAMPHENICOL
IN MIXED AQUEOUS SOLVENTS
57
-
h. 9 '9 f
$1 X 1/M 1031R~WdWWYOlW3
58
CHLORAMPHENICOL
2.23 pH E f f e c t
Since chloramphenicol i s an e s s e n t i a l l y
n e u t r a l compound, changes i n pH (over t h e pH r e g i o n 3 t o 9 )
do n o t r e s u l t i n s i g n i f i c a n t changes i n s o l u b i l i t y . The
solubility t h e a n t i b i o t i c i s i n c r e a s e d i n p r e s e n c e of
s t r o n g acidPf due t o p r o t o n a t i o n of t h e weakly b a s i c amido
nitrogen.
2.3 Distribution
As expected, t h e d i s t r i b u t i o n of chloramphenicol
between water and an immiscible s o l v e n t i s n o t markedly pH
dependent. The p e r c e n t of t o t a l chloramphenicol found i n
t h e aqueous phase a f t e r d i s t r i b u t i o n between e q u a l volumes
of water and immiscible o r g a n i c s o l v e n t s i s t a b u l a t e d i n
Table 27,16.
Table 2
% Chloramphenicol i n Aqueous Phase
Immiscible Solvent -
% Ref.
Cyclohexanone 8 7
n-Butanol 8 7
E t h y l acetate 3 16
Methyl i s o b u t y l ketone 8 7
N i trobenzene 38.0 7
Nitromethane 17.0 7
Ethyl e t h e r 20 16
Chloroform 82 16
Benzene 93 16
Petroleum e t h e r 96 16
Ethylene d i c h l o r i d e 50.0 7
Brunzell16 d i s c u s s e s t h e u t i l i t y of s i m p l e e x t r a c -
t i o n techniques i n t h e a n a l y s i s of f o r m u l a t i o n s c o n t a i n i n g
t h i s a n t i b i o t i c . The same a u t h o r provides e x t r a c t i o n
methods f o r a n a l y s i s of t h e drug i n t h e presence of hydro-
l y t i c decomposition products.
2.41 Ultraviolet
Chloramphenicol i n s o l u t i o n a b s o r b s u l t r a -
v i o l e t r a d i a t i o n over a broad range t o produce a spectrum
with a maximum near 278 nm and a minimum n e a r 240 nm
( s e e Fig. 5 f o r a t y p i c a l spectrum).
59
D A L E SZULCZEWSKI A N D FRED ENG
I.
nl
0
2
4
m
U
0
VI
m
4
0.
240 280 280 300 320 340 380 380 400

278
WAVELENGTH (nrn)
FIG. 5 - ULTRAVIOLET SPECTRUM OF CHLORAHPHENICOL I N WATER.
60
CHLORAMPHENICOL
7,17
As was e s t a b l i s h e d by Vandenbelt , this
a b s o r p t i o n i s due t o t h e p-nitrophenyl chromophore and pro-
v i d e s both a d i s t i n g u i s h i n g c h a r a c t e r i s t i c of t h e a n t i b i o t i c
and a u s e f u l method f o r a n a l y s i s .
An u l t r a v i l e t s p e c i f i c a t i o n i s i n c l u d e d i n
b o t h t h e F e d e r a l Register8 and t h e U.S.P.3 as a c r i t e r i a of
acceptability.
The u l t r a v i o l e t spectrum of chloramphenicol

i n aqueous s o l v e n t s is n o t s i g n i f i c a n t l y i n f l u e n c e d by
changes i n pH. Aqueous Borate b u f f e r s (pH 9.0) p e r t u r b
t h e spectrum, s h i f t i n g t h e maximum from 278 nm t o 284
nm as t h e r e s u l t of complex formation.
2.42 Infrared
The i n f r a r e d spectrum of chloramphenicol
(KBr d i s p e r s i o n ) i s shown i n F i g u r e 6. F u r t h e r i n f o r m a t i o n
with regard t o c o r r e l a t i o n of f u n c t i o n a l groups o i n f r a r e d
a b s o r p t i o n maxima i s given by Suzuki and Shindo" who de-
termined i n f r a r e d s p e c t r a of racemic e r y t h r o and t h r e o
isomers of t h e a n t i b i o t i c as w e l l as t h e s p e c t r a of r e l a t e d
compounds. These a u t h o r s o b t a i n e d evidence f o r i n t r a -
molecular hydrogen bonding from i n f r a r e d s p e c t r a determined
on d i l u t e s o l u t i o n s .
Assignments f o r t h e accompanying spectrum

follow:
Wavenumber
Functional Group cm-1
bonded OH, NH 3340, 3260
amide p o r t i o n of
2,2-dichlor-
acetamide moiety
amide I 1697
amide I1 1568
n i t r o group ( n i t r o -
PhenYl) 1530, 1358
hydroxyl 1068
61
WAVENUMBER (cm-1)
4000 3000 1Tw 1000 1600 1400 1100 111 118) im 900 a* in
1
J 4 C I 7 1 8 10 11 12 13 14
WAVELENGTH (microns)
FIG. 6 - INFRARED SPECTRA OF CHLORAMPHENICOL, KBr PELLET.

CHLORAMPHENICOL

A typical nuclear magnetic resonance spectrum
of chloramphenicol in deuterated acetone is given in Fig. 7.
Further information concerning the interpretation of the
NMR spectra of chloramphenicol and its diastereoisomer, the
L(+)-erythro isomer, can be obtained from Jardetsky's
careful studylg concerning the conformation of chloramphen-
icol in solution. Jardetsky's chemical shift assignments
of the chloramphenicol protons were determined from spectra
in deuterated acetone of chloramphenicol samples lyophil-
ized from D20 and H20.
Jardetsky's assignments are as follows (with

respect to benzene as external standard with 60-megacycle
NMR) Proton( s )
Associated with cps
-46.5 (center of H,B2

quartet)
R ' -CHC12 +32.6
+97.2
4-156.2
+177.5
+187.7
R1
I
H N - R ~ ~ ~ -29.3
C1 OH +99.6
C3OH +151.0
63
(Water)
b-
I
I
FIG.
3.0
I
I
I
I
LO
I
I
6.0
I
7.0
I
I
(Acetone)
7 - NMR SPECTRUM OF CHLORAMPHENICOL, PARKE DAVIS LOT H 7 0 0 1 7 1 I N OEUTERATED ACETONE CONTAINING

Lo
1
TRIMETHYLSILANE AS INTERNAL STANDARD.

CHLORAMPHENICOL

I n the chloramphenicol series c o n s i s t i n g of
four diastereoisomers, therapeutic a n t i b i o t i c a c t i v i t y re-
s i d e s i n the Dg-threo (or I R , 2R) isomer. S p e c i f i c a t i o n s
on s p e c i f i c r o t a t i o n as a c r i t e r i t of a c c e p t a b i l i t y are
contained i n t h e Federal Register as c i t e d by t h e U.S.P.
3
.
The Food and Drug Administration8 has estab-
l i s h e d the following s p e c i f i c a t i o n :
"Its (chloramphenicol' s) s p e c i f i c r o t a t i o n
i n absolute ethanol a t 2OoC i s +20 2 1.5' and a t 25' is
+18.5 - 1.5O." (NaD O r 589 nm)
The s p e c i f i c rotation2' (C=5%, ethanol, 25OC)

of chloramphenicol a t some other wave lengths follows:
578 +19.8"
546 +23.8'
436 +59.7O
Both magnitude and s i g n of o p t i c a l r o t a t i o n

are solvent dependent, i.e., i n ethanol the a n t i b i o t i c i s
dextrorotatory while i n e t h y l a c e t a t e i t is levorotatory.
Circular dichroism measurements on t h e four

chloramphenicol isomers have been made and are recorded i n
the literature21. Analysis of the a n t i b i o t i c i n combina-
t i o n with sulfonamides w a s accomplished by polarimetric
means22.
2.45 Mass Spectra

The mass s p e c t r a of chloramphenicol obtained
by conventional e l e c t r o n jmpact i o n i z a t i o n does not e x h i b i t
a parent peak. BrunnGe and associates23 obtained the m a s s
spectrum of chloramphenicol using a combined f i e l d ioniza-
t i o d e l e c t r o n impact ion source. A parent peak of only 5%
height i s obtained (see Fig. 8). The base peak is a t m / e
152 and is a t t r i b u t e d t o fragment I.
65
40' I70
20 *
oi-, . - . , . . . A . .. , . . . , .
60 I00 I50 200 250 300 340
m/e
F I G . 823 - MASS SPECTRA OF CHLORAMPHENICOL - (a) ELECTRON IMPACT I O N I Z A T I O N
(b) FIELD IONIZATIOW
66
CH LORAMPHENICOL
NH-COCHC1,
I
:i21
OZN-@-f H f- CH-CH,OH
1;;
3. Production and S y n t h e s i s
Chloramphenicol w a s o r i g i n a l l y produced by i s o l a t i n g
t h e a n t i b i o t i c from c u l t u r e s of Streptomyces Venezualae
24925.
A f t e r i t w a s demonstrated t h a t s y n t h e s i s of t h e
a n t i b i o t i c was p o s s i b l e 2 6 s e v e r a l s y n t h e t i c p r o c e s s e s
were developed f o r manufacture. A f l o w diagram f o r one
such p r o c e s s follows:
hexamethylene-
-
0 2 N B C H 2 B r -
tetramine > 0 2 N o C O C H 2 M 2
67
DALE SZULCZEWSKI AND F R E D ENG
Detailed information on various steps in this synthesis are

found in references 27-40. Other synthetic schemes are
contained in references 41-47. Processes for conversion
of erythro-p-nitrophenyl-2-amino-1,3-propanediol, produced
as a by-product, to the desired threo isomer generally pro-
ceed via oxazoline formation and are detailed in references
48-54. Resolution of the threo-(p-nitrophenyl)-2-amino-
1,3-propanediol roduced can be accomplished by conven-
tional means5 5 9 5 % or by fractional cry~tallization~~. A
rather complete review of the synthetic chemistry of chlor-
amphenicol is a~ailable~~. This review also documents
structure-activity relationships in the chloramphenicol
series.
4. Stability and Decomposition Products

4.1 Crystalline solid and solid dosage forms
Chloramphenicol in the solid state as a bulk drug
or present in solid dosage forms is a very stable anti-
biotic. Reasonable precautions taken to prevent excessive
exposure to light or moisture are adequate to prevent sig-
nificant decomposition over an extended period.
4.2 In Solution
The stability of chloramphenicol in aqueous solu-
tion is governed by the rate at which hydrolytic processes
occur. The two rimary routes of decomposition have been
determined15,59,%0,61to be (a) amide hydrolysis with the
formation of l-(p-nitrophenyl)-2-amino-1,3-propanediol
%o~N(() ~ - C - C H ~ O H +
H HN-C-CHCla
II NH, CHC l2COzH
0
and (b) hydrolysis of covalent chlorine of the dichloro-

acetamide moiety
H HN+-CHCl, OH + 2HC1
11
0
68
CHLORAMPHENICOL
The hydrolytic cleavage of t h e amide linkage5' i s the major

cause of chloramphenicol breakdown and is the only s i g n i f i -
cant r o u t e of degradation i n s o l u t i o n s below pH 7. The
r a t e of amide hydrolysis i s independent of pH over t h e pH
region 2 t o 6 and independent of the i o n i c s t r e n g t h of t h e
medium. Studies involving phosphate, acetate, and c i t r a t e
b u f f e r s i n d i c a t e t h a t the amide hydrolysis i s general acid-
base catalyzed6'.
The hydrolysis of covalent-bound c h l o r i n e is i n s i g n i f i c a n t

a t pH values below 6 but i n c r e a s e dramatically as pH in-
creases. This increase i s a t t r i b u t e d t o hydroxyl i o n
catalysis59.
Numerous secondary r e a c t i o n s can occur which g i v e rise t o a

v a r i e t y of decomposition products. Among these secondary
r e a c t i o n s are those associated with subsequent hydrolysis
of dichloroace t i c acid'' and oxidat ion-reduction r e a c t i o n s
which involve t h e n i t r o gsoup as oxidant and the s i d e chain
( p a r t i c u l a r l y t h e aminodiol s i d e chain of the primary hy-
d r o l y s i s product) as reductant. Products i s o l a t e d from
p a r t i a l l y o r completely decomposed chloramphenicol solu-
t i o n s exposed t o a v a r i e t y of conditions are given i n
Table 3.
The presence of borate b u f f e r s has been shown t o i n c r e a s e

the aqueous s t a b i l i t y of chloramphenicol. B r u n ~ e l l ~ ~
studied the s t a b i l i t y of 0.5% chloramphenicol ophthalmic
s o l u t i o n s i n pH 7.4 b o r a t e buffer and i n unbuffered aqueous
s o l u t i o n s . The r e s u l t s i n d i c a t e t h a t t h e a n t i b i o t i c i s
more s t a b l e i n the presence of h i s b u f f e r than i n i t s
absence. Heward and associates"studied the s t a b i t y of
t h e borate-buffered chloramphenicol eye drops BPCi!!j
r e s u l t s obtained are l i s t e d below.
. The
Temper a t u r e Rate Constants Calculated

OC k hrs-1 L l O L
115 0.2188 29 minutes
110 0.7413 x 10:' 85 minutes
30 0.1153 x 10 38 days
20 0.3589 x lo-' 4 months
4 0.4592 x lo-' 31 months
69
DALE SZULCZEWSKI A N D FRED ENG
Table 3
Decomposition Products of Chloramphenicol
-
No. Compound Environmental Conditions -
Ref.
1. Acidic o r b a s i c aqueous
solution
M12
2. C~,CHCO~H 11 17
3. o,N@HO Aqueous s o l u t i o n , ambient 62 9

temperature. 63 9
64
OH H
4 . H2N @--;-CH
I
HN-C
2
-CH3
OH Aqueous s o l u t i o n ,
ambient temperature . 62
II
0
65
5 R,, =Rz ‘CHz OH Aqueous a l k a l i n e s o l u - 65

t i o n , high temperature.
6. %=R2=C02H II
7. % = Rz = CHO 11
8. Rl = Rz = OH II
70
CHLORAMPHENICOL
Decomposition Products ofChloramphenico1 (Continued)
9. = C0,H; R2 = OH 65
10. 5 = C02H; Ra = CH,OH II
11
11. R,, = OH R2 = CH20H
12. €$ = OH R2 = CHO II
13. 02N@C0,H Aqueous solution after

exposure to light. 66
0
14 HOzC @“r;-@Co,H Aqueous solution after
exposure to light.
15. HC1 Aqueous solution; high

It
temperature.
71
James and Leach70,14suggested that complexation between the

antibiotic and borate ion is responsible for the increased
stability of chloramphenicol in this buffer system.
4.3 In the presence of microorganisms
Smith'l studied the decomposition of chlorampheni-
col in the presence of various microorganisms. He defined
the five routes by which chloramphenicol could undergo
degradation. These possibilities are summarized in scheme
1.
5. Metabolism and Pharmacokinetics

Biochemical changes which occur during metabolism of
chloramphenicol were determined by Glazko and associate^^^.
Isolation and identification of metabolites found in
various body fluids after administration of this anti-
biotic indicates that its metabolism can occur by routes
shown in scheme 2. Other pertinent information in this re-
gard is to be found in references 73-76.
The absorption characteristics of chloramphenicol from

oral dosage forms were determined by means of blood level
measurements and urinary excretion measurements of chloram-
phenicol and its rnetab~lites~~. Investigators concluded
that absorption occurs mainly by simple diffusion mechan-
isms in the intestinal tract with minimal absorption from
the ~ t ~ ~ n and a ~ that
h ~the
~ degree
s ~ ~of absorption was
influenced b pharmaceutical factors involved in capsule
formulation7is.
6.1 Colorimetry
6.11 Qualitative
e following color 88action is published in
the U . S . P . W', and the B.P. 1968 as part of an identi-
fication scheme.
Dissolve 10 mg. of chloramphenicol in a mix-

ture of 1 ml of diluted alcohol and 3 m l of dilute calcium
chloride T . S . (1 in 10). Add 50 mg of zinc dust, and heat
on a steam bath for 10 minutes. Decant the clear, superna-
tant liquid into a test tube, and add 100 mg of anhydrous
sodium acetate and 2 drops of benzoyl chloride. Shake the
12
/
I
r' I
Na?:-fHNH2 N H 2 ~ C O O HI
COMl
P-amlnobcnroic acid I
P-aminophenyl serine I
/
NH&-CMl&HCl2 If'
?
I
t\
\
NH~&-FH-NH,
iI
'
CW20H cn20n
I-p-aminophenyl-2-dichl~ro- I-p-aninophenyl-2-..ino- o-amino-8-hydroxy-p-
acetamido-1.3-prapanediol I.3-propanedIol
. .
aminonronioohenone
*-
ethanolamine, a m n i a
POLV MERS
( I ntermd iates)
/
Ilntermed i a t e r )
I
.'
(Carbon d i o x i d e . formaldehyde)
SCHEME I - H l C R O B l A L DECOMPOSITION PATHWAYS OF CHLORAHPHENICOL.

( C L U C U R O ~ IDATION)
1 I
I
(HYDROLYSIS)
SCHEME 2 - PATHWAYS I N THE METABOLIC DISPOSITION OF CHLDRAHPHENICOL

CH LOR AMPHEN ICOL
m i x t u r e f o r l m i n u t e , add 0.5 m l of f e r r i c c h l o r i d e T.S.

and, i f n e c e s s a r y , d i l u t e d h y d r o c h l o r i c a c i d t o produce a
c l e a r s o l u t i o n : a r e d v i o l e t t o p u r p l e c o l o r i s produced.
The formation of a yellow c o l o r by h e a t i n g t h e a n t i b i o t i c

w i t h c o n c e n t r a t e d sodium hydrox Qf;8gerved i n t h i s c a p a c i t y
i n t h e t h i r d supplement of DAB6

6.121 Reduction of N i t r o Group followed by
D i a z o t i z a t i o n and Couplinv
This approach t o a n a l y s i s of c h l o r -
amphenicol was among t h e f i r s t used € o r d e t e r m i n a t i o n of
t h e a n t i b i o t i c i n body f l u i d s 8 3 . 1 zko's o r i g i n a l pro-
cedure w a s modified s e v e r a l t i m e ~ ~ ~ ~ r~e g~a rw d it ot ht h e
reducing a g e n t used f o r r e d u c t i o n t o the corresponding a r y l -
m i n e . Since t h e n i t r o group, t h e r e a c t i v e moiety, e x i s t s
i n decomposition p r o d u c t s and m e t a b o l i t e s as w e l l as i n
chloramphenicol, t h i s procedure i s n o t s p e c i f i c when
d i r e c t l y a p p l i e d . S p e c i f i c i t y i s imparted by i n c l u d i n g a
s e p a r a t i o n s t e p p r i o r t o a n a l y s i s . The procedure i n v o l v e s
r e d u c i n g t h e n i t r o group t o an m i n e followed by d i a z o t i z a -
t i o n w i t h a c i d i c n i t r o u s a c i d and c o u p l i n g w i t h N-(1-
naphthy1)-ethylenediamine d i h y d r o c h l o r i d e .
6.122 Hydroxamic Acid Method

A h i g h e r degree of s p e c i f i c i t y i s
imparted t o c o l o r i m e t r i c a n a l y s i s by u s i n g t h e hydroxamic
a c i d r e a c t i o n . I n t h i s method chloramphenicol i s h e a t e d
w i t h hydroxylamine under b a s i c c o n d i t i o n s t o form a hydrox-
amic a c i d which $8 t h e n complexed w i t h f e r r i c i o n t o pro-
duce a r e d c o l o r . This approach h a s a l s o been used f o r
a n a l y s i s of chloramphenicol estersg7. It should be noted
t h a t t h e p r i n c i p a l h y d r o l y s i s product of chloramphenicol
does n o t y i e l d s i g n i f i c a n t c o l o r under t h e c o n d i t i o n s of
t h i s test.
6.123 I s o n i c o t i n i c Acid Hydrazide Procedure

This procedure, as developed by
Kakemi88, h a s been s t u d i e d i n some depthg9,90,91. The
b a s i s of t h e a s s a y i s t h e development of a yellow c o l o r
which r e s u l t s from mixing chloramphenicol w i t h i s o n i c o -
t i n i c a c i d h y d r a z i d e and sodium hydroxide i n aqueous solu-
t i o n . The procedure i s s i m p l e and can b e performed
75
r e l a t i v e l y rapidly. Other a n t i b i o t i c s and t h e s u c c i n a t e

and p a l m i t a t e esters of chloramphenicol have been found not
t o i n t e r f e r e with t h e assay procedure.
6.124 Miscellaneous
Other c o l o r i m e t r i c procedures have
evolved f o r a n a l y s i s of chloramphen 01which depend on
4s
r e a c t i o n of acetoneg2 o r l-naphthol with t h e a n t i b i o t i c
under b a s i c conditions. The chemistry of these methods i s
not defined and, although convenient, they o f f e r l i t t l e
advantage over those previously discussed.
6.2 Polarographic Analysis

The presence of t h e nitrophenyl group makes i t
possible t o u t i l i z e d i f f e r e n t instrumental techniques f o r
d e t e c t i o n and a n a l y s i s of t h i s a n t i b i o t i c .
Methods which depend on t h e p-nitrophenyl group

a r e not s e l e c t i v e unless preceded by a s e p a r a t i o n s t e p o r
accompanied by independent a n a l y s i s t o give assurance t h a t
the sample being analyzed contains only chloramphenicol and
i s not a mixture of the a n t i b i o t i c and degradation o r meta-
b o l i c products.
Among t h e more convenient instrumental approaches

t o a n a l y s i s of t h i s a n t i b i o t i c i s polarography. It i s
less s u s c e p t i b l e t o i n t e r f e r e n c e from o t h e r materials than
i s , f o r example, u l t r a v i o l e t spectroscopy, b u t , as dis-
cussed previously, could not be considered a s p e c i f i c pro-
cedure without modification.
A d e t a i l e d study of t h e polaro raphic behavior of

chloramphenicol w a s reported by Fossdal 84 . The a n t i b i o t i c
undergoes a 4-electron reduction a t t h e dropping mercury
e l e c t r o d e producing a well-defined diffusion-controlled
polarographic wave of a n a l y t i c a l u t i l i t y . Results ob-
tained indicated t h a t chloramphenicol could be determined
over t h e range 0.3 t o 60 mcg/ml. Previous s t u d i e ~ ~ ~ - ~ ~
reported a p p l i c a t i o n of polarography t o a n a l y s i s of chlor-
amphenicol i n pharmaceutical preparations.
The n a t u r e of polarography i m p a r t s a degree of

s e l e c t i v i t y t o the assay of chloramphenicol. 2- (2,2-
dichloracetamido)-3-hydroxy-4'-nitropropiophenone, a pos-
s i b l e t o x i c contaminant i n s y n t h e t i c chloramphenicol w a s
76
CHLORAMPHENICOL
determined by direct polarographic measurementloo. In this

case the reduction potential of the impurity is sufficient-
ly different from that of the drug's to permit direct in-
strumental analysis
6.3 Spectrophotometric
Because both spectrophotometric and polarographic
methods depend on the existence of the p-nitrophenyl group,
they are both subject to the same specificity considera-
tions. Quantitative determination via direct ultraviolet
measurement is not a specific analytical method since
decomposition products absorb over the same region
i'e''2. As previously mentioned, an ultraviolet procedure
is official as a method to determine the potency of chlor-
amphenicol'.
Ultraviolet spectroscopy has been extensively
applied to chloramphenicol determination in methods in-
volving separation prior to quantitation43~44~47,63. It
has also been employed to determine the antibiotic in
pharmaceutical preparationsa0.
6.4 Titrimetric Methods

Titrimetric methods have been developed for analy-
sis of chloramphenicol. Such procedures are dependent on
only a limited portion of the molecule, i.e., the nitro
group in the p-nitro-benzene portion or the covalent
chloride contained in the dichloracetamido moiety and hence
would not be selective unless metabolic or degradative
processes yielded products not containing these functional
groups.
Procedures utilizing covalent chloride involve

converting the covalently-bound chloride to its ionic form.
The ionic chloride is then determined by argentometric ti-
tration103,104.
Titrimetry of chloramphenicol using the nitro

group has two variations both of which require reduction to
an arylamine. Reduction of the nitro group with excess ti-
tanium chloride followed by determination of the excess
reagent by back titration with ferric ammonium sulfate con-
stitutes the titanometric methodlo5.
77
The bromatometric methodlo5 consists of a Zn-HC1

reduction to form the arylamine. The arylamine is then de-
termined by bromination in the presence of excess bromine
followed by iodometric determination of the excess bromine.
6.5 Microbiological
Microbiological procedures have been developed for
assay of chloramphenicol and appl to analysis of dosage
forms, body fluids, and bulk drugf". Although time con-
suming, these methods are as accurate as physicochemical
tests, provide analytical sensitivity equal to or surpass-
ing many, and have the advantage of being directly related
to use.
Since chloramphenicol's decomposition products or

metabolites do not possess significant antibiotic activity,
only intact chloramphenicol is measured providing that no
other antibiotics or chemotherapeutic agents (i.e., a fixed
combination dosage form, supplemental therapy) exist.
Although the basic microbiological assay procedures may be
subject to this kind of interference, the problem may be
obviated by selective inactivation of the interfering
antibiotics by using a microorganism sensitive for chlor-
amphenicol but resistent to the interfering antibiotic, by
including a separation scheme as part of the assay sequence,
or by compensating for the presence of the interfering anti-
biotic by adding it to each solution of chloramphenicol
used for the s tandard response curve1°7.
In the case of chloramphenicol, two basic micro-

biological methods are in general use viz Cylinder Plate
and Turbidimetric.
The Cylinder Plate Method for assay of chloram-
phenicol is an agar diffusion procedure using Sarcina lutea
ATOC 9341 as the test organism. The response of the assay
is produced by solutions of chloramphenicol in 1% phosphate
buffer pH 6 diffusing through an agar layer uniformly
inoculated with the test organism. To accomplish this re-
sponse, stainless steel cylinders ( 8 mm o.d., 6 mm i.d.,lO
mm long) are placed on the seeded agar surface and filled
with the chloramphenicol solutions, and then inclubated
overnight at 32OC. The responses that are produced are
clear circular zones of inhibited growth around the
78
CHLORAMPHENICOL
cylinder on the agar surface otherwise totally covered with

heavy growth. The dose-response relationship is a linear
one in restricted limits of concentration when the dose is
expressed logrithmically and the response arithmetically.
In order to determine the concentration of an unknown, a
reference standard must be used for comparison on each
petri dishlo*.
Two assay designs are commonly employed using the cylinder

plate technique: the single dilution-standard curve de-
sign and a three by three (or 2 x 2) factorial designlog.
The first assay design is the official method of the F.D.A.
'lo. The second has the advantage of being able to compare
the parallelism of the dose response line of the standard
and the unknown.
The turbidimetric method determines the concentration of

chloramphenicol by measuring the turbidity that is produced
by the actively growing test organism in a series of test
tubes containing chloramphenicol and inoculated liquid cul-
ture medium. The test is incubated in a 37OC water bath
for 2 to 5 hours. After the desired incubation, the growth
is stopped by the addition of formaldehyde or other appro-
priate means, and the responses are read in terms of
absorbance on a suitable photoelectric colorimeter or
spectrophotometer. By comparing the turbidity of the un-
known to that of the refer nce standard, the potency of
chloramphenicol is found1lP.
Like the cylinder plate assay, the assay design may vary.
The single dilution-standard curve design and 3 x 3 (or
2 x 2) factorial assay are commonly usedlog.
Several different organisms have been used for the turbidi-

metric assay of chloramphenicol. Escherichia & ATCC
10536 is the organism used in the official method of the
F.D.A. The dose-response line, log of concentration vs.
response, produced by the organism is ear with a limited
range of chloramphenicol concentration'". Shigella sonnei
ATCC 11060 has been used for the turbidimetric assay of
chloramphenicol. Because it is sensitive to lower concen-
trations of chloramphenicol than the plate assay, it is
useful in determining chloramphenicol levels in blood serum
and other clinical specimens. The dose-response line
79
obtained with t h i s organism i s n o t l i n e a r over the wide

range of concentration f o r which i t can be used. @-
bacterium tumefaciens (Parke Davis c u l t u r e No. 05057) has
a l s o been used i n place of 2. sonnei when a nonpathogenic
organism is necessary. However, i t is not as sensitive as
.
S -sonnei t o low levels of chloramphenicollo8.
-
I n general t u r b i d i m e t r i c techniques are f a s t e r and more
e a s i l y adapted t o automated techniques.
6.6 Chromatographic
6.61 Paper
The chromatographic behavior of chloramphe-
n i c o l and r e l a t e d compounds l i k e l y t o be encountered i n
metabolic s t u d i e s o r involved i n enzymatic and chemical
degradation work w a s e s t a b l i s h e d by Smith112. Whatman No.
.1 p a p e r was used together with a mobile phase c o n s i s t i n g of
water s a t u r a t e d n-butanol containing 2.5% a c e t i c a c i d .
Several reagents were used t o d e t e c t v a r i o u s compounds
a f t e r chromatography. These included p-dimethylamino-
benzaldehyde (arylamines), reduction with stannous c h l o r i d e ,
followed by p-dimethylaminobenzaldehyde (aromatic n i t r o
compounds), Ninhydrin ( a l i p h a t i c amino compounds, ammoni-
a c a l s i l v e r n i t r a t e [Formyl or Carbonyl groups]). Table 4
l i s t s Rf values and the response t o v a r i o u s d e t e c t i o n
reagents.
6.62 Thin Layer

Several t h i n l a y e r chromatographic systems
have been developed t o study t h e v a r i o u s a s p e c t s of chlor-
amphenicol chemistry. The procedures described h e r e have
been used f o r t h e 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 of chlor-
amphenicol d e r i v a t i v e s , decomposition products, and syn-
t h e t i c intermediates.
Lin113 achieved s e p a r a t i o n of chloramphenicol , chloram-

phenicol palmitate, and chloramphenicol s u c c i n a t e i n two
solvent systems using polyamide t h i n l a y e r p l a t e s . The
4 values reported are:
Solvent A Solvent B
Chloramphenicol 0.35 0.80
Chloramphenicol Palmitate 0.95 0.90
Chloramphenicol Succinate 0.25 0.72
80
IS31 HOeN 1 1 1 + 1 I I 1 I l + + l I 1
IS32 ‘WN‘ON6V l l l + l 1 1 1 + + + + 1 I +
IS31 3 N l O l Z N 3 B I I I I I 1 1 1 1 1 1 + 1 I +
IS31 N I W A H N I N + + + I + I I I I I I I I I I
IS31 3NIW 1AUV + + I l l + + I + I I I I I I
IS31 O U l N + + I + + + + + + I + + + + +
U
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81
DALE SZULCZEWSKI A N D F R E D ENG
Solvent A - n-Butanol-CHC13-acetic a c i d (10:90:0.5)

Solvent B - n-Butanol-water-acetic acid (82:18:0.5)
Schlederer114 accomplished a comparable s e p a r a t i o n using

s i l i c a g e l G p l a t e s and CHC13:MeOH ( 9 : l ) as developer.
The separation and i d e n t i f i c a t i o n of decomposition products

of chloramphenicol by t h i n l a y e r has been done mostly on
s i l i c a g e l p l a t e s using a v a r i e t y of s o l v e n t systems.
T a ~ h a r m eused~ ~ s i l i c a g e l G p l a t e s and two-dimensional
chromatography t o achieve separation. The Rf values he
obtained are l i s t e d as follows:
1st Solvent 2nd Solvent

p-nitrobenzaldehyde 0.98 ( s t r e a k ) 0.91
p-nitrobenzoic acid 0.10-0.15 0.80-0.81
Chloramphenicol 0.72 0.89-0.91
l-(~-nitrophenyl)-2-
amino-ly3-propanediol 0.08-0.10 0.35
1st Solvent - e t h y l acetate ( s a t u r a t e d with water)

2nd Solvent - n-Butanol ( s a t u r a t e d with 2.5% a c e t i c acid)
Shih63 66 used f i v e binary developers with s i l i c a g e l

p l a t e s t o d e t e c t and i d e n t i f y s e v e r a l secondary decomposi-
t i o n products as w e l l as t h e azoxy compound formed from
photolysis (see Table 3 ) .
Aromatic decomposition products (see stability-decomposi-

t i o n ) a r i s i n g from chloramphenicol a f t e r heating i n alka-
l i n e s o l u t i o n were detected by Knabe65 using s i l i c a g e l G
p l a t e s . The r e s u l t s he obtained are a s follows:
82
CHLORAMPHENICOL
Compound Rf Developer
4,4'-azodiphenol 0.25 Methylene Chloride-Ether g:1
e-n t rophenol 0.55 II
4,4 -azoxyd i pheno I 0.21 II
e-n trosophenol 0.30 II
e-[ ( hydroxypheny1)-
f-
azo benzyl alcohol 0.15 II
e-[( hydroxypheny1)-
azof-
benza 1 dehyde 0.45 II
4,41-azodibenzaldehyde 0.92 Chloroform-Ether 9:l
e- [ (a-hyd roxy-e-to1 y 1 ) -
azo] benza 1 dehyde 0.43 II
4,4'-azodibenzylalcohol 0.03 II
Thin layer chromatography has been used as part of a quan-

titative scheme of analysis. SchwarmlOl used silica gel
HF254 thin layer plates to separate chloramphenicol from
decomposition products. Once separation was accomplished,
the intact antibiotic was desorbed and photometrically de-
termined. CHC13-Isopropanol 4:l was used as developer.
The same approach was used by Kassem115 for analysis of

intact drug. In this case, silica gel plates were used
with CHC13-MeOH, 85:15, as mobile phase.
Libsovar116 developed thin layer chromatographic systems

€or uae in monitoring the classical synthesis of chloram-
phenicol. For this purpose, Aluinina plates were used with
-
binary developers of benzene-ethanol (2.5 20% ethanol).
6.63Partition (Column)
Intact chloramphenicol can be determined in
the presence of decomposition products by using a partition
c01umn''~. The column is prepared with a silicic acid-
83
DALE SZULCZEWSKI AND F R E D ENG
water as internal phase. Chloroform followed by 10% ethyl-

acetate in chloroform serve as eluents, the eluent being
monitored spectrophotometrically at 278 nm.
This analytical procedure was validated by direct compari-

son with microbiological assay before being used to study
the kinetics of degradation of chloramphenicol.
6.64 Gas Li uid

d v e l o p e d a gas chromatographic
procedure for analysis of chloramphenicol in the presence
of structurally related compounds likely to be present in
the growth medium or cell free extract of cultures of 2.
venezuelae. The procedure involved chromatography of
these compounds after conversion to their corresponding
trimethylsilyl derivatives. The column temperature was
programmed over the region 11O-26O0C and hydrogen flame
ionization was used for detection. A chromatogram of a
synthetic mixture of these compounds is included as Fig. 9.
Resnick adapted Shaw's procedure to analysis of chloram-

phenicol in serumg0. In brief, this procedure involves
extraction of chloramphenicol from serum with amyl acetate,
derivitization using Tri-Si1 reagent followed by chroma-
tography using Qf-1 on silanized Gas Chrom P. The sensi-
tivity of the method was adequate for determination of
chloramphenicol in the region of 0.6 mcg/ml. Y a m a m ~ t o ~ ~ ~
reported that chloramphenicol can be determined in body
fluids and pharmaceutical preparations by gas chromato-
graphy without prior derivatization. Two per cent diethyl-
eneglycol succinate on PVP modified Anakrom was used with
a column temperature of 195OC.
Davies"' developed a gas chromatographic method for the

determination of small amounts of the ortho and meta nitro
isomers of chloramphenicol as possible impurities in
chloramphenicol.
84
DERIVATIVES OF 2 - 1 -
PHENYL-2-AUlNO-I.3-
PROPANEDIOL
4,IO
Com-
6 pound R R‘
3,
I H H
1.
2 NH; H
3 NO 2 n
m 4 NO 2 CH ,co
2
v, 5 NO 2 OUCCO
6 NO2 CH2FC0
7 NO : (cr,)L:nco
8 NO CH;CI LO
9 NO2 CHF,CO
10 CH,O CHCllCO
I1 NOi. CF,CO
12 NO cnci2co
13 NO2 CH,BrCO
14 CH,S@, cncl,co
15 NO: C C I ,co
16 NO, C H B r C l CO
17 NO CnBr,CO
18 NO CBr,CO
10 15 20 25
TIWE IN UINUTES
118
FIG. 9 - GAS CHROHAT3GRAM OF C H L O R A n P H E N l C O L AND STRUCTURALLY R E L A T E D COYFOUNDS.
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CHLORAMPHENICOL
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91. R. C. Shah, P. V. Raman, and P. V. Sheth, I n d i a n J.
Pharm., 30, 68 (1968).
92. F. M. Freeman, Analyst, 80, 299 (1956).
93. D. Masterson, J. Pharm. S c i . , 57, 306 (1968).
94. K. Fossdal and E. Jacobson, Anal. Chim. Acta., 56, 105
(1971)
88
CHLORAMPHENICOL
95. G. B. Hess, Anal. Chem., 22, 649 (1950).

96. C. G. Macros, Chem. Chron. A , , 32 104 (1967).
97. C. Russo, I. Cruceanu, D. Monciu, and V. Barcaru,
11. Farmaco, 20, 22 (1965).
98. C. Russu, I. Cruceanu, and V. Barcaru, Pharmazie 2,
799 (1963).
99. A. F. Summa, J. Pharm. S c i . , 54, 442 (1963).
.
100. P Zuman, "Organic Polarographic Analysis", MacMillan
Co., New York, 1964, p. 186.
101. E. Schwarm, C. Dabner, J. Wilson, and M. Boghosian,
J. Pharm. S c i . 55, 744 (1966).
102. T. Higuchi, A. D. Marcus, and C. D. Bias, J. Amer.
Pharm. A s s . , S c i . Ed., 43, 135 (1959).
103. W. Awe and H. Stohlman, Arch. Pharm., 289, 61, 276
(1956).
104. M. Hadicke and G. Schmid, Pharm. Z e n t r a l h . , 95, 387
(1956).
105. W. Awe and H. Stohlman, Arzeim. Forsch., 7 (81, 495
(1959).
106. D. C. Grove and W. A. Randall, "Assay Methods of Anti-
b i o t i c s : A Laboratory Manual", 238 pp Medical
Encyclopedia, I n c . , New York, 1955
107. B. Arret, M. R. Woodard, D. M. Wintermere, and A.
Kirschbaum, A n t i b i o t . Chemother., 1 545 (1957).
108. R. Hans, M. G a l b r a i t h , and W. C. Alegnani, "Analy-
t i c a l Microbiology," F. Kavanagh, Ed., Academic P r e s s ,
1963, p 271-281.
109. United S t a t e s Pharmacopeia XVIII, 1970, pp 857-864,
Antibiotics-Microbial Assays.
110. F.D.A. Regulations T i t l e 2 1 Sec. 141.110.
111. F.D.A. Regulations T i t l e 2 1 Sec. 141.111.
112. G. N. Smith and C. S. Worrel, Arch. Biochem., 28, 1
(1950) .
113. Y. T. Lin and K. T. Wang, J. Chromatogr., 21, 158
(1966).
114. E. S c h l e d e r e r , Cosm. Pharma., 2, 1 7 (1966).
115. M. A. Kassem and A. A. Kassem, Pharm. Ztg., 48, 1972
(1966).
116. J. Lebsovar, Cesk. Farm. 11,73 (1962).
117. T. Higuchi, C. Bias, and A. Marcus, J. Amer. Pharm.
Ass. , S c i . Ed., 43, 135 (1954).
118. P. D. Shaw, Anal. Chem., 3 5 , 1580 (1963).
119. M. Yamamoto, S. I g u c h i , and T. Aoyama, Chem. Pharm.
Bull. , 15, 123 (1967).
89
120. V. Davies, Parke Davis, personal communication.
ACKNOWLEDGMENT
The authors express appreciation to
Mrs. Pat Greenwood of the Microbiology Department
at Parke, Davis & Company for assistance in pre-
paring a portion of this profile.
90
CLORAZEPATE DIPOTASSIUM
James A . Railile and Victor E. Papendick

JAMES A. RAIHLE AND VICTOR E. PAPENDICK
Contents
Analytical Profile - Clorazepate Dipotassium

1. Description

2.1 Infrared Spectrum

2.3 Ultraviolet Spectrum
2.4 Mass Spectrum
2.5 Raman Spectrum
2.7 Melting Range
2.8 Differential Thermal Analysis
2.9 Solubility
2.11 Dissociation Constant
2.12 Fluorescence
2.13 Hygroscopic Behavior
3. Synthesis
4. Stability - Degradation
5. Drug Metabolic Products and Pharmacokinetics

6.2 Phase Solubility Analysis
6.3 Chromatographic Analysis
6.31 Thin Layer Chromatographic Analysis
6.32 Gas Liquid Chromatography
6.4 Direct Spectrophotometric Analysis
6.5 Colorimetric Analysis
6.6 Non-Aqueous Titration
7, References
92
CH LORAZEPATE DI POTASSI UM
1. Description

Clorazepate dipotassium is 7-chloro-1,3-dihydro-
2-oxo-5-phenyl-1H-l,4-benzodiazepine-3-carboxylic acid,
monopotassium salt, monopotassium hydroxide.
0
H 11 0
\ II
CHCOK. KOH
CI C=N
Clorazepate Dipotassium
C16H1 1°4N2C1K2 Molecular Weight 408.93

Off white to pale yellow, fine crystalline powder
which is practically odorless.

The infrared spectrum of clorazepate dipotassium
is presented in Figure i. The spectrum was measured in the
solid state as a mull in mineral oil. The following bands
(cm-1) have been assigned for Figure 1. (1)
a. 3530 cm-l characteristic for hydroxyl
b. 1610 cm-1 characteristic skeletal stretching modes of
the aromatic ring
c. 1560 cm-1 characteristic C=O stretching mode of the
carboxyl salt
2.2 Nuclear Magnetic Resonance Spectrum (NMEl)
The NMR spectrum shown in Figure 2 was obtained
by dissolving 50 mg of clorazepate dipotassium in 0.5 ml of
D20 containing tetramethylsilane as an internal reference.
Only the aromatic protons between 7.0 and 7.6 ppm are visi-
ble. (2)
93
4c
P
Figure 2 NUCLEAR MAGNETIC RESONANCE SPECTRUM OF CLORAZEPATE DIPOTASSIUM
I I I I . . . . I . . . . I I
I . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. _. ,. .. . .
8.0 7 .O 6.0 5.0 CCM( d ) 4.0 3.0 2.0 I .o
C l o r a z e p a t e d i p o t a s s i u m when scanned between 400
and 200 nm i n 0.03% aqueous potassium c a r b o n a t e e x h i b i t s a
maximum a t 230 nm a s shown i n F i g u r e 3 , (c = 35,000) char-
a c t e r i s t i c of benzodiazepines.
2.4 Mass Spectrum

The mass spectrum shown i n F i g u r e 4 was o b t a i n e d
u s i n g an Associated E l e c t r i c a l I n d u s t r i e s Model MS-902 Mass
Spectrometer w i t h a n i o n i z i n g energy o f 50 eV and a temper-
a t u r e of 185°C. C l o r a z e p a t e d i p o t a s s i u m y i e l d s a spectrum
w i t h t h e base peak a t m / e 270 a t t r i b u t e d t o t h e decarboxy-
l a t i o n of t h e a c i d s a l t . Subsequent fragments, Table I and
F i g u r e 5, r e f l e c t t h e l o s s of p a r t s of t h e seven membered
ring o r chlorine.(3) The mass spectrum p a r a l l e l s t h a t r e -
p o r t e d f o r diazepam. (4)
2.5 Raman Spectrum

The Raman spectrum of c l o r a z e p a t e d i p o t a s s i u m , a s
shown i n F i g u r e 6 , was o b t a i n e d i n t h e s o l i d s t a t e on a
Ramalog Spectrophotometer a t Spex I n d u s t r i e s . The follow-
i n g bands (cm-l) have been a s s i g n e d f o r F i g u r e 6 . ( 1 )
a. 1595 cm-l s k e l e t a l s t r e t c h i n g mode of a r o m a t i c r i n g
b. 1565 cm-l C=N s t r e t c h i n g v i b r a t i o n o f t h e h e t e r o -
cyclic ring
c. 1495 cm'l s k e l e t a l s t r e t c h i n g mode o f a r o m a t i c r i n g
2.6 Optical Activity

C l o r a z e p a t e d i p o t a s s i u m e x h i b i t s no o p t i c a l ac-
tivity.
2.7 Melting Range

C l o r a z e p a t e d i p o t a s s i u m does n o t have a d e f i n i t e
m e l t i n g range. Typical b e h a v i o r when t h e m a t e r i a l i s slow-
l y h e a t e d i n a g l a s s c a p i l l a r y t u b e may be d e s c r i b e d i n t h e
following manner: d i s c o l o r a t i o n b e g i n s a t about 215"C,
s h r i n k i n g i s observed t o b e g i n between 225OC t o 235°C w i t h
t o t a l decomposition o c c u r i n g between 235°C and 295°C.
2.8 D i f f e r e n t i a l Thermal A n a l y s i s (DTA)

The DTA curve o b t a i n e d on a DuPont Model 900 Ana-
l y z e r a s shown i n F i g u r e 7 confirms t h e observed m e l t i n g
c h a r a c t e r i s t i c s d e s c r i b e d i n s e c t i o n 2.7.
96
CH LORAZEPATE D IPOTASS IUM
FIGURE 3.
ULTRAVIOLET SPECTRUM O F
CLO R A 2 EPATE DIPOTASSI UM
I I
1 250 300 350

WAVELENGTH (nm)
97
Figure 4 MASS SPECTRUM OF CLORAZEPATE DIPOTASSIUM
W
00
20 40 60 80 100 120 140 160 180 ZOO 220 240 260 280
vl
m d r l r l r l o r l o o o 0 0
d
UI
rl rl 0 rl 0 0 0 0 0 N
N d N N rl rl I4 d 0 0
0 m 0 rl ul m co I- m 0
rl rl rl
rn
rl
* 2
d
rn
rl
0
d
0
rl
m
I4
m
rl
\o rl
rl N rl m m 00 rl 03
m
*co0 I-
m
0
I-
03
0
*0
N
h
0
h
m
0
a\
0
0
ul
co
m
m
\D
N 4 *
N
2
4
N
ul
m
N
2
N
m
h
rl
I-
I-
rl
I-
I- *
m
u
l N
ul
I- I- *
m
*0
I-
m
0
rl
ul
0
N
U
0
h
0
co m
* 3
N
N
rl
2
hl
ul
I-
d
h
rl
h
I-
rl
I-
h *
99
CH LORAZEPATE DlPOTASSlUM
FIGURE 5.
-
FRAGMENTATION PATHWAYS OF CLORAZEPATE DIPOTASSII M
0
CI Probably on the probe
McLafferty
Rearrangement
Before or After
m/e 314 Ion Formation
not observed
..
co,
m/e 44
Bare Peak
/-". m h 270
lC1 *
m/e 235
m/e 242
rFk /-co
m/e 214 \C-
m/e 179''
100
L
-3 OOL
m
OW
-3 m
009
OOL
008
006
0001
001I
OOL I
OOEl
oopl
0091
OOLL
101
FIGURE 7.
DIFFERENTIAL THERMAL ANALYSIS CURVE

OF CLORAZEPATE DIPOTASSIUM
c
0
h)
O
m
0
25 100 175 250 325 400

TEMPERATURE; DEGREES CENTIGRADE
CHLORAZEPATE DIPOTASSIUM
2.9 Solubility
Approximate s o l u b i l i t y d a t a o b t a i n e d a t room t e m -
p e r a t u r e a r e given i n t h e f o l l o w i n g t a b l e :
Solvent S o l u b i l i t y (mg/ml)
Water > 100 < 200

Absolute Ethanol 0.6
Chloroform < 0.5
Ether < 0.5
Acetone c 0.5
Benzene < 0.5
Isopropanol 0.7
Methylene D i c h l o r i d e < 0.1
The X-ray powder d i f f r a c t i o n p a t t e r n o f c l o r a z e -
p a t e d i p o t a s s i u m was determined by v i s u a l o b s e r v a t i o n o f a
f i l m o b t a i n e d w i t h a 143.2 mm Debye-Scherrer Powder Camera.
An Enraf-Nonius D i f f r a c t i s 601 G e n e r a t o r ; 38 KV and 18 MA
w i t h n i c k e l f i l t e r e d copper r a d i a t i o n ; A = 1.5418, were
employed. (5)
2.11 D i s s o c i a t i o n Constant
Attempts t o measure t h e pKa of t h e carboxyl group
by t i t r a t i o n i n water w i t h h y d r o c h l o r i c a c i d were unsuc-
c e s s f u l . Only t h e potassium hydroxide which i s l i b e r a t e d
on d i s s o l v i n g c l o r a z e p a t e d i p o t a s s i u m i n w a t e r i s t i t r a t e d .
(6)
2.12 Fluorescence
C l o r a z e p a t e d i p o t a s s i u m does n o t e x h i b i t f l u o r e s -
c e n t p r o p e r t i e s i n an aqueous s o l u t i o n , however, i t does
e x h i b i t f l u o r e s c e n c e a t 508 nm when e x c i t e d a t 388 nm i n 9
-N a l c o h o l i c s u l f u r i c a c i d . ( 7 )
2.13Hygroscopic Behavior
Clorazepate d i p o t a s s i u m was n o t hygroscopic when
exposed t o a r e l a t i v e humidity of 30%-40% f o r 4% months.
3. Synthesis
Clorazepate d i p o t a s s i u m may be prepared by t h e r e a c t i o n
scheme shown i n F i g u r e 8 w i t h t h e r e a c t i o n of (2-amino-5-
chloropheny1)phenyl methane imine and d i e t h y l 2-amino mal-
o n a t e t o form e t h y l 7-chloro-1,3-dihydro-2-oxo-5-phenyl-lH-
103
TABLE I1
X-Ray Powder Diffraction Pattern
d-Spacings and Intensities
dA -111
1 -III
1
17.5 100 2.99 5

8.6 10 2.93 1
7.7 25 2.88 2
7.0 10 2.82 10
6.18 20 2.78 3
5.7 20 2.67 5
5.05 30 2.62 5
4.82 75 2.57 1
4.35 10 2.46 10
4.20 5 2.42 2
4.11 1 2.27 1
4.05 1 2.17 5
3.95 5 2.13 2
3.80 5 2.07 1
3.66 25 2.03 1
3.52 40 1.99 1
3.48 1 1.97 1
3.40 10 1.93 1
3.30 60 1.87 1
3.25 65 1.81 1
3.14 1 1.78 1
3.06 15 1.71 1
104
CH LORAZEPATE DlPOTASSl UM
FIGURE 8.
SYNTHESIS OF CLORAZEPATE DIPOTASSIUM
//O
C-OCzH,
I
H,N-CH
I
CI C=NH
(2-Amino-5-chlorophenyl) Diethyl-2-aminomalonate
phenyl methane imine
Ethyl 7-cidoro-l,3-dihydro-2-oxo- Potassium

5-phenyl-1H-1,4-benzodiazpine-3- Hydroxide
car boxylate
/p
\
CHC02K * KOH
CI
105
JAMES A. RAIHLE AND VICTOR E . PAPENDICK
lY4-benzodiazepine-2-carboxylate. This intermediate is

then converted to the drug substance with alcoholic potas-
sium hydroxide. (8)
4. Stability -Degradation
The hydrolysis products for clorazepate dipotassium are
shown in Figure 9. The final hydrolysis product is the
same as that reported for the acid hydrolysis of clordiaz-
epoxide and the major metabolite of diazepam.(4) The kinet-
ics of the decomposition of clorazepate dipotassium in buf-
fered aqueous solution at different temperatures was stud-
ied over the pH range of 2-11. (9) The extent of degrada-
tion was determined by a dichloromethane extraction and
spectrophotometric measurement. Ring opening was negligi-
ble under the conditions of pH and temperature used.
The degradation to N-desmethyl diazepam is first order

with respect to clorazepate dipotassium. The relationship
log, (Ct/Co) = Kt was verified where Ct is the concentra-
tion of clorazepate dipotassium at time t, Co is the con-
centration of clorazepate dipotassium at time 0, and k is
the reaction rate constant. Plots of loge (Ct/Co) as a
function of time t were linear. The slopes of these plots
give the reaction rate constants k. Some rate constants,
sec-1, at different pH and temperature values are shown in
Table 111.
The data shows that in the degradation of clorazepate

dipotassium the reaction rate constant, k, increases with
temperature. The activation energy of the degradation re-
action, Ea (obtained from the slope of the plot of loge k
as a function of 1/T where T is the absolute temperature),
is about 20.3 kilocalories/mole and is independent of pH.

The major metabolites of clorazepate dipotassium are
shown in Figure 10. The drug substance is decarboxylated
in acidic media to N-desmethyl diazepam, which can undergo
hydroxylation to oxazepam and conjugation in the urine to
the glucuronide. (10) Some clorazepate dipotassium (2-6%)
is excreted into the urine unmetabolized.(ll) Acid hydro1
ysis of the drug substance and its metabolites affords 2-
amino-5-chlorobenzophenone. Analytical procedures have
been published for the metabolites using gas chromatogra-
phy (12), thin layer chromatography ( 7 ) , high pressure
106
TABLE I11
Rate Constant vs pH f o r C l o r a z e p a t e Dipotassium
t oc+ -
2 2" 27.5" -
37"
PH
f
2.0 3. 60X10'3 6. 46X10-3 1. 93X10'2
3.0 3.40~10-~ 5.96~10-~ 1.42xlO-2
4.0 1. 16X10-3 1.98~10'~ 6.33~10'~
5.0 2.17~10-4 4.10~10-4 1.14~10-3
6.0 2.23xlO-5 --- 1. 18X10'4
6.5 3.9ox10-6 --- 8. 33X10'5
c
0 7.0 2. 33X10-6 --- 2.14xlO-5
4
7.5 1. 05X10'6 --- 1.32~10'5
8.0 --- - -- 1. 27X10'5
8.5 1. 05X10'6 --- 5. 61X10'6
9.5 9.10~10-7 --- 4. 83X10'6
11.0 8.64XlO-7 --- 4. 55X10m6
FIGURE 9.
DEGRADATION PATHWAYS OF
CLO RAZEPAT E D IPOTASSI UM
0
II
NH-C,
CHZ + KzCO,
CI
N-Desmethyl Diazepam
CI a:b II -
+
0
II
H2NCHZCOH
0
2-Amino-5-chlorobentophenone Glycine
108
FIGURE 10
METABOLIC PATHWAYS 0 F CLOR AZEPATE D IPOTASSI UM
-
0
- aNH'
H II
Strong
CHCOK Acid-
KOH
CI Hydrolysis H,NCHZCOOH
C= N CI
c=o
0
I
Clorazepate Dipotassium 2-Amino-5-chlorobenzophenone + Glycine

c
0
\D
0
0
H II \
CHOH
Hydroxylation CI C=N
CI C=N
N-Desmethyl Diazepam Oxazepam

liquid chromatography (13) and colorimetry.(l4)
Typical Result
Reported for
E1ement % Theory Lot 849-648
Carbon 46.99 46.82

Hydrogen 2.71 2.65
NF trogen 6.85 6.95
Chlorine 8.67 8.85
Potas sium 19.12 19.48, 19.34*
* Values determined by atomic absorption analysis

Data is not available due to the instability of
clorazepate dipotassium in the solvent systems screened.
6.31 Thin Layer Chromatography

Clorazepate dipotassium is partially de-
graded in numerous systems to N-desmethyl diazepam, there-
fore, thin layer chromatography is not considered a relia-
ble indicator of purity. The drug substance is readily
identified by ultraviolet light on Silica Gel GF254 in the
system methano1:acetone (1:l). Clorazepate dipotassium has
an Rf value of 0.15 while N-desmethyl diazepam has an Rf
value of 0.90.
LaFargue, et a1 (7) differentiated cloraze-

pate dipotassium from other 1,4-benzodiazepines in urine
and gastric fluid by hydrolysis to 2-amino-5-chlorobenzo-
phenone and subsequent thin layer chromatography on alumi-
num oxide in the system benzene:chloroform (3:l). The Rf
value of the hydrolysis product is 0.55. If benzophenone
is used as a reference standard, the relative Rf value of
2-amino-5-chlorobenzophenone is 0.79.
110
CH LORAZEPATE DiPOTASSl UM
Clorazepate may be directly extracted from

gastric fluid as N-desmethyl diazepam and identified on
aluminum oxide in the system ch1oroform:ethanol (29:l).
The absolute Rf is reported as 0.39 or 0.48 relative to a
diazepam reference standard.(7)
6.32 Gas Chromatography

Clorazepate dipotassium can not be directly
chromatographed, however, gas chromatography is readily ap-
plied to the metabolites and acid hydrolysis products.(7,
12) LaFargue (15) has employed 3% OV-17 (methylphenylsili-
cone) on Gas Chrom Q to separate 2-amino-5-chlorobenzophe-
none from the acid hydrolysis products of five major benzo-
diazepins.

Direct spectrophotometric analysis of clorazepate
dipotassium is applicable provided significant quantities
of interfering contaminants are not present. The drug sub-
stance may be examined directly in an aqueous carbonate
buffered media at 230 nm ( 6 = 35,000) or indirectly in al-
coholic sulfuric acid by ultraviolet absorption (16) at
388 nm or fluorescence (7) with an excitation maximum of
388 nm and an emission maximum at 508 nm.
The degradation products may be quantitated in

the drug substance by a solid-liquid extraction into di-
chloromethane. The solvent is separated from the drug and
evaporated. The residue is redissolved in alcohol and com-
pared to N-desmethyl diazepam at 230 nm.

Clorazepate dipotassium and its metabolites may
be determined by the Bratton-Marshall reaction after hy-
dolysis to 2-amino-5-chlorobenzophenone.(l4)

Clorazepate dipotassium may be potentiometrically
titrated in glacial acetic acid using perchloric acid in
glacial acetic acid and glass-calomel (0.1 LiC104 in
HOAc) electrodes. Each ml of 0.1 fi HC1O4 is equal to
136.31 mg of clorazepate dipotassium.
111
7. References
1. Washburn, W., Abbott Laboratories, Personal Com-

munication.
2. Egan, R., Abbott Laboratories , Personal Comuni-
cation.
3. Mueller, S . , Abbott Laboratories, Personal Com-
munication.
4. MacDonald, A., Michaelis, A. R., and Senkowski,
B. Z., "Analytical Profiles of Drug Substances,"
Vol. I, K. Florey, Ed., Academic Press, New York,
(1972).
5. Quick, J . , Abbott Laboratories, Personal Communi-
cation.
6. Wimer, D. C., Abbott Laboratories, Personal Com-
munica tion.
7. LaFargue, P., Meunier, J., and Lemontey, Y. , J.
Chromatog., 62, 423, (1971).
8. Schmitt, J., (Establissements Clin-Byla, 4306CB)
U. S. Patent 3,516,988.
9. Raveux, R., and Briot, M. , Chem. Therap., 4 , 3 0 3 ,
(1969).
10. Beyer, K. , Deut. Apoth. Ztg., 111, 1503, (1971).
11. LaFargue, P., Pont, P., and Meunier, J . , &
Pharm. Fran., 28, 343, (1970).
12. Viala, A . , Cano, J. P., and Angeletti-Philippe,
A . , J. Eur. Toxicol. , 2, 109, (1971).
13. Scott, C. G. , and Bomer, P. , J. Chrom. Sci. , 8 ,
446, (1970).
14. Gros, P., and Raveux, R., Chim. Ther., 4 , 312,
(1969).
15. Ibid 11, 28, 477, (1970).
16. Laguleau, J . , Crockett, R., and Mesnard, P.,
Bull. SOC. Pharm. Bordeaux, 110, 10, (1971).
112
CLOXACILLIN SODIUM
David L. Mays
D A V I D L. MAYS
TABLE OF CONTENTS
1. Description
1.2 Appearance
2.1 Infrared Spectra
2.2 Nuclear Magnetic Resonance Spectra
2.3 Ultraviolet Spectra
2.4 Mass Spectra
2.5 Cryst a1 Properties
2.6 Melting Range
2.7 Thermal Analysis
2.8 Solubility
2.9 Ionization Constant, pKa
3. Synthesis and Purification
4. Stability
5.1 Analysis of Impurities
5.2 Identification Tests
5.31 Volumetric Methods
5.32 Colorimetric Methods
5.33 Polarography
5.35 Infrared Spectroscopy
5.37 Biological Methods
5.38 Automated Methods
5.4 Thin-layer Chromatography
6. Protein Binding
7. Metabolism
8. References
114
CLOXACILLIN SODIUM
1. Description
Sodium cloxacillin is found in Chemical

Abstracts under 4-Thia-1-azabicyclo [3.2.0] heptane-2-
carboxylic acid, 6-[[[3-(2-chlorophenyl)-S-methy1-4-
isoxazolyl] carbonyl] amin0]-3,3-dimethyl-7-0~0-,
monosodium salt (1). It is more comonly known as 3-0-
chlorophenyl-5-methyl-4-isoxazolyl penicillin sodium
salt (2).
C H C1N3Na05S Molecular Weight 457.89

19 17
1.2 Auuearance
Sodium cloxa illin is a white, odorles
crystalline powder (3).
Infrared absorption frequencies were reported
for oil suspensions of cloxacillin and other penicillins
(3a). An infrared spectrogram of sodium cloxacillin
monohydrate obtained on a Perkin-Elmer Model 21
Spectrophotometer is included in the compilation of
Wayland and Weiss (4). A spectrogram of Bristol
Laboratories Primary Reference Standard recorded as a
potassium bromide disk using a Beckman Model IR9
115
Figure 1 Infrared absorption spectrum of sodium cloxacillin monohydrate
CLOXACILLIN SODIUM
Spectrophotometer is shown in Figure 1. Characteristic

absorption frequencies (cm-l) are as follows:
a. H20: 3519
b. N-H stretching band: 3370
c. beta-lactam carbonyl: 1770
d. secondary amide carbonyl: 1669
e. aromatic ring: 1619
f. carboxylate carbonyl: 1604
Within the carbonyl stretching region, the

beta-lactam frequency is most characteristic of
penicillins. Opening of the beta-lactam can be
indicated by changes in this part of the spectrum.
Proton nuclear magnetic resonance spectra for

a number of penicillins were reported by Pek and
coworkers (5) and the chemical shifts useful for
identification were tabulated. An nmr spectrogram of
Bristol Laboratories Primary Reference Standard sodium
cloxacillin monohydrate as obtained on a Varian HA-100
spectrometer is shown in Figure 2. Proton resonance
lines were measured in D20 solution with deuterated
sodium trimethylsilyl propionate as the internal
reference. Structural assignments are as follows:
Assignment Chemical Shift, (Coupling Colrstant, Hz)
aromatic 7.50 m
6-H 5.62 d (5-4.0)
5-H 5.46 d (J=4.0)
3-H 4.81 s
5-CH3 (isoxazole) 2.63 s
2-B-CH3 1.43 s
2-a-CH3 1.39 s
The ultraviolet spectrum of an aqueous

solution of sodium cloxacillin monohydrate obtained on a
Cary-14 spectrophotometer is shown in Figure 3. An
absorption maximum at 194 nm and a weak shoulder with
I17
Figure 2 NMR spectrum of sodium cloxacillin monohydrate
CLOXACILLIN SODIUM
Figure 3 Ultraviolet absorption spectrum

of sodium cloxacillin monohydrate
119
DAVID L. M A Y S
maxima at 267 and 273 nm are observed. The effect of

halogen substitution on the phenyl ring of 5-methyl-3-
phenyl isoxazole has been discussed by Doyle (6).
2.4 Mass Spectra
With few differences the mass spectrometric
behavior of cloxacillin methyl ester (Figure 4, Table I)
follows the fragmentation pattern deduced by Richter
and Biemann (7) from high resolution mass measurements.
These fragmentations have previously been discussed in
this series (8,9). The presence of the isoxazole ring
in the acyl moiety results in diagnostically useful
peaks at m/e 43, 178, 193, and 220. M/e 220 is
presumed to result from cleavage of the amide bond. The
origin of the other three peaks follows from the
discussion of the mass spectra of isoxazoles by Ohashi
and coworkers (10).

Sodium cloxacillin is a microcrystalline
powder exhibiting birefringence and extinction positions
under a light polarizing microscope (11-13).
2.6 Melting Range
Sodium cloxacillin melts with decomposition at
170' (6,ll). Cloxacillin free acid melts with
decomposition at 126-127' (14).
2.7 Thermal Analysis
Differential thermal analysis curves of
sodium cloxacillin monohydrate and cloxacillin free acid
were recorded over a range of 0 to 250' on a Perkin
Elmer Differential Scanning Calorimeter Model DSC-1B (15).
No significant transitions were observed with
cloxacillin free acid; decomposition appeared to occur
over several broad temperature ranges. With sodium
cloxacillin, broad endothermic transitions with peak
temperatures at 176' and 193' were recorded.
Dehydration probably occurred during the first transition,
since no loss of water was observed at lower temperature.
120
Figure 4 Mass spectrum of cloxacillin methyl ester
TABLE I
Low Resolution Mass Spectrum of Cloxacillin Methyl Ester
m/ e 0 1 2 3 4 5 6 7 8 9
1 .07 .05 .23 .10 .03 .16
2 .06 .10 .62 2.28 4.59 1.86
3 .98 .36 .26 .18 .36 -24 1.36 .24 .52 2.44
4 .72 3.25 2.20 48.29 2.20 2.52 .43 .80 .13 .18
5 1.02 1.19 .SO 2.85 .94 3.09 .73 .35 .60 6.31
6 .47 .38 .36 .54 .25 .24 .25 .60 1.01 1.01
7 1.10 .77 .34 .88 1.41 4.96 .90 1.20 .21 .ll
8 .2S 1.12 1.25 1.82 .61 .88 1.15 3.58 .78 .56
9 .20 .ll .07 .10 .26 .31 1.16 .72 .37 1.56
-
h)
w
10
11
12
1.63
.34
.07
.54
1.99
.07
1.29
.90
.07
.44
2.15
1.14
.20
14.35
.42
.08
2.00
.63
.08
1.06
1.02
.08
.23
.68
.15
.07
1.37
.21
.07
.31
13 .28 .08 .03 .05 .05 .08 .12 1.38 .72 1.14
14 .54 .45 3.05 .49 1.24 .15 .21 .07 .07 .23
15 4.55 .72 1.71 .31 .28 .20 .24 .ll .48 .13
16 .21 .08 .16 -23 .21 .16 .08 .05 .08 .20
17 .21 .40 .22 .65 100.00 9.59 5.24 1.54 30.89 3.41
18 10.68 1.40 .18 .08 .08 .23 .55 .20 .18 .10
19 .05 .05 .18 2.57 .36 .85 .13 .10 .07 .32
20 .12 .42 .12 .06 .06 .10 .10 .06 .06 .04
21 .05 .05 .10 2.93 .45 .13 .07 .10 .I1 .31
22 9.27 1.24 3.41 .47 .21 .23 .05 .24 .08 .05
23 .07 .03 .05 .23 2.28 .41 .76 .20 .07 .03
24 .07 .15 .21 .13 .06 .05 .48 .14 .21
2s -07
TABLE I (cont.)
m/ e 0 1 2 3 4 5 6 7 8 9
26 13.30 2.11 4.63 .70 .07
27 2.11 .44 .76 .16
28 .05 -07 - 50 -.11
~~
29 .18 .03 -07 .07 .32 .10 .ll

30
31
32
33 1.71 .31 .63 .12 .03
ZA
+
37
h, 38 .ll
39 .29 -08 .ll .02
40
41 -09 -
43
44 1.44
45 .35 .60 -16 .05
Base Peak: m / e 174 = 100.00% Relative Abundance

D A V I D L. M A Y S
2.8 Solubilitv
Sodium cloxacillin is very soluble in cold

water (11,12,16). The ratio of sodium cloxacillin
concentration in chloroform versus pH 6 buffer solution
was determined to be 0.118 (17). The solubility of
penicillin salts in nonpolar solvents is significantly
increased by the presence of a small amount of water,
even water of hydration (18). The tabulation of sodium
cloxacillin solubility shown below is taken from Marsh
and Weiss (19).
So1vent sdulu;;f ty
Water > 20 Methyl ethyl
ketone 1.771
Methanol > 20 Diethyl ether 0.086
Ethano 1 > 20 Ethylene
chloride 0.260
Isopropanol 9.158 1,4 -Dioxane 4.224
Isoamyl alcohol 5.865 Chloroform 1.820
Cyclohexane 0.028 Carbon
disulfide 0.062
Benzene 0.044 Pyridine > 20
Petroleum ether 0.0 Formamide > 20
I sooctane 0.0 Ethylene glycol > 20
Carbon tetrachloride 0.010 Propylene glycol > 20
Ethyl acetate 0.598 Dimethyl
su1foxide > 20
Isoamyl acetate 0.421 0.1 N NaOH > 20
Acetone 2.723 0.1 N HC1 4.526
2.9 Ionization Constant, pKa
Budgaard and Ilver (20) reported an apparent
pKa of 2.68 i 0.05 at 3SoC., determined by measuring the
pH of a partially neutralized 0.0025 M solution of sodium
cloxacillin. Rapson and Bird (21) obFained replicate
apparent pK values of 2.73 i 0.04 and 2.70 f 0.03 at
25OC. by titrating 0.0025 -
M*sodium cloxacillin solutions.
124
CLOXACILLIN SODIUM
Cloxacillin has 3 asymmetric carbon atoms and

is strongly dextrorotatory. Specific rotation values in
the literature are shown below:
Cloxacillin Temp. Conc. and Solvent -
Ref.
sodium salt +163O 20° 1% in water 6,11,12
sodium salt +159O 20° 1% in water 14
free acid +122O 20° 1% in acetone 14
3. Synthesis and Purification
Cloxacillin is a semisynthetic beta-lactam anti-
biotic prepared by acylation of 6-amino penicillanic acid
with 3-o-chlorophenyl-5-methyl isoxazolyl chloride (6,22,
23). The route of preparation of isoxazole acid
chlorides has been described by Doyle (6). More
recently, the conversion of Penicillin G to other
penicillins without isolation of 6-amino penicillanic
acid has been reported (24-26).
A method of purification by deionized water elution
through a G-25 Sephadex column has been described (27).
Recrystallization of the sodium salt may be accomplished
by methyl isobutyl ketone extraction of the free acid
from an acidic aqueous solution and precipitation with
sodium salts of carboxylic acids.
4. Stability
The general pattern of penicillin degradation has
been described in several reviews (18, 28). Sodium
cloxacillin is stable in water for one week at SOC. The
half-life in a solution of 50% aqueous alcohol at pH 1.3
and 35OC. was reported to be 160 minutes, approximately
the same as Penicillin V (12, 18). At a concentration of
4 mg/ml in water or saline at pH 5.5 to 6.0, cloxacillin
loses 15-25% of its biological activity in 7 hours (29).
The rates of hydrolysis of cloxacillin and other
penicillins were studied by Kinget and Schwartz (30).
By following the amount of acid formed, they showed that
125
D A V I D L. MAYS
alkaline hydrolysis was more rapid for cloxacillin than

other penicillins tested. The presence of aminoalkyl
catechols doubled the rate of hydrolysis at pH 8 but
reduced the rate at pH 6.5. The rate of cloxacillin
hydrolysis was also increased above pH 8 by the presence
of carbohydrates (31) and at neutral pH by aminoglycosides
(32) *
The kinetics of cloxacillin degradation in aqueous
solution has been studied as a function of pit, buffer
species, ionic strength, and temperature (20).
Decomposition appears first order with respect to sodium
cloxacillin content at any given pH. A rate-of-
degradation versus pH profile over a range of pH from 1 to
11 showed that decomposition is accelerated as the pH is
moved above or below 6. The rate of hydrolysis is
significantly affected by the type of buffer ions.
Citrate buffers show the least catalytic effect and
phosphate show the greatest.
Degradation by enzymes has been studied extensively.
Cloxacillin is resistant to enzymes which catalyze
hydrolysis of the beta-lactam to produce the corresponding
penicilloic acid (penicillinase or beta-lactamase
enzymes). Citri and Zyk (33) studied the effect of
different penicillin side chains on penicillinase
activity. They showed the rate of cloxacillin
inactivation by penicillinase (penicillin amidohydrolase
EC 3.5.2.6) to be about 3% the rate of penicillin G
inactivation. Smith and coworkers (34) investigated the
stability of some penicillins to penicillinase. The
initial rate of hydrolysis of cloxacillin was 0.3% and
1.1% of penicillin G hydrolysis by the penicillinases
produced by B. cereus and Staph. aureus, respectively.
Chapman (35) used the infrared absorption of the beta-
lactam at 5.6 II to observe penicillin inactivation by
beta-lactamase. Cloxacillin was more stable to
staphylococci beta-lactamase than to coliform beta-
lactamase. Combined treatment of penicillin amidase
(enzymes which deacylate penicillins to 6-amino
penicillanic acid) and beta-lactamase caused considerable
inactivation of cloxacillin in 2 hours.
126
CLOXACILLIN SODIUM
5.1 Analysis of Impurities
The penicillenic acid of cloxacillin and

other thiol-containing products such as penamaldic acid
and penicillamine can be determined by reaction of the
free thiol group with Ellman's reagent (36, 36a). The
bright yellow anion formed is measured at the 412 nm
absorption maximum. Other thiols and reducing agents
interfere. If present in sufficient quantity,
penicillenic acid may be measured directly by the
natural absorbance at 337 nm (36a).
The penicilloic acid of cloxacillin has been
separated from cloxacillin by thin-layer chromatography
(37). The penicilloic acids (and other iodine consuming
substances) of some penicillins have been estimated by
direct consumption of iodine (36a, 38).

Sodium cloxacillin is identified by the
infrared absorption spectrum and by the penicillin
characteristic purple color formed upon treatment with
chromotropic acid in sulfuric acid at 150°C. (38).
In dosage forms, cloxacillin has been
identified by the infrared absorption spectrum after
extraction from aqueous phosphoric acid solution into
chloroform and evaporation to a concentration of 2 mg/ml
(39)*
Weiss and coworkers (17) identified
penicillins in dosage forms by determining the amount of
penicillin partitioned between pH 6 buffer and organic
solvent.
5.3 Quantitative Methods
5.31 Volumetric Methods

Iodine is not consumed by penicillins
but is consumed by the hydrolysis products. The
127
DAVID L. MAYS
difference in iodine consumption before and after

alkaline hydrolysis is used as a standard method of
determining cloxacillin content (13).
Hydrolysis with a measured excess of
alkali generates an additional carboxyl group in the
penicilloic acid. Back titration of the excess alkali
with hydrochloric acid is used for determination of
cloxacillin content. (38)
Cloxacillin has been determined by
measurement of the side chain absorbance at 275 nm (39a).
Penicillins have been determined by measurement of the
absorption maximum near 340 nm produced by acid
degradation to the corresponding penicillenic acids
(38, 40-44). Formation of the penicillenic acid is
catalyzed by copper and other metals and by imidazole (44).
Penicillins have long been determined by
reaction with hydroxylamine. The hydroxamic acids
generated produce a red-colored chelate with iron (111).
Details of the procedure are available in the Federal
Register (13). Automated versions of the method have
also been used (45).
5.33 Polarography
Penicillins in serum were determined by

.
polarographic techniques (46) The analysis required
about two hours. Cloxacillin was determined at levels
of 5 vg/ml in sulfuric acid solution.
Organic acid side chains produced by
vigorous alkaline hydrolysis of penicillins were
converted to methyl esters for gas chromatographic
separation on a 3.5% SE-30 column (47). Intact
penicil 1 ins were gas chromatographed as the corresponding
methyl esters on a 2% fluoro-silicone phase by Martin
and coworkers (48). Separation of the trimethylsilyl
esters of several penicillins on a 2% OV-17 column was
128
CLOXACILLIN SODIUM
reported by Hishta, et. al. (49). Cloxacillin was

separated from several other penicillins and quantitation
was indicated by reproducibility of response factors on
reference samples.
5.35 Infrared Spectroscopy
The infrared absorption due to the beta-
lactam has been used to quantitate penicillins after
extraction into a suitable solvent (39). The
cloxacillin beta-lactam band near 1760 cm-1 was used to
measure cloxacillin inactivation by beta-lactamase as
differentiated from amidase (35). For this work
solutions were lyophilized in the presence of potassium
bromide and infrared absorption measurements were made
from the solid disks.
The change in optical rotation upon
treatment with penicillinase has been used to quantitate
penicillins (50). Penicillins which are more susceptible
to penicillinase can be determined in the presence of
resistant penicillins. Ampicillin and cloxacillin were
determined in combination.
5.37 Biological Methods

The cylinder-plate agar diffusion method
is the official microbiological method of determination
(13). Staph aureus (ATCC 6538P) is the organism of
choice (13, 51). Cloxacillin has been microbiologically
determined in the presence of ampicillin using agar
impregnated with penicillinase to destroy the ampicillin
activity (52) or after ion exchange separation on a
column of IRA-402 (53). Cloxacillin is routinely
measured by the turbidimetric method using Staph aureus
FDA-209P (ATCC 6538P) or Staph aureus BL-A9596 (54).
A microbiological paper disk procedure has been
described for measuring cloxacillin and other antibiotics
in as little as 10 p 1 of plasma (55). Hooke and Ball (56)
used an agar plate method and Sarcina lutea NCIB 8553 to
measure cloxacillin at levels below 10 pg/ml in milk.
129
DAVID L. MAYS
In a test to detect trace levels in milk, penicillin

inhibits growth of Streptococcus thermophilus B.C.,
which otherwise causes the dye 2,3,5-triphenyltetrazolium
chloride, to turn from colorless to red (57). Other
antibiotics interfere.
5.38 Automated Methods
Several chemical methods for penicillin

determination have been automated, including the
hydroxylamine method (45, 58-60), the iodometric method
(61-63), the penicillenic acid method (64), and a
colorimetric method based on enzyme deacylation to
6-amino penicillenic acid and detection with p-dimethyl-
aminobenzaldehyde (65). Cloxacillin, specifically, is
mentioned in the hydroxylamine (45) and penicillenic
acid (64) automated procedures.
5.4 Thin-layer Chromatography
Reviews on chromatography (66) and analysis

(67) of antibiotics are available. Cloxacillin has been
chromatographed on paper (68), kieselguhr G (68),
cellulose (69-71), polyamide (72), silica gel G (37, 70,
73-75), commercial silica gel plates (76, 77) and silica
gel G predeveloped with silicone (5% DC 200 in ether)
(78-80).
Cloxacillin has been separated from other
penicillins (37, 66, 75, 77-80), and from impurities (37),
from cephalosporins (79), from other antibiotics (70, 74),
and from constituents of body fluids (76, 77).
Chromatography has been used to study solvent partitioning
(78) and structure-activity relationships (79, 80).
Visualization spray reagents used include
iodine-azide solution followed by aqueous starch to give
white spots on blue purple (37, 68, 74); 10% acetic
acid in acetone followed by starch-iodine to give white
spots on blue (69); ammoniated copper sulfate (73);
0.5% bromine solution (72) ; 0.25% fluorescein (72) ;
ferric chloride and potassium ferricyanide with sulfuric
acid to give blue spots on green (70, 71); alkaline
potassium permanganate and heat to give yellow spots on
130
CLOXACILLIN SODIUM
pink (78-80); chloroplatinic acid with potassium iodide in

acetone to give white spots on red-purple (75); and
alkaline silver nitrate (68). Bacillus subtilis ATCC 6633
impregnated in agar has been used to detect penicillins
by observation of the zone of inhibition after contact
with the plates (76, 77).
6. Protein Binding
The reversible binding of protein to penicillins (81i

and drugs in general (82), has been reviewed. Schwartz's
penicillin review (28) includes a section on reversible
and irreversible protein binding. Schwartz postulated
that the high local concentration of penicillin
reversibly bound to protein accelerates aminolysis,
particularly in the case of cloxacillin which is highly
bound to serum protein. Batchelor (83) also presented
evidence that penicillins, including cloxacillin, can
become irreversibly bound to protein.
Several workers have measured the proportion of

cloxacillin reversibly bound t o serum protein. Values
of 94% (29), 95% and 96% (84), 85% (85) and 62% (86)
have been reported. Rolinson and Sutherland (87) made
a thorough study of the degree of binding of several
penicillins as a function of concentration of penicillin
and protein, of sera from different animal species, and of
temperature. Binding was shown to be essentially
reversible and competitive with other drugs. Cloxacillin
at 50 pg/ml (considered approximate blood level from a
normal dose) was 94% bound to human serum.
Affinity for Sephadex gel of a number of penicillins
was studied as a function of protein binding (88).
7. Metabolism
After oral administration of 100 mg/kg to rats,
18.7% of the cloxacillin was recovered in the urine after
24 hours, and 10.2% was recovered in the bile. Paper
chromatographic examination of the urine indicated small
amounts of two unidentified metabolites (12). Cloxacillin
was reported to be approximately 10% metabolized in man
(16, 89). One unidentified metabolite was found which
131
DAVID L. MAYS
had biological activity similar to the parent compound.

The rate of elimination of cloxacillin from young men
given oral doses was studied. (90)
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132
CLOXACILLIN SODIUM
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52. Sabath, L. D., e t . a l . , Appl. Microbiol. - 15 (3),

468-470 (1967).
53. Saccani, F., e t . a l . , Boll, Chim. Farm. - 108 (12),
777-780 (1969) .
Tylec, F . , and Kianka, J . , B r i s t o l Laboratories,
54.
p r i v a t e communication.
55. J a l l i n g , B . , e t . a l . , P h a n a c o l . Clin. -
4, 150-157
(1972).
56. Hooke, E. J., and B a l l , G. M., J. Appl. Bacteriol.
26 (2), 216-218 (1963).
-
57. S c a r l e t t , C. A., Agriculture (London) - 73 (9), 423-
426 (1966).
58. Avanzini, F., e t . a l . , Automation i n Analytical
Chemistry, 1966, Vol. 11, Mediad, Inc., White
Plains, New York, pp. 31-34.
59. -
Niedermeyer, A. O., e t . a l . , Anal. Chem. 32, 664-666
134
CLOXACILLIN SODIUM
(1960).
Stevenson, C, E., Automation in Analytical Chemistry
60.
1969, Vol. 11, Medlad, Inc., White Plains, New York,
pp. 251-256.
61.
589-595 (1965). -
Bomstein, J., et. al., Ann. N. Y. Acad. Sci. 130,
62. -
Ferrari, A , , et. al., Anal. Chem. 31 (lo), 1710-1717
(1959).
63. Russo-Alessi, F. M., et. al., Ann. N. Y. Acad. Sci.
64.
-
87, 822-829 (1960).
Celletti, P., et. al., Farmaco, Ed. Prat. 27 (12),
.
688-698 (1972) through Analyt Abs. 25 :42771973) .
65. Evans, W. G., et. al., Automation in Analytical
Chemistry, 1969, Vol. 11, Mediad, Inc., White
Plains, New York, pp. 257-259.
66. Chromatography of Antibiotics, Wagman, C. H., and
Weinstein, J. J., Elsevier Sci. Pub., New York
(1973), pp. 140-146.
67. Schmitt, J. P., and Mathis, C., Pharma. Int., Engl.
Ed., pp. 17-28 Mar. (1970).
68.
522-556 (1968) . -
Hellberg, H., J. Ass. Offic. Anal, Chem. 51 (3),
69.
313-319 (1971).
-
Bird, A. E., and Marshall, A. C., J. Chromatog. 63,
70. McGilvery, I., and Strickland, R. D., J. Pharm. Sci.
71.
-
56, 77-79 (1967).
Wayland, L. G., and Weiss, P. J., J. Pharm. Sci. - 57
( S ) , 806-810 (1968).
72. Wang, J. T., et. al., Tai-Wan K'o Hsueh, 24 (1-2)
19-20 (1970) through Chem. Abs. 74:115942r(1971).
73. Guven, D. C., and Ari, A., Eczacilik Bul. - 13 (2),
20-23 (1971).
74. Saccani, F., Boll. Chem. Farm. 106, 625-628 (1967).
75. Pokorny, M., et. al., J. Chroma=. 77, 457-460
-
(1973).
76. Nishida, M., et. al., Nippon Kagaku Ryohogakukai
Zasshi 17 (lo), 1973-1976 (1969) through Chem. Abs.
73: 127313 (1970) .
77. Murakawa, T., J. Antibiot. 23 (S), 250-251 (1970).
78. Biagi, G. L., et. al., J. Chromatog. - 41, 371-379
.
(1969)
79. Biagi, G. L., et. al., J. Chromatog. - 51, 548-552
(1970).
80. Biagi, G. L., et. al. J. Med. Chem. - 13 (3), 511-516
135
DAVID L. MAYS
(1970) .
81. Scholtan, W., Antibiot. Chemother. (Basel) - 14 53-
93 (1968).
82. Meyer, M. C., and Guttman, D. E . , J. Pharm. S c i . - 57
(6). 895-918 (1968).
83. Batchelor, F. -R., et. a l . , Nature - 206, 362-364
(1965).
84. Kunin, C. M., Antimicrob. Ag. Chemother., 1025-1034
(1965) .
85.
(1963).
-
S i d e l l , S., e t . a l . , Arch. Intern. Med. 112, 21-28
86. Kato, Y . , Nippon Kagaku Ryohogakukai Zasshi 13 ( 2 ) ,

89-93 (1965) through Chem. Abs. 64 :7214e ( 1 9 z ) .
87. Rolinson, G . N . , and Sutherland, R., B r i t . J.
Pharmacol. 25, 638-650 (1965).
88. Murakawa, T Z J. Antibiot. 23 ( l o ) , 481-487 (1970).
89. Lynn, B., Antibiot. ChemothG. 13, 125-226.
(1965) .
90. Kislak, J. W., e t . a l . , h e r . J . Med. S c i . -249 ( 6 ) ,
636-646 (1965) .
I appreciate t h e h e l p of Mr. N. Muhammad, Dr. R. D.
Brown, and M r . T. R. Marr of t h e Chemical Control
Department of B r i s t o l Laboratories f o r a s s i s t a n c e i n
obtaining and i n t e r p r e t i n g t h e spectrograms and thermal
a n a l y s i s d a t a used i n t h i s p r o f i l e .
136
DIATRIZOIC ACID
Hyam Henry Lerner

HYAM H. LERNER
TABLE OF CONTENTS
1. Description
1.1 Name , Formula, Molecular Weight

2.1 Spectra
2.11 Infrared
2.12 Nuclear (Proton) Magnetic Resonance
2.13 Ultraviolet
2.14 Mass Spectrometry

2.22 Thermal Gravimetric Analysis
2.24 Water of Crystallization
2.25 Melting Range
2.3 Solution Data
2.31 Solubility
2.32 pKa
2.33 pH
2.34 Osmotic Properties and Ionic Strength
2.35 Index of Refraction
2.36 Specific Gravity
2.37 Freezing Point Depression
2.38 Viscosity
3. Manufacturing Procedures
3.1 Synthesis
3.2 Purification
4. Stability
5. Separation Techniques and Analysis for Impurities
138
DlATRlZOlC ACID
5.1 Free Amino Compounds

5.2 Free Iodine and Free Halide
5.3 Complexometric Methods of Separation
5.4 Countercurrent Distribution

6.4 Polarography
6.5 Organically Bound Iodine
6.6 Chromatography
6.61 Paper Chromatography

6.63 Electrochromatography
6.7 Titrimetric Methods of Analysis

6.8 Radiometric Methods of Analysis
6.81 X-Ray
6-82 6- Particle Dispersion
7. Pharmacology
7.1 Drug Metabolism

7.2 Toxicity
8. References
139
HYAM H. LERNER
1. Description
1.1 Name. Formula. Molecular Weight
-
Diatrizoic Acid is 3,5- diacetamido 2,4,6 tri-
iodbenzoic acid. Chemical Abstracts listings are under the
heading benzoic acid, 3,5 - -
diacetamido 2,4,6 triiodo-. -
Among the generic and trivial names for this c m -
pound are urografin acid. Common trade names include Reno-
grafin and Hypaque.
Mol. Wt. 613.928
Diatrizoic Acid is a white, odorless and tasteless

crystalline powder. The sodium salt is colorlese and odor-
its ta te reported as both weakly salty and
slightly bitter8 .
The methylglucamine (meglumine) salt is
colorless, odorless and has a slight sweetish taste1 .
2. Phvsical ProDerties
2.1 Spectra
2.11 Infrared
The infrared spectrum in Fig. 1 was obtain-

ed on a Perkin-Elmer Model 21 infrared spectrophotmeter,
from a mineral oil dispersion, The following spectral
assigmnents were made by Toeplitz5 .
140
e
f
F i g . 1 I n f r a r e d Spectrum o f D i a t r i z o i c Acid
HYAM H. LERNER
Wavelength
-1
-cm E Assignment
2985 3.35 N-H stretch
1700 5.88 Acid C=O
1661 6.02 Amide C=O
1515 6.60 Secondary amide and
aromatic C=C stretch
This spectrum is in basic fgreement with a

published spectrum for sodium diatrizoate .
2.12 Nuclear (Proton) Magnetic Resonance
The NMU spectrum of diatrizoic acid shown

in Fig. 2, was determined on a Varian XL-100 NMR spectro-
meter at ambient probe temperature (ca.31"). The sample
was dissolved in deuterated dimethylsulfoxide, containing
tetramethylsilane as an internal reference, Spectral as-
signments are recorded in Table I2 .
TABLE I
NMR Spectral Assignments
Chemical Shift No, of Protons Assignment

(ppm,b)
2.02 6 (4 -CH3
0
9.88 1 (s) -!-OH
0
9.96 2 (s) -m-L
s = singlet
m = multiplet
2.13 Ultraviolet
The following ultraviolet spectral data

have been reported for diatrizoic acid:
142
i 1
143
HYAM H. LERNER
-
Form E
- Reference
Free Acid 0.13 NaOH 238 521 32,000 1

Sodium Salt Ethanol 239 525 33,400 4
Free Acid Ethanol 238 589 36,200 12
Free Acid Methanol 238 539 33,100 32
Free Acid .01g Methanolic 238 538 33,000 32
NaOH
Free Acid .01g Methanolic 238 531 32,600 32
HC1
Although none of the references explicity

state it, it appears the above results are reported on an
"as is" basis, except for reference 12, which appears to
be on the dried basis.
Purkiss et.a1.26, reported that the absor-

bance of sodium diatrizoate is due to the presence of the
acetamido group and is not dependent on iodine content.
Removal of iodine from the compound did not affect absor-
bance at 238 m. Neudert and R ~ p k e ~however,
~, determined
the molecular extinction coefficients (€) of related io-
dinated compounds at their peaks. They reported the for
1,3,5,-triiodobenzene at 232 nm,,is 36,600 and that for 2,
4,6-triiodobenzoic acid at 238 run, to be 33,600. They also
reported the extinction of 3-acetamidobenzoic acid at 243
tnn to be 13,500. Therefore it seems reasonable to suggest
that the strong ultraviolet absorption maximum of diatri-
zoic acid at 238 nm, is due, to a large extent, to iodine.
2.14 Mass Spectrometry
No molecular ion is observed for diatrizoic

acid. Weak peaks at m/e 569, corresponding to the loss of
-COOH at m/e 572, for the loss of -COCH2, from the unob-
served molecular ion, do occur. Major fragment ions at
m/e 487 and 486 occur, due to loss of I and HI, respective-
ly, Loss of CH3CO from the m/e 486 fragment results in a
peak at m/e 443 and loss of a second CH3CO results in the
observed peak at m/e 400
3
.
144
DlATRlZOlC ACID
Valenti34 determined the DTA of diatrizoic

acid on a DuPont 900 Thermoanalyzer at a temperature rise
of 15" per minute. The thermogram showed endotherms at 168
and 321", exotherms at 220" (small) and 330", and a shoulder
at 306". Kabadi35 reprecipitated diatrizoic acid from al-
kaline solution and dried it at roan temperature, under
vacuum. He reported DTA data under the same conditions
referred to above as follows: endotherms at 119" (large),
157" (small), 316" broad; exotherm at 234"; shoulder at 29$.
The moisture content of this specimen was <0.4%.
The work of Kabadi35 indicates that other

polymorphic forms of diatrizoic acid exist.
2.22 Thermal Gravimetric Analysis
Valenti34 determined the TGA of diatrizoic

acid on a DuPont Thermogravimetric Analyzer, The compound
was heated at a rate of 15" per minute under nitrogen sweep.
Weight loss of 5% was observed, with all volatile material
driven off before 140".
Ochs13 obtained the X-ray powder diffraction

spectrum of diatrizoic acid on a Phillips X-Ray Powder Dif-
fractometer, at a voltage of 45 kv and a current of 15 ma.
0
The sample was irradiated by a copper source at 1.54A.
Data are recorded in Table 2.
2.24 Water of Crystallization
Langecker et.al. reported that the diatri-

zoic acid crystal can contain up to 2 moles of water of
crystallization.
145
HYAM H. LERNER
TABLE I1
X-Ray Powder D i f f r a c t i o n P a t t e r n of D i a t r i z o i c
Acid Squibb Lot 1987
& Relative Intensity** *&d Relative Intensity**
13.2 0.23 3.13 0.45

9.10 0.40 3.12 0.49
7.20 0.19 3.07 0.71
6.50 0.34 3.00 0.23
5.66 0.51 2.98 0.27
5.50 0.19 2.93 0.21
5.00 0.15 2.88 0.13
4.50 0.20 2.68 0.11
4.42 0.31 2.60 0.19
4.28 1.00 2.56 0.16
4.18 0.43 2.54 0.24
4.02 0.32 2.50 0.27
3.90 0.49 2.46 0.21
3.75 0.69 2.37 0.12
3.68 0.19 2.28 0.19
3.60 0.19 2.24 0.19
3.48 0.16 2.18 0.17
3.44 0.53 2.16 0.16
3.36 0.43 2.13 0.15
3.30 0.53 2.09 0.17
3.23 0.29 1.93 0.13
3.17 0.17 1.89 0.15
1.87 0.16
* d = (interplanar distance) = 2 s i n 0
nn
where? = 1.5398
** Based on h i g h e s t i n t e n s i t y of 1.00
146
DlATRlZOlC ACID
2.25 Melting Range
Diverse values have been reported in the

literature for the melting range of diatrizoic acid. They
are shown in the table below. A possible explanation for
the wide diversity in reported values may be that different
(unidentified) polymorph forms were used in the work.
Melting Range Remarks -

Ref.
>300 -- 6
from 260 Decompositon, I2 Vapors 1
reported
>340 Crystal form: needles 11
260" - 290 Using KSfler Microblock 12

(Reichert)
2.3 Solution Data
2.31 Solubility
The data in Table I11 were reported for the

solubility of diatrizoic acid, at room temperature.
TABLE I11 - SOLUBILITY DATA
Solubility (mg/lOO ml)
Solvent Ref. 1 Ref. 12 Ref. 34
Acetone -
Benzene <lo
Chlorofo m 110 0.1
Ethanol 700 1,018
Ether <l0 0.3
Hexane - -
Me thano1 2,400 7,205
Propylene Glycol -
Water at 25" 100
Water at 50" 150
Water at 90" 270
0.1N Sodium -
hyaroxide
147
HYAM H. LERNER
.
Langecker e t a 1 .l r e p o r t e d the s o l u b i l i t y a t
20" of t h e sodium s a l t of d i a t r i z o i c a c i d t o be 60 g1100 m l
i n w a t e r and t h a t ol t h e methylglucamine s a l t t o be 89g/100
m l , Drug Standards r e p o r t e d t h a t t h e sodium s a l t i s f r e e l y
s o l u b l e i n w a t e r and dimethylformamide and v e r y s l i g h t l y
s o l u b l e i n chloroform and e t h e r . The s o l u b i l i t y i n e t h a n o l
was r e p o r t e d t o b e 2 g/100.
2.32 pKa
The pKa of d i a t r i z o i c a c i d i s 3.4
1
.
2.33 pH
R e pH of a 1%s u s p e n s i o n of t h e f r e e a c i d
i n water i s 2.1 .
The pH of a 50% s o l u t i o n of t h e sodium
s a l t i s between 7 and 94 .
Langecker e t . a l . l r e p o r t e d t h e
pH of a s o l u t i o n of t h e sodium s a l t t o b e ' 7 . 3 and t h a t of
t h e meglumine s a l t t o be 6.0.
2.34 Osmotic P r o p e r t i e s and I o n i c S t r e n g t h
Berdalen et.a1.36 determined t h e molal osmo-

t i c c o e f f i c i e n t s and i o n i c s t r e n g t h of some commercial
p r e p a r a t i o n s of d i a t r i z o a t e .
2.35 I n d e x of R e f r a c t i o n
25
The r e f r a c t i v e i n d e x (n ) of sodium d i a t r i -
g
z o a t e i n w a t e r , a t 25", i s g i v e n i n Ta l e I V .
2.36 Specific Gravity
Langecker e t , a 1 .' reported the s p e c i f i c

g r a v i t i e s (p) of w a t e r s o l u t i o n s of sodium d i a t r i z o a t e a t
25". Data are recorded i n Table I V .
2.37 Freezing Point Depression
The f r e e z i n g p o i n t d e p r e s s i o n (-AT) of
v a r i o u s c o n c e n t r a t i o n s of sodium d i a t r i z o a t e were r e p o r t e d
by Langecker e t . a l . l , and a r e r e p o r t e d i n Table I V .
148
DlATRlZOlC ACID
2.38
Viscosity
59
Schmid studied the viscosity of 0.75PJ
aqueous solutions of diatrizoate at pH 7, and 20". He
reported viscosities by Rheomat 16 and Ostwald viscometers
for sodium diatrizoate (3.29 cps), meglumine diatrizoate
(6.19 cps), and tris-(hydroxymethy1)-aminomethane diatri-
zoate (4.76 cps.).
TABLE IV
Physicochemical Data of Sodium Diatrizoate

25
Mo 1arity -
nD
0.0 0.0 0.997 - -
4.74 0.074 1.338 1.025 0.27
14.20 0.223 1.353 1.085 0.78
28.40 0.446 1.376 1.175 1.70
37.90 0.595 1.392 1.235 2.38
47.40 0.744 1.407 1.295 3.20
3. Manufacturing Procedures
3.1 Synthesis
Diatrizoic acid may be prepared from 3,5-dinitro-

benzoic acijlOby catalytic reduction with e.g. Raney nickel
in methanol or palladium (on carbon support)", to yield
the 3,5 diaminobenzoic acid. Reaction of dilute hydro-
chloric acid solutions of the latter compound with potasaturn
iodochloride or iodine monochloride yields the 3,5-diamino-
2,4,6-triidobenzoic acid7y10y11. Acylation of this com-
pound is accomplished by dissolving it in acetic anhydride,
heating and then acidifying with concentrated sulfuric
acid, added dropwise. After a brief exothermic reaction
the solution is heated on a steam bath and cooled in ice6 .
Alternatively 3,s-diacetamidobenzoic acid can be iodinated
in pH 5-6 methanolic solution with odine monochloride, at
8OoC, to yield the triiodo compound
.'1S'',' (See Fig. 3
for stepwise synthetic route)
Davidson28 acetylated the diamino compound with
acetic H3 anhydride to obtain tritiated diatrizoic acid.
149
HYAM H. LERNER
Figure 3
Synthetic Route for Diatrizoic Acid

' OOH COOH
NO A T N H b Z
CH3- e -NH H2
3.2 Purification
Crude diatrizoic acid may be purified in any of

the following ways:
The precipitate is dissolved in dilute ammonia or
sodium h droxide, charcoaled and reprecipitated by acidifi-
cation6,g, 10,ll.
Recrystallization from a 50% aqueous dimethyl-
formamide solution with charcoaling6 .
Filtration of a solution in dilute sodiumhykoxide
containing sodium hydrosulfite and charcoal. The filtrate
is passed through a medium basic anion exchange resin (in
the OH form) The eluate is acidified to reprecipitate the
pure product3 .
4. Stability
Diatrizoic acid is chemically stable at room tempera-

ture. Extrapolation o€ the activation energy and frequency
constant data, between 70" and 100" to room temperature,
indicates decomposition of the N-acyl bond to be 0.1% in
50 years37 , Evolution of iodine at 90" from diatrizoic
acid in solution at pH 9, is less than 3% in 75 hours. The
presence of impuritie,s under iodinated and free amino
compounds, enhance degradation37 .
150
DlATRlZOlC ACID
Commercial preparations of diatrizoic acid neutralized

with meglumine, and containing sodium citrate (3.2 mg/ml)
and disodium edetate (0.4 mg/ml), show no significant loss
of potency after storage for 5 years, at room temperature??
5. Separation Technique and Analvsis for ImDuritiea
Many of the chromatographic procedures described in

Section 6.6 are used to separate, detect, and estimate im-
purities in diatrizoic acid, In addition, the following
methods have been described in the literature to detect
and quantify impurities and decomposition products, and to
isolate pure diatrizoic acid.
5.1 Free Amino Compounds
Hartmann and determined free m i n o com-

pounds of iodinated contrast agents by a kinetic method
based on the greater reactivity of these impurities with
elemental bromine in acetic acid. Under these conditions,
the free amino compound quantitatively splits off iodine,
which is oxidized to iodate by the bromine. After destruc-
tion of the excess bromine, the iodate is reduced to iodine
and titrated with thiosulfate, to a starch endpoint, Free
iodides in the sample also react with bromine and give
positive results, by this procedure.
Hoevel-Kestermann and Muhlemann12 and Hartmann

and R t i ~ k edescribed
~~ a Bratton-Marshall colorimetric
reaction to quantitate free amino compound impurities.
5.2 Free Iodine and Free Halide
G ~ n d detected
a ~ ~ free iodine in diatrizoic acid
by dispersal in water, filtering and adding starch to the
filtrate. A blue coloration indicates the presence of free
iodine. An alternate p r o ~ e d u r e ~is, ~to~ acidify the fil-
trate, add chloroform and sodium nitrite and observe for
reddish coloration in the lower chloroform layer. Free
halide was detected38 by acidification of a water suspen-
sion filtrate of the drug substance with nitric acid, ad-
dition of silver nitrate and observing for the presence of
opalescence or precipitation.
151
HYAM H. LERNER
Poet3' quantitated free halide coulometrically,

with an Aminco-Cotl.ove Chloride Titrator. Hartmann and
quantitated free halide in contrast media by poten-
tiometric titration with 0.001E silver nitrate, under a
protective cover of nitrogen.
5.3 (=analexometric Mpthod o f SeDaration
Hentrich and Pfeifer40 described methods for the

precipitation of ten contrast agents as their metallic
salts or metallic complex salts. Diatrizoic acid can be
precipitated quantitatively by cadmium sulfate with thiourea,
copper sulfate with thiourea, silver nitrate, cadmium sul-
fate with pyridine and copper sulfate with pyridine. Che-
latometric methods are also described for the titration of
excess precipitantj after separation of the precipitated
salt by filtration. The molecular formulae, weights, melt-
ing range and equivalent weight of diatrizoic acid preci-
pitated by 1 ml of 0.1gsolution of the precipitant, for
the salts and complexes, are given in Table V below.
TABLE V
40
Metallic Salt and Metallic Complexes of Diatrizoic Acid
. Weight
Preci- Mol. Formula of Melting to 1ml qf 0.m
pitant Salt or Complex Mol. Wt. Range Precipitant
A
"
3 C11H813N204Ag 720.8 281-284 0.0614 g
CdSO4- {Cd [(NH2) 2CSl$
Thiourea (CllH813N204)2 1,642.7 226-227 0.0307 g
CdSO4- [Cd(C6H5N)4]* 1,656.6 288-290 0.0307 g
(C11H813N204) 2
CuSO4- f2u[(NH2)2CS$ 828.7 245 0.0614
Thiourea 11H813N204
- [cU( C6H5N) 21 1,447.5 266-267 0.0307
ine (C11H813N204)2
152
DlATRlZOlC ACID
5.4 Countercurr-
Strickler et.a1.42 separated diatrizoic acid and

other contrast agents from sera by countercurrent distri-
bution. They used a solvent system composed of E-butanol:
dil. aqueous ammonia (l:$. Both a 30-tube manual procedure
and a 200-tube automatic procedure are described. Evalu-
-
ation of other methods e.g. separation by ion exchange on
Dowex 1 column (not successful) and 2-dimensional paper
chromatography, are also discussed.
5.5 Phase Solubilitv Analvsig
K a l l ~ sreported
~~ on the use of phase solubility
analysis to determine the purity of diatrizoic acid. He
used a solvent system composed of acetone: ethanol (17:3),
with equilibration over a 23 hour period at 25". The extra-
polated solubility in this system is 3.5 mg per g.
The elemental analysis6' results were calculated

to the anhydrous basis.
Element % Theoretical % Reported60
21.52 21.98
1.478 1.78
62.01 61.69
4.562 4.26
10.42 10.29 (CALC)
Infrared (section 2.11), paper chromatography

(section 6.61), and thin-layer chromatography (section 6.62)
have been used to identify diatrizoic acid.
The evolution of intense violet fumes of libera-

ted iodine can be observed b heating a sample of diatri-
.rs,
zoic acid over an open flame 12,
153
H Y A M H. LERNER
Identification of the amide funcionality can be

made by first ascertaining the absence of free amino groups
(section 5.1) and then cleaving the acyl group, by reflux-
ing in base, and repeating the Bratton-Marshall reaction12 .
The pH (section 2.33) of a water suspension and
the ability to titrate a suspension with base, can be used
to identify the presence of the carboxylic acid12.
6.3 Direct SDectroDhotometric Analysis
Purkiss et.a1.26, determined diatrizoic acid

directly in urine and heparinized protein-free plasma at the
238 nm maximum. Samples taken from the subject prior to
administration of diatrizoic acid were used as blanks.
Dilution with water decreased blank interference.
Hoevel-Kestermann and Muhlmann12 used spectro-

photometric analysis at the 238 run peak to determine solu-
bility in various solvents.
6.4 Polarography
Kabasakalian and Mc G 1 0 t t e n ~studied

~ 10 iodin-
ated radiocontrast agents polarographically in ethanol-
water (1:l) solutions between pH 1.3 and 10.5. The effects
of pH, temperature and sample concentration on wave form
and height are examined and the analytical possibilities
of polarography as a quantitative procedure for diatrizoic
acid are discussed. Diatrizoic acid in phospha e buffer,
f
pH 8.9, exhibits two waves: -E$ at 0.90 and -E% at 1.22.
6.5 Ornanicallv Bound Iodine
Hoevel Kestermann and M"uhlemann12 reviewed three

methods for liberating organically bound iodine in contrast
agents: A. Parr bomb (fusion with sodium peroxide); B.
catalytic dehalogenation with sodium borohydride; and C.
reductive dehalogenation with zinc-sodium hydroxide. They
recommend method B because of its simplicity, short assay
time and high recision. This method was originally pro-
posed by Egli48 . wantitation is accomplished by poten-
tiometric titration with silver nitrate.
154
DlATRlZOlC ACID
Yakatan and TuckermanLC3reviewed four methods for

decomposing iodinated contrast agents to liberate organi-
cally bound iodine: A. Parr bomb (fusion with sodium per-
oxide); B. alkaline permanganate reduction; C. Zinc-sodium
hydroxide reduction; and D. oxygen flask (Schoniger) com-
bustion. Method D is recommended as a general technique
for all iodinated organic compounds because of its high
reproducibility, simplicity, and rapidity of analysis (20
min. per test). For compounds that have all iodine atoms
ortho or para to the electronegative carboxylic acid on the
aromatic ring s. diatrizoic acid, method C, zinc-sodium
hydroxide reduction, is recommended. In method C, the
iodine is reduced under reflux conditions and replaced by
hydrogen, generated by the reaction of powdered zinc with
sodium hydroxide. The iodide thus formed, is determined by
titration with silver nitrate in acidic solution, in the
presence of the absorption indicator, tetrabromophenol-
phtalein ethyl ester4 .
Ates and hall4 decomposed diatrizoic acid with
alkaline permanganate. After decoloration of the perman-
ganate with sodium nitrite and acid, they titrated the
liberated iodine with thiosulfate.
Zak and B ~ y l e
used
~ ~ chloric acid as an oxidiz-
ing digestion reagent to wet ash iodine-containing organic
compounds. The iodine present in this solution as iodate
may then be determined by titrimetry, spectrophotometry or
by polarography.
Strauss and Erhardt46 determined diatrizoic acid

directly, in urine, by splitting off the organic iodine,
followed by thiosulfate determination. They boiled urine
samples with sulfuric acid at 150" for 5 minutes, cooled
the sample, added potassium iodide, and titrated.
Many systems have been described in the

literature for separation and detection of diatrizoic acid.
These are summarized in Table VI. The drug substance may
be quantitated from the developed chromatogram by elution
155
HYAM H. L E R N E R
in aqueous solution and subsequent spectrophotometric ana-

lysis by densitometry, or by other suitable means.
Pileggi et.al.17, described a paper chroma-

tographic precedure for separation of 19 or anic iodide
compounds from blood serum. Ates and Amall' describe
systemg4for separation of 8 contrast agents. B1 fox
- - , and Turula'' described 2 dimensional paper chroma-
et.al.
tographic procedures for separation of various iodinated
contrast agents and iodinated proteins, from bile and
serum.
Key To Table VI,
Solvent Systems
I Ethanol: 25% ammonia (ratio of solvents not given).

I1 0.25u sodium citrate, pH 8.2.
I11 =-butanol: 4% ammonia (3:l).
IV toluene: ethanol: formic acid (10:3:3).
V =.-amyl alcohol: 2g ammonia (1:l)
VI water: formic acid (5:l)
-
VII n-butanol: 95% ethanol: water (4:1:5); upper phase
used for development.
VIII -
n-butanol: 1J ammonium hydroxide: ethanol USP (5: 2:9
IX water: _n-butanol: ethanol USP (5:4: 1) ; upper phase
used for development.
X a-butanol: dioxane: ammonia (ratios not reported).
Methods of Detection
A. Spray with 10% ceric sulfate and 5" sodium arsenite,

both prepared in lE sulfuric .
acid1.k
B. Compound assayed was labeled with I

? Detection
accomplished by autoradiography with Kodak Radio-
graphy film.
C. Viewed under ultraviolet light,
D. Spray with mixture of ceric ammonium sulfate and

arsenious acid, followed by spraying with 0.5%
solution of methylene blue18 .
E. Spray on both sides with a reagent containing 5
parts of a 2.7% solution of FeCl '6H 0 in 2N HC1,
5 parts of a 3.5% solution of K3$e(Ci)G and 1 part
156
TABLE VI
Paper Chromatographic Systems f o r S e p a r a t i n g D i a t r i z o i c A c i d
S o l v e n t System Paper Method of Method of -

FS Reference
Development Detection
1 Whatman No. 3 descending A, C Not r e p o r t e d 14

I1 Whatman AE 30 ascending B 0.76 16
I1 DE 20 ascending B 0.66 16
I1 Whatman No. 1 ascending B 0.73 16
I11 Whatman No. 3 descending D 0.57 17
IV Whatman No. 3 ascending E Not r e p o r t e d 19
V Whatman No. 3 descending E 0.00 19,24
VI Whatman No. 3 descending E 1.00 19
VI I Whatman No. 1 descending C 0.27 21
VIII Whatman No. 1 descending C Not r e p o r t e d 22
IX Whatman No. 4 descending C Not r e p o r t e d 23
X Not r e p o r t e d descending B Not r e p o r t e d 24
HYAM H. LERNER
of an acified 5% NaAs02. The color is allowed to

develop for 15 minutes, between glass plates, the
paper wasked with water and dried with a current of
warm air, Spraying and development are carried out
in a dim-lit or dark room20 .
Thin-layer chromatographic methods found
suitable for the separation and detection of diatrizoic
acid, are summarized in Table VII. Some of the references
cited, present sample preparation techniques and methods
for eluting the drug from the developed plate, for quanti-
tation by other means.
Hollingsworth et.al., separated iodoamino

acids and related compounds on cellulose plates with a sol-
vent system composed of E . - b u t a n o l : 2N ammonia: chloro-
form (376:70:60). They did not report this technique for
diatrizoic acid, however, it seems reasonable to expect
that this system will separate iodinated contrast agents.
Stahl and Pfeifle2’ reported on 6 systems to separate 17
iodinated contrast agents and Hoevel-Kestermann and Muhle-
mann12 separated 8 contrast agents, with a single system.
6.63 Electrochrmatography
Da~nenberg’~separated diatrieoic acid on

Whatman No. 3 paper by high voltage paper electrophoresis.
He used a pyridine: acetic acid water (100:10:890), pH 6
buffer, at a potential gradient of 45 v/cm, for 1 hour.
Diatrizoic acid in this system has a mobility of 3 cm11800
vlhr.
Turula” used a pH 8.6 diethylbarbiturate

buffer system, Whatman Nos. 1 and 3 paper, a potential
gradient of 6 v/cm, for 17 hours, to separate iodinated
contrast agents.
Gopal16 separated a number of iodinated com-

pounds including diatrizoic acid, by paper electrophoresis
on Whatman AE 30, Whatman No. 1 and DE 20 (W and R Balston
Ltd) paper. Electrophoresis was accomplished in .025u
sodium citrate buffer, at a potential gradient of 7 vlcm,
158
DlATRlZOlC ACID
Key To Table VII

Solvent System
I ethanol: ammonia, 25% (2:l)
11 benzene: acetone: formic acid (5:5:1)
111 ethylacetate: isopropanol: ammonia, 25% (11:7:4)
IV acetone: isopropanol: ammonia, 25% (
V isopropanol: ammonia 25% (4:l)
VI acetic acid: chloroform (1:19)
VII ethylacetate: methanol: diethylamine (5:4:2)
VIII chloroform: methanol: pyridine (17:1:2)
IX methanol: chloroform: ammonia (10:20:2)
X methanol: chloroform: ammonia (4:5:2)
Plate
A Silica gel G (Merck)

B Silica gel HF 254-366 (Merck)
C Silica gel GF (Brinkman)
Detection System
a. Spray with 1:l solution of 10% ceric sulfate and 5%

sodium aresenite, both in 1g sulfuric acid15.
b. Autoradiography of H3 labeled diatrizoic acid.
c. Ultraviolet light,
d. Spray with 50% acetic acid, followed by irradiation

at 254 nm for 10 min., to give blue violet spots.
159
TABLE VII
Thin Layer Systems for Separation and Detection of Diatrizoic Acid
Solvent System Plate Detection System Rf

- Reference
I A, J3 Not reported 14
I1 A Not reported 28
I11 A, B 0.12 12,29
IV A 0.30 29
V A 0.37 29
VI A 0.40 29
VII A .37 29
VIII A .oo 29
IX C 0.45 30
DlATRlZOlC ACID
in 1 hour.
6.7 Titrimetric Methods of Analysis
Sodium diatrizoate dissolved in dimethylformamide

can be litrated potentiometrically with perchloric acid in
dioxane .
Although no references appear in the literature,
it seems reasonable that a simple acid-base titration to
a phenolphtalein endpoint, of diatrizoic acid in aqueous
methanolic solution, would be successful.
6.8 Radiometric Methods of Analysis

6.81 X-Ray
Holynaka and Jankiewiczb8 used X-ray fluores-

cence and absorption techniques to determine iodine in
various commercial contrast agent preparations. In the X-
ray fluorescence work, excitatiggn of the sample was accom-
plished by irradiation from a ' ~ m source, of 5-mci
activity. The energy of excitation was 60 keV. Fluores-
cence of the K series of iodine (28.5 keV) was measured in
a scintillation counter having a 6-mm thick NaI/Tl crystal.
The characteristic radiation of iodine was separated by
means of a single-channel, pulse-amplitude analyzer cover-
ing the total width of the K-peak iodine. Measurement time
was 1 min. A calibration curve was made using HI03 as a
standard.
A 241Am source at 60 keV was used in the X-

ray absorption procedure. The detector was a scintillation
counter equipped with a 6-mm thick NaI/T1 crystal. Absorp-
tion measurements were made by means of a single-channel,
pulse-amplitude analyzer in the energy channel of 5keV,
covering 60 keV. Calibration cutges were prepared, using
H103 as a standard, The authors preferred the X-ray
fluorescence technique because of its higher precision and
accuracy.
Schiller and Synek" used radiometric

methods to determine iodine in various preparations of con-
trast agents. They used a measuring probe with a GM coun-
161
HYAM H. LERNER
t e r and scaler t o determine r e f l e c t e d r a d i a t i o n .
Buersch e t .al.'O determined t h e continuous

spectrum of X-rays on aqueous s o l u t i o n s of d i a t r i z o i c a c i d .
They r e p o r t e d t h a t Beer's l a w w i t h r e s p e c t t o l i n e a r a b s o r -
p t i o n , h o l d s , provided t h e e x c i t a t i o n beam i s s u f f i c i e n t l y
monochromatic. The a p p l i c a t i o n t o b i o l o g i c a l systems i s
a l s o discussed.
6.82 8- P a r t i c l e D i s p e r s i o n
Mikolajek et.al.51 used t h e method of r e t r o -
r a d e d i s p e r s i o n of b e t a p a r t i c l e s t o a s s a y c o n t r a s t media,
"'Tl with about 3-pCi a c t i v i t y , d e p o s i t e d on a r i n g was
used as t h e s o u r c e of r a d i a t i o n . A c a l i b r a t i o n showing t h e
number of s c a t t e r e d e l e c t r o n s vs c o n c e n t r a t i o n w a s e s t a b -
l i s h e d . R e s u l t s by t h i s method a r e i n good agreement w i t h
d e t e r m i n a t i o n s made by more c o n v e n t i o n a l methods.
7. F'harmacolonv
7.1 Drug Metabolism
D i a t r i z o i c a c i d i s e x c r e t e d r a p i d l y and un hanged
1 r e l y through t h e kidneys by glomular f i l t r a t i o n 28,46,53,
5"'- Tauxe et.alS3 s u g g e s t t h a t t h e r e i s a minor d e g r e e
of plasma p r o t e i n b i n d i n g , i n man. Woodruff and MalvinS6
i n f e r r e d about 5-10% p r o t e i n b i n d i n g of d i a t r i z o a t e , b u t
d i d not p r e s e n t d a t a . Stokes et.a1.57 r e p o r t e d t h a t 50%
d i a t r i z o a t e was p r o t e i n bound, b u t b i n d i n g was e a s i l y re-
versed. No r e p o r t s were uncovered i n t h e l i t e r a t u r e sug-
g e s t i n g m e t a b o l i t e s of d i a t r i z o i c a c i d o r c o n c e n t r a t i o n of
d i a t r i z o i c a c i d i n organs of man, o t h e r t h a n t h e kidney.
7.2 Toxicity
Langecker et.al.'
a c i d i n t h e r a t , t o b e 14.7 gfkg. Miller !a
r e p o r t e d t h e L 0 of d i a t r i z o i c
reported the
LD50 of d i a t r i z o i c a c i d t o be about 10.1 gfkg, i n t h e
mouse, SchmidS9 r e p o r t e d t h e LD50 o f sodium d i a t r i z o a t e
t o b e 14.0 gfkg i n t h e mouse and 11.4 g f k g i n t h e rat. ?he
LD50 of meglumine d i a t r i z o a t e was r e p o r t e d as 14.7 g l k g i n
t h e rat59.
162
DlATRlZOlC ACID
Acknowledpement
The author acknowledges with gratitude the
cooperation of Ms. Cheryl Pernell for her
patience in the preparation and correction
of this manuscript.
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163
HYAM H. LERNER
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L i s s i t z k y , S.,
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(1966).
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Clin. Chem., 8, 647 (1962).
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25 (1959).
19. Turula, K., Acta Endocrinol. 48, 31 (1965).
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1469 (1959).
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tion.
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cation.
23. Chow, L., Squibb Q u a l i t y C o n t r o l , p e r s o n a l c m u n i -

cat ion.
24. Blaufox, M., D. Sanderson, W. Tauxe. K. Wakim,

A. Orvis and C. Owens, h e r . J. P h y s i o l . , 204,
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DlATRlZOlC ACID
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municat ion.
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(1966).
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munication.
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(Warsaw), 14,219 (1969); Chem. Abstr., 7 l , 24774
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Pharm., Pharmacol, l8, 523 (1966); Chem. Abstr.
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munication,
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Mayo C l i n i c , 39, 761 (1964).
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167
DISULFIRAM
Norris G. Nash and Raymond D.Daley

DISULFIRAM
CONTENTS
1. Description
2 . 1 Infrared Spectra
2 . 4 Mass Spectra
2.6 Solubility
2.8 Melting Point
3. Synthesis
4. Stability-Degradation
6 . 1 Identification Tests
6 . 2 Elemental Analysis
6 . 4 Titration Methods
6 . 5 Infrared Spectroscopy
6 . 6 Proton Magnetic Resonan troscopy
6.7 Polarography
6.8 Gravimetric Methods
6 . 9 Chromatography
7. References
169
NORRIS G. NASH AND RAYMOND D. DALEY
1. DESCRIPTION
1.1 Name, Formula, Molecular Weipht

The Chemical Abstracts name f o r d i s u l f i r a m is
tetraethylthioperoxydicarbonic diamide, s t a r t i n g with
Volume 76. Previously the Chemical Abstracts name was
bis(diethylthiocarbamoy1)disulf ide. The CAS Registry No.
is [ 97-77-81. Other common names a r e tetraethylthiuram
d i s u l f i d e and TTD.
S
CH CH CH CH
3 '\N-;-S-S-C-N " / 2 3
CH,CH~' \CH~CH~
Mol. W t . : 296.54

Disulfiram is a l i g h t yellow odorless c r y s t a l l i n e
powder.
2. PHYSICAL PROPERTIES

The i n f r a r e d spectrum of d i s u l f i r a m (Figure 1)
was obtained with a Beckman IR-12 spectrophotometer.
The spe t r u m was run i n three s e c t i o n s : ( a ) from 200 t o
650 cm-I as a m n e r a l o i l m u l l on polyethylene; (b) from
500 t o 1365 cm-' a s a mineral o i l m u l l between potassium
bromide p l a t e s ; ( c ) from 1360 t o 4000 cm-l a s a perfluor-
inated o i l m u l l between potassium bromide p l a t e s . The
spectrum matches those published elsewhere (1,2).
Some of the absorption bands can be assigned t o
the a l k y l groups: 2980 t o 2870 cm'l, C-H s t r e t c h i n g ;
1460 cm", C-H bending; 1378 cm'l, methyl C-H bending
(symmetrical). The assignment of bands t o the various
v i b r a t i o n s of the remainder of the molecule is complicated
by the extensive coupling of v i b r a t i o n s of the N-C-S bonds
and the apparent conjugation of the n i t r o g e n unshared
e l e c t r o n p a i r with the C=S bond (2,3). The 1500 cm'l band
has been assigned t o a C-N s t r e t c h i n g v i b r a t i o n (3) and
the 1000 cm'l band t o a C=S s t r e t c h i n g v i b r a t i o n (9).
Other suggested assignments ( 5 ) a r e : 926 crn'l, N-C-S;
8 II
976 cm'l, C=S; 1149 and 1206 cm'l, N-C-S; 1274 cm-l, N-C.
170
Figure 1. Infrared spectrum of disulfiram, mineral oil and perfluorinated oil mulls.
NORRIS G.NASH AND RAYMOND D. DALEY

The proton magnetic resonance spectrum of disul-
firam shown in Figure 2 was run in CDCl-j, on a Varian
A-60A 60 MHz NMR spectrometer, with a tetramethylsilane
reference (6). The sweep width is 0 to 500 Hz.
The triplet at 1.40 pprn (J=7.3 Hz) is assigned to
the methyl protons, while the quartet at 4.06 ppm (J=7.3
Hz) is assigned to the methylene protons. The small bands
at 2 27 Hz of each Line are spinning sidebands (6).
The temperature dependence of the NMR spectrum of
disulfiram and similar compounds has been investigated
(78).
Figure 3 shows the ultraviolet absorption spec-
trum of disulfiram, run on a Cary Model 14 spectrophotom-
eter. The spectrum was obtained at two concentrations,
100 ,g per ml and 10 u g per ml, in methanol solution, in
1 cm cells.
2.4 Mass Spectra

The low resolution mass spectrum of disulfiram is
shown in Figure 4. This spectrum was obtained on an LKB
9000s mass spectrometer, with an ionizatio voltage of
70 eV, source temperature 25OoC (6). This spectrum is
similar to one reported and interpreted by Madsen et a1
(9); their suggested fragmentation scheme s shown in
Figure 5.

The only thermal event in the differential ther-
mal analysis curve of this compound is the melting endo-
therm at 7OoC (10).
2.6 Solubility
The solubility of disulfiram at room temperature
is as follows:-
172
Figure 2. NMR spectrum of d i s u l f i r a m , C X 1 3 s o l u t i o n .
'-4
0.9
,
O.*1
e
w
$ 0.4
.
4
P 6
m
0.3
0
m
m
6 0.2i ', ',,
'\ '/
0.1 -
\ L
-
1
'
0.0--
-c
Figure 3. Ultraviolet spectrum of disulfiram, 100 u g per ml

and 10 ,,g per ml, in methanol, 1 cm cells.
> 100-
-
I-
c
4
?W! 80-
m f-
z
- 60-
w
2 40-
I-r
I50 200 2 50 300

MASSICHARGE RAT 10
Figure 4. Mar8 spectrum of disulfiram.

N O R R I S G. N A S H AND R A Y M O N D D. D A L E Y
C H
C2H5 25\ +
N:
/
I
H C2H5
(88)
Figure 5. Mass spectrometric fragmentation (9).
176
DlSULFlRAM
Solvent Approximate S o l u b i l i t y , mg/ml
Me t hano 1 25
Ethanol (95%) 22
Ace tone 440
Water 0.3
Chloroform 740
Ether 80
Benzene 560
Petroleum e t h e r 2.9

Grabar and McCrone reported the c r y s t a l p r o p e r t i e s
of d i s u l f i r a m (11). The c r y s t a l system is-monociinic,
with a x i a l r a t i o s a:b:c of 0.870 : 1 : 0.545, i n l e r f a c i a l
angles ( 0 1 1 ~o i l ) = 47.5', p r o f i l e angle ( 0 1 1 ~011 i n 100
plane) = 123', and beta angle = 126'. The r e f r a c t i v e in-
dices (5893 A, 25'C) a r e cu=1.590 + 0.005, @=1,67+ 0.01,
and y=1.740 + 0.005. The o p t i c a x i a l angle is 2V-= 84 2
5', dispersion v > r , o p t i c a x i a l plane 010, s i g n of
double r e f r a c t i o n negative. The molecular r e f r a c t i o n is
84.2 (calculated), 85.0 (observed). Some a d d i t i o n a l in-
formation regarding c r y s t a l h a b i t , fusion behavior, and x-
ray d i f f r a c t i o n is a l s o given i n t h i s paper. No polymor-
p h i s m was observed.
Crabar and McCrone (11) a l s o reported x-ray d i f -
f r a c t i o n data f o r disulfiram. They found the c e l l dimen-
sions t o be a313.844, b=15.90A, and c=8,66A, with four
formula weights per c e l l . The d-spacings and r e l a t i v e in-
t e n s i t i e s of the p r i n c i p a l d i f f r a c t i o n l i n e s a r e tabulated.
Karle e t a 1 (12) made a complete c r y s t a l s t r u c t u r e
a n a l y s i s . The molecule contains two planar S
I1 ,c-
-S-C-N,
C-
groups, nearly perpendicular t o each other. The configu-

r a t i o n about the nitrogen atoms is planar, not pyramidal.
The space group i s P21/c, with four molecules i n the u n i t
c e l l . Their u n i t c e l l parameters were a = l l . l l 2 .02,
-
b=15.90 2 .03, c=8.66 + .02, 8=92' 42' -+
15'. The u n i t
177
TABLE I
X-RAY POWDER DIFFRACTION
PATTERN OF DISULFIRAM
-
d (A) I/I1 d(A) I/I1
9.11 LO 3.18 23
7.94 8 3.17 23
7.59 27 3.08 5
6.36 100 3.05 6
6.15 37 3.00 16
5.55 9 2.91 10
5.24 14 2.83 3
5.11 49 2.80 4
4.77 7 2.77 12
4.55 21 2.70 4
4.50 11 2.65 3
4.40 1 2.61 2
4.32 12 2.56 2
4.21 9 2.52 10
4.16 60 2.48 8
4.08 8 2.44 5
3.96 13 2.38 3
3.83 3 2.33 3
3.79 4 2.29 4
3.73 1 2.26 4
3.62 27 2.22 10
3.54 1 2.20 5
3.46 19 2.15 7
3.41 Ll 2.06 5
3.34 7 1.88 4
3.28 8 1.71 2
3.23 5
178
DlSULF IRAM
c e l l reported by Grabar and McCrone can be mathematically

transformed i n t o t h i s one.
The x-ray powder d i f f r a c t i o n p a t t e r n f o r d i s u l -
firam is shown i n Table I. This p a t t e r n was obtained on a
Norelco x-ray diffractometer w i t h n i c k e l - f i l t e r e d copper
Kcr radiation. This p a t t e r n c l o s e l y resembles t h a t r e p o r t -
ed by Grabar and McCrone (11).
2.8 Melting Point

The following melting p a i n t s have been reported:
67-71'C
70.5
68.4
74
69-70
72
69.5-70
3. SYNTHESIS
Disulfiram has been prepared i n a v a r i e t y of ways.

The usual method involves oxidation, by various means, of
diethyldithiocarbamic a c i d , i t s sodium s a l t , or some o t h e r
metal s a l t , t o give the d i s u l f i r a m product d i r e c t l y . Oxi-
dants used include hydrogen peroxide (18, 19, 2 0 , 72), so-
dium hypochlorite ( 2 1 , 2 2 ) , chlorine o r bromine ( 1 3 , 15,
2 3 ) , sodium n i t r i t e and hydrochloric a c i d ( 2 4 , 731, PO-
tassium ferricyanide (14), a i r ( 2 5 ) , and amonium persul-
f a t e (16). An a l t e r n a t i v e method is the r e a c t i o n of d i -
ethylamine monosulfide with carbon d i s u l f i d e (17).
These r e a c t i o n s are shown i n Figure 6.
4. STABILITY-DEGRADATION
No r e p o r t s of s t a b i l i t y s t u d i e s or of the forma-
t i o n of degradation products were found i n the l i t e r a t u r e ,
except f o r metabolic products (Section 5 ) . Several uni-
d e n t i f i e d degradation products a r e formed i f an a l k a l i n e
s o l u t i o n of d i s u l f i r a m i n methanol is heated t o 50°C f o r
2 hours, however, and a t least one unidentified product is
formed when a n a c i d i c methanol s o l u t i o n is heated s i m i -
l a r l y (26).
179
N
h
N
n
W
X
N
X
u +
cn-y+
W
v)
h
N
- l %
X
u
N
I X
v) U
I v
v):u I
I
F
X
rn
v ) - yI
zN
?
n
N
XN
n
+
VJ
N
N
n X
N U
X
u XW
u
.r(
W VI
3: U 0)
u
W s
R
.
v)
N..
v)
u
N 9
+
N
n
m
I X
X
+
N
rl
u
N W
v) v):u U
0 v)
+
i5N
N
zN
n
X
U
hl
X*
+
!2
n
hl
hl
+
v)
h
N
N
X U X X
0 W U U
W Pl W
X X X
0
w
U
v
u
W
.
h)
.
r( N
180
DlSULF I RAM
5. DRUG METABOLIC PRODUCTS
The reported metabolic products of disulfiram in

various species are as follows:
Metabolite Species
Die thyldithiocarbamic Acid Man

Rat
Rabbit
Cattle
Mice
Carbon Disulfide Man

Rat
Rabbit
Cattle
Guinea Pig
N,N-Diethyldithiocarbamoyl- Man (36 1

1-thio-l3-D-glucopyranosid- Rat (31932)
uronic Acid
Diethyldithiocarbamic Acid, Mice (34)

Methyl Ester
Hydrogen Sulfide
Sulfate Rat (31)
Gessner and Jakubowski have proposed the metabolic

scheme in Figure 7 ( 3 4 ) .
6. METHODS OF ANALYSIS

Disulfiram is easily identified by the physical
properties described in section 2 above. Where identifi-
cation of disulfiram in formulations is necessary, i t can
be extracted by solvents such as carbon disulfide. If
identification of very small amounts is necessary, the
colorimetric tests described in section 6 . 3 or polarog-
181
CH3CHZJ, CH2-CH3
,N-C
‘\\
,C-N,
/
CH3CH2 ‘s-s CH -CH3

2
CH CH CH3-CH S CH3-CH2, 4S
2;~-~>- 2XN-C4 +
-. ,N-C A
CH3CH2 CH3-CH2 ’ ‘SH CH3-CH2 \ s-EH- (cH&1,-dHcmH

+
1
\
\ \
cs2 S
\
\
4
CH3CH2.N-C \
CH3CH2 ’ \
22
\
\
\
\
Y =
CH3-CH2. 4S HSCH~+ s04

P C \ HCHO
CH3-CH2
..OHI
, -
A-‘ --a
Other Glucuronic Acid
Metabolites Conjugate
F i g u r e 7. Metabolic products of d i s u l f i r a m (34).

DISULFIRAM
raphy (section 6.7) may be useful.

The elemental composition of d i s u l f i r a m is as f o l -
lows :
Element 7. Theory
Carbon 40.50
Hydrogen 6.80
Nitrogen 9.45
Sulfur 43.25

The b a s i s f o r many of the colorimetric methods f o r
disulfiram i s the formation of highly colored metal-di-
ethyldithiocarbamate complexes. Very s e n s i t i v e procedures
have been developed f o r use with a v a r i e t y of types of
samples. Copper is the most frequently used complexing
metal.
Domar e t a 1 (27) developed a c o l o r i m e t r i c method
f o r disulfiram i n excreta samples, based on the r e a c t i o n
with cuprous iodide t o form the copper-diethyldithiocar-
bamate complex. Disulfiram was e x t r a c t e d with carbon t e t -
rachloride. The e x t r a c t was t r e a t e d with cuprous iodide
t o y i e l d a brown-yellow complex. Diethyldithiocarbamic
a c i d , a d i s u l f i r a m metabolite, was determined by t r e a t i n g
the carbon t e t r a c h l o r i d e extracted aqueous phase with cu-
p r i c s u l f a t e i n a buffer s o l u t i o n , followed by carbon t e t -
rachloride e x t r a c t i o n . Domar's method was adapted t o the
determination of metabolites i n blood and urine by s e v e r a l
i n v e s t i g a t o r s (29,37,38,39). Pa tzch (40) subs t i tuted cu-
p r i c chloride f o r the cuprous iodide used by Domar.
Parquot and Mercier (41) determined d i s u l f i r a m i n
f a t samples a t the 5 t o 50 u g per g level. The d i s u l f i r a m
was extracted i n t o petroleum e t h e r and reduced t o d i e t h y l -
d i thiocarbamate. The copper-dithiocarbamate complex was
formed by treatment with a cupric sulfate-hydroquinone re-
.
agent, extracted i n t o chloroform, and determined colorim-
e t r i ca 1l y
Egorova and Trukhina (42) determined d i s u l f i r a m i n
suppositories by carbon t e t r a c h l o r i d e e x t r a c t i o n , r e a c t i o n
with cuprous iodide, and colorimetric measurement a g a i n s t
a blank containing no cuprous iodide.
183
Vignoli and Cristau (43) determined disulfiram by

reduction of phosphomolybdotungstate in alkaline solution,
to form a blue color.
Fried et a1 (44) adapted the disulfiram identifi-
cation color reaction in Clarke's manual of drug identifi-
cation (45) to develop a quantitative procedure. Reaction
of disulfiram with ethanol and sodium cyanide produces a
blue color. The response is linear in the range 2 to 20
u g disulfiram per ml. Diethyldithiocarbamate and carbon
disulfide do not interfere.
Kofman and Arzamastsev (46) and Piotrowska (47) re-
ported determining disulf iram by combustion by the oxygen
flask method. The sulfur dioxide produced is bound as a
nonvolatile complex by sodium tetrachloromercurate. The
complex is determined colorimetrically by reaction with
formaldehyde and pararosaniline in hydrochloric acid solu-
tion.
6.4 Titration Methods

Disulfiram is not sufficientlv basic to be titrated
with acid even in non-aqueous media. However, several ti-
tration methods based on complex formation or oxidation
have been reported.
Ferreira (48) titrated an acidified carbon tetra-
chloride solution, containing from 15 to 25 mg of disul-
firam, with a 0.15 bromate-bromide solution. The follow-
ing reaction is given.
s, s
(C H ) -N-C-S-S-C-N-(C H )
2 5 2 2 5 2
+ 13 Br2 + 20 H20
26 HBr + 4 H2S04 + 2 HCOOH + 2 (C2H5)2NH
Analyses of the reaction mixture for sulfate and diethyl-

amine, as well as the amount of bromine consumed, support
the stoichiometry given above.
Varga (49) determined disulfiram in the drug sub-
stance and tablets by oxidation with excess 0.1l ceric
sulfate and determination of the excess reagent by back
titration with 0.1E ferrous sulfate. Sandri (50) oxidized
disulfiram with an excess of periodic acid; after 30 min-
utes the excess periodic acid was determined volumetrically
by addition of potassium iodide and titration of the io-
dine formed.
184
DlSULFlRAM
Roth ( 5 1 ) determined disulfiram i n crude products

and fungicide formulations a f t e r conversion t o carbon di-
s u l f i d e . The sample was refluxed i n formic acid and the
carbon d i s u l f i d e evolved was trapped i n an a l k a l i n e cadmium
a c e t a t e solution. The cadmium a c e t a t e s o l u t i o n was neu-
t r a l i z e d and t i t r a t e d with 0.1E methanolic iodine t o a
starch-iodine end point,
Bayer and Posgay (52,53) determined t h a t the addi-
t i o n of mercuric a c e t a t e t o a g l a c i a l a c e t i c acid s o l u t i o n
of disulfiram formed a complex t h a t consumed 2 moles of
perchloric acid per mole of disulfiram. The sample was t i -
t r a t e d t o a gentian v i o l e t end point with perchloric acid
i n g l a c i a l a c e t i c acid.
Bukreev e t a 1 (54) treated disulfiram with 0.1E
n i c k e l s u l f a t e i n an alcohol-ammonia solution. After 1
hour the excess nickel ion was t i t r a t e d with 0.lE t e t r a -
sodium edetate. The accuracy of the method was s t a t e d t o
be within 2 0.6%. Li and Li ( 5 5 ) t r e a t e d a dioxane solu-
t i o n of tetramethylthiuram d i s u l f i d e with an excess of
s i l v e r n i t r a t e s o l u t i o n , separated the p r e c i p i t a t e , and
determined the excess silver ion by t i t r a t i n g with thiocy-
anate solution. Two moles of s i l v e r reacted with 1 mole
of tetramethylthiuram d i s u l f i d e .
Fournier e t a 1 (56) determined disulfiram metabo-
l i t e s and other compounds with C-S bonds i n urine samples
by the c a t a l y t i c e f f e c t of the metabolites on the r e a c t i o n
of iodine with iodazide. Scalicka ( 5 7 ) used sodium azide
instead of iodazide t o determine d i s u l f iram metabolites by
the c a t a l y t i c e f f e c t ; q u a n t i t a t i o n was by t i t r a t i o n of ex-
was comp l e ted .

cess sodium azide with sodium arsenate a f t e r the r e a c t i o n
Stefek e t a 1 ( 5 8 ) determined the nitrogen content

by a Kjeldahl method. The ammonia was collected i n a 2%
boric acid s o l u t i o n , cooled i n an i c e - s a l t mixture, and
t i t r a t e d with 0.05t-J hydrochloric acid. The standard devi-
a t i o n f o r the determination of nitrogen was l e s s than 0.2%.
6 . 5 Infrared Spectroscopy
Salvesen e t a 1 ( 5 9 ) determined disulfiram i n tab-
l e t s by infrared spectroscopy. The s t r e n g t h was d e t e r -
mined i n a carbon t e t r a c h l o r i d e s o l u t i o n using the absorp-
t i o n bands a t 7.41, 8.32, 9.92, and 10.31 microns with a
r e l a t i v e e r r o r of 2%.
The band a t 818 cm” (carbon d i s u l f i d e s o l u t i o n )
185
NORRIS G. NASH AND RAYMOND 0.DALEY
can a l s o be used. Diethyldithiocarbamic a c i d has no ab-

s o r p t i o n bands near 818 cm'l.
6.6 Proton Magnetic Resonance Spectroscopy

Sheinin e t a1 ( 6 0 ) determined d i s u l f i r a m i n the
drug substance and t a b l e t s by proton magnetic resonance
spectroscopy. The average r e s u l t s on a t a b l e t composite
were 100.8 2 1.4% of claim by magnetic resonance spectro-
scopy and 100.7 -+
0.4% by a c o l o r i m e t r i c procedure (61).
6 . 7 Polarography
Belitskaya (62) analyzed d i s u l f irarn i n rubber
samples by micropolarographic procedure a t a 0.003M l e v e l
using the wave a t -2.5V i n an ammonia-ammonium c h l o r i d e
buffer. Taylor (63) used cathode-ray polarography t o make
the determination i n the 1.5 t o 15 ppm range on rubber
samples; a t t h i s l e v e l a l i n e a r response was obtained.
Porter e t a 1 ( 6 4 ) used cathode-ray polarography on urine
samples and increased the s e n s i t i v i t y and s p e c i f i c i t y by
the a d d i t i o n of copper t o the sample. The wave f o r t h i -
uram a t -0.5V was replaced by a wave a t -0.65V and t h a t of
the excess copper a t -0.23V. The method gave 85% recovery
on urine samples a t the 0.5 ,,g per m l l e v e l and 95% re-
covery on samples e x t r a c t e d w i t h chloroform t o give a 2 , g
per m l s o l u t i o n .
Prue e t a 1 (65) used pulse polarography f o r de-
termining d i s u l f iram i n t a b l e t samples. The pulse polar-
ographic method i s more s e n s i t i v e than d.c. polarography
and the peak is e a s i e r t o i d e n t i f y and q u a n t i t a t e . The
use of an aqueous-ethanol a c e t a t e b u f f e r r a t h e r than an
aqueous-e thanol a m o n i a b u f f e r e l i m i n a t e s the i n t e r f e r e n c e
of diethyldithiocarbamate, increasing the s p e c i f i c i t y of
the method.
6.8 Gravimetric Methods

Three papers
- - describe oxidation of d i s u l f iram i n
various ways t o produce s u l f a t e , determined gravimetric-
a l l y as barium s u l f a t e . F e r r e i r a ( 4 8 ) used bromine,
Wojahn and Wempe ( 6 6 ) used s t r o n g a l k a l i , and Parrak ( 6 7 )
used n i t r i c a c i d in the presence of amonium vanadate.
6.9 Chromatography
Parker and Berriman (68) developed a s e p a r a t i o n
system u t i l i z i n g s i l i c a g e l - C e l i t e adsorbents and 4
186
DlSULFlRAM
binary solvent mixtures to separate 32 rubber additives,

one of which was disulfiram.
S t r o m e (32) developed a method for the separation
of proteins, diethyldithiocarbamate, and disulfiram in di-
luted plasma, liver homogenate, and diluted urine using a
Sephadex G-25 gel filtration column.
Goeckeritz (69) used paper chromatography to sepa-
rate disulfiram from 4 other antimycotic drugs using paper
impregnated with one of the following: (a) heptane; (b)
heptane-xylene (1:l); (c) cyclohexane; (d) methanol-etha-
no1 (1:l); (el acetone with dimethylformamide; (f) paraf-
fin. As stationary solvents acetone or hexane was used.
For detection, iodoazide or mercury fluorescein was used.
Gessner and Jakubowski (34) used descending paper
chromatography on Whatman No. 1 or No. 4 paper to separate
disulfiram metabolites. The following systems were em-
ployed: (a) n-hexane on paper impregnated with 50% di-
methylformamide solution in ethanol; (b) cyclohexane-n-
butanol (1:l) on paper impregnated with 507. ethanol solu-
tion of dimethylformamide. Detection was by observation
of quenching spots under ultraviolet light.
Farago (70) chromatographed biological extracts on
silica gel G plates. The Rf values for disulfirarn were
(a) 0.82, using methanol-ace tone-triethanolamine (1:1 :
0.03); and (b) 0.77, using ethylene dichloride-benzene-88%
formic acid-H20. Palladium chloride was used to detect
disulfiram. The unsprayed spot can be eluted from the
silica gel with chloroform-ethanol (2:l) and the absorb-
ance determined at 272 nanometers .
Drost and Reith (71) chromatographed biological
extracts on Kieselgel G plates. Two solvent systems were
used: (a) toluene-ethyl acetate-diethylamine (7:2:1), and
(b) cyclohexane-diethylamine (9:l). The spots were de-
tected by ultraviolet light irradiation or by spraying with
a mixture of 3 m l 107. H2PtCl6, 100 ml 6% KI, and 47 ml of
water. Unsprayed spots were scraped off and extracted
with 1,2-dinitrochloroethane; the solution was evaporated
and the residue taken up in methanol for ultraviolet de-
termination.
187
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33. J. H. Stromme, Biochem. Pharmacol. 15, 287-97
(1966).
34. T. Gessner and M. Jakubowski, Biochem. Pharmacol.
-
21, 219-30 (1972).
35. E. Merlevede and J. P e t e r s , Arch. Belg. Med. SOC.,
Hyg., Med. Trav. Med. Leg. 23, 513-51 (1965); C.A.
36.
-
64, 1 8 2 2 4 ~ .
J. Kaslander, Biochim. Biophys. Acta 71, 730-2
(1963).
37. K. J. D i v a t i a , C. H. Hine, and T. N. Burbridge,
J. Lab. Clin. Med. 2, 974-82 (1952).
38. S. L. Tompsett, Acta Pharmacol. Toxicol. 2, 20-2
(1964).
39. G. Bors and N. I o a n i d , Farmacia (Bucharest) 2,
593-a (1965); C.A. 64, 8 7 8 4 ~
40. H. Patzsch, Deut. Apoth. Ztg. 94, 1284-6 (1954);
C.A. 53, 202291.
41 C. P a q u o t and J. Mercier, Rev. Franc. Corps Gras
-
6 , 695-9 (1959); C.A. 54, 6158g.
42. N. M. Egorova and V. I. Trukhina, Nauch. T r . Perm.
-
Farm. I n s t . 4, 96-7 (1971); C.A. 79, 149338e.
189
43. L. Vignoli and B. Cristau, Congr. SOC. Pharm. Fr.

C. R., 9th, 221-8 (1957); C.A. 53, 20690f.
44. R. Fried, A. N. Masoud, and M. Francis, J. Pharm.
45 . Sci. 62, 1368-9 (1973).

E. G . C . Clarke, Editor, " I s o l a t i o n and I d e n t i f i -
c a t i o n of Drugs", The Pharmaceutical Press, London,
England, 1969, p. 319.
46. M. D. Kofman and A. P. Arzamastsev, Farm. Zh.
(Kiev) 26, 52-6 (1971); C.A. 75, 91338d.
47. A. P i o t G s k a , Diss. Pharm. Pharnucol. 24, 93-7
(1972); C.A. 76, 158430~.
48. P. C. F e r r e i r a , Arquiv. biol. (Sao Paulo) 34,
103-5 (1950)
49. E. Varga, A c t a Pharm. Hung. 28, 38-43 (1958);
C.A. 53, 2541bc.
50. G , Sandri, A t t i Accad. Sci. Ferrara 35, 17-24
(1957-58); C.A. 54, 18160d.
51. H. Roth, Angew. Chem. 73, 167-9 (1961); C.A. 55,
13161e.
52. I. Bayer and Mrs. G. Posgay, Acta Pharm. Hung. 2,
43-50 (1961); C.A. 56, 7431f.
53. I. Bayer and E. Posgay, Pharm. Zentralh. 100,
65-71 (1961); C.A. 55, 14819a.
54. A. I. Bukreev, V. V. Hinakova, and V. F.
Soinikova, Med. Prom. SSSR l8, 32-3 (1964).
55. M. L i and K. L i , Chung-Shan Ta Hsueh Hsueh Pao-Tzu
Jan K'o Hsueh, 1959 (3), 26-8; C.A. 56, 6667.
56. E. Fournier, L. P e t i t and A. Lecorsier, J. Eur.
Toxicol. 2, 337-40 (1971); C.A. 76, 149631h.
57. B. Scalicka, Prac. Lek l9, 408-12 (1967); C.A.
-
68, 58318e.
58. S. Stefak, M. Blesova, and M. Zahradnicek, Cesk.
Farm. 21, 64-6 (1972); C.A. 77, 39330~.
59. B. S a l K s e n , L. Domange, J. xy, Ann. Pharm. Fr.
60
-13, 208-15 (1955); C.A. 49, 11959a.
E. B. Sheinin, W. R. Benson and M. M. Smith, Jr.,
J. Ass. Offic, Anal. Chem. 56, 124-7 (1973).
61, "Official Methods of Analysis", 1970, 11th edition,
Association of O f f i c i a l Analytical Chemists,
Washington, D. C., s e c t i o n 29.148.
62. R. M. Belitskaya, Kauch. Rezina l9, 53-5 (1960);
C.A. 55, 4025e.
63. A. F. Taylor, Talanta ll, 894-6 (1964).
190
DlSULFlRAM
64 G. S. P o r t e r and A. Williams, J. Pharm. Pharmacol.
65.
-
24, 144-145P (1972)
D. G. Prue, C. R. Warner, and B. T. Kho, J. Pharm.
Sci. 61, 249-51 (1972).
66. H. Wozhn and E. Wempe, Arch. Pharm. (Weinheim)
285, 375-82 (1952); C.A. 47, 12126g.
7
67. V. Parrak, Farmacia 22, 19-22 (1953); C.A. 49,

5776g.
68. C. A. Parker and J. M. Berrimn, Trans. Inst.
Rubber Ind. 28, 279-96 (1952); C.A. 47, 36021.
69. D. Coeckeritz, Pharmazie 2,668-74 (1966); C.A.
-
66, 98510b.
70. A. Farago, Arch. Toxikol. 22, 396-9 (1967); C.A.
71.
-
67, 79553x0
R. H. Drost and J . F. Reith, Pharm. Weekbl. 105,
1129-38 (1970); C.A. 74, 23165.
72. A. D. Cumnings and H. E. Siamons, Ind. Eng. Chem.
20, 1173-6 (1928).
c
73. J. Giral and C. Soler, Rev. SOC. Quim. Mex. 2,

167-70 (1958); C.A. 53, 11220d.
ACKNOWLEDGMENTS
The w r i t e r s wish t o thank Dr. B. T. Kho f o r h i s r e -

view of the manuscript, Dr. G. S c h i l l i n g of Ayerst Research
Laboratories f o r h i s NMR and mass s p e c t r a l data, the li-
brary s t a f f f o r t h e i r l i t e r a t u r e search, and Mrs. B. Juneau
.
f o r typing the p r o f i l e
191
ESTRADIOL VALERATE
Klaus Florey
ESTRADIOL VALERATE
CONTENTS
1. Description
1.1 Name, Fornula, Ylolecular Weight
1.2 Appearance, Color, odor
2.4 Mass Spectrum
2.5 Rotation
2.6 Melting Range
2.7 Solubility
3. Synthesis
6.2 Spectrophotometric Analysis
6.3 Spectrofluorometric Analysis
6.51 Paper
6.52 Thin-Layer
6.53 Gas - Liquid
7. Identification and Determination in Body

Fluids and Tissues
8. References
193
KLAUS FLOREY
1. Description
Estradiol Valerate is estra-l,3,5 (10)
triene-3,17p-diol-17-valerate (pentanoate).
c2 3H3203 Mol. Wt. 356.51
1.2 Appearance, Color,Odor

White, odorless, crystalline powder.
The infrared spectrum of estradiol
valeratel is presented in Figure 1.

The NMR spectrum is presented in
Figure 2. It was obtained on a 60 MHz
spectrometer in deuterochloroform containing
tetramethylsilane as an internal reference
The following proton assignments were made2 :
194
Protons at Chemical Shift Coupling Constants
T T (in Hz)
C- 1H 2.86 (doublet) 1Hy2H = 9
C-2H 3.38 (quartet) 1H, 2H = 9 ; 2 H y 4 H =2.5
C-4H 3.43 (multiplet) 2 H y 4 H = 2.5;4H, 6H =1 ;

4
C- 18H 9.19 (singlet)

r
\D
VI
C-17aH 5.25 (triplet) 16H, 17H = 7.5
ester - CH3 9.09 (triplet) 6.5
phenolic-OH 4.94 (singlet) Concentration

dependent
FREQUENCY (CM')
c
W
Q\
WAVELENGTH (MICRONS)
Figure 1. Infrared Spectrum of Estradiol Valerate in Mineral Mull.

Instrument: Perkin-Elmer 621.
I
1_ T I
F i g u r e 2. NMR Spectrum of E s t r a d i o l V a l e r a t e i n D e u t e r a t e d Chloroform.

Perkin-Elmer R-12B.
KLAUS FLOREY
EtoH 281 nm E1% 613
Xmax 1c m
Fmax 281 nm; = 20906

I m a x 287 nm; = 1800 ( s m a l l peak)
6
2.4 Mass Spectrum

The low r e s o l u t i o n mass spectrum,
shown i n F i g u r e 3 , was o b t a i n e d on an A E I MS-902
s p e c t r o m e t e r equipped w i t h a frequency modulated
analog r e c o r d e r 2.
I t demonstrates the e x p e c t e d M+ o f
m/e 356. There i s a l s o t h e M+ o f an a c i d
homolog a t m / e 370 p r e s e n t a s a minor component.
T h e h i g h mass fragment i o n a t m / e 271 o c c u r s by
the c l e a v a g e of t h e a c y l p o r t i o n of t h e e s t e r a t
t h e C-17 p o s i t i o n . There i s complement fragment
i o n a t m/e 8 5 t h a t r e p r e s e n t s t h e o t h e r p o r t i o n
of t h e molecule. The i o n s a t m/e 253-255
r e p r e s e n t t h e c l e a v a g e of t h e e n t i r e C-17 moiety.
Because i t i s an a r o m a t i c s t e r o i d , there a r e a
series of s t r o n g fragment i o n s w i t h t h e
p r o g r e s s i v e loss of f i r s t D-ring carbons, t h e n
C-ring carbons and f i n a l l y B-ring c a r b o n s , The
m/e 1 0 7 i o n c o n t a i n s t h e A-ring and t h e C-6
carbon. Phenols can a l s o e l i m i n a t e t h e oxygen
by e x p l u s i o n of CO o r HCO. Thus a second s e r i e s
of p r o g r e s s i v e loss o f carbons can o c c u r b u t
does n o t produce a s many i o n s . The assignment
o f some o f t h e d i a g n o s t i c i o n s i s d e p i c t e d
below.
198
57
H2 -CH2 -CH2 -CH3
HO
(+1H)107
1 hlhl
c
h)
0 6
0 3hl
IZI
2hl
lhl
MASSKHARGE
F i g u r e 3. Low R e s o l u t i o n Mass Spectra of E s t r a d i o l V a l e r a t e .

I n s t r u m e n t : AE1-902.
ESTRADIOL VALERATE
2.5 Rotation
[a& + 44O (dioxane)3
2.6 Melting Ranqe

Like many steroids, Estradiol Valerate
does not melt sharply. The following melting
range temperatures (OC) were reported:
144-1454 (from methanol-water)
156
146-1486
1477
142-1442
The thermomicroscopic6 and fusion properties5 of
estradiol valerate have also been described.
2.7 Solubility
Practically insoluble in water:
soluble in caster oil, in methanol, in benzyl-
benzoate and in dioxane; sparingly soluble in
sesame oil and in peanut oil8 .
No polymorphism was found7. Isomorphic
mixed crystals were formed with estradiol
propionate5 .
The powder X-ray diffraction pattern
is presented in Table 1’.
20 1
KLAUS FLOREY
Table I
Powder X-ray D i f f r a c t i o n P a t t e r n
of E s t r a d i o l V a l e r a t e
11.5 0.06 3.50 0.19

10.0 0.60 3. 3 1 0.34
6.95 0.21 3.22 0.08
6.21 0.24 3.14 0.07
5.97 0.51 3.02 0.09
4.98 1.00 2.96 0.08
4.68 0.46 2. 32 0.05
4.12 0.34
3.96 0.32
3.83 0.13
*d = i n t e r p l a n a r d i s t a n c e na
2 sin@
= 1.539A; R a d i a t i o n : K a l and K a l Copper
**
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
of 1.00
3. Synthesis
The s y n t h e s i s of e s t r a d i o l v a l e r a t e w a s f i r s t
d e s c r i b e d b y Miescher and S c h o l z 4 by
m e t h a n l y s i s of e s t r a d i o l 3 , 1 7 - d i v a l e r a t e .
202
0
II
OC- (CH2)3-CH3
Alternative1 the 3-benzoate has been removed selectively with sodium

.
borohydride 2 7
Estradiol Valerate is very stable as a solid. In solution under
certain conditions, particularily alkaline, saponification to valeric acid
and estradiol can occur.
KLAUS FLOREY
5. Druq M e t a b o l i c P r o d u c t s
An i n c r e a s e i n b l o o d and u r i n e e s t r o g e n
l e v e l s was n o t e d a f t e r a d m i n i s t r a t i o n o f
e s t r a d i o l v a l e r a t e t o o v a r e c t o m i z e d lo and
postmenopausalUwomen. N o m e t a b o l i t e s have been
i d e n t i f i e d so f a r , a l t h o u g h e s t r a d i o l v a l e r a t e
most l i k e l y f o l l o w s t h e pathway of e s t r a d i o l
metabolism.
6. Methods of A n a l y s i s
% Calc. % Found 4
C 77.49 77.54;77.42
H 9.05 9.00: 9.20
0 13.46 --
The U.V. a b s o r p t i o n a t 2 8 2 nm can be
u s e d t o d e t e r m i n e e s t r a d i o l v a l e r a t e i n sesame
o i l a f t e r e x t r a c t i o n w i t h 80% m e t h a n o l , however,
w i t h an a c c u r a c y o f n o t b e t t e r t h a n 10%
b e c a u s e o f i n t e r f e r e n c e from t h e o i l 1 5 . In the
compendia1 a s s a y t h e a b s o r b a n c e o f an a l k a l i n e
a s s a y s o l u t i o n a t 300 nm i s d e t e r m i n e d a g a i n s t
an a c i d i c a s s a y p r e p a r a t i o n 8 .
6.3 Spectroflurometric Analysis

A q u a n t i t a t i v e f l u o r o m e t r i c method f o r
determination of e s t r a d i o l v a l e r a t e i n o i l has
been d e s c r i b e d 1 6 . A f t e r column c l e a n up t h e
s t e r o i d concentration i n ethanol is determined
i n a s p e c t r o f l u o r o m e t e r ( E x c i t a t i o n wave l e n g t h :
285 nm: F l u o r e s c e n c e maximum ca. 328 nm).
E s t r a d i o l v a l e r a t e responds t o color-
imetric t e s t s used f o r e s t r o g e n s such a s t h e
phenol r e a g e n t ( F o l i n - C i o c a l t e a u reagent,phospho-
*,
molybdic p h o s p h o t u n g s t i c a c i d ) i r o n - p h e n o l solu-
t i o n 1 7 a n d t h e I t t r i c h m o d i f i c a t i o n o f t h e Kober
204
reaction (see section 7).
6.51 Paper
The quantitative determination of estradiol valerate by
paper chromatography has been described18. After spotting the paper strip
strips were impregnated with 20% diethylene glycol monoethylether(Carbitol)
in chloroform and developed for 3 hours with methylcyclohexane saturated
with diethylene glycol monoethyl ether. The steroid is located on a guide-
strip by spraying with Folin-Ciocalteau reagent, eluted with ethanol and
quantitated fluorometrically. (Activation wave length at 280 nm,
fluorescence at 310 nm). In this system estradiol stays at the origin.
6.52 Thin-Layer
t
4 Thin-layer chromatographic systems have been tabulated
0
in Table 11. Table I1
Absorbant Solvent S y s tem
Magnesium silicate Benzene-ethanol(9:l)
- -
Ref.
0.52
Ref.
19
Magnesium silicate Chloroform 0.62 19
Silica gel G Stationery phase:mineral oil
Mobile phase:50% acetic acid 0.64 20
Silica gel G Stationery phase:propylene glycol
Mobile phase:cyclohexane-pet. ether (1:l)
1 0.60 22
Silica gel G Stationery phase:tetraethylene glycol
Mobile phase:xylene 3 -- 24
Detection systems. Phosphoric acid (1:5)spray (green color);conc. sulfuric
acid, p-toluenesulfonic acid.
KLAUS FLOREY
Quantitative determination i n harmaceutical

p r e p a r a t i o n h a s b e e n d e s c r i b e d5 3 , The t e t r a -
e t h y l e n e g l y c o l - x y l e n e system24 s e p a r a t e s
e s t r a d i o l v a l e r a t e from e s t r a d i o l a n d i s t h e
b a s i s f o r a compendia1 l i m i t t e s t f o r f r e e
estradiol.
6.53 Gas-Liquid
E s t r a d i o l v a l e r a t e can b e
d e t e r m i n e d q u a n t i t a t i v e l y b y g a s chromatography
u s i n g a 3% OV-17 on 80-100 mesh V a r a p o r t 30
column a t a column t e m p e r a t u r e o f 260025 .
A l t e r n a t e l y a 3% J X R ( m e t h y l s i l i c o n e po1ymer)on
s i l a n i z e d 100-200 mesh Gas C h r o m P column a t a
column t e m p e r a t u r e of 230° c a n be u s e d f o r t h e
tetramethylsilyl derivate26.
7. I d e n t i f i c a t i o n and D e t e r m i n a t i o n i n Body
F l u i d s and T i s s u e s .
A method12 h a s b e e n d e v e l o p e d t o d e t e r m i n e
e x t r e m e l y low c o n c e n t r a t i o n o f t o t a l e s t r o g e n s
i n b o v i n e t i s s u e s , making u s e o f a s h o r t e n e d
v e r s i o n o f t h e e x t r a c t i o n p r o c e d u r e of
G o l d z i e h e r 1 3 , f o l l o w e d by f l u o r o m e t r i c r e a d o u t
u s i n g t h e I t t r i c h 1 4 m o d i f i c a t i o n of t h e Kober
reaction.
206
ESTRADIOL VALERATE
8. References
1. B. T o e p l i t z , The S q u i b b I n s t i t u t e , P e r s o n a l
Communication.
2. A. I. Cohen, The S q u i b b I n s t i t u t e , P e r s o n a l
Communication.
3. N. H. Coy a n d C. M. F a i r c h i l d , The S q u i b b
I n s t i t u t e , P e r s o n a l Communication.
4. K. Miescher a n d C. S c h o l z , H e l v . Chim.Acta
20,1237(1937): U.S.Patent, 2,233,035 (1941).
5. J. P. C r i s l e r , N. F. W i t t a n d M. H. C r i s l e r ,
Microchimica A c t a 1962,317.
6. M. K u h n e r t - B r a n d s t a t t e r , E. J u n g e r a n d
A . Kof l e r , Microchem. J. 2 , 1 0 5 ( 1 9 6 5 ) .
7. M. B r a n d s t a t t e r - K u h n e r t a n d E. J u n g e r ,
Microchimica Acta 1964,238.
8. U.S.P. X V I I I
9. Q. Ochs, The S q u i b b I n s t i t u t e , P e r s o n a l
Communication.
10. G. I t t r i c h a n d P. P o t t s , A b h a n d l . D e u t .
Akad. W i s s , B e r l i n , K1.Med. 1 9 6 5 , 5 6 ;
C.A. 64,11501 4 . (1966).
11. R. K a i s e r , Symp. D e u t . G e r . E n d o k r i n o l .
-
8 , 2 2 7 ( 1 9 6 2 ) : C.A. 6 5 , 7 5 6 5 h ( 1 9 6 6 ) .
12. H. K a d i n , The S q u i b b I n s t i t u t e , P e r s o n a l
Communication.
13. J. W. Goldzieher, R. A. B a k e r a n d E . C . R i h a ,
J. C l i n . E n d o c r . 2 l , 6 2 ( 1 9 6 1 ) .
14. G. I t t r i c h , A c t a E n d o c r . 3 5 , 3 4 ( 1 9 6 0 ) .
15. N. H. Coy a n d C. M. F a i r c h i l d , The S q u i b b
16. Th. J a m e s , J o u r n a l of t h e AOAC 5 4 , 1 1 9 2 ( 1 9 7 1 ) :
i b i d . 56,86 (1973).
17. B r i t i s h P h a r m a c o p o e i a 1973 p. 330.
18. H. R. R o b e r t s a n d M. R. S i i n o , J. P h a r m . S c i .
-
52,370 (1963).
19. V. S c h w a r z , P h a r m a z i e 1 8 , 1 2 2 ( 1 9 6 3 ) .
20. D. S o n a n i n i a n d J. A n k e r , Pharm. A c t a . H e l v .
-
42,54 (1967).
207
KLAUS FLOREY
21. T. D i a m a n s t e i n and K. Lorcher, J. Anal.Chem.

-
191,429(1962).
2 2. A. Vanden B u l c k e , Pharm. T i j d s c h r . B e l g .
-
4 6 , 2 2 1 (1969) C.A. 7 2 , 1 0 3 7 9 0 y ( 1 9 7 0 ) .
23. N. A r i , T u r k H i j . Tecr. B i y o l . Derg. 2 9 , 2 0 0
(1969) : C. A. 73,4856933 ( 1 9 7 0 ) .
24. H. K l e i n and S. Hays, Drug S t a n d a r d s
L a b o r a t o r y , P e r s o n a l Communication.
25. Th. James and B. R a d e r , F.D.A. B y - l i n e s 4,
1 6 1( 1 9 7 2 ) .
26. G. C a v i n a , G. M o r e t t i and P. S i n i s c a l c h i ,
J. Chromatog. 4 7 , 1 8 6 ( 1 9 7 0 ) .
27. K. T s u n e d a , J. Yamada, K. Yasuda and H . M o r i ,
C h e m . Pharm. Bull. (Tokyo) ll,510 ( 1 9 6 3 ) ,
J a p a n P a t e n t 2 0 , 1 6 3 ( 1 9 6 4 ) :C. A. 62,1048421
(1965).
L i t e r a t u r e s u r v e y e d t h r o u g h December 1972.
The h e l p o f H. Gonda and A . Mohr i n t h e

p r e p a r a t i o n of t h i s p r o f i l e i s g r a t e f u l l y
acknowledged.
208
HYDROXYPROGESTERONE CAPROATE
Klaus Florey
KLAUS FLOREY
CONTENTS
1. Description
1 . 2 Appearance, Color, Odor
2.4 Mass Spectra
2.5 Rotation
2.6 Melting Range
2.7 Solubility
3. Synthesis
6.4 Polarimetric Analysis
6.61 Paper
6.62 Thin-Layer
7. References
210
H Y DROXYPROGESTE RONE CAPROATE
1. Description
#
Hydroxyprogesterone caproate is
17-~-(1-oxohexyl)oxy~-4pregnene-3,20-dione;
also 17-hydroxypregn-4-one-3,2O-dione hexanoate
'iH3
20 c=o OCOCH2 (CH2)3CH3
0
C27H4004 Mol.Wt. : 428.62

White or creamy white odorless
crystalline powder.
The infrared spectrum of hydroxy-
progesterone caproate is presented in figure 1.1
2.2 Nuclear Maqnetic Resonance Spectrum

The NMR spectrum is presented in figure
2. It was obtained on a 60 MHz NMR spectrometer
in deuterochloroform containing tetramethylsilane
as an internal reference. The following proton
assignments were made: 6
Protons at Chemical Shift
c-4 4.20 singlet

C-18 9.30 singlet
c-19 8.80 singlet
c-21 7.96 singlet
Ester methyl 9.08 multiplet, coupling
constant 6.5 Hz
21 1
Figure 1. Infrared Spectrum of Hydroxyprogesterone Caproate in mineral
oil mull. 1nstrument:Perkin Elmer 621.
F i g u r e 2. NMR Spectrum o f Hydroxyprogesterone C a p r o a t e i n D e u t e r a t e d
Chloroform. I n s t r u m e n t :Perkin-E l m e r R-12B.
KLAUS FLOREY
I m a x 241 nm;E = 170008
2.4 Mass Spectrum

The l o w r e s o l u t i o n mass spectrum, shown
i n f i g u r e 3, w a s o b t a i n e d on an A E I MS-902
s p e c t r o m e t e r equipped w i t h a f r e q u e n c y modulated
analog t a p e r e c o r d e r . 6
I t d e m o n s t r a t e s t h e e x p e c t e d M+ of
m/e 428. The m/e 385 i o n r e p r e s e n t s t h e loss of
t h e C-17 a c e t y l group o r C3H7 from t h e a c y l a t e ,
probably t h e former i s t h e predominant pathway.
The loss of t h e C-17 a c y l a t e g i v e s r i s e t o t h e
m/e 330 i o n and t h e m/e 312, 313 i o n s . Prominent
&
i o n s a t m/e 7 1 and m/e 99 correspond t o t h e
a c y l a t e p o r t i o n of t h e molecule. The m/e 287 i o n
i s shown below. The m / e 269 i o n can be
envisoned a s t h e d e h y d r a t e d i o n of t h e m / e 287
ion. A s e r i e s of fragment i o n s occur from t h e
p r o g r e s sbi vu et , loss of D-ring, C-ring and B-ring
carbons
m/e 269
0
m / e 287
while present, a r e n o t p a r t i c u l a r l y in ten s e
enough t o i n d i c a t e t h e p r e s e n c e of t h e A4-3-keto
group. However, t h e i o n s a t m/e 121-124 a r e -
p r e s e n t and have t h e compositions e x p e c t e d f o r
t h e A-ring group. The assignment o f some of t h e
d i a g n o s t i c i o n s i s d e p i c t e d below. 6
214
m
0
k
pc
m
u
I Q)
C
0
k
0)
4J
10
Q)
F
0
LI
pc
h
X
0
LI
a
h
X
w
0
C
LI
Q)
4J
4J
m
o(
0
&
F
215
0 0 0
W T ( 0
Figure 3. Low Resolution Mass Spectrum of Hydroxyprogesterone Caproate.

Instrument : AE1 MS-902.
2.5 Rotation
DJ;”= + 61’ ( c = 1, i n c h l o r o f o r m ) 2
2.6 M e l t i n q Ranqe
L i k e many s t e r o i d s , h y d r o x y p r o g e s t e r o n e
c a p r o a t e d o e s n o t m e l t s h a r p l y , and t h e m e l t i n g
p o i n t depends on t h e r a t e of h e a t i n g 7 .
The f o l l o w i n g m e l t i n g r a n g e t e m p e r a t u r e s
(OC) were r e p o r t e d .
117-1228
120.0-121.09
115- 1183
119-1212
120.1-121.910
The t h e n n o m i c r o s c o p i c 8 and f u s i o n
p r o p e r t i e s 1 1 of h y d r o x y p r o g e s t e r o n e c a p r o a t e h a v e
a l s o been d e s c r i b e d .
2.7 Solubility
Insoluble i n waterlo
10
50 mg/ml i n e t h y l e t h e r
1 . 2 mg/ml i n b e n z e n e l o
25-29 mg/ml i n sesame o i l 2
350-400 mg/ml i n l e v u l i n i c a c i d
butyl ester2
S o l u b l e i n a m i x t u r e of b e n z y l
b e n z o a t e and sesame o r c a s t o r o i l
10
.
217
KLAUS FLOREY
Crystal Properties
2.8
Dense needles from isopropanol2 The .
powder X-ray diffraction pattern is presented in
Table I.
TABLE I
Powder X-ray Diffraction Pattern of
Hydroxyprogesterone Caproa te2
**
d (2: I/I 1
12.2 0.26 4.64 0.21

9.1 0.11 4.53 0.33
7.15 0.31 4.05 0.09
6.90 0.10 4.00 0.11
6.63 0.13 3.88 0.10
6.35 0.37 3.80 0.11
5.80 1.00 3.70 0.15
5.47 0.42 3.62 0.11
5.28 0.21 3.30 0.08
5.08 0.28 2.88 0.09
*d = interplanar distance n b -
2 sin0
0
A= 1.539A; Radiation; K 1 and K Copper
** Relative intensity based on highest intensity
of 1.00.
218
3.
& -:fl
Synthesis
p 3
e=0
OH anhydride
c=o
CO (CH2)4CH3
Hydroxyprogesterone caproate is synthesized by acylation of

c! 17a-hydroxyprogesterone (cf.4) with caproic acid in the presence of
\D p-toluene sulfonic acid3. Variations of this basic method have also been
reported’. It can also be prepared by oxidation of the corresponding
5-prengnen-17-01-3-one caproate2.
Hydroxyprogesterone caproate is very stable a s a solid. The
17a-esterlinkeage issterically hindered and therefore quite stable, but
can be saponified under forcing conditions. In solution photolykic
degradation of the A-ring is possible, when exposed to ultraviolet light
or ordinary fluorescent laboratory lighting (cf. 12).
KLAUS FLOREY

Excretion and tissue distribution of
labeled hydroxyprogesterone caproate was studied
.
in ratsl3, steers14 and pregnant women 16
Langeckerl5 was not able to recover unchanged
hydroxyprogesterone caproate in amounts exceeding
0.1%after administration to man. Plotzl6
incubated rat liver homogenates with 4-14C-
labeled hydroxyprogesterone caproate under
anaerobic conditions and recovered allopregnane-
38, 17a-diol-20-one-17a-caproate in 25% yield
and pregnane-3@, 17a-diol-20-one-17a-caproate in
2% yield, together with some unchanged starting
material. That the caproic acid ester was not
removed attest to the stability of the bond (see
section 4) and there is evidence that metabolites
in urine also still carry the ester group. Plotz
also found that in homogenate of human placenta
the 20-keto group was not reduced under
conditions which reduce the 20-keto group of
progesterone.
Calc. %
C 75.66
H 9.41
0 14.93

The compendia1 assay of hydroxy-
progesterone caproate is based on the U.V.
absorption maximum at 240 nm17.

Fluorescence was used for detection in
biological f luids15.
220
6.4
P o l a r i m e t r i c Analysis
Rotation has been used t o determine
hydroxyprogesterone c a p r o a t e i n o i l vehicles18.

The h a l f wave p o t e n t i a l (E-1/2 v e r s u s
s t a n d a r d calomel e l e c t r o d e ) was determined a s
-1.65 v o l t s i n 1M l i t h i u m c h l o r i d e i n 95%
methanol due t o t h e r e d u c t i o n of t h e a, B un-
s a t u r a t e d ketone22. The d i f f u s i o n c u r r e n t
c o n s t a n t ( I d ) was found t o be 5.27 f 3%.
G.6 Chromatoqraphic Analysis

6.61 Paper
The 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 and
s e p a r a t i o n from t h e - f r e e s t e r o i d of hydroxy-
progesterone c a p r o a t e by paper chromatography i n
o i l y veh4cles has been d e s c r i b e d by Roberts and
Florey19. A f t e r s p o t t i n g , t h e paper s t r i p s were
impregnated w i t h 30% d i e t h y l e n e g l y c o l monoethyl
e t h e r ( C a r b i t o 1 ) i n chloroform and developed f o r
4 hours with methylcyclohexane s a t u r a t e d with
d i e t h y l e n e g l y c o l monoethyl e t h e r . A f t e r
l o c a t i n g of t h e s t e r o i d s p o t s w i t h a f l u o r e s c e n t
paddle, t h e s p o t s a r e e l u t e d and r e a c t e d w i t h
i s o n i c o t i n i c a c i d hydrazide i n methanol c o n t a i n -
i n g a l s o h y d r o c h l o r i c a c i d . The absorbance of
t h e yellow hydrazone i s r e a d a t 415 nm.
6.62
Thin-Layer
Thin-layer
- chromatographic
systems have been compiled i n Table 11.
22 1
Table I1
Absorbent Solvent System -

Rf Ref.
Magnesium silicate Benzene-Ethanol (98:2) 0.35 20

Magnesium silicate Benzene-Ethanol (9:1) 0.61 20
Magnesium silicate Chloroform 0.44 20
Magnesium silicate Chloroform-Ethanol (98 :2) 0.53 20
Magnesium silicate Chloroform-Ethanol (96:4) 0.60 20
Magnesium s i1icate Benzene-Dioxane (2:l) 0.71 20
Magnesium silicate Ether-Ethanol (98:2) 0.83 20
Silica gel Benzene-Acetone (4:1) 0.67 21
Silica gel Benzene-Methanol (9: 1) 0.61 21
Silica gel Pet. ether, Benzene, 0.44 21
Acetic acid, Water
(67:33:85:15)
Alumina Benzene-Acetone (4:1) 0.61 21
Detection system: U.V. light; sulfuric acid (dark brown): sulfuric-acetic

acid (yellow red); vanillin-sulfuric acid (dark yellow)
7. References
Communication.
2. E. K a s p a r , K. H. Pawlowski, K a r l Junkmann
and M a r t i n Schenk, U . S . P a t e n t 2,753,360
(1956).
3. V. M. B a k s h i and Y. K. Hamied, I n d . J. Chem.
-
2 ,294 ( 1964) .
4. L. F. F i e s e r and M. F i e s e r , " S t e r o i d s "
R e i n h o l d P u b l i s h i n g Corp. 1959.
5. B r i t . P a t e n t 848,881 ( C h e m . A b s t r . 57,2348g) ;
J a p a n P a t e n t 3574(62 ) (C.A. 58,91896);
Belg. P a t e n t 661,975 (C.A. 65,5510) ; U. S. S. R.
P a t e n t 210,858 (C.A. B Y 9 6 , 9 9 8 e ) .
Communication.
7. I. Gyenes and A. L a s z l o , Magyar Kem.
F o l y o i r a t 67, 360 (1961) (C . A . 56,149506,
1962).
8. M. K u h n e r t - B r a n d s t a t t e r , E. J u n g e r and
A. K o f l e r , Microchem. J. 9,105 (1965).
9. K. Junkmann, Arch. e x p e r . P a t h . u. Pharmakol.
-
223,244 (1954).
10. G. A. B r e w e r , The S q u i b b I n s t i t u t e , P e r s o n a l
Communication .
11. J. P . C r i s l e r , N. F. W i t t a n d M. H . C r i s l e r ,
M i c r o c h i m i c a Acta 1962,317.
1 2 . D. R. B a r t o n and W. C. T a y l o r , J.Am.Chem.Soc.
-
80,244 (1958) ;J. Chem.Soc. 1958,2500.
13. H. K i e s l i n g and1 A . E l m q u i s t , Acta. E n d o c r i n o l .
-
28,502 (1958).
14. F.X. G a s s n e r , R. P. M a r t i n , W. Shimoda and
J. W. Algeo, F e r t i l i t y and S t e r i l i t y l l , 4 9
(1960).
1 5 . H. L a n g e c k e r , A. H a r w a r t a n d K. Junkmann,
Arch. e x p t l . P a t h o l . Pharmacol. 225,309 (1955).
16. M. Wiener, C. I. Lupu a n d E. J. P l o t z , A c t a .
E n d o c r i n o l . 3 6 , 5 1 1 (1961).
223
KLAUS FLOREY
1 7 . U.S.P. XVIII.
18. V. Gerosa and M. Melandri, Ann.Chim. ( R o m e )
.
4 7 , 1 3 8 8 , (C.A. 5 2 , 7 6 2 0 ( 1 9 5 8 ) )
1 9 . H. R. Roberts a n d K. F l o r e y , J . P h a r m . S c i .
51,794 (1962).
20. 7 S c h w a r z , P h a r m a z i e 1 8 , 1 2 2 (1963).
21. S. Hara a n d K. Mibe, Chem.Pharm.Bul1. 15,1036
(1967).
22. N. H. Coy a n d C. M. F a i r c h i l d , T h e S q u i b b
23. Q. Ochs, T h e S q u i b b I n s t i t u t e , P e r s o n a l
Communication.
L i t e r a t u r e s u r v e y e d through December 1 9 7 2 .
The h e l p of H. Gonda and A. Mohr i n t h e

p r e p a r a t i o n of t h i s p r o f i l e i s g r a t e f u l l y
acknowledged.
224
ISOSORBIDE DINITRATE
Luciano A . Silvieri and Nicholas J . DeAngelis

L U C I A N 0 A. SlLVlERl AND NICHOLAS J. DeANGELIS
CONTENTS
1. Description
2. Physica1 Properties
2 . 1 Infrared Spectra
2 . 4 Mass Spectra
2 . 6 Melting Range
2.8 Solubility
3. S ynthesis
4. Stability and Degradation
5. Metabolism
6. Identification
7. Aesay Methods
7.3 Infrared Analysis
7.4 Gas Liquid Chromatographic Analysis
7.6 Automated Analysis
7.7 Radiochemical Analysis
7 . 9 Paper Chromatographic Analysis
8. References
226
1. Description
Isosorbide d i n i t r a t e i s designated by t h e f o l -
lowin chemical names: 1,4: 3,6-dianhydro-~-glucitol d i n i -
f
t r a t e ; 1,4:3,6-dianhydrosorbitol 2,5-dinitrate1; d i n i t r o -
sorbidel. I n Chemical Abstracts t h e compound i s l i s t e d
under the heading (Glucitol:1,4:3,6-dianhydro, " d i n i t r a t e ,
Dtl). Some of the commonly used t r a d e o r t r i v i a l names
are: I s o r d i l , Isorbid, Vascardin, and Carvanil.
" 4
Isosorbide d i n i t r a t e i s a white, odorless, crys-
t a l l i n e powder. However, d i l u t e d isosorbide d i n i t r a t e ,
which i s a d r y mixture of isosorbide d i n i t r a t e w i t h lac-
t o s e , rnannitol, o r o t h e r i n e r t e x c i p i e n t s t o permit s a f e
handling, occurs as an ivory-white powder. The mixture
u s u a l l y contains about 25% of isosorbide d i n i t r a t e .
The i n f r a r e d (1.R.) spectrum of isosorbide d i n i -
t r a t e 4 6 is given i n Figure 1.2 The I.R. spectrum was
obtained i n a KBr p e l l e t and i s i d e n t i c a l t o t h a t pub-
l i s h e d by Sammul et.al.3 Assignment of some of t h e s i g -
n i f i c a n t absorption bands i s given i n Table I.
Table I
Infrared Spectral Assignments of Isosorbide D i n i t r a t e
Frequency (cm.-l> Vibration Mode Reference
2950-2850 a l i p h a t i c C-H s t r e t c h i n g 48
1665 and 1635 asymnetric N 4 s t r e t c h i n g 47
1460 methyl ene s c i s soring 48
vibration
1285-1270 symmetrical N 4 s t r e t c h i n g 47
rVllO0 asymnetrical C-0-C s t r e t c h i n g 48
865 0-N4 s t r e t c h i n g 47
221
Figure 1 - I.R. Spectrum of Isosorbide Dinitrate, Lot No. 5123 V 6/1. 0.25% KBr P e l l e t -
Instrument: Perkin Elmer Model 21.
Hayward et.al .4 reported t h e n i t r a t o and hydroxyl

s t r e t c h i n g bands of n i t r a t e e s t e r s of 1,4:3,6-dianhydro-
h e x i t o l s , including isosorbide d i n i t r a t e , i n d i l u t e solu-
t i o n s of benzene, a c e t o n i t r i l e , chloroform, isopropyl
e t h e r , methylal, pyridine, and carbon t e t r a c h l o r i d e .
The nuclear magnetic resonance (N.M.R-) spectrum
(Figure 2) w a s obtained by preparing a s a t u r a t e d s o l u t i o n
of isosorbide d i n i t r a t e 4 6 i n deutero chloroform containing
tetramethylsilane as i n t e r n a l r e f e r e n ~ e . ~There a r e no
exchangeable protons. The NMR proton s p e c t r a l assignments
a r e given i n Table 11.
Table I1
NMR Spectral Assignments of Isosorbide D i n i t r a t e
Chemical S h i f t (ppm.1 Pro ton
3.9-4.2 -0-%
Hopton and Thomas6 have reported on NMR s t u d i e s

of isosorbide which e l u c i d a t e the conformation of t h i s
molecule. Their findings i n d i c a t e t h e conformation i s a
composite of t h e envelope and half-chair forms.
Isosorbide d i n i t r a t e gives no eak maxima i n t h e
wavelength region of 400 nm. t o 220 run.’, but does absorb
U.V. l i g h t . The absorption begins a t about 290 nm. and
continually increases as the wavelength decreases. I n
U.S.P. alcohol a t a concentration of approximately 0.1 mg./
3
m l . an absorbance of 0.1 i s obtained a t 50 run., and an
absorbance of 1.5 i s obtained a t 220 nm.
2.4 Mass Spectra
The mass s p e c t r a of isosorbide d i n i t r a t e 4 6 were
obtained’ by d i r e c t i n s e r t i o n of the sample i n t o an MS-902
double focusing mass spectrometer modified with a SRI dual
C I E I source. Both low r e s o l u t i o n e l e c t r o n empact (EI)
and chemical i o n i z a t i o n (CI) mass s p e c t r a were obtained.
For t h e E I spectrum the ionizing e l e c t r o n beam energy w a s
70 eV and f o r the C I spectrum the beam energy was 500 eV.
The mass spectrum assignments’ of t h e prominent
229
I
N
W
0
Figure 2 - NMR Spectrum ofIsosorbide Dinitrate, Lot No. 5123 V 6/1. Solvent: deutere
chloroform, internal standard: tetramethylsilane. Instrument: Varian A-60.
ions under b a h E I and C I conditions a r e given i n Table 111.

Table I11
Mass Spectral Assignments of Isosorbide D i n i t r a t e
Chemical Ionization Electron Im act
&I R.I.(%> m/e R.I. % S ecies
23 7 15 236- 2.5 M+
190 24 W-HNQ 143 75 M-HN4, N 4
144 60 MIP-HNQ, N Q 126 100 M-HNC+, HNQ
127 100 MH+-HN%, H N4 114 27 M-HNOZ, CHON4
100 25 M - H N 4 , WCHONQ
2.5 O p t i c a l Rotation
The s p e c i f i c rotati0nL4];~ o f ' isosorbide d i n i t r a t e46
w a s determined 7 t o be +137O i n U.S.P. alcohol a t a concen-
1
t r a t i o n of 3 mg./ml. The Merck Index r e p o r t s anb];5 of
+135O. Jackson and Hayward" fpund thelo(]:O t o be +141°
and Goldberg" +134O.
2.6 Melting Range
There a r e two ranges reported i n t h e l i t e r a t u r e
f o r the melt of isosorbide d i n i t r a t e . The Merck Index1,
Wiggins12, Goldberg", and Haywa et.al.8, reporf3values of
70°C.-7L0C. J ckson and Hayward", Forman et.al. , and
Hayward et.al.'
70°C.-71.50C., ClassIJ',
report .5OC.-52OC. We found a value of
f o r isosorbide d i n i t ~ - a t e . ~This
d i f f e r e n c e i n melting point may be a r e s u l t of polymorphism.
~
The melting temperature range does n o t change s i g n i f i c a n t l y

with v a r i a t i o n s i n heating r a t e of from 1 t o 5OC./rnin.
The d i f f e r e n t i a l thermal a n a l y s i s (DTA) curve of
isosorbide d i n i t r a t e 4 6 run from room temperature t o t h e
melting point e x h i b i t s no endotherms o r exotherme o t h e r
than t h a t associated with t h e m e l t . The DTA curve7 run on
a DuPont 900 DTA using a micro c e l l and a heating r a t e of
5'C./min. i s shown i n Figure 3.
2.8 Solubility
The following s o l u b i l i t i e s have been determined
f o r isosorbide d i n i t r a t e a t room temperature.
Solvent
water
Authors
<
0.5 mg./ml.
Merck Index'
sparingly soluble
U.S.P.- 15
very s l i g h t l y
Soluble
acetone 7 1 g./ml. f r e e l y soluble very s o l u b l e
alcohol 24 mg./ml. f r e e l y soluble sparingly
soluble
23 1
h)
W
h)
Figure 3 - DTA Spectrum of Isosorbide D i n i t r a t e , Lot N o . 5123 V 6/1, heating r a t e SO/min.

Instrument: DuPont 900 DTA.
ISOSORBIDE DIN ITRATE
Solvent
ether -m
Authors
' g-m
/. .l
Merck Index1
f r e e l y soluble
-
U.S.P. 15
chloroform 330 mg./ml. f r e e l y soluble

hexane < 1 mg./ml.
The X-ray powder d i f f r a c t i o n p a t t e r n of isosorbide
d i n i t r z ~ t eobtained
~~ 7 with a P h i l i p s diffractometer16 using
CuKa r a i a t i o n i s shown i n Figure 4. The c a l c u l a t e d d
spacings' f o r the d i f f r a c t i o n p a t t e r n shown a r e given i n
Table I V .
Table I V
X-Ray Powder D i f f r a c t i o n P a t t g r n f o r Isosorbide D i n i t r a t e
-
28 (degrees)
10.0
-
d (A)
8.85
I/Io
40
16.1 5.50 25
17.3 5.13 100
19.5 4.55 3
20.2 4.40 95
21.1 4.21 1
23.2 3.83 2
23.8 3.74 9
25.1 3.55 32
25.6 3.48 20
2772 3.28 2
28.9 3.09 3
29.3 3.05 2
31.4 2.849 5
32.6 2.747 5
33.2 2.698 2
34.4 2.607 9
35.8 2.508 3
41.1 2.196 12
d(interp1anar distance) = nh
2sin4
1/10 = r e l a t i v e i n t e n s i t y (based on highest i n t e n s i t y = 100)
3. Synthesis
Isosorbide d i n i t r a t e has been prepared by routes
u t i l i z i n g both L-sorbose and D-glucitol ( s o r b i t o l ) .
I n one s y n t h e t i c rout:,() isosorbide w a s prepared from
L-aorbose by themethod of Wiggine,17 This w a r accompltshed by
hydrogenation of L-sorbose followed by dehydration t o o b t a i n
isosorbide. The n i t r a t e e s t e r of isosorbide w a s then pre-
pared by t h e method of Forman et.al.18 i n a y i e l d of about
50%; t h e y i e l d w a s increased t o 85-907- by n i t r a t i o n i n
233
N
w
p.
Figure 4 - X-Ray Diffraction Pattern of Isosorbide Dinitrate, Lot No. 5123 V 6/1.
Radiation: CuKa Instrument: Norelco p h i l i p s Diffractometer.
ISOSORBI DE DIN ITRATE
a c e t i c anhydride-ni t r i c a c i d - a c e t i c acid mixture.

I n another synthesis,19 shown i n Figure 5, i s o s o r b i d e
was prepared by dehydration of D-glucitol ( s o r b i t o l ) .
Isosorbide d i n i t r a t e was subsequently obtained by t r e a t i n g
i s o s o r b i d e with differentnitratingagents. Concentrated
n i t r i c acid (98%) gave a y i e l d of 83%, a mixture of n i t r i c
acid and s u l f u r i c acid (30 and 60% r e s p e c t i v e l y ) gave a
y i e l d of 65%.
Isosorbide dinitrate-C14 w a s prepared b anhydrization
of s o r b i t o l using p-toluene s u l f o n i c acid.28 The r e s u l t i n g
i s o s o r b i d e was n i t r a t e d using 97% n i t r i c acid.
4. S t a b i l i t y and Degradation
The s t a b i l i t y of i s o s o r b i d e d i n i t r a t e , i n t h e s o l i d
form, has been studied a t various temperatures. It w a s
found t o be s t a b l e a t a temperature of 45OC. f o r a period
of 1 2 months and a t room temperature f o r a period of 60
months. Z1.
I n a c i d i c medium, under vigorous h y d r o l y t i c condi-
tions, t s o s o r b i d e d i n i t r a t e degrades i n a stepwise
manner forming 2-mononltrate and 5-mononitrate as i n t e r -
mediates with t h e f i n a l products being i s o s o r b i d e and
inorganic nitrate.21 For example, 25% decomposition22
r e e u l t s from heating i s o s o r b i d e d i n i t r a t e i n 1 N hydro-
c h l o r i c acid a t 100OC. f o r one hour. Decomposition i n
base i s somewhat more r a p i d , i.e., a 1 N sodium hydroxide
55 .
s o l u t i o n of i s o s o r b i d e d i n i t r a
one hour, decomposes about 4 X
when heated a t 10o°C. f o r
*Jackson and Hayward” s t u d i e d t h e decomposition of

i s o s o r b i d e d i n i t r a t e i n p y r i d i n e , and found t h a t a slow
decomposition t o a polymer, n i t r o g e n oxides, and pyri-
dinium n i t r a t e took place when s o l u t i o n s were heated above
50° C.
5. Metabolism
reasoned t h a t i s o s o r b i d e d i n i t r a t e i s completely
metabolized i n man and i n t h e dog s i n c e no unchanged drug
was d e t e c t e d i n urine. L e s s than 1%of t h e dose w a s
recovered as 5-isosorbide mononitrate and 2-isoeorbide
mononitrate and t h e r e f o r e he postulated t h a t t h e removal
of t h e n i t r a t e groups i s a stepwise process ending with
t h e completely d e n i t r a t e d i s o s o r b i d e as t h e major metabo-
lite.
Sherber et.al.2L demonstrated that 86% of i s o s o r b i d e
d i n i t r a t e , intravenously administered, i s c l e a r e d from
235
CH20H
t4
I
w HCOH HCOH
al I I 0 98%HN03
HOCH OR
HCOH MIXTURE OF
I 0 1 HNO~AND o I
HCOH
I
CH2OH
H2S04 Lr,, HCONOZ
D-GLUC ITOL I SOSORBIDE I SOSORBI DE DINITRATE

(SORB ITOL)
Figure 5 -A Synthetic Route for Isosorbide Dinitrate

r a b b i t blood w i t h i n 90 seconds.
I n a l a t e r study, Reed et.al.20 found t h a t a f t e r o r a l
doses of i s o s o r b i d e d i n i t r a t a n e a r l y a l l o f the d r u g ,
w i t h carbon s k e l e t o n i n t a c t , was e x c r e t e d i n t h e u r i n e of
dogs d u r i n g t h e f i r s t 24 hours. Some 20 t o 30% o f t h e car-
bon s k e l e t o n of i s o s o r b i d e d i n i t r a t e w a s e x c r e t e d as n e u t r a l
m a t e r i a l , p r i n c i p a l l y as i s o s o r b i d e , 2-isosorbide mononitrate
and 5 - i s o s o r b i d e mononitrate and t h e e t h e r monoglucuronide
of i s o s o r b i d e .
Sisenwine and Rue1iusZ5 made a complete i n v e s t i g a t i o n
of t h e plasma c o n c e n t r a t i o n and u r i n a r y e x c r e t i o n o f i s o s o r -
b i d e d i n i t r a t e and i t s m e t a b o l i t e s i n dogs. They found t h a t
t h e i n i t i a l b i o t r a n s f o r m a t i o n occurs by d e n i t r a t i o n of t h e
two isomeric mononitrates, 2 - i s o s o r b i d e mononitrate and 5-
i s o s o r b i d e mononitrate. A s t h e mononitrate e s t e r s d i s a p p e a r
a small amount o f i s o s o r b i d e appears, probably stemming more
from h y d r o l y s i s of t h e more l a b i l e n i t r a t e group i n 2-isosor-
b i d e mononitrate than from t h e s t e r i c a l l y hindered n i t r a t e
group i n 5 - i s o s o r b i d e mononi trate. F u r t h e r t r a n s f o r m a t i o n
o f t h e i s o s o r b i d e molecule does n o t take p l a c e as demonstra-
t e d by t h e f i n d i n g t h a t o n l y unchanged i s o s o r b i d e i s e x c r e t e d
a f t e r a d m i n i s t r a t i o n of i ~ o s 0 r b i d e - C t~o ~dogs. The d i s a p -
pearance o f 5 - i s o s o r b i d e mononitrate from plasma i s probably
caused by t r a n s f o r m a t i o n o f t h e molecule t o g l u c u r o n i d e and
o t h e r conjugates. The i s o s o r b i d e glucuronide found i n u r i n e
i s a m e t a b o l i t e formed from t h e 5 - i s o s o r b i d e mononitrate
glucuronide and n o t from i s o s o r b i d e , s i n c e t h e l a t t e r does
not undergo t r a n s f o r m a t i o n o r conjugation. A p o s t u l a t e d
b i o t r a n s f o r m a t i o n scheme i s i l l u s t r a t e d i n Figure 6. These
i n v e s t i g a t o r s d i d n o t f i n d any 2-isosorbide_qononi t r a t e i n
dog2grine c o n t r a r y t o t h e f i n d i n g s of Dietzz’ and Reed et.
-.
a1
6. Identification
I s o s o r b i d e d i n i t r a t e can b e i d e n t i f i e d by v i r t u e of i t s
c h a r a c t e r i s t i c I R and X-ray s p e c t r a ( s e e 2.l-and 2.9).
7. Assay Methods
5,46
Determined
30.39
H 3.41 3.24
N 11.86 11.77
7.2 C o l o r i m e t r i c Analysis
The most comon procedures f o r c o l o r i m e t r i c a n a l y s i s
237
(-yJ-d(yj
2 - ISMN 5 - ISMN
1
- 1
ON02 OH OG I OG I
2 - ISMN GLUCURONIDE IS GLUCURONIDES 5 - ISMN GLUCURONIDE
ISDN = ISOSORBIDE DINITRATE

ISMN: ISOSORBIDE MONONITRATE
IS = ISOSORBIDE
Figure 6 - Metabolic Pathways of Isosorbide Dinitrate

ISOSORBI DE DINITRATE
of n i t r a t e e s t e r s involves a l k a l i n e hydrolysis and c o l o r i -

metric measurement of l i b e r a t e d n i t r i t e . This b a s i c proce-
dure has been used by i n v e s t i g a t o r s f o r a t l e a s t t h e l a s t 40
years .26 For isosorbide d i n i t r a t e more d i r e c t methods have
been used. Most of these involve r e a c t i o n with t h e n i t r a t e
group.
Reed et.al.*O measured isosorbide d i n i t r a t e by
r e a c t i n g i t with 1%2,6-xylenol i n g l a c i a l a c e t i c acid i n
the presence of 80% (w/v> s u l f u r i c acid f o r one-half hour
t o form nitroxylenol. The nitroxylenol w a s extracted from
the aqueous r e a c t i o n mixture using chloroform and then re-
extracted i n t o sodium hydroxide t o form t h e yellow sodium
s a l t whose absorbance w a s measured a t 420 run. This method
i s extremely s e n s i t i v e .
Jackson and Hayward” reacted isosorbide d i n i t r a t e
with 70% (v/v) s u l f u r i c acid and a 1%s o l u t i o n of 3,4-dimeth-
ylphenol f o r 30-60 minutes a t 3OOC. Water w a s then added
and t h e s o l u t i o n d i s t i l l e d c o l l e c t i n g t h e d i s t i l l a t e i n a
f l a s k containing a s o l u t i o n of 2% sodium hydroxide. The
absorbance of t h e r e s u l t i n g s o l u t i o n w a s then measured a t
435 nm.
Nagese et.a1.27 determined isosorbide d i n i t r a t e i n
pharmaceuticals by two c o l o r i m e t r i c methods. One w a s based
on t h e Griess-Romijin reagent and t h e o t h e r on t h e phenol-
d i s u l f o n i c acid reagent.
The phenoldisulfonic acid o l o r i m e t r i c method f o r
determining mannitol h e x a n i t r a t e 28-51 was adapted32 f o r
measuring isosorbide d i n i t r a t e in. t a b l e t s and i n j e c t a b l e s .
In t h i s method a sample containing 10 mg. of isosorbide
d i n i t r a t e i s extracted from an a c i d i c s o l u t i o n w i t h a t o t a l
of 100 m l . of e t h e r o r chloroform. An a l i q u o t (4 ml.) of t h e
a c e t i c acid and 2.0 m l . of phen. ’ ’ ’ a

__
e x t r a c t i s then evaporated t o dryness and 1 m l . of g l a c i a l
a r e added. A f t e r 15 minutes t h
with ammonium hydroxide and i t s _ _ .,a
nm. I n t h i s procedure it i s not necessary t o use isosor-
bide d i n i t r a t e as a standard, any of t h e inorganic n i t r a t e s ,
such as sodium o r potassium n i t r a t e , can be u t i l i z e d .
A method used f o r determining n i t r a t e 3 3 has been
adopted34 t o study s o l u t i o n s of isosorbide d i n i t r a t e . This
method involves hydrolyzing isoeorbide d i n i t r a t e with sodium
hydroxide t o form n i t r i t e ions. The n i t r i t e i s then diazo-
t i z e d with p-nitroaniline t o form a diazonium i o n which when
coupled with azulene i n t h e presence of perchloric acid
e x h i b i t s an absorbance maximum a t 515 run.
239
LUCIAN0 A. SlLVlERl AND NICHOLAS J. DeANGELlS
7.3 I n f r a r e d Analysis
Infrared spectropho om r i c procedures used f o r t h e
estimation of n i t r a t e e s t e r s 5 5 9 4 6 have been adopted32 f o r
determining isosorbide d i n i t r a t e i n t a b l e t s and i n j e c t a b l e s .
The method c o n s i s t s o f simply e x t r a c t i n g isosorbide d i n i t r a t e
from i t s dosage fo with chloroform and reading t h e absor-
bance a t 1650 cm.-'in a sodium c h l o r i d e c e l l . Carbon t e t r a -
c h l o r i d e can be s u b s t i t u t e d f o r chloroform i n t h i s proce-
dure. 7
7.4 Gas Liquid Chromatographic A n a l y s i s
The colorimetric and I R methods mentioned above a r e
v i r t u a l l y non-specific. This i s a disadvantage e s p e c i a l l y
i n cases where mixtures of e s t e r s are present due t o p a r t i a l
d e n i t r a t i o n of t h e parent e s t e r . Gas l i q u i d chromatographic
methods overcame t h i s d i f f i c u l t y , , provide adequate separa-
t i o n and, a t t h e same t i m e , maintain s e n s i t i v i t y of detec-
tion.
Sherber et.a1.24 determined i s o s o r b i d e d i n i t r a t e i n
r a b b i t blood by e x t r a c t i n g i t i n e t h y l a c e t a t e and i n j e c t i n g
t h e e x t r a c t without f u r t h e r p u r i f i c a t i o n i n a 6 f t . column
packed with 3.8% SE-30 on Gas Chrom P. The system w a s oper-
ated isothermally under t h e following conditions: oven
temperature, l l O ° C . , f l a s h h e a t e r , 130.C., d e t e c t o r , 190.C.;
nitrogen flow r a t e , 55 ml./min. These same i n v e s t i g a t o r s
a l s o used a 3% XE-60 on Gas Chrom Q column with an oven
temperature of 15OOC.; f l a s h h e a t e r a t 16OOC.; flame ioniza-
t i o n d e t e c t o r temperature a t 18OOC.; and nitrogen flow r a t e
a t 55 ml./min. The i n t e r n a l standard used was m-dinitro-
benzene. The s e n s i t i v i t y of t h i s method i s such t h a t as
l i t t l e as 0.01 pg. can be detected with a flame i o n i z a t i o n
detector.
Davideon et.a1.38 used a gas chromatographic method
f o r s e p a r a t i o n and q u a n t i t a t i o n of isosorbide d i n i t r a t e i n
t h e presence of o t h e r organic n i t r a t e e s t e r s of common
therapeutic use. They used two columns: one packed with
1%SE-30 on trimethylchlorosilylated Chromsorb P and t h e
o t h e r packed with 1%Dexsil 300 GC on dimethylchlorosilyla-
ted Chromosorb W. The i n j e c t i o n p o r t and d e t e c t o r s were
maintained a t 200OC. with nitrogen as t h e c a r r i e r gas. The
columns were programned a t a r a t e of 10°/min. with an i n i t i a l
s e t t i n g of 65OC.
A mixture of isosorbide d i n i t r a t e and i t s two mono-
n i t r a t e s w a s assayed by gas chromatography using columns
packed with e i t h e r 3% XE-60 o r 3.5% QF-1 on G a s Chrom Q. 39
240
ISOSOR B IDE DIN ITRATE
Both columns were operated isothermally, t h e QF-1 a t 110OC.

and t h e XE-60 a t 1 5 O O C . The i n j e c t i o n p o r t was maintained a t
160OC. Good separation of t h e d i f f e r e n t n i t r a t e s w a s ob-
tained on the QF-1 column. The XE-60 column was less s a t i s -
f a c t o r y as 5-isosorbide mononitrate overlapped w i t h isosor-
bide d i n i t r a t e . The minimum amount of isosorbide d i n i t r a t e
t h a t could be determined was 0.5 pg. with a flame i o n i z a t i o n
d e t e c t o r and 8 ng. i t h an e l e c t r o n capture detector. These
same investigators4' have subsequently determined isosorbide
d i n i t r a t e i n human plasma on t h e QF-1 column. I s o i d i d e
d i n i t r a t e was used as the i n t e r n a l standard.
Another gas chromatographic method f o r t h e d e t e r -
mination of isosorbide d i n i t r a t e i n t a b l e t s 2 2 uses a 5 f t . x
1/8 in. s t a i n l e s s s t e e l column packed with 1%OV-17 on Diato-
port S , 80/100 mesh operated a t 155OC. The isosorbide d i n i -
t r a t e i s extracted from an a c i d i c s o l u t i o n with chloroform,
and e t h y l pentadecanoate i s used as t h e i n t e r n a l standard.
This method w i l l separate isosorbide d i n i t r a t e from isosor-
bide and the two mononitrates.
A 4 r l a r o g r a p h i c methzg used f o r determining glyceryl
trinitrate has been adopted f o r assaying isosorbide d i n i -
t r a t e i n i n j e c t a b l e 8 and t a b l e t s . This method involves t h e
reduction of the n i t r a t e e s t e r a t t h e dropping mercury elec-
trode i n a 0.5 N t e t r a m e t h y l m n i u m hydroxide aqueous solu-
t i o n buffered a t pH 8.7 with an ammonium b u f f e r (0.5 N i n
NH,,Cl and NH,,OH). The reduction wave has a half-wave poten-
t i a l of -0.78V. N i t r a t e and n i t r i t e ions do not i n t e r f e r e .
7.6 Automated Analysis
The phenoldisulfonic acid r e a c t i o n (7.2) has been
u t i l i z e d f o r determining isosorbide d i n i t r a t e i n t a b l e t s
using an automated p r o ~ e d u r e . 4 ~ Glacial a c e t i c acid i s used
as t h e e x t r a c t i o n solvent.
7.7 Radiochemical Analysis
Isosorbide dinitrate-CI4 w a s analvzed 20925 in a
l i q u i d s c i n t i l l a t i o n s ectrometer under conditions appro-
p r i a t e f o r counting C. P4

Several TLC s y s t e m f o r t h e separation of isosor-
bide d i n i t r a t e from i t s metabolites and similar s t r u c t u r e
compoun s have been reported i n t h e l i t e r a t u r e . I n one
method2' the sample was spotted on chromatographic p l a t e s
24 1
L U C I A N 0 A. SlLVlERl AND NICHOLAS J. DeANGELlS
coated with s i l i c a g e l (-25 mm) and were developed by ascen-

ding techniques with two s o l v e n t systems, one a benzene:
ethyl a c e t a t e ( 1 : l ) mixture, and t h e second a 2-propanol:
ammonium hydroxide (4:1 ) mixture. For v i s u a l i z a t i o n t h e
p l a t e s were sprayed with 1%diphenylamine i n methanol solu-
t i o n and exposed t o u l t r a v i o l e t l i g h t f o r 5 minutes. Diphen-
ylbenzadium i n s u l f u r i c acid has a l s o been used as a spray
reagent.24 The l a t t e r reagent w as found t o b e 10 times more
s e n s i t i v e than diphenylamine f o r n i t r a t e edters. The Rf f o r
isosorbide d i n i t r a t e i s .68 i n t h e f i r s t solvent system and
.77 i n t h e second. Both of t h e s e systems separated i s o s o r -
bide d i n i t r a t e from i t s two mononitrates. Other i n v e s t i g a -
t o r s using these same solvent systems v i s u a l i z e d i s o s o r b i d e
with a metaperiodate-permanganate spray. 25
Another TLC method44 uses as an adsorbent a mixture
of s i l i c i c acid and p l a s t e r of P a r i s (70:30 w/w). Chromato-
grams were developed with mixtures of anhydrous s o l v e n t s i n
the following volume r a t i o : benzene-petroleum e t h e r (1: 1 ) ;
ether-petroleum e t h e r (3: 39) ; ether-benzene (1: 19). A 1%
s o l u t i o n of diphenylamine i n 95% ethanol, followed by expo-
s u r e t o a shortwave u l t r a v i o l e t l i g h t f o r 10 minutes w a s used
f o r detection.
Isosorbide d i n i t r a t e
21
w a s separated from i s o s o r -
bide and i t s two mononitrates on p l a t e s coated with S i l i c a
Gel G by using a mixture of carbon t e t r a c h l o r i d e and acetone
(8:2) as the solvent.
7.9 Paper Chromatographic Analysis
Jackson and Hayward21 developed s e v e r a l methanol-
hydrocarbon solvent systems u s e f u l f o r paper chromatography
of isosorbide d i n i t r a t e . For v i s u a l i z a t i o n t h e paper w a s
sprayed with 1%alcoholic diphenylamine and exposed t o W
l i g h t f o r 5 t o 10 minutes.
242
8. References
1. Merck Index, 8 t h Ed., Merck and Co., Inc.(1968).
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6. F. J. Hopton and G. H. S. Thomas, Can. J. Chm.,
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c
7. N. J. DeAngelis, Wyeth Laboratories, Unpublished
.
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-
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-
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243
LUCIAN0 A. SlLVlERl AND NICHOLAS J. DeANGELlS
(1929)
27. Y. Nagase, Y. Kanoya, A. Sugiyama, and H. Haruhiko,
Yakaguaku Zasshi, 8 5 ( 2 ) , 119-25(1965).
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29. 9- Ibid
30. 9- Ibid
-
- 39, 630(1956).
40, 815(1957).
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O f f i c i a l Agricultural Chemists", 10th Ed., 605(1965).
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38. I. W. F. Davidson. F. J. Dicarlo. and E. I. Szabo,
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-
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,440 L. D. Hayward, R. A. Kitchen, and D. J. Livingstone,
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48. L. Bellamy, "The I n f r a r e d Spectra of Complex Mole-
cules," 2nd Ed., J. Wiley and Sons, Inc., New York,
N. Y., 1964.
244
METHAQUALONE
Dahyabhai M . Patel, Anthony J. Visalli, Jerome J. Zalipsky

and Nelson H. Reavey-Cantwell
DAHYABHAI M. PATELetal.
CONTENTS
Analytical P r o f i l e - Methaqualone
1. Description
2 . 3 UI t r a v l o l e t Absorptlon Spectrum
2.4 F1 uorescence Spectrum
2.5 Mass Spectrum
2.6 M e l t i n g Range
2.7 O p t i c a l R o t a t i o n
2.8 D i f f e r e n t i a l Scanning Calorimetry
2.9 S o l u b i l i t y
2.10 Crystal P r o p e r t i e s
3. Synthesis
4. Stabi 1 ity-Degradat ion
5. Metabolism
6. Methods o f Analysis
6.1 Elemental Analysts
6.2 Phase S o l u b i l i t y Analysis
6.3 Nonaqueous T i t r i m e t r l c Analysis
6.4 Color imeiir i c Anal ys is
6.6 Spectrof 1 uorometr l c Anal ys i s
6.7 Gas Chromatographic Analysis
6.8 Thin Layer Chrornatographlc Analysls
7. References
8. Ac know1edgemen t s
246
METHAQUALONE
1. Description
1.1 Name, Formula, Molecular U e i g h t
Methaqua 1 one i s 2-methyl -3-o-to1 y 1-4 bH)-qu ina-

zol i none
16"1 4N20 Molecular Weight: 250.30
Methaqualone occurs as a w h i t e , odorless, c r y s -

t a l l i n e powder.
2.1 I n f r a r e d Spectrum (IR)
The i n f r a r e d spectrum o f methaqualone i s presented

i n Figure 1 (1). The spectrum was o b t a i n e d from a KBr
p e l l e t c o n s i s t i n g o f a 0.5% d i s p e r s i o n o f methaqualone i n
KBr. The assigned p r i n c i p a l a b s o r p t i o n bands a r e l i s t e d
i n Table I ( 1 ) .
Table I
P r i n c i p a l Bands i n t h e I R Spectrum o f Methaqualone
Wave1 eng t h ( 0 ' ' ) Ass ignmen t
3049, 2994 and 2907 CH s t r e t c h i n g

1672 Amido carbonyl s t r e t c h i n g
1603, 1595 and 1471 Aromatic s k e l e t a l v i b r a t i o n
1340 and 1326 Aromatic C-N s t r e t c h i n g
778, 760, 720 and 703 CH deformation o f aromatic r i n g
247
METHAQUALONE
2.2 Nuclear Magnetic Resonance Spectrum (NMR)
The N M R spectrum of methaqualone shown in Figure 2

was obta ned by dissolving 30 mg o f methaqualone in 0.5 ml
of CCl4 containing tetramethylsilane as the internal
reference. The assigned proton signals are listed in
Table I1 (1).
Table I1
NMR Absorption Signals of Methaqualone

No. o f Chemical Signal
Group Protons Shift (ppm) Appearance
CH (Quina-
203 i none r i ng) 3 2.06 s ing 1 et
CH (Tolyl) 3 2.15 s i ng 1 et
3
Aromatic Protons 8 7-8.4 multiplet
2.3 U1 trav iolet Absorption Spectra (UV)

Methaqualone exhibits characteristic ultraviolet
absorption properties in different solvents. These char-
acteristic properties were established using a Cary
Model 14 Spectrophotometer in 1 cm cells. The U V Spectra
are presented in Figure 3 and the relevant UV absorption
data are listed in Table I11 (1).
Table I11
UV Absorption Characteristics o f Methaqualone
Solvent X max. nm X min. nm a at x max. (€1

Methanol 225.5 214.0 140.9 (35.26 X 103)
265 249.0 38.0 ( 9.51 X 103)
Ethanol (95%) 225.5 214.0 139.6 (34.94 X lo3)
249
i
,
I
I
250
METHAQUALONE
Figure 3
Ultraviolet Absorption Spectra of Methaqualone

C
.a
A : Methaqualone In methanol
( I :200,000)
.0 B : Methaqualone i n ethanol (95%)
(I :zoo ,000)
C : Methaqualone in 1 N H C l
( I :200,000)
.7
.6
"
y.5
a
m
w
0
Y)
a
m
.4
.3
.2
.I
',/---
I I I I I 1 I 1 I I I
200 220 240 260 280 300
NANOMETERS
25 1
265.0 249.0 37.7 ( 9.43 X 103)

H C l (1 N) 235.0 215.5 133.0 (33.42 X lo3)
-270 258.5 30.8 ( 7.71 X 103)
H C l (0.1 N) 234.5 215.5 133.4 (33.39 X lo3)

-270 257.5 31.5 ( 7.88 X 103)
2.4 Fluorescence Spectrum
Methaqualone does n o t e x h i b i t any n a t i v e f l u o r e s -

cence. However, t h e reduced d e r i v a t i v e t e t r a h y d r o q u i n a -
z o l i n o n e e x h i b i t s an emission maximum a t 450 nm w i t h
e x c i t a t i o n a t 345 nm i n methanol (2).
2.5 Mass Spectrum
The mass spectrum o f methaqualone i n t h e form o f

a bar graph i s presented i n F i g u r e 4. The spectrum was
obtained on a, H i t a c h i - P e r k i n Elmer Model RMU-6D Mass
Spectrometer u s i n g t h e d i r e c t i n l e t probe w i t h sample
temperature a t 60°C. The e l e c t r o n energy was 70 ev and
t h e a c c e l e r a t i n g v o l t a g e was 1700 v o l t s . The c h a r a c t e r -
i s t i c fragmentation p a t t e r n i s presented i n Table IV ( 1 ) .
Table IV
Fragmentation P a t t e r n Produced i n Mass S p e c t r a l

Examination o f Methaqualone
Mass (m/e) Intensity Fragment Ion
250 Strong Molecular i o n (M)

235 Very Strong M-CH
145 Weak M-C t$N
144 Weak M - [ $ H + CH3]
132 Med ium M-C7$4L
91 Strong M-C H N20
76
65
50
Med iurn
Med i um
Med ium
C H9-$H
7 7
C7H7-C2
-
C 6H4 C2 H
2a
41 Very Weak C9H1 0N-C7H7
252
Figure 4
Mabs Spectrum of Hethaqualone
I00
90
80
70
2 60
-
z
0
50
-
==
Y
;40
4
z
Y
30
20
10
c
20 40 60 80 100 120 140 160 180 200 220 260
We
DAHYABHAI M. PATEL et a/.
39 Med i um
2.6 Me1 t i ng Range
The m e l t i n g range o f methaqualone i s dependent

on the r a t e o f heating, Uslng the U.S.P. X V I I I Class 1
procedure, methaqualone me1 t s between 114"-117"C ( 1 ) .
Methaqualone e x h i b i t s no o p t i c a l a c t i v i t y when
analyzed a t a s e r i e s o f mercury and sodium s p e c t r a l l l n e s
(1).
2.8 D i f f e r e n t i a l Scanning Calorimetry (DSC)
The DSC thermogram o f methaqualone i s shown I n

Figure 5. A m e l t i n g endotherm i s observed a t 385"K(l12OC)
w i t h the temperature programing a t lO"C/minute. The A Hf
was found t o be 5.55 Cal/mole C1).
2.9 Solubility
The e q u i l i b r um s o l u b i l i t y data obtained on

methaqualone a t 23°C s l i s t e d i n Table V (1).
Table V
Methaqualone Solubi 1 i t y
So 1vent S o l u b i l i t y . ( g / l O O ml)
Water 0.03
Methanol 16.58
E t ha no1 12.53
I sopropanol 4.76
Ether 3-71
Chloroform >4 0
Benzene 28.00
Acetonitrile >4 0
Petroleum Ether (B.P. 30-60") 0.33
254
a,
C
0
c
m
3
CT
(D
Jz 3
XJ
U ? Y
a,
x
rc
0
E
E
ul
0
E
a,
Jz
I-
u
In
n
0
0 0
C
W w
255
The X-ray powder d i f f r a c t i o n p a t t e r n o f methaqua-

lone was obtained on a P h i l l i p s scanning d i f f r a c t o m e t e r
u s i n g copper r a d i a t i o n coupled w i t h N i c k e l f i l t e r . The
d i f f r a c t i o n p a t t e r n i s presented I n Table VI ( 1 ) .
Table VL
X-ray D i f f r a c t i o n P a t t e r n o f Methaqualone
d (A1)*
9.4 15
8.2 25
7.76 25
7.30 50
6.95 65
6.10 25
5.53 75
4.76 100
4.13 30
4.00 20
3.75 30
3.66 30
3.53 15
3.39 60
3.24 15
3.17 15
3.12 30
3-05 25
* d = (Interplanar distance) nX
2 Sin 8
** 1/l0 = 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 1 .OO)
The pKa o f methaqualone by t h e s p e c t r o p h o t o m e t r i c

method a t i o n i c s t r e n g t h ~1 = 0.1 was found t o be 2.54 ( I ) .
256
METHAQUALONE
3. Synthesis
Methaqualone i s synthesized by t h e r e a c t i o n scheme

shown i n F i g u r e 6. A n t h r a n i l i c a c i d is reacted w i t h
acetyl chloride t o y i e l d n-acetylanthranilic acid. This
i n t e r m e d i a t e product is condensed with o - t o l u i d i n e i n t h e
presence o f PC13 t o y i e l d methaqualone (3).
4. S t a b i l ity-Degradation
On r e f l u x i n g methaqualone w i t h 1.0, 0.1 N NaOH and

1.2 N H C I , the p r i n c i p a l degradation products found a r e
a n t h r a n i l i c a c i d , o - t o l u i d i n e and a c e t i c a c i d . In
a d d i t i o n , 2-aminobenzo-o-toluidide was found on r e f l u x l n g
methaqualone w i t h 1.0 and 0.1 N NaOH. Also, n - a c e t y l -
a n t h r a n i l i c a c i d was found as a degradation product on
r e f l u x i n g methaqualone w i t h 0.1 N NaOH. Under these
s t r i n g e n t c o n d i t i o n s , a low y i e l d o f degradation products
was obtained; thereby i n d i c a t i n g good s t a b i l i t y o f t h e
drug under normal c o n d i t i o n s (4). The degradation scheme
i s presented i n F i g u r e 7.
5. Metabol i s m
Methaqualone (MTQ) and i t s h y d r o c h l o r i d e sa t i s rap-

i d l y absorbed (Ka = 0.82 h r ' l ) from t h e stomach and
duodenum 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 o f t a b l e o r
capsule dosage forms. MTQ e x i s t s i n blood p r i m a r i l y i n
t h e plasma phase bound t o serum albumin. Peak serum
l e v e l s o f 3-4 kg/ml a r e seen 1 - 1 1/2 hours a f t e r a 300 mg
dose. The serum e l i m i n a t i o n curve i s b i e x p o n e n t i a l w i t h a
d i s t r i b u t i o n a l phase (a = 0.97 h r - ' ) and an e l i m i n a t i o n
phase (p = 0.036 h r ' l ) . The p r i n c i p l e t i s s u e s o f d i s t r i -
b u t i o n a r e l i p i d t i s s u e , l i v e r and kidney. MTQ i s
metabolized by h e p a t i c microsomal oxidoreductases.
H y d r o x y l a t i o n of 2 and 2' methyl s u b s t i t u e n t s , t o l y l
sidechain and quinazolene nucleus, g i v e s r i s e t o t h e
pr i nc ip l e monohydroxy metabol i t e s . 0-g 1 ucuron i d e conj u -
g a t i o n and 0-methylation occur p r i o r t o e x c r e t i o n o f
m e t a b o l i t e s i n b i l e o r u r i n e . Ring opening i n v o l v i n g
f o r m a t i o n o f N-1-oxide and 2-nitrobenzo-o-toluidide i s a
minor metabol i t i c path. B i l l a r y e x c r e t i o n and e n t e r o -
257
Figure 6
Synthesis o f Ilethaqualone
Anthranil ic Acid n-acetyl

Methaqualone
anthranil ic acid
o-to1 u i d i ne
M
I
+
+
I
0 -
u
p
u
=
-
-
6:
o
I + N
o x
-03 -.-u
u
I
.-C
O
P , "I
.-
U N
c
Y
c
2u :oc " uz
.- .-
U E U
e
U mI mI
a ~ c
-
Y
M
259
hepatic r e c i r c u l a t i o n o f metabolites occur. A t a thera-

p e u t i c dose o f 75 t o 300 mg i n man, MTQ i s completely
biotransforrned and l i t t l e , i f any, unchanged drug appears
i n u r i n e (5).
6.1 Elemental Analvsls
The r e s u l t s from the elemental a n a l y s i s a r e

l i s t e d i n Table VII ( 1 ) .
Table VII
Elemental Analysis o f Methaqualone
Element 9: Theory 4; Found
C 76 77 76-50
H 5.64 5.74
N 11.19 10.89
6.2 Phase S o l u b i l i t y Analysis
Phase s o l u b i l i t y a n a l y s i s was c a r r i e d o u t using

n-hexane-absolute alcohol (145:5 v/v) as a s o l v e n t system.
The r e s u l t s o f the a n a l y s i s a r e shown i n Figure 8 along
w i t h the l i s t i n g s of the c o n d i t i o n s under which t h e
a n a l y s i s was performed (1).
6.3 Nonaqueous T i t r i m e t r i c Analysis
The nonaqueous t i t r a t i o n i s the o f f i c i a l method

o f a n a l y s i s i n B.P. f o r t h e b u l k methaqualone. An
a c c u r a t e l y weighed 0.5 g sample Is dissolved i n 80 m l o f
g l a c i a l a c e t i c acid, a few drops o f c r y s t a l v i o l e t T.S. i s
added and the s o l u t i o n i s t i t r a t e d t o an emerald green end
p o i n t against p r e v i o u s l y standardized 0.1 N HClO4 i n
g l a c i a l a c e t i c a c i d (6). King and Perry (7) reported t h e
a p p l i c a t i o n o f the nonaqueous t i t r i m e t r i c method f o r
assay ing met haqua 1 one tab1 e t s .
260
In
f
0
f
m
Ln
m
0
In
N
\ 0
N
-
In
-
0
In
Nn
L 0
N -
In
-0 0
I N q A l O S 6/3lfllOS 40 6m :NOIlISOdW03 N O l l f l l O S
261
DAHYABHAI M. PATEL e t a / .
6.4 Colorimetric Ana 1 ys is
Nakano et al. (8) reported a colorimetric assay

method for methaqualone in microgram quantities based upon
diazotization of the diazotizable amines formed by acld
hydrolysis of methaqualone. Pirl et al. (9) developed
assay methods for methaqualone in biological specimens as
low as 0.3 Kg/ml. The method entails isolation of
methaqualone from the biological specimen and condensing
with p-dimethylaminobenzaldehyde in alkaline media. The
resulting chromophore is measured at 503 nm in weak acid.

Akagi Masuo et al. (10) described two methods for
assaying methaqualone in blological f l u i d s and tissues.
In the first method, methaqualone is extracted from
alkalinized biologlcal material with hexane, the solvent
i s evaporated and the residue after dissolving in dil HCl
i s measured at 234 nm. The second method eliminates the
normal biological blank by geometric treatment of the
absorbances at three wavelengths. These methods are
specific for methaqualone in that they do not include
degradation products of methaqualone.
6.6 Spectrof 1 uoromet r ic Anal ys 1 s

A fluorimetrlc method of assaying therapeutic
plasma levels of methaqualone has been reported by Brown,
S. S. et al. (2). The method i s based on reducing
methaqualone to its dlhydro-derivatlve using lithium boro-
hydride as a n effective reducing agent. The resulting
dihydro-methaqualone derivative exhibits emission maximum
at 450 nm at the activation maximum o f 345 nm.
6.7 Gas Chromatographic Analysis

There are several reports regarding the gas
chromatographic analysis of methaqualone in biological
fluids (1, 10, 11, 12, 13). These methods can be applled
for formulation analysis, evaluation of quality, and
characterization of methaqualone. Table VIII summarizes
the gas chromatographic systems wl t h references.
262
METHAQUALONE
Tab1 e VIII
Gas Chromatographic Systems Used

for Methaqualone Ana ysis
Note: All systems listed used flame onization detectors.

Carrier Column Internal
Reference Col umn Gas Temp."C Standard 5
6 ft. X 4 m., I.D., N2 240" Diphen- 2.17
glass tubing, 10% 45 ml/ hyd ram i ne
SE-30 on Chromosorb min
WAW(DMCS), 100-120
mesh
4 ft. X 2 mm., I.D., N2 240" Codeine 0.53

glass tubing, 3% 30 ml/
OV-17 on Chromosorb min
W(H.P.), 80-100 mesh
4 ft. X 4 m., I.D., N2 2300 Codeine 0.56

glass t u b i n g , 3.8% 45 ml/
UC-Wg8 on Chromosorb rnin
W(H.P.), 80-100 mesh
2 ft. X 4 mm., I.D., He 180" Butyl 2.81
glass tubing, 3% XE- 50 ml/ S tea rate
60 on Gas Chrom Q, rnin
100-120 mesh
7 ft. X 4 mm., I.D., Np 200" Buto- 2.14

glass t u b i n g , 3% 55 ml/ barb i tone
Cyclohexanedimethan- rnin
01 succinate 85-100
mesh
2 ft. X 4 mm., I.D., N2 200" Codeine 0.42

glass tubing, 2.5% 40 ml/
SE-30 on Chromosorb min
G, 80-100 mesh
2-m. X 3 mm., I.D., N2 190" Bu to-

glass tubing, 4% SE- 30 ml/ barbital 0.24
30 and 6% QF-1 on min
263
DAHYABHAI M. PATEL er a/.
Chromosorb WAW
(DMCS) , 80-100 mesh Pento-
barbital 0.30
Pheno-
barbital 0.69
* R e t e n t i o n time r e l a t i v e t o i n t e r n a l standard.
6.8 T h i n Layer Chromatoqraphic A n a l y s i s
There a r e several TLC systems r e p o r t e d i n t h e

l i t e r a t u r e which a r e l i s t e d i n Table IX. These systems
a r e used f o r d e t e c t i n g t h e a s s o c i a t e d I m p u r i t i e s i n
methaqualone, i d e n t i f i c a t i o n o f methaqualone or c h a r a c t e r -
i z a t i o n o f methaqualone and i t s m e t a b o l i c products.
Table IX
T h i n Layer Chromatographtc Systems f o r

Methaqua 1one Ana 1 ys is
Reference Support Solvent System Application
6 Kieselghur G Anaesthetic Ether Detection o f

saturated w i t h a s s o c i a t e d im-
water purities
I4 S i l i c a Gel G Chloroform-acetone l d e n t f i c a t i o n
14 S i l i c a Gel G I sopropanol- ldent f i c a t ion

c h 1 o r o f orm-25%
ammonium hydroxide
(45 :45 :10)
14 S i l i c a Gel G Petroleum e t h e r Identification

(B.P. 50-75")
P y r i d i n e (75:15)
15 S i l i c a Gel G Chloroform-acetone Separation o f

-25% ammonium hy- methaqualone
d r o x i d e (50:50:2) from t h e
metabol i t e s
264
METHAQUALONE
15 Silica Gel G Cyclohexane-chloro-Separation of

form-d i ethyl ami ne methaqualone
(7:2:1) from the
metabol i tes
15 Silica Gel G Propanol-ethyl Sepa ra t ion of

aceta te-25% methaqualone
ammon i um hyd rox i de from the
(6:2:2) metabol i tes
265
7. References
1. A n a l y t i c a l and Physical Chemistry Department

contribution, Research D i v i s i o n * , W i l l i a m H.
Rorer, Inc., F o r t Washington, Pa.
2. Brown, S. S. and Smart, G. A,, J. Pharm. Pharmacol.,

-
21, 466-468 (1969).
3. Herbert, M. and Plchat, L., nthese De La Methyl-

2, 0-Tolyl-3, Quinazolone-4 "C-2, Report CEA No.
1302, Centre D'Etudes Nucleaires De Saclay, Service
De Documentation, B o l t e p o s t a l e n02-Gif-sur-Yvette,
S.-et-0 (1959).
4. Zalipsky, J., Patel, D. M. and Reavey-Cantwell,

N. H., H y d r o l y t i c Degradation o f Methaqualone,
submitted t o J. Pharm. Sci. f o r pub1 i c a t i o n .
5. Smyth, R. D., Research D i v i s i o n , W i l l i a m H. Rorer,

Inc., Personal Comnunication.
6. B r i t i s h Pharmacopoeia, 296 (1972).
7. King, E. E. and Perry, A. R., J. Ass. Pub. Anal.

-7, 55-59 (1969).
8. Nakano, M., Yakuzaigaku, 22, 267-269 (1962).
9. P i r l , Joerg, N., e t a l . Anal. Chem. -

44, 1675-1676
(1972).
10. Douglas, J. F. and Shahinian, S., J. Pharm. Sci.,

-
62, 835-836 (1973).
11. Berry, D. J., J. Chromatog., 42, 39-44 (1969).
12. F i n k l e , B. S., Cherry, E. J. and Taylor, D. M.,
J. Chromatog. Sci .,
9, 393-414 (1971).
13. Hatch, R. C., Am. J. Vet. Res., 33, 203-207 (1972).
14. Stahl, E., Thin Layer Chromatography, 2nd E d i t i o n ,

Spring-Verlag Berlin-Heideberg, New York, 539.
266
METHAQUALONE
15. H i r t z , Jean L., A n a l y t i c a l M e t a b o l i c Chemistry o f

Drugs, Marcel Dekker, I n c . , New York, 242’243.
;:Mass spectrum and X-ray d i f f r a c t i o n a n a l y s i s was

performed by Morgan Schaffer, Inc. and Walter C.
McCrone Associates, r e s p e c t i v e l y .
8. Acknowledgements
The authors wish t o thank a l l members o f t h e A n a l y t i c a l

and Physical Chemistry Department, Research D i v i s i o n ,
W i l l i a m H. Rorer, Inc. f o r t h e i r h e l p and cooperation.
Also, special thanks a r e due t o M r s . Sandy Landis f o r her
v a l u a b l e s e c r e t a r i a l h e l p i n p r e p a r i n g t h i s monograph.
267
NORETHINDRONE
Arvin P. Shroff and Ernest S. Moycr

NORETHINDRONE
1, DESCRIPTION
1.1 NAME, FORMULA, MOLECULAR WEIGHT
1 . 2 APPEARANCE, COLOR, ODOR
2, PHYSICAL PROPERTIES
2.1 INFRARED SPECTRUM
2.2 NUCLEAR MAGNETIC RESONANCE SPECTRUM
2.3 MASS SPECTRUM
2.4 ULTRAVIOLET ABSORPTION SPECTRUM
2.5 MELTING RANGE
2.6 DIFFERENTIAL SCANNING CALORIMETRY
2.7 SOLUBILITY
2.8 OPTICAL ROTATION
3. SYNTHESIS
4, METABOLISM
5. METHODS OF ANALYSIS
5 ,1 ELEMENTAL ANALYSIS
5.2 PHASE SOLUBILITY
5.3 THIN-LAYER CHROMATOGRAPHIC ANALYSIS
5.4 VAPOR PHASE CHROMATOGRAPHY
5.5 SPECTROPHOTOMETRIC ANALYSIS
5.6 COLORIMETRIC ANALYSIS
A. BLUE TETRAZOLIUM
B. ISONICOTINIC ACID HYDRAZIDE
5.7 TITRIMETRIC ANALYSIS
6. REFERENCES
269
ARVlN P. SCHROFF AND ERNESTS. MOYER
1. Description
Norethindrone is 17a-ethinyl-17fl-hydroxy-4-
estren-3-one. It is also known by the following chemical
names.
a. 17-Hydroxy-19-nor-17a-pregn-4-en-2O-yn-3-one
b. 19-Nor-17a-ethynyltestosterone
C. 17a-Ethinyl-19-nortestosterone
d . 19-Nor-17a-ethyny1-17f3-hydroxy-4-androsten-3-one
e. 19-Nor-17a-ethynylandrosten-17fl-ol-3-one
f, 17a-Ethynyl-19-nortestosterone
g. Anhydrohydroxynorprogesterone
h. 19-Norethisterone
i. Norpregneninolone
OH
C20H2602 Molecular Wt. 298.41

White to creamy white, odorless, nonhygroscopic,
crystalline powder.
The infrared absorption spectrum of norethin-
drone is presented in Figure 1. The spectrum was taken
in a KBr pellet with a Beckman IR-8 Spectrophotometer.
Some of the absorption assignments are given in Table 1.l
270
Figure 1. Infrared spectrum o f Norethindrone
A R V l N P. SCHROFF A N D E R N E S T S . MOYER
TABLE I
INFRARED ASSIGNMENTS FOR NORETHINDRONE
Vibrational
-x Intensity* Assignments
2.95 3400 v,sh,b OH s t r e t c h i n g

2.98 3300 s,sh C zCH stretching
3.05 3220 m,sh O l e f i n i c C-H s t r e t c h i n g
3.4(3.5) 2970 (2840) m,sh A l i p h a t i c C-H s t r e t c h i n g
6.1 1650 s ,sh C=O s t r e t c h i n g
6.3 1590 m,sh C=C s t r e t c h i n g
6.7(7.5) 1490 (1340) m,sh C-H deformation
6.92 1445 m,sh C-H2 bending
7.1 1390 m,sh CH3 deformation
8.7 1135 m,sh OH bending
9.42 1060 s,sh C-0 deformation
11.29 885 m,b OH wagging
14.65 680 m,b O l e f i n i c C-H wagging
* s = s t r o n g , m = medium, v = v a r i a b l e , b = broad,
sh = shoulder
272
NORETHINDRONE
Mesley2 assigned the following bands (cm-l) to

norethindrone.
a. Absorption associated with 176-hydroxyl group: 1065,
1132.
b. Characteristic absorption due to 4-ene-3-keto function:
1416, 1337, 1269, 1211, 1199, 1018, 960, 890, 764, 685.
These assi ments as well as those made by
Djerassi3 and others&"essentially agree with absorption
peaks and shoulders presented in the spectrum, Figure I.
Cross, Landis, and Murphy5 reported the

chemical shift of the C18 angular methyl group of norethin-
drone in various solvents. They observed the chemical
shifts at 54.5 cps in d-CHC13, 54.5 cps in CHCl3, 47.5 cps
in d6-DMS0 and 53.5 cps in d7-DMF.
A reference NMR spectrum of norethindrone
recorded on a Varian A-60 Spectrometer is presented in
Figure 11. An 8% solution in deuterated chloroform con-
taining tetramethylsilane as an internal reference was
used. Characteristic chemical shifts are observed at
5.87 ppm (C4 Vinyl); 2.37 ppm (C-17 OH); 2.57 ppm (C-17
C :CH); and 0.90 ppm (C-18 CH3).
2.3 Mass Spectrum

A low resolution mass spectrum of norethin-
drone on a magnetic instrument (Atlas CH-4) was reported
by De Jongh and co-workers? Major fragments were observed
&
at m/e 110, 215, and 231 representing the following
fragmentation pattern.
?H , C ECH
231
0
n
v
215
110
273
.--
, ~ " " " ' - ' ~ ' . . - ' . . . ' ~ . ." .- " . ~ . . . . ~ . ' . . ~ . . . . ~ . . . .
80 70 b0 50 M "' 40 30
1 . . . . 1 . . . . 1 .
20 10 0
F i g u r e 11. NMR S p e c t r u m o f N o r e t h i n d r o n e
a,
E
0
k
a
E
.I+
c
c,
a,
k
0
z
+I
0
5
k
c,
u
a,
a
v1
v)
v)
3!
H
H
H
a,
k
9
M
.I+
c4
E
m
275
ARVlN P.SCHROFF AND ERNESTS. MOYER
In Figure 111, t h e mass spectrum o f norethindrone

obtained on a quadrupole instrument [Finnigan Model 1015)
i s depicted. The r e l a t i v e i n t e n s i t i e s of t h e major i o n s
a r e as follows:
m/e -
%I
55 76.63
67 74.94
79 95.36
91 100.00
105 59.15
110 75.78
215 44.77
231 48.35
298 44.63
2.4 U l t r a v i o l e t Absorption Spectrum

The E ( l % , lcm) value a t 240 nm reported with t h e
f i r s t synthesis of norethindrone3 was l a t e r corrected.
Different E ( l % , lcm) values have been suggested from a
low of 525 t o a high of 590. Many of t h e s e preparations
contain foreign r e l a t e d s t e r o i d s which a f f e c t t h e E
( I % , 1cm) value.8
Nielseng reported a value of 560 which was con-

firmed l a t e r by Bastowl who isomerized norethynodrel t o
norethindrone. We have determinedl by t h i n - l a y e r chromat-
ographic s t u d i e s t h a t norethindrone, no matter how it i s
manufactured, can be p u r i f i e d chemically by first
converting t o t h e enol ether12 followed by h y d r o l y s i s ,
The u l t r a v i o l e t curve’ shown i n Figure I V was

obtained on a U.S.P. reference standard material. I t s
E ( l % , lcm) a t 240 run was 575.
276
NORETHINDRONE
6
W
0
Z
U 5
m
Q
9m 4
a
3
0 I I
2 3 220 240 260 280 300

WAVELENGTH ( n m r
Figure I V U l t r a v i o l e t Spectrum of Norethindrone
277
A R V l N P. SCHROFF AND ERNESTS. MOYER
2.5 Melting Range
The melting point a l s o varied according to

--
d i f f e r e n t i n v e s t i g a t o r s . For example, Djerassi e t a13
reported a melting p o i n t of 203-204'C, whereas Nielseng
found a range of 199-208'C with decomposition, Kofler
--
e t a l l 3 on t h e o t h e r hand, suggested a melting p o i n t
range of 202-207'C. In our experience, most of t h e
batches had a melting range of 3'C and a l l melted between
202-208'C (Class I A U . S . P . ) f 4
Figure V shows t h e DSC thermogram of norethindrone.

A Perkin-Elmer model DSC-1B employing a heating r a t e of
lO'C/minute was used.
The d i f f e r e n c e between t h e "extrapolated onset"

and t h e "peakt' f o r reference standard U,S .P. norethindrone
was 2 . S o C ,
The "extrapolated onset" and t h e "peak" a r e de-

fined as follows:
a. "Extrapolated Onset": The temperature corresponding t o

t h e i n t e r s e c t i o n of t h e extrapolation of t h e b a s e l i n e
and t h e longest, sharpest l i n e s e c t i o n of t h e low
temperature s i d e of t h e peak.
b. : The temperature of r e v e r s a l .
2.7 Solubility
Nielsen9 r e p o r t s t h e s o l u b i l i t i e s of norethindrone
t o be: almost insoluble i n water, 1 p a r t i n 80 p a r t s
ethanol, 1 p a r t i n 4 0 0 . p a r t s e t h e r , and 1 p a r t i n 15 p a r t s
chloroform. A p l o t of t h e s o l u b i l i t i e s of norethindrone
a s a function of temperature f o r eleven common organic
solvents i s i l l u s t r a t e d i n Figure V I . *
278
NORETHINDRONE
7
EXTRAPOLATED ONSET
0
x
w
n
I
a
Y
0
n
Z
W
I 1 1 I
170 180 190 200 210 22b
ARVlN P. SCHROFF AND ERNEST S.MOYER
Figure V I S o l u b i l i t y of Norethindrone i n Organic S o l v e n t s
280
NORETHINDRONE
The following r o t a t i o n s have been reported:
Ia1D -25" (Chloroform)
['ID -24' (Chloroform)
['ID -31.7' (Chloroform)
[a], -33' (Chloroform)
3. Synthesis
D j e r a s s i , Miramontes , Rosenkranz and Sondheimer3

f i r s t reported t h e synthesis (Scheme I ) of norethindrone
from 19-nortestosterone I . Chromium t r i o x i d e oxidation
afforded estr-4-ene-3,17-dione 11, Reaction of t h e
dione with ethyl orthoformate under c a r e f u l l y c o n t r o l l e d
conditions r e s u l t e d i n s e l e c t i v e enol e t h e r formation a t
C-3, Ethynylation followed by cleavage of t h e enol e t h e r
with aqueous mineral acid gave norethindrone.
Alternate methods using e s t r a d i o l 3-methyl e t h e r

o r estrone tetrahydropyranyl e t h e r have a l s o been
described.l63l7
I n a recent patent1* the preparation of 17a-

ethynyl-4-androstene-17f3,19f3-diol-3-onewas reported i n
d e t a i l . I t was mentioned t h a t t h e 19-hydroxy compound can
be converted t o the 19-nor d e r i v a t i v e with a strong base
as reported by Barber -- e t a l l 9 and Meyer?O The general
scheme o f preparing norethindrone by t h i s method i s
outlined i n Scheme 11.
4. Metabolism
Tissue d i s t r i b u t i o n of t r i t i a t e d norethindrone i n
r a t s was studied by Watanabe and co-workers?l They
observed no s e l e c t i v e uptake by any t i s s u e up t o f o u r
hours a f t e r administration. Matsuyoshi22 incubated
norethindrone with rat l i v e r and kidney homogenate and
obtained dihydro and tetrahydro d e r i v a t i v e s of
norethindrone.
281
A R V l N P. SCHROFF AND ERNESTS. MOYER
&-&
I
0 0
'I
IV Ill
Yicherne 1 Synthetic Pathway to Norethindrone
282
NORETHINDRONE
&--’”
H
Scheme I1 Alternate Synthetic Pathway to Norethindrone
283
The metabolic fate of norethindrone in humans has

been studied by several investigators.23-39
Kamyab and c o - ~ o r k e r sadministered
~~ lk-nore-
thindrone intravenously to seven women and found the
radioactivity excreted mainly in the urine. In the first
twenty-four hours 32.1%, and in five days 53.9% of the
activity given appeared in the urine. Layne and co-
w o r k e r ~found
~ ~ a similar excretion pattern with tritiated
norethindrone; namely, 50.4% in five days after oral dose
and 70.2% after intravenous administration. A radio-
activity excretion of only 33% in five days was reported
by M ~ r a t aafter
~ ~ oral administration of 100 mg of 14C or
3H labeled norethindrone to postmenopausal women.
Kamyab --
et a130 observed the urinary radioactivity
excretion in rabbits to be comparable to that found in
humans. The amount of radioactivity in the conjugated and
unconjugated states was measured31 in plasma after intra-
venous administratibn. Substantial fractions of the
administered dose were present in the circulation for up
to forty-eight hours after injection. The unconjugated
activity in the plasma decreased rapidly to less than 0.4%
of the dose per liter of plasma after twelve hours whereas
after thirty minutes, a considerable amount of activity
was in the form of conjugated metabolites.
Gerhards and c o - ~ o r k e r sstudied
~~ plasma radio-
activity as well as excretion in urine and feces after
p.0. administration of 20 mg of 14C-norethindrone of
0 . 2 5 mg 3H-norethindrone to males. The radioactive
substances in plasma were characterized as: 17a-ethynyl-
58-estrane-3~,17B-diol;17a-ethynyl-58-estrane-38,178-diol
and 17a-ethynyl-5a-estrane-3~,178-diol.
Earlier, a number of i n ~ e s t i g a t o r s ~ 3 ~ ~ ~ ~ ~ ~ J
J 3 2 >
36 had reported excretion of 17a-ethynyl-178-estradiol

after oral administration of norethindrone. These obser-
vations, however, were not supported by --
in vitro investi-
gations. As an explanation of this discrepancy, B r e ~ e r ~ ~
recently suggested that the increased estrogen content
in --
in vivo experiments probably is due to artifacts pro-
duced during work-up of the urine. Recent work of
Sti1lwe11 and co-workers substantiated this observation.
They found no ethynyl estradiol in urine after administra-
tion of norethindrone to a female.
284
NORETHINDRONE
All norethindrone metabolites characterized from

humans and rabbits possess the 17a-ethynyl group. One
exception to this was the in vitro experiments of
Palmer.37 He identified 4-estrene-3,17-dione as a
metabolite from norethindrone. The overall metabolic
pathway of norethindrone is illustrated in Scheme 111.
5.1 Elemental Analyses
The elemental analyses obtained on USP reference
standard norethindrone and that reported by Djerassi and
co-workers3 are as follows:
Element % Theory Ref. Std. Reported3
C 80.49 80.69 80.83
H 8.79 8.64 8.80
5.2 Phase Solubility

The phase solubility analysis of norethindrone
was carried out using ethyl acetate as the solvent.
Calculations as per USP XVIII were performed on a time-
sharing computer using an in-house program. The results
are illustrated in Figure VII.40
5.3 Thin-Layer Chromatographic Analysis
A TLC procedure for separating norethindrone
from possible impurities is described in the USP (XVIII)?l
One hundred micrograms of the norethindrone sample and
one microgram each of four possible impurities (19-nor-
androstendione, norethynodrel, log-hydroxy-norethynyl-
testosterone and 106-hydroxy-norandrostendione) are
spotted in individual lanes on a silica gel plate. The
TLC plate is developed to a height of 17.5 cm by ascending
chromatography employing a solvent system of 95:s
chloroform-methanol. After air drying the plate, it is
sprayed with a mixture of 1:3 H2S04 and dehydrated alcohol.
The plate is heated in an oven at 105'C for five minutes
and the intensities of the various foreign related
285
Scheme 111. Norethindrone and i t s major metabolites
NORETHINDRONE
X CQORDINATES Y CQORDJMATES
3.8489063 4.0180889
8 . 6 4 9 2 7 14 8 - 75336YO
1 1 51 38223 9.3M7186
15.8089375 9.2340285
20.6400349 9.2dTU611
I
25.5679YSt: Y. 4029951
30.0288913 9.401 9 4 9 0
3 4 - 410872V 9.6795672
42.2361355 9.6282336
5’1 * 8 5 6 0 5 19 Y. 6Y19881
.................................................
HBH K A N Y vOIIJTS TO Pt F I T T E D ? 8
SLOPE = O.GlU19193 Y INTERCEPT = 9.158532656
PERCENT P U K I l Y = 98.V80807304
Y I
0 1
0 ,
0
O t
. I
1
* t + t *
* * t
4
0
F i g u r e VII Phase S o l u b i l i t y o f Norethindrone
287
s t e r o i d spots a r e compared v i s u a l l y under long-wavelength

u l t r a v i o l e t l i g h t with the i n t e n s i t i e s produced by t h e
corresponding s t e r o i d s i n t h e norethindrone sample. No
more than two impurities t o t a l i n g a maximum of two percent
a r e allowed.
This TLC procedure t a k e s i n t o consideration

only one s y n t h e t i c method which produces t h e above
mentioned impurities. The USP Subcommittee # 6 r e c e n t l y
recognized t h i s and proposed another TLC procedure which
w i l l accommodate o t h e r methods of s y n t h e s i s .42
Thin-layer chromatography was r e c e n t l y re-

ported43 f o r q u a n t i t a t i n g norethindrone i n t a b l e t s . Here
a s i n g l e t a b l e t is e x t r a c t e d , t r e a t e d with i n t e r n a l
standard s o l u t i o n (norethynodrel) and spotted (10-20 pg)
on a s i l i c a g e l GF p l a t e . After development with 4 : l
benzene-ethylacetate, t h e p l a t e i s a i r d r i e d and scanned
a t 250 nm with a Schoeffel double-beam spectrodensi-
tometer. Quantitation i s achieved by comparing t h e
norethindrone t o i n t e r n a l standard r a t i o (area) with
t h a t of a standard obtained i n t h e same manner.
Thin-layer chromatogra hy has a l s o been used t o

i d e n t i f y norethindrone i n tablet^!^ An a l i q u o t o f a
chloroform e x t r a c t i s s p o t t e d and t h e p l a t e developed
using cyclohexane-ethylacetate (1:l) as t h e solvent
system. After drying a t 100°C f o r t e n minutes, t h e
p l a t e i s sprayed with antimony t r i c h l o r i d e i n chloroform.
Norethindrone has an Rf o f 0.47 and a v i o l e t c o l o r i n
daylight and red c o l o r under u l t r a v i o l e t l i g h t .
5.4 Vapor Phase Chromatography
Oxidation of norethindrone t o 19-norandrosten-

dione was observed45 on metal columns. Lodge46 s u b s t i -
t u t e d a g l a s s column packed with 5% QF-1 on Diatoport S
and developed a s i n g l e t a b l e t assay method. A t a b l e t
containing 1-5 mg of norethindrone i s dissolved i n acetone
containing pregnenolone a c e t a t e as an i n t e r n a l standard
and an a l i q u o t i s i n j e c t e d d i r e c t l y i n t o t h e g l a s s column
operated isothermally a t 20OoC.
288
NORETHINDRONE

K e a reported
~ ~ ~ an ultraviolet method for the
quantitative analysis of norethindrone in tablets. Here,
powdered tablets equivalent to 6 mg of norethindrone were
extracted with 100 ml of chloroform and a 20 ml aliquot
evaporated to dryness. The residue is redissolved in
100 ml methanol, its absorbance measured at 240 nm and
the norethindrone content calculated. The accuracy of
the method has been claimed to be better than 5 % . A
modification of the above procedure has been included in
the NF XII147 for determining content uniformity of
norethindrone tablets.
A number of colorimetric methods have been used
to detect and determine the quantity of norethindrone.
A. Blue Tetrazolium
The reaction of blue tetrazolium with various
steroids has been studied thoroughly by Meyer and
Lindberg ,4 8 They noted that A4-3-ketosteroids (devoid
of an a-ketol function) react with blue tetrazolium and
reduce it to form a reddish-purple colored formazan.
This reactivity is considerably enhanced in the case of
19-norsteroids and may result from a complex oxidative
mechanism resulting in aromatization of the A-ring of
these steroids. Smith and c o - w ~ r k e r sextended
~~ this
concept for the analysis of norethindrone in tablets.
They extracted the progestin with a chlorinated solvent
mixture and developed the color by adding blue tetrazolium
and tetramethylammonium hydroxide. The reaction is
allowed to proceed in absence of light for sixty minutes
at room temperature. Quantitation is achieved by
measuring the color intensities of the standard and
sample preparations at 530 run.
289
B. Isonicotinic Acid Hydrazide

Reaction of A4-3-ketosteroids with isonico-
tinic acid hydrazide (isoniazid) produces a ellow hydra-
zone which can be used for quantitation.50 8 5T* 52
Barth14 used this reaction in automating the col imetric
quantitation of norethindrone in tablets, and Wu85,54
used the manual approach for the AOAC collaborative study
on norethindrone, Recently,55 the Food and Drug Adminis-
tration Laboratory published an automatic method which is
capable of performing single tablet assays.
5.7 Titrimetric Analysis
The British P h a r m a ~ o p e i arecommends
~~ a potentio-
metric titration method for the assay of norethindrone.
Here a tetrahydrofuran solution of norethindrone, which
also contains silver nitrate, is titrated with sodium
hydroxide solution and the end point is determined
potentiometrically.
Rizk and c o - ~ o r k e r smodified
~~ the method so
that 30 mg of norethindrone can be easily titrated as
compared to 200 mg required for the Britith Pharmacopeia
method.
290
NONETHINDRONE
6. References
1. C. J. Shaw, Ortho Research Foundation, personal

communication.
2. R. J. Mesley, Spectrochimica Acta 22, 889 (1966).
3. C . Djerassi, L. Miramontes, G. Rosenkranz, and
F. Sondheimer, J. A!I?. Chem. SOC. 76, 4092 (1954).
4. W. Neudert and H. Ropke, "Atlas ofsteroid
Spectra", Springer-Verlag, Inc., New York, 1965
Spectrum #770.
5. A. D. Cross, P. W. Landis and J. W, Murphy,
Steroids 5 , 655 (1965).
6. D. C. DeJFngh, J. D. Hribar, P. Littleton, K.
Fotherby, R. W. A. Rees, S. Shrader, T. J. Foell,
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7. A. Zaffaroni, personal communication, 1961.
8. A. D. Mebane, Ortho Research Foundation, personal
communication.
9. L. S. Nielsen, Arch. Pharm. Chemi. 74,78 (1967).
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13. M. Kuhnert-Brandstatter, E. Junger and A. Kofler,
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14. H. Barth, Ortho Research Foundation, personal
communication.
15 The Merck Index 8th Edition, Merck and Co., Inc.,
a
Rahway, N.J. 1968, p.748

16. F. B. Colton, U. S. Patents 2,655,518 (1952),
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21. H. Watanabe, N. N. Saha a g D. S. Layne, Steroids
-
11, 97 (1968).
22. K. Matsuyoshi, Folia Endocr. Jap. 43, 91 (1967).
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29 1
ARVlN P.SCHROFF AND ERNESTS. MOYER
25. J. B. Brown and H. A, F. B l a i r , Proc. Roy. SOC.

Med. 53, 433 (1960).
26. K. F o z e r b y , S. Kamyab, P. L i t t l e t o n , and K. J .
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27. E. Gerhards, W. Hecker, H. Hitze, B. Nieuweboer,
and 0. Bellmann, Acta Endocr. 68, 219 (1971).
28. S. I s h i h a r a , F o l i a Endocr. Jap. g , , , 5 5 (1966).
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(1960).
30. S. Kamyab, P. L i t t l e t o n , and K. Fotherby,
J . Endocr. 39, 423 (1967).
31. S. Kamyab, K. Fotherby, and A. Klopper, J.
Endocr. 41, 263 (1968).
32. H. Langecker, Acta Endocr. 37, 14 (1961).
33. C . Lauritzen and W. D. Lehmann, Arch. Gyngk.
-
204, 212 (1967).
34. D . S. Layne, T. Golab, K. Arai, and G . Pincus,
Biochem. Pharmac. 12, 905 (1963).
35. S. Murata, Folia Endocr. Jap. 43, 1083 (1967).
36. H. Okada, M. Amatsu, S. Ishihara, and G . Tokuda,
Acta Endocr. 46, 31 (1964).
37. K. H. P a l m e r , J . F. Feierabend, B. Baggett, and
M. E . Wall, J. Pharmac. Exp. Ther. 167, 217 (1969).
38. C . A. Paulsen, Metabolism 14 ,313 ( 1 9 6 5 ) .
39. W. G . S t i l l w e l l , E . C . Horzng, M. G . Horning,
R. N. S t i l l w e l l , and Z . Z l a t k i s , J . S t e r .
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40. R. E. Huettemann, Ortho Research Foundation ,
personal communication,
41. U. S. Pharmacopeia, 18th Rev., Mack Publishing Co. ,
Easton, Pa. 1970, p.453.
42. K. Florey, U.S.P. Sub-Committee #6, personal
communication.
43. A. P. Shroff and C. J . Shaw, J. Chromatog. S c i .
-10, 509 (1972).
44. G . R. Keay, Analyst 93, 28 (1968).
45. Facts and Methods, F G M S c i e n t i f i c Research - 5,
(3) 1964.
46. B, A. Lodge, Can. J . Pharm. S c i . 5, 74 (1970).
47. National Formulary, 13th Edirion, Mack
Publishing Co. , Easton, Pa. , 1970, p.488.
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813 (1955).
292
NORETHINDRONE
49. R , V. Smith, T. H. Hassall, and S. C . Liu, J.

Ass, O f f , Anal. Chem. 53, 1089 (1970).
50. L. L. Smith and T. F o e l l , Anal. Chem. - 31, 102
(1959).
51. E . J . Umberger, Anal. Chem. 27, 768 (1955).
52. A. E r c o l i , L. deGiuseppe and P. deRuggiere,
Farm. Sci. Tec. (Pavia) 7, 170 (1952).
53. J . Wu, J. Ass. O f f . AnalTChem. 53, 831 (1970).
54. J . Wu, J. Ass, O f f , Anal. Chem. 54, 617 (1971).
55. L. K. Thornton, Public Health Service (FDA)
Drug Auto Analysis Manual, Method # 2 2 , April
1972,
56. B r i t i s h Pharmacopoeia, 1973, University
P r i n t i n g House, Cambridge 1973, p.323.
57. M. R i z k , J . J. Vallon, and A. Badinand, Anal.
-
Chim. Acta 65, 220 (1973).
293
NORGESTREL
Andrew M . Sopirak and Leo F. Cullen

NORGESTREL
CONTENTS
1. Description
2.3 U l t r a v i o l e t Spectra
2.4 Mass Spectra
2.5 Melting Range
3. Synthesis
6.2 U 1 t r a v i o l e t S pec t r o pho t ometri c Anal ye i 8
6.31 I s o n i c o t i n i c Acid Hydrazide
6.32 Dinitrophenylhydrazine
6.33 Blue Tetrazolium
6.34 2 , 6 - D i - s - b u t y l - p - c r e s o l
6.4 Fluorometric Analysis
6.5 T i t r i m e t r i c Analysis
7. References
295
ANDREW M. SOPIRAK AND LEO F. CULLEN
1. Description
Norgestrel i s designated a s &-13-ethyl-l7&-ethynyl-
17-hydroxygon-4-en-3-one and is l i s t e d i n t h e s u b j e c t i n d i c e s
of Chemical Abstracts under the heading: dl-13-ethyl-17-
hydroxy-18,19-dinor-l7&-pregn-4-en-20-yn-3~ne.
'21H28'2
Norgestrel i s a white t o off-white, p r a c t i c a l l y
odorless, c r y s t a l l i n e powder.
2.1 I n f r a r e d Spectra
An i n f r a r e d absorption spectrum of a potassium
bromide dispersion of n o r g e s t r e l (Wyeth Reference Standard
.
m a t e r i a l , Lot #C-10484) i s presented i n Figure 1. This
spectrum agrees with a published spectrum1 The s p e c t r a l
band assignments a r e l i s t e d i n Table I.
- ~~ ~~ -~ ~
Table I a. AOY4.Z
Infrared S p e c t r a l Assignments of Norgestrel

Frequency
_ _ _(cm.-1)
- Vibration Mode Reference
3350 OH s t r e t c h i n g 2
3270 Acetylenic C-H s t r e t c h i n g
2940 and 2865 A l i p h a t i c C-H s t r e t c h i n g
1655 Conjugated C=O s t r e t c h i n g
1615 C=C s t r e t c h i n g
1448 Methyl C-H asymmetrical bending
1420 and 1335 OH i n plane bending
1360 Methyl C-H synnnetrical bending
1065 Alcoholic C-0 S t r e t c h i n g
700 670 - Acetylenic C-H bending
296
WAVILINGIH IMICROhlSl
w
\D
4
3aa IWO M
WAVfNUMBfR ICM ‘I
Figure 1 - I . R . Spectrum of Norgestrel (Wyeth Reference Standard Material,

Lot #C-10484) - 1% KBr Pellet
The 60 MH, nuclear magnetic resonance (NMR) spec-

trum, shown i n Figure 2, w a s obtained by d i s s o l v i n g norges-
t r e l (Wyeth Reference Standard material, Lot tC-10484) i n
deuterochloroform containing t e t r a m e t h y l s i l a n e as an i n t e r n a l
reference. The only exchangeable proton i s t h e hydrogen
associated w i t h oxygen a t p o s i t i o n 17. The NMR proton spec-
t r a l assignments a r e l i s t e d i n Table 11.
Table I1
0
5.86 -&cH=c’
- \ singlet
2.61 -WE si ng 1e t
2.31 -
OH elnglet
1.02 -% -5 triplet
2.3 U l t r a v i o l e t Spectra
4
Fernandez and Noceda reported a Amax. of 240 nm.
f o r n o r g e s t r e l i n absolute methanol ( a = 56.0). Norgestrel
i n 95% ethanol (Wyeth Reference Standard m a t e r i a l (Lot #C-
10484) when scanned between 360 tun. and 200 nm. exhibited a
Amax. a t 240 run. ( a = 54.6). This spectrum i n 95% ethanol
is ehown i n Figure 3.
2.4 Mass Spectra
The mass spectrum of n o r g e s t r e l (Wyeth Reference

Standard m a t e r i a l , Lot 8C-10484) w a s obtained by d i r e c t
i n s e r t i o n of t h e sample i n t o an MS-902 double focusing, high
r e s o l u t i o n mass spectrometer. The sample was run a t 200°C.
and 1.0 x t o r r with t h e i o n i z a t i o n e l e c t r o n beam energy
a t 70 eV.. The high r e s o l u t i o n d a t a were compiled and tabu-
l a t e d with the aid of an on-line PDP-8 D i g i t a l Computer.
Results a r e presented as a b a r graph i n Figure 4 and t h e
high r e s o l u t i o n mass s p c t r u m assignments o f prominent ions
a r e given i n Table 111.’ This s p e c t i s i n agreement
with t h a t presented by DeJongh et.a?
298
Im, am f0 4rJ m a CPS
m
r..
ro a0
_ ._. _ _ , _ . .... . . , . _ _im.. . . . . . . . . . . . . . . . . . . . . .a.CPS
. . . . , _ .m .
t
d
W
W
Figure 2 - NMR Spectrum of Norgestrel (Wyeth Reference Standard Material, Lot #C-
10484) - Solvent: deuterochloroform
W
0
0
0
WAVELENGTH lnml
Figure 3 - Ultraviolet Spectrum of Norgestrel (Wyeth Reference Standard Material,

Lot #C-10484) - Solvent: 95% ethanol
3 2
OH
MOL. WT. 312
C17H250
229 245
110
229
20 I -
C19H2302
283
-CzHj
0 +-I
320
m/e-
Figure 4 - MassSpectrum of Norgestrel (Wyeth Reference Standard Material,

Lot #C- 10484)
ANDREW M.SOPIRAK AND LEO F. CULLEN
Table I11
High Resolution Mass S p e c t r a l Assignments of Norgestrel
Measured Mass Calculated Mass Formula
312.2082 312.2088
'21H28' 2
284.18 12 284.1775
'1gH24'2
283.1698 283.1698
' SH23' 2
1
245.1902 245.1905
'1?25'
229.1596 229.1592
c16H220
110.0730 110.0731
7H100
Norgestrel gives a molecular i o n a t nJ" 312 as i t s base
peak. The f i r s t prominent fragment occurs a t dz 297 which
corresponds t o the l o s s of t h e methyl r a d i c a l , followed by
t h e more i n t e n s e peak a t e 283 which r e p r e s e n t s cleavage
of t h e 13-ethyl r a d i c a l &C2H5.). A peak a t d= 284 i s
observed from t h e l o s s of C2H4.
Ions of 4 2 245 and d ' 229 a r e explained by the cleav-
age of the D-ring with hydrogen transfer.6 The i n t e n s e
fragment i n t h e spectrum a t d~ 110 i s i n d i c a t i v e of t h e
remaining A-ring molecular fragment w i t h t h e formula C+IloO.
2.5 Melting Range
The following melting temperature range has been
observed on n o r g e s t r e l (Wyeth Reference Standard m a t e r i a l ,
Lot #C-10484) employing t h e U.S.P. XVIII, Class I conditions:
207-210°C.8 Short et.al. r e p o r t s a melting range tempera-
t u r e of 2 0 6 - 2 0 7 ° C . 1 T m e l t i n g temperature range does n o t
change s i g n i f i c a n t l y with v a r i a t i o n s i n heating rates from
1 t o SO~./min.
The d i f f e r e n t i a l thermal a n a l y s i s (DTA) curve of
n o r g e e t r e l (Wyeth Reference Standard material, Lot 8C-10484)
obtained from room temperature t o t h e m e l t i n g point a t a
heating r a t e of 20°C./min. i s presented i n Figure 5. A
sharp endothermic change observed a t 209OC. corresponds t o
t h e m e l t of t h e drug.
2.7 Solubility
4
The f o l owing s o l u b i l i t y d a t a were obtained a t
room temperaturet
302
w
0
W
T. OC (CORRECTED FOR CHROMEL ALUMEL THERMOCOUPLES)
Figure 5 - DTA Spectrm of Norgestrel (Wyeth Reference Standard Material,

LOt #C- 10484)
ANDREW M. SOPIRAK A N D LEO F. CULLEN
chloroform 111 mg./ml.

acetone 15 mg./ml.
95% ethanol 13 mg./ml.
benzene 7 mg./ml.
ether 2 mg./ml.
water l e s s than .01 mg./ml.
The X-ray powder d i f f r a c t i o n p a t t e r n of n o r g e e t r e l
(Wyeth Reference Standard m a t e r i a l , Lot 8C-10484) obtained
with a P h i l i p s diffractometer a t a scan rate of 1°/min.
using CuKz r a d i a t i o n i s shown i n Figure 6. The c a l c u l a t e d
d-spacings for the d i f f r a c t i o n p a t t e r n a r e presented i n
Table I V . The c r y s t a l has orthorhombic synnnetry wizh space
group P212121. U n i t ~ $ 1 8parameters are a = 20.673A, b =
12.9791, and c = 6.567A.
Table I V
X-Ray Powder D i f f r a c t i o n P a t t e r n f o r Norgestrel
-
28 -
d (8) I/Io
10.95 8.08 0.02
13.9 6.37 1.00
14.2 6.24 0.53
14.5 6.11 0.81
15.2 5.83 0.20
15.8 5.61 0.72
17.4 5.09 0.17
18.0 4.79 0.06
18.9 4.69 0.06
19.8 4.48 0.42
21.2 4.19 0.04
21.8 4.075 0.05
23.3 3.817 0.19
24.9 3.575 0.24
25.3 3.520 0.12
26.0 3.428 0.09
26.3 3.389 0.09
27.6 3.232 0.07
29.05 3.073 0.07
30.4 2.940 0.10
31.8 2.814 0.10
34.7 0.07
d = (interplanar distance
1/10 = r e l a t i v e i n t e n s i t y (based on highest i n t e n s i t y of
1.001
3 04
I00 100 100
PO 90 90
80 80
80
70 70 70
60 60
60
50 50
50
w 40
0 40 40
vI
30 30
30
20 10
b
10 10
10 14
18 22 2 8 26 M 34 3a
Figure 6 - X-RayPowder Diffraction Pattern of Norgestrel (Wyeth Reference

Standard Material, Lot fC-10484)

d l - n o r g e s t r e l i s a racemate and t h e r e f o r e e x h i b i t s
no o p t i c a l r o t a t i o n . A s o l u t i o n of 500 mg. of n o r g e s t r e l
i n 10 m l . of chloroform has a reading of Oo 2 0.05O i n a
1 decimeter c e l l a t 25OC. with sodium D light.9
3. Synthesis
The b a s i c s y n t h e t i c f o r n o r g e s t r e l has been devel-
oped by Hughes and Smith €83 and i s presented i n Figure 7.
The r i n g formation i s accomplished by t h e base catalyzed
condens a t i o n of 6-(m-me thoxyphenyl) -hex-l-ene-3-one (I)
with 2-ethylcyclopentan-l,3-dione (11) t o y i e l d t h e t r i o n e
(111). Cyclodehydration of (111) with p-toluenesulfonic
acid forms gonapentaene-17-one (IV). An a l t e r n a t e s y n t h e t i c
r o u t e f o r t h i s r i n g s t r u c t u r e (IV) has been described by
Hughes et.al.12 and i s a l s o presented i n Figure 7. I n t h i s
r o u t e t h e gonapentaene-17-one (IV) i s prepared by t h e meth-
a n o l i c hydrochloric acid c y c l i z a t i o n of t h e eeco-oestra-
tetraene-14,17-dione (IIIA) prepared from t h e base catalyzed
condensation of 2-ethylcyclopentan-l,3-dione (IIA) with
t e t r a l o l CIA).
C a t a l y t i c hydrogenation of (IV) y i e l d s t h e correspon-
ding gonatetraene-17-one (V) and subsequently converted
i n t o t h e e s t r a d i o l d e r i v a t i v e (VII) by reduction i n i t i a l l y
with sodium borohydride followed by potassium-ammonia-
t e t r a h drofuran. Conversion of (VII) by t h e Wilds and
NelsonP3 modification of t h e Birch reduction14 gives t h e
1,4-dihydro d e r i v a t i v e (VIII) . An Oppenauer oxidation15
produces t h e 17-ketone (1x1, which is then ethynylated,
giving t h e enol-ether intermediate (XI. Treatment of (XI
with methanolic hydrochloric acid y i e l d s n o r g e s t r e l (XI).
A s i m i l a r syn h e t i c route i s presented i n a review a r t i c l e
by K l i m s t r a . 16
Norgestrel i s extremely s t a b l e i n t h e s o l i d s t a t e when
exposed t o long term accelerated thermal and photochemical
conditions. No decomposition has been observed f o r t h i s
compound when s t o r e d f o r periods of 5 years a t room tempera-
t u r e , 1 year a t 45OC., 1 month a t 75OC. and 1 month under
d i r e c t W light.17 Decomposition has been reported under
severe hydrolytic and more accelerated photolytic and t h e r -
mal conditions.18
Studying s t e r o i d s s t r u c t u r a l l y r e l a t e d t o n o r g e s t r e l ,
Savard et.al.19, demonstrated t h a t exposure o f 04-3-keto-
s t e r o i d s t o u l t r a v i o l e t l i g h t produced s a t u r a t i o n of t h e 4,5
double bond and/or migration of t h e double bond from t h a t
306
NORGESTREL
I
CH30
IA IIA
KOH CH30H OH- 1

C H30
lllA
CaC03
Pd
CH30
I
V
NaBH, CH30H
continued ....
Figure 7 - Synthetic Routes for Norgestrel
307
cn30&On
VI
y 3 > do
c n30
VII
Figure 7 - Synthetic Routes €or Norgeetrel (concluded)
308
NORGESTREL
p o s i t i n i n conjugation with t h e 3-ketone. Subsequent work

with ~ ‘ - 3 - k e t o progestational s t e r o i s n pharmaceutical
systems under photolytic conditions 28*2f have f u r t h e r sup-
ported degradation of s t e r o i d a l A-ring as described by
Savard et.al. However, t h e s p e c i f i c decomposition products
f o r the progestin, n o r g e s t r e l , have not been reported i n
the l i t e r a t u r e .
Administration of racemic n o r g e s t r e l t o humans has been
shown by mass spectroscopy andother analytical techniques t o
y i e l d a range of metabolites. The major metabolic product
has been i d e n t i f i e d as tetrahydronorgestrel, 13$-ethyl-1706
ethyn 1-5P-gonan-35 17$-diol; i.e., n o r g e s t r e l reduced a t
theAe-3-keto group i n the A-ring.6 By o p t i c a l r o t a t o r y
dispersion s t u d i e s , t h i s metabolite w a s shown t o be e n t i r e -
l y t h e o p t i c a l l y pure d-isomer22. Other metabolites have
been i d e n t i f i e d as 13$-ethyl-l7e~-ethynyl-5$-gonan-3$,
d i o l and mono-hydroxylated d e r i v a t i v e s of norgestrel. 4 3 3 4
The following d a t a were obtained on n o r g e s t r e l
(Wyeth Reference Standard m a t e r i a l , Lot #C-10484).
E 1ement % Theory % Determined

C 80.73% 80.66%
H 9.03% 8.92%
6.2 U l t r a v i o l e t Spectrophotometric Analysis
A 4 -3-ketosteroids produce a c h a r a c t e r i s t i c chromo-
phore s stem with maximum absorption between 230 nm. and
270 The u l t r a v i o l e t absorption maximum of n o r g e s t r e l
a t about 240 nm. i n 85% ethanol i n water (v/v) has been
u t i l i z e d f o r t h e analysis of the s t e r o i d i n t a b l e t formula-
t i o n s containing estrogens.26 In t h i s procedure a sample
equivalent t o about 0.5 mg. n o r g e s t r e l i s s t i r r e d i n 85%
ethanol i n water (v/v>. This s o l u t i o n i s d i l u t e d t o o b t a i n
a f i n a l concentration of about 10 pg./ml. i n t h e 85% ethanol.
Following c e n t r i f u g a t i o n t o remove insoluble excipient mater-
i a l , the absorbance i s spectrophotometrically measured and
compared with a standard s o l u t i o n of norgestrel. Norgestrel
has been i s o l a t e d from i n t e r f e r i n g formulation components
by an e x t r a c t i o n of the s t e r o i d with organic solvents (viz.,
chloroform and methylene c h l o r i d e ) from an a c i d i c aqueous
solution.
309
ANDREW M. SOPIRAK A N D L E O F. C U L L E N
A modified v e r s i o n of an automatic analyzer system

procedure f o r a relatedA4-3-ketosteroid27 has been applied t o
t h e q u a n t i t a t i v e a n a l y s i s of norgestre1.l’ This completely
automated technique i s capable o f d i s i n t e g r a t i n g a whole t a b -
l e t , dissolving the active constituent, f i l t e r i n g it, dilu-
t i n g a portion of t h e c l e a r f i l t r a t e t o a d e s i r e d volume
and o b t a i n i n g a complete u l t r a v i o l e t absorption spec trum.
The accuracy of t h e automated method i s comparable t o t h a t
of t h e manual spectrophotometric method f o r n o r g e s t r e l .
The absorption c h a r a c t e r i s t i c s e x h i b i t e d by nor-

g e s t r e l i n ethanol s o l u t i o n a t about 240 nm. i s due t o t h e
conjugated c h a r a c t e r of t h e OC, p-unsaturated carbonyl group
i n t h e A-ring. Any a l t e r a t i o n i n t h e conjugated c h a r a c t e r
of t h i s s y s t e m by prolonged h e a t i n g a t extreme temperatures
o r by prolonged i r r a d i a t i o n w i t h a s t r o n g u l t r a v i o l e t l i g h t
source w i l l r e s u l t i n a corresponding l o s s i n u l t r a v i o l e t
a b s o r p t i v i t y a t 240 nm. Consequently, t h i s method w i l l
measure i n t a c t n o r g e s t r e l i n t h e presence of i t s photochem-
.
i c a l and t h e ma1 degradation products and i s , thus, s t a b i l i t y -
i n d i c a t i n g 2E
Reactions of t h e 3-keto group of p r o g e s t a t i o n a l

s t e r o i d s with t h e connnon carbonyl reagents have provided
t h e b a s i s f o r s e v e r a l o t h e r spectrophotometric a n a l y t i c a l
methods. These techniques can b e applied t o t h e a n a l y s i s of
n o r g e s t r e l . Evans and G i l l i a m observed t h a t t h e absorption
maxima of t h e thiosemicarbazones of s a t u r a t e d and oC, p-unsatu-
r a t e d ketones could be determined i n t h e presence of excess
thiosemicarbazide.28 Bush29 and Talbot et.al.30 have
e
ap l i e d t h i s technique t o t h e determination of a s e r i e s of
A -3-ketosteroids which y i e l d thiosemicarbazones w i t h
absorption maxima a t 299 t o 301 nm.
Gtlr6g3l developed a procedure f o r t h e determina-

t i o n 0 € ~ ~ - 3 - k e t o s t e r o i d which
s i s based on t h e C l a i s e n con-
densation of t h e a c t i v e methylene groups of k e t o s t e r o i d s
w i t h d i e t h y l o x a l a t e l e a d i n g t o spectrophotometrically
a c t i v e glyoxalyl d e r i v a t i v e s . The development of t h e chromo-
phore was c a r r i e d o u t a t room temperature i n a mixture of
t e r t i a r y butanol and cyclohexane i n t h e presence of sodium
t e r t i a r y butoxide while t h e absorbance w a s measured i n moder-
a t e l y a c i d i c ethanol. The method i s s u i t a b l e f o r t h e char-
a c t e r i z a t i o n and q u a n t i t a t i v e determination of n o r g e s t r e l
which produces absorption maxima a t about 244 run. and 318
nm.17
310
NORGESTREL
S a l i c y l o y l hydrazide r e a c t s with A4-3-ketoeteroids

t o form c h a r a c t e r i s t i c hydrazones which absorb s t r o n g l y i n
t h e ~ l t r a v i o l e tand ~ ~m a be used f o r t h e a n a l y t i c a l d e t e r -
mination of n o r g e s t r e l . 17
Giirijg developed a simple and r a p i d u l t r a v i o l e t

spectrophotometric method f o r t h e d e t e r m i n a t i n of A4-3-
k e t o s t e r o i d s i n pharmaceutical preparation^.^' The method
i s based on t h e reduction of t h e C-3 carbonyl group with
sodium borohydride, followed by t h e determination of t h e
decrease of absorbance due t o t h e reduction ae measured by
d i f f e r e n t i a l spectrophotometry. This procedure i s a p p l i -
cable t o t h e a n a l y s i s of n o r g e s t r e l i n t a b l e t formulations.1 7
E t h i n y l e s t r a d i o l and common i n a c t i v e t a b l e t components do
not i n t e r f e r e with t h e determination.
6.31 I s o n i c o t i n i c Acid Hydrazide
Norgestrel can be assayed u t i l i z i n g t h e well-

known i soni cot i n i c acicj4hydr azid e (INH) c o l o r i m e t r i c method
described by Umberger. The chemistry of t h i s technique
i s based on t h e formation of t h e i s o n i c o t i n y l hydrazone of
n o r g e s t r e l from t h e r e a c t i o n of t h e s t e r o i d with INH rea-
gent i n absolute alcohol a c i d i f i e d w i t h hydrochloric acid.
The A 4-3-keto group r e a c t s q u a n t i t a t i v e l y a t room tempera-
t u r e i n l e s s than 1 hour. A s t a b l e colored s p e c i e s r e s u l t s
which allows measurement of i t s absorption a t about 380 nm.
I n t h e a n a l y s i s of t a b l e t formulations, n o r g e s t r e l i s extrac-
ted with chloroform from an a c i d i c aqueous suspension of t h e
t a b l e t s , INH reagent i s added t o an a l i q u o t of t h e e x t r a c t
and t h e r e s u l t a n t yellow s o l u t i o n i s spectrophotometrically
measured. Estrogens E i z . , 1 7 a e t h i n y l e s t r a d i o l and 17.C -
e t h i n y l estradiol-3-methyl e t h e r ( m e s t r a n o l x and i n a c t i v e
components t y p i c a l l y found i n o r a l estrogen-progestin combi-
n a t i o n dosage forms do not c o n t r i b u t e t o t h l o r develop-
ment; t h u s , do n o t i n t e r f e r e i n t h e assay. 28, %
The absorption c h a r a c t e r i s t i c s exhibited by
n o r g e s t r e l i n t h i s c o l o r i m e t r i c method a r e due t o t h e con-
jugated c h a r a c t e r of t h e s t e r o i d A - r i n g w i t h t h e formed
hydrazone. Thus, t h i s method w i l l measure i n t a c t norges-
t r e l i n t h e presence of i t s photochemical and thermal degra-
d a t i o n products f o r reasons analogous t o those d e s c r i ed
f o r t h e u l t r a v i o l e t spectrophotometric procedure.17, 28 Con-
sequently, t h e procedure i s a l s o s t a b i l i t y - i n d i c a t i n g .
311
Russo-Aleei describes a completely automated

INH reagent procedure f o r the content uniformity t e s t i n g of
c o r t i c o s t e r o i d t a b l e t formulation^.^^ This s e n s i t i v e , accu-
r a t e , and r a p i d automatic analyzer procedure has been adap-
ted t o the analysis ~5 n o r g e s t r e l i n o r a l contraceptive
t a b l e t formulations.
6.32 Dinitrophenylhydrazine
S t e r o i d s with an K , p-unsaturated carbonyl
group were observed by Djerassi and Ryan t o produce 2,4-
dinitrophenylhydrazones i t h absorption maxima a t 390 nm.
i n chloroform ~ o l u t i o n . ~ ’ Gornall and M a ~ D o n a l dhave ~~
described a c o l o r i m e t r i c procedure, a p p l i c a b l e t o norges-
t r e l , i n which t h e pale yellow c o l o r of the hydrazone
d e r i v a t i v e i s converted t o a s t a b l e , deep red resonating
quinoidal i o n species i n a l k a l i n e s ~ l u t i o n . ~ ’ The r e a c t i o n
with n o r g e s t r e l i s complete i n l e s s than 10 minutes and t h e
r e s u l t i n g colored species i s spectrophotometrically measured
a t about 450 nm.
I n a r e l a t e d study, Nishina et.al.40 r e p o r t
t h a t A 4-3-ketosteroids w i l l condense w i t h p-nitrophenyl-
hydrazine t o yield t h e corresponding hydrazone, which pro-
duces a reddish-purple species i n an a l k a l i n e s o l u t i o n of
dimethylformamide. Norgestrel produces a Amax a t 540 run.
6.33 Blue Tetrazolium
The u t i l i t y of t h e blue tetrazolium reagent
i n t h e anal s i s o f 4 - k e t o l and non-ketol s t e r o i d s i well
d o ~ u m e n t e d . z ~Meyer ~ ~ ~ and Lindberg43 noted t h a t ~‘-3-keto-
s t e r o i d s , devoid of an c - k e t o l function, r e a c t with blue
tetrazolium i n t h e presence of a s t r o n g organic base t o form
the reddish-purple difomazan d e r i v a t i v e with a Amax a t
530 nm. Application of t h e t e t r a z o l i u m technique t o the
s e l e c t i v e a n a l y s i s of n o r g e s t r e l i n anovulatory t a b l e t
formulations containing estrogens has been described by
-
Smith et.a1.44 This c o l o r i m e t r i c r e a c t i o n has been adap-
ted t o an automatic analyzer procedure f o t h e a n a l y s i s of
n o r g e s t r e l i n biopharmaceutical s ys tem s. 45
6.34 2,6-Di-tert-butyl-p-cresol
A c o l o r r e a c t i o n i s obtained by the a c t i o n
of 2,6-$&;~frt-butyl-p-cresol on n o r g e s t r e l i n an a l k a l i n e
medium. The concentration of n o r g e s t r e l i n t h e r e s u l -
t i n g colored s o l u t i o n can be determined spectrophotomet-
r i c a l l y i n t h e v i s i b l e region.
312
NORGESTREL
Cullen et.al.18 describe a s e n s i t i v e procedure,

based on a s u l f u r i c acid-induced fluorescence, f o r t h e
analysis of norgestrel i n t a b l e t s of low dosage, i.e., 15-
75 pg. per t a b l e t . Fluorogen formation, which i s e f f e c t e d
with an 85% s u l f u r i c acid reagent, i s measured a t an emis-
s i o n h a x of 550 nm. with an e x c i t a t i o n k ax of 418 nm.
S p e c i f i c i t y of t h e method with respect t o the a n a l y s i s of
i n t a c t n o r g e s t r e l i n the presence o f i t s photochemical and
thermal degradation products w a s demonstrated by comparison
t o q u a n t i t a t i v e thin-layer chromatography assay values.
This procedure has been completely automated t o permit
rapid content uniformity t e s t i n g of the dosage form. The
automatic analyzer procedure i s capable of analyzing 15
samples per hour with a r e l a t i v e standard deviation of
2 1.4% a t t h e 50 pg. n o r g e s t r e l per t a b l e t level.
Short and coworkers ” 48 applied fluorometry t o
the determination of n o r g e s t r e l i n b i o l o g i c a l f l u i d s . Nor-
g e s t r e l i s extracted from serum with methylene c h l o r i d e and
subsequently re-extracted i n t o an 80% s u l f u r i c acid i n
ethanol reagent which produces an i n t e n s e fluorescence. The
r e s u l t i n g f luorogen i s measured a t an emission Amax of 520
nm. and e x c i t a t i o n k a x of 460 nm. This method can be
applied t o the analysis of n o r g e s t r e l s o l u t i o n s containing
0.02 pg. per m l .
Bush and Sandberg4’ noted t h a t paper chromatograms

sprayed with sodium hydroxide developed an orange-yellow
fluorescence s p e c i f i c for&-3-ketosteroids under W i r r a d i -
ation. Subsequently, Abelson and Bond$’ found t h a t potas-
sium --butoxide could produce the a l k a l i n e fluorogenic
r e a c t i o n , and i s s u i t a b l e f o r q u a n t i t a t i v e measurement of
n o r g e s t r e l i n t h e 0.1-10.0 pg. per m l . l e v e l i n e t h a n o l i c
solu t i on. 17
6.5 T i t r i m e t r i c Analysis
The e t h i n y l group of norgeetrel r e a c t s s t o i c h i o -

m e t r i c a l l y with s i l v e r n i t r a t e i n tetrahydrofuran. The
n i t r i c acid produced can be t i t r a t e d w i t h 0.1 N sodium
hydroxide t o a poten iometric endpoint using a glass-calomel
electrode system. 51,52
313
Norgestrel e x h i b i t s a well-defined cathodic wave a t

t h e dropping mercury e l e c t r o d e i n a l k a l i n e isopropanol. The
halfwave p o t e n t i a l of n o r g e s t r e l i n t h i s system i s about
- 1 . 5 ~ and t h e d i f f u s i o n c u r r e n t i s l i n e a r with concentration
over t h e range of to M.9
Chromatography i s used q u a l i t a t i v e l y f o r t h e i d e n t i -
f i c a t i o n and q u a n t i t a t i v e l y f o r t h e determination of p u r i t y
and s t a b i l i t y of norgeetrel.
The various e l u a n t and adsorbent s y s tems

used €or thin-layer chromatography of n o r g e s t r e l are given
i n Table V. The v i s u a l i z a t i o n techniques used f o r d e t e c t i o n
of n o r g e s t r e l a r e a l s o included i n Table V.
Norgestrel has been d i r e c t l y chromatographed

on a 4 f t . x f inch g l a s s column packed with 0.5% QF-1 on
Chromosorb G (80/100 mesh) u t i l i z i n g a flame i o n i z a t i o n
detector.55 A column temperature of 215OC. i s employed
with helium a t 40 ml./min. as the carrier gas. This tech-
nique has been applied t o the s e l e c t i v e a n a l y s i s o f norges-
t r e l a t t h e 0.25 t o 1.0 mg. dosage l e v e l i n anovulatory
t a b l e t formulations. I n t h e a n a l y s i s n o r g e s t r e l i s extrac-
ted with chloroform from an a c i d i c aqueous auspeneion of the
t a b l e t s , the chloroform e x t r a c t i s concentrated by evapora-
t i o n and subsequently chromatographed using c h o l e s t e r o l as
the i n t e r n a l standard.
T h e q u a n t i t a t i v e a n a l y s i s of n o r g e s t r e l and
s t r u c t u r a l l y r e l a t e d progestational s t e r o i d s i n o r a l l y admin-
i s t e r e d anovulatory formulations hae been described u t i l i z i n g
a gel f i l t r a t i o n column technique and u l t r a v i o l e t spectrom-
e t r ~ A . ~s y n t h e t i c p l y s a c c h a r i d e (Sephadex LH-20) column
with methanol-water (17:3) as t h e e l u a n t permits t h e quanti-
t a t i v e separation of n o r g e s t r e l from formulation components.
Estrogens (viz., e t h i n y l e s t r a d i o l , mestranol, e s t r a d i o l ,
and e s t r a d i o l benzoate) and comnon t a b l e t e x c i p i e n t m a t e r i a l s
were shown not t o i n t e r f e r e i n t h i s assay procedure.
314
NORGESTREL
Table V
Thin-Layer Chromatographic System for Norgestrel
Solvent Visualization
System Adsorbent Technique Rf- Reference
A Silica Gel GF 1, 2 0.37 53
B Silica Gel GF 1; 2 0.45 53
C Silica Gel GF 1, 2 0.59 53
D Silica Gel GF 1, 2 0.53 53
E Silica Gel G 1, 2 0.47 54
impregnated with
DuPont Lumines-
cent Chemical 609
F Silica Gel G 1, 2 0.46 54
impregnated with
DuPont Lumines-
cent Chemical 609
G Silica Gel G 3 0.54 26
H Silica Gel G 4 0.73 1
I MN Silica Gel 1, 2 -- 18
G-HR/W
Solvent Systems
A Benzene:methanol (95: 5)
B Benzene:acetone (80:20)
C Chloroform:methanol (90: 10)
D Methylene ch1oride:me thanol: water (150: 9! 0.5)
E Petroleum ether: ethyl ether (10: 90)
F Petroleum ether: ethyl ether:diethylamine (4 0 : 60: 10)
G Chloroform: alcohol, U.S. P. (96: 4 )
H N-hexanet ethyl acetate (1: 3)
I Benzene:chloroform (8:2)
Visualization Techniques
1 Ultraviolet light
2 Sulfuric Acid Spray Reagent
3 10% Ethanolic Phosphomolybdic Acid Spray Reagent
4 Antimony Trichloride Spray Reagent
315
7. References
1 P. M. Short, E. T. Abbe and C. T. Rhodes, Can. J.

Pharm. S c i . , A, 8(1969).
2 L. Bellarny, I I T h e I n f r a r e d Spectra of Complex Mole-
cules," 2nd Ed., J. Wiley and Sons, Inc., New York,
N. Y., 1964, Chapter 6.
3. R. M. S i l v e r s t e i n and G. C. Bassler, "Spectrometric
I d e n t i f i c a t i o n of Organic CompoundeYf12nd Ed.,
J. Wiley and Sons, Inc., New York, N. Y., 1967,
Chapter 3.
4. A. A. Fernandez and V. T. Noceda, J. Pharm. Sci.,
5.
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T. Chang and C. Kuhlman, Wyeth Laboratories, Inc.,
Personal Communication.
6. D. C. DeJongh, J. D. H r i b a r , P. L i t t l e t o n , K. Foth-
erby, R. W. A. Reee, S. Shrader, T. J. F o e l l and
H. Smith, S t e r o i d s , 2, 649(1968).
7. W n i t ed S t a t e s Phannacopei a," 18th Revision , Mack
Publishing Co., Easton, Pa., 1970, pp. 935-936.
8. N. DeAngelis , Wyeth Laboratories, Inc., Personal
Comnunicat ion.
9. M. B. Freeman, Wyeth Laboratories, Inc., Personal
Communication.
10. H. Smith, G. A. Hughes, G. H. Douglas, G. R. Wendt,
G. C. Buzby, R. A. Edgren, J. F i s h e r , T. F o e l l ,
B. Gadsby, D. Hartley, D. Herbst, A. B. A. Jansen,
K. Ledig, B. J. McLoughlin, J. McMenamin, T. W.
Pattieon, P. C. P h i l l i p s , R. Rees, J. S i d d a l l ,
J. Siuda, L. L. Smith, J. Tokolics and D. H. P.
Watson, J. Chem. SOC., 1964, 4472(1964).
11. V. Petrow, Chem. Rev., 70, 713(1970).
12. G. H. Hughes and H. Smith, U. S. Patent 3,478,106
(1969) : i . S . Patent 3,442;920(1969).
13. A. L. Wilds and N. A.-Nelson, J. Amer. Chem. SOC.,
-975 5360(1953).
14. A. J. Birch and S. M. Mukherji, Nature, 163, 766
(1949).
15. R. V. Oppenauer, Org. Syn., 2,18(1941).
16. P. D. Klimstra, J. h e r . &am. Educ., 10, 630(1970).
17. L. F. Cullen, Wyeth Laboratories, Inc., Personal
18.
.
Communic a t ion
L. F. Cullen, J. G. Rutgers, P. A. Luccheei and
G. J. Papariello, J. Pharm. Sci., 57, 1857(1968).
19. K. Savard, H. W. Wotiz, P. Marcus and H. M. Lemon,
J. Amer. Chem. SOC., 75, 6327(1953).
316
NORGESTREL
20. R. E. Graham, P. A. Williams and C. T. Kenner, J.

Pharm. Sci., !By 1152(1970).
21. W. E. Hamlin, T. Chulski, R. H. Johnson and J. G.
Wagner, J. Amer. Pharm. A s s . , S c i . Ed., 2, 253(1960).
22. K. Fotherby and C. A. Keenan, Acta Endocrinol. Suppl.
-9138 83(1969).
23 P. L i t t l e t o n and K. Fotherby, Acta Endocrinol. Suppl.
- 119, 162(1967).
24. S. Kamyab, K. Fotherby and G. Wilson, J. Biochem.,
-9103 14(1967).
25. T. Higuchi and E. Brochmann-Hanssen, I1Pharmaceutical
Analysis,11 I n t e r s c i e n c e P u b l i s h e r s , Inc., New York,
N. Y., 1961, Chapter IV.
26. ,
G. J. P a p a r i e l l o , Wyeth L a b o r a t o r i e s , Inc. Personal
Communication.
27. A. J. Khoury and L. J. C a l i , Ann. N. Y. Acad. Sci.,
- 153, 456(1968).
28 L. K. Evans and A. E. G i l l i a m , J. Chem. SOC., 1943,
565(1943).
29. I. E. Bush, Fed. Proc., 2, 186(1953).
30. N. B. Talbot. S. Ulick. A. Koupreianow and A.
Zygmuntowicz, J. Clin. Endocrinol. Metab., 15, 301
(1955).
31. S. Gorog, Anal. Chem., 42, 560(1970).
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33. S. Gerog, J. Pharm. S c i . , 57, 1737(1968).
34. E. J. Umberger, Anal. Chem., 27, 768(1955).
35. Y. P. John, J. A s s . Offic. Anal. Chem., 53, 83l(1970).
36. F. M. Russo-Alesi, Ann. N. Y. Acad. S c i , , 153, 511
(1968).
37. C. D j e r a s s i and E. Ryan, J. h e r . Chem. SOC., 71,
lOOO(1949).
38. A. G. Gornall and M. P. MacDonald, J. Biol. Chem.,
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F. D. Chattaway and G. R. Clemo,,J. Chem. SOC., 1923,
3041(1923).
40. T. Nishina, Y. Sakai and M. Kimura, S t e r o i d s , 4,
255(1964).
41. W. J. Mader and R. R. Buck, Anal. Chem., 24,. 666
(1952).
42. J. E. Sinsheimer and E. F, Salim, Anal. Chem., 37,
566(1965).
43. A. S. Meyer and M. C. Lindberg, Anal, Chem., 27,
813(1955).
44. R. V. Smith, T. H. Hassall and S. C. Liv, ,J. A s s .
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317
46. E. P. Schultz and J. D. Neuss, Anal. Chem., 2,

1662(1957).
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49 . 26(19731_ -
-
I. E. Bush and A. A. Sandberg, J. Biol. Chem., 205,
783(1953).
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-'
57 208(1955).
51 "United S t a t e s Phanacopei a," 1 8 t h Revis ion, Mack
Publishing Co., Easton, Pa., 1970, pp. 454-455.
52. J. G. Rutgers, Wyeth Laboratories, Inc., Personal
Communication.
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517(1970).
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I1 Farmaco; 27, 537(1972).
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munication.
The L i t e r a t u r e Search was conducted up t o July, 1974.
318
PHENFORMIN HYDROCHLORIDE
Joseph E. Moody
JOSEPH E. MOODY
CONTENTS
Analytical P r o f i l e - Phenfonnin Hydrochloride

1, Description
1.1 N a m e , Formula, Molecular Weight
2.4 Mass Spectrum
2.5 Melting Range
2.6 Solubility
2.7 Distribution Coefficients
2.8 I o n i z a t i o n Constant pKa,
2.9 D i f f e r e n t i a l Scanninq Calorimetry
2.10 Thennogravimetric Analysis
3. Synthesis
4, Stability
5. Metabolism
6.1 E l e m e n t a l Analysis
6.2 Ultraviolet
6.3 Fluorometric
6.4 Colorimetric
6.5 Chromatographic
6.51 Paper
6.52 Thin Layer
6.53 P a p e r E l e c t r o p h o r e s i s
6.54 Gas-Liquid
7. Rsferences
320
1. Description
1.1 N a m e , Formula, Molecular Weight

P h e n f o d n hydrochloride is named i n Chemical
Abstracts as 1-phenethylbiguanide hydrochloride. Other
names occasionally used are N ’ - P -phenethylformamidinyl-
iminourea hydrochloride and DBI.
CH2-CH2-NH-C-NH-C-NH2. HC1
I1 II
N-H N-H
Mol. W t . 9 241.73
C10H16N5C1
Phenfonnin hydrochloride i s a white o r p r a c t i c a l l y
white, o d o r l e s s , c r y s t a l l i n e powder.
The i n f r a r e d spectrum of p h e n f o d n hydrochloride
(USP Standard) i s shown i n f i g u r e 1. The spectrum w a s ob-
t a i n e d on a Perkin-Elmer 621 Spectrophotometer from a KBr
pellet, The s t r u c t u r a l assignments have been c o r r e l a t e d
with t h e following band frequencies.
Frequency (cm’l) Ass iqnment
3400 - 3250 N-H stretching
3200 - 3100 Amine s a l t N-H s t r e t c h i n g
1660 - 1610 ;C=N (a-P-unsaturated)
1590 - 1510 Amine s a l t bending
756 & 695 Phenyl (monosubstitutedl

Phenformin hydrochloride displayed t h e NMR spectrum
shown i n f i g u r e 2. The sample w a s d i s s o l v e d i n dimethyl
sulfoxide-d6. The spectrum was measured on a Varian A-60 NMR
Spectrometer with TMS as t h e i n t e r n a l reference. The follcrw-
i n g s t r u c t u r a l assignments have been made f o r f i g u r e 2.
321
W
N ?
t
4
Figure 1. Infrared Spectrum of Phenformin Hydrochloride (USV House Standard batch 22574)
taken i n KBr Disc. Instrument: Perkin E l m e r 621.
PHE NFORMl N HYDROCHLORIDE
Chemical S h i f t ( 6 1 Assignment
2.90 and 3.35 -C;rCErNH-
7.05 Exchangeable
7.30 Aromatic
S h a p i r o (1) r e p o r t e d t h e a b s o r p t i o n s p e c t r a f o r
phenformin hydrochloride. The s p e c t r a are shown i n T a b l e 1.
Samples were measured i n a c i d i c , n e u t r a l and a l k a l i n e medium
a s w e l l a s i n methanol.
TABLE 1
U l t r a v i o l e t S p e c t r a of Phenformin Hydrochloride
Solvent X max (nm) E x 10-3
H 20 233 14.5
1~10'~NHCl 233 11.2
1~10'~NHCl 233 14,s
'0 'NN aOH

1x1 2 32 12.7
1 N NaOH 225 - 228 12,o
methanol 2 34 17.7
2.4Mass Spectrum
Phenformin hydrochloride w a s found t o be u n s t a b l e
under electron impact-mass spectrometry c o n d i t i o n s . The
major fragments peaks were observed a t m/e 205, 146, 104,
and 101 ( 2 ) . The molecular ion d i d n o t appear.
2.5 Melting Range

Phenformin h y d r o c h l o r i d e h a s a m e l t i n g p o i n t of
176-178' (1).
323
w
w
P
F i g u r e 2. NMR Spectrum of Phenformin Hydrochloride (USV House Standard batch 22574) in

deuterated DMSO. Instrument: V a r i a n A-60.
2.6Solubility
The s o l u b i l i t y of p h e n f o d n hydrochloride was de-
termined a t room temperature i n t h e following s o l v e n t s .
Solvent mg/ml S o l v e n t < 0.02 m q / d
Water 83 Chloroform
Ethanol Abs. 34 Acetone
Ethanol 959 64 E t h y l Acetate
Methanol 225 Ether
Isopropanol 3.8 Hexane
D i m e thy1f ormamide 415
2.7 Distribution Coefficients

The d i s t r i b u t i o n c o e f f i c i e n t s , P, of p h e n f o d n
hydrochloride were determined i n t h e oil/water systems, A
and B. The values a r e shown below as a function of sodium
hydroxide concentration ( 3 ) . The p a r t i t i o n i n g of t h e drug
i n t o t h e organic phase i n c r e a s e s w i t h higher pH levels.
(NaOH) (NaOH)
pB
0.32 0.20 1.92 0.24
0.57 0.80 2.37 0.64
System A = methylene chloride/water
System B - (111) chloroform t-amylalcohol/water
2.8 I o n i z a t i o n Constant, p K a
P h e n f o d n i s a s t r o n g l y b a s i c substance and con-
sequently e x i s t s i n d i and mono i o n i c f o r m . Ray (4) has
reported t h e i o n i z a t i o n c o n s t a n t s , pKa'
2.7 a t 32'. ,
I n our l a b o r a t o r y
-
the apparent pKa" - -
11.8 and pKa"
3.1 w a s
measured a t 25' by p o t e n t i o m e t r i c t i t r a t i o n . The second
,
i o n i z a t i o n c o n s t a n t , pKa' could n o t be r e l i a b l y measured
325
JOSEPH E . MOODY
i n aqueous m e d i u m by our method. However, Garrett (3) has

c a l c u l a t e d an approximate pKa' of 13.0 from plots of the re-
c i p r o c a l s of the apparent p a r t i t i o n c o e f f i c i e n t s a g a i n s t t h e
hydrogerl ion concentration.

The thermal s t a b i l i t y of phenformin hydrochloride
w a s determined on a Perkin-Elmer Model DSC-1B Thermal
Analyzer ( 2 ) . The sample is stable when heated t o loo below
i t s melting p o i n t for 30 minutes. A m e l t endotherm w a s ob-
served a t 175 -179O. However, phenformin hydrochloride i s
unstable on h e a t i n g above i t s melting p o i n t . The following
data shows t h e h e a t of fusion and melting p o i n t of p h e n f o d n
.
hydrochloride as determined by DSC Analysis on a sample of
99.8% p u r i t y
Heat of Fusion - 39.8 cal/gram

9600 cal/mole
Melting Range - 175 - 179OC
Melting P o i n t - 176.5OC
2.10 Thermogravimetric Analysis
Phenformin hydrochloride i s weight stable through
i t s melting point. At-temperatures above 230°, the sample
loses approximately 30% of i t s i n i t i a l weight. The thermal
a n a l y s i s d a t a was determined on a Perkin-Elmer TGS Thermal-
analyzer (2).
3. Synthesis
Phenformin hydrochloride can be prepared by the a d d i t i o n
of 2-phenylethylamine hydrochloride t o dicyanodiamine a t
l5Oo (1). Phenformin hydrochloride i s then r e c r y s t a l l i z e d
from the r e a c t i o n mixture with isopropanol.
4. Stability
Phenformin can be recovered as t h e d i a c i d s a l t a f t e r
prologned h e a t i n g with 3 and 6N hydrochloric acid, 50% s u l -
f u r i c acid o r polyphosphoric acid. Phenformin hydrochloride
i s s t a b l e i n d i l u t e sodium hydroxide a t room temperature.
However, t h e compound hydrolyzes i n s t r o n g sodium hydroxide
326
t o p-phenethylguanidine and small amounts o f P-phenethyiurea

and p-phenethylamine (1).
5. Metabolism
Wick (5) i d e n t i f i e d t h e major metabolites of phenformin
hydrochloride i n t h e e x c r e t e d u r i n e o f male rats as p-hydroxy
- p - phenethylbiguanide and i t s glucuronide conjugate.
Beckmann (6) d i s c o v e r e d t h a t m a n also m e t a b o l i z e s
phenformin hydrochloride t o p-hydroxyphenformin. Nearly
33% o f t h e dose was e x c r e t e d as t h i s m e t a b o l i t e .
H a l l (7) s t u d i e d t h e metabolism o f phenfonnin w i t h an

--
i n v i t r o l i v e r system. The b i o t r a n s f o r m a t i o n t o p-hydrox-
phenformin w a s i n h i b i t e d by a known m e t a b o l i c i i b i t o r .
T h i s evidence and t h e t i s s u e d i s t r i b u t i o n o f Cl'labeled
phenformin s u g g e s t t h a t t h e l i v e r is t h e major s i t e of
metabolism.
Element % Theory 0 Reported
C 49.7 49.6
H 6.7 6.7
N 29.0 29.3
c1 14.6 14.3
6.2 Ultraviolet
Phenformin hydrochloride can be analyzed by
measuring i t s absorbance i n water a t 233 nm ( 8 ) .
6.3 Fluorometric
Bailey (9) treated s o l u t i o n s o f phenformin hydro-
c h l o r i d e with a l k a l i n e ninhydrin and measured t h e f l u o r e s -
cence of t h e phenformin-ninhydrin complex a t 518 nm on a
Turner fluorometer. The f l u o r e s c e n t complex showed e x c i -
t a t i o n peaks a t 304 and 392 nm. The a c t i v a t i o n monochrometer
w a s s e t a t 390 nm. This method can detect 0.025 pg/ml of
phenformin.
321
JOSEPH E . MOODY

Phenformin hydrochloric forms an e x t r a c t a b l e ( 1 1 1 )
i o n p a i r with bromthymol blue. Garrett (10) found t h a t the
i o n p a i r i s e x t r a c t e d q u a n t i t a t i v e l y i n methylene c h l o r i d e
from aqueous s o l u t i o n . - The absorbance of t h e ion p a i r was
then measured i n methylene c h l o r i d e a t 413 nm. Garrett
discovered a more s e n s i t i v e procedure by t r e a t i n g t h e
phenfodn-bromthymol blue i o n pair with excess tetra-
butylammonium hydroxide t o form a ( 2 r l ) tetrabutylammnium-
bromthymol blue ion pair. The absorbance of t h i s (211) i o n
pair w a s determined a t 630 nm. This l a t t e r method w a s
applied t o t h e a n a l y s i s of phenformin i n human urine. Minor
i n t e r f e r e n c e from n i c o t i n e i n some u r i n e samples was ob-
served.
Shepherd and McDonald (11) developed a c o l o r i m e t r i c

assay based on t h e c o l o r r e a c t i o n of phenfonnin hydro-
c h l o r i d e with t h e 1-naphthol-diacetyl reagent. The q u a n t i t y
of phenformin was determined from t h e absorbance of the
colored s o l u t i o n a t 565 nm. Beer's Law w a s obeyed up t o a
concentration of 20 pg of phenfonnin per m l of s o l u t i o n .
This method was applied t o t h e a n a l y s i s of phenformin i n
b i o l o g i c a l f l u i d s (12). The U. S. Pharmacopeia (13) de-
s c r i b e s t h i s c o l o r i m e t r i c procedure.
To achieve t h e s p e c i f i c i t y required o f modern

a n a l y t i c a l methods, t h e s e c o l o r i m e t r i c procedures should be
coupled with a suitable chromatographic s e p a r a t i o n technique.
6.5Chromatographic Methods
Phenformin hydrochloride can be determined by t h e
following chromatographic methods.

The following d a t a show t h e s o l v e n t system
and response f a c t o r s , (Rf) f o r p h e n f o d n hydro-
chloride. For t h e q u a l i t a t i v e d e t e c t i o n of phen-
formin, t h e chromatograms can be sprayed with the
l o c a t i o n reagents. pentacyanoaquoferriate (PCF)
Sakaguchi, and 1-naphthol-diacetyl reagents (14,
.
15). 1-Naphthol-diacetyl reagent i s the most
s e n s i t i v e , with a d e t e c t i o n l i m i t a t i o n of 0 -05
pg phenfonnin.
328
Solvent system -Rf Reference
Acetone :water 1:1 0.76 9,16
Isopropanol :water: ammonium

hydroxide 40 t 9 t 6 0.73 16
Pyridinerbutanoltwater 1:l:i 0.79 16
Ethyl a c e t a t e rethanolrwater
6r3tl 0.76 13,16
Butanol :water: ammonium

hydroxide 4 t 5 :1 0.49 16
Butanol s a t u r a t e d with water 0.43 16

A TLC system was developed by Bailey (9) f o r
phenformin hydrochloride on s i l i c a - g e l GF-g l a s s
p l a t e s . The TLC plates were e l u t e d w i t h t h e upper
layer of e q u i l i b r a t e d n-butanolracetic acid:H20
(40:5 :55) . The migration rate w a s approximately
14 cm i n 5 hours w i t h an Rf o f 0.45.
Phenformin hydrochloride is q u a n t i t a t i v e l y de-

termined i n our l a b o r a t o r y on c e l l u l o s e p l a t e s
which are pre-washed i n t h e developing s o l v e n t ,
isopropanol:HZO:acetic a c i d , 80120:s. After
development i n t h e above s o l v e n t system, phenformin
hydrochloride i s e x t r a c t e d i n methanol and t h e ab-
sorbance of t h e s o l u t i o n determined a t 233 nm.
6.53 Paper E l e c t r o p h o r e s i s
Bailey (9) h a s developed an e l e c t r o p h o r e s i s
method using Whatman N o . 3 paper a t 400 v o l t s . With
lN acetic a c i d , phenformin showed a migration rate
of 8.25 c m i n 1 hour. Phenformin w a s e x t r a c t e d from
t h e paper chromatogram and determined by U.V.
spectroscopy i n t h e usual manner.
6.54 Gas Liquid Chromatography

Wickramasinghe and Shaw (17) have reported
t h e thermal i n s t a b i l i t y of phenformin hydrochloride
t o gas chromatographic conditions. A s i n g l e peak
of r e t e n t i o n t i m e about 22 minutes was observed on
a g l a s s s p i r a l column (122 cm long) packed with
0.5% Carbowax 20M on g l a s s beads (80-100 mesh) a t
329
JOSEPH E. MOODY
a column oven temperature of 225O. The analysis of

the s i n g l e GC peak on a LKB-9000 gas chromatograph-
mass spectrometer showed nrominent GC-MS s i s n a l s a t
m/e 230, 139, 110, 91 and68. This GC-MS data is
consistent with the s-triazine structure shown
below.
330
7. References
1. S.L. S h a p i r o , V.A. P a r r i n o and L. Freedman, J. Am.

Chem. SOC., E l , 2220 (1959).
2. F. T i s c h l e r , Ciba-Geigy Pharm. Corp., personal

comunication.
3. E.R. Garrett, J. Tsau, and P.H. H i n d e r l i n g , J. Pharm.

Sci
-.I -
61, 1411 (1972).
4. -
P. Ray, Chem, Rev., 61, 313-359 (1961)
5. P.J. Murphy and A.N. Wick, J. Pharm. S c i . , 57, 1125

(1968).
6. R. Beckmann, Ann. N.Y. Acad. S c i . , 820-832148,

,
(1968) t Chem. Abstr. 69, 50656p (1968).
H. Hall, G. Ramachander and J.M. G l a s s m a n , Ann. N.Y.

7.
-
Acad. S c i . -11968 148, 6 0 1 - 6 1 1 ~ Chem. Abstr. 69,
65872e (1968).
,
8. U. S. Pharmacopeia X V I I I , p. 488.
9. R.E. B a i l e y , C l i n . Biochem., f,23-31 (1970).
10. E.R. Garrett and J. Tsau, J. Pharm. SCi., 61, 1404

(1972).
11. H.G. Shepherd, Jr., and H.J. McDonald, C l i n i c a l

- A,
Chem., 496 (1958).
12. L. Freedman, M. B l i t z , 3%. Gunsberg, and S. Zak,

J. Lab. Clin. Med., 58, 662-666 (1961).
13. U. S. Pharmacopeia X V I I I , p. 487.
14. R. E. B a i l e y and D. A. Durfee, J. Chromatogr. , l.6,

546 (1964).
15. ,
I. Smith "Chromatographic and E l e c t r o p h o r e t i c
,
Techniques" I n t e r s c i e n c e , New York (1960) p. 226.
16. J. L. Faymon, C. J. S t e w a r t and A. N. Wick, Appl.

-, 4, 378-381 (1962).
331
JOSEPH E. MOODY
17. J. Wickramasinghe and S. R. Shaw, J. Chromatogr.,

-
71, 265-273 (1972).
332
PROCAINAMIDE HYDROCHLORIDE
Raymond B. Poet and Harold Kadin

RAYMOND 6. POET AND HAROLD KADIN
TABLE OF CONTENTS
1. Description


2.12 Nuclear (Proton) Magnetic
Resonance Spectra
2.14 Mass Spectra
2.15 Fluorescence

2.22 Differential Scanning Calorimetry
2.23 Melting Range
2.25 Hygroscopicity
2.3 Solution Data
2.31 Solubility
2.32 pKa
2.33 Surface Chemistry
2.34 Complex Formation
3. Synthesis
4. Stability
5. Analysis of Impurities, Degradation Products

and Residual Intermediates
334
PROCAl NAMl DE HYDROCHLORIDE
6. Analytical Tests and Methods

6.3 Spectrophotometric Methods
6.4 Isolation and Chromatographic Methods
6.41 Solvent Extraction

6.42 Paper and Thin-Layer
Chromatography
6.44 Ion-Exchange Chromatography
6.5 Spectrophotofluorometric Methods

6.6 Titrimetric Methods
6.7 Microbiological Methods
7. Analysis in Biologic Fluids and Tissues
8. References
335
RAYMOND 6. POET AND HAROLD RADlN
1. Description
Procainamide hydrochloride is benzamide,

~-amino-N-~2-diethylamino)ethy~monohydro-
chloride with Chem. Abstr. registry number
614-3 9-1.
Among the generic and trivial names for

this compound are procaine amide hydrochloride,
amidoprocain and procainamide. Common trade
names are Pronestyl, Procamide, Procardyl,
Novocamid, Novocainamid and Supicaine Amide.
PH2CH3
H2N @ !-NHCH~CHZN HC1
'CH2CH3
C13H21N30'HCl Molecular Weight 271.8
1.2 Appearance, Color,Odor
Procainamide hydrochloride is a
white-to-tan, odorless crystalline powder.
Samples of procainamide hydro-

chloride were dispersed in a potassium bromide
pellet or in mineral oil to obtain the infrared
spectra given in Figures la and lb from a
Perkin-Elmer Model 21 prism instrument12 The .
potassium bromide and mineral oil spectra are
essentially identical.
336
W
W
4
Figure l a . I n f r a r e d Spectrum of Procainamide H y d r o c h l o r i d e from K B r

p e l l e t . I n s t r u m e n t : P. E. Model 2 1 I n f r a r e d S p e c t r o p h o t o m e t e r .
w
w
Do
F i g u r e lb. I n f r a r e d S p e c t r u m o f P r o c a i n a m i d e H y d r o c h l o r i d e from
mineral o i l mull. I n s t r u m e n t : P.E. Model 2 1 I n f r a r e d
S p e c t r o p h o tome t e r .
S t r u c t u r a l l y s i g n i f i c a n t bands
w e r e i n t e r p r e t e d b y T o e p l i t z l 2 a s f o l l o w s (see
Figures l a and l b ) .
Wave l e n q t h ( c m - l ) Interpretation
3389,3279,3205 NH2 and NH
2564,2457 HC 1
1639 Amide C=O
1538 S e c o n d a r y Amide
1600 NH2 a n d A r o m a t i c C=C
1511 A r o m a t i c C=C
840 Para-Substituted Aromatic
2.12 Nuclear (Proton) Magnetic

Resonance S p e c t r a
Cohen59 o b t a i n e d b o t h 60- a n d
100-MHz s p e c t r a of p r o c a i n a m i d e and i t s h y d r o -
chloride. The 6O-MHZ NMR s p e c t r a were o b t a i n e d on
a V a r i a n A s s o c i a t e s A-60 s p e c t r o m e t e r of CD30D
s o l u t i o n s c o n t a i n i n g t e t r a m e t h y l s i l a n e a s an
i n t e r n a l reference. The 100-MHz NMR s p e c t r a were
o b t a i n e d on a V a r i a n A s s o c i a t e s XL-100 s p e c t r o -
meter d e u t e r i u m - l o c k e d t o t h e s o l v e n t (CDC13,
CD30D, o r d g - p y r i d i n e ) c o n t a i n i n g t e t r a m e t h y l -
s i l a n e a s i n t e r n a l reference.
Because procainamide hydro-

chloride is not soluble i n chlorinated
h y d r o c a r b o n s , t h e 60-MHZ NMR s p e c t r u m was
obtained i n tetradeuteromethanol (Figure 2 ) :
t h i s s p e c t r u m i s compared w i t h t h a t o f t h e f r e e
b a s e i n t h e same s o l v e n t ( F i g u r e 3 ) . A l t h o u g h
t h e s p e c t r a a r e q u a l i t a t i v e l y t h e same, t h e
s p e c t r u m of t h e h y d r o c h l o r i d e s a l t d i f f e r s
because of protonation a t t h e alkylamine s i t e ,
r e s u l t i n g i n a g r e a t d o w n f i e l d s h i f t of t h e
p r o t o n s on t h e m e t h y l e n e g r o u p s d i r e c t l y
a t t a c h e d t o t h e a l i p h a t i c amine n i t r o g e n a n d a
339
w
P
0
Figure 2. 6O-MHZ NMR Spectrum of Procainamide Hydrochloride in

Tetradeuteromethanol. Instrument: Varian Associates A-60
Spectrometer. Tetramethylsilane as Internal Standard.
Figure 3. 60-MHz NMR Spectrum of Procainamide Base in Tetra-
deuteromethanol. Instrument: Varian Associates A-60
Spectrometer. Tetramethylsilane as Internal Standard.
T a b l e 1. NMR S p e c t r a l A s s i g n m e n t s
43
a 0
TT
n
H2 0 Or
or I1 D /H 2-CH 3
D2* C-N-CH2- CH 2-
@ O C
' H2 -CH3
@ 8
C h e m i c a l S h i f t (6) ppm (No. o f Peaks***, C o u p l i n g C o n s t a n t ( J )i n Hz)
c
60-MHz S p e c t r u m
~ssignment ( F i g u r e 2 ) ( F i g u r e 3)
P
N o f Prot o n H C 1 S a l t Base i n
P o s i ti o n i n HOCG3and HOCD3 a n d
DOCD3 DOCD3
0 1.33 (t, 7.3) 1 . 2 2 ( t , 7.3)
Q * 3 . 2 9 ( 9 , 7 . 3 ) 2 . 6 1 (9, 7 . 3 )
0 %3.35 (m)
*3 . 7 1 ( t , 7 )
*'L2.60 (m)
* 3.47 ( t , 9 )
CD
@ 7.70 (d, 8.6) 7.65 (d, 8 . 6 )
@ 6.71 (d, 8 . 6 ) 6.68 (d, 8.6)
I Table 1, c o n t i n u e d . . ..
Assignment (Figure 4 ) (Figure 5 ) (Figure 6 )
of Proton Base in HOCD3 Base in Base in
Position and DOCD3 CDC13 Perdeuteropyridine
1.08(t, 7 ) 1.04 (t, 7 ) 0.95 (t, 7 )

2 . 6 1 (9,7 ) 2.57 (9, 7 ) 2.48 (q, 7)
** 2 . 6 6 (m) 2.62 (m) 2.67 (t, 7)
** 3 . 4 8 (m, 8 ) 3.46 (m) 3.71 (9, 7)
7.58 (d, 8 . 5 ) 7.59 (d, 8 ) 7.13 (d, 8 )
6 . 6 5 (a, 8 . 5 ) 6.59 (d, 8 ) 5.93 (d, 8 )
- 6.79 (Broad) 6.79 (Broad)
- 4.04 (Broad) 4.04 (Broad)
*Hydrogens not distinguishable.
**Hydrogens are distinguishable.
***No. of peaks is designated t = t r i p l e t , d = doublet, q = quartet, and
m = multiplet.
". I
w
P
P
10 I 2 3 4 ?., , 9 , , ? , P
<-
- ,,.
Figure 4. 100-MHz NMR Spectrum of Procainamide Base in Tetra-

deu teromethano 1. Instrument : Varian Associate s XL- 100
Spectrometer, Deuterium-Locked to the Solvent Containing
Tetramethylsilane as Internal Reference.
.. I
& I
nb
,k
i
1
w
P
VI
Figure 5. 100-MHz NMR Spectrum of Procainamide Base in Deutero-

chloroform. Instrument: Varian Associates XL-100
Spectrometer, Deuterium-Locked to the Solvent Containing
Tetramethylsilane as Internal Reference.
W
P
m
F i g u r e 6. 100-MHz NMR S p e c t r u m of P r o c a i n a r n i d e B a s e i n P e r d e u t e r o p y r i d i n e .
I n s t r u m e n t : V a r i a n A s s o c i a t e s XL-100 S p e c t r o m e t e r , D e u t e r i u m -
Locked t o t h e S o l v e n t C o n t a i n i n g T e t r a m e t h y l s i l a n e a s
I n t e r n a l Reference.
s l i g h t d o w n f i e l d s h i f t of t h e p r o t o n s on t h e
@-carbons. T h a t t h e s i t e of p r o t o n a t i o n i s n o t
on t h e a r o m a t i c amine i s d e m o n s t r a t e d b y t h e
s i m i l a r i t y of t h e a r o m a t i c p r o t o n r e s o n a n c e s o f
t h e two compounds. Protonation of t h e aromatic
amine would have r e s u l t e d i n a l a r g e d o w n f i e l d
s h i f t of t h e o r t h o p r o t o n s and a small d o w n f i e l d
s h i f t of t h e meta p r o t o n s , b e c a u s e t h e -NH3 +
g r o u p i s n o t a s s t r o n g an e l e c t r o n - d o n a t i n g
g r o u p a s i s t h e -NH2group. The a s s i g n m e n t s
of t h e chemical s h i f t s shown i n T a b l e 1 a r e g i v e n
i n D e l t a v a l u e s ( 6 ) and t h e c o u p l i n g c o n s t a n t s ,
J, a r e g i v e n i n Hz w i t h t - t r i p l e t , d - d o u b l e t ,
q - q u a r t e t and m - m u l t i p l e t .
I t i s e v i d e n t t h a t t h e 100-MHz
spectrum o f t h e b a s e i n CD30D y i e l d s a d d i t i o n a l
r e s o l u t i o n ( F i g u r e 4 and T a b l e 1). The 100-MHz
NMR spectrum of procainamide b a s e ( F i g u r e 5)
shows t h e amine and amide p r o t o n s . There are
a d d i t i o n a l l i n e s f o r t h e methylene p r o t o n s
a t t a c h e d t o t h e amide n i t r o g e n due t o c o u p l i n g
of t h e amide p r o t o n , which i s Ja7Hz. The
a s s i g n m e n t s a r e shown i n T a b l e 1. When t h e
100-MHz NMR spectrum o f p r o c a i n a m i d e b a s e i s
obtained i n perdeuteropyridine, considerable
changes i n c h e m i c a l s h i f t s o c c u r t h a t r e s u l t i n
separation of a l l t h e proton resonances
( F i g u r e 6 ) . The a s s i g n m e n t o f t h e s p e c t r u m i s
d e p i c t e d i n T a b l e 1.
D ~ n h a mhad ~ ~ indicated t h a t the

u l t r a v i o l e t s p e c t r a of p r o c a i n a m i d e h y d r o c h l o r i d e
s h o u l d b e measured i n 0.1g ammonium h y d r o x i d e i n
e i t h e r methanol o r e t h a n o l . S p e c t r a f o r t h e
compound d i s s o l v e d i n e i t h e r s o l v e n t a l o n e can
have v a r i a b l e w a v e l e n g t h s of maximum a b s o r b a n c e ,
341
RAYMOND 6 . POET AND HAROLD KADIN
a s indicated by t h e following s p e c t r a l d a t a
o b t a i n e d w i t h a Cary 1 5 S p e c t r o p h o t o m e t e r .
Solvent max (nm) E (l%, 1 cm)

MeOH c o n t a i n i n g 282 667
0.13 NH40H 282 670
282 676 ( N o t e )
MeOH 2 91 6 94
MeOH 291 690
MeOH c o n t a i n i n g
N HC1 293 68 2
- HC1 2 93 3 99
EtOH, 95% 286 656
EtOH, 95% 286 6 54
EtOH, 95% 282 6 58

containing
0.1N NH4OH 282 654
N o t e : The s p e c t r u m o f t h i s s o l u t i o n , d e t e r m i n e d
a g a i n 5 h r . l a t e r , showed max = 282 nm,
E (l%,l c m ) = 670.
The d a t a i n d i c a t e t h a t t h e wave-
l e n g t h of maximum a b s o r b a n c e i s pH d e p e n d e n t .
Both m e t h a n o l i c and e t h a n o l i c s o l u t i o n s of pro-
c a i n a m i d e h y d r o c h l o r i d e h a d a % max of 2 8 2 nm i n
0.1N ammonium h y d r o x i d e , a l t h o u g h t h e E ( l % , l c m )
v a r i e d between t h e t w o s o l v e n t s . A d d i t i o n of
a c i d t o t h e methanolic s o l u t i o n s h i f t s the
maximum t o 293 nm. S p e c t r a of t h e compound
d i s s o l v e d i n e i t h e r s o l v e n t a l o n e c a n show h m a x
v a l u e s t h a t a r e q u i t e v a r i a b l e , d e p e n d i n g on t h e
p o s s i b l e c o n t r i b u t i o n of s l i g h t r e s i d u a l a c i d o r
348
PROCAlNAMl DE HYDROCHLORIDE
b a s e on t h e g l a s s w a r e o r from i m p u r i t i e s i n t h e
b a t c h of s o l v e n t used t o make t h e s o l u t i o n s and
measurements. When N hydrochloric acid i n
methanol i s used a s s o l v e n t , t h e r e i s a s l i g h t
d e c l i n e ( a b o u t 1.5%) i n a b s o r b a n c e , w i t h a 2-nm
upward s h i f t i n ~ m a x ,r e l a t i v e t o t h e a b s o r b a n c e
i n methanol a l o n e . However, when t h e a c i d
concentration is increased t o 3, t h e
a b s o r b a n c e d e c l i n e s a p p r e c i a b l y ( a b o u t 42%),
r e l a t i v e t o a b s o r b a n c e i n methanol a l o n e . T h i s
d e c l i n e h a s been a s c r i b e d t o p r o t o n a t i o n of t h e
aromatic-NH2 group55, 65.
The e f f e c t s of t h e s u b s t i t u t i o n
of amino and c a r b o n y l g r o u p s on t h e s h i f t o f t h e
a b s o r p t i o n o f t h e benzene u l t r a v i o l e t chromophore
have been discussed51. The u s e of u l t r a v i o l e t
s p e c t r o p h o t o m e t r y f o r d e t e r m i n a t i o n of r o c a i n -
P
amide h y d r o c h l o r i d e h a s been d e s c r i b e d 5 9 5 5
(see Section 6.3).
2.14 Mass S p e c t r a
C0hen5’ h a s r e p o r t e d t h e
f o l l o w i n g c o n c e r n i n g t h e mass s p e c t r a o f p r o -
cainamide h y d r o c h l o r i d e .
The l o w - r e s o l u t i o n mass spectrum

(see F i g u r e 7 ) i s dominated by t h e m / e 86 i o n
( b a s e peak) r e s u l t i n g from t h e c l e a v a g e o f t h e
bond b e t a t o t h e t e r t i a r y amine n i t r o g e n . T h i s
cleavage i s a n t i c i p a t e d i n t h e fragmentation of
amines. The M+ of t h e compound i s o b s e r v e d a t
m / e 235. H i g h - r e s o l u t i o n peak matching, T a b l e 2 ,
and h i g h - r e s o l u t i o n d a t a a c q u i s i t i o n , T a b l e 3,
were employed t o d e t e r m i n e t h e c o m p o s i t i o n o f
e a c h fragment i o n . Assignment o f t h e fragmenta-
t i o n i s shown b e l o w 3 ~ 5 .
349
RAYMOND B. POET AND HAROLD K A D l N
only
The, low-and high-resolution mass spectra were

obtained on an AEI MS-902 Spectrometer equipped
with a frequency-modulated analog tape recorder
The low-resolution mass spectrum was processed
on a Digital Equipment Corporation PDP-11
computer using Squibb programs62, while the
high-resolubion mass spectrum was processed by
W ~ m a c kon
~~an EAI computer. Peak-matched
data were obtained from the AEI MS-902
spectrometer equipped with a wide-ratio
accessory.
350
PROCAINAM I DE HYDROCHLORIDE
RELATIVE INTENSITY
ru
m
u
LJl
m
0
ru
.--
LN
LN
n
i
0
0
rl
ru
nl
I
F
I
u
a
u3
m
ru
Lhl
ul
I
m
m
nl
-
LN
m
0
m
-
m
ru
e,
LN
m
I
m
LJl
PERCENT TOTAL I O Y I Z A T I O N
39
Fig. 7 Low Resolution Mass Spectrum of Procainamide Hydrochloride.

Instrument: AEI MS-902 Spectrometer Equipped with
Frequency Modulated Tape Recorder;Spectrum Processed
on Digital Equipment Corporation PDP-11 Computer
351
RAYMOND 6 . POET AND HAROLD KADlN
Table 2
H i g h - R e s o l u t i o n Peak-Match D a t a of P r o c a i n a m i d e
Found Ca l c d . -
C -
H -
N -
0
M+ 235.1685 235.1685 13 21 3 1
206.1293 206.1293 11 16 3 1
205.1215 205.1215 11 15 3 1
189.1266 189.1266 11 15 3 0
188.1188 188,1188 11 14 3 0
174.1031 174.1031 10 12 3 0
163.0871 163.0872 9 11 2 1
148.0678 148.0637 8 8 2 1
136.0627 136.0637 7 8 2 1
120.0451 120.0449 7 6 1 1
99.1041 99.1048 6 13 1 0
92.0507 92.0500 6 6 1 0
86.0963 86.0970 5 12 1 0
71.0735 71.0735 4 9 1 0
70.0653 70.0657 4 8 1 0
352
PROCAINAMIDE HYDRDCHLORIDE
Table 3
High-Resolution Mass Spectrum of Procainamidea
Rel.
Found Calcd. c/c13 -H N 0 Int.---
M+ 235.1667 235.1685 13/0 21 3 1 4
233.1530 233.1529 13/0 19 3 1 4
189.1241 189.1266 11 15 3 0 4
188.1137 188.1188 11 14 3 0 1
187.1151 1
164 5
149.0705 149.0715 8 9 2 1 4
148.0700 148.0637 8 8 2 1 5
137.0646 137.0715 7 9 2 1 3
136.0597 136.0637 7 8 2 1 7
121.0479 121.0482 6/1 6 1 1 12
120.0458 120.0449 7 6 1 1 141
119.0368 119.0371 7 5 1 1 2
100.1078 100.1064 5/1 13 1 0 20
99.1031 99.1048 6 13 1 0 242
93.0554 93.0533 5/ 1 6 1 0 6
92.0488 92.0500 6 6 1 0 57
91.0421 91.0422 6 5 1 0 8
87.0974 87.1003 4/1 12 1 0 78
86.0944 86.0970 5 12 1 0 999
72.0799 72.0813 4 10 1 0 22
71.0732 71.0735 4 9 1 0 25
70.0662 70.0657 4 8 1 0 13
65.0395 65.0391 5 5 0 0 55
58.0687 58.0657 3 8 1 0 90
57.0625 57.0578 3 7 1 0 11
56.0501 56.0500 3 6 1 0 36
a
High-Resolution FM analog tape processed by
Dr. J. Womack, E A I , Princeton, N . J .
353
RAYMOND E. POET AND HAROLD KADIN
A s i m i l a r m a s s - s p e c t r a l fragmen-
t a t i o n p a t t e r n was r e p o r t e d b y A t k i n s o n e t a139.
These a u t h o r s commented t h a t t h e most p r o m i n e n t
i o n ( m / e 8 6 i o n ) r e s u l t s from t h e a b i l i t y o f t h e
f r e e e l e c t r o n p a i r o n t h e t e r t i a r y amine n i t r o g e n
atom t o s t a b i l i z e a p o s i t i v e c h a r g e on t h e
a d j a c e n t c a r b o n atom. A t k i n s o n e t a 1 . 3 9 a s c r i b e d
t h e m / e 99 f r a g m e n t t o a M c L a f f e r t y
rearrangement66.
2.15 Fluorescence
Procainamide h a s been r e p o r t e d 1 9
t o e x h i b i t maximum f l u o r e s c e n c e a t 385 nm, w i t h
maximum a c t i v a t i o n a t 295 nm, a t pH 11. The
s p e c t r o p h o t o f l u o r o m e t e r u s e d employs a xenon a r c
source e m i t t i n g a continuum from 200 t o 800 nm
a s t h e a c t i v a t i n g l i g h t source. This instrument
y i e l d s spectra uncorrected f o r the non-linear
i n t e n s i t y of e m i s s i o n o f t h e xenon a r c and t h e
uneven p h o t o m u l t i p l i e r r e s p o n s e o v e r t h i s
continuum. T h i s may e x p l a i n the same maximum
a c t i v a t i o n a t 295 nm, b u t a d i f f e r e n t
f l u o r e s c e n c e maximum a t 360 nm, a t pH 11,t h a t
was r e p o r t e d b y Koch-Weser 8 .
R e f e r t o s e c t i o n s 6 . 5 and 7 . 0
f o r a p p l i c a t i o n s of t h i s p h y s i c a l p r o p e r t y .

2 . 2 1 D i f f e r e n t i a l Thermal A n a l y s i s
D i f f e r e n t i a l thermal a n a l y s i s
(DTA) of p r o c a i n a m i d e h y d r o c h l o r i d e b y V a l e n t i 3 0
y i e l d e d a s h a r p endotherm a t 169OC. A DuPont
900 Thermoanalyzer programmed f o r a t e m p e r a t u r e
r i s e o f 15O p e r min produced t h e thermogram o f
F i g u r e 8.
354
w Figure 8. Differential Thermogram of Procainamide Hydrochloride
Instrument: DuPont 900 Thermoanalyzer, lSO/min
RAYMOND B. POET AND HAROLD KADIN
2.22 D i f f e r e n t i a l Scanninq
C a l o r i m e t r y (D. S. C. )
Valenti3' a l s o r e p o r t e d the
D.S.C. p u r i t y o f t h e same l o t of procainamide
h y d r o c h l o r i d e t o be 99.94 mole p e r c e n t . A
P e r k i n E l m e r D.S.C. Model 1B was u s e d .
2.23 M e l t i n g Range
The m e l t i n g r a n g e f o r U.S.P.
procainamide h y d r o c h l o r i d e i s s p e c i f i e d a s
1 6 5 . 0 t o 169.0°C1. The s a m p l e o f p r o c a i n a m i d e
h y d r o c h l o r i d e u s e d f o r most of t h e s t u d i e s re-
p o r t e d h e r e i n h a s a U.S.P. m e l t i n g r a n g e b e t w e e n
1 6 8 . 4 and 169.2OC. T h i s narrow, high-melting
r a n g e s i g n i f i e s e x c e l l e n t p u r i t y , which i s
c o n f i r m e d b y t h e s h a r p 169OC D . T . A . endotherm
and t h e 99.94% D.S.C. p u r i t y ( S e c t i o n s 2 . 2 1 a n d
2.22). Various p r e p a r a t i o n s of procainamide
h y d r o c h l o r i d e a r e reported t o h a v e the
following melting ranges:
356
Melting Range Author ( s ) Ref. N o .

(OC)
165-9 K. Iwaya and 98
K. Yoshida
165-7 M. Yamazaki, 85
Y. Kitagawa,
S. H i r a k i , and
Y. Tsukamoto
164 G. Ghielmetti 99
167 H. Najer and 86
M. Joannic
166-7 V. Hack and 87
E. Koppovs
166-6.5 Z. Led6chowski , 89
M. Bogucka,
“A. Ledbchowski , and
A. Chimiak
167-8 E. Pavlovsk a, 100
J. Lukac and
M. Borovicka
165-8 K. Radushkevich 72
165-8 P. Egli 93
165-8 Y. Tashika and 84
M. Kuranari
2.24 X-Ray Powder D i f f r a c t i o n
The X-Ray powder d i f f r a c t i o n of

procainamide h y d r o c h l o r i d e was o b t a i n e d by
0cl-1~ a t~ a~ v o l t a g e of 25 kv and a c u r r e n t of
1 0 ma, u t i l i z i n g a P h i l l i p s X-Ray Powder
Diffractometer. The sample was i r r a d i a t e d by a
copper s o u r c e a t 1 . 5 4 A O . Data d e r i v e d from t h e
spectrum a r e l i s t e d i n Table 4; t h e i n t e n s i t y of
each peak i n t h e spectrum i s shown r e l a t i v e t o
t h e i n c i d e n t r a d i a t i o n (Io), a s 1/10 x 100, a t
twice t h e a n g l e of i n c i d e n c e o r r e f l e c t i o n of the
r a d i a t i o n ( 2 0 ) for t h e d i s t a n c e , d , between
t h e atomic p l a n e s i n t h e c r y s t a l .
357
RAYMOND B. POET AND HAROLD KADIN
Table 4
X-Ray Powder D i f f r a c t i o n P a t t e r n o f Procainamide
Hydrochloride
Instrument:
P h i l l i p s X-Ray Powder Dif f r a c t o m e t e r
1 dl (Ao) * 1/10 x loo** I (20)
***
8.30 25.26 24
7.40 12.63 12
6.43 7.37 07
5.76 10.53 10
5.40 14.74 14
5.26 100.00 95
4.82 14.74 14
4.52 40.00 38
4.30 27.37 26
4.10 30.53 29
4.01 90.53 86
3.92 66.32 63
3.74 49.47 47
3.48 52.63 50
3.34 18.95 18
3.23 8.42 08
3.14 18.95 18
2.98 28.42 27
2.95 14.74 14
2.86 8.42 08
2.77 20.00 19
2. 50 10.53 10
2.36 7.37 07
*d ( i n t e r p l a n a r d i s t a n c e ) = n A
2 sin 8
= 1.539A0
** Based on h i g h e s t i n t e n s i t y o f 100.
*** Twice t h e a n g l e o f i n c i d e n c e o r r e f l e c t i o n .
358
PROCAINAMIDE HYDROCHLORIOE
2.25 Hygroscopicity
WaltOn6’ r e p o r t e d t h a t s o l i d
procainamide h y d r o c h l o r i d e is r e a d i l y converted
t o a s o l u t i o n i n a n a t m o s p h e r e o f 65% r e l a t i v e
humidity.
2.3 S o l u t i o n Data
Valenti3O r e p o r t e d t h e f o l l o w i n g
s o l u b i l i t i e s (U. S. P. d e f i n i t i o n ) o f p r o c a i n a m i d e
h y d r o c h l o r i d e i n v a r i o u s s o l v e n t s , a t room
temperature.
P a r t s of S o l v e n t / O n e
Solvent Solubility P a r t of S o l u t e
Water very soluble less t h a n 1
0.1g Hydro-
c h l o r i c acid very soluble less than 1
0 . 1 g Sodium
hydroxide very soluble less t h a n 1
95% E t h a n o l soluble 1 0 t o 30
Propy l e n e sparingly 30 t o 100

Glycol soluble
Chloroform slightly 100 t o 1000

soluble
Acetone slightly 100 t o 1000

soluble
Ether insoluble more t h a n 10000
Benzene insoluble more t h a n 10000
Hexane i n so l u b 1e more t h a n 10000
359
RAYMOND 6 . POET AND HAROLD K A D I N
The s o l u b i l i t y of procainamide h y d r o c h l o r i d e i n
a c e t o n e a t 32OC was r e p o r t e d t o be 3.43 mg p e r g
of solvent".
R a d u ~ h k e v i c hr~e ~
ported the
s o l u b i l i t y of procainamide h y d r o c h l o r i d e i n
a b s o l u t e e t h a n o l t o be 6% a t 20° and 70% a t t h e
b o i l i n g p o i n t of ethanol. Procainamide hydro-
c h l o r i d e was r e p o r t e d t o be i n s o l u b l e i n 1 , 2 - d i -
chloroethane o r i n d i e t h y l ether72.
2.32 pKa
The pKa o f procainamide hydro-
c h l o r i d e , determined by a t i t r i m e t r i c procedure,
was 9.24 ( 2 0.10)77. The pKa r e f e r s t o t h e
following type of d i s s o c i a t i o n :
R-NH-3' R-NH2 + H+
2.33 S u r f a c e Chemistry
The d i e l e c t r i c p o t e n t i a l s and
s u r f a c e t e n s i o n s of s o l u t i o n s o f p r o c a i n e and o f
procainamide h y d r o c h l o r i d e were compared o v e r a
wide pH range97. A t pH 1 0 . 3 and 1 0 . 5 t h e
following d a t a on d i e l e c t r i c p o t e n t i a l were
obtained :
C o n c e n t r a t i o n and Druq Ell D i e l e c t r i c P o t e n t i a l
(mv)
0.0066PJ P r o c a i n e 10.5 477
0.02M - Procainamide 10.3 268
Although t h e s o l u t i o n of procainamide was t h r e e

times a s c o n c e n t r a t e d a s t h a t o f p r o c a i n e , t h e
d i e l e c t r i c p o t e n t i a l of t h e former was less.
A t lower pH's, t h e d i e l e c t r i c p o t e n t i a l o f t h e
procainamide s o l u t i o n was a l s o l e s s than t h a t o f
procaine, although t h e d i f f e r e n c e w a s n o t a s
360
pronounced a s a t pH 10.3 and 10.5.
Similarly, the surface tension

f o r t h e procainamide s o l u t i o n was less t h a n t h a t
f o r p r o c a i n e , over t h e pH range t e s t e d .
The a u t h o r s g 7 concluded t h a t t h e
t o x i c i t y of procainamide i s less t h a n t h a t o f
p r o c a i n e because procainamide i s l e s s s u r f a c e
active.
2.34 Complex Formation
Complex formation by p r o c a i n e and

procainamide w i t h adenosine t r i p h o s p h a t e (ATP) i n
aqueous s o l u t i o n was s t u d i e d by o p t i c a l r o t a t i o n
and NMR methodsg6. These s t u d i e s i n d i c a t e d t h a t
hydrophobic s t a c k i n g o f t h e a r o m a t i c i t y , r a t Q e r
than h o r i z o n t a l hydrogen bonding, i s i m p l i c a t e d
i n t h e formation o f t h e s e complexes. A t pH 7.0,
ATP b i n d s one drug molecule t i g h t l y o r two
molecules weakly. The formation c o n s t a n t s f o r
t i g h t l y bound complexes a s determined by b o t h
o p t i c a l r o t a t i o n and NMR measurements, were i n
good agreement. The weak-association c o n s t a n t s
were o b t a i n e d by t r i a l - a n d - e r r o r procedure.
The complex-formation c o n s t a n t s
were :
ATP Complex
Strenqth Procaine Procainamide
Tight 1430 f 38Og-l 3.60 f 90M-l
Weak 1.1 f 0.7x105g-2 4.7 f 2.8F103g-2
According t o Thymm e t a l . 96 t h e
a c t i v i t y of t h e s e drugs on n e r v e membranes may be
a s c r i b e d t o complexing w i t h membrane-bound ATP.
361
3. Synthesis
E-Nitrobenzoic a c i d i s c o n v e r t e d t o t h e a c i d
c h l o r i d e by r e a c t i o n w i t h s u l f o n y l c h l o r i d e .
E-Nitrobenzoyl c h l o r i d e i s t h e n r e a c t e d w i t h
diethylaminoethylamine. The r e s u l t a n t n i t r o
condensation p r o d u c t i s t h e n t r e a t e d w i t h
h y d r o c h l o r i c a c i d and reduced t o y i e l d t h e f i n a l
product85.
02N -@ *
+ NH CH CH N2;:5;
COOH + S 0 C l 2
0,N
-+ 02N e c o c 1
- -
CONHCH,CII,N
,C H
‘C,tIs
7 5
2 2 2 2 5 -
HC 1
red. ’ H2N -@CONHCH2CH2N. ,c c2H5
tI
2 5
* HC 1
A l t e r n a t i v e s y n t h e s e s of procainamide have
been d e s c r i b e d by s e v e r a l workers72,86-90,95,98.
P r e p a r a t i o n of procainamide by e l e c t r o l y t i c r e -
d u c t i o n of N- ( 2 - d i e t h y l a m i n o e t h y l ) -E-nitro-
benzamide h a s a l s o been r e p o r t e d 8 4 .
The s y n t h e s i s of 14C-procainamide hydro-

c h l o r i d e , r e p o r t e d by E g l i g 3 , u t i l i z e d
E-aminobenzoic a c i d l a b e l l e d i n t h e c a r b o x y l
carbon. The s y n t h e s i s o u t l i n e d above was
followed. A p u r i t y a n a l y s i s was performed by
r a d i o s c a n n i n g a t h i n - l a y e r chromatogram o f t h e
f i n a l p r o d u c t , u s i n g t h e s u p p o r t and s o l v e n t
system I1 of F a i r b r o t h e r and Shand68as d e s c r i b e d
i n s e c t i o n 6.42. The f i n a l p r o d u c t c o n t a i n e d
4.83 yCi/mg, w i t h l e s s t h a n 0.5% of r a d i o c h e m i c a l
i m p u r i t i e s . E g l i g 3 r e p o r t e d t h a t i t was p o s s i b l e
t o m a i n t a i n t h e p r o d u c t a t o n l y 0.5% i m p u r i t y b y
keeping t h e s p e c i f i c a c t i v i t y a s low a s p o s s i b l e
and growing t h e c r y s t a l s a s l a r g e a s p o s s i b l e ;
t h i s procedure a p p a r e n t l y minimizes t h e s u r f a c e
362
oxidation that is common with labelled compounds.
4. Stability
Bulk samples of procainamide hydrochloride

were stored at 25 and 5OoC for as long as 1 year.
Samples stored at these temperatures showed no
significant differences in intravenous or oral
toxicity in mice. The purity, measured by
titration with perchloric acid, ranged from
98.9-100.1% over the period of one year.
Procainamide hydrochloride bulk appeared to be
stable under the conditions employed91,92.
5. Analysis of Impurities, Degradation Products

and Residual Intermediates
N- (2-diethylaminoethyl)-p-nitrobenzamide, an
intermediate in the synthesiz of procainamide,
has been determined polarographically73. The pH
4.0 buffer contained 250 ml of 1M sodium acetate,
750 ml of 1M acetic acid, 7.46 g of potassium
chloride and 530 mg of dodecyltrimethyl ammonium
chloride. A dropping-mercury electrolysis cell
was used in which the average half-wave reduction
potential (vs. Hg) was found to be -0.405 volts.
Use of a gravimetric method69 has also been
reported, in which the nitrobenzamide compound
was extracted into ether from alkaline solution;
the solvent was removed by evaporation and the
residue was dried and weighed.
The determination of pnitrobenzoic acid, a

possible process contaminant, was accomplished
gravimetrically69 after extraction from acid
solution into ether and removal of the solvent
by evaporation. The stability of p-nitrobenzoic
acid can be monitored by thin-laye? chromatogra-
phy83 on silica gel plates (with indicator) with
363
RAYMOND 6. POET AND HAROLD KADlN
a s o l v e n t system p r e p a r e d by mixing 10 m l s w a t e r
w i t h 20 m l s of U.S.P. e t h a n o l t h e n d i l u t i n g t o
1000 m l s w i t h methyl i s o b u t y l k e t o n e .
C o n c e n t r a t i o n s of N,N-diethylethylenediamine
( a p r e c u r s o r diamine) i n t h e range o f 0.01-0.3%
have been determined i n procainamide hydro-
c h l o r i d e by a c o l o r i m e t r i c method7' by Whigan
and Kadin. I n t h i s t e s t , based upon a r e p o r t by
J. Bartos94, a 50-mg sample of procainamide
h y d r o c h l o r i d e i s d i s s o l v e d i n dimethylformamide
and r e a c t e d w i t h a 0.1% s o l u t i o n of a s c o r b i c
a c i d , d i s s o l v e d i n t h e same s o l v e n t , by h e a t i n g
t h e mixture f o r 10 min i n a boiling-water bath.
The absorbance of t h e c o l o r e d r e a c t i o n p r o d u c t
i s measured a t 530 nm. The c o l o r r e a c t i o n
between t h e diamine and a s c o r b i c a c i d i s
enchanced by t h e p r e s e n c e of procainamide. A
s i m i l a r enhancement by p a m i n o b e n z o i c a c i d (PABA)
had been observed. T h e r e f o r e , 30 mg of PABA
were added t o b o t h t h e r e a g e n t b l a n k and t h e
diamine s t a n d a r d t o c o r r e c t f o r t h i s enhancement.
A s l i t t l e a s 0.1% N,N-diethylethylenediamine
i n procainamide c o u l d a l s o be determined q u a n t i -
t a t i v e l y by paper e l e c t r o p h o r e s i s and densitome-
try7'. Samples and s t a n d a r d s w e r e s p o t t e d 1 2 c m
from t h e a n o d i c edge of 1 2 x 36 c m Whatman 3MM
paper. E l e c t r o p h o r e s i s i n a pyridinium-formate
b u f f e r was c a r r i e d o u t f o r 30 m i n a t a v o l t a g e
g r a d i e n t of 1 0 V/cm. The s h e e t was a i r d r i e d ,
sprayed w i t h a n i n h y d r i n r e a g e n t c o n t a i n i n g
sym-collidine, and developed f o r 3 h r . The
c o l o r e d s p o t s were e v a l u a t e d b y d e n s i t o m e t r y .
E-Aminobenzoic acid i n s o l u t i o n s of
procainamide h a s been detected by c i r c u l a r paper
chromatography60. A f t e r development w i t h
ammonia-saturated b u t a n o l , t h e paper was d r i e d
364
and s p r a y e d w i t h sodium n i t r i t e i n 5% hydro-

c h l o r i c a c i d , f o l l o w e d by 1 - n a p h t h o l and
e t h a n o l i c potassium h y d r o x i d e , Procainamide and
i t s c l e a v a g e p r o d u c t g i v e r e d c i r c u l a r zones.
p-Aminobenzoic a c i d , a p o t e n t i a l c l e a v a g e
p r o d u c t , h a s been s e p a r a t e d by e l e c t r o p h o r e s i s
on paper78, e l u t e d i n d i s t i l l e d water, t h e n
d e t e r m i n e d c o u l o m e t r i c a l l y . p-Aminobenzoic a c i d
may be s e p a r a t e d from p r o c a i n a m i d e i n an ex-
t r a c t i o n system79. The a l k a l i n i z e d sample 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 t h e E- aminobenzoic
a c i d remaining i n t h e aqueous phase a s t h e sodium
s a l t can be d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y
(Section 6.3).
The p h a s e - s o l u b i l i t y of p r o c a i n a m i d e i n
a c e t o n e a t 23 and 32OC h a s been used t o d e t e r m i n e
t h e p u r i t y of t h e compound 52980. P u r i t y was a l s o
e v a l u a t e d by D i f f e r e n t i a l Scanning C a l o r i m e t r y
(Section 2.22).
6. A n a l y t i c a l T e s t s and Methods
6 . 1 Elemental A n a l y s i s
Element Theory Reported64

C 57.45% 57.45
H 8.16% 8.16
N 15.46% 15.46
0 5.89% 5.88
c1 13.04% 13.05
6.2 I d e n t i f i c a t i o n Tests
A t e s t b a s e d o n t h e p r e p a r a t i o n of
b e n z o y l procainamide and t h e d e t e r m i n a t i o n of i t s
m e l t i n g p o i n t i s d e s c r i b e d i n t h e U.S.P. 1 .
3 65
RAYMOND 6. POET AND HAROLD KADlN
Clarke has reported results for micro-

crystals and Vitalit s color testsll. Crystal-
reaction identification tests have been
described58. A number of spot-plate tests to
identify procainamide have been reported133 l7 9
18. Identification has been achieved by paper
and test-tube chromatography,16, as well as by
gas chromatography41. Color reactions with
barbituric acid and 2-thiobarbituric acid have
been reported33.
Infrared spectroscopy (Section 2. ll),

spectrophotometry (Section 6.3), paper and thin-
layer chromatography (Section 6.42), spectro-
photofluorometry (Section 6.5) and gas
chromatography (Section 6.43) provide alternate
methods for purposes of identification.
6.3 Spectrophotometric Methods
Procainamide and its decomposition

product E-aminobenzoic acid have been determined
by measuring their ultraviolet absorption55
(see Section 2.13).
The color developed upon the reaction

of procainamide with vanillin in acid solution
has been used to measure its c o n ~ e n t r a t i o n ~ ~ j ~ ~ ,
54. A reaction with thyme camphor after
diazotization has also been reported49. Reaction
with sulphonphthalein dyes50 has been used to
give a colored product for measurement.
Refer to Section 7 for spectrophoto-

metric methods applicable to the determination of
procainamide in biologic fluids and tissues.
366
6.4 I s o l a t i o n and C h r o m a t o g r a p h i c Methods

6.41 Solvent Extraction
Procainamide h a s been i s o l a t e d by
s o l v e n t e x t r a c t i o n (see S e c t i o n s 5 . 0 and 7 . 0 ) for
s u b s e q u e n t d e t e r m i n a t i o n b y a v a r i e t y of methods
(see S e c t i o n s 6 . 4 3 , 6 . 6 and 7 ) .
6.42 P a p e r and Thin-Layer Chroma to-

graphy
Procainamide h a s been assayed by

p a p e r c h r o m a t o g r a p h i c method^^^,^^, 60. R o b e r t s 4 7
u s i n g S&S 597 f i l t e r p a p e r , d e v e l o p e d chromato-
grams f o r 1 6 h r i n t h e s o l v e n t - r i c h u p p e r p h a s e
of a s o l v e n t s y s t e m composed o f e q u a l volumes of
i s o b u t a n o l and 0.05N p h o s p h o r i c a c i d . The z o n e s
on t h e d e v e l o p e d chromatograms were l o c a t e d b y
t h e i r u l t r a v i o l e t absorbance.
A number o f t h i n - l a y e r chromato-
g r a p h i c s y s t e m s h a v e b e e n u s e d t o examine pro-
c a i n a m i d e u a l i t a t i v e l 20, 2 1 22, ~ 38 and q u a n t i t a -
t i v e l y2 2 , 3 2 9 4 0 , 6 8 , 8 2 9 g 3 . The q u a n t i t a t i v e TLC
s y s terns369 82 c a n s e p a r a t e p r o c a i n a m i d e from s u c h
known i m p u r i t i e s a s p a m i n o b e n z o i c a c i d a n d
p n i t r o b e n z o i c acid. The most u s e f u l t h i n - l a y e r
systems are given i n t h e n e x t t a b l e .
6.43 G a s Chromatography
Gas c h r o m a t o g r a p h y h a s b e e n u s e d
t o s e p a r a t e and q u a n t i t a t e procainamide ( s e e
S e c t i o n 7 . 0 ) , a s w e l l a s f o r i d e n t i f i c a t i o n of
t h e compound (see S e c t i o n 6 . 2 ) .
367
Thin-Layer Chromatographic Systems
Ref. Support Solvent Composition

38 Silica Gel GF250 Benzene:Ethyl Acetate:Ethanol:
Ammonium Hydroxide (15:15:5:1)
36,38,40 Silica Gel GF250 Benzene:Ethyl Acetate:Methanol:

Ammonium Hydroxide(160:80:160:1)
82 Kieselgel GF254 (Merck) Benzene:Ethyl Acetate:Methanol:

Ammonium Hydroxide (20:10 :20 :1)
W
QI
W
93,68 Silica Gel QlF250 I. Benzene:Ammonium Hydroxide:
(Quantum Industries) 1,4-Dioxane (10:5:80)
1I.Isopropanol:Chloroform:Ammonium
Hydroxide (45:45 :5)
6.44 I o n Exchange Chromatography
The u s e of ion-exchange chroma-

tography f o r t h e s e p a r a t i o n of procainamide from
m i x t u r e s h a s been r e p o r t e d 24,29,74,75
6.5 Spectrophotofluorometric Methods
F l u o r e s c e n c e o f procainamide i n t h e
u l t r a v i o l e t h a s been r e p o r t e d 19 . It exhibited
an u n c o r r e c t e d a c t i v a t i o n maximum a t 295 nm,with
an u n c o r r e c t e d f l u o r e s c e n c e maximum t o 385 nm a t
pH 11. The minimal d e t e c t a b l e c o n c e n t r a t i o n was
r e p o r t e d t o be 0 . 0 1 p,g/ml.
The c o n c e n t r a t i o n of procainamide i n
b i o l o g i c f l u i d s h a s been measured s p e c t r o p h o t o -
f l u o r o m e t r i c a l l y 8 2 4 3 (see S e c t i o n 7 . 0 ) .
6.6 T i t r i m e t r i c Methods
Procainamide h a s been determined by

t i t r a t i o n w i t h IC126. The most g e n e r a l l y u s e f u l
methods f o r t h e d e t e r m i n a t i o n o f procainamide
have b e e n t h o s e i n v o l v i n g t i t r a t i o n w i t h n i t r i t e
37,42
2 3 J 2 5 J 2 7 9 2 8 J 4 2 and w i t h p e r c h l o r i c a c i d
KadinG1 r e c e n t l y r e p o r t e d t h a t pro-
cainamide, a s w e l l a s some o t h e r a r o m a t i c amines,
r e a d i l y undergo p a r t i a l a c e t y l a t i o n i n g l a c i a l
a c e t i c a c i d c o n t a i n i n g a c e t i c anhydride. The
a c e t y l a t e d a r o m a t i c amines a r e n o t t i t r a t a b l e
w i t h a c e t o u s p e r c h l o r i c a c i d . The a d d i t i o n of
a c e t i c anhydride t o g l a c i a l a c e t i c a c i d i s
commonly employed t o remove water when t h e
a c e t o u s p e r c h l o r i c a c i d i s c o n s t i t u t e d . Indeed,
q u i t e s a t i s f a c t o r y t i t r a t i o n s may be o b t a i n e d
when t h e a c e t i c a n h y d r i d e i s n o t added t o t h e
a c e t o u s p e r c h l o r i c a c i d . However, r e a g e n t g r a d e
369
RAYMOND 6. POET AND HAROLD K A D I N
g l a c i a l a c e t i c a c i d may a l r e a d y c o n t a i n o f f e n d i n g
a c e t i c anhydride. KadinG1 h a s shown t h a t a c e t i c
a n h y d r i d e i n t e r f e r e n c e can b e e l i m i n a t e d by p r i o r
r e a c t i o n of t h e t i t r a t i o n s o l v e n t w i t h an aroma-
t i c amine. He t i t r a t e d an a l i q u o t o f s o l v e n t ,
which had been c l e a r e d o f a c e t i c a n h y d r i d e
through r e a c t i o n w i t h t h e a r o m a t i c amine
benzocaine, t o n e u t r a l i t y j u s t p r i o r t o
d i s s o l v i n g t h e procainamide sample f o r t i t r a t i o n .
However, he s t r o n g l y recommended abandonment o f
t h i s r a t h e r cumbersome nonaqueous t i t r a t i o n i n
f a v o r of a s i m p l e r , more s e l e c t i v e n i t r i t e
t i t r a t i o n of procainamide u s i n g t h e s t a b l e
i n t e r n a l i n d i c a t o r ferrocyphen.
Procainamide, i s o l a t e d by e x t r a c t i o n
i n t o chloroform from ammoniacal aqueous s o l u t i o n ,
was d i s s o l v e d i n d i l u t e h y d r o c h l o r i c a c i d a f t e r
removal of t h e s o l v e n t by e v a p o r a t i o n . Pro-
cainamide was t i t r a t e d w i t h 0 . 1 g sodium n i t r i t e
by o x i d a t i o n - r e d u c t i o n p o t e n t i o m e t r y , u s i n g
platinum electrodes3’. The d i r e c t conductometric
t i t r a t i o n of procainamide h y d r o c h l o r i d e w i t h
sodium hydroxide h a s been d e s c r i b e d 3 4 . The
u s u a l change i n t h e s l o p e o f t h e conductance
response w i t h volume of t h e sodium h y d r o x i d e
t i t r a n t o c c u r s a f t e r t h e n e u t r a l i z a t i o n of t h e
amine h y d r o c h l o r i d e . I n t e r s e c t i o n of t h e two
l i n e a r p l o t s , r e p r e s e n t i n g t h e changes i n
conductance b e f o r e and a f t e r n e u t r a l i z a t i o n ,
then a c c u r a t e l y d e l i n e a t e s the endpoint.
6.7 M i c r o b i o l o g i c a l Methods
A microbiological assay of
procainamide w i t h A c e t o b a c t e r suboxydans h a s
been r e p o r t e d 32 .
370
7. A n a l y s i 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
C o n c e n t r a t i o n s of procainamide i n b i o l o g i c a l
media have been measured c o l o r i m e t r i c a l l y a f t e r
d i a z o t i z a t i o n and coupling t o t h e
Bratton-Marshall r e a g e n t ( N (1-naphthyl) - e t h y l e n e -
d i a m i n e ) t o form a c o l o r e d product. These methods
have involved p r o t e i n p r e c i p i t a t i ~ on r ~s o~l v e n t
e x t r a c t i o n 2 4 3 9 4 6 9 56 t o i s o l a t e procainamide
J
p r i o r t o t h e c o l o r i m e t r i c measurement.
C o n c e n t r a t i o n s of procainamide i n blood and

u r i n e were e s t i m a t e d a f t e r r e a c t i o n of t h e drug
w i t h 4 - d i r n e t h y l a m i n o ~ i n n a m a l d e h y d e ~t ~
o form a
c o l o r e d product.
A r a p i d procedure f o r measuring plasma pro-

cainamide c o n c e n t r a t i o n s by g a s chromatography
h a s been r e p o r t e d by Atkinson e t a139. They used
a 2 m x 2 mm i . d . g l a s s c o i l packed w i t h 0.2%
OV-17 on a 100/120 mesh g l a s s bead (Corning GLC-
1 1 0 ) . The temperature o f t h e i n j e c t o r p o r t was
23OoC, t h a t of t h e column 225OC and of t h e
flame i o n i z a t i o n d e t e c t o r , 2400C. )
A flow r a t e of
30 m l / m i n N 2 c a r r i e r g a s and H2 was used. The
a i r - f l o w r a t e f o r t h e d e t e c t o r was 300 m l / m i n .
Procainamide was e x t r a c t e d from a l k a l i n e s o l u t i o n
i n t o methylene c h l o r i d e , w i t h p-amino-N- (2-
dipropyl-aminoethy1)benzamidine HC1 a s t h e
i n t e r n a l s t a n d a r d . The r e s i d u e r e s u l t i n g from
t h e e v a p o r a t i o n of t h i s methylene c h l o r i d e
s o l u t i o n was d i s s o l v e d i n e t h y l a c e t a t e and
i n j e c t e d i n t o t h e chromatograph.
The d e t e r m i n a t i o n o f procainamide i n serum8t

43by use of i t s f l u o r e s c e n t p r o p e r t i e s h a s
provided an a d d i t i o n a l t o o l . Procainamide was
e x t r a c t e d from a l k a l i n i z e d s a l t - s a t u r a t e d serum
i n t o benzene c o n t a i n i n g 1.5% i s o p e n t y l a l c o h o l .
371
I t was re-extracted i n t o d i l u t e hydrochloric

acid. I t s f l u o r e s c e n c e was measured a f t e r t h e
pH had been a d j u s t e d t o 11 by t h e a d d i t i o n o f
sodium hydroxide.
A c o l o r i 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-acetylprocainamide, a m e t a b o l i t e of
procainamide, h a s been r e p o r t e d by Poet44. In
t h i s method2 Y ~ N-acetylprocainamide
~ , is
e x t r a c t e d from a l k a l i z e d serum i n t o benzene
c o n t a i n i n g 1.5% isoamyl a l c o h o l . I t i s t h e n
r e - e x t r a c t e d from t h e s o l v e n t i n t o 13-hydro-
c h l o r i c acid. An a l i q u o t is heated i n a
b o i l i n g - w a t e r b a t h t o h y d r o l y z e any N-acetyl-
procainamide p r e s e n t . N-acetylprocainamide i s
then determined a s t h e d i f f e r e n c e i n procainamide
c o n t e n t o f t h e sample e x t r a c t b e f o r e and a f t e r
h y d r o l y s i s , a s measured by t h e
r e p o r t e d c o l o r i m e t r i c method2 5 ireviou
.
The metabolism and d i s t r i b u t i o n of
procainamide h y d r o c h l o r i d e i n man and dog was
s t u d i e d by Mark e t a12. They r e p o r t e d t h a t
50-60% of a s i n g l e i n t r a v e n o u s dose a d m i n i s t e r e d
t o man was e x c r e t e d unchanged i n t h e u r i n e ; t h a t
about 2-10% was accounted f o r a s p a m i n o b e n z o i c
a c i d ; and t h a t t h e drug d i d n o t accumulate when
given i n r e p e a t e d o r a l doses. Procainamide
c o n c e n t r a t i o n s i n t h e s e s t u d i e s w e r e measured
a f t e r i s o l a t i o n of t h e drug from a l k a l i z e d
b i o l o g i c a l m a t e r i a l by e x t r a c t i o n i n t o benzene,
augmented by s a t u r a t i o n of t h e aqueous phase
w i t h sodium c h l o r i d e . The drug was r e t u r n e d
t o d i l u t e a c i d , d i a z o t i z e d , and coupled w i t h
N(1-naphthy1)ethylene diamine. The r e s u l t i n g
c o l o r e d dye was determined s p e c t r o p h o t o m e t r i -
c a l l y (see Section 7.0). The s p e c i f i c i t y of t h e
method was demonstrated by t h e c a l c u l a t i o n of
comparative d i s t r i b u t i o n r a t i o s 6 .
312
Dreyfuss e t a l . 3 9 4 s t u d i e d t h e b i o t r a n s f o r -
mation of procainamide h y d r o c h l o r i d e i n r h e s u s
monkey, man, and dog, u s i n g I 4 C - l a b e l l e d d r u g .
I n t h e dog, 50-67% o f t h e 14C 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 was unchanged p r o c a i n a m i d e ; f o u r
m e t a b o l i t e s were r e c o g n i z e d . I n monkey, 22-49”/0
of t h e 1 4 C a c t i v i t y was e x c r e t e d i n t h e u r i n e as
unchanged p r o c a i n a m i d e ; t w o metabolites w e r e
r e c o g n i z e d . N-acetyl p r o c a i n a m i d e , i s o l a t e d and
i d e n t i f i e d ‘ b y a c o m b i n a t i o n o f TLC, NMR and mass
s p e c t r o m e t r y , was a major m e t a b o l i t e e x c r e t e d by
monkey: a l t h o u g h p r e s e n t i n man t h i s metabolite
d i d n o t a p p e a r t o o c c u r i n dog. The c o m b i n a t i o n
of these a n a l y t i c a l techniques helped t o provide
e s s e n t i a l d a t a i n t h e s e m e t a b o l i c s t u d i e s . These
s t u d i e s a l s o s u g g e s t e d t h a t compounds c o n t a i n i n g
r e a d i l y a c e t y l a t e d a r o m a t i c amino g r o u p s s h o u l d
be s t u d i e d i n p r i m a t e s r a t h e r t h a n i n d o g s , b u t
p r e f e r a b l y i n b o t h species. Weily and Genton‘
have r e p o r t e d on t h e r o u t e s and mechanism o f
p r o c a i n a m i d e e x c r e t i o n : t h e e f f e c t o f u r i n e pH;
and t h e e f f e c t s o f f l o w r a t e and t h e p r e s e n c e o f
r e n a l o r h e p a t i c impairment on t h e plasma h a l f
time and on t h e r e n a l and h e p a t i c c l e a r a n c e and
e x c r e t i o n of procainamide.
The p h a r m a c o k i n e t i c s o f p r o c a i n a m i d e hydro-
c h l o r i d e i n man h a s b e e n s t u d i e d . e x t e n s i v e l y 7 ’ 8, 9.
Koch-Weser d e m o n s t r a t e d t h a t plasma c o n c e n t r a t i o n s
between 4 and 8 mg/L a r e found a f t e r t h e u s u a l l y
e f f e c t i v e t h e r a p e u t i c doses. H e showed t h a t
knowledge o f plasma c o n c e n t r a t i o n s i s h e l p f u l i n
e s t a b l i s h i n g t h e optimal dosage i n i n d i v i d u a l
p a t i e n t s . Procainamide c o n c e n t r a t i o n s were
d e t e r m i n e d s p e c t r o p h o t o m et r i c a l l y 8 and s p e c t r o -
p h o t o f l u o r o m e t r i c a l l y 8 b y methods t h a t a r e w e l l
within the capability of h o s p i t a l laboratories
(see S e c t i o n 7 ) .
373
A recent report'' indicated that N-acetyl

procainamide was detected in the plasma of two
patients receiving procainamide. This compound
was identified by a combination of TLC, gas
chromatography and mass spectrometry. This
metabolite was demonstrated to have anti-
arrhythmic activity in mice. The report
concluded that the observed anti-arrhythmic
activity of procainamide in man may be due, in
part, to its conversion to N-acetylprocainamide
(see also Section 7).
8. References
1. United States Pharmacopeia, XVIII,

pgs. 8,541 (1970).
2. L. C. Mark, H. J. Kayden, J. M. Steele,

J. R. Cooper, I. Berlin, E. A. Rovenstine,
and B. B. Brodie, J. Pharmacol. Exp.
Ther., z,5-15 (1951).
3. J. Dreyfuss, J. T. Bigger, A. I. Cohen

and E. C. Schreiber, Clin. Pharmacol.
--
Ther. 13, 366-71 (1972).
4. J. Dreyfuss, J. J. Ross, and

E. C. Schreiber, Arzneim-Forsch. ,=(7),
948-51 (1971).
5. A. I. Cohen,Squibb Institute, Personal

Communication.
6. H. S. weily and E. Genton, Arch. Intern.

Med
- 0 9 -
130, 366-69 (1972).
7. J. Koch-Weser, Ann. N.Y. Acad. Sci. ,

-
179, 370-82 (1971).
374
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/
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RAYMOND 6. POET AND HAROLD KADIN
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/
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/
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/
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RAYMOND B. POET AND HAROLD KADlN
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63. J. Womack, E.A.I., Princeton, N.J.,

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64. J. Alicino and J. Hydro, Squibb

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47, 676 (1958).
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Communication.
68. J. Fairbrother and S. Shand, Squibb

International Development Laboratories,
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Communica tion.
70. D. Whigan and H. Kadin, Squibb

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RAYMOND 6 . POET AND HAROLD K A D I N
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84. Y. Tashika and M. Kuranari, J. Pharm.

SOC. Japan, 73, 1069-71 (1953), Chem.
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Y. Tsukamoto, J. Pharm. SOC. Japan, 73,
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Franc, l3, 556-9(1955), Chem. Abstr.,
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50, 9331d (1956).
/
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88. K. Iwaya and K. Yoshida, Ann. Rep.

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A. Led6chowski and A. Chimiak,
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W. Luh, Yao Hsueh T' ung. Pao,, 8,319-21
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91. M. Blaich and A. Restivo, Squibb

92. M. Blaich and A. Restivo, Squibb

93. P. Egli, Squibb Institute, Personal
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Communication.
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(1964).
95. J . O r l o s k i , S q u i b b I n s t i t u t e ,
96. P. T. Thymm, R. L u c h i , H. L. Conn.,

J. Pharmacol. Exp. T h e r . , 164, 239-251
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97. J . Kruk, Zesz, Nauk. Uniw. J a g i e l l o n .

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383
RESERPINE
Roger E. Schirmer
RESERPINE
COWENTS
1. Descript ion
2.1 Melting Point
2.2 Electronic Spectra
2.2.1 Ultraviolet Absorption Spectrum
2.2.2 Optical Activity
2.2.3 Fluorescence and Phosphorescence
2.5 Crystal Form and X-Ray Powder Pattern
2.6 Solubility and Partition Coefficients
3. Degradation of Reserpine
3.1 Chemistry of Reserpine Degradation
3.1.1 Hydrolysis
3.1.2 Epimerization
3.1.3 Oxidation
3.2 Degradation of Reserpine in Pharmaceutical Dosage
Forms
4. Metabolism of Reserpine
5. Identification of Reserpine
6. Elemental Analysis
7. Chromatographic Methods of Analysis
7.4 Electrophoresis
7.5 Countercurrent Distribution
8. Titrimetric Determination
9. Electrochemical Analysis
10. Spectrophotometric Analysis
10.1 Infrared
10.2 Ultraviolet
10.3 Colorimetric
10.4 Ion-Pair Extraction with Spectrophotometric
Detection
10.5 Fluorescence
11. Analysis of Reserpine in Biological Systems
385
ROGER E . SCHIRMER
1. Description
Reserpine is 1,2-didehydro-2,7-dihydro-ll,l7a-dimethoxy-
3f3,20a-Yohimban-l6f3-~arboxylic
acid methyl ester, 18B-tri-
methoxybenzoate ester.
Reserpine is generally obtained by extraction of the

roots of certain species of Rauwolfia (Apocynaceae),
-
prin5igjally R. Serpentina and R. Vomitoria, or by eynthe-
sis. It occurs as a white or pale buff to slightly
yellow, odorless, crystalline substance. It is weakly basic,
with a pK of 6.6.
2.1 Melting Point 1

Reserping melts at 264-265°C with decomposition.
Hochstein s. u
&. have reported that reserpine melts at
284-286°C in an evacuated tube, and that the apparent
melting point is less dependent on heating rate when measured
under vacum.
The melting pqints of several salts of reserpine
are listed in Table 1, and melting points for several
stereoisomers have been included in Table 2.
386
RESERPINE
Table 1
Melting P o i n t s of Several Reserpine S a l t s 7
Salt E m p i r i c a l Formula Melting Point*
hydrochloride C33H40N2040 HCll H 2 0 224"
sulfate 242-244"
c33H40N209' H2S04
perchlorate C3 H40N209* HC 1O4 238-23 9"
nitrate 235 "

C33H40N209'm03
oxalate C33H40N209* C2H204- 1/2H20 206"
methylbromide C H N 0 *CH3Br 271-272"

33 40 2 9
maleat e 226-227"
3 3 H4ON 2 '9' c4H404
picrate
'3 3 H40N2'9' H2 -
183 186"
387
Table 2
Sodim D-line Rotations for Reserpine

and Several of Its Configurational Isomers
Melting
Cornpound Isomer* Point "C U D Conditions Ref
-
reserpine - 264-265 -118"
-164"
CHCl , 23°C
pyriaine, 26°C
798
7
-168" d imethyl-
foramide, 26°C 7
-100" dilute acetic
acid, 25°C 16
- 125" Dioxane, 25°C 16
16-epireserpine 16a 180 + 44" CHC13 1
18-epireserpine 18a 141-145 + 38" CHC13 10
16-epi-17-epi-reserpine
(neoreserpine) 16a,178 -
163 170 + 29" CHC13 11
3- isoreserpine 3a 152-165 -164" CHC13 899
18-epiisoreserpine 182 245-248 CHC13 10
W h e entries in this column give the configuration o f the carbons which differ from the
configuration in reserpine.
RESERPINE
2.2 Electronic Spectra
2.2.1 Ultraviolet Absorption Spectrum

The ultraviolet spectra of indole alkaloids
generally consist of three bands, one band in the region of
225 nm and two bands, often unresolved, in the region of
280 run. These bands have been assumed to have the same
origin as the low enfsgy transitions of benzene, and so thp3
benzene nomenclature is used for the indole chromophore.
As shown in Figure 1, all thfee bafds are distinct in the
spectrum of reserpine. lThe F a c A band maximum occurs at
216 p" (€=35,700), the La t A band at4267 nm (€=15,700) and
the I,,* A band at 296 nm (€=9,660). The absorption
maxima and extinctions are essentially identical in chloro-
form and methanol.
2.2.2 Optical Activity

Carbons 3 , 15, 16, 17, 18, and 20 of
reserpine are asymmetric, resulting in 64 poseible configu-
rational isomers. Reserpine itself ha rotation of -118"
(sodium D line) at 23°C in chloroform.'18 The D-line
rotations for reserpine and several of its isomers are
listed in Table 2. The circular dichroism (CD) and magnetic
circularlgichroism (MCD) spectra of reserpine have been
reported and are reproduced in Figure 2. The magnetic
circular dichroism is of special analytical interest because
the sign pattern can be used to distinguish indole alkaloids
from alkaloids bearing the indoline or oxindole chromophores:
chromophores show a positive effect at low wave-
transition) and negative effects at higher wave-
transition), whereas indole alkaloids have
fects at low wavelength and positive effects at
higher wavelength.
2.2.3 Fluorescence and Phosphorescence

The absorption band at 296 nm in resgz&;19
gives rise to strong fluorescent emission at 375 nm.
In water, the fluorescence intensity is maximum at low pH: 17
a spectrum in water at pH 1,O is reproduced in Figure 3 .
Fluorescence is typical of compounds closely related to
reserpine, including alkaloids which occur naturally along
with reserpine and degradation products of reserpine (e
infra). The fluorescence characteristics of several of these
are summarized in Table 3. In reserpine, deserpidine, and
rescinnamine, it has been shown that light energy absorbed by
the indole c9tjoypphore is emitted from the trimethoxybenzoate
chromophore. ' The intramolecular singlet-singlet exci-
389
ROGER E. SCHIRMER
*
b
P 3
X
W
200 250 300 350 400

Clm
Figure 1. The Ultraviolet Absorption Spectrum of Reserpine
390
RESERPINE
20
c3
I
0
v
10 x
W
I I 0
250 300
Yigure 2. Magnetic Circular Dichroism (-), Circular

Dichroism (----), and Absorption Spectra (.....) of Reserpine
in Methanol
391
loo
90 -
1
80 -
70 -
200 250 300 350 400 450 500 550 600 650 700 750 800
W AVELENGTH-MILLIMICRONS
Figure 3. Fluorescence Spectrum of Reserpine in Water at pH 1.
RESERPINE
tation transfer occurs with 100% efficiency, and iyolves

transfer from the indole S (v,TT*) state to the
(TT,TT*) 3
state of the trimetioxybenzoate acceptor. T e 22
energy transfer appears to occur bF3an exchange mechanism
rather than a resonance mechanism.
Table 3
Fluorescence Characteristics o
Compounds Related to Reserpine €9
Excitation Emission Relative
Compound Maximum Maximum Sensitivity
Reserpine 280 360 6
3-is0 Reserpine 390 5 10 18
Dehydroreserpine perchlorate 390 510 75
Tetradehydroreserpine
chloride 340 440 50
Rescinnamine 310 440 2
Rescinnamine N-oxide 310 340 2-3
Deserpidine 280 360 15
Reserpine N-oxide 280 360 8
Methyl Reserpate 300 360 19
Reserpinine 300 360 2
Methyl-0- (35-dimethoxy-4-
hydroxybenzoyl reserpate) 300 360 8
Syrosingopine 300 360 4
Syrosingopine N-oxide 290 350 6
Trimethoxybenzoic Acid 280 360 2
Trimethoxycinnamid Acid 300 400 <1
Indole 280 340 36
Hannaline HC1 390 490 150
Phosphozgscent emission from reserpine occurs at about
460 nm.
The infrared spectrZ-96 reserpine has been
reported by several workers. The spectrum in chloro-
form is reproduced in Figure 4, and the assignments of
several bands in the spectrum are as follows:
3480 cm" >N-H stretch

2840-3039 ~m-1 >C-H stretch
1732 cmI1 >C=O stretch, acetyl group
1713 cm >C=O stretch, trimethoxybenzoate
group
393
101
90
80
40
u
k 30
20
10
0 ~ 1 1 1 1 ~ 1 1 1 1 1 1 1 ~ ~ ~ ~ ~ ~ '
4001 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 ll00 1800 1700 1601 1500 1400 1300 1200 1100 1000 900 800 700 600 500 400
WAVENUMBER CM-'
Figure 4 . The Infrared Spectrum of Reserpine i n Chloroform

RESERPINE
At one time Wenkert3O suggested that the presence of t o or

-Tfr
more bands or distinct shoulders between 2700-2900 cm on
the low wave number side of the major band at about 2900 cm-;
indicated an a hydrogen on carbon 3, whereas the absence of
these bandf8indicated a p hydrogen on carbon 3. It has since
been shown that these bands are associated with axial
hydrogens at C-3, and are therefore indicative of the confor-
mation rather than the configuration of the molecule.

The n.m.r. spectra of methyl 3-isoreserpate,methyl
reserpate, methyl neoreserpate, and the 3',4',5'-trimethoxy-
benzoate esters of eacb of these compounds have been reported
by Rosen and Shoolery. The 60 MH2 spectrum of reserpine is
reproduced in Figure 5, and the assignments of several of the
resonances in the spectrum are given in Table 4.
As axial protons are generally found at higher
fields in the n.m.r. spectrum than the corresponding equa-
torial protons, the resonances of the C-3 and C-18 protons
may be used to distinguish reserpine from isoreserpine or
neoreserpine. In the most stable conformation of reserpine,
the C-3 proton is equatorial and the C-18 proton is axial.
In 3-isoreserpine,however, the C-3 proton is axial and is
shifted to high enough field that it is obscured by the -WH3
resonances: the axial C-18 resonance is essentially un-
changed. In neoreserpine the C-3 proton is again axial
(therefore not discernable in the spectrum) and the C-18
proton is equatorial and therefore shifted to lower field
than in reserpine (5.83 p.p.m. compared with 5.05 p.p.m.,
respectively).
395
I I I I I I I I I
8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.o 0

PPM I61
Figure 5. The 60 MHz NMR Spectrum of Reserpine in CDC13. See Table 5 for Assignments.
RESERPINE
Table 4
Assignment of the N.M.R. Spectrum

of Reserpine in CDC13
Chemical
Functional shift
Group @.p.m. from tetramethylsilane)
NH 7.85 p.p.m. (broad singlet)
Indole aromatic H Two groups of lines at about
((3-9,C-lO,C-12) 6.7 and 7.3 p.p.m.
Trimethoxybenzoic acid
aromatic H 7.34 p.p.m. (sharp singlet)
C-18 H 5.05 p.p.m. multiplet
C-3 H 4.43 p.p.m. multiplet
Trimethoxybenzoic acid,
-OCH3 3.92 p.p.m. singlet
(2-11, -OCH3 3.79 p.p.m. singlet
0
C-16, -C-OC% 3.79 p.p.m. singlet
C-17, -OCH3 3.46 p.p.m. singlet
2.5 Crystal Form and X-Ray Powder Pattern

CrystaJiographic data on reserpine has been re-
ported by Rose, The crystals are monoclinic with axial
ratios, a:b:c, of 1.654:1:1.537 and B = 115'8'. They occur
orthopinacoid 100 or basal pinacoid 001 .
as blades or needles elongated parallel to b and lying on the
The crystals
sometimes show the clinodome 011 and the hemipyramid:
blades are generally terminated by the clinopinacoid 010
TQe density of the crystals wag determined to be
.
1.298 g/cm by flotation and 1.293 g/cm by x-ray. The
refractive indexes of the crystal at 25°C for 5893A radiation
are as follows: ~ 1 . 5 1 ,p=1.568 + 0.002 andY=1.687 2 0.004.
The unit Sell contsins two molegules and has
dimensions A=14.57AI b=8.8lA, and c=13.5A. The powder
pattern is summar ed in Table 5.
Hamilto$ has reported that reserpine is one of the
largest acentric molecules whose structure has been de-
termined by direct x-ray techniques, but he did not report
the details of this study.
391
ROGER E. SCHIRMER
Table 5
31
X-Ray Powder P a t t e r n of Reserpine
-d -I/I
1 -
hkl d (calculated)
13.14 0.40 100 13.11
12.22 0.40 001 12.18
7.44 0.20 101 7.48
7.19 1.oo 011 7.14
5.73 0.40 111 5.70
5.34 0.40 210 5.26
5.02 0.40 012 5.01
4.78 0.60 102 4. 80
4.49 0.80 103 4.45
4.21 0 -40 112 4.21
4.18 0.20 021 4.15
3.71 0.40 202 3.74
3.56 0 -40 401 3.57
3.47 0.20 103 3.48
2.6 S o l u b i l i t y and P a r t i t i o n C o e f f i c i e n t s
Reserpine i s only soluble t o t h e extent of 6 mcg/ml
i n 0.1 N HC1 at 37"C, and i t s s o l u b i l i t y i n aqueous solvents
decreases a t high pH. Reserpine i s much more soluble i n less
polar solvents, with a s o l u b i l i t y of about 0.56 mcg/ml 13
ethanol and 167 mg/ml i n chloroform a t room temperature,
E
D i s t r i b u t i o n c o e f f i c i e n t s f o r r e s e r p i n e and seve a1
r e l a t e d a l k a l o i d s have been reported by Hochstein e t . a l .
and are reproduced i n Table 6.
Table 6
D i s t r i b u t i o n C o e f f i c i e n t s f o r Reserpine
6
and Related Alkaloids
Solvent system
Compound A B C
Reserpine 9.0 0.01 8.0

Yohimbine 9.0 0.01 1.5
Rauwol sc i n e 9.0 0.01 0.7
Ajmalicine 11.0 0.01 8.0
Heterophyllin 20.0 0.01 8.0
Serpentine 0.01 11-13 7
A jmaline 0.01 11-13 1
398
RESERPINE
system Solvent 1 Solvent 2
A benzene .05M pH 7
phosphate buffer
B 50% CH OH in H20 CHC13

3
C n-butano1 15% CH3COOH in H20
Distribution Coefficient = conc. solvent lfconc. solvent 2
3. Degradation of Reserpine
3.1 Chemistry of Reserpine Degradation
3.1.1 Hydrolysis
The reserpine molecule contains two ester
groups, both of which are susceptible to hydrolysis, as
shown in Figure 6.
Hydrolysis of the trirnethoxybenzoate ester
group occurs2~~sg,ygre rapidly than hydrolysis of the methyl
ester group. As with all esters, the hydrolysis of
reserpine is catalyzed by both acid and b e with basic
catalysis generally being most efficient," Although the
full pH profile of reserpine hydrolysis has apparently not
been studied, one would expect the rate of hydrolysis to be
minimum in the pH range of 3-4.
3.1.2 Epimerization
Although reserpine has six asymmetric
carbons, only C-3 is inverted easily enough for isomeri-
zation to be a significant route of degradation. Epimeri-
zation of reserpine t 3-gsoreserpine occurs gsincipally in
strong acid solution.'6-3 Gaskell and Joule have shown
that epimerization under these conditions is initiated by
protonation of C-2 with subsequent ring opening to give an
intermediate in which C-3 is planar and properly oriente
99
66
for e icient reclosure of the system to 3-isoreserpine.
Bayer has reported that epimerization of reserpine in
solution in chloroform is a380 promoted by heat and light.
Hakkesteegt has reported that the ratio
of epimerization to hydrolysis was 2.2, 5.6 and 8.1 for so-
lutions of reserpine at pH 1.3, 2.2 and 3.0, respectively,
after storage of the solutions for 48 hours at 100°C.
399
OCH3 OPlMERlZATlON [C-3)
3-ISORESERPINE
HYOROLYSIS.
ACID OR BASE
P
0
0
METHYL RESERPATE 18-(3.4,5-TRIMETHOXYBENZOYl) RESERPIC ACID 3 ,I-DIDEHYDRORESERPIWE
RESERPIC ACID
Figure 6 . Principle Routes for Degradation of Reserpine

RESERPINE
3.1.3 Oxidation
Oxidation of reserpine may be induced either
photolytically or chemically, and it is usuallglthe major
route of degradation for reserpine. Ljungberg found that
photo-oxidation of reserpine first produces a yellow
substance with yellow-green fluorescence, but that further
photolysis gives rise to a reddish brown solution with blue
fluorescence (due to lumireserpine) which eventuphly retunes
through red and orange to yellow. Banes s. e. first
suggested that the initial oxidation product was 3,4 di-
dehydrore$5rpine, and this was later confirmed by Kzgbs and
Futscher. Lunireserpine was shown by Hakkesteegt to be
3,4,5,6-tetradehydroreserpine, a resul which has bee re-
confirmed recently by Wright and Tang.65 Hakke~teegt'~ has
suggested that some of the non-fluorescent brown and yellow
products formed during oxidative degradation of reserpine are
polymers. The oxidations of reserpine are also summarized in
Figure 6.
The oxidation of reserpine is rapid in the
presence of 1' ht and oxygen. The oxidation is also cata-
lyzed by acid38 , and by certain metal ions. The efficiency
of several metal ions in catalyzing the oxidation of
reserpine decreases in the following order: Cu Mn Fe Al.
46
* 3.2 Degradation of Reserpine in Pharmaceutical Dosage

Forms
Hydrolysis of4reserpine appears to be very unlikely
in solid dosage forms , and there apparently have been no
reports in the literature of significant levels of hydrolysis
in reserpine izdectables or elixirs.
Bayer has reported that reserpine does undergo
epimerization in both solution and crystalline pure drug
under the influence of heat and light, but did not provide
any tqformation on the extent of conversion observed. Weis-
Fogh studied the degradation of a 0.25% reserpine solution
at pH 2.5 under the influence of sunlight. After three
months at room temperature, he found that the reserpine
content had decreased to 70% of initial, and that there was
5% 3,4-didehydroreserpine present. Weis-Fogh felt that the
remaining 25% of the reserpine was present largely as 3-iso-
reserpine, but this conclusion was probably incorrect because
his assay method would have measured most of the -
reserpine along with the reserpine. Hakkesteegt3 P was
0 unable
to find 3-isoreserpine in crystalline reserpine or in
reserpine tablets from one to seven years old and estimated
from his kinetic studies that it would take 26 years for 10%
of the reserpine in a pH 3.0 solution to be lost through
401
ROGER E. SCHIRMER
cornbivsd hydrolysis and epimerization at 20°C. Wright and

Tang found evidence of 3-isoreserpine in several com-
mercial brands of reserpine tablets by TLC, but it may be
concluded that epimerization is not likely to be a signifi-
cant route of degradation for reserpine in most pharmaceuti-
cal preparations.
Oxidation appears to be the most importag3 route of
degradation for reserpine in formulations, Banes examined
several reserpine injections, elixirs and tablets. All
injections and elixirs examined contained significant levels
of oxidation products, whereas only one of the eight lots of
tablets exaytned showed any evidence of these products.
Haddesteegt found levels of oxidation products as high as
35% in one lot 85 reserpine tablets, and more recently,
Wright and Tang found that 3,4-didehydroreserpine was
present in all commercially available reserpine tablets that
they examined (3,4,5,6-tetradehydroreserpine was not detected
in any of them).
4. Metabolism of Reserpine
The distribution, excretio~ a - ~ ~ ~ 6 ~ ~ 5 ~ ~ ~ ~ ~ ~ ~
havZglqp6ftudied ig2the mous89 ,62 , rat54,63 ¶
' , monkey , rabbit Y gljinea Pig Y
man , and Busycon canaliculatum.

The met&43sm of reserpine is qualitatively similar in
all species , with oxidative demethylat&;59f6fjhe 4 po-
sition of the 3,4,5-trimethoxybenzoyl group
&rlgglysis of the trimethoxybenzoate ester linkage 4 9 5 3 y
being the major identified routes of metabolism
metabolic products therefore4&&$j ,$&y#1
,?Be
re erpat so Y
3,4,5-trimethoxybenzoic acid and its glucu-
ronide or sulfate Sgnjugates67 syringoyl methyl reserpate54,
and syringic acid. Sheppard has made special note of the
absence of 3-isoreserpine, reserpine N-oxide, didehydro-
reserpine, and tetradehydroreserpine in guinea pig brain at
doses which produced measurable quantities of unmetabolized
reserpine in these tissues.
5. Identification of Reserpine
Reserpine can be identified by its NMR, IR, and W
spectra and by melting point (See section 2). Reserpine
forms a rose-pink color upon addition of about 1 mg. to a
few tenths of a 1-2% solution of vanillin in7g"f: the color
will deepen upon standing or gentle heating. A green
color is produced when 0.5 mg, of reserpine is treated with
5 mg dimethylaminobenzaldehyde, 0.2 ml glacial acetic acid,
and 0.2 ml of sulfuric acid: the color will change from green
402
RESERPINE
to red upon addition of 1 ml of glacial acetic acid.77 1 mg

of reserpine treated with 0.1% solution of sodium molybdate
in sulfuric acid produces7;) yellow color which turns to blue
within about two minutes. If 1 mg of reserpine i s dis-
solved in 0.5 ml of dilute acetic acid and 5 drops of sodium
chlorigg test solution are added, a white precipitate
forms. A yellow color accompanied by greenish fluorescence
develops in a solution of 1 mg reserpine in 5 ml of chloro-
form upon addition of 5 m17gf a 10% solution of trichloro-
acetic acid in chloroform.
54
Identi cation of reserpine by
fonnatio of crystalline derivatives , formation of eut
80
mixtures , and by use of a variety of other color tests
S€%
have been reported.
6. Elemental Analysis
Carbon 65.12%
Hydrogen 6.62%
Nitrogen 4.60%
Oxygen 23.66%
7. Chromatographic Methods of Analysis

A variety of thin layer systems have -een developed
for reserpine and a number of these are summarized in Table 7.
Reagents used for detection and identification of reserpine
on the plate are summarized in Table 8 . Thin layer systems
have also been described in references 102-111.
Quantitation of reserpine following thin layer
chromatography is described in references 50, 92, 96-101,
104, 107 and ,110-111.

Several paper chromatography systems for reserpine
are summarized in Table 10 and methods for visualizing
reserpine on the paper are summarized in Table 11. Paper
chromatography systems for f57erpine are also discussed in
references 119-127. Becker has applied elatography to
reserpine, elatography being the technique in which a sample
is spotted on the paper, treated with an appropriate reagent,
and the reaction products are then separated chromatographi-
cally.
Quantitation of reserpine by ultraviolet and infra-
red spectrophotometry, colorimetry, titration, and polar-
ography after isolation by paper chromatography are discussed
in references 116 and 125.
403
Table 7
Thin Layer Chromatography Systems for Reserpine
Solvent System Sorbent -
Rf Application and Comnents Reference
1. CHC13:Me2CO:NH40H, Silica gel-gypsum Separation from other 87

plates pretreated at tranquilizers
80:20:1
120°C for 30 minutes
2. CHC13:Me2C0, 85:15 Alumina 0.60 Separation from other 88, 90,
alkaloids of R. serpentina 94
3. CHC13 :EtOH:Me2C0 , Alumina 0.89 Separation from other
P alkaloids of R. serpentina 88, 90
0
P
90:5:5
4. CHC13:Me2CO:Et2NH, Silica Gel G 0.72 Separation of Rauwolfia 89-91
and opium alkaloids
5:4:1
5. CHC13:Et2NH, 9:1 Silica Gel G 0.80 Separation of Rauwolfia 90, 91
alkaloids
6. Cyclohexane:CHC13: Silica Gel G 0.25 Separation of Rauwolfia 50, 90-
FT2NH, 5:4:1 alkaloids 92
7. Cyc1ohexane:CHCl3’ Alumina G 0.35 Separation of Rauwolfia 90, 91

alkaloids
3:7 and 0.05% Et2NH
8. Methanol Silica Gel G, 0.1N 0.69 Separation of Rauwolfia 90, 91
NaOH impregnated alkaloids
Solvent System Sorbent Rf
- Application and Coments Reference
9. Heptane:Me-CO-Et, Cellulose, formamide 0.59 Separation of Rauwolfia 90, 93

1:l in atmosphere impregnated alkaloids
of annnonia
10. Heptane:Me-CO-Et: Whatman SG 41 Silica 0.25 Separation of 10 Rauwolfia 95

MeOH, 60:30:10 Gel alkaloids
11. Heptane:Me-CO-Et: Kieselgel G (Silica 0.32 Separation of 10 Rauwolfia 95

MeOH, 60:30:10 Gel) alkaloids
12. Heptane:Me-CO-Et: Aluminum oxide G 0.63 Separation of 10 Rauwolfia 95

MeOH, 60:30:10 alkaloids
P
0
13. Heptane:Me-CO-Et:n- Whatman SG 41 0.47 Separation of 10 Rauwolfia 95
BuOH, 60:30:10 (Silica Gel) alkaloids
14. Heptane:Me-CO-Et:n- Kieselgel G (Silica 0.52 Separation of 10 Rauwolfia 95

BuOH, 60:30:10 Gel) alkaloids
15. Heptane:Me-CO-Et:n- Aluminum oxide G 0.78 Separation of 10 Rauwolfia 95

BuOH, 60:30:10 alkaloids
16. Heptane:Me-CO-Et: Whatman SG 41 (Silica 0.07 Separation of 10 Rauwolfia 95

Pyridine, 70:15:15 Gel) alkaloids
17. Heptane:Me-CO-Et: Kieselgel G (Silica 0.05 Separation of 10 Rauwolfia 95

Pyridine, 70:15:15 Gel)
- Appliaation and C m e n t s Referenoe
18. Heptane:Me-CO-Et: Aluminum oxide G 0.90 Separation of 10 Rauwolfia 95
Pyridine, 7 0 : 1 5 : 1 5 alkaloids
19. n-Bu0H:glacial Whatman SG 4 1 (Silica 0.64 Separation of 10 Rauwolfia 95

HOAc:H20, 4 : l : l Gel) alkaloids
20. n-BuOH:glacial Kieselfel G (Silica 0.75 Separation of 10 Rauwolfia 95
HOAc:H20, 4 : l : l Gel) alkaloids
21. n-Bu0H:glacial Aluminum oxide G 0.97 Separation of 10 Rauwolfia 95
H0Ac:H 0, 4 : l : l alkaloids
2
P 22. 1sooctane:Et 0: Silica Gel G 0.00 Separation of Rauwolfia 9 6 , 97
0
Q\ xylene:EtOAc2, alkaloids for quantitative
45:40:15:5 analysis
23. Ethylene dichloride: Silica Gel G 0.58 Separation of Rauwolfia 9 6 , 97
EtOAc:n-BuOH, alkaloids for quantitative
6 0 : 3 0 : 10 analysis
24. Me C0:Pet. Ether: Silica Gel G 0.32 Separation of Rauwolfia 9 6 , 97
C C ~:isooctane, alkaloids for quantitative
3 5 : $0:20:15 ana1ysis
25. 1sooctane:Me C0:n- Silica Gel G 0.40 Separation of Rauwolfia 96
BuOH, 58:33.8: 8.4 alkaloids for quantitative
analysis
- Application and Comments Reference
26. Me C0:Pet. Ether: Silica Gel G 0.22 Separation of Rauwolfia 96, 97

2
glacial HOAc, 45:45:10 alkaloids for quantitative
analysis
27. Me C0:MeOH:glacial Silica Gel G 0.80 Separation of Rauwolfia 96, 97

H d c , 70:25:5 alkaloids for quantitative
analysis
28. Pet. Ether:Me CO: Silica Gel G 0.60 Separation of Rauwolfia 96

Et2NH, 70:2O:fO alkaloids for quantitative
analysis
0 29. Me2CO:MeOH:Et2NH, Silica Gel G 0.96 Separation of Rauwolfia 96

4 70:20: 10 alkaloids for quantitative
analysis
30. Me CO:CC14:Pet. Silica Gel G 0.43 Separation of Rauwolfia 97

Etzer , 45:45:30 alkaloids for quantitative
analysis
31. H O:EtOH:CHC13, Cellulose 1.00 Detection of cholinesterase 98

58:42:2 inhibitors at low levels
32. C6H6:EtOH, 9:l Aluminum oxide Quantitative analysis 100
33. n-Bu0H:Me-CO-Et : Silica Gel G 0.95 Separation of reserpine 34

H20, 65:25:25 from its degradation
products
Solvent System Sorbent -
Rf Application and Comments Reference
34. CHCl3:Me2C0, 70:30 Silica Gel G 0.35 Separation of reserpine 101

from other drug substances
P
Table 8
Visualization of Reserpine on Thin Layer Plates
No. Treatment Result Reference

1 Iodine Vapor 87
2 Dragendorff Reagent brown spot 88
3 Acetylchloride, then heat 89
4 1% Ceric Sulfate in 10% H2S04 greenish brown, turning brown 95

after 5 minutes at 105°C
P
0
\o
5 Frohde's Reagent (Sulphomolybdic yellow-brown 95
acid)
6 5% Ferric Chloride in 50% HN03 yellow-green turning green- 95

brown after 5 minutes at 105°C
7 Iodoplatinate pink 95
8 0.5% Phosphomolybdic acid in 50% yellow-green 96

m03
9 1% Ammonium Vanadute in 50% HN03 yellow-green 96
-
10 Spray with source of cholinesterase blue spots on yellow 98, 99

(human blood plasma suitable), background
followed by 1 part 0.6% bromthymol
blue in 0.1N NaOH and 15 parts
aqueous 1% acetylcholine chloride
11 500 mg p-dimethylaminobenzaldehyde greenish-black spot 101

in 50 ml conc. H SO
2 4
Table 9
Paper Chromatography Systems for Reserpine
No. Solvent Systems Immobile Phase Rf

- Application and C m e n t s Reference
1. C6H6 50% ethanolic formamide .96 Distinguish reserpine from 112, 116
synthetic precursors and
related compounds. De-
tection lhit, 1 mcg.
2. C6H6 50% ethanolic fonnami.de .75 Distinguish reserpine from 112

containing 5% ammonium synthetic precursors and
formate related compounds. De-
2 tection limit, 1 mcg.
c
3. C6H6:C6H12, 1:l 50% ethanolic formamide .61 Distinguish reserpine from 112
synthetic precursors and
related compounds. De-
tection limit, 1 mcg.
4. C6H6:C6H12, 1:l 50% ethanolic formamide .20 Distinguish reserpine from 112
containing 5% amnonium synthetic precursors and
5 * ‘gH12 50% ethanolic formarni.de .OO Distinguish reserpine from 112

containing 5% aunnonium synthetic precursors and
No. Solvent Systems
- Immobile Phase Rf
6. Shake isooctane: Me CO: formamide, .56 Identification of reserpine 113, 114

C H :Formamide, 106:30 and related compounds in
6 drug formulations
180:50:5 together,
discard lower layer,
add 2 parts C H
and filter. eatirate
chamber with N 3 vapor
7. C6H6:C6H12, 1:1 Formamide:MeOH, 70:30 Estimation of reserpine in 115

Rauwolfia root
8. C6H6:CeHa2, .i:l Propylene glycol: Quantitative analysis of 116

satura e wi h MeOH;HOAc, 50:50:1 raw materials
propylene glycol
9. Et-CO-Me:Me CO: Whatman No. 1 .86 117

HCOOH:H20 , $0:2:1:6
10. Et-CO-Me:Me2NH:H 0, Whatman No. 1 .95 117

2
921:2: 77
11. i-Bu-CO-Me:HCOOH: Whatman No. 1 .44 117

H20, 10 parts
ketone saturated
with 1 part 4% formic
acid
-
No. Solvent S y s t e m I m m o b i l e Phase -
Rf A p p l i c a t i o n and C o m m e n t s Reference
12. CHC13:MeOH:HCOOH: Whatman No. 1 .91 117

H 0 , 10 p a r t s CHC13
2
saturated w i t h a
m i x t u r e of 1 p a r t
MeOH and 1 p a r t
4% HCOOH
13. C H :Et-CO-Me:HCOOH: Whatman No. 1 .12 117

6 6
H 0 , 9 parts C H6
2
and 1 p a r t Et-6O-Me
saturated w i t h 1
p a r t 2% HCOOH
e
1J 14. C H :HCOOH:H20, 10 W h a t m a n No. 1 .05 117
pP&s c H satu-
rated w f t f : 1 p a r t
2% f o r m i c acid
15. A c e t i c A c i d : 5%
aqueous s o d i u m
acetate, 10: 90
a. Shake w i t h n- Whatman No. 5 4 2 .34 D e t e c t less than 1 m c g 118

BuOH, added i n paper
small portions,
u n t i l saturation
of t h e aqueous
phase i s j u s t achieved
-
No. Solvent System Immobile Phase Rf
b. Same as a except Whatman No. 542 .34 Detect less than 1 mcg 118
replace n-BuOH paper
with i-pantanol
16. n-Bu0H:C H6: equal Whatman No. 1 .90 Radio-assay for reserpine 48
parts 1.5~ NH OH in biological samples
and 1.5N (NH4f2C03,
80:5:15
Table 10
Visualization of Reserpine on Paper Chromatograms
-
1. ObsCmre under low or high pressure Strong green fluorescence 112

Hg lamp
2. Observe under low pressure Hg lamp Strong green 112
after spraying with .0025% solution
of fluorescein in 0.5M annnonia
3. Observe under W lamp after spraying Strong green 112

P
c with 3% solution of sodium nitro-
VI
prusside in 50% trichloroacetic acid
4. Spray with mixture containing 5 ml Brown spot, reaction weak 112

HC1, 95 ml ethanol, and 1 g p-
dimethylaminobenzyldehyde, heat
briefly at lOO"C, respray with nitro-
prusside solution (No. 3 above), and
heat again at 100°C for 3-5 minutes
5. Dragendorff Reagent (2% potassium 117

bismuth tetraiodide in 0.01N HC1)
ROGER E . SCHIRMER
System Description Reference
1, column: Solka-Floc and Celite 545, 1:l 128
Eluting Solvent: 5N acetic acid
Application: Quantitative determi-

nation - pharmaceutical preparations.
2. Column: A 200 x 22 m id column packed in 129-131

four layers is used. 1) bottom layer 1 g -
celite +0.5 ml of fresh 2% NaHC03, 2 ) 1 g
celite +0.5 ml of fresh 0.5% citric acid
solution, 3) 0.5 g celite +0.5 ml H 0, and 4)
2
1 m g equivalent of reserpine sample and 1 m l
dimethylsulfoxide and 2 g celite.
Eluting Solvent: Chloroform
Application: Quantitative determi-

nation of reserpine in formulations.
3. Column: 1) Bottom layer consisting of 113, 114

1.5 g celite 545 and 0.4 ml ethanol and 1 ml
2% NaHCO , and 2) top layer of 10 g celite
545 and 30 ml of a solution prepared by
dissolving 1.05 g citric acid in water, diluting
to 50 ml with water, and adding 20 ml ethanol.
Eluting Solvent: Shake together 100 ml

CHC13, 200 ml isooctane, 100 ml water and 40 ml
ethanol. Discard aqueous layer and filter.
Application: Quantitative determination of

reserpine in formulations.
4. Column: Celite 450 and fonnamide layer 132, 133

resulting from shaking together 15 ml heptane,
110 ml CHC13, 1 ml morpholine, and 25 ml
formamide.
Eluting Solvent: CHCl layer obtained

as described under "column."
416
RESERPINE
System Description Reference
Application: Separation and determi-

nation of reserpine, deserpidine, and
rescinnamine.
5. Colnmn: Acid activated aluminum oxide 134
Eluting Solvent: Methanol
Application: Separation and quanti-

tation of reserpine in Rauwolfia extracts.
6. Column: Dowex 50-X-2 cation exchanger 135
Eluting Solvent: Methanol/ammonia,

411
Application: Quantitation of reserpine
in formulations.
7. Column: Aluminum oxide equilibrated 136

with CHC13.
Eluting Solvent: Ethanol/CHC13 , 1/99

Application: Separation of reserpine
from weakly basic alkaloids in R. Serpentina
extract.
8. Column: Alumina 137
Eluting Solvent: Gradient elution

with benzene containing 0-40% ethanol.
Electrophoresis
7.4
Reserpine has been separated from rescinnamine,
reserpic acid and serpentine by electro49fy33s on paper
with 5N acetic acid as the electrolyte. Under the
conditions employed (8 volts/cm, 1.2 mA, 5 hours) res-
cinnamine was not completely separated from reserpine.
Electrophoretic separations of reserpine from other
Rauwolfia alkaloids were also reported in references 6, 121
and 136.
417
ROGER E. SCHIRMER
7.5 Countercurrent D i s t r i b u t i o n
The t h r e e solvent systems described i n Table 7
have been used t o s e p a r a t e r e s e r p i n e from o t h e r Rauwolfia
a l k a l y j g s by countercurrent d i s t r i b u t i o n . ‘ Kidd and
Scott a l s o reported several solvent systems, but found
t h e u s e of d i e t h y l e t h e r : chloroform ( 3 : l ) as t h e mobile
phase and a pH 3.1 b u f f e r (16.3 g c i t r i c a c i d and 16.1 g
Na HPO *12H 0 i n 1 l i t e r water) a s t h e s t a t i o n a r y phase
2
gave tke be& separation of t h e a l k a l o i d s of R. Serpentina
and R. Vomitoria. Other countercurrent systems may be
found i n references 140-142.
8. T i t r i m e t r i c Determination
Reserpine has been determined by t i t r a t i o n i n C H C l

3
s o l u t i o n using 0.1N H C l O i n dioxane as t i t r a n t and de-
4
t e c t i o n of t h e endpy&tl~&th methyl r e d , methyl yellow, o r
potentiometrically. ’ T i t r a t i o n s with anionic
surf ac t an its , y g h - as sodium l a u r y l s u l f a t e , have a l s o been
reported .
Reserpine has been determined t i t r i m e t r i c f&+y i n formu-
l a t i o n s a f t e r an appropriate e x t r a c t i o n . Sun extracted
with 0.3N c i t r i c a c i d , alkalyzing with NH OH, back
extracting i n t o CHCl drying, taking up i n t o .01N H SO4,
3’
and f i n a l l y back-tifEgting with 0.01N NaOH using met?iyl red
i n d i c a t o r . Sakurai e x t r a c t e d r e s e r p i n e with CHCl from
i n j e c t i o n s o l u t i o n s a l k a l i n i z e d with NH OH and then 3
4
t i t r a t e d with 0.002N p-toluenesulfonic a c i d dissolved i n
ethylene glycol: 2-propanol ( 1 : l ) .
Reserpine has a l s o been determined by p r e c i p i t a t i o n as
i t s tetraphenylborate s a l t , d i s s o l u t i o n of t h e p r e c i p i t a t e
i n acetone,lttd t i t r a t i o n with AgNO using silver
electrodes; j q ~ dby micro-Zeisel J e t e m i n a t i o n of i t s s i x
methoxy groups.
9. Electrochemical Analysis
I n a c i d i c media, a l k a l o i d s containing t h e 6-methoxy-

indole nucleus undergo a one e l e c t r o n oxidation which
probably involves i n s e r t i o n of a hydroxyl group i n t o t h e
aromatic portion of t h e molecule. I f t h e molecule contains
a v a i l a b l e n i t r o g e n with an unshared p a i r of e l e c t r o n s , a
two e l f S i r o n oxidation occurs with formation of a n N-
oxide. 15$Leserpine does not undergo polarographic re-
duct ion.
418
RESERPINE
Reserpine has been analyzed coulometrically by reaction

with chlorine generated electrochemically from HC1. l'Qe
electrochemical coefficient was 0.0007885 mgfma-sec.
10. Spectrophotometric Analysis
10.1 Infrared
The infrared absorption of reserpine in the 5.0-
6.5 micron region has beef5tsed for quantitation of this
compound in formulations. The band intensity was
measured following extraction of the reserpine into chloro-
form.
10.2 Ultraviolet
The ultravio€p6,f~f"~~fP~20f85eserpinecan be
used for quantitation, but the possi-
bility of interference from related alkaloids, excipients,
and degradation products requires that the reserpine be
isolated from these other substances prior to measurement.
The separation from roots and crude pfsga$g5ions has been
accomplished by extensive extraction ; from formy16
lations by paper chromatography (System No. 8, Table 9) ;
and from crude extracts, raw materials, and formulations by
electrophoref$3,~3~paper using 5N acetic acid as the
electrolyte. The absorbancels{ the final sample is
generally measured at 268 nm. Page has reported a semi-
automated procedure for reserpine in tablets using a
Technicon Auto-Analyze@ system with both ultraviolet and
colorimetric detection.
Ultraviolet spectrophotometry has also been
evaluated for the deteytgation of foreign alkaloids in
reserpine preparations.
10.3 Colorimetric
The most commonly used colorimetric procedures
for reserpine involve oxidation of the compound to 3,4-
didehydroreserpine with nitrite and measurement of the
absorbance of the oxidation product at about 390 nm. The
reaction is carried out in solutions containing 5-15 pgfml
reserpine in efkbef6.ethanol
- or ethanol acidified with
sulfuric aci This procedure has been appli
f8??$bt ,ions
96,160,165
, cry& Rauwolfia preparationsf2lF0
and animal feeds. An automated version of this
assay for determining reserpine in tablets has been
419
ROGER E . SCHIRMER
183
reported by Page.
An a l t e r n a t e procedure employs oxidation of
-
reserpine with n i t r i t e inlgrjef& acid followed by ex-
t r a c t i o n i n t o chloroform. The absorbance of t h e
chloroform l a y e r i s measured a t 465 nm. This procedure is
s u i t a b l e f o r 50-300 pg of r e s e r p i n e , and has t h e advant
t h a t hydrolysis products of r e s e r p i n e do not i n t e r f e r e . 887
A v a r i a t i o n of t h i s procedure i n which t h e sample i s
t r e a t e d with amyl n i t r i t e r a t h e r than with aceySfj a c i d -
sodium n i t r i t e reagent has a l s o been reported.
Reserpine has a l s o been analyzed c o l o r i r n e t r i c a l l y
by reactiof7yiflj2vanillin (absorbafyg of 0.1 a t 532 nm w i t h
17 pg/ml) , ’ amifqgy~+yidine,
plggylisocyanate, iodine,977
xanthydrpS6 (measur
50-500 pg a t 500 nm),
and sodium glyoxalate FeC13. -
10.4 Ion-Pair Extraction with Spectrophotometric
Detecy {p
Booth reported a procedure f o r t h e a n a l y s i s of
r e s e r p i n e i n formulations by e x t r a c t i n g t h e r e s e r p i n e i n t o
chloroform from pH 4.0 phosphate b u f f e r as an ion-pair w i t h
bromcresol purple. A f i n a l s o l u t i o n concentration of
2.8 pg/ml gave an absorbance of 0.188 a t 402 nm. Pro-
181
cedures employing bromcresol green and methyl orange
i n place of bromcresol purple have a l s o been reported.
10.5 Fluorescence
Since 3,4-didehydroreserpine i s s t r o n g l y f l u o -
rescent (Section 2.2.3), t h e s e n s i t i v i t y of t h e c o l o r i -
metric methods employing oxidation of r e s e r p i n e t o t h i s
product (Section 10.3) can be increased by using f l u o r i -
metric detection. N i t r i t e oxidation and f l u o r i m e t r i
determination have been used f o r a n a l y s i s of t a b
f 84
h?f-9188
f o r feeds containing r e s e r p i n e a t t h e ppm level.
automated s i n g l e - t a P & p l g a s a y based on t h i s procedure has
a l s o been reported.
Reagents o t h e r than n i t r i t e have been us
@i8f?90-
fsyelop f luorescencf93fp$tuding hydrogen peroxide,
sg+zpious a c i d , pgpiygnesulfonic acid i n a c e t i c
a c i d , 2 0 0 ~ y ~ ~ v a n a adcii cd , and vanadium pent-
oxide. The vanadium pentoxide p r o c ~ $ ~ ~ ~ Obeen ljas
automated f o r use i n s i n g l e t a b l e t a s s a y s , The
selenious acid procedure has beey9ysyd4to determine
r e s e r p i n e i n b i o l o g i c a l samples. ’
420
RESERPINE
Kollistrat~h s a~s ~r e~p o r t e d t h a t ThCl g r e a t l y

enhances t h e f l u o r e s c e n c e of r e s e r p i n e i n e t h a n o t ,
methanol, a c e t o n e , and dioxane, b u t d i d not determine t h e
mechanism r e s p o n s i b l e f o r t h e enhancement.
11. Analysis of Reserpine i n B i o l o g i c a l Systems

Reserpine levels hayGgbeen deteyg&ned f l u o r h e t r i c a l l y
i n t i s s u e samples. Poet and Hess a d j u s t e d t h e pH of
samples t o 8.5 w i t h b o r a t e b u f f e r , e x t r a c t e d w i t h heptane
o r petroleum e t h e r , back-extracted i n t o d i l u t e s u l f u r i c a c i d
and f i n a l l y developed f l u o r e s s 5 n c e u s i n g t h g 4 s e l e n i o u s a c i d
o x i d a t i o n procedure, Glaszko and Maronde adjusted t h e
pH o f plasma and u r i n e samples t o 3.7-4.0 w i t h c i t r a t e
b u f f e r , e x t r a c t e d w i t h e t h y l e n e d i c h l o r i d e , and developed
f l u o r e s c e n c e w i t h odium n i t r o p r u s s i d e followed by hydrogen
peroxide. Zsoter” analyzed p l f A a and u r i n e samples u s i n g
a m o d i f i c a t i o n of J a k o v l j e v i c ‘ s f l u o r i m e t r i c procedure.
The most widely used methods f o r detygminins
r e s e r p i n e i n t i s s u e s a r e t h o s e employing C o r H labeled
r e s e r p i n e , S p e c i f i c i t y i s obtained he r a d i o a c t i v e
d e t ermina 53’65 t h i n l a y e r chroma-
%a!:i!
togre”Xg’54965’66
PhY *
, ~ i r , ~ t j t g s , g 9 5 t ;o~r ~paper
~ ~ ~ chromatogra-
~2
Other procedures may b e found i n t h e
r e f e r e n c e s c i t e d i n S e c t i o n 4.
The b i o a v a i l a b i l i t y of r e s e r p i n e a d m i n i s t e r e d o r a l l y
as a c o p r e c i p i t a t e w i t h p o l y v i n y l p y r r o l i d o n e o r w i t h b i l e
a c i d s has been determined using t h e b l e p h a r o p t o t
of t h e p r e p a r a t i o n a s a measure of a v a i l a b i l i t y .
3655% P i t y
212
P t o s i s w a s r a t e d using t h e s c a l e devised by Rubin g . &.
These s t u d i e s included examination of t h e d i s s o l u t i o n rates
of t h e c o p r e c i p i t a t e s and t h e r e l a t i o n s h i p between d i s s o -
l u t i o n rate and b l e p h a r o p t o t i c a c t i v i t y .
42 1
ROGER E. SCHIRMER
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430
SPIRONOLACTONE
John L . Sutter and Edward P. K. Lau

JOHN L. SUTTER AND EDWARD P. K . LAU
Contents
1. Description
2.1 Infrared Spectm
2.2 Nuclear Magnetic Resonance Spectm
2 . 3 Ultraviolet Spectm
2.4 Mass Spectm
2.6 Melting Range
2 . 7 Differential Scanning Calorimetry
2.8 Solubility
3. Synthesis
6.1 Partition Coefficient
6.61 High Pressure Liquid Chromatography
7. Acknow1edgements
8. References
432
SPI RONOLACTONE
1. Description
Spironolactone is 17-hydroxy-7a-mercapto-3-oxo-
17a-prep-4-ene- 21-carboxylic acid y- lactone 7-
acetate,
C24H3204S Molecular Weight: 416.59

Spironolactone is a yellowish-white crystalline
powder, with a faint mercaptan odor.
2. Phvsical Prouerties
The infrared absorption spectrum of a spirono-
lactone reference standard compressed in a KBr
disc is shown in Figure 1. Essentially the same
infrared spectrum is observed in chloroform solu-
tion. The following assignments have been made
for absorption bands in Figure 1:
Wavenumber, an- Assignment
1775 5-membered lactone carbonyl
1670 - 1690 3-ketone, 7-thioester car-
bony1
1620 4,5 - double bond
433
P
W
A
Figl. I n f r a r e d Spectrum of S p i r o n o l a c t o n e
SPIRONOLACTONE
2.2 Nuclear Mametic Resonance Smctrwn

The NMR spectrum of a spironolactone reference
standard i n deuterated chloroform is shown i n
Figure 2. Spectral assignments are as follows:
Chemical Shift
(PPm) Type Assignment
5.7 Broad quartet 4 - H
4.0 Broad s i n g l e t 7 - H
2.4 Singlet 7-thioacetate pro-
tons
1.2 Singlet 19-methyl protons
1.0 Singlet 18-methyl protons
The u l t r a v i o l e t absorption spectrum of a spirono

lactone reference standard i n methanol is shown
i n Figure 3. The molar absorptivity of spirono-
lactone i n methanol i s 19.6 X l o 3 a t the
absorbance maximum a t about 238 nm.
2.4 Mass S D e c t m
The low resolution m a s s spectrum of spironolac-
tone shown i n Figure 4 was obtained with an AEI
Model Ms-30 mass spectrometer. A molecular ion
was observed a t m/e 416. The base peak i n the
spectrum was a t m/e 341, corresponding t o loss
of .SCOCH3. Structure assignments are as
follows :
m/e Assignment % Relative Intensity
43 CH3CO+ 89
26 7 340 - Lactone 26
ring
325 340 - methyl 12
340 W - HSCOCH3 43
341 M? - .SCOCH3 100
359 IvP - (CH2CO+CH3) 20
435
P
w
Q\
Fig. 2 N u c l e a r Magnetic Resonance Spectrum o f S p i r o n o l a c t o n e

Fig. 3 Ultraviolet Spectrum of Spironolactone
Fig. 4 Mass Spectrum of Spironolactone
SPI RONOLACTONE
374 M-f - (CH co) 36

383 M-f - (H26 + CH3) 1
416 M-f 0.4
The following s p e c i f i c rotation values i n

chloroform' have been reported f o r spironolactone :
[a]& = -403.8'
CCt1;g6 = -110.00
c.15';~ = - 45.8O
[~ti;;~
= - 38.8'
C ~L Ji . -1 ~ -~ 36.3'
~
2.6 Melting Range
The USP l a melting range of spironolactone is

1980 - 2070C.* A reference standard l o t of the
compound was found t o melt from 205.40 t o 206.90C.
Melting and r e s o l i d i f i c a t i o n a t lower temperatures
is sometimes noted.
The DSC thermogram of a spironolactone reference

standard c r y s t a l l i z e d from ethanol and water
shown i n Figure 5 was obtained on a Perkin-Elmer
DSC-1B d i f f e r e n t i a l scanning calorimeter a t a
heating rate of 2OoC/minute i n an atmosphere of
nitrogen. A s l i g h t exothermic peak was observed
beginning a t about 160OC, and an endothermic
melting peak was found a t about 210°C.3
Spironolactone chemical c r y s t a l l i z e d from
d i f f e r e n t solvents can y i e l d d i f f e r e n t thermo-
grams. Samples c r y s t a l l i z e d from methanol have
439
I
P
P
0
I I 1 1 I I I
140 150 160 170 180 190 200 210 220
TETPERATURE, O C
Fig. 5 DSC Thermogram of Spironolactone
SPI RONOLACTONE
been shown to give thennograms with two trans-

ition peaks: an endotherm at about 120°C and an
exothenn at about 14OOC. These transition geaks
can be removed by heating the sample at 105 C or
refluxing in water for four hours. A sample
crystallized from n-propanol, however, gave only
one endothermic peak at 2100C. Thermograms of
spironolactone crystallized from ethyl acetate
have shown two transition peaks: one endotherm
at about llOOC and an another at about 207OC.
This behavior suggests that spironolactone can
exist in a number of polymorphic f o m .
2.8 Solubility
Solubilities of spironolactone in various sol-
vents at 25OC are given in the following table:4
Solvent Solubility, mg./ml.
Water 2 . 8 x 10-2
Methanol 6.9
Ethanol (USP) 27.9
Chloroform 50
Heptane 2.4 x 10-1
3. Synthesis
The synthetic route to spironolactone shown in Figure 6
has been reported in papers by Cella, Brown and
Burtner', and Cella and Tweit.
Carbonation of the Grignard reagent of l7a-ethynyl-5-
androstene-38,178-diol (I) yielded an acetylenic acid
(11). Selective reduction of the acetylenic bond was
accomplished by catalytic hydrogenation over palladium
on calcium carbonate, using dioxane and pyridine as
solvents. Treatment of the product with mineral acid
yielded the unsaturated lactone, 3- (38,178-dihydroxy-
- propenoic acid lactone (111) ,
5-androstene-17a-yl)
441
JOHN L . SUTTER AND EDWARD P. K. LAU
Figure 6. Spironolactone Synthesis
442
SPI RONOLACTONE
which was easily reduced to the saturated lactone (IV)

by hydrogen over palladium on charcoal. oppenauer
oxidation of the product afforded 3-(3-0x0-178-
hydroxy-4-androsten-17a-yl) propionic acid lactone
(V). maturation at c6 was then introduced by
treatment with chloranil. Finally, the resulting
17-hydroxy-3-0x0-17a-pregna-4,6-diene-21-carboxylic
acid-y-lactone (VI) w a s reacted with thioacetic acid,
yielding 17-hydroxy-7a-mercapto-3-0x0-17a-prep-4-
ene-21-carboxylicacid y-lactone 7-acetate, spirono-
lactone (VII).
Spironolactone has been found to decompose to the
dienone, canrenone. The reaction is not a facile one
in either the pure chemical or its dosage forms,
since canrenone forms only to the extent of 1% or
less over a period of five years at 400C.'
5. Drug Metabolic Products and Phannacokinetics
The structures of spironolactone metabolites in man
identified so far are summarized in Figure 7.
Gochman and Gantt, using the fluorimetric method de-
scribed in Section 6.5, demonstrated that spirono-
lactone MI)is readily converted to the dethio-
acetylated metabolite, canrenone (VI) , in humans. '
Subsequent studies by Zicha et a19 and by Wagner
"
et all showed the presence of at least six meta-
bolites in the urine of humans after ingestion of
spironolactone. Tentative structures for the un-
known metabolites were proposed by these investi-
gators;9, "however, rigorous identification of the
metabo l ites was not attempted.
Karim and Brown established that elimination of the
quantitative.
-
thioacetate group of spironolactone in vivo is not
Besides canrenone, nese mvestiga-
tors identified a major sulfur-containingmetabolite,
3-(3-0x0-7a-methylsulfinyl-6~,17~-dihydroxy-4-andros-
ten-17a-yl) propionic acid y-lactone (Metabolite C,
(VIII), Figure 7), in the urine of humans. Tentative
443
Figure 7. Spironolactone Metabolism
444
SPI RONOLACTONE
structures were also proposed for three other minor

sulfur-containing metabolites, (IX) , (X) , and (XI).
Under the gas chromatographic conditions reported by
Chamberlain for the determination of canrenone,
it was found that Metabolite C (VIII) was converted
to A4-3,6-dioxo steroid (XII). Further, under these
conditions, spimnolactone (VII) as well as labile
metabolites (IX), (X), and (XI), were converted to
canrenone (VI).
Further unpublished observations have shown that
metabolite C (VIII) had the same retention time as
an unknown urinary polar metabolite observed by
Chamberlain.
The pharmacokinetics of spironolactone and canreno?:
were investigated by Sadee, Dagcioglu and Schroder .
Determination of plasma levels following equivalent
oral administration of spironolactone or canrenone
led to the conclusion that 79% of spironolactone is
dethioacetylated to canrenone. The terminal log-
linear phase half-life of canrenone in plasma follow-
ing oral administration of spironolactone ranged from
17 to 22 hours. Fluorescence assay revealed that
from 14 to 24% of oral doses of spironolactone were
excreted in the urine as fluorogenic metabolites
within five days.
6.1 Partition Coefficient
Spironolactone is preferentially extracted into
heptane from water. The partition coefficient
is 3.5 at 25%.
Phase solubility analysis of spironolactone can
be carried out by equilibration in methanol at
25%. Figure 8 shows the phase solubility dia-
gram of a spironolactone reference standard.
445
JOHN L.SUTTER AND EDWARD P. K. LAU
Sample: Spironolactone
Solvent: Methanol
Equilibration: 24 h r . @ 25OC
Extrapolated Solubility: 8.7 mg./g.
_ - -
I I I I 1 I I I I
SYSTEM CCBPOSITION (mg./g. solvent)
Figure 8. Phase Solubility Diagram of Spironolactone
446
SPI RONOLACTONE

The u l t r a v i o l e t absorption spectrum of spirono-
lactone is the basis f o r t h e USP XVIII assay f o r
the compound. The absorbance maximum a t abo?
238 nm. in methanol i s used f o r quantitation.
Reaction of spironolactone with methanolic
hydroxylamine hydrochloride and ferric perchlo-
r a t e yields a red ferric hydroxamate complex
having an absorbance maximum a t about 515 nm.'
The absorbance a t this wavelength is linear
with concentration over a range of 5 mg./ml. t o
29 mg./ml. The colored complex is s t a h l e f o r
up t o 2 hours. The method is used f o r analysis
of t h e compound and a variety of i t s dosage
forms. Canrenone, the principal degradation
product of spironolactone, does not interfere.
Isonicotinic acid hydrazide may be reacted with
spironolactone i n methanolic solution, yielding
a soluble yellow product whose absorbance rises
t o a plateau beginning a t about 375 m. "The
molar absorptivity i s 2.2 X 103 a t about 380 nm.
The method has not yet been adopted f o r quanti-
t a t ive analysis.
6.5 Fluorometric Analvsis

Spironolactone may be dethioacetylated under mild
acid o r alkaline conditions, yielding the cor-
responding 4,6 -dienone, '' canrenone, which,
i n 62% s u l f u r i c acid, i s converted t o a fluor-
escent trienone. This compound has an excit-
a t i o n maximum a t 483 nm. and an emission maxi-
mum a t 525 m. The fluorescence is useful f o r
quantitation of canrenone in plasma over the
range 40-160 ng./ml.' The procedure may be used
t o determine spironolactone in the presence of
i t s major dethioacetylated metabolites, by mea-
suring fluorescence in 62%H2SOq,$oth with and
without p r i o r dethioacetylation.
441
JOHN L. SUTTER A N D E D W A R D P. K . L A U

Spironolactone may be separated from
canrenone on an octadecyl silane column
using methanol - water mobile phase, and
detected with the aid of a continuously
variable wavelength W detector. Spirono-
lactone, which is eluted first, may be
detected at about 238 nm. and canrenone
may be observed at \$s absorption maxi-
nnun at about 283 nm.
Thin-layer chromatographic systems and
corresponding R values for spironolactone
are summarized in the following table:
Solvent System Adsorbent Detection -
R
Ethyl acetate, Silica Gel 1,293 0.53
100% GF (Woelm)
4
Benzene: Ethyl Silica Gel G 4 0.67
acetate: Methanol
73: 25: 2
Detection:
1. Observe under short wave W.
2. Spray with 50% H SO , heat at 80 C for
10 minutes, observe under long wave W.
3. spray with Phosphamolybdic Acid.
4. Spray with Phosphamolybdic Acid, heat
at 80 C. for 10 minutes.
448
SPI RONOLACTONE
7. Acknowledgements
The authors wish t o express t h e i r appreciation t o
Mr. E. Brown, Dr. H. Dryden and Mr. R. Tweit f o r
t h e i r helpful discussions on synthesis; t o Dr. R.
Bible, Mr. A. Damascus, Dr. J. Hribar and Miss
Lydia Swenton for t h e i r assistance i n the preparation
of the sections on I R , NMR and Mass Spectra; t o Dr.
A. Karin f o r sharing with them his knowledge of
spironolactone metabolism and phaxmacokinetics ; and
t o Mrs. Lorraine Wearley f o r searching the l i t e r a t u r e
and assembling various sections of this manuscript.
The expert s e c r e t a r i a l assistance of Mrs. Corey Leone
is also gratefully acknowledged.
449
JOHN L. SUTTER AND EDWARD P. K. L A U
8. References
1. Anthony, G., Searle Laboratories, personal
c o m i c a tion.
2. "The United States Pharmacopeia" XVIII,
p. 681.
3. Marshall, S . , Searle Laboratories, personal

comunication.
4. Aranda, E., Searle Laboratories, personal
comication.
5. Cella, J. A., Brown, E. A. and Burtner, R. R.,
J. Org. Chem. -
24, 1109 (1959).
6. Cells, J. A. and Weit, R. C., J. Org. Chem.
24, 1109 (1959).
-
7. Baier, M. E., Searle Laboratories, personal
comication.
8. Gochman, N. and Gantt, C.L., J. Pharmacol.
-
Exp. Ther. 135, 312 (1962).
9. Zicha, L., Weist, F., Scheiffarth, F. and

Schmid, E., Arzneimittel-Forsch. -
14, 699
(1964).
10. Wagner, I]., Weist, F. and Zicha, L., Arzneimittel-
Forsch. -
17, 415 (1967).
11. Karim, A. and Brown, E. A., Steroids 20, 41 -
(1972).
12. Chamberlain, J., J. Chromatog. 55, 249 (1971).
L
13. Karim, A,, Searle Laboratories, personal

commication.
450
SPI RONOLACTONE
14. Sadee, W., Dagcioglu, M. and Schroder, R.,

-
J. Pharmacol. Exp. "her. 185, 686 (1973).
15. Seul, J., Searle Laboratories, personal

connnunication.
16. Wearley, L. , Searle Laboratories, personal

connnunication.
17 Sadee, W., Dagcioglu, M., and Riegelman, S.,
J. Phan. Sci. -
61, 1126 (1972).
18. Sadee, W., Riegelman, S. and Johnson, L. F.,
Steroids -1 7 , 595 (1971).
19. Runser, D. J. and Fortier, C., Searle Lab-

oratories, personal c o m i c a t i o n .
45 1
TESTOSTERONE ENANTHATE
Klaus Florey
CONTENTS
1. Description
2.4 Mass Spectrum
2.5 Rotation
2.6 Melting Range
2.8 Solubility
3. Synthesis
6.41 Paper
6.42 Thin-Layer
6.43 Column
7. References
453
KLAUS FLOREY
1. Description
1.1 Name, F o r m u l a , M o l e c u l a r Weiqht
Testosterone enanthate (heptanoate) is
17-~~(1-oxoheptyl)oxy~-4-androsten-3-one.
OCO ( C H 2 ) 5CH3
I
c2 6H4Oo 3 Mol. W t . 400.61
1.2 A p p e a r a n c e , C o l o r , Odor
White, c r y s t a l l i n e , o d o r l e s s powder,
2 . 1 I n f r a r e d Spectrum
T h e i n f r a r e d s p e c t r u m (KBr p e l l e t of
t e s t o s t e r o n e e n a n t h a t e i s p r e s e n t e d i n F i g u r e 11
.
2.2 Nu clear M a g n e t i c Resonance S p e c t r u m
The NMR s p e c t r u m i s p r e s e n t e d i n
Figure 2. I t w a s o b t a i n e d on a 60 MHz s p e c t r o -
meter i n deuterochloroform c o n t a i n i n g t e t r a -
m e t h y l s i l a n e a s an i n t e r n a l r e f e r e n c e . The
f o l l o w i n g p r o t o n a s s i g n m e n t s were made2 :
Protons a t Chemical S h i f t Coupling
Constants
T ( i n H2)
C-4H 4.20 s i n g l e t -
C-17aH 5.38 t r i p l e t 16H, 17H = 8 . 5
C-18H 9.17 s i n g l e t -
C-19H 8.82 s i n g l e t -
ester
methyl 9.13 m u l t i p l e t -
454
Figure 1. Infrared Spectrum of Testosterone Enanthate(KBr Pellet).
Instrument: Perkin-Elmer 621.
F i g u r e 2. N M R S p e c t r u m of T e s t e r o n e E n a n t h a t e in D e u t e r a t e d C h l o r o f o r m .
I n s t r u m e n t : P e r k i n - E l m e r R12B.
21378 5139815 782L15
MASS/CHARGE
Figure 3. Low R e s o l u t i o n Mass S p e c t r u m of T e s t o s t e r o n e E n a n t h a t e .
I n s t r u m e n t : AEI-902.
KLAUS FLOREY

Squibb House Standard (Lot 78245) gave
a single band, due to the 3-keto-4 ene system3:
A max 240 nm E E m 42g3

A max 240 nm El%
1cm
4107
2.4 Mass Spectrum

The low-resolution mass spectrum,shown
in Figure 3, demonstrates the expected M+ of
m/e 400. There is progressive loss of carbons
from the alkyl portion of the acylate which gives
rise to the ions at m/e 315, 316 from the hydro-
carbon portion of the chain and the m/e 288 ion '
with the additional l o s s of the acylate carbonyl.

The loss of the entire C-17 group yields the
m/e 270 and 271 ions. The ions at m/e 85 and
113 represent the hydrocarbons, CgH13, and
C7H130, respectively. Fragment ions occur through
a progressive l o s s of D-ring, C-ring and B-ring
carbons. The m/e 147 and 124 ions are
diagnostic for A4-3-ketones. The assignment of
some of the diagnostic ions is depicted below2 .
(-1H)288
458
2.5 Rotation
The following rotation were reported:
+78O (dioxan)4
+78.0° (Squibb House Standard Lot
78245)
7
+7 go
+7 5-7608
2.6 Melting Ranqe

The following melting range temperature
temperatures (OC) were reported:
36-37. !j4
3
37-38 (Squibb House Standard Lot
78245)
35 (from alcohol-ether)
366
36-377
36-378

Endotherm at 37OC.
2.8 Solubility
Insoluble in water; very soluble in
ether, soluble in vegetable o i l s 2 3 .

The powder X-ray diffraction pattern is
presented in Table 19. It is not too well
resolved, since due to the low melting point of
testosterone enanthate, the sample partially
liquified during analysis.
459
KLAUS FLOREY
Table I
Powder X-ray Diffraction
Pattern of Testosterone Enanthate’
14.5 0.39 4.80 1.00

9.20 0.15 4.69 0.25
8.00 0.12 4.60 0.30
7.30 0.15 4.49 0.27
6.40 0.25 4.20 0.45
6.30 0.25 4.11 0.27
6.18 0.25 4.03 0.27
6.00 0.25 3.72 0.22
5.80 0.30 3.52 0.20
5.46 0.28
5.27 0.33
5.10 0.77
*d = interplanar distance n k
2 sine
A= 1.539 0
A;
Radiation:Kal and Ka2 Copper
**
Relative intensity based on highest intensity
of 1.00
460
3. Synthesis
T e s t o s t e r o n e e n a n t h a t e i s p r e p a r e d by a c y l a t i o n o f t e s t o s t e r o n e w i t h
enanthic anhydride i n pyridine4.
E n a n t h i c a c i d and an i o n exchanger8 and e n a n t h i c a c i d c h l o r i d e 6 j 7

have a l s o been used i n s t e a d o f e n a n t h i c a n h y d r i d e . I t can a l s o be
p r e p a r e d by t r a n s e s t e r i f i c a t i o n o f t e s t o s t e r o n e p r o p i o n a t e and methyl
enan t h a te5.
4. S t a b i l i t y , Degradation
T e s t o s t e r o n e e n a n t h a t e i s s t a b l e as a s o l i d . I n solution photolytic
d e g r a d a t i o n of t h e A-ring i s p o s s i b l e when exposed t o u l t r a v i o l e t l i g h t
o r o r d i n a r y f l u o r e s c e n t l a b o r a t o r l i g h t n i n g ( c f . 1 6 ) . The k i n e t i c s o f
s a p o n i f i c a t i o n have been s t u d i e d ' ' 9 18. Mucor f u n g i p o s s e s s e s t e r a s e s
a b l e t o s a p o n i f y t e s t o s t e r o n e e n a n t h a t e among o t h e r s t e r o i d e s t e r s l g .
KLAUS FLOREY

Generally, the metabolic fate of testosterone
enan thate is deacylation to testosterone .
It
then follows testosterone metabolism, mostly to
17-keto steroids such as 4-androsten-3,20-dione
and estrogenic substances. Data have been
reported for human urine after intramuscular
injectionlO,ll, rats12, their prostates and
livers13. The fate in maternal peripheral blood
and cord venous blood has also been studiesl4.
Tissue distribution and excretion of labeled
testosterone enanthate was studied in steers15.
C H 0
Calc.% 77.95 10.07 11.98

The U . V . maximum at 240 nm can be used
for quantitation determination in pharmaceutical
preparations20,21,22,

Reaction with isoniazid and determina-
tion of the resulting hydrazone at 380 nm is the
basis of the compendia1 assay23.

6.41 Paper
The quantitative determination and
separation from testosterone by paper chromato-
graphy in oily vehicles has been described by
Roberts and Florey26. After spotting, the paper
strips were impregnated with 30% diethylene
glycol monoethyl ether (Carbitol) in chloroform
and developed for 2-1/2 hours with methyl
cyclohexane saturated with diethylene glycol
462
monoethyl e t h e r . After locating of the s t e r o i d spots with a fluorescent
p a d d l e , t h e s p o t s a r e e l u t e d and r e a c t e d w i t h i s o n i c o t i n i c a c i d h y d r a z i d e
i n methanol c o n t a i n i n g a l s o h y d r o c h l o r i c a c i d . The a b s o r b a n c e of t h e
y e l l o w h y d r a z o n e i s r e a d a t 415 nm.
A s i m i l a r procedure, using impregnation with propylene glycol, phenylglycol
e t h y l e t h e r and m e t h a n o l ( 1 : 1 : 2 ) and p r o p y l e n e g l y c o l , p h e n y l g l y c o l e t h y l
e t h e r and h e p t a n e (1:1:200) a s d e v e l o p i n g s o l v e n t and 2 , 4 - d i n i t r o p h e n y l -
hydrazone a s s p r a y r e a g e n t h a s a l s o been r e p o r t e d 2 7 .
6 . 4 2 Thin-Layer
T h i n - l a y e r chromatographic systems have been compiled i n
T a b l e 11.
T a b l e I1
%
w
Absorbent S o l v e n t System Rf. Ref.
Silica gel G 50% A c e t i c A c i d 31 24
impreg. w i t h m i n e r a l o i l 50% E t h a n o l 26 24
Silica gel Benzene-Acetone ( 4 :1) -- 25
Silica gel Benzene-Methanol(9: 1) -- 25
Silica gel P e t - e t h e r , Benzene , A c e t i c
A c i d , W a t e r (67:33:85: 35)
Alumina Benzene-Acetone (4: 1)
Silica gel Methanol-Water (9: 1)
impreg.with corn o i l
D e t e c t i o n systems: U.V. light, sulfuric acid - alcohol.

KLAUS FLOREY
6.43 Column
Column c h r o m a t o g r a p h y o n s i l a n i z e d
c h r o m a t o g r a p h i c s i l i c e o u s e a r t h u s i n g 95%
e t h a n o l - h e p t a n e s o l v e n t system f o l l o w e d b y t h e
i s o n i a z i d r e a c t i o n i s t h e b a s i s o f t h e compendia1
a s s a y of t e s t o s t e r o n e e n a n t h a t e i n o i l
.
p r e p a r a t i o n s 23
7. References
Communication.
Communication.
3. G. A. B r e w e r , J r . , The S q u i b b I n s t i t u t e ,
4. K. Junkmann, J. K a t h o l , H. R i c h t e r , U. S.
P a t e n t 2 , 8 4 0 , 5 0 8 (1958) : K. Junkmann, Arch.
Expt. P a t h o l . Pharmacol. 2 1 5 , 8 5 ( 1 9 5 2 ) .
5. S. A . A l t e r , S p a n i s h P a t e n t 241,206 ( 1 9 5 8 ) ;
C.A. 54,3532a ( 1 9 6 0 ) .
6. T. Kasugai, J a p a n . P a t e n t 1 4 , 0 8 9 ( 1 9 6 2 ) :
C.A. 59,10182a ( 1 9 6 3 ) .
7. H. C i e s l i k , H. Koprowska, A. Walczak,
Pol. P a t e n t 56,358(1968)C.A.71,39286~(1969).
8. M. U l r i c h , A. Novacek a n d F. S t e j s k a l ,
Czech. P a t e n t 95,825(1960)C.A. 55,15551d
.( 1 9 6 1 ) .
9. Q. Ochs, The S q u i b b I n s t i t u t e , P e r s o n a l
Communication.
10. J. A . S c h n e i d e r and A. S c h u c h t e r , A r z t l .
Wochschr. 9 , 3 9 2 ( 1 9 5 4 )
11. G. D o e r n e r , F. S t a h l and R. Z a b e l ,
Endokrinologie 45,121(1963).
12. M. Wenzel, L . P i t z e 1 a n d P. E. S c h u l z e ,
Angew.Chem. I n t . E d . E n g 1 . 1 , 2 1 1 ( 1 9 6 8 ) .
13. J. S h i m a z a k i , H. K u r i h a r a , Y. I t o , K.Shida
a n d K. Kirao, Gunma J. Med. S c i . 1 4 , 3 1 3
464
(1965) : C . A . 66,263296 (1967) i b i d .

14,326 (1965) ; F A . 66,622631 ( 1 9 6 7 ) .
14. H. H. Simmer, M. V r F r a n k l a n d and
M. G r e i p e l , S t e r o i d s 2 , 2 2 9 ( 1 9 7 2 ) .
15. F . X . G a s s n e r , R. P. M a r t i n , W. Shimoda and
J. W. Algeo, F e r t i l i t y and S t e r i l i t y
-
11,49(1960).
16. D.R. B a r t o n and W. C. T a y l o r , J.Am.Chem.
SOC. 8 0 , 2 4 4 (1958) : J. Chem. SOC.1958,2500.
17. M. Schenk and K. Junkmann, Arch.Exper.
P a t h . and Pharmacol, 227,210 ( 1 9 5 5 ) .
18. 2. V e s e l y , J. P o s p i c e k and J . T r o j a n e k ,
C o l l e c t . Czech. Chem. Commun. 3 4 , 6 8 5 ( 1 9 6 9 ) .
19. K.A. Kohcheenko, G. K. S k r y a b i n ,
B. K. E r o s h i n , L. M. Kogan and J.V.Torgor,
P r i k l . Biokhim. i . M i c r o b i o 1 . L, 181 (1965) :
C.A.63,9018~(1965).
20. N. H. Coy, The S q u i b b I n s t i t u t e , P e r s o n a l
Communication.
21. A . R e g i n a t o , A n a l e s f a c . quim. y. f a r m . ,
Univ. C h i l e lO,48(1958)C.A.54,6028f ( 1 9 6 0 ) .
22. S. Kanna, M. Takuma and S. Watanabe,
Yakuzaigaku 25,145(1965)C.A. 64,15676d
(1966).
23. U. S. P. 1 8 t h e d i t i o n .
24. D. S o n a n i n i and L. Anker, Pharm. Acta
Helv. 4 2 , 5 4 ( 1 9 6 7 ) .
25. S. Hara and K. Mibe, Chem. Pharm. B u l l .
-
15,1036 ( 1 9 6 7 ) .
26. H. R. R o b e r t s and K. F l o r e y , J . P h a r m . S c i .
-
51,794 ( 1 9 6 2 ) .
27. A. N i l u f e r , Turk. I j i y e n T e c r u b i . B i y o l .
-
D e r g i s i 2 l , l 4 9 ( 1 9 6 1 ) C . A . 59,6200f ( 1 9 6 3 ) .
L i t e r a t u r e s u r v e y e d t h r o u g h December 1972.
The h e l p of H. Gonda and A. Mohr i n t h e p r e p a r a -

t i o n of t h i s p r o f i l e i s g r a t e f u l l y acknowledged.
465
THEOPHY LLINE
Jordan L . Cohen
THEOPHYLLINE
Table of Contents
Page
1. Description 3
1.1 Name: Theophylline 3
1.2 Formula and Molecular Weight 3
1.3 Hydrates 3
1.4 Salts 3
1.5 Appearance, Color, Odor and Taste 4
2. Physical Properties 4
2.1 Spectra 4
2.11 Infrared Spectrum 4
2.12 Nuclear Magnetic Resonance Spectrum 6
2.13 Mass Spectrum 6
2.14 Ultraviolet Absorption Spectrum 6
2.2 Optical Rotation 10
2.3 Me 1t ing Range 10
2.4 Solubility 10
2.5 Dissociation Constant 12
2.6 Dipole Moment 12
3. Synthesis 12
4. Isolation and Purification 14
5. Theophylline Stability and Compatibility 14
6. Methods of Analysis 15
6.1 Identification Tests 15
6.2 Ouantitative Analytical Methods 16
6.21 Ultraviolet Spectrophotometry 16
6.22 Visible Spectrophotometry 17
6.23 Gas-Liquid Chromatography 17
6.24 High Pressure Liquid Chromatography 17
6.25 Thin Layer Chromatography 18
6.26 Paper Chromatography 19
6.27 Column Chromatography 19
6.28 Polarography 19
6.29 Titrimetry 19
6.291 Complexometric Titration 20
6.292 Photometric Titration 20
6.293 Non-Aqueous Titration 20
6.294 Miscellaneous Titrations 20
6.3 Bioassay Methods 20

7. Pharmacokinetics 20
8. References 23
467
JORDAN L. COHEN
1. Description
1.1 Name: Theophylline

Theophylline1,2,3 is designated by Chemical
Abstracts as 1, 3-dimethylxanthine. It is also known as
theocin.
1.2 Formula and Molecular Weight
180.17
1.3 Hydrates
Theophylline has been reported to exist in
both anhydrous and monohydrate forms. The anhydrous form
is obtained by drying finely powdered drug at 15OoC for
three hours4.
1.4 Salts
Sodium and potassium salts as well as a large
number of less basic salts and/or complexes have been pre-
pared to increase the water solubility of theophylline for
parenteral administration. The ethylenediame salt (amino-
ph~lline)~,~ is the most widely utilized form of this drug.
Other theophylline salts which have been prepared inc ude
4
sodium and alumi urn glycinates7, h xamethylene iamine ,
8 8
monoethan lamine8, trietha olamine , glucamine methyl-
F
glucamine8 ethylglucamine , 2-diethylaminoethanol10,
choliy5" and sodium acetatel2. Theophylline aminoisobu-
tanol and 2-carbamoylphenoxyacetic acid, sodium salt14
have been prepared and studied clinically as has a niacin-
amide-theophylline complex 15,16. Theophylline has also
been shown to form stable, complexes with saccharinl7,
phenobarbita118, papaverine19, caffeinel5, sulfosalicylic
468
THEOPHYLLINE
acid15, benzyl alcohol15, and d -, and

tate15.
is- naphthalene ace-
1.5 Appearance, Color, Odor and Taste
Theophylline occurs as a white, odorless, crys-
talline powder with a bitter taste.
2.1 Spectra

The IR spectrum of anhydrous theophylline is
shown in Figure 1, This was recorded on a KBr pellet with
a Perkin-Elmer model 21 spectrophotometer20. This spec-
trum is consistent with the literature interpretation which
is presented in Table I21,
Table I
Infrared Spectrum of Theophylline
IR Absorption Band h) Interpretation

1
2.90 N-H (stretch)

5.86, 5.98 C=O (stretch)
6.20 C=C (stretch)
6.40 C-N (stretch)
6.92 C-H (bending)
7 . 7 , 8.0 C-N, C-0 (vibration)
The spectrum is generally consistent with

those of other purine bases although the placement of the
methyl groups prevents enolization and is reflected in
differences in the 2.5-3.5Pregion of the spectra. The
region from 5.8-8.2 is nearly identical for the three
methylated xanthines; i.e. theophylline, theobromine and
caffeine, however, the fingerprint region between 8.5 and
13.0kis quite dissimilar and could be used for differen-
t iation.
469
Figure 1. Infrared Spectrum of Theophylline Reference Standard.
THEOPHYLLINE

The 60 MHZ proton magnetic resonance spectrum
run in deuterodimethylsulfoxide o a Perkin-Elmer R-12 B
spectrometer is shown in Figure 2$0 The. of theo-
phylline has been reported in the leteratuT2 and the
assignments are given in Table 11.
Table I1
NMR Spectral Assignments for Theophylline
Proton Assignment No. Chemical Shift (h
N-CH (position 3) 3 6.83 ( 8 )

N-CH (position 1) 3 6.63 (s)
N-H 1 -3.4 (b)
C=C-H 1 2.01 ( 8 )
Impurity - 7.55
s= singlet, b= broad
2.13 Mass Spectrum

The low resolution, electron-impact mass
spectrum of theophylline us&g a 70 e.v. ionizing energy
is illustrated in Figure 3. The resultant fragmentation
represented by this spectrum is not readily explainable
and is only in Frtial agreement with a literature mass-
spectral report 3. The peak at 180 m/e corresponds to the
base peak, which is also the molecular ion and the peak
at m/e 123, which represents loss of CONCH3, are c o m n
to both spectra. other peaks observable in Figure 3 occur
at m/e 95, 68, 53 and 41 while the literature report in-
dicates major peaks at 137, 109, 82, 67 and 55 m/e.
Another, more recent, literature report24 suggests that
fragmentation from m/e 1 2 3 9 9 5 is due to loss of CO and
that m/e 95-68 is due to loss of HCN from metastable ion
calculations. Apparently ring opening occurs which results
in a further complex fragmentation pattern.
2.14 Ultraviolet Absorption Spectrum

The ultraviolet spectrum of theophylline, K
and K Laboratories, recrystallized from water, recorded on
a Perkin-Elmer Hitachi, Model 124 Recording Spectrophoto-
47 1
Figure 2. NMR Spectrum of Theophylline Reference Standard in DMSO-dfj
THEOPHYLLINE
“v-m
Figure 3. Low Resolution Mass Spectrum of Theophylline
473
P
4
P
WAVELENGTH ( nm)
Figure 4. Ultraviolet Spectrum of Theophylline in 0.1 N NaOH.
THEOPHY L L l N E
meter in 0.1 N NaOH is illustrated in Figure 4. In addi-

tion the spectra was also recorded in 0.1 N HC1, pH 6.3
phosphate buffer and CHC13 with all of the results given
in Table 111.
Table I11
Ultraviolet Absorption of Theophylline

Amax. (nm) 1oLymax. Solvent
271 1.02 0.1 N HC1

271 1.04 pH 6.3 buffer
274 1.28 0.1 N NaOH
271 1.04 CHC13
All of these spectra exhibited a single max-

imum in the spectral region between 230-300 nm. The na-
ture of the spectra, as well as the calculated molar ab-
sorptivities are in good agreement with published liter-
ature datz. Turner and 0 ~ 0 reported
1 ~ ~ a n 6 at 271 nm. of
1.02 x 10 in pH 6.3 phosphate buffer, while the value of
6 at 274 nm. in 0.1 N NaOH calculated from the data of
Schach and Waxler26 compares closely with the observed
value above.
475
JORDAN L. COHEN

Theophylline exhibits no optical activity.
2.3 Melting Range

The original synthetic literature27 reported a
c
melting point 0 ; 264O for theophylline subsequently the
melting range has been reported to be between 271° and
274O C.28
2.4 Solubility
The reported solubility of theophylline is 8.3
mg/ml in water; 12.5 mg/ml in ethanol; 11.f,Tg/ml in
chloroform; and sparingly soluble in ether , Several
basic salts have been prepared which enhance the water
solubility of theophylline and have been utilized ther-
apeutically intended for oral, rectal and parenteral
administration. (See Section 1.4) In addition, there
are numerous literature reports of altered theophylline
solubility due to specific interactions wi a variety
of chemical substances, Leuallen and 0 ~ 0 1 ~ ’
have dem-
onstrated marked increases in theophylline solubility
in the presence of a wide variety of primary, secondary
andd tertiary aliphatic amines. They attributed this
increased solubility to salt formation as well as hydro-
phobic interactions. Paruta and Irani3O reported a
three-fold increase in theophylline solubility in alco-
hol-water and dioxane-water mixtures with maximal solu-
bility occurring at a dielectric constan of 40 in these
mixed solvent systems. A British patent” reports a six-
fold increase in theophylline water solubilit in the
presence of guiacol. Several other paper^^^'^^
have re-
ported specific interactions of theophylline with a mul-
titude of planar organic molecules in aqueous solution
usually resulting in an increase in the solubility of
of both interacting species, For a thorough discussion
of the nature of these specific molecular interactions
and the magnitude of this solubility effect see Cohen
and Connors. 37 Not surprisingly there has been some
NMR evidence suggesting that theophylline also forms
1:1 complexes with itself (dimerizes). 38,39
Theophylline also demonstrates altered solubility
in the presence of several inorganic salts as illustrated
in Table IV.
476
THEOPHYLLINE
Table IV
Effect of Inorganic Salts on Theophylline Solubility
-
Salt Effect
NaI, KI, NaCNS, KCNS Increased theophylline sol-

ubility
NaC1, KC1, Na2SO4 Decreased theophylline sol-

ubility
NaBr, KBr No effect on theophylline

solubility
2.5 Dissociation Constant

Theophylline is a weakly acidic compound with
the proton on the nitrogen in position 7 being dissoci-
able. The pKa of 8.6 determined both potentiometrically
and spectrophotometri all is in good agreement with
other literature datah5y4y,42 reporting a pKa-8.6 in
aqueous solution, The0 hylline is "'$8
very weakly
basic and pKb's of 13.555.43 and 11.5 have been re-
ported in the literature from aqueous potentiometric
mea surements .
2.6 Dipole Moment
The dipole moment of theophylline has been deter
mined in three different solvents with the following
values being reported (all in D ye units):& = 3.94 in
dio~ane~= ~ ;4.6
P in 90% phenol"; an@,= 3.94 in ben-
zene47.
3. Synthesis
Although present in coffee, tea and cocoa as well
as other natural sources, theophylline is made available
commercially by total synthesis. Theophylline was first
syntliesized by Traube27 as 'an intermediate in the total
synthesis of caffeine starting from urea, This classic
procedure, which is still utilized, is illustrated in
Figure 5. Several subsequent syntheses which are mod-
ifications of this procedure and are claimed to either
increase the reaction y Id or simplify the process,
have also been reportedfig,49.
411
JORDAN L. COHEN
CH3-NH
Ie o
CH3-N
I
-I C=O
o=O CH+THp,, NCCH7COOH C=O CH2

I I I
CH3-NH CH3-NH CN
0 J alkalai
cH3-k3N0
(HNOr, cH3k(
)
0 NH 0 NH
I I
m3
j. 0
C H 3 k 3 NH2 C H 3 ~ ~ " ' NH3

* "
0 NH2 0
dH3
0
H
I
k:ki
cH3"jii
0 I
CH3
Figure 5. Chemical Synthesis of Theophylline
478
THEOPHYLLINE
Uric acid has been used as the starting c o m p ~ u n d . ~ ~ , ~ ~

In addition various intermediates have been prepared, in-
cluding 1,3-dimethyl-4-amino-5-formylaminouraci152 1,3-
dimethyl-4,5-diamino-2,6-dih dro~yformylpyrimidine~3,1,3-
dimethyl-5,6-diaminourscily5~ and 1,3-dimethyl-4-imino-2.6-
dio~yhexahydropyrimidine~5, from which theophylline has
been synthesized.
4. Isolation and Purification

Because of its significantly increased solubility in
hot water2, theophylline can be purified by recrystalli-
zation from water. The anhydrous form can be obtained by
finely powderin the resulting solid and drying at 150° C.
for three hours 8. Adjusting the pH of an aqueous solution
to 4-6 allows optimal extraction with chloroform while pH
5-7 is required using 1,2-dichloroethane.56 Saturation
of the aqueous phase with sodium or amonium sulfate and
addition of 5-15% isopropyl alcohol to the organic phase
has been shown to markedly increase the extraction effi-
ciency26,57,58.
Sephadex G-10 adsorption chromatography has been

used to isolate theophylline from biological samples also
containing several closely-related xanthines59. Thin
layer chromatographic separation of theophylline, caffeine
and theobromine on silica gel-G using an ethylacetate,
acetone, butanol, 10% NH,!+OH(5:4: 3: 1) mixture has also
been reported60. Cohen and Garrettson have demonstrated
base line separation of these same three compounds using
reverse-phase high performance liquid ~hromatography~8.
5. Theophylline Stability and Compatibility

Theophylline solutions are generally quite stable
over the entire pH range. Strongly alkaline solutions
(pH) 12) show decom osition and apparent ring opening
after several weeks e.Solutions of theophylline have
been shown to be susceptible to oxidation at position 8
forming 1,3-dimethyluric acid in the presence of methylene
blue which acts as a photosensitizing dye61. Because of
its low solubility and relatively high pKa theophylline
will precipitate from aqueous solutions if the pH drops
below 9 unless present in concentrations less than the
419
JORDAN L. COHEN
water solubility. All acidic salts, therfore, are poten-

tially incompatible with theophylline in solution, As
discussed earlier theophylline forms generally soluble
complexes with a variety of other compounds. In the solid
state however, these complexes may lead to formation of
eutechtic mixtures as in the cases of theophylline, pheno-
barbital and papaverine hydrochloride62 and theophylline
riboflavin interactions63 which have been reparted.
Theophylline ethylenediame (aminophylline), which is

commonly employed in alkaline parenteral solutions in con-
centrations considerably greater than the solubility of
theophylline, has many incompatibilities associated with
it. Since the solutions are alkaline, but essentially
unbuffered, all acidic substances produce precipitation
of the free theophylline when mixed together with amino-
phylline solutions. Limited exposure to air results in
carbon dioxide absorption and also leads eventually to
precipitation of the free drug. For a more complete dis-
cussion and listing of the incom atibilities of amino-
phylline Remmington3, MartindalePl or a series of papers
in the hospital pharmacy may be consulted.

Theoph lline has been qualitatively identified
!!
by infrared21, "MR 2 and mass spectroscopy23, thin-layer60
and paper chromatography69 using color localization reac-
tions as well as by a multitude of specific color forma-
tion tests designed to distinguish it from caffeine, theo-
bromine and other xanthines and purines, Brown needles
are produced from the aqueous reaction of theophylline and
ammoniacal AgN03 70 while reaction with ammoniacal silver
and subsequent treatment with thallium acetate yields
light brown ball shaped masses71. A combination of 2,6-
dichloroquinone chlorimide and theophylline in sodium
borate solution gives a blue color and subsequently forms
a red-violet precipitate72. Sanchez73 has reported theo-
phylline to produce a semi-quantitative red color when
reacted with diazo-p-nitroaniline. More recently Feigl,
et. a1.74, have shown that microgram quantities of theo-
480
THEOPHYLLINE
phylline can be detected in the presence of other xanthines

by heating at 15Oo-18O0 C. with excess Hg(CN)2.
6.2 Quantitative Analytical Methods
6.21 Ultraviolet Spectrophotometry

Shack and Waxler described a remarkable ultra-
violet spectrophotometric method for the analysis of theo-
phylline in aqueous solutions as well as biological fluids
in 1949 26. This procedure involves extraction from the
sample adjusted to pH 7.4 using 5% isopropyl alcohol-
chloroform followed by back-extraction of the drug into
0.1 N NaOH and measurement of absorbance at 274 nm. They
found the method to be sensitive to about 1 mcg/ml and
demonstrated its applicability to theophylline analysis in
water, plasma, urine and even tissues. Although it lacked
specificity, it was rapid and sensitive and is still widely
utilized for routine blood level determinations of theo-
phylline. Gupta and L ~ n d b e r greported
~~ a modified pro-
cedure using differential spectrophotometry and estimating
the theophylline concentration from absorbance readings at
285 nm and a standard curve. Although their method was
somewhat less sensitive, 10 mcg/ml in plasma, it was
specific for theophylline in the presence of phenobarbital;
a drug frequently taken with theophylline and a serious
interference in the Shack and Waxler procedure.
A similar ultraviolet procedure was developed

for the analysis of theophylline in dosage forms which
avoided the back-extraction into NaOH. The absorbance of
the organic extract was measured at 271 nm and the theo-
phylline concentration determined from a beers 1 w plot
of standards which was linear from 2-15 m~g/ml.~' A me-
thod utilizing extraction and combination of two succes-
sive volumes of CHClg-isopropyl alcohol (20:l) from an
aqueous sample to which sodium chloride had been added
reports improved sensitivity of 0.5 mcg/ml from aqueous
sarnple~'~, A method involving oxidation of theophylline
with K2Cr2q in acid and steam distillation of the product
which is measured at 257 nm has also been reported appli-
cable to plasma samples78. It is, however, not specific
for theophylline and marginally sensitive for therapeutic
JORDAN L. COHEN
plasma levels of 10-20 mcg/ml.
6.22 Visible Spectrophotometry

Several methods involving derivitization
of theophylline and subsequent colorimetric analysis
have been reported, Coupling reactions have been des-
cribed with p-nitroaniline79, and p-aminobenzenesulfonic
acidso exhibiting colorimetric absorption maxima at 510
nm and 482 nm respectively, While the sensitivity was
less than ultraviolet methods, acceptable standard curves
enabled quantitation of 50-5,000 mg. Chemical reaction
of theophylline with murexide and thiophene enabled quan-
itation of 300-2,500 mg. amounts by measuring absorbance
at 6601m.~~
6.23 Gas-Liquid Chromatography
A gas chromatographic method for theophyl-
line in dosage forms using a 6 f-ot, 1.5%, SE-30 chromo-
.
sorb W column has been reported88 Helium was used as
the carrier gas and temperature program was required to
separate the individual xanthines. Elefant, et a183
reported a GLC procedure for tablets containing ephedrine,
phenobarbital and thophylline. Using a 6', o.d., 3%
HI-EFF 8 BP on 100/120 mesh Gas Chrom Q, theophylline
eluted as a moderately sharp peak after 14 minutes at a
column temperature of 250° C.
Recently, Shah and R i e g e l m a ~reported
~~~ a
GLC procedure applicable to therapeutic plasma and saliva
determinations of theophylline with a sensitivity of 1
mcg/ml. The dipropylated analog was formed by deriviti-
zation using tetrapropylamnonium hydroxide and chromato-
graphed on a 3% OV-17 column at 190'. The retention
time was 3.6 minutes with a sharp peak resulting.

The author has developed a high performance
liquid chromatographic method for the analysis of theo-
phylline in biological fluids58. A reverse-phase 2',
Phenyl Corasil column with a 5% CH CN in pH 8.5 phosphate
2
buffer mobile phase permits determ nation of as little
as 0.5 mcg/ml using an ultraviolet detector. Baseline
separation of theophylline, theobromine and caffeine is
also achieved,
482
THEOPHYLLINE

Several thin layer chromatographic systems for
the separation of theophylline, theobromine and caffeine
and subsequent quantitation have been developed and are
summarized in Table V.
Table Va4
Summary of Thin Layer Chromatographic

Literature Data
Rf
Solid Solvent Theophyl- Caffeine Theobromine
Silica Gel CHCl Ether .35 .20 .40

G 3'(85: 15)
11
CHC13-CH30H .50 .23 ,26
(95:5)
11
CC14-CHC1 -CH30H .54 -46 .31
3:
(8: 1)
11
CHClg-acetone- .50 .49 .44
CH30H (1: 1:1)
Aluminum CHC13-BuOH .54 .30 .15
Oxide (98:2)
11
CHC13-C2H50H 1.00 .10 .10
(99:1)
II
CgHg'C2H OH .15 .06 .02
(93: 2)
II
C ~ H ~ - C Z H ~ O H .47 .08 .04
(95:5)
11
C6Hg-C H OH .58 .11 .06
f98: 10)
11
CgHg'C2H OH .75 .27 .19
(88:20)
483
JORDAN L. COHEN
wantitation was most often achieved by densitometry at

254nm. Fluorescence inhibition scanning at 254 nm. en-
abled quantitation of 50 mcg/ml of theophylline on a
sili a gel plate and a 95:5 CHC13-C2H50H developing sol-
ventg5. Two papers report the use of Draggendorfs Reagent
for the quantitative estimation of theophylline in amounts
as low as 0.5 mcg. Schunack, et alg6 utilized two differ-
ent solvent systems, benzene-acetone (30:70), Rf= 0.06
and chloroform-ethanol-formic acid (88:10: 2), Rf= 0.45
while Senanayake, et alg7 utilized an 8:5:1 mixture of
chloroform, carbontetrachloride and methanol and found an
Rf for theophylline of 0.31.

There'areseveral references in the literature
to paper chromatographic syst or the separation and
quantitation of theophylline.
illustrated by Berger and Hedrickg9 who developed two dif-
ferent percentage mixtures of acetonitrile and aqueous buf-
fer to achieve separations of theophylline from theobro-
mine and caffeine. Quantitation by diazotization and
fluorescence quenching densitometry following a paper
chrgvatographic separation has been described by Kala, et
al.

Theophylline has been separated and quanti
tively determined by column adsorption chromatography.H-
A silicic acid column with 5% butanol in chloroform was
utilized and the eluent analyzed by ultraviolet spectro-
photometry .
6.28 Polarograph
Dusinsky an: Cavanakg2 report on a non-speci-
fic polarographic determination of theophylline involving
the bromine oxidation to methyl parabanic acid. The half-
wave potential was reported to be -0.72 v. and accuracy
for dosage form analysis was of the order of 3%.
6.29 Titrimetry
There are numerous literature reports describ-
ing titrimetric quantitative analytical methods for theo-
phylline due to its acidic properties and reactivity with
a variety of compounds.
484
THEOPHY LLlNE
6.291 Complexometric Titration

Theophylline has been determined by
reaction with excess copper (11) ions, usually as amine
salts and then back-titration with omplexon I11 (Murexide)
to a visual indicator endpoint.939 st Theophylline also
forms molecular complexes with silver ions and has been
determined b both direct titration with si ver nitrate
using visualJ5 or potentiometric detection" and by back-
titration after the addition of excess anrmoniacal silver
nitrate using potassium thiocyanate9 7.
6.292 Photometric Titration

Fleischery6 has reported a photometric
titration determination of theophylline by coupling with
Fast Blue. Sensitivity from aqueous solutions is claimed
to be 2 mcg/ml.

Theophylline can be determined by ti-
tration in a nonaqueous solvent using perchloric acid in
acetic acid as a titrant.99 Endpoint determination can
be done either wi h a visual indicator such as Nile BlueYID
or Crystal Violet or potent iometricallylo2. Theophyl-
line has also been titrated as an acid in dimethylforma-
mide using sodium methoxide and thymol blue as an indica-
tor.103
6.294 Miscellaneous Titrations

Other titrimetric procedures re orted
for theophylline determination include iodometric114,105,
bromometriclo6 amperometric1°7, coulometriclo8 and
.
radiometric109'
6.3 Bioassay Methods

The diuretic activity of theophyllig$_was de-
termined on a relative scale by Lipshitz, et "
'l
a in
dogs by plotting the log dose versus the log of the diur-
etic effect. With urea normalized to 1.0, thophylline
was found to be 115 timesas effective.
7. Pharmacokinetics
Because of the availability of methodology for
the analysis of theophylline in plasma as early as 194926
there are numerous literature reports detailing the dispo-
485
JORDAN L. COHEN
sition of this drug in humans following administration by

several routes. Although theophylline is generally well
absorbed following oral administration, considerable var-
iation does occur depending upon dosage form, formulation
and which salt form of theophylline being administered.
Hydroalcoholic solutions produce peak plasma levels in 60
minutes which decline below acceptable thera ti levels
four hours after single dose administration.pfy, lE2 Tablets
demonstrate peak levels from 2-4 hours with availability
dependent upon formulation, while sustained release tablets
provide peak levels at 6 hours and provide therapeutic
levels up to 10 hours‘13. A non-alcoholic liquid suspen-
sion administered as a soft gelatin capsule showed absorp-
tion characteristics between those for a hydroalcoholic
solution and a sustained release tablet114. This same
study suggests that there is significantly slower absorp-
tion of theophylline ethylenediamine (aminophylline) than
plain theophylline in comparable oral doses in the same
subjects on a cross-over study desi n. This contradicts
earlier findings by Waxler and and additional stu-
dies are being performed.
Theophylline is conrmonly administered rectally,

particularly in asthmatic children,but has been shown to
be poorly and erratically absorbed from normal suppository
base preparations showing peak plasma levels considerable
lower than from comparable oral doses,and occurring 1-3
hours after administration116, Theophylline retention
enemas, however, have been shown to produce plasma consi-
deration--time profiles similar to those following an
intravenous dosel14. The drug appears to be fully bio-
available from this route if allowance is made for a nor-
mal 30-60 minute absorption dela and subsequent effect of
partial first-pass metabolism. Correlation of plasma
levels and clinical response is readily achievable and
.
this route has been proposed as the o timal method of theo-
phylline administration in children118
Theophylline disposition following intravenous,

intramuscular and oral administration can be described by
an open two-compartment okinetic mode1119 with an
average plasma half-life -phase dissappearance of
486
THEOPHY LLlNE
4 . 4 hours, Wide patient-to-patient variation with half-

lives ranging from 2.5-9.5 hours presents possible ser-
ious management roblems for asthmatics on chronic theo-
.
phylline therapyP20 The drug is relatively widely and
rapidly distributed into all tissues with high rates of
blood flow and has a volume of distribution (Vd) of 0.3v
kg. and a half-life for distribution (o(-phase) of 0.12
hours, Erythrocytes apparently also take up significant
amounts of theophylline rapidly. While there was found
to be no statistically significant differences in the
pharmacokinetic parameters between rmal volunteers and
asthmatic subjects, Maselli, et allf' showed that asth-
#
matic children evidenced significantly shorter -phase
half-lives than adults with a mean of 2.65 hours com-
pared to the 4;4 hour half-life for the adult population.
A recent study122 has shown theophylline to

be bound about 60% to plasma proteins at therapeutic
plasma concentrations of 12-15 mcg/ml This same study,
as well as that of Shah and RiegelmanS$ indicates that
saliva levels of theophylline are approximately 50% of
those in plasma and the disappearance of theophylline
from saliva parallels that from plasma over the normal
therapeutic concentration range. This may lead to a
simplified method for monitoring theophylline concen-
trations in clinical pharmacokinetic studies for patients
undergoing chronic therapy.
Theophylline has been shown to be extensively

metabolized in vivo with 13% of the administered dose
appearing in the urine as 3-methylxanthine) 35% as 1,3-
dimethyl-uric acid and 19% as l-methyluric acid123.
Although considerably more work needs to be performed
in this area, the variations in plasma half-life in
adults and the marked decrease in plasma half-life in
children is probably related to differences in meta-
bolic activity in individual subjects.
487
JORDAN L.COHEN
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49 1
JORDAN L. COHEN
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492
THEOPHYLLINE
116. 237, 585 (1959).

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136 (1971).
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-
228, 315 (1957).
493
TYBAM ATE
Philip Reisberg, John Kress, and Jerome I. Bodin

TYBAMATE
CONTENTS
1. Description
1.2 Appearance, Color, Odor,Mode of Occurrence
2 . Physical Properties
2.3 Mass Spectrum
2.4 Thermal Characteristics
2.41 Melting Range
2.42 Boiling Range
2.5 Solubility Characteristics
2.51 Solubility in Several Solvents
2.52 Partition Coefficients
3 . Synthesis
3.1 Purification and Crystallization
4. Stability
6. Methods of Identification and Analysis
6.2 Identification ‘rests
6.21 Derivative Formation
6.22 Colorimetric Identification Test
6.3 Cobalt Cyanate Colorimetric Assay
6.4 Hypochlorite Colorimetric Assay
6.5 Volumetric Analysis
6.61 Infrared Identification
6.7 Gas Chromatographic Methods
6.71 Tybamate in Dosage Forms
6.72 Tybamate in Biological Fluids
6.8 Thin-Layer Chromatographic Methods
6.81 Identity and Purity of Commercial
Tybamate
6.82 Identification in Presence of Other
Drugs
7. Pharmacokinetics
8. References
9. Acknowledgements
495
PHILIP REISBERG e t a /

Tybamate i s 2 - (hydroxymethyl) -2-methyl-
p e n t y l b u t y l c a r b a m a t e 'carbamate. It is also
known a s 2-methyl-2-propyltrimethylene b u t y l c a r -
bamate carbamate, and as N-butyl-Z-methyl-2-
propyl-1,3-propanediol dicarbamate.
0 CH3 0
II I II
H2N-C-O-CH2-C-CH2-O-C-NH-CH2-CH2-CH-3
I
C13H26N204, M o l e c u l a r Weight: 274.36

1 . 2 Appearance;Color, Odor, Mode of
Occurrence
Tybamate o c c u r s as a w h i t e , c r y s t a l l i n e
powder or as a c l e a r , c o l o r l e s s , v i s c o u s l i q u i d ,
which may c o n g e a l t o a s o l i d form on s t a n d i n g .
I t h a s a m i l d c h a r a c t e r i s t i c o d o r and a b i t t e r
taste .
2 . 1 I n f r a r e d Spectrum
The i n f r a r e d s p e c t r u m o f N. F. Tybamate
Reference S t a n d a r d (1) i s shown i n F i g u r e 1. The
spectrum was o b t a i n e d on a 0.5% d i s p e r s i o n o f
tybamate, p r e v i o u s l y d r i e d u n d e r vacuum a t 30° t o
35OC for 4 h o u r s , i n a p o t a s s i u m bromide d i s c .
The f o l l o w i n g band a s s i g n m e n t s have been
made ( 2 ) :
Wave Number, cm. -1 C h a r a c t e r i s t i c of
3350 NH s t r e t c h i n g
1720 Amide I band
1540 Amide I1 band
1255 Amide I11 band
620 Amide IV band
496
P
W
4
Figure 1 - IR spectrum of N.F. Tybamate Reference

Standard in KBr dispersion.
PHILIP RElSBERGetaL
2 . 1 I n f r a r e d Spectrum Cont'd.
A mjneral o i l (Nujol) mull of c r y s t a l l i n e
tybamate can be p r e p a r e d much more e a s i l y t h a n a
s a t i s f a c t o r y K B r d i s c , mainly because o f t h e low
m e l t i n g r a n g e o f tybamate.
The i n f r a r e d s p e c t r u m o f a 1%m i n e r a l o i l
mull of tybamate i s shown i n F i g u r e 2 .
2.2 N u c l e a r Magnetic Resonance Spectrum
The NMR s p e c t r u m as p r e s e n t e d i n F i g u r e 3
was o b t a i n e d by k i n g a 12.5% s o l u t i o n o f tybam-
a t e i n carbon t e t r a c h l o r i d e c o n t a i n i n g t e t r a -
m e t h y l s i l a n e as t h e i n t e r n a l s t a n d a r d . The
f o l l o w i n g peak a s s i g n m e n t s have been made:
Chemical S h i f t ,ppm. P r o t o n No. of P r o t o n s
(a) 0.9 CH3-C 9
(b) 1.3 C-CH2 -CH2- C a
(c) 3.05 N-CH2 2
(d) 3.8 C-CH2-0 4
(e) 5.2 - 5.7 N -H 3

N-H2
26
The peak a s s i g n m e n t s i n d i c a t e d i n F i g u r e 3
are shown on t h e f o l l o w i n g s t r u c t u r a l r e p r e s e n t a -
t i o n o f tybamate:
498
Figure 2 - IR spectrum of N.F. Tybamate Reference
Standard in Nujol mull.
VI
0
0
Figure 3 - NMR spectrum of N.F. Tybamate Reference Standard.

TYBAMATE
2.3 Mass Spectrum

The low r e s o l u t i o n mass spectrum o f tybam-
a t e ( 3 ) , shown i n F i g u r e 4 , e x h i b i t s t h e e x p e c t e d
M+ o f m/e 2 7 4 . Long c h a i n a l k y l g r o u p s , p a r t i c u -
l a r l y t h o s e a t t a c h e d t o n i t r o g e n , fragment on t h e
carbon-carbon bond p t o t h e n i t r o g e n f u n c t i o n
w i t h r e t e n t i o n o f t h e c h a r g e on t h e a l k y l moiety.
Cleavage of t h e N-butyl group r e s u l t s i n an m/e
43 i o n . The m/e 4 1 i o n , C3H5, arises i n t h e
c l e a v a g e s of t h e branched hydrocarbon p o r t i o n o f
t h e molecule. L i k e w i s e , an m/e 43 i o n arises i n
t h e f r a g m e n t a t i o n o f t h e C3H7 moiety o f t h e
branched hydrocarbon. The m/e 55-57 i o n s have
t h e composition C 4 H 7 t o C Hg. The fragment i o n s
r e s u l t i n g from d i r e c t bon8 c l e a v a g e are: m/e 2 9 ,
43, 4 4 , 7 2 , 7 4 , 158, 2 0 0 , 2 1 4 , 231, and 245.
The d i a g n o s t i c peak f o r c a r b a m a t e s , m/e 62,
was p r e s e n t and was shown by C o u t t s ( 4 ) t o be
NH2COOH2+. Through r e a r r a n g e m e n t , HOCONH2 i s
l o s t from t h e M+ t o form t h e m/e 213 i o n . The
same loss can o c c u r from t h e m/e 213 i o n t o form,
t h e m/e 1 7 0 i o n . I n a p a r a l l e l f r a g m e n t a t i o n
mode, t h e e l e m e n t s of OCONHz are l o s t from t h e
m/e 231 i o n t o y i e l d t h e m/e 1 7 1 i o n . The v e r y
i n t e n s e h i g h mass m/e 158 i o n can be formed from
e i t h e r t h e m/e 200 i o n through l o s s o f CH3-CHmCHz
( 4 2 amu) o r from t h e M+ by t h e l o s s o f OCONHC4Hg.
F i n a l l y , t h e m/e 184 i o n can a r i s e through t h e
loss o f C 2 H g from t h e m / e 213 i o n .
The mass spectrum i s i n agreement w i t h t h e
s t r u c t u r e proposed f o r tybamate i n 1.1.
2 . 4 Thermal C h a r a c t e r i s t i c s
2 . 4 1 Melting Range
C r y s t a l l i n e tybamate i n f i n e powder
form, d r i e d under vacuum f o r 4 h o u r s a t 3Oo-35'C,
melts w i t h i n a range o f 2' between 49' and 54'C.
2.42 Boilin Ran e
m s a t a p p r o x i m a t e l y 1500-
152'C a t a p r e s s u r e o f 0.06 mm. o f mercury.
501
t
t
t
n
0
N
MASS/CHARGE
Figure 4 - Mass Spectrum of N.F. Tybamate Reference Standard.
TYBAMATE
2 . 4 Thermal C h a r a c t e r i s t i c s C o n t ' d .
2.43 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
f e
T i r e 5
was o b t a i n e d w i t h a P e r k i n I E l m e r DSC-lb i n s t r u -
ment by f i r s t c o o l i n g t h e e n c a p s u l a t e d sample t o
O°C w i t h a m i x t u r e o f d r y i c e and methanol. The
sample was t h e n h e a t e d a t t h e r a t e o f 10°C p e r
minute u s i n g a r e c o r d e r c h a r t s p e e d o f 1 i n c h
p e r m i n u t e , and an o r d i n a t e s e n s i t i v i t y o f 8
m i l l i c a l o r i e s p e r s e c o n d . A s h a r p m e l t i n g endo-
therm o c c u r s a t a b o u t 46OC. P u r i t y c a n b e e s t i -
mated by comparison w i t h a r e f e r e n c e s t a n d a r d .
2.5 S o l u b i l i t y C h a r a c t e r i s t i c s
2.51 S o l u b i l i t y i n S e v e r a l S o l v e n t s
S o l u b i l i t y of tybamate i n s e v e r a l
s o l v e n t s was d e t e r m i n e d a t room t e m p e r a t u r e .
Approximate s o l u b i l i t y d a t a i s shown i n T a b l e I .
2.52 P a r t i t i o n C o e f f i c i e n t s
The p a r t i t i o n c o e f r i c i k n t s f o r tybam-
a t e were d e t e r m i n e d f o r s e v e r a l s o l v e n t s y s t e m s
a t room t e m p e r a t u r e . These a r e shown i n T a b l e 11.
2.6 O p t i c a l R o t a t i o n
A 5 % a l c o h o l i c s o l u t i o n o f tybamate i s
o p t i c a l l y i n a c t i v e when t e s t e d by t h e USP p r o -
cedure.
3. S y n t h e s i s
Tybamate h a s been s y n t h e s i z e d ( 5 , 6 ) by t h e
s e q u e n c e o f r e a c t i o n s shown i n F i g u r e 6 . The
f i r s t s t e p i s the p r e p a r a t i o n of 2-methyl-2-
p r o p y l - 1 , 3 - p r o p a n e d i o l by r e a c t i n g 2 - m e t h y l -
p e n t a n a l and formaldehyde i n t h e p r e s e n c e o f
p o t a s s i u m h y d r o x i d e . The d i o l is t h e n r e a c t e d
w i t h d i e t h y l c a r b o n a t e i n t h e p r e s e n c e o f sodium
m e t h y l a t e t o form 5-methyl-5-propyl-2-m-dioxan-
one. T h i s i s d i s t i l l e d and r e a c t e d w i t K 28%
aqueous ammonia s o l u t i o n forming 2 - m e t h y l - 2 -
503
PHILIP REISBERG eta/.
Figure 5 - DSC thermogram o f N . F . Tybamate

Reference Standard .
504
TYBAMATE
TABLE I
Approximate S o l u b i l i t y o f Tybamate
m l s . S o l v e n t Required
Solvent per g . o f Tybamate
Water 2000
Ethyl Alcohol 0.6
Chloroform 0.5
Ethyl Ether 1
Propylene Glycol 2
TABLE I 1
P a r t i t i o n C o e f f i c i e n t s f o r Tybamate
S o l v e n t System Part i t i on C oe f f i ci en t
Cottonseed Oil/Water 28
Chloroform/Water >loo
Carbon Tetrachloride/Water 10
Benzene/Water 43
Heptane/Water 0.2
505
CH3
I
CH3-CH2-CH2-C-CH0
I
H
-
CH 2O
KOH
“””\
C
ICH2OH
CH3 - CH2 - CH2

(C2Hs0) 2C=0
( 2 -methy 1pent an a1 ) (2-methyl-2-propyl- J.

1,3-propanediol)
0
-
I
CH3\ /CHZ-O-C-NH2
NH40H
m
0 CH3-CH2-CH2 CH3-CHZ-CH2
CH3 1/ CH2 -0- C -NH2

3 2
FIG. 6: SYNTHESIS OF TYBAMATE

TYBAMATE
3. S n t h e s i s C o n t ' d .
k r o x y p r o p y l carbamate. This i n t u r n
ProPY
i s t r e a t e d w i t h b u t y l i s o c y a n a t e t o form N - b u t y l -
2-methyl-2-propyl-l,3-propanediol d i c a r b a m a t e
(tybamate) .
3.1 P u r i f i c a t i o n and C r y s t a l l i z a t i o n
T h e m p u r e s y n t h e s i z e d tybamate p r o d u c t i s
c r y s t a l l i z e d from h e x a n e - b u t a n o l , o r x y l e n e -
naptha solvent mixtures.
S u c c e s s i v e c r y s t a l l i z a t i o n s o f tybamate
from t r i c h l o r e t h y l e n e , SDA-3A a l c o h o l , and from
h o t w a t e r by d i l u t i o n w i t h c o l d water w i l l r e -
move most i m p u r i t i e s .
The l i q u i d form o f tybamate may b e con-
v e r t e d t o t h e c r y s t a l l i n e form by d i s s o l v i n g t h e
f o r m e r i n a m i x t u r e o f one p a r t o f t r i c h l o r e t h y -
l e n e and two p a r t s o f h e x a n e , and c o o l i n g .
4. S t a b i l i t
d i s v e r y s t a b l e c h e m i c a l l y ( 7 ) as a
s o l i d o r as a l i q u i d . I f d e g r a d a t i o n s h o u l d
o c c u r , i t c o u l d be v i a t h e r m a l o r h y d r o l y t i c
c l e a v a g e o f t h e carbamate g r o u p . As N - s u b s t i t u -
t i o n i n c r e a s e s , s t a b i l i t y i n c r e a s e s and t h u s
tybamate would be e x p e c t e d t o b e more s t a b l e
t h a n meprobamate which h a s no N - s u b s t i t u t i o n . It
has been shown t h a t e v e n c a r b a m a t e s o f t h e mepro-
bamate t y p e do n o t d e g r a d e r e a d i l y u n d e r t h e
above c o n d i t i o n s . ( 7 ) I t i s s t a b l e i n d i l u t e
a c i d o r i n d i l u t e a l k a l i , and i s n o t b r o k e n down
a t 37OC i n g a s t r i c o r i n t e s t i n a l f l u i d . Hot
a l k a l i o r s t r o n g a c i d h y d r o l y s e tybamate t o
y i e l d t h e c o r r e s p o n d i n g d i o l , ammonia, b u t y l a m i n e
and c a r b o n d i o x i d e .
Dosage forms o f tybamate ( t a b l e t s , soft g e l a -
t i n c a p s u l e s ) have shown no loss o f p o t e n c y
a f t e r a 5 y e a r p e r i o d a t room t e m p e r a t u r e .
507
PHILIP REISBERG eta/.
5, Drug Metabolic Products

Douglas, Ludwig, Schlosser, and Edelson (8)
have identified 4 compounds in the urine of both
dogs and cats after administration of tybamate.
These are: the unchanged drug, meprobamate (I),
hydroxymeprobamate (11), and the major metabolite,
hydroxytybamate (111). The metabolites are
shown in Figure 7.
6. Methods of Identification and Analysis
A typical result obtained on medicinal
grade tybamate using the F & M Model 185 CH & N
Analyser (9) is shown below:
E lemen t % Theoretical 0 Found
Carbon 56.90 56.51
Hydrogen 9.55 9.45
Nitrogen 10.21 10.30
6.21 Derivative Formation
A crystalline derivative of tybamate
with xanthydrol in glacial acetic acid can read-
ily be prepared. This reaction is characteristic
of carbamates.(lO)
H O
\ II
H OH H N-C-R
R-8-N-H2
‘ 0
/ 0
+
H20
R = balance of Tybamate molecule
508
TY BAMATE
(I) Meprobamate
(11) Hydroxymeprobamate
0
II
CH2-0-C-NH2
/
O HcH3\
-I /\
CH3 C- CH2 CHz-O-C-NHz
I II
0
H
(111) Hydroxytybamate
0
II
FIG. 7: METABOLIC PRODUCTS OF TYBAMATE
509
PHILIP REISBERG e t a / .
6 . 2 2 C o l o r i m e t r i c I d e n t i f i c a t i o n Test
A red c o l o r is formed when tybamate
i s h e a t e d w i t h p-dimethylaminobenzaldehyde, a n t i -
mony t r i c h l o r i d e , and a c e t i c a n h y d r i d e . (11)
6 . 3 Cobalt Cyanate C o l o r i m e t r i c Assay
Tybamate i s h y d r o l y s e d i n a l k a l i n e a l c o -
h o l i c medium t o y i e l d c y a n a t e i o n which forms a
b l u e complex w i t h c o b a l t i o n . The a b s o r p t i o n i s
determined a t 590 nm. (12) An automated method
b a s e d on t h e c o b a l t complex h a s been d e v e l o p e d
f o r N - u n s u b s t i t u t e d carbarnates and c a n be a p p l i e d
t o tybamate. (13)
6 . 4 H y p o c h l o r i t e C o l o r i m e t r i c Assay
A t pH 10.5 tybamate reacts w i t h hypoch-
l o r i t e t o form an " a c t i v e " c h l o r i n e d e r i v a t i v e .
The e x c e s s h y p o c h l o r i t e i s decomposed w i t h p h e n o l
i n d i l u t e a c i d . The c h l o r i n a t e d compound i s r e -
a c t e d w i t h e x c e s s p o t a s s i u m i o d i d e and t h e l i b e r -
a t e d i o d i n e i s measured c o l o r i m e t r i c a l l y a t 357
nm. (14)
6 . 5 Volumetric A n a l y s i s
s- upon t h e s o l v o l y s i s
o f t h e u n s u b s t i t u t e d carbamate group by sodium
methoxide i n a nonaqueous medium. The e x c e s s
sodium methoxide i s t i t r a t e d w i t h 0.1N hydroch-
l o r i c a c i d u s i n g p h e n o l p h t h a l e i n T.S. as t h e
i n d i c a t o r . (11) C e r r i , e t a1 (15) have shown
t h a t t h i s method i s s p e c i f i c for t h e non-N-sub-
s t i t u t e d c a r b a m a t e s . They have p o s t u l a t e d t h e
f o l l o w i n g mechanism f o r t h e r e a c t i o n :
(1) 0 OH
II
R-0-C-NH2 R-0-C=NH
I
(2) OH ONa
I I
R-0-b-NH + CH3ONa+R-O-b=NH + CH30H
510
TYBAMATE
6.5 Volumetric Analysis Cont'd.

(3) ONa
I
R-O-CmNH-R-OH + NaO-CEN
I n anhydrous p y r i d i n e , t h e e q u i l i b r i u m shown i n
the f i r s t equation is strongly s h i f t e d t o the
r i g h t . The e n o l i c s p e c i e s r e a c t s w i t h t h e sodium
methoxide. I n aqueous medium, t h e k e t o form i s
f a v o r e d and t h e r e a c t i o n w i t h a l k a l i i s no l o n g e r
quantitative.
6.6 S p e c t r o p h o t o m e t r i c A n a l y s i s
6.61 I n f r a r e d I d e n t i f i c a t i o n
The i n f r a r e d a b s o r p t i o n s p e c t r u m o f a
1 0 % s o l u t i o n o f tybamate i n c h l o r o f o r m i s com-
p a r e d w i t h t h e s p e c t r u m o b t a i n e d w i t h N.F. Tybam-
a t e R e f e r e n c e S t a n d a r d . No e x t r a n e o u s bands
s h o u l d be found. M i n e r a l o i l ( N u j o l ) m u l l s have
a l s o been u s e d f o r o b t a i n i n g tybamate I R s p e c t r a .
6.62 N u c l e a r Magnetic Resonance
An NMR method was d e v e l o p e d f o r t h e
a n a l y s i s o f meprobamate t a b l e t s u s i n g m a l o n i c
a c i d as an i n t e r n a l s t a n d a r d . (16) A s i m p l e
a d a p t i o n o f t h i s method c a n be u s e d f o r tybamate.
(17) P o s s i b l e d e g r a d a t i o n o f tybamate t o
N-butyl-2-methyl-2-propyl-3-hydroxypropylcarbam-
a t e can be d e t e c t e d by o b s e r v i n g t h e NMR s i g n a l s
o f t h e p r o t o n s o f t h e methylene g r o u p s a t t a c h e d
t o oxygen, I n t y b a m a t e , t h e r e a r e 2 s u c h g r o u p s
and t h e y a p p e a r as a s i n g l e t . I f h y d r o l y s i s
o c c u r s , t h e s i n g l e t w i l l d e c r e a s e and a new s i g -
n a l w i l l s t a r t a p p e a r i n g down f i e l d from t h e
methylene s i n g l e t .
6.7 Gas Chromatographic Methods
6 . 7 1 Tybamate i n Dosage Forms
A method f o r t h e d e t e r m i n a t i o n o f
tybamate i n dosage forms i s made a v a i l a b l e by
a d a p t a t i o n o f t h e procedure o f Rabinowitz, e t
511
PHILIP RElSBERGetaL
6 . 7 1 Tybamate i n Dosage Forms Cont'd.

a1.(18) T h i s i n v o l v e s a simple e x t r a c t i o n p r o -
cedure, followed by chromatography on a 3.8% OV-
1 7 column w i t h meprobamate a s t h e i n t e r n a l s t a n d -
a r d . Other methods u s i n g d i f f e r e n t i n t e r n a l
s t a n d a r d s have been r e p o r t e d . (19,ZO)
6.72 Tybamate i n B i o l o g i c a l F l u i d s
A method for t h e d e t e r m i n a t i o n o f
tybamate i n b i o l o g i c a l f l u i d s has been r e p o r t e d
by Douglas, e t a 1 , ( 2 1 ) which employs g l a s s c o l -
umns packed w i t h 3.89 UC-W98 methyl s i l i c o n e
on 80-100 mesh D i a t a p o r t S. A l i n e a r r e l a t i o n -
s h i p has been e s t a b l i s h e d u s i n g d i b u t y l phtha-
l a t e a s t h e i n t e r n a l s t a n d a r d . Other carbarnates
a r e determined s i m i l a r l y .
6.8 Thin-Layer Chromatographic Methods
6.81 I d e n t i t y and P u r i t y o f Commercial
f -F a 1 0 % s o l u t i o n of tybamate
i n chloroform i s s p o t t e d on a s i l i c a g e l p l a t e .
The chromatogram i s developed w i t h a mixture of
chloroform and acetone ( 4 : 1 ) , t h e p l a t e i s a i r -
d r i e d f o r s e v e r a l minutes, sprayed w i t h a s a t u r a -
t e d s o l u t i o n of antimony t r i c h l o r i d e i n c h l o r o -
form and then w i t h a 31 s o l u t i o n o f r e d i s t i l l e d
f u r f u r a l i n chloroform. A f t e r 10 t o 1 5 minutes
dark s p o t s a r e v i s i b l e on a w h i t e t o grey back-
ground which slowly darkens. Pure tybamate shows
o n l y one s p o t , Commercial m a t e r i a l may have
t r a c e q u a n t i t i e s of meprobamate and 2-methyl-2-
propyl-3-hydroxypropyl carbamate w i t h Rf v a l u e s
o f 0.36 and 0 . 5 1 r e l a t i v e t o tybamate. (22)
6.82 I d e n t i f i c a t i o n i n Presence o f Other
&%a1 o t h e r TLC methods a r e a v a i l -
a b l e f o r t h e i d e n t i f i c a t i o n of tybamate i n t h e
presence of o t h e r p s y c h o t r o p i c drugs i n blood or
u r i n e . P o s i t i v e i d e n t i f i c a t i o n has been o b t a i n -
e d by t h e u s e of 5 d i f f e r e n t chromatographic
systems, (19,ZO)
512
TYEAMATE
7 . Pharmacokinetics
Peak serum c o n c e n t r a t i o n s o f tybamate were ob-
t a i n e d a b o u t 1 hour a f t e r o r a l a d m i n i s t r a t i o n .
The major m e t a b o l i t e found i n t h e u r i n e was h y d r o -
xytybamate. Serum l e v e l s o f tybamate were d e t e r -
mined i n male mongrel dogs by t h e c o l o r i m e t r i c
method o f Hoffman and Ludwig (25) a f t e r o r a l
a d m i n i s t r a t i o n of 1 5 mg./Kg. Metabolic s t u d i e s
were c a r r i e d o u t a f t e r t y b a m a t e , 100 mg./Kg.,
was g i v e n d a i l y f o r 5 d a y s . The d i s t r i b u t i o n of
tybamate i n r a t t i s s u e was o b s e r v e d 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 3 t o 7 mg. o f c a r b o n - 1 4 l a b e l e d
d r u g . Tybamate was r a p i d l y a b s o r b e d ; a maximum
b l o o d serum l e v e l i n dogs o f a b o u t 10 mcg./ml.
occurred within 1 hour a f t e r administration.
Serum h a l f - l i f e was a b o u t 3 h o u r s and no d r u g was
d e t e c t a b l e a f t e r 24 h o u r s , N e i t h e r tybamate n o r
i t s m e t a b o l i t e s were r e t a i n e d by r a t t i s s u e , b u t
were e x c r e t e d a l m o s t e n t i r e l y i n t h e u r i n e d u r i n g
a 24 h o u r p e r i o d . (5)
513
PHI LIP RE ISBE R G e t a / .
8 . References
1. A. Z . Hayden, W. L. Brannen, and

C . A. Yaciuw, J . Ass. O f f i c . Agr. Chem.,
49, 1109 (1966).
2 . K J . Bellamy, "The I n f r a r e d S p e c t r a o f
Complex Molecules", 2nd e d . , J o h n Wiley and
S o n s , I n c . , New York, N . Y . , 1 9 6 4 , chap. 1 2 .
3. K . F l o r e y and P. T. Funke, S q u i b b I n s t i t u t e
f o r M e d i c a l R e s e a r c h , p e r s o n a l communica-
tion.
4 . R. T. C o u t t s , J . Pharm. S c i . , 6 2 , 7 6 9 ( 1 9 7 3 ) .
5 . F. M. Berger and B. J , L u d w -i g. , T . S . P a t e n t
2,724,720-(1955).
6. B. J. Ludwig and E. C. P i e c h , J . Amer.
Chem. SOC., 73, 5779 (1951).
7. ams and F. A. Baron, Chem. Revs., - 65,
!67*:1965).
8. J. F. Douglas, B. J . Ludwig, A. S c h l o s s e r ,
and J . E d e l s o n , B i o c h e m i c a l Pharmacology,
1 5 , 2087, ( 1 9 6 6 ) .
9. A. Brenner, Carter-Wallace, I n c . , un-
published data.
1 0 . E . F. S a l i m , J . I . Bodin, H. B. Zimmerman,
and P. R e i s b e r g , J. Pharm. S c i . , - 5 5 , 1439
(1966).
11. "The N a t i o n a l Formulary", 1 3 t h e d . , Mack
P u b l i s h i n g Co., E a s t o n , P a . , 1 9 7 0 , p.749.
1 2 . G . Devaux, P. Mesnard, and J . Cren, P r o d .
Pharm., 1 8 , 2 2 1 (1963).
13. C u n e n . L. J . Heckman. a n d G. J .
P a p a r i e l l o , J . Pharm. S c i . , 58, 1537 (1969)
1 4 . G. H. E l l i s and C. A. Hetzel. Anal. Chem..
~ .
3 1 , 1090 ( 1 9 5 9 ) .
1 5 . K C e r r i , A. S i a l t i n i , and U. G a l l o ,
Pharm. Acta Heyv., 34, 1 3 (1959)
16. 3. W. T u r c z a n a n d T. C . K r a m , J . Pharm.Sci.
56, 1 6 4 3 ( 1 9 6 7 ) .
1 7 . TT A. B r e n n e r a n d P. R e i s b e r g , Carter-
Wallace, I n c . , u n p u b l i s h e d d a t a .
1 8 . M. P. R a b i n o w i t z , P. R e i s b e r g , a n d J. I .
Bodin, J . Pharm. S c i . , 6 1 , 1974 ( 1 9 7 2 ) .
1 9 . R. C . G r a n t , F.D.A. B y - L i n e s , 2 ( 1 ) : 1 0 - 1 4 ,
J u l y (1971).
514
TY BAMATE
20. C . C a r d i n i , V. Q u e r c i a , a n d A. C a l o , J-.
Chromato r 37, 190 ( 1 9 6 8 ) .
21. Y77PT' OUI!" . .sa B. S m i t h . and J . A .
S t o c k a g e , J . Pharm. S c i . , 5 8 , 145 ( 1 9 6 9 ) .
2 2 . A . E. Martin, A. H. R o b b i n s C o . , p e r s o n a l
communication.
23. I . Z i n g a l e s , J. Chromatogr., 31, 405 ( 1 9 6 7 ) .
2 4 . T. W. McConnell, J. C h r o m a t o g r , - 29, 283
(1967).
25. A. J . Hoffman and B. J . Ludwig, J. A m e r .
Pharm. A s s . , S c i Ed., - 4 8 , 740 ( l g r
General References
1. B . J . Ludwig, L.S. P o w e l l , and F.M. B e r g e r ,

J . Med. Chem., 1 2 , 462 (1969).
2 . B . J . Ludwig a n d 7 . R . P o t t e r f i e l d , Adv.
Pharmacology and Chemotherapy, - 9,
1971).
9 . Acknowledgements
The a u t h o r s wish t o e x p r e s s t h a n k s t o D r . K.
F l o r e y and D r . P.T. Funke o f t h e Squibb I n s t i t u t e
f o r Medical Research f o r p r o v i d i n g t h e mass
s p e c t r a l d a t a , t o Urs. B . J . Ludwig and D . R e i s n e r
o f Wallace L a b o r a t o r i e s f o r s u g g e s t i n g improve-
ments i n t h e m a n u s c r i p t , and t o Mr. I . A . Brenner,
Miss R. Rader, and Mrs. M. B e l a s o n f o r a s s i s t a n c e
i n its preparation.
515
ADDENDA
ADDENDA
Ampicillin
6.21 Spectrophotometric determination of

ampicillin sodium in the presence of
its degradation and polymerization
products. H. Bundgood, J. Pharm.
Pharmac. 26, 385 (1974).
Chloramphenicol
3. Biosynthesis of Chloramphenicol.
Origin and Degradation of the
Aromatic Ring.
W. P. O'Neill, R. F. Nystrom,
K. L. Rinehart Jr. and D. Gottlieb,
Biochemistry l2, 4775 (1973).
Chlordiazepoxide Hydrochloride
6.6 Determination of Chlordiazepoxide

Hydrochloride and its Major
Metabolites in Plasma by
Differential Pulse Polarography.
M. R. Hackman, M. A. Brooks,
J. A. F. de Silva and T. S. Ma,
Anal. Chem. 46,1075 (1974).
Diazepam
6.33 Quantitative Determination of

Medazepam, Diazepam and Nitiazepam
in Whole Blood by Flame-Ionization
Gas-Liquid Chromatography.
M. S. Graeves, Clin. Chem. 2 0 ,
141 (1974),
518
ADDENDA
Dexamethasone
5. Radioimmunoassay for Dexamethasone

in Plasma.
M. Hichens and A. F. Hogans,
Clin. Chem. 20,266 (1974).
Fluphenazine Hydrochloride
5. Metabolism in rats; in vitro and

urinary metabolites.
H. J. Gaertner, U. Breyer and
G. Liomin, Biochem.Pharmac. 23,
303 (1974).
Formation of identical metabolites

from piperazine and dimethylamino-
substituted phenothiazine drugs in
man, rat and dog.
U. Breyer, H. J. Gaertner and
A. Prox, Biochem. Pharmac. 23,313
(1974).
6.72 Thinlayer chromatography of
phenothiazine derivative and
analogues.
A. deleenheer, J. Chromatog. 75,
79 (1973).
6.74 Gas-liquid chromatographic analysis

of fluphenazine and fluphenazine
sulfoxide in the urine of chronic
.
schizophrenic pa tien ts
M. I. Kelsey, A. Keskiner,
E. A. Moscatelli, J. Chromatog.
-
75,294 (1973).
519
ADDENDA
Meprobamate
6.63 GLC Determination of Meprobamate

in Water, Plasma and Urine.
L. Mortis and R. H. Levy,
J. Pharm. Sci. 63,834 (1974).
Methadone Hydrochloride
5.4 Biliary excretion of methadone by

the rat; identification of a
para-hydroxylated major metabolite.
R. C. Baselt and M. H. Bickel,
Biochem. Pharmac. 22,3117 (1973).
Methaaualone
5. Identification of Free and

Conjugated Metabolites of
Methaqualone by Gas Chromatography-
Mass Spectrometry
R. Bonnichsem, Y. Marde and
R. Ryhage, Clin. Chem. 20,230 (1974).
Qualitative and Quantitative

Determination of Methaqualone in
Serum by Gas Chromatography.
M. A . Evenson and G. L. Lensmeyer,
Clin. Chem. 20,249 (1974).
Propoxyphene Hydrochloride
4. Fluorometric Determination of
Propoxyphene.
J. C. Valentour, J. R. Monforte
and I. Sunshine, Clin. Chem. 20,
275 (1974).
520
ADDENDA
Sulfamethoxazole
5. Modification of an Automated Method

for Measurement of S ulfamethoxazole
and its Major Metabolite in
Biological Fluids.
A. Bye and A. F. J. Fox, Clin.Chem.
-
20,288 (1974).
Triamcinolone
2.4 Mass spectrometry of some

corticosteroids and related
compounds of pharmaceutical
interest. P. Taft, B. A . Lodge and
M. B. Simard, Can. J. Pharm.Sci.
-7,53 (1972).
Triamcinolone Acetonide
5. 6@-Hydroxylation of trimcinolone
acetonide by a hepatic enzyme
system.
D. Kupfer and D. Partridge,
Arch. Biochem. Biophys., 140, 23
(1970).
Triflupromazine Hydrochloride
5. Formation of identical metabolites

from piperazine and dimethylamino-
substitu ted phenothaizine drugs in
man, rat and dog.
U. Breyer, H. J. Gaertner and
A. Prox, Biochem. Pharmac. 23,313
(1974).
52 1
ADDENDA
6.72 Thinlayer Chromatography of

phenothiazine derivatives and
analogues.
A. deleenheer, J. Chromatog. 75
(1973).
522
ERRATA
ERRATA
Fluphenazine Enanthate
6.2 Vol. I1 p. 258 Neutralization

equivalent: calc. 275. not 285.
Triamcinolone
1.1 Vol.1 p.369 Molecular weight:

394.45 not 434.49
524
CUMULATIVE INDEX
Italic numerals refer to Volume numbers.
Acetaminophen, 3, 1 Fluphenazine Enanthate, 2, 245; 4, 523

Acetohexamide, 1, 1;2, 573 Fluphenazine Hydrochloride, 2, 263; 4,
Alpha-Tocopheryl Acetate, 3, 1 1 1 5 I8
Amitriptyline Hydrochloride, 3, 127 Flurazepam Hydrochloride, 3, 307
Ampicillin, 2. 1 ;4, 5 17 Halothane, I , 119;2, 573
Cefazolin, 4, 1 Hydroxyprogesterone Caproate, 4 , 209
Cephalexin, 4, 2 1 lodipamide, 3, 333
Cephalothin Sodium, I , 319 Isocarboxazid, 2, 295
Chloral Hydrate, 2, 85 Isopropamide,2, 315
Chloramphenicol, 4, 4 7 , 5 17 Isosorbide Dinitrate; 4, 225
Chlordiazepoxide, 1, 15 Levartereno1 Bitartrate, 1, 49; 2, 573
Chlordiazepoxide Hydrochloride, I , 39; 4, Levallorphan Tartra?e, 2, 339
517 Meperidine Hydrochloride, I , 175
Chlorprothixene, 2, 6 3 Meprobamate, I , 209; 4, 519
Clidinium Bromide, 2, 145 Methadone Hydrochloride, 3, 365;4, 519
Clorazepate Dipotassium, 4, 9 1 Methaqualone, 4 , 2 4 5 , 5 19
Cloxacillin Sodium, 4 , 1 1 3 Methyprylon, 2, 363
Cycloserine, I , 5 3 Norethindrone, 4, 268
Cyclothiazide, I , 66 Norgestrel, 4, 294
Dexamethazone, 2, 163; 4, 5 18 Nortriptyline Hydrochloride, I , 233; 2,
Diatrizoic Acid, 4, 137 573
Diazepam, 1, 79; 4 , 5 17 Oxazepam, 3 , 4 4 1
Digitoxin, 3, 149 Phenazopyridine Hydrochloride, 3 , 4 6 5
Dioctyl Sodium Sulfosuccinate, 2, 199 Phenelzine Sulfate, 2, 383
Diphenhydramine Hydrochloride, 3, 173 Phenformin Hydrochloride, 4, 3 19
Disulfiram, 4, 168 Phenoxymethyl Penicillin Potassium, I , 249
Echothiophate Iodide, 3, 233 Phenylephrine Hydrochloride, 3,48 3
Erythromycin Estolate, I , 101;2, 573 Primidone, 2,409
Estradiol Valerate, 4, 192 Procainamide Hydrochloride, 4, 333
Ethynodiol Diacetate, 3, 253 Propiomazine Hydrochloride, 2,439
Fludrocortisone Acetate, 3, 28 1 Propoxyphene Hydrochloride, I , 301 ;4,
Fluorouracil, 2, 22 I 519
525
CUMULATIVE INDEX
Reserpine, 4, 384 Triamcinolone Diacetate, I, 423

Secobarbital Sodium, 1, 343 Triclobisonium Chloride, 2, 507
Spironolactone, 4, 431 Triflupromazine Hydrochloride, 2,523; 4,
Sulfamethoxazole, 2, 467; 4, 520 520
Sulfisoxazole, 2 , 4 8 7 Trimethaphan Camsylate, 3, 545
Testosterone Enanthate, 4, 452 Trimethobenzamide Hydrochloride, 2, 55 1
Theophylline, 4, 466 Tropicamide, 3, 565
Tolbutamide, 3, 5 13 Tybamate, 4 , 4 9 4
Triamcinolone, I, 367;2, 571;4, 520,523 Vinblastine Sulfate, 1, 443
Triamcinolone Acetonide, 1,397;2, 57 1 ; Vincristine Sulfate, I, 463
4,520
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Analytical Profiles

Norman W.Atwater Salvatore A. Fusari

Compiled under the auspices of the

Academic Press New York San Francisco London 1975

Norman W. Atwater David ElGuttman'

* Until his death in 1974.

Academic Press Rapid Manuscript Reproduction

ACADEMIC PRESS. INC.

United Kingdom Edition published by

Library of Congress Cataloging in Publication Data

Analytical profiles of drug substances.

Includes bibliographical references.

PRINTED IN THE UNITED STATES OF AMERICA

J. I. Bodin, Carter-Wallace Inc., Cranbury, New Jersey

G. A . Brewer, Jr., The Squibb Institute for Medical Research, New

L. C h j e t z , Warner-Lambert Research Institute, Morris Plains, New

E. M . Colien, Merck, Sharp and Dohme, West Point, Pennsylvania

J. L. Cohen, School of Pharmacy, University of Southern California,

J. P. Comer, Eli Lilly and Company, Indianapolis, Indiana

L. F. Cullen, Wyeth Laboratories, Philadelphia, Pennsylvania

R . D. Daley, Ayerst Laboratories, Rouses Point, New York

N. J. DeAngelis, Wyeth Laboratories, Philadelphia, Pennsylvania

F. Eng, Parke, Davis and Company, Detroit, Michigan

K. Florey, The Squibb Institute for Medical Research, New Brunswick,

S. A. Fusari, Parke, Davis and Company, Detroit, Michigan

D.E. Guttman, School of Pharmacy, University of Kentucky,

W. W.Holl, Smith, Kline and French Laboratories, Philadelphia,

E. H. Jensen, The Upjohn Company, Kalamazoo, Michigan

H. Kadin, The Squibb Institute for Medical Research, New Brunswick,

B. T. Kho, Ayerst Laboratories, Rouses Point, New York

J. Kress, Carter-Wallace Inc., Cranbury, New Jersey

E. P. K.Lau, Searle and Company, Chicago, Illinois

H. H. Lerner, The Squibb Institute for Medical Research, New Brunswick,

L. P. Marrelli, Eli Lilly and Company, Indianapolis, Indiana

D. L. Mays, Bristol Laboratories, Syracuse, New York

A. F. Michaelis, Sandoz Pharmaceuticals, East Hanover, New Jersey

J. E. Moody, USV Pharmaceutical Corporation,Tuckahoe, New York

E. S. Moyer, Ortho Research Foundation, Raritan, New Jersey

N. G. Nash, Ayerst Laboratories, Rouses Point, New York

G. J. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvania

V. E. Papendick, Abbott Laboratories, North Chicago, Illinois

D. M. Patel, William H. Rorer Inc., Fort Washington, Pennsylvania

R. B. Poet, The Squibb Institute for Medical Research, New Brunswick,

A . Post, Smith, Kline and French Laboratories, Philadelphia, Pennsylvania

J. A . Raihle, Abbott Laboratories, North Chicago, Illinois

N. H.Reavey-Cantwell, William H. Rorer Inc., Fort Washington, Pennsylvania

P. Reisberg, Carter-Wallace Inc., Cranbury, New Jersey

R. E. Schirmer, Eli Lilly and Company, Indianapolis, Indiana

A . P. Schroff. Ortho Research Foundation, Raritan, New Jersey

B. Z. Senkowski, Hoffmann-LaRoche, Inc., Nutley, New Jersey

L. A . Silvieri, Wyeth Laboratories, Philadelphia, Pennsylvania

A . M. Sopirak, Wyeth Laboratories, Philadelphia, Pennsylvania

J. L. Sutter, Searle and Company, Chicago, Illinois

D. Szulczewski, Parke, Davis and Company, Detroit, Michigan

F. Tishler, Ciba-Geigy, Summit, New Jersey

A . J. Visalli, William H. Rorer Inc., Fort Washington, Pennsylvania

J. J. Zalipsky, William H. Rorer Inc., Fort Washington, Pennsylvania

A . F. Zappala, Smith, Kline and French Laboratories, Philadelphia,

Alfred F. Zappala, Walter W . Holl, and Alex Post

2.2 Nuclear Magnetic Resonance Spectrum