Analytical Profiles of Drug Substances Volume 4 Edited b y
Klaus Florey The Squibb Institute for Medical Research New B...
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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 Gerald J. Papariello Jack P. Comer Frederick Tishler
Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences
Academic Press New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers
1975
EDITORIAL BOARD
Norman W. Atwater Olenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen Jack P. Comer Klaus Florey Salvatore A. Fusari
*
David ElGuttman' Erik H. Jensen Boen T. Kho Arthur F. Michaelis Gerald J, Papariello Bernard 2. Senkowski Frederick Tishler
Until his death in 1974.
Academic Press Rapid Manuscript Reproduction
COPYRIGHT 0 1975, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.
ACADEMIC PRESS. INC. 111 Fifth Avenue, New York. New
York 10003
United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON)LTD. 24/28 Oval Road. London NWI
Library of Congress Cataloging in Publication Data 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, 111. Academy of Pharmaceued. 11. Brewer, Glenn A. tical Sciences. Pharmaceutical Analysis and Control [DNLM : 1. Drugs- Analysis-Yearbooks. QV740 Section. AA1 ASS] 615'.1 70-187259 RM300.A5 6 lSBN 0-12-260804-6 (v.4)
PRINTED IN THE UNITED STATES OF AMERICA
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 Pharmaceutical 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 compendial drugs but, increasingly, in the industrial research laboratories. Analytical Profiles are being prepared and periodically updated to provide physico-chemical 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 possible. 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
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
UV Hydroxylamine Method
CEFAZOLIN
1. D e s c r i p t i o n
1.1 Name, Formula, Molecular Weight C e f a z o l i n is 3-[ [(5-Methyl-l,3,4-thiadiazol-2y 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 . C14H14NgOqS3 (Na, -HI
1.2
454.512 ( a c i d ) 476.495 ( s a l t )
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 .
2.
Physical Properties 2.1
I n f r a r e d Spectrum
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
1500
1000
.O
7 0
S O
VO
10
11
I4
Ib I I 10
15 303540
DO
01 0 Y
01
L
P
0
03
::
04 05
10
4000
3500
3000
IS00
1600
1400
1100
1000
SO0
b00
WAVENUMBER 1CM-l)
Figure 1:
Infrared Spectrum of Cefazolin Reference Standard, mineral o i l dispersion. Instrument: Perkin-Elmer 4578
400
150
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
2.2
Nuclear Magnetic Resonance Spectrum 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
9
1
1
I
I
I
I
1
I
1
J
I
I
I
I
I
I
I
I
I
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 Chemical S h i f t (ppm)
-
N M R S p e c t r a l Assignments f o r Cefazolin
Multiplicity
C h a r a c t e r i s t i c of
I n t e g r a t i o n of of Protons
No.
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 s i n g l e t & doublet
protons a t and
5 6
3
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
ALFRED F. ZAPPALA eta/.
2.3
U l t r a v i o l e t Spectrum The u l t r a v i o l e t 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.
2.5
Melting 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
Solvent
Cefazolin
mg Cef a z o l i n / m l
ac e t one acetone:water ( 4 : l v:v) chlorof orm 95% e t h a n o l e t h y l acetate isobutylacetate isopropyl alcohol methanol methylene c h l o r i d e m e t hyl i sobut y lk eto n e sodium c h l o r i d e , h a l f -satu rated sodium c h l o r i d e , s a t u r a t e d water
8
5.7 21.2 0.02 1.1 0.24 0.05 0.21 1.7 0.02 0.25 0.83 0.44 1.1
CEFAZOLIN
pH Solubility Profile The pH solubility profile of cefazolin is presented in Figure 5. 2.71
2.8
pKa
The pKa is 2.15 determined spectrophotometrically. A pKa of 2.05 determined titrimetrically has been reported (3). 2.9
Crystal Properties Several crystal forms of sodium cefazolin have been reported (4). The pathways of conversion of one form to another are shown below.
4
gain of water
treat with methanol or ethanol (loss of water)
8-f o m 312 moles of water.
n-f orm
9
ALFRED F . ZAPPALAetal.
U.V. ABSORPTION SPECTRUM
0.9
COMPOUND: CEFAZOLIN CODE: REFERENCE STANDARD
0.8
CONCENTRATION: 0.0294 m g / m l SOLVENT: 0.7
0.1M NaHCOj
CELL PATH: 1 cm
0.6 Y
U
4 m w
0.5
0
2 4:
0.4
0.3
0.2
0.1
0.0
2 00
250 300 WAVELfNGTH nm
350
-
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
0
1
I
1
50
100
150
I
200
250
300
350
1
I
400
450
T,"C
Figure 4 :
DTA of Cefazolin, Reference Standard Instrument:
DuPont 900
1
500
I In 0 0
I
o n
I
o In N
I
0
0 0 0
lU/
0
0 (Y
I
0
I
0
1
In
2 h
2
0
NllOZV333 s
12
G
N
rl rl 0
U
a
a w 0
w
a 4 rl Ll
0
w
h
c4
U
rl
l-4
rl
1
a
UY
0
I-l
X
m
n W
..a
u z 00
t-l
G
rl rl rl
0
91
rcI
5 h P
a 91
a
t-l (d
a
a
t-l
a
I
0=& I 0 I
o=u N
5I
fll
V
rf
o=v
I
0
+
!
o=vI
0
91
V
2-2
”9 II
I
N
P h
X V
(d
B G
rl rl
N (d
W
H
T m
74 1
cn
z-1”
*& w o
R
X
+
H
H H
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.
- - r0 24 48 -
- 'C 72 24 72
-
10% d e x t r o s e i n H20
100
101
98.2
95.8
104
99.1
5% d e x t r o s e i n H20
100
98.3
98.2
94.5
99.4
99.7
5% d e x t r o s e w i t h
100
98.3
96.6
94.4
98.6
97.8
5%d e x t r o s e i n 100 Ringer's i n j e c t i o r
101
100
96.3
100
93.2
5% d e x t r o s e i n 0 . 9 % NaCl
100
100
99.4
98.5
102
97.4
5% dextrose i n 0.45% NaCl
100
100
100
96.0
9 8 . 0 100
10%d e x t r o s e i n 0.9% NaCl
100
100
97.7
97.7
99.4
99.7
0.9% NaCl
100
101
99.7
98.9
105
100
l a c t a t e d Ringer lnje c t i o n
100
103
98.9
36.9
LO1
39.4
linger injection
too
102
101
36.9
LOO
99.7
lactated Ringer
kExpressed as a percent of i n i t i a l concentration
15
ALFRED
Table V
-
F. ZAPPALA er a/.
Comparison of Chemical and Microbiological Met hod s
*
Months
1
3
6
9
Chem. assay
100
101
97.3
Micro. assay
99.4
103
97.5
12
18
24
99.0
96.0
99.0
100
98.2
97.0
101
98.0
*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.
Methods of Analysis 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) 0.05N (2) Tetrabutylamonium hydroxide (TBAH) 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 dimethyls 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.
-
6.4
Thin-Layer Chromatography 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. 86.5 g of sodium (3) A l k a l i n e Sodium Acetate 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. "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. 6.7
19
ALFRED F. ZAPPALA eta/.
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 (1970)
5.
J. Hill and H. Winicov, SmithKline Corp., Personal Communication
6.
L. Ravin and E. Rattie, SmithKline Corp., Personal Communication
7.
L. Ravin and E. Rattie, SmithKline Corp., Personal Comunication
8.
W. W. Holl et al, SmithKline Corp., Personal Comunication, to be published
9. 10,
-
Antimicrobial Agents & Chemotherapy, Vol. 5 , - 227 (1974)
NO. 3, 223
J. of Antibiotics, Vol. XXV, NO. 2, 86 (1972)
20
-
93
136
CEPHALEXIN
Louis P.MarreUi
LOUIS P. MARRELLI
TABLE OF CONTENTS Page 1. Description 1.1 Name: Cephalexin 1.2 Formula and Molecular Weight 1.3 Isomers 1.4 Hydrates 1.5 Appearance 2. Physical Properties 2.1 Spectra 2.11 Infrared Spectrum 2.12 Nuclear Magnetic Resonance Spectrum 2.13 Ultraviolet Absorbance 2.2 Crystal Properties 2.21 X-Ray Powder Diffraction 2.22 Differential Thermal Analysis 2.3 Solubility 2.4 Dissociation Constant 2.5 Optical Rotatory Dispersion 3 . Cephalexin Stability 4. Synthesis 5. Methods of Analysis 5.1 Identification Tests 5.2 Quantitative Methods 5.21 Titration 5.22 Colorimetric Determination 5.23 Thin Layer Chromatography 5.24 Paper Chromatography 5.25 Column Chromatography 5.26 Electrophoresis 5.27 Microbiological Assays 5.3 Assay Methods for Intermediates and Imp uritie8 5.31 7-Aminodesacetoxycephalosporanic Acid (7-ADCA) 5.32 Phenylglycine 6. Protein Binding 7. Pharmacokinetics 8. References
22
3 3 3 4 4
4 4 4 4 b
7 7 7 7 7 10 10 10 10 11 11 11 11 12 14 15 15 16 16 16 16 17 18 18 20
CEPHALEXIN
1.
Description 1.1
Name: Cephalexin Chemical Abstracts
designates cephalexin as 5-thia1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid, 7-( 2-amino2-ph eny1-ac etamido) -3-methyl-8-ox0 Ce halexin monohydrate is also known as 5-thia-1azabicyclo f4.2.01 oct-2-ene-2-carboxylic acid, 7-[(aIUinophenylacetyl) amino]-3-methyl-8-oxo monohydrate' * 7-( D-2amino-2-phenylacetamido) -3-me thyl-3-cephem-4-carboxylic acid monohydrat3 * and 7-( D-cr-aminophenylacetamido)-3-methyl-3cephem-4-carboxylic acid monohydrate'
.
1.2
Formula and Molecular Weight
c16
365.41
17 N304S'H20
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. Cephal 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 monohydrate 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
I n f r a r e d Spectrum The i n f r a r e d spectrum of cephalexin monohydrate 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. 2.11
Nuclear Magnetic Resonance Spectrum 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) -propanes 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 2.12
.
H(7)
24
4000
3600
3200
2800
2400
2000
1800
1600
1400
1200
1000
800
WAVENUMBER CM-'
FIGURE 1. Infrared spectrum of cephalexin monohydrate (potassium bromide disc).
600
400
7.0
5 .O
4.0 3.0 2.0 1.0 PPM (a) FIGURE 2. NMR spectrum of cephalexin monohydrate (020 + trifluoroacetic acid). 8.0
6.0
0
TABLE I
Infrared Spectrum of Cephalexin Monohydrate
-1
Wavelength (cm )
- 3000 (series of broad bands)
3500
2600 (broad)
Assignment OH from H 0 and amide NH stretch 2
+
Y3
1760
@-lactam c
1690
h i d e c = o stretch
(1600 [very broad] (1400
C-0
1550 (unresolved) 820
- 690
0
-
=
o stretch
(carboxylate stretching)
h i d e I1 band Mainly skelectal vibrations including out-of-plane aromatic hydrogen bending, characteristic of monosubstituted aromatic ring
TABLE I1
Proton Magnetic Resonance Spectrum Peak Assignments p .p .m. (6)
Relative Intensity
2.07
Mu1t ip 1icity
Assignment
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 Pcm W absorption maximum a t 262 nm (Figure 3 ) . The El% 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 = Cchromophore 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 solvated 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 , formamid 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 Methanol N-oc tan01 Chloroform Ether
13-5 3- 4 0.03 4.01 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
250
300
monohydrate. 49 mcg per ml in H20
30
I
350
CEPHALEXIN
TABLE I11
of Cephalexin Monohydrate Norelco De Bye-Scherrer Camera
X-Ray Powder Diffraction Pattern
Radiation; Cu/Ni.
Cephalexin Monohydrate
d
1/11 0.40 1.00 0.30 0.20 0.50 0.50 0.20 0.40 1.00 0.50 0.40 0.40 0.60 0.60 0.70 0.70 0.80 0.30 0.60 0.60 0.40 0.60 0.40 0.20 0.40 0.10 0.30 0.15 0.30 0.30 0.10 0.05 0.02 0.05 0.05 0.05 0.10 0.05 0.02 0.02
-
-
15.15 11.85 11.00 9.36 8.55 7.86 6.89 5.98 5.39 4.97 4.76 4.57 4.39 4.22 4.00 3.86 3.60 3.46 3.24 3.10 2.98 2.90 2.81 2.73 2.68 2.63 2.47 2.41 2.31 2.25 2.12 2.09 2.01 1.93 1.87 1.85 1.82 1.72 1.66 1.62 31
TABLE IV
Solubility
PH
- pH Profile of Cephalexin in Water
(37°C)
Cephalexin Monohydrate mg I d .
Cephalexin Monohydrate mg Iml
.
. .
~
W
N
2.3
13
2.5
16
3 .O
.o
24
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
rn
66% 66% DMF
h0
Carboxyl
Amino
5-2
7.3
4
5.3
7.3
17
7.1
Reference
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 anhydrous 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 dependent 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 The 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 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 Cephalexin i n serum w a s found t o l o s e lo%, 5% and dayg2 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
/
@:b.lii>
CHzOAc
N
C02H
/
COzH
I, R-H II. A=Boc
111. R-Boc
V. R-H
SCHEME I1
CO2H
COZH
V
IV
FIGURE 4. Synthasis routes for eaphelexin.
34
CH3
CEPHALEXIN
butoxy-carbonyl (BOC) group was removed from I11 with trifluoroacetic 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 The nucleus, 7-aminodesused t o obtain cephaloglycid' acetoxycephalosporanic a c i d ( 7-ADCA)25 was prepared, acylated with BOC-protected D-phenylglycine employing a mixed anhydride 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 charact 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 The been used f o r the determination of c e p h a l e x i d 6 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-hydrolysis 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 solut 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 thereof4 The procedure incorporates a sample hydrolysis step 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 automated system gives excellent l i n e a r i t y of response f o r t h e
!A;:(
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 cephal 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 compounds 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 advantage 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. Colorimetric Determination Reaction with hydroxylamine has been u t i l i z e d f o r t h e colorimetric determination of cephaThe method i s based on t h e f a c t t h a t hydroxylexid’ ’32 lamine cleaves t h e 8-lactam r i n g (pH 7.0) t o form a hydroxamic 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 8lactam r i n g r e a c t as well. 5.22
.
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 cephradine’ 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 procedure 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-(lq4c 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 developed 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 A t the 1 mg. per ml. l e v e l , t h i s red chromophores (520 nm.). 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-aminophenylacetamido) 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
Silica Gel
Ethyl Acetate/Acetone/Acetic Acid/ Water (5:2:2:1)
0.22
35
Cellulose Chromatogram Sheet
Butanol/Acetic Acid/ Water (3:l:l)
-
36
Sheet
Ethyl Acetate/Acetic A c i o a t e r (3:l:l)
-
36
Sheet
Acetonitrile/Ethyl Acet at e/Water ( 3 :1 :1)
-
36
Cellulose
Acetonitrilefiater (3:1)
0.50
37
Cellulose
Butanol/Acetic Acid/ 0.70 Water (3:l:l)
37
(3: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 chromatography 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 Butanol/Ac e t i c AcidJWate r (3:l:l)
Rf -
Ref. -
0.60
13, 36, 38
-
36
Ethyl Acetate/Acetic Acid/ Water (3:l:l)
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 chromatographic 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 cephradine 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 phenylglycine 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 test f o r impurities by the B r i t i s h Pharmacopoeia' as 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 cephalexin. 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 The prolow l e v e l s of 7 - A K A i n cephalexin (0.4 1.5%). 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 n Section 5.31 f o r the determination o f 7 - m A i n cephal 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 phenylglycine 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'. I
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 satur 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 microb 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. Naumann and Fedde9O a l s o found and 4196 a t 0.2 yg./ml. 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 animals 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 Wells e t . al.5' a blood serum l e v e l of 18 pg./ml. reported a similar value (17 Fg./ml.) with a 10 mg./kg. 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 '5' 'J and 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.' 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. al.5 found t h a t probenecid increased the Thornhill et. 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 aneph41.0 r i c p a t i e n t s . H a l f - l i f e values ranged from 23.5 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, Personal Communication, (1968). 16. Pfeiffer, R.R., Eli Lilly Analytical Development, Personal Communication, (1970). 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. 207 (1972). 44. Mann, J.M. , Anal. Microbiol., Simmons, R.J., Anal. Microbiol., s, 193 (1972) 45. 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.
a,
45
LOUIS P. MARRELLI
57. 58. 59. 60. 61. 62. 63.
Chemother., 353 (1968). Perkins, R.L., Apicella, M.A., Lee, I., Cuppage, F.E., and Saslaw, S., J. Lab. Clin. Med., 7 l , 75 (1968). Thornhill, T . S . , Levison, M.E., Johnson, W.D., and Kaye, D., Appl. Microbiol., l7, 457 (1969). Gower, P.E. and Dash, C.H., Br. J. Pharmac., 37, 738 (1969). O'Callaghan, C.H., Footill, J.P.R., and Robinson, W.D., J. Pharm. Pharmac., 23, 50 (1971). Kirby, W.M.M., de Maine, J.B., and Serrill, W.S., Postgrad. Med. J., 47, February Suppl., 46 (1971). Kabins, S.A., Kelner, B., Walton, E., and Goldstein, E., her. J. Med. Sci., 259, 133 (1970). 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 microbiological 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. Physical Properties 2.1 Crystal Properties 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 determinationdifferential scanning calorimetry 2.2 Solubility 2.21 Single Solvents 2.22 In mixed solvents or as a result of complexation 2.23 pH Effect 2.3 Distribution 2.4 Spectral Properties 2.41 Ultraviolet 2.42 Infrared 2.43 Nuclear Magnetic Resonance 2.44 Optical Rotation 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. Methods of Analysis 6.1 Colorimetric 6.11 For identification 6.12 Quantitative Analysis 48
CHLORAMPHENICOL
CONTENTS (Cont ' d) 6.2 6.3 6.4 6.5 6.6
Polarographic Spectrophotometric Titrimetric Microbiological Chromatographic
49
DALE SZULCZEWSKI AND FRED ENG
1. Description 1.1 Nomenclature 1.11 Chemical Names a. Dg-(-)-threo-2 ,2-dich1oro-N- [B-hydroxya- (hydroxymethyl)-p-nitrophenethyl]acetamide b. Dg-(-)-threo-l-(~-nitrophenyl)-2- (2,2dich1oroacetamido)-l,3 propanediol
.
1.12 Generic Name Chloramphenicol 1.13 Trade Names Chloromycetin (The Merck Index' other trade names.)
lists 45
1.2 Formulae 1.21 Empirical C11H12C12N205 1.22
Structural and Stereochemical CH20H
I
C12CHCONHCc
r l ~ D
-(or Ls)-threo-l-(p nitrophenyl)-2-(2,2dichloroacetamid )-1,3 propanediol9 I3
I )Lc
*OH
bI
or
N02
(lR, 2R) l-(E-nitrophenyl)-2-(2,2dich1oroacetamido)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. Physical Properties 2.1 Crystal Properties 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 chloramphenicol 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. Measured Density.
51
2V. 7 8 O . 1.49.
DALE SZULCZEWSKI AND FRED ENG
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
11.08 8.76 8.15 6.87 5.64
7.98' 10.10 10.85 12.89 15.72
12 1 32 64 57
4.98 4.68 4.47 4.37 4.28
17.81 18.98 19.85 20.30 20.74
32 61 57 79 100
113 204 311 400 401,114
3.95 3.88 3.70 3.66 3.61
22.50 22.91 24.05 24.31 24.63
11 11 18 64
205 313 115,006 020 021
3.44 3.38 3.34 3.28 3.23
25.93 26.37 26.68 27.17 27.61
54 7 7 7 18
404
3.16 3.13 3.05 2.97 2.92
28.25 28.55 29.25 30.05 30.64
4 br* 510,315 10 br* 511,405 7 024 7 207 600,513 79
2.89 2.87 2.82
30.90 50 31.19 14 31.69 89 plus other lines
*
-1 111
4
broad 53
h k-&-
002 200 201 202 203
220 221 023 222
601 316,117 406,602,025
DALE SZULCZEWSKI AND FRED ENG
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 analysis 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
k.
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 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. 2.132
2.133
I 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 chloramphenicol follows as Figure 2. 2.2
Solubility
2.21 Merck Index1:
Single Solvents The following d a t a are a b s t r a c t e d from the
S o l u b i l i t y a t 25' i n water = 2.5 mg./rnl.; i n propylene glyc 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 . Solub i l i t y i n 50% acetamide soln. about 5%. S o l u b i l i t i e g determined by Weiss e t a l . , A n t i b i o t i c s & Chemotherapy 7,374 Water 4.4; methanol 720; (1957) i n mg./ml. a t about 28': ethanol >20; isopropanol >20; isoamyl alcohol 17.3; cyclohexane 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; chloroform 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 mg
4.421
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
I n mixed s o l v e n t s o r as a r e s u l t of complexation 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. 2.22
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 ' , "-tetramethylphthalamides 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 approximately 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 inc 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 . Res 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 Concentration
0 0.0001 0.001 0.005 0.01 0.02 0.05 0.11 0.125 0.15
Solubility
PH
%
of Solution
0.375 0.391 0.438 0.614 0.732 1.23 2.14 3.46 3.67 3.87
4.70 7.15 8.65 8.65 8.65 8.65 8.70 8.90 8.90 9.00
56
CHLORAMPHENICOL
401
h
0,
\
F
v
> I-
-Tm
301
3
.J
0
v)
20(
101
~~
50
1 10
CONCENTRATION OF PURE SOLVENT ( w t . %)
I. II. I I I. IV. V.
VI.
Dimethylacetamide, 3 1 ° C Acetone, O°C Methanol Ethanol N-Propanol Propylene Glycol
FIG. 3
10
-
SOLUBILITY CURVES OF CHLORAMPHENICOL IN MIXED AQUEOUS SOLVENTS 57
$1
-
h.
X
1/M
9
'9 f 1031R~WdWWYOlW3
58
CHLORAMPHENICOL
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. 2.23
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 Cyclohexanone n-Butanol E t h y l acetate
Methyl i s o b u t y l ketone N i trobenzene Nitromethane Ethyl e t h e r Chloroform Benzene Petroleum e t h e r Ethylene d i c h l o r i d e
% -
Ref.
8 8 3 8 38.0 17.0 20 82 93 96 50.0
7 7 16
7 7 7 16 16 16 16
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 hydrol y t i c decomposition products. 2.4
Spectral Properties 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
280 278
300
320
340
380
WAVELENGTH (nrn)
FIG. 5
-
ULTRAVIOLET SPECTRUM OF CHLORAHPHENICOL I N WATER.
60
380
400
CHLORAMPHENICOL
As was e s t a b l i s h e d by Vandenbelt
7,17
, this a b s o r p t i o n i s due t o t h e p-nitrophenyl chromophore and prov 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 determined 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: Functional Group
Wavenumber cm-1
bonded OH, NH
3340, 3260
amide p o r t i o n of 2,2-dichloracetamide moiety amide I amide I1 n i t r o group ( n i t r o PhenYl) hydroxyl
1697 1568 1530, 1358 1068
61
WAVENUMBER (cm-1) 3000
4000
1Tw
1000
1400
1600
111
1100
118)
im
a*
900
in
1
J
4
C
I
7
1
8
10
WAVELENGTH (microns) FIG. 6
-
INFRARED SPECTRA OF CHLORAMPHENICOL, KBr PELLET.
11
12
13
14
CHLORAMPHENICOL
2.43
Nuclear Magnetic Resonance
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 chloramphenicol in solution. Jardetsky's chemical shift assignments of the chloramphenicol protons were determined from spectra in deuterated acetone of chloramphenicol samples lyophilized 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-
(Acetone)
3.0 I
I
I
I
I
I
FIG.
LO I I
6.0
7.0
I
7 - NMR SPECTRUM OF CHLORAMPHENICOL, PARKE DAVIS LOT H 7 0 0 1 7 1 I N OEUTERATED ACETONE CONTAINING TRIMETHYLSILANE AS INTERNAL STANDARD.
Lo
I
I
1
CHLORAMPHENICOL
2.44
Optical Rotation 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 res 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 3 contained i n t h e Federal Register as c i t e d by t h e U.S.P.
.
The Food and Drug Administration8 has establ 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 combinat i o n with sulfonamides w a s accomplished by polarimetric means22.
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 ionizat 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. 2.45
65
DALE SZULCZEWSKI AND FRED ENG
40'
I70
20 *
oi-, 60
. - . , I00
.
.
. A . .. 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, OZN-@-f
3.
:i21 H
f-
I CH-CH,OH
1;;
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: hexamethylenetetramine > 0 2 N o C O C H 2 M 2 0 2 N B C H 2 B r
-
-
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 proceed via oxazoline formation and are detailed in references 48-54. Resolution of the threo-(p-nitrophenyl)-2-amino1,3-propanediol roduced can be accomplished by convenA tional means5 5 9 5 % or by fractional cry~tallization~~. rather complete review of the synthetic chemistry of chloramphenicol 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 antibiotic. Reasonable precautions taken to prevent excessive exposure to light or moisture are adequate to prevent significant decomposition over an extended period. 4.2
In Solution The stability of chloramphenicol in aqueous solution 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
NH,
II
+ CHC l2COzH
0
and (b) hydrolysis of covalent chlorine of the dichloroacetamide moiety
H
OH
HN+-CHCl, 11
0
68
+ 2HC1
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 acidbase 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 increases. 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 hyd 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 solut 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 The r e s u l t s obtained are l i s t e d below.
.
Temper a t u r e OC
Rate Constants k hrs-1
Calculated L l O L
115 110 30 20 4
0.2188 0.7413 0.1153 0.3589 0.4592
29 minutes 85 minutes 38 days 4 months 31 months
x 10:'
x 10 x lo-' x lo-'
69
DALE SZULCZEWSKI A N D FRED ENG
Table 3 Decomposition Products of Chloramphenicol
No.
Compound
1.
Environmental Conditions
Ref. -
Acidic o r b a s i c aqueous solution
M12 2.
C~,CHCO~H
3.
o,N@HO
17
11
Aqueous s o l u t i o n , ambient temperature.
62 9 63 9 64
H
OH
4 . H2N @--;-CH
OH 2
I
Aqueous s o l u t i o n , ambient temperature
-CH3
HN-C
.
62
II
0
65
5
R,, =Rz ‘CHz
6.
%=R2=C02H
II
7.
%
11
8.
Rl = Rz = OH
OH
Aqueous a l k a l i n e s o l u t i o n , high temperature.
= Rz = CHO
65
II
70
CHLORAMPHENICOL
Decomposition Products ofChloramphenico1 (Continued)
= C0,H;
9.
10.
5
11.
R,, = OH
12.
€$ = OH R2
R2 = OH
65
= C02H; Ra = CH,OH
II
11
R2 = CH20H = 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 temperature.
71
It
DALE SZULCZEWSKI AND FRED ENG
James and Leach70,14suggested that complexation between the antibiotic and borate ion is responsible for the increased stability of chloramphenicol in this buffer system. In the presence of microorganisms Smith'l studied the decomposition of chloramphenicol in the presence of various microorganisms. He defined the five routes by which chloramphenicol could undergo degradation. These possibilities are summarized in scheme 4.3
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 antibiotic indicates that its metabolism can occur by routes shown in scheme 2. Other pertinent information in this regard 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 chloramphenicol and its rnetab~lites~~. Investigators concluded that absorption occurs mainly by simple diffusion mechanisms 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. Methods of Analysis 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 identification scheme. Dissolve 10 mg. of chloramphenicol in a mixture 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, supernatant liquid into a test tube, and add 100 mg of anhydrous sodium acetate and 2 drops of benzoyl chloride. Shake the 12
/
Na?:-fHNH2 COMl P-aminophenyl serine
/
? If'
acid I
I
t\
\
iI '
NH~&-FH-NH,
cn20n
I-p-aminophenyl-2-dichl~ro-
acetamido-1.3-prapanediol
N H 2 ~ C O O HI P-amlnobcnroic
I
NH&-CMl&HCl2 CW20H
I I
r'
I-p-aninophenyl-2-..inoI.3-propanedIol
o-amino-8-hydroxy-paminonronioohenone . .
*-
Ilntermed i a t e r )
( I ntermd iates)
/
I
SCHEME I
-
ethanolamine, a m n i a (Carbon d i o x i d e . formaldehyde)
.'
H l C R O B l A L DECOMPOSITION PATHWAYS
OF CHLORAHPHENICOL.
POLV MERS
( 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 6.12
Quantitative Analysis 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 procedure 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-(1naphthy1)-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 hydroxamic a c i d which t h e n complexed w i t h f e r r i c i o n t o produce 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.
$8
.
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 solut i o n . The procedure i s s i m p l e and can b e performed
75
DALE SZULCZEWSKI AND FRED ENG
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 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.
4s
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 metab 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 discussed previously, could not be considered a s p e c i f i c procedure 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 obtained 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 chloramphenicol 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- (2,2dichloracetamido)-3-hydroxy-4'-nitropropiophenone, a poss 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 sufficiently different from that of the drug's to permit direct instrumental 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 considerations. Quantitative determination via direct ultraviolet measurement is not a specific analytical method since decomposition products absorb over the same region As previously mentioned, an ultraviolet procedure is official as a method to determine the potency of chloramphenicol'
i'e''2.
.
Ultraviolet spectroscopy has been extensively applied to chloramphenicol determination in methods involving 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 analysis 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 titration103,104. 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 titanium chloride followed by determination of the excess reagent by back titration with ferric ammonium sulfate constitutes the titanometric methodlo5.
77
DALE SZULCZEWSKI AND FRED ENG
The bromatometric methodlo5 consists of a Zn-HC1 reduction to form the arylamine. The arylamine is then determined 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 to analysis of dosage assay of chloramphenicol and appl forms, body fluids, and bulk drugf". Although time consuming, these methods are as accurate as physicochemical tests, provide analytical sensitivity equal to or surpassing 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 chloramphenicol 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 antibiotic by adding it to each solution of chloramphenicol used for the s tandard response curve1°7. In the case of chloramphenicol, two basic microbiological methods are in general use viz Cylinder Plate and Turbidimetric
.
The Cylinder Plate Method for assay of chloramphenicol 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 response, 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 design 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 culture 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 appropriate means, and the responses are read in terms of absorbance on a suitable photoelectric colorimeter or spectrophotometer. By comparing the turbidity of the unknown 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 turbidimetric 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 concentrations 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
DALE SZULCZEWSKI AND FRED ENG
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 chloramphen 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-dimethylaminobenzaldehyde (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, ammonia 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 chloramphenicol 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 chloramphenicol d e r i v a t i v e s , decomposition products, and synt h e t i c intermediates. Lin113 achieved s e p a r a t i o n of chloramphenicol , chloramphenicol 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 0.35 0.95 0.25
Chloramphenicol Chloramphenicol Palmitate Chloramphenicol Succinate
80
Solvent B 0.80 0.90 0.72
l
1
l
1
l
1
+
+
IS31 HOeN
IS32 ‘WN‘ON6V I
I
I
I
I
IS31 3 N l O l Z N 3 B +
I
+
+
IS31 N I W A H N I N +
u
n
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e
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C 1
l
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+
+
l
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>. r I
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c
.-
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n
+
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+
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-
81
DALE SZULCZEWSKI A N D F R E D ENG
Solvent A Solvent B
-
n-Butanol-CHC13-acetic
- n-Butanol-water-acetic
a c i d (10:90:0.5) 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 0.98 ( s t r e a k ) 0.91 0.10-0.15 0.80-0.81 0.72 0.89-0.91
p-nitrobenzaldehyde p-nitrobenzoic acid Chloramphenicol l-(~-nitrophenyl)-2amino-ly3-propanediol 1st Solvent 2nd Solvent
-
0.08-0.10
0.35
e t h y l acetate ( s a t u r a t e d with water) 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 decomposit 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-decomposit i o n ) a r i s i n g from chloramphenicol a f t e r heating i n alkal 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 e-[ ( hydroxypheny1)-
0.30
fe-[( hydroxypheny1)benza 1 dehyde azofazo benzyl alcohol
II
0.15
II
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 quantitative 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 determined. 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 chloramphenicol. For this purpose, Aluinina plates were used with binary developers of benzene-ethanol (2.5 20% ethanol).
-
6.63
Partition (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% ethylacetate in chloroform serve as eluents, the eluent being monitored spectrophotometrically at 278 nm. This analytical procedure was validated by direct comparison 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 chloramphenicol in serumg0. In brief, this procedure involves extraction of chloramphenicol from serum with amyl acetate, derivitization using Tri-Si1 reagent followed by chromatography using Qf-1 on silanized Gas Chrom P. The sensitivity 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 chromatography without prior derivatization. Two per cent diethyleneglycol 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.3PROPANEDIOL
4,IO
Com-
6
3,
m
2
v,
10
I
1.
15
R
pound
2
3
4
5 6
7 8
9
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n
CH ,co
OUCCO CH2FC0
(cr,)L:nco CH;CI LO CHF,CO CHCllCO
12
NO2 CH,O NOi. NO
13
NO2
CH,BrCO
CH,S@, NO: NO, NO NO
cncl,co
10
I1
14
15 16 17 18
20
H NH; NO 2 NO 2 NO 2 NO2 NO : NO
R‘
CF,CO
cnci2co C C I ,co C H B r C l CO CnBr,CO CBr,CO
25
TIWE IN UINUTES
FIG.
9
118
-
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.
-
DALE SZULCZEWSKI AND FRED ENG
References 1. The Merck Index, 8th ed., Merck and Co., Inc., Rahway, N.J. (1968), p. 233. 2. W. H. Hartung and J. Andrako, J. Pharm. Sci., 50, 805 (1951). 3. The United States Pharmacopeia, 18th revision, Mack Publishing Co., Easton, Pa. (1970). 4. J. Krc, Parke Davis, personal communication. 5. J. Krc, Parke Davis, personal comunication. 6. J. D. Dunitz, J. Am. Chem. SOC., 74, 955 (1952). 7. Q. R. Bartz, J. Biol. Chem., 172, 445 (1948). 8. Federal Register, 146 d. 301, July (1961). 9. C. Johnston, Parke Davis, personal communication. 10. H. Negoro, et al., Annual Report, Takanime Research Institute, 9, 77 (1957). 11. H. Kostenbauder and T. Higuchi, J. Am. Pharm. Assoc., Sci. Ed., 45, 518 (1956). 12. E. Regdon-Kiss, Pharmazie, 18,755 (1963). 13. M. Suzuki, J. Antibiot., Ser. B., 2 323 (1961). 14. K. James and R. Leach, J. Pharm. Pharmacol., 22, 612 (1970). 15. T. Higuchi and A. Marcus, J. her. Pharm. Ass., Sci. Ed., 43, 530 (1954). 16. A. Brunzell, J. Pharm. Pharmacol., 8, 329 (1956). 17. M. Rebstock, H. Crooks, J. Controulis, and Q. Bartz, J. her. Chem. SOC., 71, 2458 (1949). 18. M. Suzuki and H. Shindo, Yakugaku Zasshi, 76, 927 (1956). 19. 0. Jardetsky, J. Biol. Chem., 238, 2498 (1963). 20. D. Szulczewski, unpublished information. 21. L. A. Mitscher, F. Kautz, and J. Lapidus, Can. J. Chem., 47, 1957 (1969). 22. S. C. Ray, Ann. Biochem. Exp. Med., 23, 411 (1963). 23. C. Brunnee, G. Kappus, and K. H. Maurer, Z . Anal. Chem., 232 17 (1967). 24. U. S. Pat. 2,438,871 25. U. S. Pat. 2,483,892 26. J. Controulis, M. Rebstock, H. Crooks, J. her. Chem. SOC., 71, 2463 (1949). 27. L. M. Long, H. D. Troutman, J. her. Chem. SOC., 71, 2469 (1949). 28. ibid 71, 2473, (1949). 29. ibid 73, 481 (1951). 86
CHLORAMPHENICOL
30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48.
i b i d 73, 542 (1951). U. S . P a t . 2,681,364 U. S. P a t . 2,687,434 U. S. P a t . 2,562,107 U. S. P a t . 2,515,239 U. S. P a t . 2,515,240 U. S. P a t . 2,515,241 U. S. P a t . 2,546,762 U. S. P a t . 2,483,885 U. S. P a t . 2,692,897 U. S. P a t . 2,677,704 U. S. P a t . 2,538,763 U. S. P a t . 2,515,377 U. S. P a t . 2,686,788 U. S. P a t . 2,751,413 U. S. P a t . 2,543,957 U. S. P a t . 2,483,884 U. S. P a t . 2,699,451 G. Moersch, D. Hylander, J. h e r . Chem. SOC., 76, 1703 (1954). 49. S. Igiguma, Yakugaku Z a s s h i , 75, 673 (1952) 50. M. Myamoto, i b i d , 72, 673 (1952) 51. U. S. P a t . 2,562,113 52. U. S. P a t . 2.718,520 53. U. S. P a t . 2,807,645 54. U. S. P a t . 2,562,114 55. U. S. P a t . 2,734,919 56. U. S. P a t . 2,727,063 57. U. S. P a t . 2,586,661 58. M. Suzuki, J. A n t i b i o t . , Ser. B., 14,323 (1961). 59. T. Higuchi and C. Bias, J. Amer. Pharm. Ass., S c i . Ed., 42, 707 (1953). 60. T. Higuchi, A. Marcus, and C. Bias, i b i d , 43, 129 (1954). 61. C. Trolle-Lassen, Arch. Pharm. Chemi., 60, 689 (1953). 62. R. Saba, D. Monnier, and F. R. Khalil, Pharm. Acta. Helv. , 42, 335. 63. K. K. Shih, J. Pharm. S c i . , 60, 786 (1971). 64. J. Lacharme and D. Netien, Bull. Trav. SOC. Pharm. Lyon. 4, 122 (1964). 65. J. Knabe and R. K r a u t e r , Arch. Pharm., (1962) 190. 66. I. K. S h i h , J. Pharm. S c i . , 60, 1889 (1971). 67. A. B r u n z e l l , Sv. Farm. T i d s k r . , 61, 129 (1957).
87
D A L E SZULCZEWSKI A N D F R E D E N G
68. 69. 70.
71. 72. 73. 74. 75. 76.
77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94.
M. Heward, D. A. Norton, and S. M. Rivers, Pharm. J., 386 (1970). B r i t i s h Pharmaceutical Codex 1968 London, The Pharmac e u t i c a l P r e s s , 1968. K. C. James and R. H. Leach, Pharm. J., 204, 472 (1970) G. Smith and C. Worrel, Arch. Biochem., 28, 232 (1950) A. Glazko, Antimicrob. Ag. Chemother., (1966) 655. W. A. D i l l , E. M. Thompson, R. A. F i s k i n , and A. J. Glazko, Nature, 185, 535 (1960). A. Glazko, W. A. D i l l , A. Kazenko, L. M. Wolf, and H. E. Carnes, A n t i b i o t . Chermother., 8, 516 (1968). A. J. Glazko, W. A. D i l l , and L. M. Wolf, J. Pharmacol. Exp. Ther., 104,452 (1952). A. J. Glazko, L. M. Wolf, W. A. D i l l , and A. C. B r a t t o n , Jr., i b i d , 96, 445 (1949). A. J. Glazko, A. W. Kinkel, W. C. Alegnani, and E. L. Holmes, C l i n . Pharmacol. T h e r . , 2, 472-483(1968) A. J. Aguiar, L. M. Wheeler, S. F u s a r i , and J . Zelmer, J . Pharm. S c i . , 57, 1555 (1968).
204,
United S t a t e s Pharmacopeia XVI. 1960 B r i t i s h Pharmacopeia 1968. Deutsche Apotheker. 5 D b l l , Arzeim. Forsch., 5, 97 (1955). A. Glazko, L. Wolf, and W. D i l l , Arch. Biochem., 23, 411 (1949) S. P. Bessman and S. Stevens, J. Lab. C l i n . Med., 35, 1 2 7 (1950). J. Levine and H. Fishback, A n t i b i o t . Chemother., 1, 59 (1951). T. H. Aihara, H. Machida, and Y. Yoneda, J. Pharm. SOC. J a p . , 77, 1318 (1957). M. S. Karawya and M. G. Ghourab, J. Pharm. S c i . , 2, 1331 (1970). K. Kakemi, T. A r i t o , and S. Ohasaki, Yakugaku Z a s s h i , 82, 342 (1962). D. Hughes and L. K. Diamond, Science, 144,296 (1964) G. L. Resnick, D. Corbin, and D. H. Sandberg, Anal. Chem., 38, 582 (1966) R. C. Shah, P. V. Raman, and P. V. Sheth, I n d i a n J. Pharm., 30, 68 (1968). F. M. Freeman, Analyst, 80, 299 (1956). D. Masterson, J. Pharm. S c i . , 57, 306 (1968). K. Fossdal and E. Jacobson, Anal. Chim. Acta., 56, 105 (1971)
88
CHLORAMPHENICOL
G. B. Hess, Anal. Chem., 22, 649 (1950). C. G. Macros, Chem. Chron. A , , 32 104 (1967). 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 Antib 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, "Analyt 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). 95. 96. 97.
.
.
89
DALE SZULCZEWSKI AND FRED ENG
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 preparing 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 1.
Description 1.1 1.2
2.
- Clorazepate Dipotassium
Name, Formula, Molecular Weight Appearance, Color, Odor
Physical Properties 2.1 2.2 2.3 2.4
Infrared Spectrum Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Raman Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 2.10 Crystal Properties 2.11 Dissociation Constant 2.12 Fluorescence 2.13 Hygroscopic Behavior
3.
Synthesis
4. Stability
-
Degradation
5.
Drug Metabolic Products and Pharmacokinetics
6.
Methods of Analysis 6.1 6.2
6.3
6.4 6.5 6.6
Elemental Analysis Phase Solubility Analysis Chromatographic Analysis 6.31 Thin Layer Chromatographic Analysis 6.32 Gas Liquid Chromatography Direct Spectrophotometric Analysis Colorimetric Analysis Non-Aqueous Titration
7, References
92
CH LORAZEPATE DI POTASSI UM
1.
Description
1.1
Name, Formula, Molecular Weight Clorazepate dipotassium is 7-chloro-1,3-dihydro2-oxo-5-phenyl-1H-l,4-benzodiazepine-3-carboxylic acid, monopotassium salt, monopotassium hydroxide.
0
11
H
0
\ C=N
CI
C16H1 1°4N2C1K2
II
CHCOK. KOH
Clorazepate Dipotassium Molecular Weight 408.93
1.2
Appearance, Color, Odor Off white to pale yellow, fine crystalline powder which is practically odorless. 2.
Physical Properties 2.1
Infrared Spectrum 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 visible. (2) 93
4c
P
Figure 2 NUCLEAR MAGNETIC RESONANCE SPECTRUM OF CLORAZEPATE DIPOTASSIUM
I . . 8.0
. , . . . .
I I I I . . . . I . . . . I I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. _. ,. .. 7 .O
6.0
5.0
CCM(
d
)
4.0
3.0
2.0
I .o
. .
JAMES A. RAIHLE AND VICTOR E. PAPENDICK
2.3
U l t r a v i o l e t Spectrum 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) chara 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 tempera 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 decarboxyl 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 followi 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 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 b. 1565 cm-l 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 slowl 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 Anal 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 WAVELENGTH (nm)
97
350
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
UI
d
d
r
o 0
0
o
N
o
0
l
0
r
0
o
0
l
0
r
rl
l
0
0
r
rl
0
l
rl
d
0
I4
m
rl
I-
rl
co
N
m
N
ul
d
rl rl
N
0 rl
rl
rl
rl
03 ul
m *
m *
co m
00
I-
Ih
I-
0
a\
0 I-
h rl
I-
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I-
0
h
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rl
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m
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m
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m
m N
N
2
*0
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N
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03
I-
rl
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IN
U 0
Irl
ul 0
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* 2
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m N
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ul
0
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rn
rl
rl
*co0 m
\D N
u l
I-
*0
rl
hl
N
N
* 3
99
CH LORAZEPATE DlPOTASSlUM
FIGURE 5. FRAGMENTATION PATHWAYS OF CLORAZEPATE DIPOTASSII M
-
0
Probably on the probe
CI
McLafferty Rearrangement Before or After Ion Formation
m/e 314
..
not observed
co, m/e 44 Bare Peak
/-".
lC1
m h 270
*
m/e 235
m/e 242
rFk
m/e 214
/-co
\C-
m/e 179''
100
L
-3
-3
101
OOL
m OW
m 009
OOL
008
006
0001
001I
OOL I OOEl
oopl
0091
OOLL
FIGURE 7. DIFFERENTIAL THERMAL ANALYSIS CURVE OF CLORAZEPATE DIPOTASSIUM
c
0
h)
O
m
0
25
100 175 250 325 TEMPERATURE; DEGREES CENTIGRADE
400
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 : S o l u b i l i t y (mg/ml)
Solvent Water Absolute Ethanol Chloroform Ether Acetone Benzene Isopropanol Methylene D i c h l o r i d e
> 100 < 200 0.6 0.5 0.5 0.5 0.5 0.7 < 0.1
< < c
100-
-
Ic
4
m
?W!
80-
f-
z - 60w
2 40-
I-r
I50
200
MASSICHARGE RAT 10
Figure 4.
Mar8 spectrum of disulfiram.
2 50
300
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 25\
C2H5 H
I
/
+
N:
C2H5
(88)
H
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
2.7 Crystal Properties 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 in0.005, @=1,67+ 0.01, dices (5893 A, 25'C) a r e cu=1.590 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 information regarding c r y s t a l h a b i t , fusion behavior, and xray 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 polymorp 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 dimensions 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 int 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
-S-C-N,
,c-
C-
groups, nearly perpendicular t o each other. The configur 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
NORRIS G. NASH AND RAYMOND D. DALEY
TABLE I
X-RAY POWDER DIFFRACTION PATTERN OF DISULFIRAM
d (A) 9.11 7.94 7.59 6.36 6.15 5.55 5.24 5.11 4.77 4.55 4.50 4.40 4.32 4.21 4.16 4.08 3.96 3.83 3.79 3.73 3.62 3.54 3.46 3.41 3.34 3.28 3.23
d(A)
I/I1 LO
3.18 3.17 3.08 3.05 3.00 2.91 2.83 2.80 2.77 2.70 2.65 2.61 2.56 2.52 2.48 2.44 2.38 2.33 2.29 2.26 2.22 2.20 2.15 2.06 1.88 1.71
8 27
100 37 9 14 49 7 21 11 1 12 9 60 8 13 3 4 1 27 1 19
Ll 7 8 5
178
I/I1 23 23 5 6 16 10 3 4 12 4 3 2 2 10 8
5 3 3 4 4 10 5
7 5 4 2
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 . Oxidants used include hydrogen peroxide (18, 19, 2 0 , 72), sodium 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, POtassium ferricyanide (14), a i r ( 2 5 ) , and amonium persulf 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 format 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 unid 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
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. 9
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-Diethyldithiocarbamoyl1-thio-l3-D-glucopyranosiduronic Acid
Man Rat
Diethyldithiocarbamic Acid, Mice Methyl Ester
(36 1 (31932) (34)
Hydrogen Sulfide
Sulfate
Rat
(31)
Gessner and Jakubowski have proposed the metabolic scheme in Figure 7 ( 3 4 ) . 6.
METHODS OF ANALYSIS
6.1 Identification Tests Disulfiram is easily identified by the physical properties described in section 2 above. Where identification 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, ,N-C CH3CH2
CH CH 2;~-~>CH3CH2
‘s-s
‘\\ / ,C-N,
CH2-CH3 CH -CH3 2
CH3-CH
CH3-CH2,
S 2XN-C4 + -. CH3-CH2 ’ ‘SH
1
+ cs2
CH3CH2.N-C 4 CH3CH2 ’ \
CH3-CH2
,
I
A-‘
Other Metabolites
F i g u r e 7.
A
\
s-EH- (cH&1,-dHcmH
\ \ \
S
\
\
\
22 ..
CH3-CH2. 4S P C \ CH3-CH2 OH
4S
,N-C
\
\
HSCH~+
\ \ Y
=
s04 HCHO
-
--a
Glucuronic Acid Conjugate
Metabolic products of d i s u l f i r a m (34).
DISULFIRAM
raphy (section 6.7) may be useful.
6.2 Elemental Analysis The elemental composition of d i s u l f i r a m is as f o l -
lows : 7. Theory
Element
40.50 6.80 9.45 43.25
Carbon Hydrogen Nitrogen Sulfur
6.3 Colorimetric Methods 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-diethyldithiocarbamate 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-diethyldithiocarbamate 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 cup 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 cup 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 reagent, extracted i n t o chloroform, and determined colorime 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
NORRIS G. NASH AND RAYMOND D. DALEY
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 identification color reaction in Clarke's manual of drug identification (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) reported 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 solution. 6.4 Titration Methods Disulfiram is not sufficientlv basic to be titrated with acid even in non-aqueous media. However, several titration methods based on complex formation or oxidation have been reported. Ferreira (48) titrated an acidified carbon tetrachloride solution, containing from 15 to 25 mg of disulfiram, with a 0.15 bromate-bromide solution. The following reaction is given.
s,
s
(C H ) -N-C-S-S-C-N-(C H )
2 5 2
26 HBr
2 5 2
+ 13
Br2
+ 4 H2S04 + 2 HCOOH + 2
+ 20
H20
(C2H5)2NH
Analyses of the reaction mixture for sulfate and diethylamine, as well as the amount of bromine consumed, support the stoichiometry given above. Varga (49) determined disulfiram in the drug substance 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 minutes the excess periodic acid was determined volumetrically by addition of potassium iodide and titration of the iodine formed.
184
DlSULFlRAM
Roth ( 5 1 ) determined disulfiram i n crude products and fungicide formulations a f t e r conversion t o carbon dis 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 neut 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 addit 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 solut 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 thiocyanate 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 metabol 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 excess sodium azide with sodium arsenate a f t e r the r e a c t i o n was comp l e ted 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 devia 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 absorpt 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 abs 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 spectroscopy 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% recovery 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 determining d i s u l f iram i n t a b l e t samples. The pulse polarographic 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 gravimetrica 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 diluted plasma, liver homogenate, and diluted urine using a Sephadex G-25 gel filtration column. Goeckeritz (69) used paper chromatography to separate 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-ethano1 (1:l); (el acetone with dimethylformamide; (f) paraffin. 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 employed: (a) n-hexane on paper impregnated with 50% dimethylformamide solution in ethanol; (b) cyclohexane-nbutanol (1:l) on paper impregnated with 507. ethanol solution 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 absorbance 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 detected 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 determination.
.
187
NORRIS G. NASH AND RAYMOND D. DALEY
7.
REFERENCES
I.
C. J. Pouchert, "The Aldrich Library of Infrared Spectra", Aldrich Chemical Company, Milwaukee, Wisconsin, 1970, spectrum 369D. 2. A. T. Pilipenko and N. V. Mel'nikova, Zh. Neorg. Khim. 14 ( 7 ) , 1843-6 (1969); C.A. 71, 7 5 8 1 5 ~ . 3. L. J. C l l a m y , "Advances i n I n f r a r e d Group Frequencies", Methuen & Co. Ltd., London, 1968, pp. 212-214. 4. H. C. Brinkhoff and A. M. Grotens, Rec. Trav. Chim. Pays-Bas 90 ( 3 1 , 252-7 (1971). 5. M. L. Shankaranarayana and C. C. P a t e l , Spectrochim. Acta 21 (11, 95-103 (1965). 6. G. S c h i l l i n g y A y e r s t Research Laboratories, p r i va t e communicat ion. 7. H. C. Brinkhoff, A. M. Grotens, and J . J. Steggerda, Rec. Trav. Chim. Pays-Bas 89 ( l ) , 1117 (1970). 8. A. M. Grotens and F. W. P i j p e r s , Rec. Trav. Chim. Pays-Bas 92 (5), 619-27 (1973). 9. J. 0, Madsen, S.-0. Lawesson, A. M. Duffield, and C. D j e r r a s i , J. Org. Chem. 32, 2054-8 (1967). 10. C. E. Orzech, Ayerst Laboratories Lnc., p r i v a t e communication. 11. D. J. Grabar and W. C. McCrone, Anal. Chem. 22 (4), 620-1 (1950). 12. I. L. Karle, J. A. E s t l i n , and K. Britts, Acta Crystallogr. (2), 273-80 (1967). 13. R e H. Cooper, U.S, Patent No. 2,375,083, May 1, 1945; C.A. 40, 1875. 14. R. R o t h s t e i n a n d K. Binovic, Rec. Trav. Chim. Pays-Bas 73, 561-2 (1954). 15. M. M. Miville, Ger. Patent No. 1,023,030, Jan. 23, 1958; C.A. 54, 17274~. 16. B. L. R i c h a z s , Belg. Patent No. 648,878, Sept. 30, 1964; C.A. 63, 14714a. 17. E. S. Blake, J. Am. Chem. SOC. 65, 1267-9 (1943). 18. H. S. Adams and L. Meuser, U.S. Patent No. 1,782, 111; C.A. 25, 303. No. 281,099, Nov. 6, 1962; C.A. 59, 19. Span. Pate; 73 78a. 20. J. P. Zumbrunn, Fr. Patent No. 2,038,575, Jan. 8, 1971; C.A. 75, 87658r.
-
188
DlSULF I RAM
21.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
33. 34. 35. 36. 37.
38. 39. 40. 41 42.
P a t e n t No. 1,796,977; C.A. 25, 2598. S. L. Kemichrom, Span. P a t e n t No. 354,918, NOV. 16, 1969; C.A. 72, 110847a. J. C. Counts, R. T. Nelson, and W. R. T r u t n a , U.S. P a t e n t No. 2,777,878, Jan. 15, 1957; C.A. 2,968li. J. L. Eaton, U.S. P a t e n t No. 2,464,799, Mar. 22, 1949; C.A. 43, 4690h. W. L. Cox and A. Gaydash, Fr. P a t e n t No. 1,322,579, Mar. 29, 1963; C.A. 59, 9812. G. L. Boyden, Ayerst L a b o r a t o r i e s Inc., p r i v a t e communication. G. D o m a r , A. Eredga, and H, Linderholm, Acta Chem. Scand. 3, 1441-2 (1949); C.A. 44, 10025f. L. E l d j y r n , Scand. J. Clin. Lab. Invest. 2, 202-8 (1950); C.A. 45, 6298c. H. Linderholm and K. Berg, Scand. J. C l i n . Lab. I n v e s t . 3 , 96-102 (1951); C.A. 45, 10286e. R. FischTr and H. Brantner, Naturwissenschaften 50, 551 (1963). J. H. StrOmme, Biochem. J.-92, 25P (1964). J. H. Stromme, Biochem. Pharmacol. 2,393-410 (1965). J. H. Stromme, Biochem. Pharmacol. 15, 287-97 (1966). T. Gessner and M. Jakubowski, Biochem. Pharmacol. 21, 219-30 (1972). 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. 64, 1 8 2 2 4 ~ . J. Kaslander, Biochim. Biophys. Acta 71, 730-2 (1963). K. J. D i v a t i a , C. H. Hine, and T. N. Burbridge, J. Lab. Clin. Med. 2, 974-82 (1952). S. L. Tompsett, Acta Pharmacol. Toxicol. 2, 20-2 (1964). G. Bors and N. I o a n i d , Farmacia (Bucharest) 2, 593-a (1965); C.A. 64, 8 7 8 4 ~ H. Patzsch, Deut. Apoth. Ztg. 94, 1284-6 (1954); C.A. 53, 202291. C. P a q u o t and J. Mercier, Rev. Franc. Corps Gras 6 , 695-9 (1959); C.A. 54, 6158g. N. M. Egorova and V. I. Trukhina, Nauch. T r . Perm. Farm. I n s t . 4, 96-7 (1971); C.A. 79, 149338e. G. C. B a i l e y , U.S.
-
-
-
-
189
NORRIS G. NASH AND RAYMOND D. DALEY
43. 44. 45
.
46. 47. 48. 49.
50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60 61, 62. 63.
L. Vignoli and B. Cristau, Congr. SOC. Pharm. Fr. C. R., 9th, 221-8 (1957); C.A. 53, 20690f. R. Fried, A. N. Masoud, and M. Francis, J. Pharm. 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. M. D. Kofman and A. P. Arzamastsev, Farm. Zh. (Kiev) 26, 52-6 (1971); C.A. 75, 91338d. A. P i o t G s k a , Diss. Pharm. Pharnucol. 24, 93-7 (1972); C.A. 76, 158430~. P. C. F e r r e i r a , Arquiv. biol. (Sao Paulo) 34, 103-5 (1950) E. Varga, A c t a Pharm. Hung. 28, 38-43 (1958); C.A. 53, 2541bc. G , Sandri, A t t i Accad. Sci. Ferrara 35, 17-24 (1957-58); C.A. 54, 18160d. H. Roth, Angew. Chem. 73, 167-9 (1961); C.A. 55, 13161e. I. Bayer and Mrs. G. Posgay, Acta Pharm. Hung. 2, 43-50 (1961); C.A. 56, 7431f. I. Bayer and E. Posgay, Pharm. Zentralh. 100, 65-71 (1961); C.A. 55, 14819a. A. I. Bukreev, V. V. Hinakova, and V. F. Soinikova, Med. Prom. SSSR l8, 32-3 (1964). 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. E. Fournier, L. P e t i t and A. Lecorsier, J. Eur. Toxicol. 2, 337-40 (1971); C.A. 76, 149631h. B. Scalicka, Prac. Lek l9, 408-12 (1967); C.A. 68, 58318e. S. Stefak, M. Blesova, and M. Zahradnicek, Cesk. Farm. 21, 64-6 (1972); C.A. 77, 39330~. B. S a l K s e n , L. Domange, J. Ann. Pharm. Fr. 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). "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. R. M. Belitskaya, Kauch. Rezina l9, 53-5 (1960); C.A. 55, 4025e. A. F. Taylor, Talanta ll, 894-6 (1964).
-
xy,
-
190
DlSULFlRAM
64 65. 66.
S. P o r t e r and A. Williams, J. 24, 144-145P (1972) D. G. Prue, C. R. Warner, and B. G.
Pharm. Pharmacol.
T. Kho, J. Pharm. Sci. 61, 249-51 (1972). H. Wozhn and E. Wempe, Arch. Pharm. (Weinheim) 285, 375-82 (1952); C.A. 47, 12126g. V. Parrak, Farmacia 22, 19-22 (1953); C.A. 49, 5776g. C. A. Parker and J. M. Berrimn, Trans. Inst. Rubber Ind. 28, 279-96 (1952); C.A. 47, 36021. D. Coeckeritz, Pharmazie 2,668-74 (1966); C.A. 66, 98510b. A. Farago, Arch. Toxikol. 22, 396-9 (1967); C.A. 67, 79553x0 R. H. Drost and J . F. Reith, Pharm. Weekbl. 105, 1129-38 (1970); C.A. 74, 23165. A. D. Cumnings and H. E. Siamons, Ind. Eng. Chem. c 20, 1173-6 (1928). J. Giral and C. Soler, Rev. SOC. Quim. Mex. 2, 167-70 (1958); C.A. 53, 11220d. 7
67. 68. 69. 70.
71. 72. 73.
-
-
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 library 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.
Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectrum 2.5 Rotation 2.6 Melting Range 2.7 Solubility 2.8 Crystal Properties
3.
Synthesis
4.
Stability
5.
Drug Metabolic Products
6.
Methods of Analysis 6 . 1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Spectrofluorometric Analysis 6.4 Colorimetric Analysis 6.5 Chromatographic Analysis 6.51 Paper 6.52 Thin-Layer 6.53 Gas - Liquid
7.
Identification and Determination in Body Fluids and Tissues
8.
References
-
Degradation
193
KLAUS FLOREY
1.
Description 1.1 Name, Formula, Molecular Weight Estradiol Valerate is estra-l,3,5 (10) triene-3,17p-diol-17-valerate (pentanoate).
Mol. Wt. 356.51
c2 3H3203
1.2
Appearance, Color,Odor White, odorless, crystalline powder.
2.
Physical Properties 2.1 Infrared Spectrum The infrared spectrum of estradiol valeratel is presented in Figure 1. 2.2
Nuclear Magnetic Resonance Spectrum 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 T
C- 1H
2.86
C-2H
3.38 (quartet)
1H, 2H = 9 ; 2 H y 4 H =2.5
C-4H
3.43
2 H y 4 H = 2.5;4H, 6H =1
C-
18H
(doublet)
Coupling Constants T (in Hz)
(multiplet)
1Hy2H = 9
9.19 (singlet)
r \D
VI
C-17aH
5.25
(triplet)
ester - CH3
9.09 (triplet)
6.5
phenolic-OH
4.94 (singlet)
Concentration dependent
16H, 17H = 7.5
; 4
FREQUENCY (CM')
c
W
Q\
WAVELENGTH (MICRONS)
Figure 1.
Infrared Spectrum of Estradiol Valerate in Mineral Mull. Instrument: Perkin-Elmer 621.
1_
T
I
F i g u r e 2.
I I
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
2.3
U l t r a v i o l e t Spectrum EtoH
Xmax
281 nm E1% 613 1c m
Fmax 281 nm; I m a x 287 nm;
= 20906 = 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 Thus a second s e r i e s by e x p l u s i o n of CO o r HCO. 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 benzylbenzoate and in dioxane; sparingly soluble in sesame oil and in peanut oil8
.
2.8
Crystal Properties 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
3.50 3. 3 1 3.22 3.14 3.02 2.96 2. 32
0.06 0.60 0.21 0.24 0.51 1.00 0.46 0.34 0.32 0.13
11.5 10.0 6.95 6.21 5.97 4.98 4.68 4.12 3.96 3.83
*d = i n t e r p l a n a r d i s t a n c e
0.19 0.34 0.08 0.07 0.09 0.08 0.05
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
.
Stability - Degradation Estradiol Valerate is very stable as a solid. In solution under certain conditions, particularily alkaline, saponification to valeric acid and estradiol can occur.
4.
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 6.1 Elemental Analysis % Calc. C 77.49 H 9.05 0 13.46
% Found 4 77.54;77.42 9.00: 9.20
--
6.2
Spectrophotometric Analysis 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). 6.4
Colorimetric Analysis E s t r a d i o l v a l e r a t e responds t o colorimetric 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,phosphomolybdic 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 solut 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
t 4 0
reaction (see section 7). 6.5 Chromatographic Analysis 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 guidestrip 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 Thin-layer chromatographic systems have been tabulated in Table 11. Table I1
- -
Solvent S y s tem Ref. Ref. Absorbant Magnesium silicate Benzene-ethanol(9:l) 0.52 19 Magnesium silicate Chloroform 0.62 19 Stationery phase:mineral oil Silica gel G 0.64 20 Mobile phase:50% acetic acid Silica gel G Stationery phase:propylene glycol 0.60 22 Mobile phase:cyclohexane-pet. ether (1:l) Stationery phase:tetraethylene glycol Silica gel G -24 Mobile phase:xylene Detection systems. Phosphoric acid (1:5)spray (green color);conc. sulfuric acid, p-toluenesulfonic acid.
1
3
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.
.
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. 7.
206
ESTRADIOL VALERATE
8. 1.
2. 3. 4. 5. 6. 7. 8. 9. 10.
11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
References 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. 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. 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. 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). 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. 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 ) 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. U.S.P. X V I I I 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. 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). 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 ) . 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. 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 ) . 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 ) . 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. 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). B r i t i s h P h a r m a c o p o e i a 1973 p. 330. 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). 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 ) . 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). B.
.
-
-
-
207
KLAUS FLOREY
21. 2 2.
23. 24. 25. 26.
27.
T. D i a m a n s t e i n and K. Lorcher, J. Anal.Chem. 191,429(1962). 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 ) . 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 ) . 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. 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 ) . 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 ) . 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. 2.
3. 4.
5. 6.
7.
Description 1.1 Name, Formula, Molecular Weight 1 . 2 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Spectrum 2.4 Mass Spectra 2.5 Rotation 2.6 Melting Range 2.7 Solubility 2.8 Crystal Properties Synthesis Stability - Degradation Drug Metabolic Products Methods of Analysis 6 . 1 Elemental Analysis 6.2 Spectrophotometric Analysis 6.3 Spectrofluorometric Analysis 6.4 Polarimetric Analysis 6.5 Polarographic Analysis 6.6 Chromatographic Analysis 6.61 Paper 6.62 Thin-Layer References
210
H Y DROXYPROGESTE RONE CAPROATE
1.
Description 1.1 Name, Formula, Molecular Weight Hydroxyprogesterone caproate is 17-~-(1-oxohexyl)oxy~-4pregnene-3,20-dione; also 17-hydroxypregn-4-one-3,2O-dione hexanoate
# 20
'iH3 c=o OCOCH2 (CH2)3CH3
0
Mol.Wt. : 428.62
C27H4004 1.2
Appearance, Color, Odor White or creamy white odorless crystalline powder. 2.
Physical Properties 2.1 Infrared Spectrum The infrared spectrum of hydroxyprogesterone 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 C-18 c-19 c-21 Ester methyl
4.20 singlet 9.30 singlet 8.80 singlet 7.96 singlet 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
2.3
U l t r a v i o l e t Spectrum 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 carbons r o g r e s sbi vu et , loss of D-ring, C-ring and B-ring
&
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
I
215
0
m
m
0 k pc Q)
u C 0
0)
k 4J 10 Q)
F
h
0 LI pc X
0 LI
h
a 0
X w C
LI
Q)
4J
m
4J
o(
&
F
0 W
Figure 3.
0 0 T ( 0
Low Resolution Mass Spectrum of Hydroxyprogesterone Caproate. Instrument : AE1 MS-902.
HYDROXYPROGESTERONE CAPROATE
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 10 b e n z o a t e and sesame o r c a s t o r o i l
217
.
KLAUS FLOREY
Crystal Properties Dense needles from isopropanol2 The powder X-ray diffraction pattern is presented in Table I. 2.8
.
TABLE I Powder X-ray Diffraction Pattern of Hydroxyprogesterone Caproa te2
**
(2:
I/I 1
12.2 9.1 7.15 6.90 6.63 6.35 5.80 5.47 5.28 5.08
0.26 0.11 0.31 0.10 0.13 0.37 1.00 0.42
d
*d
0.21 0.28
4.64 4.53 4.05 4.00 3.88 3.80 3.70 3.62 3.30 2.88
0.21 0.33 0.09 0.11 0.10 0.11 0.15 0.11 0.08 0.09
-
= 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
c=o
e=0
OH
anhydride
CO (CH2)4CH3
0
c!
\D
Hydroxyprogesterone caproate is synthesized by acylation of 17a-hydroxyprogesterone (cf.4) with caproic acid in the presence of p-toluene sulfonic acid3. Variations of this basic method have also been It can also be prepared by oxidation of the corresponding reported’. 5-prengnen-17-01-3-one caproate2. 4. Stability - Degradation 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
5.
Drug Metabolic Products 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 4-14Clabeled hydroxyprogesterone caproate under anaerobic conditions and recovered allopregnane38, 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.
.
6. Methods of Analysis 6.1 Elemental Analysis Calc. % C 75.66 H 9.41 0 14.93
6.2
Spectrophotometric Analysis The compendia1 assay of hydroxyprogesterone caproate is based on the U.V. absorption maximum at 240 nm17. 6.3
Spectrofluorometric Analysis Fluorescence was used for detection in biological f luids15.
220
HYDROXYPROGESTERONE CAPROATE
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. 6.5
Polarographic Analysis 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 uns 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 hydroxyprogesterone 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 Magnesium silicate Magnesium silicate Magnesium silicate Magnesium silicate Magnesium silicate Magnesium s i1icate Magnesium silicate Silica gel Silica gel Silica gel Alumina
Solvent System Benzene-Ethanol (98:2) Benzene-Ethanol (9:1) Chloroform Chloroform-Ethanol (98 :2) Chloroform-Ethanol (96:4) Benzene-Dioxane (2:l) Ether-Ethanol (98:2) Benzene-Acetone (4:1) Benzene-Methanol (9: 1) Pet. ether, Benzene, Acetic acid, Water (67:33:85:15) Benzene-Acetone (4:1)
Rf -
Ref.
0.35 0.61 0.44 0.53 0.60 0.71 0.83 0.67 0.61 0.44
20
0.61
20 20 20 20 20 20
21 21 21
21
Detection system: U.V. light; sulfuric acid (dark brown): sulfuric-acetic acid (yellow red); vanillin-sulfuric acid (dark yellow)
HYDROXYPROGESTERONE CAPROATE
7.
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. 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 ) . 6. 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. 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 I n s t i t u t e , P e r s o n a l Communication. 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 .
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.
The h e l p of H.
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. 2.
3. 4. 5. 6. 7.
8.
Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physica1 Properties 2 . 1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectrum 2 . 4 Mass Spectra 2.5 Optical Rotation 2 . 6 Melting Range 2.7 Differential Thermal Analysis 2.8 Solubility 2.9 Crystal Properties S ynthesis Stability and Degradation Metabolism Identification Aesay Methods 7.1 Elemental Analysis 7.2 Colorimetric Analysis 7.3 Infrared Analysis 7.4 Gas Liquid Chromatographic Analysis 7.5 Polarographic Analysis 7.6 Automated Analysis 7.7 Radiochemical Analysis 7.8 Thin Layer Chromatographic Analysis 7 . 9 Paper Chromatographic Analysis References
226
ISOSORBIDE DINITRATE
1.
Description 1.1
Name, Formula, Molecular Weight
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 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.
f
1.2
" 4
Appearance, Color, Odor
Isosorbide d i n i t r a t e i s a white, odorless, cryst 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 lact 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 .
2.
Physical Properties
2.1
Infrared Spectra
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 pubAssignment of some of t h e s i g l i s h e d by Sammul et.al.3 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 47 1665 and 1635 asymnetric N 4 s t r e t c h i n g 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 0-N4 s t r e t c h i n g 47 865 221
Figure 1
-
I.R. Spectrum of Isosorbide Dinitrate, Lot No. 5123 V 6/1. Instrument: Perkin Elmer Model 21.
0.25% KBr P e l l e t
-
ISOSORBIDE DINITRATE
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-dianhydroh 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 solut 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 .
2.2
Nuclear Magnetic Resonance Spectra
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. 2.3
U l t r a v i o l e t Spectrum
Isosorbide d i n i t r a t e gives no eak maxima i n t h e but does absorb wavelength region of 400 nm. t o 220 run.’, 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./ 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.
3
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 of
Isosorbide Dinitrate, Lot No. 5123 V 6/1. Solvent: deutere chloroform, internal standard: tetramethylsilane. Instrument: Varian A-60.
ISOSORBIDE DINITRATE
ions under b a h E I and C I conditions a r e given i n Table 111.
&I
23 7 190 144 127
Table I11 Mass Spectral Assignments of Isosorbide D i n i t r a t e Chemical Ionization Electron Im act R.I.(%> m/e R.I. % S ecies 15 2362.5 M+ 24 W-HNQ 143 75 M-HN4, N 4 MIP-HNQ, N Q 126 60 100 M-HNC+, HNQ M-HNOZ, CHON4 27 N 4 114 100 MH+-HN%, H 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 concen1 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" and Goldberg" +134O. 2.6
fpund thelo(]:O
t o be +141°
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.' report .5OC.-52OC. We found a value of 70°C.-71.50C., Class 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.
,
IJ',
2.7
D i f f e r e n t i a l Thermal Analysis
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. 15 U.S.P. Solvent Authors Merck Index' 0.5 mg./ml. sparingly soluble very s l i g h t l y water Soluble f r e e l y soluble very s o l u b l e 7 1 g./ml. acetone f r e e l y soluble sparingly 24 mg./ml. alcohol soluble
-
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 reextracted 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-dimethylphenol 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 phenold 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 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 c e t i c acid and 2.0 m l . of phen. ’ ’ ’ 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 isosorbide 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
__
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 diazot 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 absorbance 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 procedure. 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 separat 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 detection. 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 operated 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 ionizat i o n d e t e c t o r temperature a t 18OOC.; and nitrogen flow r a t e The i n t e r n a l standard used was m-dinitroa t 55 ml./min. 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 dimethylchlorosilylated 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 monon 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 obtained 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 isosorbide 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 Diatoport 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 isosorbide and the two mononitrates. 7.5
Polarographic Analysis
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 electrode i n a 0.5 N t e t r a m e t h y l m n i u m hydroxide aqueous solut 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 potent 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 approp r i a t e f o r counting C. P4 7.8
Thin Layer Chromatographic Analysis
Several TLC s y s t e m f o r t h e separation of isosorbide 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 ascending 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 solut 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. Diphenylbenzadium 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 Chromatoof 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). 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 expos 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.
21
Isosorbide d i n i t r a t e 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 methanolhydrocarbon 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
ISOSORBIDE DINITRATE
8.
References 1. 2.
Merck Index, 8 t h Ed., Merck and Co., Inc.(1968). Benjamin K. A y i , Wyeth Laboratories, Personal Communication. 3. 0. R. Sammul, W. L. Brannon, and A. L. Hayden, J. A s s Off. Agr. Chem., 47 (51, 918(1964). 4. L. D. Hayward, D. J. Livingstone, M. Jackson, and V. M. Csizmadia, Can. J. Chem.,*, 2191(1967). 5. Bruce Hoffmann, Wyeth Laboratories, Personal Communication. 6. F. J. Hopton and G. H. S. Thomas, Can. J. Chm., 47, 2395 (1969). c 7. N. J. DeAngelis, Wyeth Laboratories, Unpublished r e s u lts 8. L. D. Hayward, R. A. Kitchen, and D. J. Livingstone, Can. J. Chem.. 40, 434(1962). 9. Charles Kuhlman, Wyeth Laboratories, Personal Communication. 10. M. Jackson and L. D. Hayward, Can. J. Cham, 38 496(1960). 11. L. Goldberg, Acta Physiol. Scand., IS, 173(1948); C.A. 42, 5564d. 12. L. F. Wiggins, Advances i n Carbohydrate Chem., 5, 206 (1950). 13. S. E. Forman, C. J. Carr, and J. C. Krantz, J. Am. Pharm. h s o c . , 30, 132(1941). 14. USP XVIII, Mack P r i n t i n g Co., P. 935(1970). 15. USP XIX, i n print. 16. North American P h i l i p s Company, Mount Vernon, New York. 17. L. F. Wiggins, J. Chem. SOC., 4, (1945). 18. S. E. Forman, C. J. Carr, and J. C. Krantz, J. Am. Pharm. ASSOC., 30, 132(1941). 19. P. M. Kochergin and R. M. Titkova, Med. Prom.S,S.S.R. 13, NO. 8, 18-20(1959). 20. D. E. Reed, J. F. May, L. G. Hart, and D. H. McCurdy, Arch. I n t . Pharmacodyn. , 318-336(1971). 21. J. Rutgers, Wyeth Laboratories, unpublished r e s u l t s . 22. K. Dilloway, Wyeth Laboratories, unpublished r e s u l t s . 23. A. J. Dietz Jr., Biochem. Pharmacol., 2 (121, 24478(1967). 24. D. A. Sherber, M. Marcus, and S. Kleinberg, Biochem. Pharmacol. , l9, (2) , 607-12(1970). 25. S. F. Sisenwine and H. W. Rueliue, J. Pharmacol. Exp. Ther., 1 7 6 ( 2 ) , 296-301(1971). 26. L. A. Crandall, C. D. Leake, A. S. Leovenhart, and C. W. Muehlberger, J. Pharmacol. Exp. Ther., 2, 283
.
-
191,
-
243
LUCIAN0 A. SlLVlERl AND NICHOLAS J. DeANGELlS
(1929) Y. Nagase, Y. Kanoya, A. Sugiyama, and H. Haruhiko, Yakaguaku Zasshi, 8 5 ( 2 ) , 119-25(1965). 28. E. Sornoff, J. A s s . Off. Agr. Chem., 38, 637(1955). 39, 630(1956). 29. 9- Ibid 40, 815(1957). 30. 9- Ibid 31. "Official Methods of Analysis of the Association of O f f i c i a l Agricultural Chemists", 10th Ed., 605(1965). 32. D. Gutekunst, Wyeth Laboratories, unpublished r e s u l t s . 33. E. E. Garcia, Anal. Chem., 39, 1605(1967). 34. J. Poole, Wyeth Laboratories, unpublished r e s u l t s . 35. Jonas Carol, J. Ass. Off. Agr. Chem., 42, 468(1959). Ibid , 43, 259(1960). 36. 2 37. D. Woo, J. K. C. Yen, and P. Sofronas, Anal. Chem., 45(12), 2144-45(1973). 38. I. W. F. Davidson. F. J. Dicarlo. and E. I. Szabo, J. Chromatogr., S?, 345-52(1071): 64 39. M. T. Rossell and M. G. Bogaert, J. Chromatogr., -' 364-7(1972). M. T. Rossell and M. G. Bogaert, J. Pharm. Sci., 62 40 (51, 754(1973). 41. B. C. Flann, J. Phann. Sci., 58(11), 122(1969). 42. J. Bathish, Wyeth Laboratories, unpublished r e s u l t s . 43. C. Davis, Wyeth Laboratories, unpublished r e s u l t s . ,440 L. D. Hayward, R. A. Kitchen, and D. J. Livingstone, Can. J. Chem., 40, 434-40(1962). 45. M. Jackson and L. D. Hayward, ,J. Chromatogr.,>, 166-69(1961) 46. Lot No. 5123 V 6/1, Wyeth Laboratories. 47. R. D. Guthrie and H. Spedding, J. Chem. S O C . , (1960). 48. L. Bellamy, "The I n f r a r e d Spectra of Complex Molecules," 2nd Ed., J. Wiley and Sons, Inc., New York, N. Y., 1964.
27.
-
-
-
-
953
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 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor
2.
Physical Properties 2.1 I n f r a r e d Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 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 2.11 D i s s o c i a t i o n Constant
3.
Synthesis
4.
Stabi 1 ity-Degradat ion
5.
Metabolism
6.
Methods o f Analysis Elemental Analysts Phase S o l u b i l i t y Analysis Nonaqueous T i t r i m e t r l c Analysis Color imeiir i c Anal ys is Spectrophotometric Analysis Spectrof 1 uorometr l c Anal ys i s Gas Chromatographic Analysis Thin Layer Chrornatographlc Analysls
6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8
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
zol i none
16"1 4N20 1.2
Molecular Weight:
bH)-qu ina-
250.30
Appearance, Color, Odor
Methaqualone occurs as a w h i t e , odorless, c r y s t a l l i n e powder.
2.
Physical P r o p e r t i e s 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 1672 1603, 1595 and 1471 1340 and 1326 778, 760, 720 and 703
CH s t r e t c h i n g Amido carbonyl s t r e t c h i n g Aromatic s k e l e t a l v i b r a t i o n Aromatic C-N s t r e t c h i n g CH deformation o f aromatic r i n g
247
METHAQUALONE
Nuclear Magnetic Resonance Spectrum (NMR)
2.2
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 Protons
Chemical Shift (ppm)
Signal Appearance
203 i none
(Quinar i ng)
3
2.06
s ing 1 et
CH
(Tolyl)
3
2.15
s i ng 1 et
Aromatic Protons
8
Group
CH
3
2.3
7-8.4
multiplet
U1 trav iolet Absorption Spectra (UV)
Methaqualone exhibits characteristic ultraviolet absorption properties in different solvents. These characteristic 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
225.5 265
214.0 249.0
140.9 (35.26 X 103) 38.0 ( 9.51 X 103)
Ethanol (95%)
225.5
214.0
139.6 (34.94 X
249
lo3)
i ,
I
250
I
METHAQUALONE
Figure 3 Ultraviolet Absorption Spectra of Methaqualone C .a
A : Methaqualone In methanol
( I :200,000)
.0
B : Methaqualone i n ethanol
(I
:zoo ,000)
(95%)
C : Methaqualone in 1 N H C l ( I :200,000)
.7
.6
"
y.5
a m w
Y) 0
a m
.4
.3
.2
.I
',/--I 200
I
I 220
I
I
1
I 260
240 NANOMETERS
25 1
1
I 280
I
I 300
DAHYABHAI M. PATELetal.
265.0
249.0
37.7 ( 9.43 X 103)
H C l (1 N)
235.0 -270
215.5 258.5
133.0 (33.42 X lo3) 30.8 ( 7.71 X 103)
H C l (0.1 N)
234.5 -270
215.5 257.5
133.4 (33.39 X lo3) 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) 250 235
145 144 132
91
76 65 50
41
Intensity Strong Very Strong Weak Weak Med ium Strong Med iurn Med i um Med ium Very Weak
252
Fragment Ion Molecular i o n (M) M-CH M-C t$N M - [ $ H + CH3]
M-C7$4L
M-C H N20 C H9-$H 7 7 2 C7H7-C2 C 6H4 C2 H
-
a
C9H1 0N-C7H7
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 We
160
180
200
220
260
DAHYABHAI M. PATEL et a/.
Med i um
39 2.6
Me1 t i ng Range
The m e l t i n g range o f methaqualone i s dependent X V I I I Class 1 on the r a t e o f heating, Uslng the U.S.P. procedure, methaqualone me1 t s between 114"-117"C ( 1 ) . 2.7
Optical Rotation
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) The A Hf w i t h the temperature programing a t lO"C/minute. 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
Water Methanol E t ha no1 I sopropanol Ether Chloroform Benzene Acetonitrile Petroleum Ether (B.P.
S o l u b i l i t y . ( g / l O O ml) 0.03
16.58 12.53
4.76 3-71
>4 0
28.00
>4 0 30-60")
254
0.33
a,
0
C
3
m
c
CT (D
a,
U
Jz
x rc
E
0
E
0
ul
E
a, Jz
I-
u In n
255
0 0 W C
0 w
3 XJ
?
Y
DAHYABHAI M. PATEL et a/.
2.10
Crystal Properties
The X-ray powder d i f f r a c t i o n p a t t e r n o f methaqualone 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)*
15
9.4
8.2
25 25
7.76 7.30
50
65
6.95
6.10
25
75
5.53 4.76 4.13
100
30 20 30 30
4.00
3.75 3.66 3.53 3.39
15
60
15 15
3.24
3.17
3.12 3-05
*
**
30 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)
2.11 D i s s o c i a t i o n Constant 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 rapi 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 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 phase (p = 0.036 h r ' l ) . 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 0-g 1 ucuron i d e conj u pr i nc ip l e monohydroxy metabol i t e s 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 anthranil ic acid
Methaqualone o-to1 u i d i ne
o
+
M
I
-
+ I 0
-
=
N
u
p u
o I +
x
6:
M 259
Y
U
.-
c
Y
-03 -.-u u .-C I P , "I O
c
N
mI
E
~
mI
U
2u :oc " uz .- .e U
U
a
c
DAHYABHAI M. PATEL et a/.
hepatic r e c i r c u l a t i o n o f metabolites occur. A t a therap 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.
Methods o f Analysis
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 C
H N
6.2
9: Theory
76 77 5.64 11.19
4; Found
76-50
5.74 10.89
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 King and Perry (7) reported t h e g l a c i a l a c e t i c a c i d (6). 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
L Nn
\
N 0
In
-
I N q A l O S 6/3lfllOS 40 6m
261
0
-
:NOIlISOdW03 N O l l f l l O S
f In
f 0
m Ln
0 m
N In
N 0
In
-
0
-
In
0
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.
6.5 Spectrophotometric Analysis 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 borohydride 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.
Reference
Col umn
Carrier Gas
Column Internal Temp."C Standard
5
X 4 m., I.D., N2 glass tubing, 10% 45 ml/ SE-30 on Chromosorb min WAW(DMCS), 100-120 mesh
240"
Diphen- 2.17 hyd ram i ne
4 ft. X 2 mm., I.D.,
240"
Codeine
0.53
4 ft.
X 4 m., I.D., N2 glass t u b i n g , 3.8% 45 ml/ UC-Wg8 on Chromosorb rnin W(H.P.), 80-100 mesh
2300
Codeine
0.56
He 2 ft. X 4 mm., I.D., glass tubing, 3% XE- 50 ml/ rnin 60 on Gas Chrom Q, 100-120 mesh
180"
Butyl
2.81
7 ft. X 4 mm., I.D., Np glass t u b i n g , 3% 55 ml/ Cyclohexanedimethan- rnin 01 succinate 85-100 mesh
200"
Buto2.14 barb i tone
N2 2 ft. X 4 mm., I.D., glass tubing, 2.5% 40 ml/ SE-30 on Chromosorb min G, 80-100 mesh
200"
Codeine 0.42
2-m. X 3 mm., I.D.,
190"
Bu tobarbital 0.24
6 ft.
N2
glass tubing, 3% 30 ml/ OV-17 on Chromosorb min W(H.P.), 80-100 mesh
N2
glass tubing, 4% SE- 30 ml/ 30 and 6% QF-1 on min
263
S tea rate
DAHYABHAI M. PATEL er a/.
Chromosorb WAW (DMCS) , 80-100 mesh
*
Pentobarbital Phenobarbital
0.30 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 saturated w i t h water
Detection o f a s s o c i a t e d impurities
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 sopropanolldent 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 (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
Identification
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 aceta te-25% ammon i um hyd rox i de (6:2:2)
265
Sepa ra t ion of methaqualone from the metabol i tes
DAHYABHAI M. PATELetal.
7.
References
1.
2.
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. Brown, S. S. and Smart, G. A,, 466-468 (1969).
J. Pharm. Pharmacol.,
21,
3.
nthese De La MethylHerbert, M. and Plchat, L., 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.,
-7,
8.
Nakano, M.,
9.
P i r l , Joerg, N., (1972).
10.
J. Ass. Pub. Anal.
55-59 (1969). Yakuzaigaku,
22, 267-269
(1962).
e t a l . Anal. Chem. 44, 1675-1676
Douglas, J. F. and Shahinian, S., J. Pharm. Sci.,
62,
835-836 (1973).
42,
39-44 (1969).
11.
Berry, D. J., J. Chromatog.,
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.,
14.
Stahl, E., Thin Layer Chromatography, 2nd E d i t i o n , Spring-Verlag Berlin-Heideberg, New York, 539.
.,
Am. J. Vet. Res.,
266
33,
203-207 (1972).
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 NAME, FORMULA, MOLECULAR WEIGHT 1 . 2 APPEARANCE, COLOR, ODOR 1.1
2,
PHYSICAL PROPERTIES 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8
INFRARED SPECTRUM NUCLEAR MAGNETIC RESONANCE SPECTRUM MASS SPECTRUM ULTRAVIOLET ABSORPTION SPECTRUM MELTING RANGE DIFFERENTIAL SCANNING CALORIMETRY SOLUBILITY OPTICAL ROTATION
3.
SYNTHESIS
4,
METABOLISM
5.
METHODS OF ANALYSIS 5 ,1 5.2 5.3 5.4 5.5 5.6
5.7
6.
ELEMENTAL ANALYSIS PHASE SOLUBILITY THIN-LAYER CHROMATOGRAPHIC ANALYSIS VAPOR PHASE CHROMATOGRAPHY SPECTROPHOTOMETRIC ANALYSIS COLORIMETRIC ANALYSIS A. BLUE TETRAZOLIUM B. ISONICOTINIC ACID HYDRAZIDE TITRIMETRIC ANALYSIS
REFERENCES
269
ARVlN P. SCHROFF AND ERNESTS. MOYER
1. Description 1.1 Name, Formula, Molecular Weight Norethindrone is 17a-ethinyl-17fl-hydroxy-4estren-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
Molecular Wt. 298.41
C20H2602
1.2 Appearance, Color, Odor White to creamy white, odorless, nonhygroscopic, crystalline powder. 2. Physical Properties 2.1
Infrared Spectrum
The infrared absorption spectrum of norethindrone 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
-x
Intensity*
Vibrational Assignments OH s t r e t c h i n g
2.95
3400
v,sh,b
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
14.65
680
m,b
OH wagging 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. 2.2 Nuclear Magnetic Resonance Spectrum Cross, Landis, and Murphy5 reported the chemical shift of the C18 angular methyl group of norethindrone 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 containing 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 norethindrone 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
n v
0
110 273
215
.--
, ~ " " " ' - ' ~ ' . . - ' . . . ' ~ . ." .- " . ~ . . . . ~ . ' . . ~ . . . . ~ . . . . 80
1 . . . . 1 . . . . 1 .
70
b0
50
F i g u r e 11.
M
"'
40
30
20
NMR S p e c t r u m o f N o r e t h i n d r o n e
10
0
E m
275
a, E 0
k E
a .I+
c k
a,
c,
0 z 0
+I
5 k
a,
u
c,
a v1
v) v)
3! a,
H H H
k 9 M
.I+
c4
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:
2.4
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
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 confirmed 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 chromatographic 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
9 0
7 6 W
0
Z
U m
5
Q
9m
a
4
3
2 1
0
2 3
Figure I V
I
I
220 240 260 280 WAVELENGTH ( n m r
300
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 In our experience, most of t h e range of 202-207'C. 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
--
--
2.6
D i f f e r e n t i a l Scanning Calorimetry
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 defined 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. 2.7
The temperature of r e v e r s a l .
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
170
180
1
1
190 200
I
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
2.8
Optical Rotation The following r o t a t i o n s have been reported:
3.
Ia1D
-25"
(Chloroform)
['ID
-24'
(Chloroform)
['ID
-31.7'
(Chloroform)
[a],
-33'
(Chloroform)
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 Chromium t r i o x i d e oxidation from 19-nortestosterone I . 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 17aethynyl-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 The general as reported by Barber -e t a l l 9 and Meyer?O 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 They r a t s was studied by Watanabe and co-workers?l 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
&-&
0
0
I
'I
IV Yicherne 1
I
Ill Synthetic Pathway to Norethindrone
282
NORETHINDRONE
&--’”
H
Scheme I1
Alternate Synthetic Pathway to Norethindrone
283
ARVlN P. SCHROFF AND ERNESTS. MOYER
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-norethindrone 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 cow 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 radioactivity 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. et a130 observed the urinary radioactivity Kamyab -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 intravenous 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 radioactivity 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-ethynyl58-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 had reported excretion of 17a-ethynyl-178-estradiol after oral administration of norethindrone. These observations, however, were not supported by -in vitro investigations. 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 produced during work-up of the urine. Recent work of Sti1lwe11 and co-workers substantiated this observation. They found no ethynyl estradiol in urine after administration of norethindrone to a female. J 3 2 >
36
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.
Methods of Analysis 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 timesharing 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-norandrostendione, norethynodrel, log-hydroxy-norethynyltestosterone 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 3.8489063 8 . 6 4 9 2 7 14 1 1 51 38223 15.8089375 20.6400349 25.5679YSt:
I
30.0288913 3 4 - 410872V 42.2361355 5’1 * 8 5 6 0 5 19
Y CQORDJMATES 4.0180889 8 - 75336YO 9.3M7186 9.2340285 9.2dTU611 Y. 4029951 9.401 9 4 9 0 9.6795672 9.6282336 Y. 6Y19881
................................................. HBH K A N Y vOIIJTS TO
SLOPE
Pt F I T T E D ? 8
O.GlU19193
=
PERCENT P U K I l Y =
Y INTERCEPT
=
9.158532656
98.V80807304
I
Y
I
0
1
0
1
0 ,
O t .
I
*
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
ARVlN P. SCHROFF AND ERNESTS. MOYER
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 reported43 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 spectrodensitometer. 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 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 .
tablet^!^
5.4
Vapor Phase Chromatography
Oxidation of norethindrone t o 19-norandrostendione 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
5.5
Spectrophotometric Analysis
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. 5.6 Colorimetric Analysis 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
ARVlN P. SCHROFF AND ERNESTS. MOYER
B.
Isonicotinic Acid Hydrazide
Reaction of A4-3-ketosteroids with isonicotinic acid hydrazide (isoniazid) produces a ellow hydrazone 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 Administration Laboratory published an automatic method which is capable of performing single tablet assays. 5.7
Titrimetric Analysis
a potentioThe British P h a r m a ~ o p e i arecommends ~~ 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, and H. Smith, Steroids 11,649 (1968). 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). 10. R. A. Bastow, J. Pharm. Pharmac. 19, 41 (1967). 11. A. P. Shroff and G. Karmas, unpublished data. 12. H. J. Ringold, C. Djerassi, and A. Bowers, U.S. Patent 3,138,589. 13. M. Kuhnert-Brandstatter, E. Junger and A. Kofler, Microchem. J. 9 , 105 (1965). 14. H. Barth, Ortho Research Foundation, personal communication. 15 The Merck Index 8th Edition, Merck and Co., Inc., Rahway, N.J. 1968, p.748 16. F. B. Colton, U. S. Patents 2,655,518 (1952), 2,691,028 (1953) , and 2,725,378 (1955). 17. C. Gandolfi and P. deRuggieri, Gazz. Chim. Ital. 94, 675 (1964). Bowers, Canadian Patent 746,499. 18. 19. G. W. Barber and M. Ehrenstein, J. Org. Chem. 2 0 , 1253 (1955). 20. A . S. Meyer, Experientia 11, 99 (1955). 21. H. Watanabe, N. N. Saha a g D. S. Layne, Steroids 11, 97 (1968). 22. K. Matsuyoshi, Folia Endocr. Jap. 43, 91 (1967). 23. H. Breuer, U. Dardenne, and W. Nocke, Acta. Endocr. 33, 10 (1960. 24. H. Breuec Lancet 11, 615 (1970). a
29 1
ARVlN P.SCHROFF AND ERNESTS. MOYER
25. 26. 27. 28. 29. 30. 31. 32. 33.
34. 35. 36. 37. 38. 39. 40. 41.
42. 43. 44. 45.
46. 47. 48.
J. B. Brown and H. A, F. B l a i r , Proc. Roy. SOC. Med. 53, 433 (1960). K. F o z e r b y , S. Kamyab, P. L i t t l e t o n , and K. J . Dennis, Acta Endocr. Suppl. 2,136 (1966). E. Gerhards, W. Hecker, H. Hitze, B. Nieuweboer, and 0. Bellmann, Acta Endocr. 68, 219 (1971). S. I s h i h a r a , F o l i a Endocr. Jap. g , , , 5 5 (1966). R. Kaiser and H. Stecher, Arch. Gynak. 194,146 (1960). S. Kamyab, P. L i t t l e t o n , and K. Fotherby, J . Endocr. 39, 423 (1967). S. Kamyab, K. Fotherby, and A. Klopper, J. Endocr. 41, 263 (1968). H. Langecker, Acta Endocr. 37, 14 (1961). C . Lauritzen and W. D. Lehmann, Arch. Gyngk. 204, 212 (1967). D . S. Layne, T. Golab, K. Arai, and G . Pincus, Biochem. Pharmac. 12, 905 (1963). S. Murata, Folia Endocr. Jap. 43, 1083 (1967). H. Okada, M. Amatsu, S. Ishihara, and G . Tokuda, Acta Endocr. 46, 31 (1964). K. H. P a l m e r , J . F. Feierabend, B. Baggett, and M. E . Wall, J. Pharmac. Exp. Ther. 167, 217 (1969). C . A. Paulsen, Metabolism 14 ,313 ( 1 9 6 5 ) . 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 . Biochem. 3, 699 (1972). R. E. Huettemann, Ortho Research Foundation , personal communication, U. S. Pharmacopeia, 18th Rev., Mack Publishing Co. , Easton, Pa. 1970, p.453. K. Florey, U.S.P. Sub-Committee #6, personal communication. A. P. Shroff and C. J . Shaw, J. Chromatog. S c i . 10, 509 (1972). G . R. Keay, Analyst 93, 28 (1968). 5, Facts and Methods, F G M S c i e n t i f i c Research (3) 1964. B, A. Lodge, Can. J . Pharm. S c i . 5, 74 (1970). National Formulary, 13th Edirion, Mack Publishing Co. , Easton, Pa. , 1970, p.488. A. S. Meyer and M. C . Lindberg, Anal. Chem. 27, 813 (1955).
-
292
NORETHINDRONE
49. 50. 51. 52. 53. 54. 55. 56. 57.
R , V. Smith, T. H. Hassall, and S. C . Liu, J. Ass, O f f , Anal. Chem. 53, 1089 (1970). L. L. Smith and T. F o e l l , Anal. Chem. 31, 102 (1959). E . J . Umberger, Anal. Chem. 27, 768 (1955). A. E r c o l i , L. deGiuseppe and P. deRuggiere, Farm. Sci. Tec. (Pavia) 7, 170 (1952). J . Wu, J. Ass. O f f . AnalTChem. 53, 831 (1970). J . Wu, J. Ass, O f f , Anal. Chem. 54, 617 (1971). L. K. Thornton, Public Health Service (FDA) Drug Auto Analysis Manual, Method # 2 2 , April 1972, B r i t i s h Pharmacopoeia, 1973, University P r i n t i n g House, Cambridge 1973, p.323. 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.
2.
3. 4. 5. 6.
7.
Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 U l t r a v i o l e t Spectra 2.4 Mass Spectra 2.5 Melting Range 2.6 D i f f e r e n t i a l Thermal Analysis 2.7 S o l u b i l i t y 2.8 C r y s t a l Properties 2.9 Optical Rotation Synthesis S t a b i l i t y and Degradation Drug Metabolic Products Methods of Analysis 6.1 Elemental Analysis 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 6.3 Colorimetric Analysis 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 6.6 Polarographic Analysis 6.7 Chromatographic Analysis 6.71 Thin-Layer Chromatography 6.72 Gas Chromatography 6.73 Column Chromatography References
295
ANDREW M. SOPIRAK AND LEO F. CULLEN
1.
Description 1.1
Name, Formula, Molecular Weight
Norgestrel i s designated a s &-13-ethyl-l7&-ethynyl17-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-17hydroxy-18,19-dinor-l7&-pregn-4-en-20-yn-3~ne.
'21H28'2 1.2
Appearance, Color, Odor
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.
Physical Properties 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.
.
-
~~
~~
-~ ~
a. Table I Infrared S p e c t r a l Assignments of Norgestrel Frequency Vibration Mode Reference _ _ _(cm.-1) 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 A l i p h a t i c C-H s t r e t c h i n g 2940 and 2865 Conjugated C=O s t r e t c h i n g 1655 1615 C=C s t r e t c h i n g Methyl C-H asymmetrical bending 1448 OH i n plane bending 1420 and 1335 1360 Methyl C-H synnnetrical bending 1065 Alcoholic C-0 S t r e t c h i n g Acetylenic C-H bending 700 670 AOY4.Z
-
296
WAVILINGIH IMICROhlSl
w
\D
4
3aa
IWO WAVfNUMBfR ICM ‘I
Figure 1
-
I . R . Spectrum of Norgestrel (Wyeth Reference Standard Material, 1% KBr Pellet Lot #C-10484)
-
M
ANDREW M. SOPIRAK AND LEO F. CULLEN
2.2
Nuclear Magnetic Resonance
The 60 MH, nuclear magnetic resonance (NMR) spectrum, shown i n Figure 2, w a s obtained by d i s s o l v i n g norgest 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 spect r a l assignments a r e l i s t e d i n Table 11. Table I1 0
5.86
-&cH=c’
2.61
-WE
si ng 1e t
2.31
OH
elnglet
1.02 2.3
-
-
\
-% -5
singlet
triplet
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 #C10484) 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 tabul 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
am
Im,
m
ro
r..
f0 a0
4rJ
m
a CPS
. . . . , _ .m _ ._. _ _ , _ . .... . . , . _ _im.. . . . . . . . . . . . . . . . . . . . . .a.CPS .
t d W W
Figure 2
- NMR Spectrum of Norgestrel (Wyeth Reference 10484) - Solvent: deuterochloroform
Standard Material, Lot #C-
W
0 0
0 WAVELENGTH lnml
Figure 3
-
Ultraviolet Spectrum of Norgestrel (Wyeth Reference Standard Material, Solvent: 95% ethanol Lot #C-10484)
-
3 2 OH
MOL. WT. 312
C17H250 245
229 110 229
I
20
-
C19H2302 283
-CzHj
+-I
0
320
m/eFigure 4
- Mass
Spectrum 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 1 ' SH23' 2 245.1902 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 297 which corresponds t o the l o s s of t h e methyl r a d i c a l , followed by e 283 which r e p r e s e n t s cleavage t h e more i n t e n s e peak a t A peak a t 284 i s of t h e 13-ethyl r a d i c a l &C2H5.). observed from t h e l o s s of C2H4.
dz
d=
Ions of 4 2 245 and d ' 229 a r e explained by the cleavage 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 temperat 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. 2.6
D i f f e r e n t i a l Thermal Analysis
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 acetone 95% ethanol benzene ether water 2.8
111 mg./ml. 15 mg./ml. 13 mg./ml. 7 mg./ml. 2 mg./ml. l e s s than .01 mg./ml.
C r y s t a l Properties
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 13.9 14.2 14.5 15.2 15.8 17.4 18.0 18.9 19.8 21.2 21.8 23.3 24.9 25.3 26.0 26.3 27.6 29.05 30.4 31.8 34.7
8.08 6.37 6.24 6.11 5.83 5.61 5.09 4.79 4.69 4.48 4.19 4.075 3.817 3.575 3.520 3.428 3.389 3.232 3.073 2.940 2.814
0.02 1.00 0.53 0.81 0.20 0.72 0.17 0.06 0.06 0.42 0.04 0.05 0.19 0.24 0.12 0.09 0.09 0.07 0.07 0.10 0.10 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
40
40
40
30
30
w
0 vI
30
20
10
b
10
10
Figure 6
14
- X-Ray
18
22
2 8
10
26
M
34
3a
Powder Diffraction Pattern of Norgestrel (Wyeth Reference Standard Material, Lot fC-10484)
ANDREW
2.9
M.SOPIRAK AND LEO F. CULLEN
Optical Rotation
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 developed by Hughes and Smith 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 metha n o l i c hydrochloric acid c y c l i z a t i o n of t h e eeco-oestratetraene-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 corresponding 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-ammoniat 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
€83
.
4.
S t a b i l i t y and Degradation 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 temperat 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-ketos 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
KOH
I
CH30
IA
OH-
CH30H
C H30
IIA
1 lllA
CaC03
Pd
CH30 V
NaBH,
I
CH30H
continued Figure 7
- Synthetic Routes for Norgestrel 307
....
ANDREW M. SOPIRAK A N D LEO F. CULLEN
cn30&On
y
3
>
do
c n30
VI
Figure 7
VII
- Synthetic Routes €or Norgeetrel
308
(concluded)
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 supported 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 .
5.
Drug Metabolic Products
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
6.
Methods of Analysis 6.1
Elemental Analysis
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 C
H
% Theory 80.73% 9.03%
6.2
% Determined 80.66% 8.92%
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 chromophore 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 formulat 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 materi 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, dilut 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 norg 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 photochemi 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 These techniques can b e applied t o t h e a n a l y s i s of methods. 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-unsatur 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 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.
e
Gtlr6g3l developed a procedure f o r t h e determinai s based on t h e C l a i s e n cont i o n 0 € ~ ~ - 3 - k e t o s t e r o i d which s 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 chromophore 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 modera 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 chara 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 A4-3The method k e t o s t e r o i d s i n pharmaceutical 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.
preparation^.^'
6.3
Colorimetric Analysis 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 wellknown 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 reagent 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 temperat 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 extracted 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 combil o r developn a t i o n dosage forms do not c o n t r i b u t e t o t h 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 conjugated 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 norgest r e l i n t h e presence of i t s photochemical and thermal degrad 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 Consequently, 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
ANDREW M. SOPIRAK A N D LEO F. CULLEN
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 , accur a t e , and r a p i d automatic analyzer procedure has been adapted t o the analysis n o r g e s t r e l i n o r a l contraceptive t a b l e t formulations.
~5
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 2,4dinitrophenylhydrazones 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 norgest 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-nitrophenylhydrazine t o yield t h e corresponding hydrazone, which produces 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-ketos 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 adapted 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 spectrophotometr i c a l l y i n t h e v i s i b l e region.
312
NORGESTREL
6.4
Fluorometric Analysis
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., 1575 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 emiss i o n h a x of 550 nm. with an e x c i t a t i o n k a x 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 . Norg 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 potassium --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
ANDREW M. SOPIRAK A N D LEO F. CULLEN
6.6
Polarographic Analysis
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 6.7
Chromatographic Analysis
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. 6.71
Thin-Layer Chromatography
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.
6.72
Gas Chromatography
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 technique 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 norgest 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 extracted 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 evaporat 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. 6.73
Column Chromatography
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 admini 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 spectrome 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 quantit 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 Technique Rf Reference System Adsorbent 1, 2 0.37 53 Silica Gel GF A B Silica Gel GF 1; 2 0.45 53 C 1, 2 0.59 53 Silica Gel GF D 1, 2 0.53 53 Silica Gel GF E 1, 2 0.47 54 Silica Gel G impregnated with DuPont Lumines cent Chemical 609 F Silica Gel G 1, 2 0.46 54 impregnated with DuPont Luminescent Chemical 609 G Silica Gel G 3 0.54 26 Silica Gel G 4 0.73 1 H MN Silica Gel 1, 2 18 I
-
-
--
G-HR/W
A B C D E F G H
I 1 2 3
4
Solvent Systems Benzene:methanol (95: 5) Benzene:acetone (80:20) Chloroform:methanol (90: 10) Methylene ch1oride:me thanol: water (150: 9! 0.5) Petroleum ether: ethyl ether (10: 90) Petroleum ether: ethyl ether:diethylamine (4 0 : 60: 10) Chloroform: alcohol, U.S. P. (96: 4 ) N-hexanet ethyl acetate (1: 3) Benzene:chloroform (8:2) Visualization Techniques Ultraviolet light Sulfuric Acid Spray Reagent 10% Ethanolic Phosphomolybdic Acid Spray Reagent Antimony Trichloride Spray Reagent
315
ANDREW M. SOPIRAK AND LEO F. CULLEN
7.
References
1
2 3.
4. 5. 6.
7. 8. 9. 10.
P. M. Short, E. T. Abbe and C. T. Rhodes, Can. J. Pharm. S c i . , A, 8(1969). L. Bellarny, I I T h e I n f r a r e d Spectra of Complex Molecules," 2nd Ed., J. Wiley and Sons, Inc., New York, N. Y., 1964, Chapter 6. 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. A. A. Fernandez and V. T. Noceda, J. Pharm. Sci., 58, 740(1969). T. Chang and C. Kuhlman, Wyeth Laboratories, Inc., Personal Communication. D. C. DeJongh, J. D. H r i b a r , P. L i t t l e t o n , K. Fotherby, 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). W n i t ed S t a t e s Phannacopei a," 18th Revision , Mack Publishing Co., Easton, Pa., 1970, pp. 935-936. N. DeAngelis , Wyeth Laboratories, Inc., Personal Comnunicat ion. M. B. Freeman, Wyeth Laboratories, Inc., Personal Communication. 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., 4472(1964). V. Petrow, Chem. Rev., 70, 713(1970). G. H. Hughes and H. Smith, U. S. Patent 3,478,106 (1969) : i . S . Patent 3,442;920(1969). A. L. Wilds and N. A.-Nelson, J. Amer. Chem. SOC., -975 5360(1953). A. J. Birch and S. M. Mukherji, Nature, 163, 766 (1949). R. V. Oppenauer, Org. Syn., 2,18(1941). P. D. Klimstra, J. h e r . &am. Educ., 630(1970). L. F. Cullen, Wyeth Laboratories, Inc., Personal Communic a t ion L. F. Cullen, J. G. Rutgers, P. A. Luccheei and G. J. Papariello, J. Pharm. Sci., 57, 1857(1968). K. Savard, H. W. Wotiz, P. Marcus and H. M. Lemon, J. Amer. Chem. SOC., 75, 6327(1953).
,
-
1964,
11. 12.
13. 14. 15. 16. 17. 18. 19.
10,
.
316
NORGESTREL
20.
21.
22. 23 24. 25. 26.
27. 28 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.
R. E. Graham, P. A. Williams and C. T. Kenner, J. Pharm. Sci., !By 1152(1970). W. E. Hamlin, T. Chulski, R. H. Johnson and J. G. Wagner, J. Amer. Pharm. A s s . , S c i . Ed., 253(1960). K. Fotherby and C. A. Keenan, Acta Endocrinol. Suppl. -9138 83(1969). P. L i t t l e t o n and K. Fotherby, Acta Endocrinol. Suppl. 119, 162(1967). S. Kamyab, K. Fotherby and G. Wilson, J. Biochem., -9103 14(1967). 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. 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. A. J. Khoury and L. J. C a l i , Ann. N. Y. Acad. Sci., 153, 456(1968). L. K. Evans and A. E. G i l l i a m , J. Chem. SOC., 565(1943). I. E. Bush, Fed. Proc., 186(1953). N. B. Talbot. S. Ulick. A. Koupreianow and A. 301 Zygmuntowicz, J. Clin. Endocrinol. Metab., (1955). S. Gorog, Anal. Chem., 42, 560(1970). 292(1959). P. S. Chen Jr., Anal. Chem., S. Gerog, J. Pharm. S c i . , 57, 1737(1968). E. J. Umberger, Anal. Chem., 27, 768(1955). Y. P. John, J. A s s . Offic. Anal. Chem., 53, 83l(1970). F. M. Russo-Alesi, Ann. N. Y. Acad. S c i , , 511 (1968). C. D j e r a s s i and E. Ryan, J. h e r . Chem. SOC., lOOO(1949). A. G. Gornall and M. P. MacDonald, J. Biol. Chem., 201, 279(1953). F. D. Chattaway and G. R. Clemo,,J. Chem. SOC., 3041(1923). T. Nishina, Y. Sakai and M. Kimura, S t e r o i d s , 4, 255(1964). W. J. Mader and R. R. Buck, Anal. Chem., 24,. 666 (1952). J. E. Sinsheimer and E. F, Salim, Anal. Chem., 37, 566(1965). A. S. Meyer and M. C. Lindberg, Anal, Chem., 27, 813(1955). R. V. Smith, T. H. Hassall and S. C. Liv, ,J. A s s . Offic. Anal. Chem., 53, 1089(1970). P. M. S h o r t and C. T. Rhodes, S t e r o i d s , 6 .l, 217(1970).
2,
-
,
-
1943,
2,
15,
2,
153,
71,
-
1923,
317
ANDREW
46. 47. 48. 49
.
50. 51 52. 53.
54. 55.
M.SOPIRAK AND
LEO F. CULLEN
2,
E. P. Schultz and J. D. Neuss, Anal. Chem., 1662(1957). E. P. Schultz, M. A. Diaz and L. M. Guerrero, J. Pharm. Sci., 53, 1119(1964). M. P. Short and C. T. Rhodes, Can. J. Pharm. Sci., 26(19731_ I. E. Bush and A. A. Sandberg, J. Biol. Chem., 205, 783(1953). D. Abelson and P. K. Bondy, Arch. Biochem. Biophys., -' 57 208(1955). "United S t a t e s Phanacopei a," 1 8 t h Revis ion, Mack Publishing Co., Easton, Pa., 1970, pp. 454-455. J. G. Rutgers, Wyeth Laboratories, Inc., Personal Communication. M. B. Simard and B. A. Lodge, J. Chromatogr., 51, 517(1970). G. Moretti. G. Cavina, P. P a c i o t t i and P. Siniscalchi, I1 Farmaco; 27, 537(1972). K. Dilloway, Wyeth Laboratories, Inc., Personal Communication.
S,
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 1.2 Appearance, Color, Odor 2.
Physical P r o p e r t i e s 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 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.
Methods of Analysis E l e m e n t a l Analysis 6.1 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
PHENFORMIN HYDROCHLORIDE
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 -phenethylformamidinyliminourea hydrochloride and DBI.
CH2-CH2-NH-C-NH-C-NH2. I1 II N-H
HC1
N-H
Mol. W t .
C10H16N5C1
9
241.73
Appearance, Color, Odor 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. 1.2
2.
Physical P r o p e r t i e s
2.1
I n f r a r e d Spectrum 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 obt a i n e d on a Perkin-Elmer 621 Spectrophotometer from a KBr The s t r u c t u r a l assignments have been c o r r e l a t e d pellet, with t h e following band frequencies. Frequency (cm’l)
3400 3200 1660
Ass iqnment
- 3250
-
N-H
stretching
Amine s a l t N-H s t r e t c h i n g
3100
- 1610
;C=N
(a-P-unsaturated)
1590
-
1510
Amine s a l t bending
756
&
695
Phenyl (monosubstitutedl
Nuclear Magnetic Resonance Spectrum 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 follcrwi 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. 2.2
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
U l t r a v i o l e t Spectrum 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. 2.3
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
1x1 '0 'NN aOH
2 32
12.7
1 N NaOH
methanol
225
- 228
2 34
12,o 17.7
Mass 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. 2.4
2.5 176-178'
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
(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.
PHENFORMIN HYDROCHLORIDE
2.6
Solubility The s o l u b i l i t y of p h e n f o d n hydrochloride was determined a t room temperature i n t h e following s o l v e n t s .
mg/ml
Solvent
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
Isopropanol
Ether
Hexane
3.8
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 consequently 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' 11.8 and pKa" 2.7 a t 32'. I n our l a b o r a t o r y the apparent 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 rec 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. 2.9
D i f f e r e n t i a l Scanning Calorimetry 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 observed 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
-
.
- 39.8 cal/gram 9600 cal/mole Melting Range - 175 - 179OC
Heat of Fusion
Melting P o i n t
- 176.5OC
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 Thermalanalyzer (2). 2.10
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.
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 4.
326
PHENFORMIN HYDROCHLORIDE
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-hydroxphenformin 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.
-6.
Methods o f Analysis 6.1
Elemental Analysis 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 hydroc 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
Colorimetric Methods 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 tetrabutylammonium hydroxide t o form a ( 2 r l ) tetrabutylammniumbromthymol 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 observed. 6.4
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 hydroc 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) des 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. 6.5
Chromatographic Methods Phenformin hydrochloride can be determined by t h e following chromatographic methods. 6.51
Paper Chromatography 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 hydrochloride. 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 phenformin, 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
PHENFORMIN HYDROCHLORIDE
-
Solvent system Acetone :water
Rf
1:1
Reference
0.76
9,16
Isopropanol :water: ammonium hydroxide 40 t 9 t 6
0.73
16
Pyridinerbutanoltwater
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
1:l:i
6.52
Thin Layer Chromatography 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 The migration rate w a s approximately (40:5 :55) 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 determined 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 absorbance 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 chromatographmass 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
PHENFORMIN HYDROCHLORIDE
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., comunication.
3.
E.R.
Sci -.I
Garrett, J. Tsau, and P.H.
-
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S. Pharmacopeia X V I I I , p. 488.
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332
PROCAINAMIDE HYDROCHLORIDE
Raymond B. Poet and Harold Kadin
RAYMOND 6. POET AND HAROLD KADIN
TABLE OF CONTENTS 1.
Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor
2.
Physical Properties 2.1
Spectral Properties 2.11 2.12 2.13 2.14 2.15
2.2
Crystal Properties 2.21 2.22 2.23 2.24 2.25
2.3
Infrared Spectra Nuclear (Proton) Magnetic Resonance Spectra Ultraviolet Spectra Mass Spectra Fluorescence
Differential Thermal Analysis Differential Scanning Calorimetry Melting Range X-Ray Powder Diffraction Hygroscopicity
Solution Data 2.31 2.32 2.33 2.34
Solubility pKa Surface Chemistry 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 6.1 6.2 6.3 6.4
Elemental Analysis Identification Tests Spectrophotometric Methods Isolation and Chromatographic Methods 6.41 6.42
Solvent Extraction Paper and Thin-Layer Chromatography 6.43 Gas Chromatography 6.44 Ion-Exchange Chromatography 6.5 6.6 6.7
Spectrophotofluorometric Methods Titrimetric Methods Microbiological Methods
7.
Analysis in Biologic Fluids and Tissues
8.
References
335
RAYMOND 6. POET AND HAROLD RADlN
1.
Description 1.1 Name, Formula, Molecular Weight
Procainamide hydrochloride is benzamide, ~-amino-N-~2-diethylamino)ethy~monohydrochloride 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. H2N
@
C13H21N30'HCl
PH2CH3 !-NHCH~CHZN HC1 'CH2CH3 Molecular Weight 271.8
1.2 Appearance, Color,Odor Procainamide hydrochloride is a white-to-tan, odorless crystalline powder. 2.
Physical Properties 2.1 Spectral Properties 2.11 Infrared Spectra
Samples of procainamide hydrochloride were dispersed in a potassium bromide pellet or in mineral oil to obtain the infrared spectra given in Figures la and lb from a The Perkin-Elmer Model 21 prism instrument12 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 .
PROCAINAMIDE HYDROCHLORIDE
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 NH2 and NH 3389,3279,3205 2564,2457 HC 1 1639 Amide C=O 1538 S e c o n d a r y Amide NH2 a n d A r o m a t i c C=C 1600 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 hydrochloride 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 Tetradeuteromethanol. 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
C-N-CH2-
D2*
CH 2-
@ O
@
/H
C ' H2
2-CH 3 -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) 60-MHz S p e c t r u m ( F i g u r e 2 ) ( F i g u r e 3) ~ssignment H C 1 S a l t Base i n o f Prot o n i n HOCG3and HOCD3 a n d P o s i ti o n DOCD3 DOCD3 1.33 (t, 7.3) 1 . 2 2 ( t , 7.3) ( 9 , 7 . 3 ) 2 . 6 1 (9, 7 . 3 ) 3 . 2 9 Q * %3.35 (m) *'L2.60 (m) 0 * 3.47 ( t , 9 ) *3 . 7 1 ( t , 7 ) 7.70 (d, 8.6) 7.65 (d, 8 . 6 ) @ c
P N
0
CD
@
6.71
I
(d, 8 . 6 )
6.68
(d, 8.6)
Table 1, c o n t i n u e d . .
..
Assignment of Proton Position
(Figure 4 ) Base in HOCD3 and DOCD3 1.08(t, 7 ) 2 . 6 1 (9,7 ) ** 2 . 6 6 (m) ** 3 . 4 8 (m, 8 ) 7.58 (d, 8 . 5 ) 6 . 6 5 (a, 8 . 5 )
-
(Figure 5 ) Base in CDC13 1.04 2.57 2.62 3.46 7.59 6.59 6.79 4.04
(Figure 6 ) Base in Perdeuteropyridine
(t, 7 ) (9, 7 ) (m)
0.95
(t, 7 )
2.48 2.67
(t, 7)
(m) (d, 8 ) (d, 8 ) (Broad) (Broad)
3.71
(9,
7.13
(d, 8 ) (d, 8 ) (Broad) (Broad)
5.93 6.79 4.04
(q, 7) 7)
*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
I
w
P
P
10
I