Analytical Profiles of Drug Substances Volume 10

Analytical Profiles of Drug Substances
Volume 10
Edited by
Klaus Florey
The Squibb Institute for Medical Research New Brunswick, New Jersey
Contributing Editors
Rafik Bishara Lee T. Grady Glenn A. Brewer, Jr. Hans-Georg Leemann John E. Fairbrother Joseph A. Mollica Bruce C. Rudy
Compiled under the auspices of the Pharmaceutical Analysis and Control Section Academy of Pharmaceutical Sciences
ACADEMIC PRESS
1981
A Subsidiary of Harcourt Brace Jovanovich, Publishers
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EDITORIAL BOARD
Norman W. Atwater Rafik Bishara Jerome I. Bodin Glenn A. Brewer, Jr. Lester Chafetz Edward M. Cohen John E. Fairbrother Klaus Florey
Salvatore A. Fusari Lee T. Grady Boen T. Kho Hans-Georg Leeman Joseph A. Mollica Gerald J . Papariello Bruce C . Rudy Milton D. Yudis
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Library o f C o y r e s s C a t a l o g i y i n Publication Data Main e n t r y under t i t l e : Analytical p r o f i l e s of drug substances. Compiled under t h e auspices o f t h e Pharmaceutical Analysis and Control Sect ion. Academy o f Pharmaceutical Sciences. Includes bibliographical r e f e r e r c e s and index. 1. Drugs--Analysis--Collected works. 2. Chemistry, Pharmaceutical--Collected works. I. Florey, Klaus. 11. Brewer, Glenn A. 111. Academy of Pharmaceutical Sciences. Pharmaceutical Analysis and Control Section. [ONLM: 1. Drugs--Analysis--Yearbooks. QV740 A A 1 A551 RS189.A58 615' .1 70-187259
ISBN
0-12-260810-0
(v. 10)
AACRl
PRINTED I N THE UNITED STATES OF AMERICA
81 82 83 84
98 76 5 4 3 2 1
AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS
H . Y. Aboul-Enein, Riyadh University, Riyadh, Saudi Arabia A . A . Al-Badr, Riyadh University, Riyadh, Saudi Arabia N . W. Atwater, E. R. Squibb and Sons, Princeton, New Jersey S. A . Benezra, Burroughs Wellcome Company, Research Triangle Park, North Carolina W. F . Beyer, The Upjohn Company, Kalamazoo, Michigan R . Bishara, Eli Lilly and Company, Indianapolis, Indiana J . I . Bodin, Carter Wallace, Inc., Cranbury, New Jersey G. A . Brewer, The Squibb Institute for Medical Research, New Brunswick, New Jersey H . Brik, Gist-Brocades, Delft, Holland L. W. Brown, The Upjohn Company, Kalamazoo, Michigan L . Chafetx, Warner-Lambert Research Institute, Morris Plains, New Jersey C . C . Chiu, The United States Pharmacopeia, Rockville, Maryland H . P. Deppeler, Ciba-Geigy Ltd., Basel, Switzerland H . A . El-Obeid, Riyadh University, Riyadh, Saudi Arabia J. Fairbrother, Stiefel Laboratories Ltd., Sligo, Ireland L. V. Feyns, The United States Pharmacopeia, Rockville, Maryland K. Florey, The Squibb Institute for Medical Research, New Brunswick, New Jersey P. R. B . Foss, Burroughs Wellcome Company, Research Triangle Park, North Carolina S. A . Fusari, Parke-Davis, Inc., Detroit, Michigan L. T . Grady, The United States Pharmacopeia, Rockville, Maryland M . M . A . Hassan, Riyadh University, Riyadh, Saudi Arabia S. E . Ibrahim, Riyadh University, Riyadh, Saudi Arabia A . I . Judo, Riyadh University, Riyadh, Saudi Arabia
AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS
T . Kho, Ayerst Laboratories, Rouses Point, New York J. Kirschbaum, The Squibb Institute for Medical Research, New Brunswick, New Jersey K . Krummen, Sandoz, Basel, Switzerland H . G. Leemann, Sandoz, Basel, Switzerland G. G. Liuersidge, University of Nottingham, Nottingham, England M . A. Loutfy, Riyadh University, Riyadh, Saudi Arabia F . M a d , Sendoz, Basel, Switzerland J. Mollica, Ciba-Geigy Corporation, Suffern, New York 1. S. Mossa, Riyadh University, Riyadh, Saudi Arabia F. J. Muhtadi, Riyadh University, Riyadh, Saudi Arabia F . Nachtmann, Sandoz, Basel, Switzerland G. R . Padmanabhan, Ciba-Geigy Ltd., Suffern, New York G. Papariello, Wyeth Laboratories, Philadelphia, Pennsylvannia E . Riemer, Sandoz, Rasel, Switzerland B . C. Rudy, Mary Kay Cosmetics, Dallas, Texas R. W. Souter, Eli Lilly, Indianapolis, Indiana S. Sun, The United States Pharmacopeia, Rockville, Maryland P . G. Takla, University of Wales Institute of Science and Technology, South Wales, United Kingdom W . P . Wilson, Burroughs Wellcome Company, Research Triangle Park, North Carolina D. K . Wyatt, The United States Pharmacopeia, Rockville, Maryland M . D. Yudis, Schering-Plough, Inc., Rloomfield, New Jersey M . U . Zubair, Riyadh University, Riyadh, Saudia Arabia
PREFACE
Although the official compendia list tests and limits for drug substances related to identity, purity, and strength, they normally do not provide other physical or chemical data, nor do they list methods of synthesis or pathways of physical or biological degradation and metabolism. For drug substances important enough to be accorded monographs in the official compendia, such supplemental information should also be made readily available. To this end the Pharmaceutical Analysis and Control Section, Academy of Pharmaceutical Sciences, has undertaken a cooperative venture to compile and publish Analytical Profiles of Drug Substances in a series of volumes of which this is the tenth. The concept of analytical profiles is taking hold not only for compendia1 drugs but, increasingly, in the industrial research laboratories. Analytical profiles are being prepared and periodically updated to provide physiochemical and analytical information of new drug substances during the consecutive stages of research and development. Hopefully, then, in the not-too-distant future, the publication of an analytical profile will require a minimum of effort whenever a new drug substance is selected for compendial status. The cooperative spirit of our contributors has made this venture possible. It is gratifying to note that increasingly profiles are being written not only in industrial laboratories but also in academic institutions worldwide. All those who have found the profiles useful are requested to contribute a monograph of their own. The editors stand ready to receive such contributions. The goal to cover all drug substances with comprehensive monographs is still a distant one. It is up to our perseverance to make it a reality. Klaus Florey
xi
AMINOSALICYLIC ACID
Mahmoud M . A . Hassan, Ahmad I . Jado, and Muhammad Uppal Zubair
1. Description 1 . 1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Taste, Odor 2. Physical Properties 2.1 Crystal Properties 2.2 Solubility 2.3 Identification 2.4 Spectral Properties 3. Synthesis 4. Metabolism 5. Methods of Analysis 5.1 Nonaqueous Titration 5.2 Diazometric Assay 5.3 Spectrophotometry 5.4 Combined TLC and Colorimetry 5.5 Ultraviolet Method References
2 2 2 3 3 3 3 3 6 7 7 17 19 21 21 22 23 23 23 25
MAHMOUD M. A. HASSAN e t a ! .
1. DESCRIPTION 1.1 Nomenclature 1.1 1 Chemical Names a. 4-Amino-2-hydroxybenzoic acid.
b. 4-Aminosalicylic acid.
c. Benzoic acid, 4-Amino-2-hydroxy. The CAS Registry No. is [65-49-61. 1.1 2 Generic Name p-Aminosalicylic acid. 1.1 3 Trade Names Apas, Apacil, Deapasil, Hellipidyl, PAS,. PAS-C, Pamcyl, Pamisyl, Parasil, Pasorbic, Pasolac, Parasalicil, Parasalindon, Pasnodia, Propasa, Rezipas, Sanipirol-4,Para-Pas, Pasem. 1.2 Formulae 1.2 1 Empirical
c7 H7 N03 1.2 2 Structural

COOH
1.2 3 Wiswesser Line Notation ZR CQ DVQ
AMINOSALICYLIC ACID
1.3 Molecular Weight 153.13 1.4 Elemental Composition C,54,90%; H, 4.61%; N, 9.5%; 0, 31.34%. 1.5 Pppearance, Color, Taste, Odor White, o r yellowish white, bulky powder or crystals darkens on exposure to light and air, odorless or has slight acetous odor.
2 ., Physical Properties
2.1 Crystal Properties 2.1 1 X-Ray Diffraction Crvstal data Monoclinic, a = 7.209 (2), b = 3.786 (l), co= 25.109 (9) A o , B = 103.22 (3)O, U = 6.67.14 A 3, Z = 4, Dc = 1.53, F (000) = 320. Cu-Ka radiation, A = 1.5418 A'; u (Cu-Ka) = 1 0 . 2 0 ~ m - ~ . Systematic absences = h01, 1 = 2n + 1, OkO, k = 2n + 1, space g r m p P21/C from systematic absences (1). Optical goniometry It crystallises from ethanol in at least two habits. The interfacial angles of habit I were measured with a Huber two circle optical goniometer and conpared with angels calculated from unit-cell dimentions for all faces having Miller indices between (and including) +2 and -2. A unique set of assignments f o r the faces was obtained and confirmed by precision photography. The h k o net was in approximately reflecting position on the precession camera when the faceassigned indices (001) were approximately normal to X-ray beam. Fig. 1 shows a schematic drawing of habit I with assigned faces. The end faces of habit I1 did not have the indices (011) but precession photography and optical goniometry showed that (001) and (103) were its two largest faces.
MAHMOUD M. A. HASSAN etal.
Fig. 1 : Schematic diagram of crystals of p-Aminosalicylic acid in habit I. Crystal Structure Two different crystal structures have been reported for p-aminosalicylic acid. Structure 11 has been reported before the advent of modern computers (2) while structure I has been developed very recently (1). Table 1 and 2 list the bond lengths and angels and Table 3 atom positions. Intramolecular contacts and angels involving the 0(1)-H(21). . .0(2) hydrogen bond are also included. Data for p-aminosalicylic acid are consistant with the idea that resonance structure (Ib) and (Ic) contribute significantly to its structure.
P;
I1
AMINOSALICYLIC ACID
Table 1 Bond l e n g t h s (A) i n p - a m i n o s a l i c y l i c a c i d ( l ) , w i t h s t a n d a r d d e v i a t i o n s i n p a r e n t h e s e s . I n t r a m o l e c u l a r cont a c t s i n v o l v i n g t h e 0 ( 1 ) - H ( 2 1 ) . . 0 ( 2 ) hydrogen bond a r e included.
O ( 1 ) -C(2> 0 (2) -c (7) 0 (3) -C (7) O(2). . 0 ( 1 ) N (1 1-c (4 1 0(1)-H(21) O ( 3 ) -H(71) 0 ( 2 ) . .H(21) N-Ff (4 1) N-H(42)
1 . 3 6 1 (2) 1.243 (2) 1.311(2) 2.620 (2) 1 .364 (2) 0.98 (3) 0.95(3) 1.73(3) 0.91(3) 0 . 8 3 (3) Table 2
1.414(2) 1.400 (3) 1.447 (2) 1 .3 7 1 ( 2 ) 1.392 (3) 1 .4 0 6 ( 3 ) 1.362 ( 2 ) 0 .9 8 (2) 0.98(2) 0.94 (2)
Bond a n g l e s (") i n p - a m i n o s a l i c y l i c a c i d ( 1 ) , w i t h e s t i m a t e d s t a n d a r d d e v i a t i o n s i n p a r e n t h e s e s . Angles i n v o l v i n g t h e O(1)-H(21). . 0 ( 2 ) hydrogen bond a r e i n c l u d e d .
O(2) -C(7)-0(3) 0 (2) -C (7) -C (1) 0 (3) -C(7) - C ( l ) C (7) - C ( l ) -C(2) C(7) -C(1) -C(6) C (2) - C ( l ) -C (6) C (1) -C(2) -0 (1) C(1) -C(2) -C(3) H(71) -0 (3) -C(7) H(21)-0(1)-C(2) O(2). .H(21)-O(1) C ( 7 ) - 0 ( 2 ) . . .H(21) H(3) -C (3) -C (2) H(3)-C(3)-C(4) H(41) -N(l) -H(42)
121.1(1) 123 (2) 115.8 (2) 1 2 0 . 8 (2) 121.7(2) 117.4 ( 1 ) 1 2 1 . 3 (1) 120.6 (2) 113 ( 2 ) 107(2) 147(3) lOO(1) 118 (1) 121 (1)
c ( 2 ) -c (3) -c (4)
0 (1)-C (2) -C (3)
C(3) -C(4) -C(5) C (3) -C (4) -N(1) C ( 5 ) -C (4) -N( 1 ) C(6)-C(S)-C(4) C(1) -C(6) -C(5) H(4l)-N(l)-C(4) H(42)-N(1) -C(4) H(5) -C(5) -C(4) H(S)-C(S)-C(6) H (6) -C (6) -C (1) H (6) -C (6) -C(5)
118.2 (2) 1 2 1 . 1 (2) 1 1 8 .7 ( 1 ) 120.7 (2) 120.6 (2) 1 2 0 .1 ( 2 ) 122.0 (2) 120(2) 115 (2) 119 (1) 121(1) 119 (1) 119 (1)
MAHMOUD M. A. HASSAN e t a [ .
Table 3
4 3 F i n a l atomic p o s i t i o n s (x10 ; f o r H x 10 ) f o r p-aminos a l i c y l i c acid ( I ) , with standard deviations i n parentheses.

X
Y
539(4) 178(4) 345(4) 453(5) 353(5) 138(5) 531(5) 175(5) 5 457(5) 4 OS8(5) 1 SSO(5) 241 (9) 37(8) 789 (8) 782(8) 602 (6) 595 (6) 354 (5) 3 1 1 7 3 4 5 6
z 1 641.0(5) 65 1 . 0 ( 5 ) 58.2 ( 5 )
6 5 7 13 8 8 10 11 11 10 7
1 1
1 1
882(2) 572(2) 438(2) 290(3) 718(2) 539(2) 041(3) 784(2) 966(3) 474(2) 136(2) 601 (4) 637 (4) 316(4) 427(4) 958 (3) 319(3) 064(2)
2 111.6(8) 946.6 (6) 1 483.1(6) 1 860.5(7) 1 728.6(7) 1 193.8(7) 819.9 (7) 547.1(6) 133 (1) -19(1) 246 (1) 200 (1) 223 (1) llO(1) 47(1)
2 . 1 2 Melting Range
The m e l t i n g p o i n t o f 4 - a m i n o s a l i c y l i c a c i d i s u n c e r t a i n (3) : 135'-140 w i t h decomposition ( 4 ) , 148' (dec.) ( 5 ) , 149-151(dec.) ( 6 ) . 150-151' with e f f e r v e s c e n c e ( 7 , 8 ) , 1 3 9 - 1 4 l 0 ( d e c . ) (9) and 220" (dec.) (10,ll) have been r e p o r t e d . Seaman e t a 1 (3) have concluded t h a t t h e most n e a r l y c o r r e c t melti n g p o i n t i s about 240' and t h e m e l t i n g p o i n t i s n o t a good c r i t e r i o n o f p u r i t y .
2.2 Solubility
1 g i n about 600 m l of water and about 2 1 m l o f a l c o h o l ; s l i g h t l y soluble i n ether; practically insoluble i n benzene. S o l u b i l i t y i s i n c r e a s e d with a l k a l i n e s a l t s o f a l k a l i metals (NaHC03) and i n weak n i t r i c a c i d , t h e amine s a l t s of h y d r o c h l o r i c and s u l p h u r i c a c i d s a r e i n s o l u b l e . The aqueous s o l u t i o n s have a pH o f about 3.2 and when h e a t e d t h e a c i d decomposes ( 1 2 ) .
AMINOSALICYLIC ACID
2.3 Identification
1. p-Aminosalicylic acid gives an intense orange-brown color when reacted with potassium ferricyanide in alkaline solution (13).
2. It gives a green color which changes first to orange and then to orange-red on reaction with hexamine and sulphuric acid at room temperature (14). 2.4 Spectral Properties 2.4 1 Infrared Spectrum The infrared spectrum of 4-aminosalicylic acid is recorded as a nujol mull on Unicam SP 1025 Spectrophotometer and is shown in Fig. 2. The assignments or the characteristic bands in the infrared spectrum listed in Table 4. Table 4 Frequency cm 3520 3400 1630
890
-1
Assignment
NH2
NH2;,OH bonded C = 0
isolated C-H out of plane deformation. C-H out of plane deformation.
820 800 770
Other characteristic finger print bands are: 1305, 1230, 1200, 1170, 1110, 970, 725 and 690 c m ' . Other values for PAS in potassium bromide disc (15) are, 3571, 3448, 3030, 1667, 1613, 1515, 1449, 1299, 1220, 1190, 1163, 813 and 775. 2.4 2 Ultraviolet SDectrum IUVI Cary, 219 spectrophotometer ; from 400 to 200 nm(16), three maxima and two minima were observed. The maxima are located at 235, 274 and 303 nm.
UV spectrum of PAS in ethanol was scanned using
1 ,
AMINOSALICYLIC ACID
The minima occur at 252 and 289 nm. The spectrum is shown in Fig. 3. The UV spectral data of PAS have also been reported earlier (17). 2.4 3 Nuclear Magnetic Resonance Spectrum (NMR) PMR The proton NMR spectra of PAS in DMSO-d6 and in acetone-d6 are shown in Fig. 4 and 5 . These were recorded on Varian T-60A, 60 MHz NMR Spectrometer, using tetramethylsilane as internal reference ( 1 8 ) . The PMR spectral data of PAS are given in Table 5. Table 5 : PMR Chemical Shifts of PAS
Chemical shifts (6)
DMSO-d6 Acetone-d6
8.07
8.07
6.08
6.08
6.13 7.50 6.20 7.56
6.10
6.10
(s) = singlet, (d) = doublet.

Long range coupling between the C(~)-B and C(5)-H is observed in the 200 MHz spectrum i r l .DMSO-d6 (Fig. 6) (18). 13C NMR Hassan and Uppal Zubair (19) have investigated the NMR spectrum of PAS, and determined its carbon shifts. The spectrum (Fig. 7) shows seven singlets. The carbon chemical shifts of are as PAS in hexadeuterodimethylsu.1foxide follows : CO : 172.17, C(l) : 100.46, C ( 2 ) : 163.56, C(3) : 98.81, C(4) : 155.73, C ( 5 ) : 106.34, C(6) : 131.56. The off-resonance decoupled spectrum F i g . 8 and 9 shows four singlets representing COY C ( 1 ) , C(2)
F i g . 3 : UV Spectrum o f p-Aminosalicylic a c i d i n E t h a n o l .
10
.a 0
-0
-0
11
F i g . 4 : PMR Spectrum of p - h i n o s a l i c y l i c a c i d i n DMSO-d a n d 6 TMS .
OD
12
Fig. 5 : PMR Spectrum of p-Aminosalicylic acid in Acetone-d
and TMS.
5.4 4
5.20
F i g . 6 : 200 Mttz PMR S p e c t r u m of p-Aminosalicylic acid in L)i.!SO-d
6.
14
4
s
Fig. 7 : 13C
NMR
Spectrum of p-Aminosalicylic acid in DMSO-d6.
15
3
3 N
is
>
F i g . 8 : I3C NMR o f f Resonance Decoupled Spectrum o f p - h i n o s a l i c y l i c a c i d i n DMSO-d6.
b 0
E 3 k w
V
c,
c n a
.I+
4
e ,
c c rdrd c c
o x
u a
( c 1 0
o c
AMINOSALICYLIC ACID
I7
and C(4) and three doublets representing C(3), C(5) and C ( 6 ) . The carbon chemical shifts are as follows:
CO : 172.08, C(1)
: 100.49, C ( 2 ) : 163.59, C3 : 99.29, and 98.33, C(4) : 155.77, C(5) : 106.84 and 105.85, C ( 6 ) : 132.12 and 131.03.
2.4 4 Mass SDectrum The mass spectrum of PAS obtained by conventional electron impact ionisation shows a molecular in M+ at m/e 153. The base peak is at m/e 135. The MC ion peak has about 62.1% relative intensity (Fig.10). The m/e f o r the most prominent fragments are listed in Table 6. Tateniatsu et al, have also reported the mass spectrometry of mixed drugs including 4-aminosalicylic acid (20). Table 6.
m/e 52 79 107 135 136 153 3. Svnthesis
Relative Intensitv 14.3 14.3 24.6 100.0 15.1 62.1
Several synthetic routes to 4-aminosalicylic acid have been reported (21-30). Two of these are illustrated below. Route I : Modified Kolbe-Schmidt Reaction of 4-aminosalicylic acid have been obtained by heating dry finely divided m-aminophenol and potassium carbonate under anhydrous conditions at 150-190' in C02 atmosphere (27). The yield is 90%. Route 11: This route describes the synthesis of C14-carboxyl-labelled 4-aminosalicylic acid by Sandmeyer Reaction
AMINOSALICYLIC ACID
19
using potassium radio-cyanide to synthesise p-nitrosalicylic acid which was then reduced by Catalytic hydrogenation at room temperature. The yiels is 62% (29). COOH
NH2
Route I
NH2
N02
COOH
*
FOOH
NH2
Route I1 4. Metabolism The metabolism of PAS has been studied in both rabbits and humans. Bray et a1 (31)have studied in great detail the metabolism of PAS in the rabbit and found that approximately 50% of a dose of 1-2 gms is excreted unchanged and 50% as 4-acetamido-salicylic acid (m.p. 238-23g0), which has been isolated and characterised. Also they have isolated 4-acetamido-salicylic acid from human urine after oral administration of 3 gms sodium 4-aminosalicylate. Considerable amounts were excreted unchanged. This has also been proved by others (32). Zini (33) has studied the fate of 4-aminosalicylic acid in humans, the
20
MAHMOUD M . A. HASSAN et al.
+ ; H
H COOH
Q
NH2
+ NH2CH2 COOH
Glycine
_____3
NH2
Salicyluric acid
Acetyl at ion
@ +HoQ
\
NHCOCH3
COOH
--+
OH
00
co H
OH
OH Glucuronic acid
NHCOCH Estere?ucuronide COOH
OH
NHCOCH~
Ether-glucuronide Scheme I
AMINOSALICYLIC ACID
21
urinary metabolites of PAS were acetylated-PAS, unchanged PAS, glycine-PAS and glucuronic acid-PAS conjugated compounds. Way et a1 (34), have reported the quantitative determination of the various metabolites of PAS excreted in the urine of human subjects by using countercurrent distribution and paper chromatography. They found that of the total dosage o f PAS, 14 to 33% was excreted unchanged, 28 to 63% as acetyl-PAS, 0 to 26% as p-aminosalicyluric acid, 2 to 10% as unknown free amines and 3 to 10% as unknown bound amine. Lehman (35) have reported the occurrance of N-acetyl-PAS and N-(4-aminosalicyloyl) glycine in human blood plasma and urine after oral administration of PAS. He concluded that concomitant administration of high dosage of PAS with isoniazid probably depletes CO-A and thereby inhibits the acetylation of isonizid. Wan et al., (36), have reported that the metabolism of PAS is mainly by acetylation which accounts for 50 to 70% of the absorbed dose and glycine conjugation to p-aminosalicyluric acid accounts for up to 25% of the dose. These two metabolites together constitute greater than 90% of the metabolites found in urine (37, 38). Metabolites of PAS are shown in scheme I. 5. Methods of Analysis
5.1 Non-aqueous titration
Kucharsky et a1 (39) and Chatten (40) have described a non-aqueous titration technique for the determination of PAS and Sodium PAS, both in pure form and in tablet formulation. Determination of pure PAS is based on titration of anhydrous acetone solution of the acid with 0.1N potassium hydroxide solution in anhydrous methanol using 0.5% of thymol blue solution in anhydrous methanol until the color of the indicator changes to blue. For tablet formulation the above determination is preceded by extraction of the specified amount of the tablet powder with anhydrous acetone.
For the determination o f Sodium PAS the method is based on dissolving the specified amount of the substance on anhydrous methanol and titrated with 0.05N perchlo-. ric acid solution in dioxane using 0.5% thymol blue solution as indicator, until its color changes to peach. For the same in tablets the above determination is preceded by extraction of the specified amount of the powdered tablets with anhydrous methanol. These methods were reported to be specific even in the
22
MAHMOUD M. A. HASSAN e t a / .
presence of m-aminophenol (MAP). Butter and Ramsay (41) titrated PAS and its sodium salt potentiometrically with perchloric acid in glacial acetic acid and acetic acid. Carbon tetrachloride solvent mixture served as the titration medium. Stockton and Zuckerman (42) determined sodium PAS and its solutions by potentiometric titration with perchloric acid in propylene glycol and isopropyl alcohol (l:l), using the same solvent mixture as the titration medium. The decomposition products MAP and sodium bicarbonate did not interfere. Das and Pate1 (43) employed the same titrant and solvent system. Hunt and Blake (44) have described a non-aqueous titration method for the analysis of PAS and its salts and dosage forms. This method was reported to be specific in the presence of MAP. The method is based on titration with sodium methoxide in benzene-methanol using dimethylformamide as titration solvent. The end point is detected visually using thymol blue as indicator o r potentiometrically. PAS and its decomposition product, m-aminophenol may be differentiated with this titration system. Salts of p-aminosalicylic acid are converted to the acid form by ion-exchanged chromatography prior to titration. 5.2 Diazometric Assay USP XVIII method (45)for the determination of PAS, its salts and dosage forms, involves the diazotisation reaction and is based on procedures developed by Tarnoky and Bews (46) and Pesez (47,48). Blake et a1 (49) have described a method for determination of sodium p-aminosalicylate in the presence of m-aminophenol. m-Aminophenol, the major breakdown produced p-aminosalicylic acid, if present, is also diazotised and constitutes an interference in the official assay procedure. In this method the PAS content and mixtures containing MAP is determined by the modification of the official assay procedure. The MAP is removed by passing the solution of the mixture in dimethylformamide through a column containing a strong cation exchange resin. The elute is then treated according to the official method.
AMINOSALICYLIC ACID
23
5 . 3 Spectrophotometry Coccia (50) has d e s c r i b e d d e t e r m i n a t i o n o f PAS, m-aminophenol and p-aminophenol c o l o r i m e t r i c a l l y by u t i l i s i n g t h e i r r e a c t i o n w i t h sodium n i t r o p e n t a c y a n o c o b a l t a t e t o g i v e an orange compound. The c o l o r p r o duced obeys B e e r ' s law a t 440 nm i n t h e range of 0 t o 0.75 1 . 1 8 o f PAS p e r m l . The compound o b t a i n e d w i t h PAS was p r e p a r e d and i t s formula and molecular weight were obtained. Rieder (51) has r e p o r t e d a n o t h e r c o l o r i m e t r i c method f o r d e t e r m i n a t i o n o f f r e e PAS i n blood. The method i s based on t h e coupling o f PAS w i t h d i a z o t i s e d s u l p h a n i l i c a c i d i n a s t r o n g l y a l k a l i n e a l c o h o l i c medium The r e s u l t i n g s o l u t i o n shows maximum e x t i n c t i o n a t 600 nm, b u t a n a l y s i s were c a r r i e d out a t 630 n m in o r d e r t o avoid i n t e r f e r e n c e . The c o l o r i s s t a b l e f o r 30 minutes and t h e maximum e r r o r i s f 5% i n t h e range of 5 t o 20mg of PAS p e r 100 m l . Another c o l o r i m e t r i c method has a l s o been r e p o r t e d (52) u t i l i s i n g r e a c t i o n o f PAS and MAP with n i n h y d r i n solution. 5.4 Combined TLC and Colorimetry Kinze (53) has r e p o r t e d t h e s e p a r a t i o n o f PAS and MAP on l a y e r s o f Alumina oxide by u s i n g e t h a n o l o r methan o l a s a d e v e l o p e r . PAS remains on t h e base l i n e i n b o t h i n s t a n c e s . The s p o t s a r e d e t e c t e d by s p r a y i n g with 1%p-dimethylaminobenzaldehyde s o l u t i o n i n ethanol t r e a t e d w i t h 5% h y d r o c h l o r i c a c i d . After e x t r a c t i o n from t h e p l a t e 2-60 m g of MAP can be determined c o l o r i m e t r i c a l l y a t 420 n m with 1%f u r f u r y l a l c o h o l s o l u t i o n i n anhydrous a c e t i c a c i d . 5 . 5 U l t r a v i o l e t method Moussa (54) h a s r e p o r t e d a U . V . method f o r determinat i o n o f PAS i n t h e p r e s e n c e o f i t s d e g r a d a t i o n product MAP. The f i n e l y powdered t a b l e t s a f t e r e x t r a c t i o n w i t h e t h a n o l i s f i l t e r e d and t h e f i l t r a t e i s d i l u t e d and t r e a t e d w i t h b o r a t e b u f f e r s o l u t i o n o f pH3 and t h e absorbance i s measured a t 300 nm a g a i n s t t h e b u f f e r s o l u t i o n . There i s no i n t e r f e r e n c e from MAP i n amounts u p t o a t l e a s t twice t h a t of PAS.
24
MAHMOUD M. A. HASSAN et a / .
PAS can be analysed spectrophotometrically by dissolving the sample in ethanol (95%) to give a concentration of about 15 Ug/ml and the absorbance of the solution so produced is measured at 303 nm. The log 5 values are given in Table 7 (16).
Table 7
X max nm
2 35
274
303
Log
2.765 3.622
3.624
AMINOSALICYLIC ACID
25
REFERENCES
1.
Chung-Tang Lin, Pik-Yen Siew and S.R. Byrn, J. Chem.Soc., Perkin 11, 957 (1978).
2. 3. 4.
F. Bertinotti, G. Giacomello and A.M. Liquori, Acta Cryst., - 7, 808 (1945).

J . Am. Chem. SOC., 71, 2940 (1949).
W. Seaman, W. Allen, R.L. Pasternak and A. Pollara,
Remington Pharmaceutical Sciences, Arther Osol et al, XV Edition, Mack Publishing Co., Easton, Pennsylvania, U.S.A. p. 1149 (1975). Kolb, U.S. Patent 427, 564; German Patent 50, 835. Erlenmeyer, et al, Helv. Chim. Acta, 31, 988 (1948). The Merck Index, An Encyclopedia of Chemicals and Drugs, Martha Windholz et al, IX Edition, Merck and Co. Inc., Rahway, N.J., U.S.A., p. 66 (1976). O'Connor, Lancet, -254, 191 (1948). Whittel, Lancet, -254, 268 (1948).
5.
6.
7.
8.
9.
Ber.,34, 4351 (1901). 10. Seidel, 11. Seidel and Bittner, Monatsh, 23, 415 (1902). 12. Textbook of Organic, Medicinal and Pharmaceutical Chemistry, C.O. Wilson et al, VII Edition, J.B. Lippincott. Co., Philadelphia, U.S.A. p. 236, 1977. 13. Yu M. Ostroskil, Aptechnoe Delo, 4(6),
10 (1955).
4, No.6, 8, (1955). 14. A.M. Gal'perina, Aptechnoe Delo, 15. I.R. Grating Collection, Sadtler, Research Laboratories, SADG 9560. 16. M.Uppa1 Zubair, M.M.A. Hassan, unpublished results. 17. Spectral Collection data, Sadtler Research Labs.SADG 3162. 18. M.M.A. Hassan and M. Uppal Zubair, unpublished results.
26
MAHMOUD M. A. HASSAN et al.
19. 20.
M.M.A.
Hassan and M. Uppal Z u b a i r , unpublished d a t a .
A. Tatematsu, T . Nadai, T. Goto, Y . Nakajima, H. 87 ( 4 ) , 329 (1967). Tsuyama and H. Doi, Yakugaku Z a s s h i , German P a t e n t 50, 835, F r i e d l a n d e r , 2 , 139 (1887-90).
21.
22.
23. 24.
J . T . Sheehan, J . Am. Chem. SOC. 70, 1665, (1948).

A. Wander, A . G . Swiss 265, 516, Dec., 15 (1949).
D . D . M a r t i n , D . E . Seymour and F.S. S p r i n g , B r i t . P a t . 636, 333, Apr. 26 (1950). D . D . M a r t i n , D . E . Seymour and F.S. S p r i n g , B r i t . P a t . , 697, 965, Oct 7 (1953).
25.
26.
G . F . Felemons and R . A . Aug 26 (1953). R . P . P a r k e r and J . M . June 30 (1953).

Brit.,
Wilkinson, B r i t . Pat.696, 132,
27.
Smith. Jr. U.S. P a t . 2 , 644, 011,
28. 29.
P a t . 693, 386, J u l y 1 (1953). Roth, J . Am. Pharm. Assoc.
L . C l e r k , A. H e l l e r and L . J .
4 4 , 328 (1955).
30.
I . H i r a o , Y . Kosugi, T . Matsuura, Y . Hironaka and Y. Gosei, Kagaku Kyokai S h i , 25 ( 5 ) , 417 (1967).

H . G . Bray, B . E . 64 (1948). Ryman and W.V. Thorpe, ~Nature, 162, Lewis,
31.
32.
A . Venkataraman, P . R . Venkataraman and H . B . J . B i o l . Chem., 173, 641 (1948).
33. 34.
F . Z i n i , Riv. C r i t . C l i n . Med. 5 3 , 308 (1953).
E . L . Way, G . Peng, N . Allawala and T . C . D a n i e l s , J . Am. Pharm. Assoc., 4 4 , 65 (1955).

J . Lehman, Scand. J . Resp. D i s . ,
35. 36.
50 (3), 169 (1969). -
Sci.,
S.H. Wan, P . J . P e n t i k a i n e n and D . L . Azarnoff, J.Pharm. 63, 708 (1974).
AMINOSALICYLIC ACID
27
37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53.
54.
E . L . Way, P.K. Smith, D . L . Howe, R. Weiss and R . Swanson, J . Pharmacol. Exp. T h e r . , 93, 368 (1948).
J . Kawamata and J . Kashiwagi, Med. J . Osaka Uni., 6 , 119 (1955).

T i t r a t i o n s i n non-aqueous s o l v e n t s , J . Kucharsky and L . S a f a r i c k , E l s e v i e r , New York, N . Y . , p . 182 (1965).
L.G.
Chattan, (1956) .
J . Am. Pharm. Ass.
S c i . Ed.
,45, 556
A.Q.
B u t l e r and J . C . Ramsay, i b i d , 42, 338 (1953). S t o c k t o n and R . Zuckerman, i b i d , 43, 273 (1954).
J.R.
M . N . Das and S.R. P a l i t , J . Ind. Chem. S O C . , 31, 34 (1954). J . Hunt and M.I. Blake,
J . Pharm. S c i . , 5 9 , 683(1970).
The Pharmacopeia of t h e United S t a t e s of America, p . 3 6 , 1 8 t h r e v . , Mack Pub.Co.Easton, Pa,USA, p.36 (1970).

A. Tarnoky and B . A .
Brews, Biochem. J . , 45, 508 ( 1 9 4 9 ) .
M. Pesez, B u l l . SOC. Chim. F r . , 30, 918 (1949).

M. Pesez, B u l l . SOC. Chim. B i o l . , 31, 1369 (1949).
M.I. Blake, K . Makris and J . Hunt, J. Pharm. S c i . , 60 ( l l ) , 1695 (1971).

P . A . Cuccia, Anal. Chem., 3 1 ( 8 ) , 1306 (1959).
H . P . R i e d e r , Kiln Wochscher, 39 ( 1 5 ) , (1961).

K . N . Gaind, R . N . Dar and S.C. Bapna, I n d i a n J . Pharm., 26 ( 9 ) , 248 (1964). W.
Kinze, Pharm. Z e n t r a l h a l l e D t l . ,
105 ( 6 ) , 365 (1966)
A. Moussa, Pharmzie, 33 ( 7 ) , 460 (1978).
AZATHIOPRINE
Wendy P . Wilson and Steven A. Benexra
1 . Description 1 . 1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Odor 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectrum 2.4 Mass Spectra 2.5 Melting Point 2.6 Solubility 2.7 Dissociation Constant 3. Synthesis 4 . Stability 5. Metabolism and Pharmacokinetics 5.1 Metabolism 5.2 Excretion 5.3 Tissue Distribution 6. Methods of Analysis 6.1 Elemental Analysis 6.2 Nonaqueous Titration 6.3 Polarography 6.4 Microbiological Assay 6.5 Phosphorescence Spectroscopy 6.6 Fluorimetric Analysis 6.7 Chromatography References
30 30 30 30 30 32 34 37 37 37 39 39 39 39 39 41 42 42 42 43 43 43 44 44 44 51
ANALYTICAL PROFILES OF DRUG SUBSTANCES, 10
29
30
WENDY P. WILSON AND STEVEN A. BENEZRA
1.
Description
1.1
Name, Formula, Molecular Weight
Azathioprine is 6-[(l-methyl-4-nitroimidazol-5-yl)thio]purine
9H7N702S
277.3
1.2 Appearance, Color, Odor Azathioprine is a pale yellow, odorless powder.
2.
Physical Properties
2.1
Infrared Spectrum
The infrared spectrum of azathioprine i s shown in Figure 1. The spectrum was obtained as a 0.4% dispersion of azathioprine in KBr with a Nicolet Model 7199 FT-IR spectrophotometer. The infrared assignments consistent with the structure of azathioprine are given in Table 1 . ' Table I Infrared Spectral Assignments for Azathioprine Band Frequency (Wavenumbers) Structural Assignment C-H deformation attributable to the purine nucleus. C-H deformation attributable to the imidazole ring.
921 and 857 831 and 637
? !
3
3
#. .
Ln
N
0
0
Ln
I
0 Ln
r
I
0 0
NUl
g
: $
6 0
Ln N
0
>
0 3
I n
r
N
0
0 0
In
0 0
lP
P7
In
. r
m
0 '3
.
32
1233 1470 and 1390 1537 and 1374 1595 and 1570 1893 and 1807
2810 2976 3109 3191
C-N stretching from a tertiary amine and a purine nucleus. C-H bending from a methyl group. C-NO, stretching (asymmetric and symmetric, respectively). C=N stretching characteristic of the amidine groups in substituted purine and imidazole structures. C-H deformation overtones attributable to the substituted purine and imidazole functions. C-H stretching indicative of a CH3-N group. C-H stretching characteristic of a pyrimidine group. C-H stretching characteristic of imidazole groups. N-H stretching characteristic of a purine function.
2.2
Nuclear Magnetic Resonance (NMR) Spectra
The 'H NMR spectrum o f azathioprine is shown in Figure 2 . The spectrum was obtained in deuterated dimethyl sulfoxide with a Varian XL-100A NMR spectrometer at 100 MHz. Chemical shifts referenced to DMSO at 2 . 5 1 ppm and consistent with the structure of azathioprine are presented in Table II.2 Table I1
NMR Assignments for Azathioprine

Proton a
No. of Protons 1
Shift (ppm)
8.59. 8.55 8.25 3.70 13.8
Mu1tip licit y singlet singlet quartet doublet broad singlet
14
I
1 3
I '
12
I
11
1 '
1 0
I
. ..
i i
. . .. . . . ...~... .
-!
5
PPm
Figure 2 - ' H Nuclear Magnetic Resonance Spectrum of Azathioprine
34
WENDY P. WILSON AND STEVEN A . BENEZRA
e
The 13C NMR of azathioprine, shown in Figure 3 , was obtained with a Varian CFT-20 NMR spectrometer at 80 MHz. Deuterated dimethyl sulfoxide was used as the solvent with tetramethylsilane a s an internal standard. Carbon assignments for the 13C NMR are given in Table III.3 Table I11 Carbon No. 2 4 5 6
8 2' 4' 5'
Chemical Shift (ppm) 151.6 150.6 130.0 154.6 144.5 139.4 149.7
117.1
CH3
32.9
2.3 Ultraviolet (W) Spectrum

The ultraviolet spectrum of azathioprine in methanol was obtained with a Beckman ACTA CIII W spectrophotometer and i s shown i n Figure 4 . Table IV gives UV data f o r azathioprine in various solvents.
34
e
The 13C NMR of azathioprine, shown in Figure 3 , was obtained with a Varian CFT-20 NMR spectrometer at 80 MHz. Deuterated dimethyl sulfoxide was used as the solvent with tetramethylsilane a s an internal standard. Carbon assignments for the 13C NMR are given in Table III.3 Table I11 Carbon No. 2
4 5 6
8 2' 4' 5'
Chemical Shift (ppm) 151.6 150.6 130.0 154.6 144.5 139.4 149.7
117.1
CH3
32.9
2.3 Ultraviolet (W) Spectrum

The ultraviolet spectrum of azathioprine in methanol was obtained with a Beckman ACTA CIII W spectrophotometer and i s shown i n Figure 4 . Table IV gives UV data f o r azathioprine in various solvents.
0
-v)
0
7
I
I W
I
I
N
0 0
0
0
d 0
AZATHIOPRINE
37
Table IV
W Spectral Data for Azathioprine
Solvent methanol 0.1N NaOH 0 . 1 N HC1

2.4
m a x
(-1
276 285 280
&
max
1 . 8 2 x 104
1.55 x 104
1.73
x 104
Mass Spectra
The low resolution electron impact and field desorption6 mass spectra of azathioprine are shown in Figures 5 and 6 . The electron impact spectrum was obtained with a Varian MAT CH5-DF mass spectrometer. The sample was introduced into the ion source y & direct probe at 285OC. The electron energy was 70 eV. The major fragment ions formed on electron impact are consistent with those found by Brent _ et a L 7 Loss of NO2 yields CSH7N6S, m/z 231 (100%). Cleavage between sulfur and the purine ring with retention of charge on the purine ring results in (Pur)+, m/z 119 ( 4 2 % ) . The fragment m/z 152 (10%) is formed by fission of the sulfur imidazole bond with fearrangement of a hydrogen to the purine moiety (PurS + H)., and m/z 42 (45%) is C2H4N. The field desorption spectrum was obtained with a Varian MAT 731 mass spectromFter at an emitter heating current of 18 ma. The (M+1) ion (m/z 2 7 8 ) , while absent from the electron impact spectrum, appears in the field desorption spectrum ( 4 . 6 % ) . Other fragments prTsent in the field desorption spectrum are m/z 231 (loo%), M.-NO2 and m/z 2 7 7 .
2.5
Melting Point Azathioprine melts and decomposes at approximate-
ly 240C.8
2.6
Solubility
Azathioprine is very slightly soluble in water It is also slightly soluble in (-0.01% w/v at 25).8 chloroform, ethanol and dilute mineral acids. Azathioprine i s soluble in dilute solutions of alkali hydroxides with slow decomposition, dimethyl sulfoxide and poly-
38
1
J
80 cn 6 0 Z
u
>
40-
140 180 m/z
220
260
200
Figure 5 - Electron Impact Mass Spectrum of Azathioprine
m/z
Figure 6 - Field Desorption Mass Spectrum of Azathioprine
AZATHIOPRINE
39
ethylene glycol 400. 2.7
'
Dissociation Constant The pKa2 of azathioprine is 8.2 at 25OC.'
3.
Synthesis
Azathioprine is synthesized by the synthetic route shown in Figure 7 . Diethylsuccinate ( 1 ) is reacted with methylamine (2) to give N,"-dimethylsuccinamide (3) which in turn is reacted with-PCl,/POCl, to ring close to 1-methyl-5-chloroimidazole ( 4 ) . The imidazole, 4, is converted to its salt with nitric acid to give l-methyl5-chloroimidazole nitrate (5). The imidazole nitrate, 5 , is then converted to l-methyl-4-nitro-5-chloroimidazole ( 6 ) . Ethyl cyanoacetate (7) is nitrosated, reduced and acetylated to give e t h y l a c e t a m i d o c y a n o a c e t a t e ( 8 ) . The ring closure of 8 is done with formamide to give hypoxanthine ( 9 ) which is reacted with phosphorous pentasulfide to give 6-mercaptopurine ( 1 0 ) . The imidazole (6) and 6-mercaptopurine (10) are condensed to yield azathioprine. lo 4. Stabilitv
Bulk samples of azathioprine are stable for at least two years at temperatures between 5OC and 37OC and one year at 5OoC when stored in well closed, light resistant containers.' ' ' I 2 The drug develops a dark orange surface after four weeks when exposed to fluorescent or ultraviolet light." Azathioprine is stable in neutral and acidic solutions but is hydrolyzed to 6-mercaptopurine by alkali.13'14 5. Metabolism and Pharmacokinetics
5 . 1 Metabolism
Azathioprine is initially split by glutathione in the liver to 6-mercaptopurine and l-methyl-4-nitro-5(5-glutathiony1)imidazole. To a much lesser extent azathioprine may be split between the purine ring and the sulfur to yield the metabolite l-methyl-4-nitro-5-thio-
m I
V
2
I
$1
i
A
z
N
I
V z N
r-
AZATHIOPRINE
41
imidazole. l5 The metabolism o f the 6-mercaptopurine moiety follows two known pathways. It can be inactivated by xanthine oxidase to 6-thiouric acid or it can be converted to its active form, the ribonucleotide 6-thioinosinic acid, by hypoxanthine-guanine phosphoribosyl transferase in tissues. 16 l7 l8 The major urinary metabolite of the l-methyl-4nitro-5-(S-glutathionyl)imidazole moiety in man and in dogs is N , N - [5-(methyl-4-nitro)imidazolyl]cysteine. The major metabolite in the rat, 1-methyl-4-nitro-5(N-acetylS-cysteinyl)imidazole, accounted for only a small-percentage of the dose in dogs and in man. Other metabolites o f the methylnitroimidazole moiety include several 5-substituted amino-1-methyl-4-nitroimidazoles one of which, a glycine derivative, indicates that 6-mercaptopurine may also be displaced from azathioprine by nucleophilic attack o f amino acids. 192021 5.2 Excretion In a human study using 35S-azathioprine to follow the fate o f the purine moiety, over 50% of the radioactive dose was excreted in the urine in twenty-four hours indicating a good absorption o f the drug. Seventy percent of the 35S had been excreted in forty-eight hours. Twelve percent unabsorbed material was excreted in the forty-eight hour stool specimens. Very little of the drug was eliminated unchanged. The major urinary metabolite was thiouric acid with less than 1%of the dose eliminated as 6-mercaptopurine and from 10% to 20% inorganic ~ u 1 f a t e . l ~ Similar ~~ studies done in rats and in dogs gave similar results with the exceptions of relatively larger quantities of 6-mercaptopurine being excreted by rats and both rats and dogs excreted slightly more unchanged azathioprine.2324 Clearance of the methylnitroimidazole portion of the drug is much slower than that of the purine moiety. Following an oral dose of 90 mg o f 14C-azathioprine the patient excreted only 20% of the 14C in the first twentyfour hours. In forty-eight hours only 37% of the 14C had been excreted in the urine in contrast to the 70% excretion of 35S in forty-eight h o u r ~ . ~ Similar ~ ~ results were obtained in the rat and dog studies with 14C-azathio~ r i n e . ~ ~ Forty-two percent of the 14C had been excret-
42
ed by the dogs in 32 hours with very little radioactivity excreted after 32 hours. l 9
5.3
Tissue Distribution
The peak plasma radioactivity of the purine portion of azathioprine occurred at 2 hours in a patient treated with 35S-azathioprine. The half-life of the plasma radioactivity was 4 . 5 to 5 hours and after 1 0 hours, when most of the remaining 35S was inorganic sulfate, the clearance of radioactivity was much slower.15 Another patient was treated with 14C-azathioprine. Plasma radioactivity of the methylnitroimidazole moiety peaked at 4 hours at which time the plasma radioactivity was twice that found in the blood cells. After 12 hours the radioactivity had equilibrated between the plasma and the cells. At twelve hours the level o f radioactivity was 40% of the peak value and this level persisted for 36 hours.15 The concentration of 35S was determined in several organs of rats treated with 35S-azathioprine. The highest concentration o f 35S was found in the liver 6 hours after administration of the drug. This concentration was five times that found in the blood plasma. Only traces of radioactivity were found in the fat-rich organs.23 Another rat study showed that there is rapid hepatic extraction of azathioprine. After only 5 minutes a high proportion of the radioactive dose was recovered in the liver.25 Radioactivity levels rapidly attained a maximum in the blood cells and then declined rapidly in dogs treated with 14C-azathioprine. The peak plasma radioactivity was reached about 5 hours after drug administration and after 8 hours the radioactivity had equilibrated between the plasma and the blood cells. The radioactivity then declined gradually over 48 hours. l 9
6.
Methods of Analysis
6.1 Elemental Analysis

The elemental analysis of azathioprine is given in Table V . 2 6
AZATHIOPRINE
43
Table V Elemental Analvsis of Azathiofirine Element

0
Theory (%>
38.98 2.55 3.5 .36 11.54 11.57
H
N
0
5.2
Nonaaueous Titration
An accurately weighed sample of azathioprine is dissolved in dimethylformamide. The solution is titrated with standardized 0 . 1 N tetrabutylammonium hydroxide to the thymol blue endpoint. Precautions must be taken to prevent the absorption of atmospheric carbon dioxide. Each milliliter o f 0.1 N tetrabutylammonium hydroxide is equivalent to 27.73 mg of azathioprine.'
6.3
Polarography
A differential pulse polarographic analysis is used to assay azathioprine tablets and azathioprine sodium for injection. The samples are dissolved, diluted with 0.1 N sulfuric acid and de-aerated with nitrogen. Using a dropping mercury electrode with a saturated calomel reference electrode, the polarogram is recorded from -0.60 volt to -1.00 volt. The height of the diffusion current is compared to that of a reference standard prepared in a similar manner to obtain the concentration of azathioprine in the formulations.
6.4
Microbiological Assay
Harber and Maddocks described a method o f estimating nanogram quantities of azathioprine by measuring the extent of growth inhibition o f Lactobacillus casei. A modified folic acid assay medium containing between 20 and 200 ng azathioprine was inoculated with 2 drops o f a stock solution of Lactobacillus casei. The cultures were incubated at 37OC for 18 hours and turbidity was then measured at 560 nm. A range of standards were similarly prepared and a standard curve was drawn from which the concentration of azathioprine was read.27
44
6.5 Phosphorescence Spectroscopy Azathioprine has been analyzed phosphorimetrically at -196OC. In alkaline ethanol, with excitation and phosphorescence wavelengths of 311 nm and 451 nm, respectively, azathioprine had a detection limit of 2 . 6 vg/ml and the concentration to phosphorescence relationship was linear over at least two orders of magnitude of concentration. Phosphorescence in neutral ethanol was observed at 442 nm with an excitation wavelength of 300 nm. The detection limit of azathioprine under these conditions was 10 pg/m1.28 6.6 Fluorimetric Analysis Azathioprine and its metabolite 6-mercaptopurine have been successfully quantitated in plasma using a fluorimetric assay. The 6-mercaptopurine was first derivatized with phenyl mercuric acetate. This derivative could then be extracted from the plasma with toluene. The derivative was convert.ed back t o 6-mercaptopurine with 0.1 N hydrochloric acid and the toluene was removed. The 6-mercaptopurine was then oxidized to purine 6-sulfonate with potassium chromate followed by sodium metabisulfate and sodium hydroxide solutions. The fluorescence of the solution was measured at 398 nm with an excitation wavelength of 288 nm. Azathioprine was hydrolyzed to 6-mercaptopurine with 5 N sodium hydroxide. After neutralization with 5 hydrochioric acid the derivatization, extraction, oxidation and fluorimetric analysis steps were followed as for the 6-mercaptopurine. The concentration of azathioprine was calculated from the difference in the 6-mercaptopurine concentration in the hydrolyzed and non-hydrolyzed samples.2 9 6.7 Chromatography 6.71 Column Chromatography Nelson and coworkers have separated several azathioprine metabolites by column chromatography on DEAESephadex columns. The metabolites were eluted with pH 4.7 triethylammonium acetate buffer. 1 0 mM f3-mercaptoethanol was added to the mobile phase to prevent oxidation of the thiopurines. Azathioprine was converted to 6-mercaptopurine on the column under these conditions and could not
AZATHIOPRINE
45
be separated. Detection was W at 254 Measurement of azathioprine, 6-mercaptopurine and 6-thiouric acid in urine was achieved on the cation exchange resin Zeo Karb 225. 6-Thiouric acid was eluted first with 30-40 ml water which was then evaported to dryness. 6-Mercaptopurine was eluted next with 15 ml of 20% ammonium hydroxide and then evaporated to dryness. Azathioprine was converted to 6-mercaptopurine by the addition of glutathione to the pH 8.9 adjusted urine, which was then chromatographed as described above. The eluates were dissolved in 5% perchloric acid and concentrations were determined by the decrease in extinction measured after the addition of mercuric chloride. 6-Thiouric acid was measured at 345 nm and 6-mercaptopurine was measured at 330 run. The concentration of azathioprine was determined by the difference in 6-mercaptopurine concentration before and after the addition of glutathione.3 1 Azathioprine has been separated from other purines on Sephadex G-10. The mo.bile phase was 0.05M, pH 7 phosphate buffer. The recovery of the chromatographed purines was quantitative. 3 2 6.72 High Performance Liquid Chromatography (HPLC) Table VI gives various HPLC systems used for azathioprine and its metabolites. 6.73 Paper Chromatography Azathioprine and several methylnitroimidazole metabolites have been separated on Whatman no. 3 filter paper. The two solvent systems used were p-butano1:acetic acid:water ( 4 : 1 : 5 ) , the top layer was used, and p-propanol: water (7:3). The chromatograms were developed for 20 hours and the compounds were detected under ultraviolet light. The R values for azathioprine were 0.75 in the p-butanol f system and 0.86 in the 2-propanol 6.74 Thin Layer Chromatography (TLC) TLC systems used for azathioprine and its metabolites are given in Table VII. Ito and Fujita describe the use of 3,5-di-tertbutyl-1,2-benzoquinone-iron (111) chloride as a TLC spray
reagent for the detection of thiols.
Fifty nanomoles of
Table VI HPLC Svstems for Azathiourine

Column
Mobile Phase gradient from 0 . 0 3 M to 4.0 M ammonium acetate pH 4.7 11% Acetonitrile in 0.01 M sodium acetate buffer pH 4.0
Flow (ml/min)
Retention Time (min) AZA- 18 6-MP- 4 6-TU- 78 AZA- 8
Detection
Ref
30
PA-38 pellicular anion-exchange resin (3m x lmm i.d.) PBondapak C 1 8 (30cm x 3.9mm i.d.)
0.4
254 nm or 350 nm
280 nm
33
5~ ODs-Hypersil (10cm x 5mm i.d.)
Methanol:25 mM potassium dihydrogen phosphate : glacial acetic acid (20:79.5 :O.5) pH 4.50 Water : Methanol (70: 30) Acetonitri1e:water: glacial acetic acid (15:85:0.02)
1.5
6-MP- 2 MNTI- 2.5 "HI- 1
AZA- 4
240 nm
34
PBondapack C18 (30cm x 4mm i.d.) VBondapak Cl,
AZA- 3 . 2 AZA- 11
280 nm 280 nm
35
36
Table VI continued Column Aminex-27 (100cm x 1.24mm i.d.) Mobile Phase gradient from 0.015 M to 6.0 M sodium acetate pH 4.0 0.02 M ammonium formate pH 4.9 Flow (ml/min) 0.13 Retention Time (min) Detection 254 run or 280 nm 280 nm
Ref
19
AZA- 300
N,N'-MNIC- 925 MNTI- 1020

0.4
PA-38
pellicular anion exchange resin (3m x lmm id)
AZA- 15 6-MP- 7
20
AZA 6-MP
M N H I MNT I
azathioprine 6-mercaptopurine l-methyl-4-nitro-5-hydroxyimidazole

l-methyl-4-nitro-5-thioimidazole N,N'-[5-(methyl-4-nitro)imidazolyl)cysteine _ _ 6-thiouric acid
N,N'-MNIC 6 -TU
Table VII TLC Systems for Azathioprine Adsorbent Silica gel 60 F 254 Mobile Phase aceti (1:9) acid:ethanol
-
Detection and Comments Azathioprine metabolites were converted to phenyl mercury derivatives before chromatograpy. Following conversion back to the parent thiols by spraying with 2 N HC1, compounds were detected by low temperature (-196OC) phosphorescence at 254 nm and 366 nm. Detection of mercury can also be achieved by spraying chromatogram with 0.1 N acetic acid followed by a dithiozone solution. Low temperature (-196OC) phosphorescence detection was used with excitation and phosphorescence wavelengths of 342 nm and 485 nm respectively and 320 nm and 448 nm respectively.
Ref 39
6-MP- 0.59 6-TU- 0.30

6-MP- 0.63
6-TU- 0.37
ammon a :butanol : water (1 :60: 39) heptane :chlorofo rm : ethanol (1:l:l)
6-MP- 0.58 6-TU- 0.04
Cellulose
0.1 M hydrochloric acid
AZA- 0.66 6-MP- 0.44
28
Table V I I continued Adso rb ent Cellulose

0.1
Mobile Phase
Rf AZA- 0 . 7 0 6-MP- 0.43 6-TU- 0.24 AZA- 0.70 6-MP- 0.26 6-TU- 0 . 7 5 AZA- 0.87 6-TU- 0.25 6-MP- 0 . 5 5 AZA- -0.8 6-MP- 0.45 AZA- 0.47 6-MP- 0.36
Detection and Comments
Ref
~
N hydrochloric acid
low temperature (-196OC) luminescence detection at 366 nm
37
water
isopropano1:methanol: water:ammonia (60:20:20:1)
ECTEOLAcellulose
acet0ne:O.l M sulfuric acid:ethyl acetate
(45 : 10 :45)
acetone:water (20:80)
Viewed under an W lamp. 6-Mercaptopurine fluoresced at 254 nm and 366 nm. Azathioprine quenched fluorescence at the same wavelengths.
38
AZA 6- MP 6- TU
azathioprine 6-mercaptopurine 6-thiouric acid
50
6-mercaptopurine was detected on a cellulose TLC plate with this reagent.4 0 6.75 Thin Layer Electrophoresis
The separation of azathioprine from other thiopurine derivatives has been achieved on both silica gel and ECTEOLA-cellulose thin layer chromatography plates with the use of low-voltage thin layer electrophoresis. A 0.7% triethanolamine buffer adjusted to pH 9.5 with acetic acid was used with the silica gel plates and a 5% pyridine buffer adjusted to pH 6.0 with acetic acid was used with the ECTEOLA-cellulose. The electrophoresis w a s carried out at 300 V at 4OC for 3 hours for the silica gel plates and 3.5 hours for the ECTEOLA-cellulose plates. After electrophoresis the plates were air dried then dipped into an ammonia fume chamber for 30 seconds. Low temperature (-196OC) phosphorescence detection was performed at 254 nm and 366
AZATHIOPRINE
51
References
1.
H. Powell, Burroughs Wellcome Co., personal communication, 1980.
2. A. Ragouzeos, Burroughs Wellcome Co., personal communication, 1980.

3.
B.S. Hurlbert, R . Crouch, Burroughs Wellcome Co., personal communication, 1981. W.P. Wilson, Burroughs Wellcome Go., unpublished data, 1981. cation, 1980.
4.
5. R. Johnson, Burroughs Wellcome Co., personal communi6. B. Soltman, Burroughs Wellcome Co., personal communication, 1980.
7.
D.A. Brent, P . de Miranda, H.R. Schulten, J. Pharm. Sci., 6 3 , 1370 ( 1 9 7 4 ) . G.R. Griffith, Wellcome Foundation Ltd., personal communication, 1980.
U.S.P.
8.
9. 10.
XX, Mack Printing Co., 1 9 7 9 .
H.N. Yeowell, G.B. Elion, J. Heterocyc. Chem.,

1017 ( 1 9 7 3 ) .
lo ,
11.
R.C. Thompson, R.I. Poust, Burroughs Wellcome Co., personal communication, 1 9 7 9 . R.C. Thompson, S. Cliett, Burroughs Wellcome Go., unpublished data, 1979. G.B. Elion, Burroughs Wellcome Go., personal communication, 1 9 6 7 . Medical Department, Burroughs Wellcome Co., unpublished data, 1969. G.B. Elion, Proc. Roy. SOC. Med.,
12.
13.
14.
15.
16.
9, 257
(1972)
G.B. Elion, S . Bieber, G.H. Hitchings, Ann. N.Y. Acad. Sci., 6 0 , 297 ( 1 9 5 4 ) .
52
17.
P.R.B. FOSS,S.A. Benezra In "Analytical Profiles of Drug Substances", v o l . 7 ; K . Florey, Ed.; Academic Press: New York, 1 9 7 8 ; p. 3 5 5 . N. Kaplowitz, J. Pharmacol. Exp. Ther.,
(1977).
18.
200
( 3 ) , 479
19.
P. de Miranda, L.M. Beacham, 111, T.H. Creagh,

G.B. Elion, J . Pharmacol. Exp. Ther.,
195,50
(1975).
20.
P. de Miranda, L.M. Beacham, 111, T.H. Creagh, G.B. Elion, J. Pharmacol. Exp. Ther., 187, 5 8 8
(1973).
21.
G.B. Elion, F.M. Benezra, L.O. Carrington,

R . A . Strelitz, Fed. Proc., Fed. h e r . SOC. Exp.
2 9 , 2027 ( 1 9 7 0 ) . Biol., -
22.
G.B. Elion In "International Symposium on Immunopathology", Vth, Punta Ala, Italy, June 1 9 6 7 ; P.A. Miescher, P. Grabar, Ed.; Grime and Stratton: New York, 1 9 6 8 ; p. 3 9 9 .
23.
U. Bar, H. Becker, B. May, D. Mayer, S . Ohlendorf,

7 9 , 943 ( 1 9 7 3 ) .
P. Otto, F.W. Schmidt, Verh. Dtsch. Ges. Inn. Med.,
24.
Medical Department; Burroughs Wellcome Co., unpublished data, 1 9 6 3 . N. Kaplowitz, J. Kuhlenkamp, Gastroenterology, 74
90 (1978).
25.
26. Merk Index, Ninth Edition, Merck and Co. Inc: Rahway, N.J., 1 9 7 6 , p. 9 1 2 .
27.
M.J. Harber, J.L. Maddocks, J. Gen. Microbiol.,

351 ( 1 9 7 3 ) .
2,
28.
A.I. Al-Mosawi, J.N. Miller, J . W .

~
Bridges, Analyst,
1 0 5 , 448 ( 1 9 8 0 ) .
29. 30.
J . L . Maddocks, Br. J. Clin. Pharmac.,
8,273
(1979).
D.J. Nelson, C.J.L. Bugge, H.C. Krasny,

T.P. Zimmerman, J. Chromatogr.,
E,1 8 1
(1973). (1975).
31.
A.H. Chalmers, Biochemical Medicine,
12, 2 3 4
AZATHIOPRINE
53
32.
L . Sweetinan, W.L. Nyhan, J . C h r o m a t o g r . , (1968).

T . L . Ding, L . Z . B e n e t , 3. Chromatogr., (1979). A.F. F e l l , S.M. P l a g , J . M . 691 ( 1 9 7 9 ) .
32,
662
33.
163,281
34.
N e i l , J . Chromatogr.,
35.
N . Hobara, A . Watanabe, A c t a . Med. Okayama, 239 ( 1 9 7 9 ) .
2,
36.
S . - N . Lin, K . J e s s u p , M. F l o y d , T.-P.F. Wang, C . T . Van Buren, R.M. C a p r i o l i , B.D. Kahan, T r a n s plantation, 2 9 , 290 ( 1 9 8 0 ) .
J . L . Maddocks, B r . J , C l i n . P h a r m a c o l . , (1975).
37.
2,
359
38.
M . J . H a r b e r , J . L . Maddocks, J. Chromatogr., 231 (1974).
101,
39. 40.
41.
R . C . T h a p l i y a l , J . L . Mahdocks, J. Chromatogr., 239 (1978).

S. I t o , K . F u j i t a , J . C h r o m a t o g r . ,
160,
187, 418
(1980).
P.C.-P. Wong, J . L . Maddocks, J. C h r o m a t o g r . , 491 (1978).
150,
BENZYL BENZOATE
Mahmoud M . A. Hassan and Jaber S. Mossa
1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Taste, Odor 2. Physical Properties 2.1 Boiling Range 2.2 Melting Point 2.3 Density 2.4 Refractive Index 2.5 Solubility 2.6 Identification 2.7 Spectral Properties 3 . Synthesis 4. Metabolism 5. Methods of Analysis 5.1 Titrimetric Method 5.2 Spectrophotometric Methods 5.3 Spectrotitrimetric Methods 5.4 Gas Chromatographic Method 5.5 Proton Magnetic Resonance Method 6. References 56 56 56 56 56 57 57 57 57 57 57 57 57 58 64 67 68 68 68 68 68 69 73
ANALYTICAL PROFILES OF DRUG SUBSTANCES. 10
55
Copyright l Y X l byAcademicPre,s. Inc All rights of reptoduction in an) h r m rewved. ISBN 0-12-260810-0
56
MAHMOUD M. A. HASSAN AND JABER S. MOSSA
1. Descrivtion 1.1 Nomenclature 1.11 Chemical Names a) Benzoic acid phenylmethyl ester. b) Benzoic acid benzyl ester. c) Benzylbenzenecarboxylate. 1.12 Generic Name Benzyl benzoate. 1.13 Trade Names Ascarbin; Ascabiol; Benylate; Vanzoate; Venzonate. 1.2 Formulae 1.21 Emperical C14H1202
1.22 Structural
1.23 CAS No. 120-51-4 1.24 Wiswesser Line Notation RVOIR. 1.3 Molecular Weight 212 - 2 5 1.4 Elemental Composition
C,
79.22%; H, 5.70%;
0, 15.8%.
BENZYL BENZOATE
57
1.5 Appearance, Color, Taste,OdorLeaflets or colorless oily liquid, faint, pleasant aromatic odour, sharp burning taste. 2. Physical Properties 2.1 Boiling Range 323 - 324C (1) , (2) . bp16 189 - 191C (2); bpll 1 7 0 ' C bpqe5 1 5 6 ' C
(2).
(1)
2.2 Melting Point 21C. 2.3 Density di5 1.118 (2) , dt5 1.1121 (1).
2.4 Refractive Index

n2' D nZo
D
1.5681 (2) 1.5680 (1)

1.568 - 1.570 ( 3 ) .
n20
2.5 Solubility Insoluble in water o r glycerol, mescible with alcohol (95%), chloroform, ether, oi.ls, acetone, benzene, methanol, petroleum ether (1-3) . 2.6 Identification Boil 2 g with 25 ml of alcoholic potassium hydroxide solution for 2 hours under a reflux condenser. Remove the alcohol on a water-bath, add 50 ml of water, and distill until the liquid distilling is no longer turbid. The liquid remaining in the flask, after acidification with dilute hydrochloric acid, yields a white crystalline precipitate of benzoic acid.
58
To the distillate add 2.5 g of potassium permanganate and 2 ml of sodium hydroxide solution, boil for 15 minutes under a reflux condenser, cool, and filter. The filtrate after acidification with dilute hydrochloric acid, yields a white crystalline precipitate of benzoic acid ( 3 ) . 2.7 Spectral Properties 2.71 Infrared Spectrum The infrared spectrum of benzylbenzoate is recorded as a film on a Unicam SP 3-300 Spectrophotometer and is shown in Fig. 1. The assignments for the characteristic bands in the infrared spetrum are listed in Table 1. Table 1
IR Characteristics of Benzvlbenzoate -1 Assignment Frequency CM

1720 1601 1590 1275 1110 710, 700 C = C = C = c c Aromatic (ester) aromatic aromatic - c 0 - c monosubstitution.
0 C C 0
Other finger print bands characteristic of benzylbenzoate are 3060, 3030, 1500, 1450, 1380, 1315, 1180, 1070, 1025 and 740. The IR spectral data have also been reported (1,4), 2.72 Ultraviolet Spectrum (UV) The UV spectrum of benzylbenzoate in ethanol was scanned from 400-200 nm using Varian Cary 219,six maxima and six minima were observed (Fig.2). The maxima were located at 229, 256, 263, 266, 272 and 280 nm. The minima occur at 215, 254, 260, 265, 270 and 277. The UV spectral data of benzylbenzoate have also been reported (1,s). The El%, 1 cm = 843 at 230 nm ( 6 ) .
0 LD
8
0
ol
60
Fig. 2 .
Ultraviolet spectrum of benzyl benzoate in ethanol.
BENZYL BENZOATE
61
2.73 Nuclear Magnetic Resonance Spectrum 2.731 Proton Spectrum The proton NMR spectra of benzylbenzoate in deuterated chloroform and in DMSO-D6 are shown in Fig. 3a and Fib. 3b. These were recorded on a Varian T-60A, 60 MHz NMR spectrometer, using tetramethylsilane as an internal reference. The PMR spectra assignment of benzylbenzoate are given in Table 2. Table 2 PMR Characteristics of Benzylbenzoate Protons -CH2
0
Chemical shifts DMS0-D 6 CDCl3 5.26 5.39 7.47 8.03
-2H, 6H (adjacent to C) 7.23 Other aromatic Drotons. 8.00
Other PMR spectral data have also been reported (7,8). 2.732 I3C Suectrum I3C NMR spectrum of benzylbenzoate in carbon tetrachloride using tetramethylsilaine as an internal standard was recorded using Jeol FX 100 MHz instrument at ambient temperature and using 10 mm sample tube. The data consist of 8192 data points over a 5000 Hz spectral width Fig. 4 . The carbon chemical shift values areshown in Table 3. (9-11). 10 11
62
F i g . 3 a . PMR s p e c t r u m of b e n z y l b e n z o a t e a n d TMS i n CDCl?. F i g . 3b. PMR s p e c t r u m of b e n z y l b e n z o a t e a n d TMS i n DMSO-D6.
-r
4
u
-4
c,
(u
0
N
R
r i
a,
>r
N
R w
a,
5
c,
u 0) a
m
a z
m
7 .
-r
4
tn
63
64
MAHMOUD M. A. HASSAN A N D JABER S . MOSSA
Table 3 13C NMR Characteristics of Benzylbenzoate Carbon

No.
Chemical Shift. 165.63 130.30 129.56 128.20 132.59 128.20 129.56
Carbon
No.
Chemical Shift. 66.40 136.29 128.40 128.01 128.01 129.01 128.40
1 2 3 4 5
6
8 9 10 11 12 13 14
2.74 GC/Mass Spectrum The GC/Mass spectrum was recorded on Ribermag R 10-10 GC/Mass spectometer using 3% SE 30, packed glass column. The GC trace shows a retention time o f 6.57 minutes. The mass spectrum was obtained by conventional electron impact ionisation at 70 eV, shows a molecular ion M+ at m/e 212 and shown in Fig. 5. Other prominent fragments and their relative intensity are shown in Table 4. Table 4 m/e 77 91 105 65 51 Relative Intensity 100.00 77.3 95.9 42.9 57.4 Fragment ' g H ; C H CH2 6 5 C6H5 CO+
+
The mass spectrum o f benzyl benzoate has also been reported (1, 9). 3. Synthesis Three main methods are used f o r preparation of benzyl benzoate.
B B l SCAN 156 SICMA=S ~ r = 6 : 5 7 BACKGD= 15x100 100t/.=4046848 TITLE: SAHPLE BENZYL BENZOATE: 150-230 DEG(t0 DEG/ M I N I ; 3XSE30;E.I.
Figure 5. Mass spectrum of benzyl benzoate.
66
MAHMOUD M. A. HASSAN AND JABER S.MOSSA
I) Estrification of benzoic acid with benzylalcohol(12,13)
h2504
H20
11) Transposition between sodium benzoate and benzyl-
chloride. (12, 13).
Et3N 120
14OOC ( 1 h r ) ?
+ NaCl
BENZYL BENZOATE
67
111) Condensation of two molecules of benzaldehyde in the presence of sodium hydroxide (13).
4. Metabolism Benzylbenzoate is rapidly hydrolysed in vivo to benzoic acid and benzylalcohol. Benzylalcohol in turn is oxidised to benzoic acid which is then conjugated with glycine to form hippuric acid (Scheme 1 ) . , (5,6).
In V i v o
Oxidation
CH2 NH2 COOH
il C
-N -C
I H
- COOH
68
5. Methods of Analysis
5.1 Titrimetric Method The U.S.P. XVIII (14) describes a titrimetric method for determination of benzylbenzoate. The method is based on the hydrolysis of a weighed amount of the ester with aknown volume of O . 5 N alcoholic potassium hydroxide by boiling under reflux for an hour. Then the reaction mixture is cooled, phenolphthalin T.S. as indicator is added and the excess alkali is back titrated with 0.5N Hydrochloric acid. A blank determinanation is also performed. 1 ml of 0.5N alcoholic potassium hydroxide Z 106 mg of benzylbenzoate(C 14H 120 2) . 5.2 Spectrophotometric Methods Quantitative determination of benzylbenzoate as pure drug and in benzylbenzoate lotion by a spectrophotometric methods have been reported (15,16). The methods involve heating the sample under reflux with 10% alcoholic potassium hydroxide for 5 minutes and measuring the extinction of the cooled reaction mixture after dilution with water or ethanol at 268 nm. Beer's law is obeyed for up to 250 g ml-1 of hydrolysed benzylbenzoate. Interference from other ingredients of the sample (e.g., oleic acid and triethanolamine) is negligible.
5.3 Spectrotitrimetric Methods
Benzylbenzoate and dibutylphthalate are determined in mixtures by measurement of the absorbancy at 250 nm and the quantity of alkali required to saponify the esters (17). The concentration of the esters are calculated by application of a differential equation for which the absorptivity and the saponification contents of the components are required. Analysis of five know mixtures showed average recoveries of 100.8% for benzylbenzoate and 99.97% for dibutylphthalate. Application to cloth patches impregnated with insect repellents containing these esters gave average recoveries of 101.4% for benzylbenzoate and 99.66% for dibutylphathalate.
5.4 Gas Chromatographic Method
A gas-chromatographic method has been described for the determination of benzylbenzoate as a product of catalytic oxidation of toluene (18). The determination was
BENZYL BENZOATE
69
carried out on a column ( 2 m) of 20% of carbowax 20 M on chromosorb W (60 to 80 mesh) operated at 2000 with N ( 4 4 ml min-l) as carrier gas, flame ionisation detection and acetophenone or-benzylalcohol as nternal standard. 5.5 Proton Magnetic Resonance Method
An accurate, simple and precise PMR procedure has been developed in our laboratory for the quantitat on of benzylbenzoate and benzylcinnamate as pure drugs and in Peru and Tolu balsams (19). The method is based on the integration of the benzylmethylene protons of benzylbenzoate appearing at 5.30 ppm (Fig. 6 ) . In Peru and Tolu balsam the corresponding peak appears at 5.32 ppm (Figs. 7 8).
Ethylbenzoate is chosen as the internal standard, since it has methylene protons that provide comparable area of integration. Acetone, rather than acetone-D6, is employed as the solvent, since it is inexpensive and dissolves all balsam constituents as well as the internal standard. The average recovery of pure benzylbenzoate in standard mixture is 100.2 0.38 w/w. This method also offers the advantage of individually quantitating the esters, rather than the total ester contents in the medicinally used balsams. Moreover the spectrum of the balsam provides a useful mean for estimating the exact ratio of benzylbenzoate and benzylcinnamate, by simply measuring their corresponding benzylmethylene protons integrals. A l s o the PMR spectra of the esters and balsams are specific means of identification.
so
Fig.
7.0
6.0
6:
A.
PMR s p e c t r l l m of b e n z y l b e n z o a t e i n a c e t o n e - D 6 .
B.
P a r t of t h e P M R s p e c t r u m of b e n z y l b e n z o a t e a n 6 e t h y l benzoate i n acetone.
M 4
TMS
A
I ,
I I
I -
I
C . . l
I
B . . * i
I
* * *
a0
7.0
6.0
5.0 P P M ( b )
4.0
3.0
2.0
1.0
Fig. 7:
A. B.
PMR spectrum of Feru balsam in acetone-D6. Part of the PMR spectrum of Peru balsam and ethyl benzoate in acetone,
Fig.
t 3
-4
3:
A.
0.
PMR s p e c t r u m o f T O ~ J balsam i n acetone-D6.
P a r t ?f t h e PMR s p e c t r l i n of Tolu balsm a n d e t h y - b e n z o a t e i n acetorc.
BENZYL BENZOATE
73
References
1.
Atlas of Spectral data and Physical constants of Organic Compounds, edited by J . G . Grasselli and M 7 . M . Ritchey. Volume 2, CRC Press 1975, page 414. Merck Index, ninth edition, Merck 6 Co., Inc., Rahaway, N . . J . , {J.S.A., 1976, page 148. British Pharmacopoeia, London, Her Majesty's Stationery Office, 1973, page 51. The Aldrich Library of Infrared Spectra, Charles S. Pouchert, Second edition, Aldrich Chemical Company Tnc., 1975, page 90OC. F.C.G. Clarke "Isolation and Identification of Drugs". The Pharmaceutical Press, London, 1971, page 217. The Pharmaceutical Codex, Eleventh edition, The Pharma ceutical Press, London, 1979, page 91. Hi'gher resolution W R Spectra Catalog, Vol. 2, Spectrum No.627 compiled by Y . S . Rhacca, D.P. IJollis, L . F . ,Johnson and F.A. Pier of the Instrument Division of Varinn Associates, 1963. The Aldrich Library of NFlR Spectra. S. Pouchert and J.R. Compbele, \'olurr.e 7 , Aldrich Chemical Company, 1g7A, page 27D.
F . Stenhagen, S. Ahrahamsson and F.W. Mclafferty, "Req-
2. 3.
4.
5.
6.
7.
8.
9.
istry of )lass Spectral Data", .John Wiley and Sons, London, 1974, page 1122, A.A. 1511-1.
10.
Rruker 1.3-CData Rank, Volume 7, Rr000218. Sadtler Standard Carbon-13 NMR Spectra SAD 02833.
11.
12. Remington's Pharmaceutical Sciences, Fifteenth edition, Mack Publishing Co., Faston, Pa., 18042, 1975, paeel179 13. L.M. Atherden, "Bentley and Driver's Textbook of Pharmaceutical Chemistry", Eighth edition, London, Oxford University Press, 1969 page 571.
14.
The Pharmacopeia o f the U.S.A., Eighteenth revision, The [J.S. Pharmacopeial Convention Inc., 1970, page 76.
74
15.
V . Das Gupta and Hon. W. No, Am. J . Hosp. Pharm. 33 ( 7 ) , 665 (1976). V . Das Gupta and Hon. W . Ho, Am. J . Hosp. Pharm. 34 ( 6 ) , 453 (1977).
16.
17.
18.
J.O.
Page, Anal. Chem. 27 ( 8 ) , 1233 (1955).
Mager, S o r i n ; Hoparlean, I o n e l ; Toranu, Ruxandra and 22(2), P a i n , F l o r i c a , S t u d . Babes-Bolyai, Ser. Chem. 45 (1977).
A . H . Al-Obeid, M . M . A . Hassan and J . S . Mossa, S p e c t . 1 3 ( 6 ) , page 361 ( 1 9 8 0 ) . Letters, -
19.
CLINDAMYCIN HYDROCHLORIDE
Leo W. Brown and William F. Beyer
1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Color, Taste, Odor 2. Physical Properties 2.1 Approximate Solubility 2.2 Melting Range 2.3 Specific Rotation 2.4 pKa 2.5 Crystal Properties 2.6 Infrared Spectrum 2.7 Nuclear Magnetic Resonance Spectrum 2.8 Mass Spectrum 3. Synthesis and Proof of Structure 4. Drug Metabolites 5. Pharmacokinetics and Toxicity 6. Antibacterial Activity 7. Methods of Analysis 7.1 Microbiological 7.2 Paper Chromatographic 7.3 Gas Chromatographic 7.4 Liquid Chromatographic 7.5 Titrimetric 7.6 Radioimmunoassay 7.7 Thin Layer Chromatographic 8. References
76 76 76 76 76 77 77 77 77 77 79 80 80 80 82 82 83 83 83 84 84
R7
87 89 90
76
LEO W. BROWN AND WILLIAM F. BEYER
1.
Description
1.1
Name, Formula, Molecular Weight
C1 i ndamycin hydrochloride is methyl 71S)-chloro-6,7 ,atrideoxy-6-trans-(1 -methyl-4-propyl-L-2-pyrrolidinecarboxarnido)-1 -thio-L-threo-a-D-galacto-octopyranoside monohydrochl oridel, also 7 C1-7-deoxylincomycin.
CH3 HCCl CH3 I
HC I
C1gH33C1 N205S.HC1
Mol Wt. 461.44
1.2 Appearance, Color, Taste, and Odor Clindamycin hydrochloride monohydrate is a white or practically white, crystalline powder. It is odorless or has a faint mercaptan-like odor and has a bitter taste.
2.
Physical Properties 2.1 Approximate Sol ubil i ty2 Sol vent Water Pyridi ne Met ha no1 Ethyl Acetate Solubility, mg/ml
<1000, A00 <200,>loo <200,>100 <1
77
2.1 Approximate Solubility (con't) Sol vent Acetone Chloroform Oimethyl formamide Benzene Cycl ohexane Ethanol 2.2 Melting Range3 C1 indamycin Hydrochloride 141-143C Epicl indamycin Hydrochloride 164-166C (7-positi on) 2.3 Specific Rotation3 C1 indamycin ydrochloride Epicl indamyc n Hydroch oride 2.4 pKa The pKa of c indamycin hydrochloride has been reported as 7.64, the same as lincomycin5. 2.5 Crystal Properties6 Crystals of cl indamycin hydrochloride momhydrate were determined to be monocl inic with cell parameters: 0 0 a = 9.47A, b = 9.91A, c = 13.50A, p = 104.5". The crystal structure i s stabilized by complex hydrogen bonding involving the chloride ion and the water of hydration.
0
Sol ubi 1 i ty , mg/ml

<1 <l
c500,>250
<1 <1
<lo,>5
+144"(water) +122"(water)
2.6 Infrared Spectrum
A typical mineral oil mull spectrum of clindamycin hydrochloride monohydrate is shown in Figure 1. Anhydrous cl indamycin hydrochloride is hygroscopic, consequently, different crystal forms have been observed due to the degree of hydration7. Solvation with ethanol or acetone is also possible, producing variation in the infrared spectrum.
100
90
80
70
60 50
40
30
20 10
3800 3000
2000
1800
1600
1400
F RE QUE NCY C M
Figure 1.
-'
1200
1000
800 700
Infrared spectrum o f clindamycin hydrochloride monohydrate.
79
2.7
Nuclear Magnetic Resonance Spectrum
l o g s including c l indamycin (7-chloro-7-deoxy-l incomycin) . They found the nmr spectrum o f lincomycin t o be complex, containing many superimposed m u l t i p l e t s which were d i f f i c u l t t o f a c t o r . Consequently, t h e compound was hydrolyzed a t the amide 1 inkage i n t o an amino a c i d (L-trans-4-n-propyl hygric a c i d ) and an amino t h i o sugar. Values obtained f o r the sugar moiety of clindamycin a r e shown i n Table I . The nmr spectrum of clindamycin i s quite s i m i l a r t o t h a t o f lincomycin w i t h the exception of a downfield s h i f t o f the doublet a t t r i b u t a b l e t o the hydrogens of the terminal methyl(C-8) due t o the c h l o r i n e atom a t the 7p o s i t i o n of clindamycin i n s t e a d of the hydroxyl a t t h i s pos i t i o n i n l incomycin. 3 TABLE I Chemical s h i f t s and coupling c o n s t a n t s obtained on the sugar moiety o f clindamycin i n D20 w i t h sodium 2,2dimethyl -2- s i 1apentane- 5- sul f o n a t e a s r e f e r e n c e . Chemical S h i f t P o s i t i o n Frequency, cps Coupling Constant Jxy Frequency, cps 1 2 2,3 34 45 5.5 10.5 3.5 1 10.5
m r t o study the s t r u c Slomp and MacKellar* used n ture of lincomycin, i t s degradation products, and some ana-
c- 1 c- 2
c-.3 c- 4 c- 5 C- 6 c- 7 C-8
325 249 21 9 235 260
2 64
272
87
Carbon - 1 3 nmr s p e c t r a l a n a l y s i s and s p i n l a t t i c e r e l a x a t i o n times f o r c l indamycin hydrochloride were r e p o r t e d by Mizsak e t a1 .9
80
2.8 Mass Spectrum
Kagan and GrosticlO used mass spectrometry as a technique for structural determination of lincomycin, its degradation products and analogs including cl indamycin. They found cl indamycin to have essentially the same fragmentation pattern as 1 incomycin. Some characteristic m/e values and their percent relative intensity (in brackets) were reported as follows: 424( 0.3) 426(0.2)
"I+
(a) 377( 0.4) 379(0.1)
275( 3.4)
(b)
(c) (d) 126(100.0) 82( 3.5)
277(1.3)
Fragments containing the chlorine atom produce two values due to the two isotopes of the chlorine atom ( 17C135,!7Cl 3 7 ) . Figure 2 shows the structures of the characteristic fragment ions.
3.0 Synthesis and Proof of Structure
C1 indamycin( 7(S)-chloro-7-deoxyl incomycin) can be prepared b y treating 1 incomycin hydrochloride with thionyl chloride or with triphenylphosphine (along with chlorine or carbontetrachloride). The reactions place a chlorine atom at the 7-position of lincomycin replacing the hydroxyl at that position with an inversion o f configuration. Configuration of the hydroxyl at C-7 in lincomycin was previously established as 7(R). Since the methods used for synthesis of clindamycin are known to cause inversion, the expected configuration of the chlorine at the 7-position was 7(S). Oxidation of clindamycin produced an acidic fragment identified as L-chloropropionic acid giving unequivocal evidence as to the configuration at C-7.
4.0 Drug Metabol i tes
Microbial transformations of cl indamycin were studied by Coats and Argoudelis. l 1 They showed that clindamycin added to whole-cell cultures of Stre tom ces coelicolor resulted in production of the inactive against Sarcina lutea) metabolite cl indamycin 3-phosphate. C1 indamycin sulfoxide was the major transformation when clindamycin was added to fermentations of Streptomyces armentosus. Ndemethylation was observed in fermentations of Streptomyces punipal us.
81
HCCl
R2
HCCl
Figure 2.
Fragmentation pattern of clindamycin. R 1 = n-propyl, R 2 , R 3 = methyl
82
4.0 Drug Metabol i tes (con't)
In human subjects, a metabolite believed to be the N-demethylation compound was detected b y Brodasky et a1 l 3 using thin layer chromatography. Using radioactive cl indamycin given orally to a dog, Sun14 determined that the following radioactive materials were excreted in the dog urine: 36% intact drug, 28% as the sulfoxide, and 28% as the glucuronide conjugate of the intact drug. In an experiment using the rat, he found that the following were excreted in the urine: 53% as intact drug, 31% as the sulfoxide, and 15% as the N-demethyl metabol i te.
5.0 Pharmacokinetics and Toxicity
Absorption, excretion and half-1 ive of cl indamycin in normal adult males was studied b y Wagner et al.l5 Clindamycin was absorbed extremely rapidly in man following oral administration. The half-1 ife of cl indamycin, estimated from serum activities, averaged 2.38 hours. Approximately oneeighth of the dose of clindamycin administered orally was recovered as active drug in the urine in 24 hours.16 Pharmacokinetic studies in humans were also made by DeHaan et a1.l7 They found no evidence of drug accumulation or that it stimulates its own metabolism during repeated dosing. Common untoward effects of clindamycin given orally in man were loose stools, cramps, excessive flatus, and nausea. Oral toxicity of clindamycin in laboratory animals was studied by Gray et a1.18 They found the maximum daily tolerated dose in the dog and rat for as long as one year was greater than 300 mg/kg but less than 600 rng/kg. At 600 mg/kg in short term studies, the effects of foca? irritation in the mucosa of the stomach and gallbladder of the dog were in evidence.
6.0 Antibacterial Activity
in-vitro antibacGarrison et al l 9 have compared -terial activities of cl indamycin, 1 incomycin, and erythromycin against a number o f strains of several genera of clinical isolates of bacteria. Oppenheimer and Turck20 carried out both laboratory and clinical evaluation of clindanycin showing it to be active against Staphylococcus aureus and Diplococcus pneumoniae but of minimal activity against Enterobacteriaceae. Magerlein21 has reviewed the antibacterial activity of clindamycin and its microbial modifications.
83
Structure-acti vi t y re1 ationshi ps of 1 i ncomyci n , cl i ndamyci n , and related a n t i b i o t i c s have been reviewed by Pyke.22
7.0
Methods of Analysis 7.1 Microbiological
Hanka e t al 2 3 developed a microbiological assay f o r lincomycin which i s also applicable t o clindamycin. I t i s a disk-plate agar diffusion method using Sarcina lutea as the assay organism. The procedure i s sensitive t o approximately 0.25 mcg/ml i n blood-level determinations. Sarcina lutea is grown f o r 18 hours on the surface o f agar i n Roux bottles, harvested, and suspended i n a broth a t a concentrat i o n of about 3 x 1O1O c e l l s per ml. Penassay Seed Agar i s inoculated w i t h the cell suspension a t 0.5 ml of the culture per l i t e r of medium. E i g h t ml of the seeded medium i s poured i n t o a p l a s t i c petri dish and allowed t o s o l i d i f y . A sample of a buffered solution of clindamycin, approximately 5 mcg/ml , i s applied on an assay disk and the plate incubated f o r 1618 hours a t 30?C. The zone of inhibition i s then measured and compared w i t h a reference.
7.2
Paper Chromatographic
Paper chromatography has been widely used i n identification of a n t i b i o t i c s from fermentation beers. Mason e t a124 used s i x solvent systems (Table 11) t o characterize lincomycin, a precursor of clindamycin. Zones were located and quantitated by bioautography on Sarcina 1 utea.
TABLE I 1
Solvent systems used f o r identification of a n t i b i o t i c s by paper chromatography. Devel o p i ng Sol vent Developing Time, h r .
16 16
16
I 1- butanol -water( 84: 1 6 ) I1 l-butanol-water(84:16) plus 0.25% p-toluenesul fonic acid I I I 1 -butanol -acetic acid-water (2:l:l) I V 1 - bu tan01 -water ( 84 :16 ) pl us 2% pi peri di ne
16
84
7.2
Paper Chromatographic ( c o n ' t ) Developing Time, hr. 5 5
Developing Sol vent

V
VI
1- bu tan01 -water ( 4 :96) 1- butanol -water ( 4 :96) pl us 0.25% p-toluenesulfonic a c i d 7.3 Gas Chromatographic
S t a b i l i t y of clindamycin i n aqueous s o l u t i o n was determined by Oesterl i n g 2 5 using gas chromatography. Brownz6 developed a gas chromatographic a s s a y f o r clindamycin i n hard f i l l e d c a p s u l e s . For s a t i s f a c t o r y chromatography, d e r i v a t i z a t i o n ( t r i m e t h y l s i l y l , a c e t y l , t r i f l u o r o a c e t y l ) was u s u a l l y employed t o n e u t r a l i z e the p o l a r groups of the clindamycin molecule. The t r i f l u o r o a c e t i c a c i d d e r i v a t i v e was prepared by adding 0.5 ml of t r i f l u o r o a c e t i c anhydride t o approximatel y 5 mg of clindamycin hydrochloride in 2.0 ml o f chloroform c o n t a i n i n g hexacosane a s i n t e r n a l standard. The s o l u t i o n was m heated a t 45?C f o r 30 min. and chromatographed on a 61 c column packed w i t h 3% OV-17 on 60-80 mesh Gas Chrorn Q. Figure 3 shows a chromatogram obtained u s i n g the following condit i o n s : column temperature 170C, FID d e t e c t o r temperature 2OO0C, helium c a r r i e r gas a t a flow o f 60 m l / m i n . , and 1 micro1 i t e r sample volume i n j e c t e d d i r e c t l y i n t o the g l a s s column. 7.4 Liquid Chromatographic
Brown27 chromatographed c l i ndarnyci n hydrochloride by HPLC u s i n g i o n - p a i r chromatography. Figure 4 shows a chromatogram obtained u s i n g the following c o n d i t i o n s : Column:
30 cm x 4 mm ID prepacked w i t h C18 UBondapa k
Mobile Phase:
q.s.
1 g. sodium d i o c t y l s u l f o s u c c i n a t e 1 . 0 m l formic a c i d 125 m l water 500 m l w i t h anhydrous methanol
Detection:
r e f r a c t i v e index d e t e c t o r ( 9 . 6 x RI u n i t s f u l l s c a l e )
85
w
v)
z
0
v)
a a 0 c u
YI
L
0
a b
1 0
Figure 3.
4 8 TIME (MINUTES)
12
Gas chromatogram of: (a) trifluoroacetylated epilincomycin, (b) trifluoroacetylated clindamycin B, (c) trifluoroacetylated clindamycin, and (d) hexacosone as internal standard.
86
e
C
b
CT 0
0
Figure 4.
10 MINUTES
15
20
HPLC chromatogram of: (a) injection, (b) solvent, (c) lincomycin, (d) clindamycin, (e) testosterone propionate as internal standard.
87
7.4 Liquid Chromatographic (conlt)

Column Pressure: 1000 psi
An improved ion-pairing HPLC method has been developed by LandisZ8 which separates clindamycin, cl indamycin B y and 7-epic1 indamycin. (In Brown's method27, epiclindamycin and clindamycin elute as a single peak.) RI detection was utilized with the mobile phase consisting of a 60/40 ratio methanol/water, 2 ml glacial acetic acid per liter (0.035 M), and 0.005 M D,L-10-sodium camphor sulfonate, adjusted to pH 6.0. k 30 cm x 3.9 mm Water's u Bondapaks C1, column was utilized at a flow rate of 1.0 ml/min. (approximately 900 psig). Data are given for the assay of capsules, syrups, and clindamycin hydrochloride bulk drug. The RSD for the assay is approximately 1%. A chromatogram of a synthetic mixture containing lincomycin, lincomycin By clindamycin B y and 7-epiclindamycinY and clindamycin is given in Figure 5. The author stated that if additional sensitivity is required UV 214 nm detection can be employed, utilizing a mobile phase comprised of a 60/40 ratio methanol/ water, 0.01 M phosphate buffer, and 0.005 M sodium pentane sul fonate.
7.5 Ti trimetric
0.5 N potassium hydroxide using a potentiometric t i t r a t ~ r . ~ ~
C1 indamycin hydrochloride has been titrated with
The endpoint is the point at which A E / A V is greatest. 7.6 Radioimmunoassay
A radioimmunoassay was developed by Gil bertson and Stryd30 which is as sensitive as the microbiological assay . 1 mcg/ml). Tritiated clindamycin was pre(approximately 0 pared for the assay by reacting N-demethyl clindamycin with formaldehyde and tritium sodium borohydride. C1 indamycin-2hemisuccinate was prepared and coupled with bovine serum albumen. Rabbits were injected with the conjugate along with Freund's Complete Adjuvant at monthly intervals to product antiserum. Serum samples to be assayed for clindamycin were extracted with chloroform and the chloroform extract evaporated to dryness. Standards were made in the range of 1 to 250 nanograms of clindamycin hydrochloride per 0.1 ml of di sti 1 1 ed water.
88
F 2
a LL
c
I, II
IV A
L
12
14
0
Figure 5.
6 a 10 TIME (MINUTES)
Chromatogram of a synthetic mixture of lincomycin (l), lincomycin B (11), clindamycin B (111), 7-epiclindamycin (IV) , and clindamycin (V)
89
7.6
Radioimmunoassav (can't)
The assay procedure i n v o l v e s t h e doubl e a n t i body method i n which both unlabeled and t r i t i a t e d clindamycin compete f o r b i n d i n g s i t e s on t h e a n t i b o d i e s o f t h e antiserum produced from t h e r a b b i t s . A second antibody i s added t o form an i n s o l u b l e primary-secondary antibody complex. The p r e c i p i t a t e i s washed and s c i n t i l l a t i o n c o c k t a i l added. A f t e r d i s p e r s i o n o f t h e p r e c i p i t a t e i n t h e c o c k t a i l , t h e mixt u r e i s counted i n a l i q u i d s c i n t i l l a t i o n counter. 7.7 Thin Layer Chromatographic
S i l i c a gel H t h i n l a y e r p l a t e s w i t h developing sol vent methyl e t h y l ketone-acetone-water(186:52:20) were used by Brodasky e t a l l 3 t o q u a n t i t a t e t h e N-demethyl c l i n damycin metabol it e . The zones were detected by bioautography on Sarcina l u t e a . S i l i c a gel GF254 t h i n l a y e r p l a t e s have been used w i t h developing s o l v e n t o f methanol-chloroform ( 1 : 3 ) . 2 I n t h i s system, clindamycin has an approximate Rf o f 0.70 w h i l e l i n c o m y c i n has an R o f 0.65. Detection i s u s u a l l y by c h a r r i n g , i o d i n e vaporg, o r permanganate spray. The l a t t e r c o n s i s t s o f 10 g. o f potassium carbonate, 8 g. o f sodium periodate, and 1 cj. o f potassium permanganate i n 500 m l o f water. The m i x t u r e i s l e f t standing f o r 16 hours and f i l t e r e d . Clindamycin gives a y e l l o w s p o t on a p u r p l e background u s i n g t h i s spray.
90
9.0
References 1. " U n i t e d States Pharmacopeia," 1 9 t h Ed. P r i n t i n g Co., Easton, PA. Humphrey, L.M. data.
, Mack
2.
3. 4.
, The
Upjohn Company, unpublished J.Med. Chem.
Birkenmeyer, R.D.
and Kagan, F.J.,
13, 616(1970).
Novak, E., Wagner, J.G. , and Lamb, D.J. C1 in. Pharm. 3, 201 (1970). "Merck Index" 8 t h Ed., Merck and Co.,
, Int. J.
5.
6.
Rahway, N.J.
Duchamp, D.J., A b s t r a c t s o f t h e American C r y s t a l lography Assoc. , Summer Meeting, Aug. 20-25, 1967, Minneapolis, Minn., Paper D5. Meulman, P.A.
7. 8. 9. 10. 11. 12. 13. 14. 15.
, The Upjohn Company, unpublished data.

J. Am. Chem. SOC.
89,
Slomp, G. and MacKellar, F.A., 2454( 1967).
Mizsak, S., Lukacs, G.,
Slomp, G., Neszmely, A., Gero, S.D. and Tetrahedron L e t t e r s , 8, 721 (1977). Org. Mass Spectrometry
J
Kagan, F. and G r o s t i c , M.F.,
6, 1217(1972).
Coats, J H. and Argoudel i s , A1 exander D., Bact. 108, 459(1971). Argoudelis, A.D. , Coats, J.H., Mason, D.J., Sebek, O.K., J. A n t i b i o t . 22, 309(1969). Brodasky, T.F., Argoudelis, A.D., J. A n t i b i o t . 21, 327(1968).
Sun, F. F.
.
and
and Eble, T.E., (1970).
Fed. Proceedings
29,
Wagner, J.G., Novak, E., Patel, N.C., Chidester C.G., and Lummis, W.L. , Am. J. Med. Sci. 256, 25( 1968). McGehee, R.F., Smith, C.B., Wilcox, C., M., Am. J. Med. S c i . 256, 279(1968). and F i n and ,
16.
91
9.0
References ( c o n ' t ) 17. DeHaan, R.M., Metzler, C.M., Schellenberg, D . , VandenBosch, W . D . , and Masson, E . L . , I n t . J . Clin. Pharmacol. 6 , 105(1972). Gray, J.E., Weaver, R.N., Feenstra, E.S., Toxicol. 51 6( 1972). B o l l e r t , J.A., and 21, Appl. Pharmacol. -
18.
19. 20. 21. 22.
Garrison, D.W., DeHaan, R.M., and Lawson, J.B., Antimicrob. Agts. and Chemother. p. 397(1967).
256,
Oppenheimer, S. and Turck, M., 314(1968).
Am. J . Med. S c i .
Magerlein, B.J., Adv. Appl. Microbio ocy 14, 185(1971).

L
Pyke, T . R . , Progress i n Antimicrobia and Anticancer Chemotherapy, Proceedings o f 6th I n t . Cong. Chemotherapy, p. 254(1969). Hanka, L.J., Mason, D.J., Burch, M.R., and Treick, R. W . , Antimicrob. Ag. Chemother., p. 565( 1962). Mason, D.J., Dietz, A . , and DeBoer, C . , Antimicrob. Ag. Chemother. , p. 554( 1962). O e s t e r l i n g , T.O., J . Pharm. S c i . 59, 63(1970).
23. 24. 25. 26. 27. 28. 29.

30.
63, 1597(1974). Brown, L . W . , J . Pharm. Sci Brown, L.W., Landis, J.B.

Knight, N.H.,
J . Pharm. S c i . , 67,1254 (1978).
, submitted
f o r pub1 i c a t i o n .
The Upjohn Company, unpublished d a t a .

C l i n . Chem
G i l b e r t s o n , T.J. and S t r y d , R.P., 828( 1976).
2,
CODEINE PHOSPHATE
Farid J. Muhtadi and Mahmoud M . A. Hassan
1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste 2. Physical Properties 2.2 Solubility 2.3 Dissociation Constant 2.4 Optical Rotation 2.5 Sptctral Properties 3. Preparation 3.1 Isolation of Codeine 3.2 Formation of Codeine Phosphate 4. Synthesis of Codeine Phosphate 4.1 Total Synthesis of Codeine 4 2 Partial Synthesis of Codeine 5 . Biosynthesis of Codeine 6. Metabolism 7. Methods of Analysis 7.1 Identification Tests 7.2 Microcrystal Tests 7.3 Titrimetric Methods 7.4 Complexometry 7.5 Spectrophotometry 7.6 Chromatography 8. References 94 94 94 96 96 97 97 101 101 101 103 110 110 112 112 112 113 117 120 120 120 122 123 124 125 127 134
ANALYTICAL PROFILE5 OF DRUG SUBSTAKCES, 10
93
Copyright @) 1981 b) Academic Press. Inc All rights of reproduction iii any form rerencd. ISBN 0-12-260810-l1
94
FARID J. MUHTADI AND MAHMOUD M. A. HASSAN
1. Description
1.1.
Nomenclature
1.1.1
Chemical Names (a) 7,8-Didehydro-4,5 C I -epoxy-3-methoxy17-methylmorphinan-6 a -01 -phosphate
(1 : 1) (salt).
(b)
Morphinan-6-01, 7,8-didehydro-4,5epoxy-3-methoxy-l7methyl-, ( 5 a , 6 a)
phosphate (1 : 1) (salt). 1.1.2 Generic Names Codeine phosphate ; Morphine-3-methyl ether phosphate; Methylmorphine phosphate; Morphine monomethyl ether phosphate. 1.2. Formulae 1.2.1 Emprical: C18H24N07P. C H NO P. $H20 18 24 7 C H NO P. 312 H20 18 24 7 1.2.2 Structural (Anhydrous) (Hemihydrate) (Sesquihydrate)
CODEINE PHOSPHATE
95
More t h a n twenty s t r u c t u r e s were proposed f o r morphine and o t h e r r e l a t e d p h e n a n t h r e n e a l k a l o i d s . The c u r r e n t l y a c c e p t e d s t r u c t u r e i s t h a t proposed i n 1925 by Gulland and Robinson ( 1 ) . The proposed s t r u c t u r e w a s confirmed by t h e t o t a l s y n t h e s i s of morphine i n 1956 which w a s a c h e i v e d by G a t e s and T s c h u d i ( 2 ) . 1.2.3 CAS no.
[ 52-28-81
codeine phosphate tr ih emih y d r ate H3P04.3/2 H20)
(C18H21N03. 1.2.4
Wiswesser L i n e N o t a t i o n
TB6 566 B6/CO 4ABBC R
BX H
0 PN
DU GHT & & T T J
F Q J O 1 P & QH 6r H3-P-04 1.2.5 Stereochemistry Codeine p o s s e s s e s f i v e d i f f e r e n t asymmetric c g n t r e s ( a t C 59 c69 cg9 c13 and c 1 4 ) , b u t s i n c e t h e b r i d g e d r i n g system imposes some r i g i d i t y upon t h e system, t h e t h e o r e t i c a l number of o p t i c a l i s o m e r s i s l i m i t e d t o s i x t e e n . The d e d u c t i o n of r e l a t i v e conf i g u r a t i o n s a t t h e v a r i o u s c e n t r e s by c h e m i c a l methods h a s been w e l l summarized by Ginsburg ( 3 ) . The a b s o l u t e s t e r e o c h e m i s t r y h a s been deduced from a c o m b i n a t i o n of X-ray c r y s t a l l o g r a p h y and c h e m i c a l d e g r a d a t i o n and c o r r e l a t i o n I. From t h e X-ray d a t a of morphine hydroi o d i d e d i h y d r a t e (4) and of c o d e i n e hydrobromide d i h y d r a t e ( 5 , 6 ) i t w a s concluded t h a t t h e m o l e c u l e s a r e a p p r o x i m a t e l y T-shaped, w i t h atoms of r i n g s A and B and t h e o x i d e r i n g l y i n g n e a r o n e p l a n e , and t h e atoms of r i n g s C(cycloa1kene r i n g ) and D ( p i p e r i d i n e r i n g ) l y i n g c l o s e t o a second p l a n e a t r i g h t a n g l e s t o t h e f i r s t . The B / C r i n g j u n c t i o n w a s shown t o b e c i s , and p i p e r i d i n e r i n g , D ,
96
t o be a n almost r e g u l a r chair-shape, w i t h t h e methyl group a t t a c h e d t o n i t r o g e n by a n e q u a t o r i a l bond. The c y c l o a l k e n e r i n g C i s a l m o s t boat-shaped, and t h e C -oxygen and C 5 6 hydroxyl group are c i s t o e a c h o t h e r ( 7 ) . The a b s o l u t e geometry of t h e e n t i r e m o l e c u l e 11, follows from i t s r e l a t i v e s t e r e o c h e m i s t r y and h a s been confirmed by optical rotatory dispersion studies ( 8 ) .
OH
I I
1.3
M o l e c u l a r Weight:Anhydrous Hemihydrate Sesqu ihyd ra t e
397.37 406.36 424.37
1.4
Elemetal Composition: Anhydr Gus

C , 54.41% ; N, 3.56%. H , 6.05%
0, 28,18% ;
P, 7.80%
Hemihydrate C , 53.20% ; H , 6.16 P , 7.63%

S e squ i h y d r a t e
; N , 3.45%
0 , 29.56%
C , 50.94% ;
H , 6.37% ;
N , 3.30% ; 0 , 3 2 . 0 8 ; P,7.31%
CODEINE PHOSPHATE
97
1.5
Appearance, C o l o r , Odor and T a s t e : Codeine p h o s p h a t e o c c u r s i n two forms, one c o n t a i n i n g % m o l e c u l e of water of c r y s t a l i z a t i o n and t h e o t h e r c o n t a i n i n g 1%m o l e c u l e of water of c r y s t a l i z a t i o n . I t i s o d o r l e s s and h a s a b i t t e r taste. H e m i hyd r a t e F i n e , w h i t e , n e e d l e shaped c r y s t a l s o r w h i t e c r y s t a l l i n e powder. Sesquihydrate Very e f f l o r e s c e n t , small c r y s t a l s o r c r y s t a l l i n e powder.
2.
Physical Properties
2.1.1
X-ray D i f f r a c t i o n The s t e r e o c h e m i c a l c o n f i g u r a t i o n of t h e c o d e i n e m o l e c u l e w a s d e t e r m i n e d by Lindsey and Barnes (5) by a two d i m e n s t i o n a l s t u d y of c o d e i n e hydrobromide d i h y d r a t e . A t h r e e d i m e n s t i o n a l s t u d y of t h e s a l t h a s been a l s o c a r r i e d o u t by K a r t h a e t a 1 ( 6 ) . I n t e r a t o m i c d i s t a n c e s and bond a n g l e s a r e l i s t e d i n T a b l e s 1 and 2 r e s p e c t i v e l y . The c o d e i n e m o l e c u l e i n i t s a b s o l u t e c o n f i g u r a t i o n i s r e p r e s e n t e d i n F i g . 1. T a b l e 1. I n t e r a t o m i c D i s t a n c e s (A ) Intramolecular
0
CHI-O
1.443 1.399 1.372 1.401 1.387 1.399 1.369

1.388
(1.58) (1.49) (1.45) (1.34) (1.30) (1.40) (1.32) (1.37)
C14-C8
C8-C7
1.497 1.324 1.531 1.516 1.427 1.561 1.539 1.496
(1.48) (1.31) (1.49) (1.59) (1.46) (1.59) (1.63) (1.52)
'1-'3
'3-'2 c2-c1
cl-cll cll-c12
C7-Cg
C6-C5 C6-OH C14-C9 C9-Cl0
1 2-'4 '
c4-c3
Cl0-Cl1
CODEINE PHOSPHATE
98
F i g . 1.
Model showing t h e a b s o l u t e c o n f i g u r a t i o n of t h e c o d e i n e m o l e c u l e ( o n l y t h o s e H ' 5 on C 5' C 6 , C g , C I 4 a r e shown).
CODEINE PHOSPHATE
c4-02 0 -c 2 5 c5-c13
' 1 3 ' 1 2 ' 1 3 ' 1 4
1.370 1.472 1.532 1.504 1.564
(1 -45)
C13-C15 c15-c16 C16-N N-Cg N-CH3"
1.535 1.530
1.468
(1.62) (1.54) (1.58)
1.521 1.506
Intermolecular
c1-c7
7 ' 2 C3-H20' C4-H20' C5-Br C6-N C -H 0" 8 2
3.716 3.728 3.561 3.528 3.728 3.817 3.638 3.393
Cg-OH C11-C7 C12-C7
3.577 3.722 3.638
C10-H20" 3.680
16 2 ' CH3-H20"
c~~'l-0~ N-C 6 ' 1 ' 1 5 02-H20' OH-CH;'
C13-H20' 3.834 C14-H20' 3.543 C15-OH 3.644
c7-c 12
rable 2. Bond angles CH -0 -C 3 1 3 1 3 4 117.2 114.5 127.3 118.2 120.2 122.5 115.2 125.8
118.6
(112) (111)
(g)
105.5
108.4
-c -c 0 -c -c 1 3 2 c4-c3-c2 c3-c2-c1 c2-c1-c11

0
c12-c13-c14 c15-c13-c14
c5-Cl3-Cl4
(129)
(119)
116.9 112.2
c12-c13-c1 5
c13-c14-c8 cl3-cl4-Cg C8-Cl4-C9
(115) (129) (113) (129)
108.1
107.8 112.5 112.6 110.7 113.6
Y C 1 l-cl 2 o-cll-cl
c1 0-cll-c12 1 ' 1 ' 1 2 ' 4

13 ' 1 2 ' 1 1
122.7 127.3 109.4 120.8 111.9
C13-C15-C16 (118) C15-C16-N (1 25) C16-N-Cg (126) (118) (115) C16-N-CH3" N-Cg-C14 N-C -C 9 10
110.4
113.1 105.2 113.0
1 ' 3 ' 1 2 ' 4 1 ' 2 ' 4 ' 3 1 ' 3 ' 1 2 ' 2
(1 09) C9-N-CH3"
100
c3-c 4-0 2 c4-0 2-c 5

O2-'5-'13
127.0 107.7 105.1 112.8 111.0 101.3
(126) (102) (108) (118) (119) (96) (119)
Cl4-Cg-Cl0
C -C
114.0
11
115.4 112.5 111.2 113.9 119.8 120.6
(109)
10
-C
(119)
(104) (107) (100) (123) (118)
C5-C6-OH
c6-c 5-0 2
'5-'13-'12 c5-c13'c1
c6'c5-c13
C7-C6-OH
C5-Cg-C7
C6-C7-C8 C7-C8-C14
112.4
The i n t r a m o l e c u l a r v a l u e s i n p a r e n t h e s e s have been r e c a l c u l a t e d w i t h an I B M 650 computer f o r t h e u n i t c a l l dimensions a t atomic p a r a m e t e r s of Kindsey and Barnes (1955). The i n t e r a t o m i c d i s t a n c e s and a n g l e s s u g g e s t i v e of hydrogen bonding a r e l i s t e d i n Table 3 . Table 3 . 0-H.. .N
H-O'-H...O-H
H-oI-H..
I n a t e r a t o m i c d i s t a n c e s and a n g l e s i n v o l v i n g p o s s i b l e hydrgen bonds (. .)
2.772 A
H20'
...H-Br
.H20'.
3.262 A 3.395 3.397 112.9' 102.5 137.8 353.2'
11
.O1
2.762 2.852 105.6' 109.3 104.1 113.6) 110.4) 113.1)
H-Br...H-0
H-BY.
O1..
..H-0"-H
..OH ...H B r
H20'
...N-C16 OH. . .N-C 9 OH ...N-CH3

OH
01...H20'...HBr
OH...
(C 16-N-C9
16 (C~-N-CH 3
Mean
(c
-N-CH~II
109.3'
H20'. H20'
. ..N H20' . . .OH-C6

H20'. .OH.
C6-OH..
.N
105.5' 122.3
.. . H B r ...H20" H20". . .H B r . . .H20"
.. H B r . .
.H20"
176.6' 100.4 82.3 -~ 359.3O
127.8
CODEINE PHOSPHATE
101
The system of p o s s i b l e hydrogen bond i s r e p r e s e n t e d by t h e b r o k e n l i n e s i n F i g . 2 .
2.1.2.
Melting P o i n t s Codeine p h o s p h a t e m e l t s a t : -
245 - 248O 225 - 240' 220 2.1.3.
(9)
by h o t b a r method by h o t s t a g e method
(9)
235'
w i t h d e c . (10)
Eu tec t i c Tempra t u r e
Sal
Dic
187' 149'
187'
(9)
by h o t bar method
Sal
Dic
143O
(9)
by h o t s t a g e method
D i c = dicyandiamide
S a l = acetaminosalol
2.2.
Solubility F r e e l y s o l u b l e i n water ( l g i n 4 m l ) ; v e r y s o l u b l e i n h o t water ( l g i n 0 . 5 m l ) ; s l i g h t l y s o l u b l e i n a l c o h o l ( l g i n 4 5 0 ml) b u t more s o l u b l e i n b o i l i n g a l c o h o l ( l g i n 125 ml) (11, 1 2 )
2.3.
D i s s o c i a t i o n Constant The pKa v a l u e of c o d e i n e p h o s p h a t e a t 20' 8.2 ( 1 3 ) .

is
2.4. Optical Rotation

D
- 98' t o - 102'
( 2 % aqueous s o l u t i o n ) ( 1 3 , 1 4 ) .
The o p t i c a l r o t a t i o n of c o d e i n e p h o s p h a t e as.013% aqueous s o l u t i o n and a s .013 % e t h a n o l i c s o l u t i o n have been d e t e r m i n e d i n o u r l a b o r a t o r y u s i n g a Perki n E l m e r P o l a r m a t i c model 241 MC and found t o b e :
[a] D
24'
- 110'
102
Fig.
2.
P r o j e c t i o n showing p o s s i b l e h y d r o g e n b o n d s (broken l i n e s ) .
103
2.5.
Spectral Properties 2.5.1

U 1 t r a v i o l e t Spectrum
The UV spectrum of c o d e i n phosphate i n water w a s scanned from 200 t o 400 nm u s i n g Varian c a r r y 119 s p e c t r o p h o t o m e t e r . I t e x h i b i t s a c h a r a c t e r i s t i c UV spectrum ( F i g . 3 ) w i t h a maximum a t 284.8 nm (1570). Other UV s p e c t r a l d a t a of c o d e i n e phosp h a t e have a l s o been r e p o r t e d : -
max 284 nm
(1585)
i n water (15) in
A max 284 nm water. (16).

2.5.2 I n f r a Red Spectrum
( E l % , 1 cm about 52.3)
The I R spectrum of c o d e i n e phosphate a s KBr-disc was recorded on a Unicum SP 1025 s p e c t r o m e t e r ( F i g . 4 ) . The s t r u c t u r a l assignments have been c o r r e l a t e d w i t h t h e f o l l o w i n g hand f r e q u e n c i e s i n T a b l e 4 . T a b l e 4.
I R c h a r a c t e r i s t i c s of c o d e i n e phosphate
Frequency Cm 3500 2500 1645
-1
Assignment
-OH (broad)
-N H
C7=C8
(alkene)
1618, 1515
1280, 1090
C=C ( a r o m a t i c )
c-0-c
Two a d j a c e n t H (aromatic) Other c h a r a c t e r i s t i c a b s o r p t i o n bands are: 2990, 1460, 1335, 960 880 and 845 Cm-l.
790,
760
Fig.3.
T h e U V s p e c t r u m o f c o d e i n e p h o s p h a t e in w a t e r .
aD
8
N
f
c
m
0 J =
a l
.4
a, C
a,
73
U
u . 4
E
.L
Y
>
P
K
e :
c
c a ! 9)
>
106
2.5.3
Nuclear Magnetic Resonance S p e c t r a
2.5.3.1
P r o t o n Spectrum The PMR spectrum of c o d e i n e phosphate i n deuterium o x i d e w a s recorded on a v a r i a n XL 200, 200 M H z NMR spectrometer using tetramethyl s i l a n e as a reference s t a n d a r d (Fig. 5 ) . The f o l l o w i n g s t r u c t u r e assignments have been made (Table 5 ) .
T a b l e 5.
PMR c h a r a c t e r i s t i c s of c o d e i n e phosphate.
Chemical S h i f t (6 ) 6.78 6.95 5.78 5.40 4.40 3.87 3.00 (d) (d) (d) (m) (m) (s) (s)
Assignment
1H 2H 7H 8H 9H 3-OCH3 N -CH3
s = s i n g l e t , d = doublet,
multiplet.
2.5.3.2
C-NMR
3C-NMR completely d ec o u p l ed and o f f r e s o n a n c e s p e c t r a are shown i n F i g . 6. ( A & B r e s p e c t i v e l y ) . Both were r e c o r d e d o v e r 5000 Hz r a n g e , i n d e u t e r i u m o x i d e (conc. 575 mg/2 m l D20) on FT-80 A-80 MHz NMR s p e c t r o m e t e r . Using 10 mm sample t u b e and tetramethyls i l a n e as reference standard, a t ambient.
The carbon chemical s h i f t a r e a s s i g n e d on t h e b a s i s of t h e
4J
a;
c
a
m
s a aJ e
.ri
u a
M
.ri
Lr,
CODEINE PHOSPHATE
109
a d d i t i v i t y p r i n c i p a l s and o f f r e s o nance s p l i t t i n g p a t t e r n ( T a b l e 6 ) .
.cn3
l7
Table 6: Carbon No.
Carbon c h e m i c a l s h i f t s Chemical S h i f t PPm
of c o d e i n e phosphate. Chemical S h i f t ppm.
Carbon No.
c-1 c-2 c-3 c-4 c- 5 C- 6 c-7

C-8
121.1
115.1
c-9
s
=
142.7 147.2 66.7 91.6 126.4 134.1 61.3

singlet ; d
=
d d s s d d d d d
c-10 c-11 C-12 C-13 C-14 c-15 C-16 C-17 C-18

t = triplet,
33.4 125.0 129.9 42.4 41.90 21.8 47.9 39.2 57.2
s
s s
s
t t q q
doublet,
q = quartet.
Carbons 3 , 4 , 11, 1 2 and 1 3 chemic a l s h i f t s were a s s i g n e d , based on r e l a x a t i o n d a t a of t h e q u a t e r n a r y carbons (17).
2.5.4
Mass Spectrum
The mass spectrum of c o d e i n e phosp h a t e o b t a i n e d by e l e c t r o n impact i o n i z a t i o n which w a s r e c o r d e d on Ribermag R-1010 mass s p e c t r o m e t e r eqquibed w i t h
110
d i r e c t i n l e t probe. The spectrum ( F i g . 7 ) shows a molecular i o n peak M+ a t m / e 299 w i t h a r e l a t i v e i n t e n s i t y 100%. The most prominent fragments and their relative intensities are listed i n Table 7 . Table 7:
Mass fragments of c o d e i n e phosphate.

Relative I n t e n s i t y %
100
m/e
299 298 229 214 188 162 124 115 81
70
( b a s e peak)
15 20
10 10
35 20 15
10 10 15
59
3.
Preparation
3.1.
I s o l a t i o n of Codeine Codeine o c c u r s i n opium which i s t h e d r i e d l a t e x o b t a i n e d from t h e u n r i p e c a p s u l e s of Papaver somnif erum Linn. (Family Papaveraceae) Opium c o n t a i n s about 2% of codeine.
S e v e r a l methods have been used f o r t h e i s o l a t i o n of codeine from opium. One of t h e s e i s a s follows : Powdered opium i s e x t r a c t e d w i t h w a r m water t o complete e x h a u s t i o n . The e x t r a c t i s c o n c e n t r a t e d under vacuum, t r e a t e d w i t h a s o l u t i o n of calcium c h l o r i d e (1 : l ) , l e f t f o r 48 hours and t h e n f i l t e r e d . The f i l t r a t e c o n t a i n i n g t h e h y d r o c h l o r i d e s of t h e a l k a l o i d s i s c o n c e n t r a t e d when morphine and codeine h y d r o c h l o r i d e s d e p o s i t i n t h e form of double compound known as "Gregory salt''. T h i s s a l t i s d i s s o l v e d i n w a r m water and n e u t r a l i z e d w i t h d i l u t e
50
60
70
80
90
100 110
120
130
140
150
160
170 1 8 C
190 200
210 220
Fig. 7.
230 240
250
260
270 280
290
300 310
The mass spectrum of c o d e i n e phosphate.
112
ammonia t o p h e n o p h t h a l e i n end p o i n t (pH 9 ) . Morp h i n e p r e c i p i t a t e s , w h i l e c o d e i n e remains i n s o l u t i o n a s ammonium-codeine c h l o r i d e . The s o l u t i o n i s c o n c e n t r a t e d , t r e a t e d w i t h 30% sodium hydroxide and c o d e i n e i s t h e n e x t r a c t e d w i t h chloroform. For f u r t h e r p u r i f i c a t i o n of c o d e i n e , t h e chloroformic l a y e r i s extracted with d i l u t e s u l f u r i c a c i d and t h e aqueous l a y e r i s d e c o l o r i z e d w i t h c h a r c o a l and f i l t e r e d . The f i l t r a t e i s r e n d e r e d a l k a l i n e w i t h sodium hydroxide s o l u t i o n and e x t r a c t e d w i t h benzene, which i s evaporated t o dryness t o a f f o r d codeine. 3.2. Formation of Codeine Phosphate: T h i s i s formed by n e u t r a l i z i n g c o d e i n e w i t h phosphoric a c i d and p r e c i p i t a t i n g t h e s a l t from aqueous s o l u t i o n w i t h a l c o h o l ( 1 8 ) .
4 . S y n t h e s i s o f Codeine Phosphate
The f i r s t s y n t h e s i s of t h e s k e l e t o n of t h e morphine a l k a l o i d s was achieved by Grewe e t a 1 ( 1 9 ) , whose method was i n f a c t a v e r s i o n of t h e b i o g e n e t i c approach and i n v o l ved an a c i d c a t a l y s e d c y c l i z a t i o n of benzylhexahydroisoq u i n o l i n e t o a morphine d e r i v a t i v e .
4 . 1 T o t a l S y n t h e s i s o f Codeine (Scheme 1)
The f i r s t t o t a l s y n t h e s i s of codeine was achieved i n 1952 by Gates and Tschudi ( 2 ) . The key i n t e r m e d i a t e "4-cyanomethyl-1,2-naphthoquinone" [8] was r e a c t e d w i t h b u t a d i e n e (Diels-Alder r e a c t i o n ) t o g i v e [91 which when reduced w i t h copper chromite underwent r i n g c l o s u r e t o t h e k e t o l a c t a m [lo]. Reduction o f [lo] (Wolf-Kischner method) gave [ll] which was N-methylated t o [I21 and reduced t o (t)-B-A6-dihydrodesoxy c o d e i n e [13]. Resol u t i o n was e f f e c t e d w i t h d i b e n z o y l t a r t a r i c a c i d t o g i v e t h e ( + ) - b a s e . The r e s o l v e d ( + ) - b a s e was h y d r a t e d w i t h d i l u t e s u l f u r i c a c i d t o [14], followed by p a r t i c a l d e m e t h y l a t i o n and o x i d a t i o n t o [15]. T h i s was brominat e d t o [16] and i s o m e r i z e d t o t h e more s t a b l e l-bromot h e b a i n o n e [17]. Reduction o f [17] gave d i h y d r o t h e bainone [18]. F i n a l l y , [18] was brominated and t r e a t e d w i t h 2,4-dinitrophenylhydrazine when oxide r i n g c l o s u r e occured t o g i v e t h e hydrazone [191, which upon h e a t i n g w i t h p y r i d i n e and s p l i t t i n g w i t h a c i d gave l-bromo-
CODEINE PHOSPHATE
113
codeinone [20]. codeine [21].
Reductive removal of 1-bromo gave
A rather different approach was adopted by Elad and Ginsberg (20) who synthesized (-)-dihydrothebainone. This constitutes a formal total synthesis of codeine since (-)-dihydrothebainone is transformed into codeine by Gates and Tschudi (2).
4.2 Partial Synthesis of Codeine
Codeine can be prepared by partial synthesis from morphine. Morphine is dissolved in theoretical amount of potassium hydroxide dissolved in absolute alcohol, the required quantity of the methylating agent (usually phenyltrimethylammonium hydroxide) added, and the solution is heated at about 130". After cooling, water is added, the solution is acidified with sulfuric acid, the dimethylanilline formed is separated, and the alcohol is removed by distillation. Treatment with caustic soda solution precipitates the codeine, while any unreacted morphine is held in solution by the sodium hydroxide. The crude codeine is purified by crystallization as the sulfate (21).
HO
Methylation
HO
Morphine
H O
Codeine
Codeine is also prepared from thebaine by appropri(22). ate reduction and demethylation
114
Scheme 1:
T o t a l S y n t h e s i s of Codeine.
%
0
0 0-C.
O.C.Ph
-~11%
FeC13
1.
1 so2
:
[41
1.
Ph
0 . C. Ph II 0
[31
O K
Me
Me%
[81-I:;p
[51
O K
HCOOEt CN
[71
CN
CODEINE PHOSPHATE
115
Me0 Butad iene & / O e M

CO;:;: Chr o m i t e
Me0
NaH/MeI
-11
LiA1H4
Me 0
HO
116
KOHIdiethyleneglycol. Oppenauer o x i dation
(3 ) CH3C0 CH3 /H+
CODEINE PHOSPHATE
117
5.
B i o s y n t h e s i s of Codeine P o s t u l a t i o n of t h e b i o s y n t h e t i c pathway o f opium a l k a l o i d s s t a r t e d i n 1910 w i t h t h e s u g g e s t i o n o f W i n t e r s t e i n and T r i e r (23) t h a t t h e b e n z y l i s o q u i n o l i n e a l k a l o i d s were b u i l t up i n n a t u r e from two u n i t s of 3,4d i h y d r o x y p h e n y l a l a n i n e (DOPA). These m o l e c u l e s , w i l l g i v e r i s e t o 3,4-dihydroxyphenylethylamine and 3,4-dihydr o x y p h e n y l a c e t a l d e h y d e by u n d e r g o i n g d e c a r b o x y l a t i o n and o x i d a t i v e deaminat i o n r e s p e c t i v e l y . Gulland and Robinson (1) proposed t h a t morphine arises i n t h e p l a n t from a s u i t a b l e b e n z y l i s o q u i n o l i n e p r e c u r s o r ( n o r l a u d n o s o l i n e ) by r o t a t i o n o f t h i s p r e c u r s o r f o l l o w e d by o x i d a t i v e r i n g c l o s u r e . The v a l i d i t y of s u c h schemes remained u n t e s t e d u n t i l t h e a d v e n t of r a d i o c h e m i c a l t e c h n i q u e s , when i n 1958 - 1960 e x p e r i m e n t s w i t h l a b e l l e d t y r o s i n e administ e r e d t o poppy c a p s u l e s d e m o n s t r a t e d t h a t t h e biosynt h e t i c r o u t e s proposed above do i n f a c t t a k e p l a c e i n living plants. B a t t e r s b y and Co-workers i n 1958 and 1961 ( 2 4 , 25) and Leete (26) e s t a b l i s h e d t h a t when 2-I4C t y r o s i n e was f e d t o poppy p l a n t , r a d i o a c t i v e t h e b a i n e , c o d e i n e and morphine w e r e o b t a i n e d . These a l k a l o i d s were shown t o b e l a b e l l e d e q u a l l y and s p e c i f i c a l l y a t c a r b o n s 9 and 16 a s e x p e c t e d . By f e e d i n g l - 1 4 C dopamine, B a t t e r s b y and F r a n c i s (27) found t h a t o n l y C-16 of t h e a l k a l o i d s w a s l a b e l l e d b u t n o t C-9. F u r t h e r m o r e , B a t t e r s b y e t a 1 (28) have shown t h a t l - 1 4 C n o r l a u d a n o s o l i n e w a s i n c o r p o r a t e d i n t o morphine m o l e c u l e by p l a n t w i t h l a b e l l i n g a t C-9 only. These r e s u l t s i n d i c a t e d t h a t t h e p h e n a n t h r e n e a l k a l o i d s were l a b e l l e d a t t h e a n t i c i p a t e d s i t e s . B a t t e r s b y e t a 1 (29) have f u r t h e r found t h a t (-)r e t i c u l i n e i s t h e most e f f i c i e n t p r e c u r s o r t o morphine s k e l e t o n , and t h i s a l k a l o i d w a s found t o b e p r e s e n t i n opium ( 3 0 ) . Barton e t a1 (31) e s t a b l i s h e d t h a t s a l u t a r i d i n e which do e x i s t i n trace amounts i n opium, i s formed by p h e n o l i c o x i d a t i v e c o u p l i n g of ( - ) - r e t i c d i n e . The e x i s t e n c e of c o d e i n o n e as a n i n t e r m e d i a t e between t h e b a i n e and c o d e i n e w a s confirmed ( 3 2 ) . It w a s s u g g e s t e d by B a r t o n and Cohen (33) and B e n t l e y and Cardwell (8) t h a t t h e b a i n e i s formed f i r s t i n t h e p l a n t and t h a t c o d e i n e , t h e n morphine a r i s e d from i t . Rapaport ( 3 4 , 35) h a s shown
118
Scheme 2:
Biosynthesis of Codeine
Shikimic acid Pathway

O J II
p-hydroxy phenyl pyruvic acid
0 Q
CH2-C-COOH
c____,
OH
OH
NH2 I CH2-CH-COOH
Tyros ine
HO
0 -0
OH
HO
i:
CH2-C-COOH
CH2-CH-COOH
NH2 I
OH
3,4-dihydroxy phenyl pyruvic acid
DOPA (3,4-dihydroxy phenylalanine)
HO
H
H
HO
HO
3,4-dihydroxy phenylethylamine.
Norlaudanosoline carboxylic acid
CODEINE PHOSPHATE
119
-9F!$
HO H HO H
NH
OH
Norlaudanosoline
Nor laudanosoline.
HQCO
H3C0 " $ N t
HH:$!32NcH3
0
(+) S a l u t a r i d i n e
OH
(-) Reticuline
Thebaine
Codeinone
Codeine
1%
by exposing Papaver somniferum p l a n t s t o 1 4 c 0 2 f o r v a r y i n g l e n g t h of t i m e , t h a t r a d i o a c t i v e t h e b a i n e was f i r s t formed i n t h e p l a n t and w a s c o n v e r t e d i n t o r a d i o a c t i v e c o d e i n e and t h i s w a s c o n v e r t e d i n t o r a d i o a c t i v e morphine b u t n o t i n t o t h e b a i n e . B a t t e r s b y (36) h a s i n d e p e n d e n t l y r e a c h e d t h e same c o n c l u s i o n . The b i o s y n t h e s i s of c o d e i n e i s i l l u s t r a t e d i n scheme 2 . 6. Metabolism The a b s o r p t i o n of c o d e i n e i s r e l a t i v e l y r a p i d a f t e r p a r e n t e r a l a d m i n i s t r a t i o n and e r r a t i c a f t e r o r a l medicat i o n ( 3 7 ) . Codeine i s m e t a b o l i s e d m a i n l y i n t h e l i v e r . The predominent m e t a b o l i c changes a r e N-demethylation t o n o r c o d e i n e , 0-demethylation t o morphine and c o n j u g a t i o n w i t h g l u c u r o n i c a c i d a t t h e 6-hydroxyl t o 6-0-glucuronide. Experiments w i t h e i t h e r 0-methyl o r N-methyl 1 4 C l a b e l l e d c o d e i n e have shown t h a t r a p i d d i s p o s a l o c c u r s i n man and t h a t 24 h o u r s a f t e r i n j e c t i o n , t h e maxium of morphine (4-13%) , n o r c o d e i n e (8%) , bound c o d e i n e (35 40%) and unchanged c o d e i n e ( 5 - 12%) i s p r e s e n t i n t h e u r i n e ; n e g l i g i b l e amounts are found i n t h e f e c e s and o n l y p a r t of t h e d e t a c h e d 0-methyl and N-methyl r a d i c a l s c a n be r e c o v e r e d a s e x p i r e d c a r b o n d i o x i d e (38, 3 9 ) . I n 2 4 h o u r s 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 of 4 mg/Kg i n d i v i d e d d o s e s , 4% of t h e d o s e was e x c r e t e d i n t h e u r i n e a s f r e e n o r c o d e i n e , 14% a s c o n j u g a t e d n o r c o d e i n e , 1%a s f r e e morphine and 7% a s c o n j u g a t e d morphine ( 1 6 ) . The metabolism of c o d e i n e is p r e s e n t e d in"scheme 3 .
7.
Methods of A n a l y s i s
7.1
I d e n t i f i c a t i o n Tests The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e t h o s e mentioned i n t h e U.S.P.XX(11):A)
The i n f r a r e d a b s o r p t i o n spectrum of a p o t a s s i u m bromide d i s p e r s i o n of i t , p r e v i o u s l y d r i e d , e x h i b i t s maxima o n l y a t t h e same w a v e l e n g t h s as t h a t of a s i m i l a r p r e p a r a t i o n of USP Codeine Phosphate R e f e r e n c e S t a n d a r d .
CODEINE PHOSPHATE
121
Scheme 3 : Metabolism
of
Codeine.
m
I
OH
4 2
CH3
I
Morphine
0
/
'b
" ' ,
GC
OH
'"'
OH
Normorphine
FARID J. MUHTADI AND MAHMOUD M . A. HASSAN
D i s s o l v e 100 m g i n 15 m l of water. Render t h e s o l u t i o n a l k a l i n e w i t h ammonia TS, e x t r a c t witli t h r e e 5-ml p o r t i o n s of chloroform, f i l t e r t h e combined chloroform e x t r a c t s through f i l t e r paper t h a t p r e v i o u s l y h a s been washed and moistened w i t h chloroform, and e v a p o r a t e t h e combined chloroform e x t r a c t s on a steam b a t h j u s t t o t h e d i s a p p e a r a n c e of t h e chloroform odor: t h e r e s i d u e of c o d e i n e m e l t s between 154' and 158'.
To 1 m g contained i n a porcelain c r u c i b l e o r small d i s h add 1 d r o p of s u l f u r i c a c i d c o n t a i n i n g , i n each m l , 5 m g of s e l e n i o u s a c i d : a green c o l o r i s produced a t once, and i t r a p i d l y changes t o b l u e , t h e n s l o w l y t o d a r k o l i v e green.
To a s o l u t i o n of 5 m g i n 5 m l of s u l f u r i c a c i d c o n t a i n e d i n a t e s t t u b e add 1 drop of f e r r i c c h l o r i d e TS, mix, and h e a t i n b o i l i n g water f o r 2 m i n u t e s : a b l u e c o l o r i s producted and upon t h e a d d i t i o n of 1 d r o p of n i t r i c a c i d changes t o red-brown. N e u t r a l i z e a s o l u t i o n (1 i n 50) w i t h ammonia TS, and add s i l v e r n i t r a t e TS: a y e l l o w p r e c i p i t a t e of s i l v e r phosphate i s formed, and i t i s s o l u b l e i n d i l u t e d n i t r i c a c i d and i n ammonia TS. Other i d e n t i f i c a t i o n t e s t s (16, 40) a r e as follows : P l a c e a l i t t l e i n powder, on t h e s u r f a c e of a d r o p of n i t r i c a c i d , a yellow c o l o r i s produced. Add t o a l i t t l e of c o d e i n e , 1 m l of s u l f u r i c a c i d c o n t a i n i n g d r o p s of formaldehyde, a p u r p l e c o l o r i s formed ( s e n s i t i v i t y 0.05 u g ) . Add d r o p s of ammonium m o l i b d a t e t o c o d e i n e phosphate, a g r e e n c o l o r i s produced ( s e n s i t i v i t y 0.1 u s ) .
7.2
M i c r o c r y s t a l Tests Add potassium cadmium i o d i d e s o l u t i o n t o c o d e i n e
CODEINE PHOSPHATE
123
phosphate, g e l a t i n o u s r o s e t t e s c r y s t a l s a r e formed, changing t o a g g r e g a t e s of s m a l l t a b l e t s (16). Add potassium t r i - i o d i d e s o l u t i o n t o c o d e i n e , f e a t h e r y r o s e t t e s c r y s t a l s formed o v e r n i g h t ( 1 6 ) . 7.3 T i t r i m e t r i c Methods The o f f i c i a l methods of d e t e r m i n i n g c o d e i n e phosphate a r e d e s c r i b e d by t h e B.P. (40) and U.S.P.XX(11). 7.3.1 Aqueous T i t r a t i o n The B.P. d e s c r i b e s a method f o r a s s a y i n g c o d e i n e phosphate i n t a b l e t s a s follows:Weigh and powder 20 t a b l e t s . D i s s o l v e a q u a n t i t y of t h e powder e q u i v a l e n t t o 0.3 g of Codeine Phosphate a s completely a s p o s s i b l e i n 20 m l of 0.5 N s u l f u r i c a c i d , f i l t e r , w i t h 0.5N s u l f u r i c a c i d u n t i l complete ext r a c t i o n of t h e a l k a l o i d i s e f f e c t e d . Make a l k a l i n e w i t h d i l u t e ammonia s o l u t i o n , and e x t r a c t w i t h s u c c e s s i v e q u a n t i t i e s of c h l o r o form u n t i l complete e x t r a c t i o n of t h e a l k a l o i d i s e f f e c t e d . Wash each chloroform s o l u t i o n w i t h t h e same 1 0 m l of water. Evapor a t e t h e chloroform. To t h e r e s i d u e add 5 m l of a l c o h o l (95 p e r c e n t ) p r e v i o u s l y n e u t r a l i s e d t o methyl r e d s o l u t i o n and remove t h e a l c o h o l by e v a p o r a t i o n . D i s s o l v e t h e r e s i d u e i n 1 m l of n e u t r a l i s e d a l c o h o l (95 p e r c e n t ) , add 1 0 m l of 0 . 1 N h y d r o c h l o r i c a c i d and 1 0 m l of water, and t i t r a t e w i t h 0.1N sodium hydr o x i d e , u s i n g methyl r e d s o l u t i o n as i n d i c a t o r . Each m l of 0 . 1 N h y d r o c h l o r i c a c i d i s e q u i v a l e n t t o 0.04064 g of C18H21N03,H3P04,%fl. Other methods have been a l s o r e p o r t e d u t i l i z i n g 0.01 M aqueous sodium d i o c t y l s u l f o s u c c i n a t e as a t i t r a n t u s i n g dimethyl yellow ( 4 1 ) , o r a m i x t u r e of 0.1% d i m e t h y l yellow and 0.1% m a l a c h i t e g r e e n s o l u t i o n i n c h l o r o form ( 4 2 ) a s i n d i c a t o r s .
124
7.3.2
Non-Aqueous T i t r a t i o n The U.S.P. method:(XX) d e s c r i b e s t h e f o l l o w i n g
D i s s o l v e a b o u t 1 g of Codeine P h o s p h a t e , a c c u r a t e l y weighed, i n 20 m l of g l a c i a l acet i c a c i d , warming s l i g h t l y i f n e c e s s a r y t o e f f e c t s o l u t i o n , and t i t r a t e w i t h 0 . 1 N p e r c h l o r i c a c i d , d e t e r m i n i n g t h e end-point p o t e n t i o m e t r i c a l l y . Perform a b l a n k d e t e r m i n a t i o n and make a n y n e c e s s a r y c o r r e c t i o n . Each m l of 0.1 N p e r c h l o r i c a c i d i s equival e n t t o 39.74 mg of C H NO .H FO 18 2 1 3 3 4' Another method i s as f o l l o w s (43) :Codeine p h o s p h a t e i s t r e a t e d w i t h NaOH o r Na2C03 e x t r a c t i n g t h e l i b e r a t i n g b a s e i n t o CHC13 e v a p o r a t i n g most of t h e s o l v e n t and t i t r a t i n g w i t h H C 1 0 4 i n d i o x a n u s i n g d i m e t h y l y e l l o w and m e t h y l e n e b l u e a s i n d i cator.
A t h i r d method f o r m i c r o - d e t e r m i n a t i o n of c o d e i n e p h o s p h a t e i n t a b l e t s i s d e s c r i b e d ( 4 4 ) . 60 mg of c o d e i n e p h o s p h a t e i s d i s s o l v e d i n water, r e n d e r a l k a l i n e w i t h sodium hydr o x i d e s o l u t i o n and e x t r a c t e d w i t h e t h a n o l f r e e chloroform, f i l t e r t h e e x t r a c t through c o t t o n wool and anhydrous sodium s u l f a t e i n t o a 50 m l f l a s k and d i l u t e w i t h c h l o r o f o r m t o t h e mark. To an a l i q u o t of 10 ml of c o d e i n e s o l u t i o n , add 0.1% d i m e t h y l y e l l o w s o l u t i o n i n c h l o r o f o r m and t i t r a t e w i t h 0.005 N t o l u e n e - p a r a - s u l f o n i c a c i d i n c h l o r o f o r m and carry out a blank determination.
7.4
Complexometry a) Codeine p h o s p h a t e i s d i s s o l v e d i n w a t e r , e t h a n o l and queous NaOH a r e added. To t h e r e s u l t i n g m i x t u r e add 0.2 M copper p i c r a t e ( 3 0 m l ) , mix and a f t e r 30 min. c o l l e c t t h e p r e c i p i t a t e on a s i n t e r e d - g l a s s f i l t e r (G4) and wash w i t h w a t e r (5 x 5 m l ) . To t h e combined f i l t r a t e and washi n g s add 0.2 M EDTA (20 ml) and ammonia b u f f e r s o l u t i o n of pH 10.4 ( 0 . 3 ml) and murexide-NaC1
CODEINE PHOSPHATE
125
(1 : 200) and t i t r a t e t h e e x c e s s of EDTA w i t h 0.2 M ZnSO4 u n t i l t h e s o l u t i o n i s g r e e n ( 4 5 ) .

b) E x c e s s of S t a n d a r d MgS04 s o l u t i o n i s added t o t h e h o t s l i g h t l y ammonical c o d e i n e p h o s p h a t e s o l u t i o n , which ( a f t e r f i l t e r i n g o f f t h e p r e c i p i t a t e d MgNH4P04) i s back t i t r a t e d w i t h EDTA (disodium s a l t ) u s i n g Eriochrom b l a c k T a s i n d i c a t o r (46).
7.5
Spectrophotometry 7.5.1 Colorimetry

A c o l o r i m e t r i c procedure w a s d es cr ib ed f o r t h e a s s a y of c o d e i n e p h o s p h a t e u s i n g p i c r i c a c i d ( 4 7 ) . An aqueous s o l u t i o n (1.0 ml) c o n t a i n i n g a b o u t 0 . 5 m g of c o d e i n e phosphate is t r e a t e d w i t h 0 . 5 m l b u f f e r solut i o n (300 g i n Na2H2P04.2H20 and 9 g of NaOH made up t o 750 m l w i t h w a t e r ) , 1 . 0 m l of p i c r i c a c i d s o l u t i o n and 10 m l of c h l o r o f o r m ( a l c o h o l f r e e ) . The m i x t u r e i s shaken f o r 30 s e c o n d s and t h e e x t i n c t i o n of t h e f i l t e r e d c h l o r o f o r m p h a s e i s measured a t 430 nm. A c a l i b r a t i o n c u r v e i s c o n s t r u c t e d from r e a d i n g s obtained with standard preparations.
Another method i s based on t h e r e a c t i o n w i t h bromothymol b l u e ( 4 8 ) . Codeine p h o s p h a t e i s d i s s o l v e d i n 0.5% H C 1 ( v / v ) and t r e a t e d w i t h a 0.04% s o l u t i o n of bromothymol b l u e u s i n g M c I l v a i n e b u f f e r s o l u t i o n of pH 8 . The sample i s t h e n e x t r a c t e d w i t h c h l o r o f o r m and t h e e x t i n c t i o n of t h e e x t r a c t i s measured a t 410 nm.
A t h i r d method i n v o l v i n g t h e f o r m a t i o n of molybdophosphoric a c i d from c o d e i n e phosp h a t e w i t h a m i x t u r e of H2SO4 and HNO3. Chrom e t h y l p y r a z o l i s added and t h e e x t i n c t i o n of t h e r e s u l t i n g c o l o r i s measured a t 620 nm.(49). A c a l i b r a t i o n c u r v e i s p r e p a r e d u s i n g 0.5 t o 3 . 5 m l of 64.5 ( M-KH2P04 add 0 . 5 m l of 5% ammonium molybdate s o l u t i o n , 3 m l of 2 M HNO3, 1 m l of 1%s t a r c h s o l u t i o n and ( a f t e r 3 t o 4 m i n u t e s ) 3 m l of 0.4 mM. of c h r o m e t h y l p y r a z o l , d i l u t e e a c h s o l u t i o n t o 25 m l , warm a t 30 f o r
126
40 m i n u t e s , and measure t h e a b s o r b a n c e a t
620 nm. 7.5.2
A s t a b i l i t y i n d i c a t i n g a s s a y h a s been r e p o r t e d (50) f o r t h e d e t e r m i n a t i o n of c o d e i n e p h o s p h a t e i n s y r u p s . The s y r u p s were subj e c t e d t o a c c e l a r a t i n g a g e i n g a t 60, 70and 80' and t h e c o n t e n t of c o d e i n e p h o s p h a t e w a s then determined spectro p h o to metr ically a t 285 nm, a f t e r s e p a r a t i o n of c o d e i n e p h o s p h a t e M M paper by chromatography on Whatman No. 3 impregnated w i t h 0.5 M-KH2POq, t h e d e v e l o p i n g solvent is isobutyl alcohol/water/ethanol/ a c e t o n e / e t h y l a c e t a t e (8:5:2:2:1).
Another method w a s r e p o r t e d f o r t h e d e t e r m i n a t i o n of c o d e i n e p h o s p h a t e i n m i x t u r e s (51), w i t h mean r e c o v e r i e s i n t h e r a n g e 98.5 t o 99.85%. The powdered sample i s mixed w i t h water and d i l u t e d t o 50 m l , t h e n c e n t r i f u g e d and a p p l i e d a s 20 m l p o r t i o n s t o a column (35 x 2 cm) of a l g i n i c a c i d . E l u t e c o d e i n e p h o s p h a t e w i t h 0.01 N HC1 and measure t h e e x t i n c t i o n a t 250 nm and 273 nm. 7.5.3 NMR -
A r a p i d , a c c u r a t e and p r e c i s e P M R method i s r e p o r t e d (52) f o r t h e q u a n t i t a t i v e d e t e r m i n a t i o n of c o d e i n e and c o d e i n e p h o s p h a t e a s b u l k d r u g s and i n t a b l e t d o s a g e form. The d e t e r m i n a t i o n i s based on t h e i n t e g r a t i o n of t h e C-3 m e t h y p r o t o n s o r t h e two a r o m a t i c p r o t o n s of c o d e i n e o r i t s s a l t r e l a t i v e t o t h a t of t h e n i n e p r o t o n s of t - b u t a n o l . Stand a r d d e v i a t i o n s of f 1.39, 0.27 and 0.65% w e r e obtained f o r codeine, codeine phosphate b u l k d r u g and c o d e i n e p h o s p h a t e t a b l e t s r e s p e c t i v e l y . The sample powder ( o r powdered t a b l e t s i s mixed w i t h 2 m l of t h e i n t e r n a l s t a n d a r d s o h . (10 mg ml-1 of t - b u t y l a l c o h o l i n ethanol-free chloroform f o r codeine o r i n water f o r c o d e i n e p h o s p h a t e ) , w i t h s h a k i n g f o r 3 min. A f t e r c e n t r i f u g i n g t h e m i x t u r e , 0.5 m l of t h e c l e a r s o h . i s s u b j e c t e d t o n.m.r. a t 60 MHz. Repeated i n t e g r a t i o n s a r e
CODEINE PHOSPHATE
127
made of t h e peaks a t 3 . 8 and 1 . 2 3 p.p.m. ( c o r r e s p o n d i n g t o t h e C-3 methyl p r o t o n s of c o d e i n e and t h e n i n e p r o t o n s of t - b u t y l a l c o h o l ) f o r c o d e i n e and of t h o s e a t 6.83 and 1.27 p.p.m. (two a r o m a t i c p r o t o n s of c o d e i n e phosphate; n i n e p r o t o n s of t h e a l c o h o l ) f o r c o d e i n e phosphate. No i n t e r f e r e n c e i s observed from e x c i p i e n t s i n t h e t a b l e t s . The d e t e c t i o n of morphine i n c o d e i n e and i t s s a l t s i s p o s s i b l e , as t h e aromatic-proton d o u b l e t f o r morphine i s a t 6.7 p.p.m.
7.5.4.
Mass Opium a l k a l o i d s c a n be determined from t h e i r mass s p e c t r a by comparison of t h e p a r e n t peak of each i n d i v i d u a l a l k a l o i d w i t h t h a t of a r e f e r e n c e compound added i n known amount ( 5 3 , 5 4 ) and by r e f e r e n c e t o a c a l i b r a t i o n graph. A t a low i o n i s a t i o n v o l t a g e , t h e & i o n c a n be d e t e c t e d c l e a r l y , b u t t h e peak i s small. A t h i g h e r i o n i s a t i o n v o l t a g e s , t h e & i o n peak becomes h i g h e r , b u t many o t h e r fragment peaks o v e r l a p i t . The r e l a t i o n s h i p between t h e i n t e n s i t y of t h e M+ i o n peak and i o n i s a t i o n v o l t a g e w a s s t u d i e d , and a p p r o p r i a t e i o n i s a t i o n v o l t a g e s were determined. Tatematsu e t a1 ( 5 5 ) , have found t h a t t h e mass s p e c t r a of c o d e i n e s a l t s ( h y d r o c h l o r i d e , hydrobromide, s u l f a t e , phosphate, o x a l a t e , malonate, s u c c i n a t e , t a r t a r a t e , c i t r a t e , meconate and p i c r a t e ) showed t h e spectrum of t h e f r e e b a s e ( c o d e i n e ) w i t h an M peak a t m / e 299. The r e l a t i v e i n t e n s i t y of t h e main fragment w a s t h e same f o r a l l s a l t s . I t w a s c o n s i d e r e d t h a t t h e t e m p r a t u r e a t which t h e M i o n peak appeared w a s r e l a t e d t o v o l a t i l i t y and t h e a c i d r a d i c a l and t h e h e i g h t of t h e M i o n peak t o t h e s t r e n g t h of t h e a c i d .
+ +
7.6
Chromatography
7.6.1
Paper Chromatography a) Q u a l i t a t i v e Paper Chromatography The Rf v a l u e s of c o d e i n e i n d i f f e r e n t
128
FARID J. MUHTADI A N D MAHMOUD M . A. HASSAN
s o l v e n t s y s t e m s a r e l i s t e d i n t a b l e 8.
Table 8 :
Rf v a l u e s of c o d e i n e on paper chromatography.
Chromatogram
Solvent
Rf v a l u e of c o d e i n e
Locat ion Reagent
1.
Whatman No. 1 , b u f f e r e d by d i p p i n g i n 5% s o l u t i o n of sodium d i h y d r o g e n citrate, blotti n g and d r y i n g a t 25O f o r 1 hour (16). Reversed phase chromatography. Whatman No.1 impregnated w i t h 10% s o l u t i o n of t r i b u t y r i n i n acet o n e and d r y i n g i n a i r (16). Whatman No. 1 , imregnated w i t h 0.5 M KH2P04 (56) (PH 4 . 2 )
4.8 g of c i t r i c acid i n a mixture of 130 water & 8 7 0 m l of n-butanol
0.16
- Examination u n d e r UV ( 2 5 4 mu). Iodoplatinate reagent Dragendorf f reagent
2.
Acetate b u f f e r (pH 4 . 5 8 ) o r Phosphate buff e r (pH 7 . 4 )
0.89 0.22
- I o d i n e vapor
5.
Cyclohexane/ chloroform/ diethylamine (7:2:1)
0.56
b)
Q u a n t i t a t i v e Paper Chromatography
A d e s c e n d i n g t e c h n i q u e on Whatman No. 1 paper s t r i p s . The p a p e r i s impregn a t e d w i t h a s o l u t i o n of ammonium s u l f a t e (2%). F r e s h l y prepared i s o b u t a n o l l a c e t i c a c i d l w a t e r ( 1 0 : 1 : 2 . 4 ) , i s used
CODEINE PHOSPHATE
129
a s s o l v e n t ( 5 6 ) . The aqueous s o l u t i o n of codine phosphate (or e t h a n o l i c s o l u t i o n of t h e a l k a l o i d ) i s s p o t t e d by means of a n "Alga" micrometer s y r i n g s . F i v e microl i t e r e s , which s h o u l d c o n t a i n 5-50 U g of t h e alkaloid is applied, yielding a spot n o t l a r g e r t h a n 5 mm i n d i a m e t e r . The paper i s e q u i l i b r a t e d f o r s i x h o u r s i n a j a r which i s s a t u r a t e d w i t h s o l v e n t vapors
Chromatography t a k e s p l a c e o v e r n i g h t (16 h o u r s ) , i n which t i m e t h e s o l v e n t f r o n t t r a v e l s a b o u t 38 cm. A f t e r d r y i n g t h e chromatogram i s sprayed on b o t h s i d e s , t h o r o u g h l y and u n i f o r m l y w i t h p o t a s s i u m i o d o p l a t i n a t e r e a g e n t and d r i e d a g a i n o r 15 min. i n a c u r r e n t of a i r . The t o t a l c o l o r d e n s i t y of t h e b l u e s p o t s on w h i t e background are scanned d i r e c t l y by u t i l i zing a s e l f - i n t e g r a t i n g densitometer. The s t a n d a r d c u r v e of c o d i n e phosp h a t e i s p r e p a r e d by p l o t t i n g t h e concent r a t i o n s ( i n U g . v s . t o t a l d e n s i t y of t h e spot). 7.6.2 Thin Layer Chromatography (TLC) The Rf v a l u e s of c o d e i n e p h o s p h a t e i n d i f f e r e n t s o l v e n t systems are l i s t e d i n T a b l e 9. T a b l e 9: Rf v a l u e s of Codeine Phosphate.
-
Chromatograms
S o l v e n t System
Rf v a l u e of c o d e i n e 0.35 (16)
Locat i o n Reagent
1. S i l i c a g e l G
Methanollstrong ammonia solut ion (100 : 1.5)
- Acideified iodoplatinate spray Dragendorf f spray Examinat i o n under
130
FARID J. MUHTADI AND MAHMOUD M . A. HASSAN
Benzenefdioxanf ethanolfstrong ammonia s o l u t i o n (50:40:5:5) Acetic a c i d / e t h a n o l 1wa t er (30:60:10) Xylenefethylmethylketone/ methanolldi ethylamine (20:20:3:1) Xylene / a c e t o n e 1 methanol f ammonia 0.88 (20:20:3:1)
0.39 (16)
- U.V.
(254 nm.)
0.29 (16)
0.30 (57)
0.24 (58)
2% HgC12 solution containing 0.01% methyl r e d (59) o r 2% HgC12 solution, drying i n a n oven and spraying with K I solution
A q u a n t i t a t i v e e v a l u a t i o n of c o d e i n e p h o s p h a t e i n d r u g m i x t u r e s u s i n g TLC by measuring i t s r e m i s s i o n and f l u o r e s c e n c e h a s been r e p o r t e d ( 6 0 ) . A powdered t a b l e t c o n t a i n i n g c o d e i n e p h o s p h a t e , phenobarbit o n e , c a f f e i n e , a s p i r i n and p h e n a c e t i n i s extracted with methanolfdiethylamine ( 9 9 : l ) ( 2 x 5 m l ) and t h e n w i t h d i c h l o r o methane ( 2 x 5 ml), and t h e combined ext r a c t s a r e d i l u t e d t o 25 m l w i t h d i c h l o r o methane. T h i s s o l u t i o n i s a p p l i e d t o a pre-coated K i e s e l g e l G (Merck) p l a t e , which i s d e v e l o p e d w i t h cyclohexane-CHClg-
CODEINE PHOSPHATE
131
a c e t i c a c i d (6:3:1) and / o r acetone-CHC1325% aqueous NH3 (65:35:4) and t h e rem i s s i o n s p e c t r a are measured d i r e c t l y on the plate. Other TLC d a t a have been a l s o r e p o r t ed (61-64).
7.6.3
Column Chromatography D i f f e r e n t column chromatograms have been employed f o r t h e s e p a r a t i o n , p u r i f i c a t i o n and q u a n t i t a t i o n of c o d e i n e phosp h a t e i n d r u g m i x t u r e s (65-67).
7.6.4
G a s Chromatography (GC)
The g a s chromatograms f o r c o d e i n e a r e l i s t e d i n T a b l e 9. T a b l e 9: The GC of Codeine.
Column CondiCarrier Gas Detec- S t a n d a r d tions. tor
Retention time
1. 2.5% SE-30 on
80-100 mesh chromosorb W ( 5 f t x 4 mm i n t e r n a l diameter) column temp. :
Nitrogen, 50 ml/min.
FID
Diphenhy-
4.65 (16)
H2 dramine 50 m l /
min. air 300 m l /
min.
225O.
2. 3% XE-60 s i l i - N i t r o g e n , cone n i t r i l e 50 ml/min.

polymer on 100-120 mesh chromosorb W , column temp. : 225'.
DiphenhyFID H2 dramine 50 m l / min. air 300 m l /
9 . 6 (16)
min.
FID
3. 3.8% of s i l i -
Helium cone gum (Lin- 55 ml/min. d e W 98) on
Peak height. The Coeff.
132
Diatoport C (80-100 mesh), column temp.: 225'.
of v a r i t i o n 2 4.93% (68) Nitrogen 60 m l / m i n . FID
4. 2% d e x i l 300 on v a r a p o r t 30 ( 6 f t x 2 mm) column temp.: 280'.

5. 3% DV-17 on silanised chromosorb W AW ( 8 0 t o 100 mesh) (100 c m x 0.64 cm 0.d.) column temp.: 240.
Peak height o r area ( 6 9 ) .
Helium 28 ml/min
FID
6. 3% OV-17 on g a s N i t r o g e n chrom Q (100 35 ml/min. 120 mesh) column (1.8 m x 2 mm), column temp. : 1200.
FID
Propyl-4hydroxybenzoate
Cofficient of v a r i tion f 0.42% f o r codeine phosphate and a c e t y l codeine phosphate (71)

f
7.6.5
High Performance Liquid Chromatography (HPLC) Codeine phosphate w a s determined i n a cough syrup by h i g h - p r e s s u r e l i q u i d chromatography (72) a s f o l l o w s : The cough s y r u p i s d i l u t e d w i t h 0.05 MKH2P04 s o l u t i o n i n aqueous 13% methanol and determined d i r e c t l y by HPLC on a Bondapak-C18 column (30 cm x 4 mm) w i t h KH2PO4 s o l u t i o n as mobile phase (2 ml/min) and d e t e c t i o n a t 254 nm.
A second method i s employed f o r t h e
CODEINE PHOSPHATE
133
s e p a r a t i o n of opium a l k a l o i d s by highperformance l i q u i d chromatography, on P a r t i s i l , w i t h methanol/2N-NH3/1N NH4N03 (30:2:1) a s mobile phase o r on S i l i c a RP-18 w i t h a c e t o n i t r i l e / O . 01N -(NH4) 2C03 (2:3) as mobile phase. The e x t i n c t i o n of each e l u a t e i s measured a t 278 nm. Stepw i s e - g r a d i e n t e l u t i o n can be used t o a c h i e v e more r a p i d e l u t i o n of t h e slowermigrating contaminants (73).
A t h i r d method i s a l s o r e p o r t e d i n 1980 (74) f o r t h e s e p a r a t i o n and d e t e r m i n a t i o n of opium a l k a l o i d s by HPLC a s follows : A r a p i d and s i m p l e method f o r t h e r o u t i n e d e t e r m i n a t i o n of morphine, codeine, c r y p t o p i n e , t h e b a i n e , n a r c o t i n e and papav e r i n e i n gum opium i s u s e d . The sample i s e x t r a c t e d w i t h 2.5% a c e t i c a c i d and an a l i q u o t of t h e e x t r a c t i s d i l u t e d t h r e e f o l d w i t h methanol b e f o r e a n a l y s i s on Nucleosil-1OCN w i t h 1%ammonium acetate buffer solution (adjusted t o p H 5.8 w i t h a c e t i c acid)/acetonitrile/dioxan (8: 1 :1) a s mobile phase (1.5 mlfmin.) d e t e c t i o n i s a t 254 nm. For a sample of gum opium from I n d i a , t h e c o e f f . of v a r i a t i o n (16 r e s u l t s o r each a l k a l o i d ) w e r e < 1 . 5 % ; t h e c r y p t o p i n e c o n t e n t w a s below t h e d e t e c t i o n limit.
A s t a b i l i t y indicating assay f o r c o d e i n e phosphate by u t i l i z i n g HPLC h a s been r e p o r t e d ( 7 5 ) . The b e s t r e s u l t s w e r e o b t a i n e d by HPLC on a column (30 c m x 4 mm) of Bondapak -C18, w i t h aqueous 0.1 M-KH2P04/,nethanol (21 : 19) a s m o b i l e phase (21 ml/niin.) and d e t e c t i o n a t 254 nm.
134
8.
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B.P. (1973) "The B r i t i s h Pharmacopeia" Her M a j e s t y S t a t i o n e r y O f f i c e , Cambridge ( 1 9 7 3 ) .

I . M . R o u s h . d i , S.A. Soliman and Y.A. B e l t a g y , J. Chem. Un. Arab Repub., 2 ( 4 ) , 4 3 9 (1970).
I. Matolcsy, Acta Pharm. Hung.,
38,
( l ) , 23, (1968).
L. N y i t r a y , Acta Pharm. Hung., 34, ( 4 ) , 159, (1964).

L . S a f a g i k , E s l k a ' Farm.,
15, ( 7 ) ,
360, ( 1 9 6 6 ) .
S. R a l s k i , M. Gajewska and E. Matusak, Farmacja P o l . , 25, ( 2 ) , 111, ( 1 9 6 9 ) .

J . Zyka, Ceskosl. Farmac.,
4,
( l ) , 9, (1955).
K. Howorka, Pharm. Z e n t r a l h a l l e D t l . ,
(1969)
108, ( 5 ) ,
322,
D.L. Robertson, M. Fumi, and W.N. Sci, ( 2 ) , 47, (1972).
1,
French Can. J . Pharm.
CODEINE PHOSPHATE
137
4 9 . Z.G. Kalugina, L.p. F i l i p p o v a , L. B. K r i s t a l e v a and V. I. S o t i n a , Farmatsiya (Mascow), 2, ( 5 ) , 7 2 , ( 1 9 7 7 ) . 50. 51. 52.

R. Wachowiak, Herba P o l . ,
2, (4),
356, (1974). ( 4 ) , 644, (1968).
F. D e F a b r i z i o , J. Pharm. S c i . ,
57,
E.A. Aboutabl, J . S . Mossa and M.M.A. lett., ( 7 , 8 ) , 579, (1979).
12,
Hassan, Spectroscop.
53.
Tatematsu and T. Goto, J . Pharm. SOC. J a p a n , ( 9 ) , 778 (1965).
85,
54. 55.
A. Tatematsu and T. Goto, I b i d , 85, ( 9 ) , 786, (1965). A. Tatematsu, T. Goto, T . Nakamura and S. Yamaguchi, Ibid, 86, ( 3 ) , 195, (1966).
56.
C.P. S t e w a r t and A. Stalman "Toxicology, Mechanisms and A n a l y t i c a l Methods" V o l . 11, Ch. 7 , Academic P r e s s , New York ( 1 9 6 0 ) . I. Bayer, J. Chromat.,
J.W.
57. 58.
16,2 3 7 ,
(1964).
F a i r b a i r n , M. D j o t e , and A. P a t e r s o n , Phytochem.,
7 , 2111, (1968).
59. 60.
P. Miodrag, Arh. Farm.,
2, (5-6),
435, (1976).
E. E i c h , H. Geissler, E. Mutschler and W. Schunack, Arzneimittel-Forsch., (11), 1 8 9 5 , ( 1 9 6 9 ) .
19,
61.
H. G a n s h i r t and A. Malzacher, Arch, Pharm., B e r l i n , 293, ( l o ) , 925, (1960). M. B a c h r a t a , J. Cerna and S. Szucsova, E s l k a Farm., -18, (l), 18, (1969).
62.
63.
B.E. Qaroe and K.E. Rasmussen, Meddr n o r s k . farm. S e l s k . , 38, ( l ) , 1 3 , ( 1 9 7 6 ) .
64.
P. Gundermann and R. Pohloudek-Fabini, (5-6), 296, (1980).

R. H y a t t , J. A s s . O f f . A g r i c , Chem.,
H.W.
Pharmazie,
35,
65. 66.
3, ( 3 ) ,
475 ( 1 9 6 4 ) .
Dibbern and G. S c h o l z , Arch. Pharm., B e r l i n , ( 3 ) , 175, (1965).
298,
138
67. 68. 69.

70.
D. J. Smith, J. A s s . O f f . Analyt. Chem.
3, (3),
536 ( 1 9 6 6 ) .
H.J.
Wesselman and W.L. Koch, J . Pharm. S c i , 57, (5),
845, (1968).
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M.R.
S t e v e n s , J . Pharm. S c i . ,
64 ( l o ) ,
1686, (1975).
71. 72. 73. 74. 75.
R.N. G a l a n t e , A . J . V i s a l l i and D.M. S c i . , 68 ( 1 2 ) , 1 4 9 4 , (1979).

V.D.
P a t e l , J. Pharm.
Gupta and A . G . Ghanekar, J . Pharm. S c i , 895, (1977).
66,
(6),
I. F e h e r , L. Szepesy and J. S z a n t o , Magy. Kern. F o l y ,
85, ( 9 ) , 337, (1979).

Y. Nobuhara, S. Hirano, N. S h i o r i and M. Hashimoto, J . Chromat. 1 9 0 , ( l ) , 251, ( 1 9 8 0 ) .
V.D.
Gupta and O.H.
Shek, Am. J. Hosp. Pharm.,
1086 ( 1 9 7 6 ) .
z, (lo),
COLCHICINE
Dorothy K . Wyatt, Lee T. Grady, and Sy-rong Sun
1. History 2. Description 2.1 Name, Formula, Molecular Weight 2.2 Appearance, Color, Odor 3. Physical Properties 3.1 Infrared Spectrum 3.2 Nuclear Magnetic Resonance Spectra 3.3 Ultraviolet Spectrum 3.4 Mass Spectrum 3.5 Melting Range 3.6 Solubility 3.7 Moisture Content 3.8 Specific Rotation 3.9 pKa 3.10 Crystal Properties 4. Isolation 5. Biosynthesis of Colchicine 6. Synthesis 7. Stability and Degradation 8. Metabolism 9. Pharmacokinetics 10. Methods of Analysis 10.1 Elemental Analysis 10.2 Color Tests 10.3 Aqueous Titrimetric Analysis 10.4 Nonaqueous Titrimetric Analysis 10.5 Spectrophotometric Analysis 10.6 Fluorescence Analysis 10.7 Polarographic Analysis 10.8 Thin-Layer Chromatography 10.9 Paper Chromatography 10.10 Gas Chromatography 10.11 Liquid Chromatographic Analysis 10.12 Related Alkaloids 10.13 Determination of Occluded Solvents 11. Determination in Biological Fluids 11.1 Bile 11.2 Urine 11.3 Plasma 11.4 Blood 11.5 Microbial Cultures 11.6 DNA 11.7 Tubulin-Colchicine Complex 12. Determination in Pharmaceuticals References
ANALYTICALPROFILES OFDRUG SUBSTANCES, 10
140 140 140 140 141 141 141 144 147 147 149 149 150 150 150 150 157 158 161 163 164 165 165 165 166 166 166 167 170 170 170 170 172 172 172 172 172 175 175 175 175 175 175 176 177
Copyright G 1981 by Academic P r m . Inc
13
All right? of reproduction
~nan) form reserved
ISBN 0-12-2fiOR10~0
140
DOROTHY K. WYATT at a / .
1 .
History
Colchicine in impure form (Colchicum) has been known to man for thousands of years [l]. I t is the active ingredient of one of eighteen plants still in use of the approximately 700 listed in the Ebers Papyrus of ancient Egypt (1550 B . C . ) . Dioscorides [23,1], Nero's personal physician, provided the earliest remaining complete botanical description of Colchicum autumnale, the autumn crocus or meadow saffron, whose seeds, powdered corm, and dried flowers contain sufficient colchicine to effect relief of pain. Colchicum use for the treatment of gout was documented in approximately 560 A.D. and use appears to have been widespread until the eleventh century. Although relief of pain was obtained quickly, its high toxicity led to disuse (see # 9 ) . British formularies did list, then discard, colchicum in the early 1600's (London Pharmacopoeia and Complete English Dispensatory) but it was not until the early 1800's that colchicine use again became widely established. Colchicine's other biological characteristics, namely, its highly specific association with microtubule proteins and its effects on basic cell functions such as mitosis, secretion, cell morphology, motility, intracellular transport of macromolecules, microtubular assembly, and mitogenic activation [51j, make it a highly studied and widely applicable compound for use medicinally and for biochemical and biomedical research. 2. Description 2.1 Name, Formula, Molecular Weight
Colchicine is N-(5,6,7,9-tetrahydro-l,2,3,10t~etramethoxy-9-oxobenzo[~]heptalen-7-yl)-, (S)-acetamide. The CAS registry number 7 s 64-86-8 [2,3].
'2 2H2 gN06 molecular weight 399.44
2 . 2
Appearance , Color, Odor Pale yellow, amorphous scales, or powder.
Is
COLCHICINE
141
odorless or nearly so, and darkens on exposure to light [4,5,6, 9,101 *

3.
Phvsical ProDerties
3.1
Infrared Spectrum
The infrared spectrum is presented in Figure 1 . The spectrum was obtained from a potassium bromide dispersion of previously dried material ( 1 0 5 ' , 3 hours) using a Beckman 5260 grating infrared sp ctrophotometer. Principal bands are 1248, 1566, 1589 [7].
3.2
Nuclear Magnetic Resonance Spectra
b?H3
3.2.1
Proton NMR Spectrum
The proton NMR spectrum is presented in Figure 2 and spectral assignments in Table I . The spectrum was obtained using a Varian FT-80A instrument. Sample concentration: 500 mg/2.5 ml; tube diameter: 5 mm; spectral width: 1 0 0 0 Hz; acquisition time: 1.023; pulse width: 60 sec. Table I
H ' NMR Spectral Assignments for Colchicine [ 8 ]

Chemical Shift* PPm
6.57 7.67 6.93 7.40
Multiplicity singlet singlet doublet (J = 11) doublet (J = 11)
Characteristic of Proton
4 -H 8-H 11-H 12-H
100 MHz; J
*Chemical shifts
=
(6) in CDC13 (TMS internal standard) at coupling constant in Hz.
,
I
s - -
4 i .
__= <
___i
144
DOROTHY K . WYATT et uZ.
3.2.2 Figure 3.
Carbon-13 NMR Spectrum The carbon-13 NMR spectrum is presented in
Spectrometer: Varian FT-80A; sample concentration: 500 mgl2.5 ml; tube diameter: 10 mm; spectral width: 4000 Hz; acquisition time: 1.023 sec.; pulse width (flipping 4 5 ' ) ; number of data points: 10,230. angle): 8 sec. ( Spectral assignments are listed in Table I1 [15,16,18]. Table I1 13C N M R Spectral Assignments for Colchicine Chemical Shift (6 in ppm relative to TMS) 60.9 60.7 56.0 168.9 22.4 55.9 125.7 150.7 141.1 153.2 108.0 134.4 29.4 36.0 51.7 151.2 134.7 178.4 163.8 112.3 130.7 135.6 3 . 3 Carbon Number 13 14 15 16 17
Ring
[ C12-( 15,18) ]
I6
7 7a
Ring C [C8-(15,18)]
Ultraviolet Spectrum
The ultraviolet absorption spectrum of colchicine obtained from a 1 in 100,000 solution in chloroform is shown . One absorption maximum was observed at about in Figure 4 350 nm with an absorptivity of about 45 [8]. The uftra-
rc-
i
i
E 0
..
COLCHICINE
147
violet absorption spectrum of colchicine obtained from a 1 in 100,000 solution in 95% ethanol is shown in Figure 5 [5,8]. TWO absorption maxima were observed at about 351 and 243 nm with absorptivity values of 45 and 81, respectively. 3.4
Mass Spectrum
The mass spectrum is shown in Figure 6, and the fragmentation pattern is presented in Table I11 and IV. The mass spectrum was measured using a CEC 21-103C mass spectrometer equipped with a "direct" sample inlet system with the isotron temperature at 270". The ionization energy is maintained at 70 eV, the ionizing current at 50 VA [20,40]. Table I11
Mass Spectrum Fragmentation Pattern of Colchicine
mle 399 371 312 297 281
OH CH3-b=NH M+( 312)-CH3 M+( 312 ) -0CH3

M+
S ecies M$ M+ - CO
Table IV Mass Spectrum Fragmentation Scheme for Colchicine [ (m/e) , speciesI (399)
M+
3.5
Melting Range
The melting range of a colchicine sample, determined after drying at 105C for 3 hours is between 140" and 141.5"C according to the United States Pharmacopeia XX Class I procedure [2]. Additional melting points using unspecified procedures and colchicine of unspecified purity or hydration are given in Table V.
I
I
I
1;zz
Figure 5: Ultraviolet spectrum of colchicine in 95% ethanol
40
20
I
mk
Figure 6: Mass Spectrum of Colchicine
148
COLCHICINE
149
Table V Melting Point ( ' C ) 142-150 153-157 155-157 143-147 145 155 3.6 Solubility Reference 5 7, 1 0 21 23 6
1
The approximate solubilities obtained at room temperature are listed in Table VI [5,6,7,9,10]. Anhydrous colchicine decreases in solubility with increasing temperature. The reverse occurs with the sesquihydrate [lo]. Table VI Solubility Data of Colchicine at Room Temperature Solvent water ether benzene alcohol chloroform petroleum ether 3.7 Approximate Solubility (g/ml) $ 2 2 ml 151 g/20 ml [7,10] g/25 ml [6] g/220 ml [5,6] g/160 ml ( 1 5 . 5 ' ) [7,9,10] 1 g/100 ml [5] freely soluble [5,6,7,9,10] freely soluble [5,6,7,9,10] practically insoluble [5,10] 1 1 1 1 1
Moisture Content
The Karl Fischer titration procedure is the official United States Pharmacopeia XX [2] method for the drug substance. for 3 hours has been reported Colchicine has also been dried over phosphorus pent[4]. oxide at a pressure not exceeding 0.7 kPa (about 5 torr) for 24 hours [9].
Loss on drying at 1 0 5 ' C
150
DOROTHY K.WYATT et al.
3.8
Specific Rotation
The specific rotation measured at the sodium D line (approximately 589 nm), determined in an aqueous solution containing 100 mg in each 10 ml at 25"C, is between -435" and -460" calculated on an anhydrous, solvent-free basis according to the United States Pharmacopeia XX [ 2 ] . The specific rotation given in the British Pharmacopoeia [9] for a 1% aqueous solution at 19.5"-20.5"C is -425" to -450". I t has been reported as -410" to -435" at 25C [6]. Speci. 9 fic rotation in chloroform is determined as -121" (C = 0 m) at 17C [5]; -119.9" (C = 0.878) at 13C [l].
3.9
p H of a
pKa The pKa at 20" is 12.35 [5,6]. 5% solution = 5.9 [5]. Crystal Proverties
= 4.5
3.10
The crystal structure [ll] , conformation, bond distances, bond angles, and atomic coordinates of colchicine are presented in Figures 7, 8, 9, 10, and Table V I I , respectively. Crystals grown by slow evaporation of an aqueous solution containing tris(hydroxymethy1)aminomethane (tris) buffer were found to be the dihydrate C22H2 NO .2H20, . 00 f monoclinic, space group P21 a = 17.08 f 0.01, b = 0 0.007, c = 13.88 f 0.018, f3 = 117.9 f 0.1". Unit-cell parameters and standard deviations were obtained from least squares analysis of diffractomet r angle measurements. The measured density (Dm = 1.32 /cm ) indicated Z = 4 (calculated density Dx = 1.29 g/cm ); therefore, the asymmetric unit consists of two independent colchicine and four water molecules. Intensity data were collected from a 'crystal of 0.3 x 0.3 x 0.1 nm. A Syntex P2 diffractometer was used with monochromated Cu Ka(A = 1.5i178 a) x-rays.
? 4
4.
Isolation
Natural product: Colchicine is the medicinally active component in Colchicum autumnale L (Liliaceae) as well as more than fifty species of the Liliaceae family (Melanthoideae subfamily, tribe: Colchiceae). Alcoholic Extraction The corm or seed is extracted with alcohol. After distilling off the alcohol, the syrupy residue is diluted
C 0L C H I CI N E
151
Fig. 7. Projection of the crystal structure down b. Circles are water molecules. Hydrogen atoms are omitted for clarity. Lightly inked molecules are related to heavily inked ones by a twofold screw operation. Dashed lines indicate hydrogen bonds. Symbol indicates hydrogen bonds to molecules related to molecules shown by translation up or - are connected by a down in y. 0(14), O ( l ) ( f i ) , and 0(2)(a) single bifurcated hydrogen bond.
152
DOROTHY K. WYATT etal.
Fig. 8 . Experimentally determined conformations of molecule a (left) and molecule b (right), viewed normal to the planes defined by C(5), C(87, C(15). Torsion angles ( " ) are given for rings B and C, for O(1)-C(1)-C(Z), for C(6)C(16)-N-C(17), and fTr the-methoxy groups. The latter are taken to be the angles between the planes COCH3 and the least-squares planes or rings A and 2.
COLCHICINE
153
Fig. 9 . Bond distances (a); those for molecule a are listed above those for molecule b . Standard deviations are approximately 0.01 for each bond. Numbered atoms without element symbol are carbon, unlabelled atoms are hydrogen.
Table VII.
Atomic coordinates (x104 )
Standard deviations estimated from the least-squares calculations, assuming random errors in the intensity data, are given in parentheses as deviations in the last significant figure.
X -
Y
(6) (6) (6) (6) (5) (5) (6) (6) (6) (6) (6) (6) (6) (5) (6) (5) (6) (6) (6) (7) (6) (6)
z -
X -
Y (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (5) (6) (5) (6) (6) (8) (6) (7) (6) 5109 6198 6316 5422 4187 3556 3965 3519 2368 1749 2277 3376 3984 1778 1299 2228 2066 5077 3362 491 8328 1488 (8) (8) (8) (8) (7) (7) (8) (8) (8) (8) (9) (8) (8) (8) (8) (8) (10) (12) (12) (9) (9) (11)
z -
5867 5910 5520 5004 4768 5027 5495 4191 3401 2830 3048 3869 4413 3166 3728 4718 5947 5890 4210 1623 6581 6359
8131 8237 7449 6367 5714 6080 7089 4623 4547 3540 2582 2621 3636 5556 5478 5213 4648 2991 467 1627 9462 4831
(10) (9) (9) (9) (10) (11) (9) (10) (12) (11) (10) (10) (10) (10) (9) (12) (10) (10) (13) (10) (12)
3535 2512 1637 1488 2165 3296 3823 1727 1775 1317 815 802 1222 2337 3563 3903 5658 1792 748 85 1623 6872
(7) (7) (6) (6) (6) (7) (7) (6) (6) (7) (7) (7) (6) (7) (7) (6) (8) (9) (8) (10) (7) (7)
528 903 1230 1362 1099 598 387 1345 1794 2052 1878 1423 1160 1963 1092 313 -1276 -200 580 2526 1342 -2012
988 1674 2747 3543 3477 2435 1436 4520 4693 5686 6492 6291 5321 3835 2882 2570 1448 4647 7256 7704 1543 455
(7) (7) (6) (6) (6) (6) (6) (6) (6) (7) (6) (6) (6) (6) (7) (6) (7) (10) (9) (7) (8) (7)
T a b l e VII Molecule a
X -
-.- Cont'd.
b Molecule X -
Y (5) (4) (4) (4) (4) (4) (4) (4) (5) 5309 8991 9247 3706 1683 1555 3998 3645 2203 (9) (7) (6) (6) (6) (8) (8) (6) (7)
Y (5) (4) (3) (4) (4) (4) (4) (5)

(9)
Z -
5216 6228 6395 5165 4073 2507 6286 8016 8179
5072 4236 2524 1089 263 300 5254 5401 3685
(6) (5) (4) (4) (4) (5) (5) (5) (5)
-471 304 898 736 1255 2126 -1398 5459 6893
1787 5149 7179 5119 3920 1708 2783 1839 1142
(6) (6) (6) (5) (6) (6) (7) (9) (11)
1590 -6 1033 5177 7095 7479 2067 3814 4086
(5) (5) (4) (4) (4) (4) (5) (5) (9)
156
DOROTHY K. WYATT et al.
with water to precipitate the insoluble fats and resins, and filtered. The aqueous solution is then repeatedly extracted with chloroform [l] or digested with lead carbonate, refiltered, evaporated to a small volume, and extracted with chloroform [ 6 ] . The colchicine is recovered as a crystalline addition complex with chloroform. The chloroform is then distilled off in steam or alcohol. Amorphous colchicine is produced upon evaporation of the residual qolution. Amorphous colchicine may be crystallized from ethyl acetate as pale yellow needles. Modifications
1.
2.
3 .
h.
Chromatographic purification of the chloroform solution on alumina [l]. Extraction of the dried powder derived from saffron plant parts with petroleum ether to remove fats followed by alcoholic extraction [l]. Wax and paraffin wax for the removal of resin [ 4 2 ] . Soxhlet apparatus [ 4 2 ] .
Fig. 1 0 . Bond angles ("); those for molecule a are listed above those for molecule b . Standard deviations are approximately 1 ' for each angle. Numbered atoms without element symbol are carbon, unlabelled atoms are hydrogen.
hvdroxylationoxidation
H
v)
phenylalanine
cinnanic acid
l-phenyltetrahydroisoquinoline
m
0
P
A H 3
C CH30 CH3 H
Ho
pyclization methylation
\ W
r c H -
- : 3"
autumnaline
9 ' O-methylandrocymbine
hydroxylation
'
\,
cH3
/OH
w
Y
ID 0
..
N
I u I u
ellmlnation
5 CH3 cH30w%p~3 CH3 .s/

c CH30 H 3 T A
'H3
\
expansio c H n 3 % iC ,H 3
r i z
CH30
.\I
salt
~
hydrolysis'
a cH30
\
H
3H3immonium
CH30
demcthylation acetylation
cH3
\
demecolchicine
OCH3
colchi cine
OCH3
158
DOROTHY K. W A T T e t a ! .
6.
[221
Synthesis
6.1
Synthesis from 7,8,9-Trimethoxy-9-benzosuberone
resolution. N-acetylation, and 0-rnethylation.
COLCHICINE
159
6.2
[221.
Synthesis from Purpurogallin trimethyl ether
: ) N N ! H i
(+)-trirnethylcolchicinic acid
OCH3
colchicine
160
DOROTHY K. WYATT et a[.
6.3
[221.
Synthesis from a Substituted Isothiazole

I) 03
Br-CH2
CH3CQC
zs
P, 2 ) NoOCH3
3)3,4,5-(CH30)3-OCH0
$mN
I)HN=NH
I
cw c w
o=c \
0
CH3
I S
2)LiAIHq 3)MnOz
HOOC
OAc
b H
methylation
t
(t)colchiceinz
Acid hydrolysis
co lchicine
,
I
with diazomethane
c
isocolchicine
(~)-trimethylcolchicinic acid
co lc h I ci n c
COLCHICINE
161
7.
Stability and Degradation
7.1
Reaction with Ultraviolet Light [ 2 2 , 2 3 ]
Colchicine is converted into a mixture of three photoisomers in the presence of ultraviolet light. A tetracyclic structure is formed with loss of the tropanoid ring
colchicine
f.
OCH3
8-1umicolc h icin e
7-lumicolchicine
+
NHAc
cn,o
a-lumicolchicine
OCH,
162
7.2
Acid Hydrolysis [ 2 3 , 1 I
CH3 3 i I ? CH30
A H 3
0
CCH3
mild c o n d i t i o n s d i l u t e HCI
0
colchicine
colchiceine
HC 1 150, 6 hours
c o l c h ic i n ic acid
NOTE: Conversion of c o l c h i c i n e t o c o l c h i c e i n e and o t h e r products a l s o occurs during a l k a l i n e hydrolysis p H >13 [ 2 4 ] , t h e r e i s no a p p r e c i a b l e h y d r o l y s i s t o c o l c h i c i n e o c c u r r i n g i n n e u t r a l o r s l i g h t l y a l k a l i n e (pH 8.1) s o l u t i o n s even a f t e r 2 months s t o r a g e [9].
COLCHICINE
163
7.3
Hydrogeiia'iion [23,1]
colchicine 7.4 Oxidation (23)
hexahydrocolchicine
warm potassium permanganate
'
CH30
CH30
OCH3
colchicine 8. Metabolism
3,4,5-trimethoxyphthalic
acid
Little is known regarding colchicine absorption from the gastrointestinal tract [7]. A number of studies do indicate that a metabolite is the active form of the drug. A study of metabolism in gout patients suggests that a major portion of the drug is deacetylated but it is not known whether deacetylcolchicine is the human metabolite [18]. Colchicine and desmethycolchicine have been found in the bile 1391. There is no evidence of specific accumulation in certain tissues [7]. However, colchicine's ability to disrupt mitosis is well known and post-mortem examination indicates arrested metaphases in the lymph glands, spleen,
164
DOROTHY K. WYATT et a / .
and liver, as well as in bone marrow and in duodenal mcosa [l]. After two hours, the highest concentrations of colchicine have been found in the liver and kidney in the dog, rat, rabbit, and hamster; the brain had the lowest concentration [39]. The ability to arrest dtosis is believed to result from strong binding of the drug to the protein tubulin which prevents the assembly of the tubulin into microtubules which form the mitotic spindle [ll]. Although metabolites are not well known, it has been postulated that colchicine inhibits the acute gouty attack by inhibiting phagocytosis of urate crystals by leukocytes, thus diminishing the inflammatory reaction [35]. Colchicine also induces polyploidy in plants and malformations of embryos [1,11].
9. Pharmacokinetics
Humans excrete 5-50% of the injected dose within 48 hours [39]. About 50% of the injected dose is recovered from the mouse within 16 hours. The bulk of the colchicine is excreted within the first 24 hours, especially at high blood levels. Colchicine may be excreted in the bile or directly into the gastrointestinal tract [7,39], although bile seems to be an important pathway for the excretion of colchicine [39j. In mice, 2.4-f4% of the dose is excreted in the feces [7]. Higher amounts are excreted in the feces of rats. Dose -- 1 mg initially; subsequent doses of 500 pg every two hours until pain is relieved or until toxic effects such as vomiting or diarrhea are experienced; IP max in 24 hours is 3 mg. Colchicine frequently causes nausea, vomiting, and diarrhea. Larger doses may cause dehydration and hypotension. Hair l o s s may result after prolonged treatment. Symptoms of poisoning may be observed after 3-6 hours [9,34]. Fatality has resulted from ingestion of as little as 7 mg [34]. Death generally results in 7 to 36 hours [9].
COLCHICINE
165
10.
Methods of Analvsis
10.1
Elemental Analysis [ 6 4 ] theory carbon hydrogen nitrogen oxygen

66.15 6.31 3.51 24.03
found
65.40 6.30 3.35 23.97
10.2
Color Tests Color intense yellow violet slowly changing to yellow then to green yellow (sensitivity 0.25 Pg) yellow (sensit ivity 0 - 2 5 Pg) green (sensitivity 0.25 pg) yellow + purple/ brownlred-brown (sensitivity 0.25 1 1 8 ) garnet red lemon-yellow greenish-blue + reddish + yellow or almost colorless red yellow Reference
1
Aeent
1
dilute mineral acids and alkalis nitric acid
3
4
sulfuric acidformaldehyde ammonium molybdate ammonium vanadate (Vitali's test)
7
7
ferric chloride T . S . alcohol followed by nitric acid
4Y9
7 sulfuric acid
8 excess of sodium hydroxide

9
4Y9 9
water (color intensified by adding mineral acids)
166
DOROTHY K. WYATT et nl.
Agent
10
Color orange red violet yellow brownlred + orangelred
Reference 34
nitric acid-watersodium hydroxide
11 concentrated nitric acid; addition of water; followed by sodium hydroxide 12 hydroxylamine-sodium hydroxide (warm the solution) 10.3
-+
42
orange
43
Aqueous Titrimetric Analysis (Residual Titration)
An accurately weighed sample of colchicine is . 0 2 N hydrochloric acid. The excess dissolved in excess 0 acid is titrated with 0.07 N sodium hydroxide using methyl orange as indicator [47].
10.4
Non-aqueous Titr imetric Analysis
The non-aqueous titration procedure is the official United States Pharmacopeia XX [2,9] and the British Pharmacopoeia method for the drug substance. An accurately weighed sample of colchicine is dissolved in a mixture of acetic anhydride-toluene ( 1 : 2 ) . The end-point is determined potentiometrically using 0.02 N perchloric acid as the titrant. An additional non-aqueous titration procedure was presented [47] in which glacial acetic acid containing 3-4 drops of acetic anhydride is used to dissolve colchicine. Titration is accomplished using either crystal violet or potentiometric determination using calomel and glass electrodes; 0.01 N perchloric acid is used as the titrant. 10.5 Spectrophotometric Analysis
The official USP XX [2] method for the analysis of Colchicine Tablets is spectrophotometric. A portion of powdered tablets is weighed and colchicine is extracted with chloroform from an aqueous solution. The W spectrum of the chloroform solution is recorded and compared to the USP reference standard (diluted to the same final concentration with chloroform) at the maximum absorbance at about 350 nm. Spectrophotometric analysis also was conducted using nitric acid to dissolve the drug, followed by sodium hydroxide T . S . and dilution with water; the solution was
COLCHICINE
167
read at 350 nm, with an additional maximum observed at about 510 nm [42]. Analysis using the hydroxylamine-sodium hydroxide color reaction (orange color) was accomplished using readings taken at 500 n m wavelength [43]. Ferric chloride solutions, after acid hydrolysis, were read at 470 nm [48, 491. Colchicine was also analyzed after lithium aluminum hydride reduction and extraction from 1% hydrochloric acidammonia-acetic acid solution into carbon disulfide. The organic layer was removed and combined with benzene. Readings are at 445 nm [47]. Isonicotinic hydrazide in alkaline media has also been used for reaction with colchicine for a colorimetric determination 1341. 10.6 Fluorescence Analysis
The emission and excitation spectra are provided in Figure 11. The emission maximum is 422 nm. I t was obtained using a Perkin Elmer MPF-2A spectrofluorometer with a slit at 6 nm [50] and the excitation wavelength at 350 nm [67]. The emission maximum is shifted towards the red as the solvent polarity is decreased [67].
WAVELENGTH
,
L
nrn.
300
1
400
I
500 600
I
t v )
-1.0
?
z a
I-
-0.5
I -
2
B a
0.0
WAVENUMBER, ( ern-'. lo-' )
Fig. 11. Absorption (000,296 K), excitation (--, 7 7 K ) and emission (----, 77K) spectra for colchicine in EPA. The excitation is not corrected. The inset shows the polarization of the excitation and emission spectra.
Table VIII Thin-layer Chromatography of Colchicine Plate silica gel F-254 silica gel F-254 silica gel F-254 aluminum oxide F-254 silica gel F-254 silica gel F-254 Solvent chloroform-acetonediethylamine (5:4:1) chloroform-methanolacetic acid (85:15: 1) chloroform-methanol (9:1) chloroform-acetoneaqueous ammonia (25: 20: 0.4) chloroform-methanoldiethylamine (5:4:1) toluene-ethanolaqueous* ammonia (170:28:2) benzene-acetoneether-10% aqueous ammonia (4:6:1:0.3) Method of Detection A,B,C,D,E A,B,C,D,E A,B,C,D,E A,B,C,D,E
Rf x 100
Ref.
8
47
75
59
54,55
8
64 68 64
8 39
A,B,C,D
98
8 8
silica gel G
--------
15
54
Table V I I I -- Cont'd. Plate silica gel G Solvent benzene-acetoneether-25% aqueous ammonia (4:6:1:0.3) chloroform-diethylamine (9:l) chloroform Method of Detection Rf x 100 20 Ref. 54
--------
silica gel G alumina G
41
11
54,55 54,55 54,55
G G
silica gel G pre- methanol . 1 treated with 0 N sodium hydroxide silica gel G benzene-ethyl acetatediethylamine (5:4:1) + 8% methanol chloroform-acetonediethylamine (7:2:1) silica gel G methanol-aqueous ammonia (100: 1.5)
57
B,G,H
56 46 61 60 62
56,5
39
B,G,H
56,5
39
170
DOROTHY K . WYATT c't
a/.
10.7
Polarographic Analysis
A polarographic analysis of colchicine was accomplished using a dropping mercury working electrode and a silver wire reference electrode in 0.1 M tetrabutylammonium iodide [53,52]. The dc and ac polarographic responses are recorded. Half wave p tential is -1.47 volts. Detection o ' moles per lite ac: 1 x 1--5 moles limits for dc: 3 x l per liter; ac second harmonic: 5 x 10-5;moles per liter. 10.8 Thin-Layer Chromatography
Thin-layer chromatography has frequently been used for the analysis of colchicine. Methods of detection and solvent systems are listed in Table V I I I . 10.9 Paper Chromatography
Ascending paper chromatography was accomplished using Whatman #1 paper which was dipped in a 5% solution of sodium dihydrogen citrate and dried. The solvent consisted of 4 . 8 g of citric acid in a mixture of 130 ml of water and 870 ml of 1-butanol (Rf x 100 = 83). Examination was conducted using shortwave ultraviolet light [7]. An additional analysis was obtained using formamide/benzene-chloroformformamide (7:3:1) and longwave ultraviolet detection [57]. 10.10 Gas Chromatography
The following GLC systems have been used for analysis; however, thermal instability of colchicine has been observed. Detector FID Phase and Column 5% SE-30 on 60-80 mesh Chromosorb WAW 5-ft x 1/8-inch stainless steel col. (31 ml/min nitrogen)
1% Hi-Eff-8B on 1001 120 mesh silanized Gas Chrom P 0.9x 3 . 2 glass columns (approximately 60 ml/ min nitrogen)
Temperature ("C)
230
Ref. 7,54 54
250, 270
HFI
220,
250
54
COLCHICINE
171
Methods of Detection
A. B.
C. D. E.
F. G. H . I.
shortwave ultraviolet light longwave ultraviolet light 0.5% iodine in chloroform 40% sulfuric acid in methanol followed by heat ( 1 0 5 ' ) 40% sulfuric acid in methanol followed by heat (105" C) and longwave ultraviolet light acidified potassium iodoplatinate potassium iodoplatinate antimony (111) chloride p-dimethylaminobenzaldehyde
172
DOROTHY K. WYATT et a / .
10.11
High-Performance Liquid Chromatographic Analysis
High-performance liquid chromatography has been used extensively for the analysis of colchicine. The various HPLC systems used for the analysis are given in Table IX. 10.12 Related Alkaloids
The related alkaloids procedure is the official USP XX method [2] for the drug substance. Two solutions of differing concentrations of colchicine in alcohol are compared by thin-layer chromatography using an alumina plate with a fluorescent indicator (254 run) and a mobile solvent consisting of chloroform-acetone-aqueous ammonia (25:20:0.4). Comparison of the two samples is accomplished using shortwave ultraviolet light. 10.13 Determination of Occluded Solvents (chloroform and ethyl acetate) The occluded solvents procedure is the official USP XX limit test for the drug substance 121. A gas chromatographic procedure using a flame-ionization detector, 20% polyethylene glycol (950-1050 average molecular weight) on flux-calcined diatomaceous earth, and nitrogen carrier gas is used. The column is maintained at 75". A n aqueous colchicine solution with added internal standard (n-propyl alcohol) is compared to known quantities of aqueous ethyl acetate-chloroform solution (n-propyl alcohol added).
11.
Determination in Biolortical Fluids
11.1
Bile
Colchicine has been determined in bile by thinlayer chromatography on silica gel F-254 using ultraviolet detection and the systems listed below [39]. Colchicine was separated from colchiceine, desacetylcolchicine, and desmethycolchicine. Systems methanol-chloroform (9:l) Rf = 0.68 benzene-ethyl acetate-diethylamine (5:4:1) and 8 % methanol Rf = 0.46 chloroform-acetone-diethylamine (7:2:1) Rf = 0.60.
Table IX High Performance Liquid Chromatography Systems for Colchicine Column Zorbax-Si1 Partisil 10/25 Chromanetics C8 LiChrosorb RP-8 Partisil ODs UBondapak C18 pBondapak C18 Mobile Phase 87-89% methylene chloride-2-propanol 85-90% methylene chloride-2-propanol methanol-water (1: 3) Flow/ Temperature ambient ambient ambient ambient ambient ambient ambient
UV Detection Pressure
42 atm 1000 psi
1000 psi
Wavelength 254 254 254 254 254 254 350
Ref.
8 8 8 8
8
30% acetonitrile-water
methanol-water (1: 1,l: 2 ) 20% and 35% acetonitrile-water acetonitrile-methanol -phosphate buffer pH 7 . 6 (17:5:78) -phosphate buffer pH 6.0 (17:5:78) -phosphate buffer pH 6 . 0 (16:5:79) -phosphate buffer pH 6 . 0 (15:5:80)
925-1550 psi 1000 psi 925-1550 psi 1390 psi (2 ml/min)
8
58
Table IX -- Cont'd. Column LiChrosorb Si-60 Mobile Phase gradient : acetonitrile - 10% acetonitrile in water (0-30%) 5 mM pentanesulfonic acid in methanolacetonitrile-phosphate buffer ( 0 . 1 M pH 7.6), (41: 15: 4 4 ) . The mobile phase was adjusted to a final pH of 6.45 by addition of glacial acetic acid dichloromethane2-propanol acetonitrile-methanolphosphate buffer pH 6 . 0 (16:5:79) Temperature ambient Flow/ Pressure 2 d/min
W Detection
Wavelength 254
Ref. 59
Octadecylsilane reversed phase column (Waters)
40"
1.5 ml/min
2 54
62
Hypersil (5 um) LiChrosorb RP-18
ambient ambient
2 ml/min
240 350
63
3.0 ml/min
60
COLCHICINE
11.2
Urine
Colorimetric analysis after acid hydrolysis followed by addition of ferric chloride reagent has been used. Readings are at 630 nm t o minimize interference by colored urine components [49]. Paper chromatography using benzene-formaldehyde on Whatman fl or f4 paper and concentrated hydrochloric acid spray for detection (yellow green band) after extraction has also been used [49]. 11.3 Plasma
HPLC at 240 nm using a Hypersil column and dichloromethane/isopropanol solvent was reported. The plasma was extracted from an aqueous ammonia solution into dichloromethane. Ethanol was added to the dichloromethane after removal of the aqueous layer. The solution was evaporated to dryness and dichloromethane added to dissolve the residue [63].
11.4
Blood
Analysis by HPLC was conducted using octadecylsilane reverse phase columns eluted with pentanesulfonic acid in methanol-acetonitrile-phosphate buffer, pH 7.6 (4:15:44) adjusted to pH 6.45, and UV detection at 254 nm. The colchicine was extracted from sodium bicarbonate solution into dichloromethane [62].
11.5 Microbial Cultures

HPLC using a LiChrosorb W-18 column with acetonitrile-methanol-phosphate buffer pH 6.0 (16:5:79) mobile phase and UV detection at 350 nm was used f o r determination of colchicine and colchiceine in microbial cultures after extraction from sodium hydroxide solution into chloroform. The chloroform extract was evaporated to dryness and reconstituted in the mobile phase [60]. 11.6 DNA -
Spectrophotometric analysis in pH 7, 10, and 12 phosphate buffer and proton NMR in D20 was used to determine the interaction between colchicine and DNA [37]. 11.7 Tubulin-Colchicine Complex Fluorescence was measured in PMC (sodium phosphate
176
pH 7.0-magnesium chloride) buffer solutions [67]. 12. Determination in Pharmaceuticals
The official USP and BP methods are spectrophotometric HPU: and other metholis can readily be adapted for quantitative use. Additional procedures such as HPLC involve analysis of the powdered seeds [59].
[2,9].
Acknowledgements The authors wish to thank the chemists of the USP Drug Research and Testing laboratory for their experimental contributions and Ann K . Ferguson for providing the . Wyatt and computerized literature search, and William K Barbara A . Bowman for their assistance.
COLCHICINE
References 1 . 2 .
3.
. , Colchicine, The Iowa Eigsti, O.J., Dustin, P State College Press, Ames, Iowa (1955). The United States Pharmacopeia, 20th revision, Mack Publishing Company, Easton, Pennsylvania (1980). USAN and the USP Dictionary of Drug Names, United States Pharmacopeial Convention, Inc., Rockville, Maryland (1980) p. 100.
4 . The United States Pharmacopeia, 19th revision (1975) p . 102; 18th revision (1970) p . 142, Mack Publishing, Company, Easton, Pennsylvania.
5.
The Merck Index, Ninth Edition, Merck . 318. Rahway, New Jersey (1976) p
Co., Inc.,
6. 7 .
8.
Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania (1975) p. 1049. Clarke, E.G.C., Isolation and Identification of Drugs, The Pharmaceutical Press, London (1969) p. 269. The United States Pharmacopeial Convention, Inc., Drug Research and Testing Laboratory, 12601 Twinbrook Parkway, Rockville, Maryland 20852.
9. British Pharmacopoeia, Her Majesty's Stationery Office, University Printing House, Cambridge, England (1980) p. 124.
10.
Martindale, The Extra Pharmacopoeia, Twenty-sixth Edition, The Pharmaceutical Press, London, England (1975) p. 448. Lessinger, L., Margulis, T.N., "The Crystal Structure of Colchicine. A New Application of Magic Integers to Multiple Solution Direct Methods," Acta Cryst. B34 578 (1978). Lessinger, L., Margulis, T.N. (1978)
11.
12.
"The Crystal Structure
of Isocolchicine an Inactive Isomer of the Mitotic Spindle Inhibitor Colchicine," Acta Cryst. B34 1556
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13.
Iorio, M.A.,
Brossi, A., Silverton, J.V.,
"7-
deacetamidocolchicine: W o Novel Procedures from the Base Catalyzed Reaction of (-)-N-Benzylideneh i ; . Acta 61 1213 (1978). deacetylcolchiceine, Helv. C
"
0x0-deacetamidocolchiceine and 7-Benzylimino-
14.
Capraro, H.G., Brossi, A., "Simple Conversion of Colchicine into Demecolcine," Helv. Chim. Acta 62 965 (1979). Hufford, C.D., Capraro, H.G., Brossi, A,, ''13C and 'H -NMR Assignments for Colchicine Derivatives," Helv. 63 50 (1980). Chim. Acta Blade-Font, A., Muller, R., Elhuero, J., Faure, R., Vincent , E. J., "Carbon-13 Nuclear Magnetic Resonance Spectrum of Colchicine: A Reassignment," Chemistry Letters 233 (1979). Singh, S.P., Pamar, S . S . , Stenberg, V.I., Farnum, S.A., "Carbon-13 Nuclear Magnetic Resonance Spectrum of 10 (12) 1001 (1977). Colchicine," Spectroscopy Letters Hufford, C.D., Collins, C.C. , Clark, A.M. , "Microbial Transformations and I3C NMR Analysis of Colchicine,'' J.Pharm.Sci. 68 1239 (1979). Danieli, B., Palmisano, G., Ricca, G., Severini, G., '*13CN M R Analysis of Colchicine and Isocolchicine: A Revision of Colchicine Assignments," Gazz. Chim. Ital. 110 351 (1980). Wilson, J.M., Ohashi, M. , Budzikiewicz, H., Santavy, F., Djerassi, C., "Mass Spectrometry in Structural and Stereochemical Problems -- XXXIII: Colchicine Alkaloids," Tetrahedron 19 2225 (1963). Glasby, J.S., Encyclopedia of the Alkaloids, Vol. 1, Plenum Press, New York (1975) p. 312. Pelletier, The Chemistry of the Alkaloids, Van Nostrand-Reinhold, New York (1970) p. 199. Dalton, David R., The Alkaloids, Marcel Dekker, Inc., New York (1979).
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21. 22. 23.
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24.
Wilczok, T., Buzzman, E., Sulkowska, A., Iubas, B . , "The Effect of pH on Colchicine Conformation and Structure," Z.Physiol.Chem. 360 59 (1979). Blade-Font, A., "New Chemistry of Colchicine and Related Compounds," Tetrahedron Letters 47 4097 (1977). Carlassare, M., "Titolazione della Colchicina nel Prodotto Officinale, nel Colchico e suoi Preparati," Boll.Chim.Farm. 118 343 (1979). Evans, D.A., Hart, D.J., Koelsch, P.M., "A New Approach to the Synthesis of Tropolones: Synthesis of Colchicine and 6-Dolabrin," J. Am. Chem.Soc. 100 (14) 4593 (1978). Burger, A,, Medicinal Chemistry, 32nd edition, Wiley Interscience, New York (1970). Hartwell, J.L., Nadkarni, M.V., biter, J., "NSubstituted Colchiceinamides," J. Am. Chem. ! & . 74 3180 (1951) Horowitz, R.M., Ullyot, G.E., "Colchicine. Some 74 587 (1952). Reactions of Ring C," J. Am. Chem. SOC. Rapoport, H., Williams, A.R., Cisney, M.E., "The Synthesis of d,l-Colchinol Methyl Ether," J. Am. Chem. SOC. 72 3324 (1950). -Rapoport, H., Williams, A.R., Cisney, M.E., "The Synthesis of d,l-Colchinol Methyl Ether," 3. Am. Chem. S O ~ .73 1414 (1951). -Doering, W. von E., Knox, L.H., Chem. SOC. 73 828 (1951). Wallace, S.L., (1961) "Tropolone, J. Am.
'I
25. 26.
27.
28. 29.
30. 31.
32.
33. 34. 35.
"Colchicine," Am. J. Med. 30 439
Krakoff, I.H., "Clinical Pharmacology of Drugs which Influence Uric Acid Production and Excretion," Clinical Pharm. "her. 8 124 (1967). Friend, D.G., (1968). "Uricosuric Drugs," Practitioner 200 153
36.
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37.
Buszman, E., Wilczok, T., Witman, B., Siebert, G., "Interaction of Colchicine with DNA Molecules," Hoppe. Physiol. Chem. 358 819 (1977). Seyler's Z Chang, Yi-Han, "Mechanism of Action of Colchicine," J. Pharmacol. Exp. Ther. 194 154 (1975). Hunter, A.L., Klaassen, C.D., "Biliary Excretion of 192 605 (1975). Colchicine," J. Pharmacol. Exp. Ther. Schonharting, M., Pfaender, P., Rieker, A., Siebert, G., "The Oxidative Formation of Products from Colchicine in the Udenfriend System," Hoppe-Seyler's 2. Physiol. Chem. 354 421 (1973). Hasenmuller, B . , Schonharting, M., Siebert, G., "A Study of Microsomal Oxidative Functions with the Aid of a Series of N-Colchiceyl Derivatives," Hoppe-Seyler's 2. Physiol. Chem. 359 725 (1978). Smolenski, S.J., Crane, F.A., Voigt, R.F., "A Chromatographic-Spectrophotometric Method for the Separation and Determination of Colchicine," J. Am. Pharm. Assoc. XLVII 359 (1958). Mack, H., Finn, E.J., "A Colorimetric Method for the Estimation of Colchicine in Pharmaceutical Preparations," J. Am. Pharm. Assoc. 2 532 (1949). Uffer, A., Schindler, O., Santary, F., Reichstein, T., "Teilsynthese des Demecolcins und Einiger Anderer 37 18 (1954). Colchicinderivate," Helv. Chim. Acta Santavy, F., Winkler, R., Reichstein, T., "Zur Konstitution von Demecolcin aus Colchicium Autumnale 36 1318 (1953). L," Helv. Chim. Acta Dessouky, Y.M., Ismaiel, S . A . , "Colorimetric Determination of Piperazine in Pharmaceutical Formulations," Analyst 99 482 (1974). Karawya, M.S., Diab, A.M., "Colorimetric and Volumetric Assays of Colchicine in Galenicals and in Pharmaceutical Preparations," J.AOAC 58 1171 (1975).
38. 39.
40.
41.
42.
43.
44.
45.
46.
47.
48. King, J.S., "A Colorimetric Method for the Estimation of Colchicine," J. Am. Pharm. Assoc. XL 424 (1951).
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49.
Pearce, E.M., "The Quantitative Colorimetric Determination of Colchicine in Aqueous Solution, and 2 108 Studies on Its Application to Urine,"J. Chrom. (1959)
50.
Croteau, R., LeBlanc, R.M., "Photophysical Processes in Tropolone, a-Methoxy-tropone, and Colchicine," Photochemistry and Photobiology 2 8 33 ( 1 9 7 8 ) . Clark, J.I., Garland, D., "Fluorescein Colchicine,"J. Forensic Sci. 7 6 619 ( 1 9 7 8 ) .
of Organic Pharmaceuticals Using Aprotic Organic Solvents," J. Electroanal. Chem. 100 1 4 5 ( 1 9 7 9 ) .
51.
52.
Schaar, J.C., Smith, D.E.,
"A.C. Polarographic Analysis
53.
Woodson, A.L., Smith, D.E., "Direct Current and Alternating Current Polarographic Response of Some Pharmaceuticals in an Aprotic Organic Solvent System," Anal. Chem. 42 242 ( 1 9 7 0 ) . Zweig, G., Handbook of Chromatography, CRC Press, Ohio
(1972).
54.
55.
Kirchner, J.G., Thin-layer Chromatography, Interscience Publishers, New York ( 1 9 6 7 ) . Stahl, E., Thin-layer Chromatography, Springer-Verlag, Berlin ( 1 9 6 5 ) . Macek, K. , Pharmaceutical Applications of Thin-layer and Paper Chromatography, Elsevier Publishing Co., Amsterdam ( 1 9 7 2 ) . Davis, P.J., Klein, A.E., "High-Performance Liquid Chromatographic Separation of Colchicine and Its Phenolic and N-Desacetylated Derivatives," J. Chrom.
188 280 (19807.
56.
57.
58.
59.
Forni, G., Massarani, G., "High-Performance Chromatographic Determination of Colchicine Colchicoside in Colchicum (C. Autumnale L . ) Home-made Stationary Phase," J. Chrom. 131
-4 4 4
Liquid and Seeds on a

(1977).
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60. Klein, A.E., Davis, P.J., "Determination of Colchicine and Colchiceine in Microbial Cultures by Highperformance Liquid Chromatography," Anal.Chem. 52
2432 (1980). 61.
McLinden, V. J., Stenhouse, A.M., "A Chromatographic . System for Drug Identification,'' Forensic Science International 13 7 1 (1979). Caplan, Y.H., Orloff, K.G., Thompson, B.C., "A Fatal 4 153 Overdose with Colchicine," J. Anal. Tox. (1980).
62.
63.
Jarvie, D., Park, J., Stewart, M.J., "Estimation of Colchicine in a Poisoned Patient by Using HighPerformance Liquid Chromatography," Clinical Tox. 14
375 (1979).
64. 65. 66.
USP Private communication, November, 1973. Private communication, May, 1972. Boyland, E., Mawson, E., "The Conversion of Colchicine into Colchiceine," Biochem. J. 32 1205 (1938). Arai, T., Okuyania, T., "Fluorometric Assay of TubulinColchicine Complex," Anal. Biochem. 69 443 (1975). Walaszek, E.J., Kocsis, J.J., et al., "Studies on the Excretion of Radioactive Colchicine." Arch. Int. Pharmacodyn Ther. 125 371 (1960).
67.
68.
CYANOCOBALAMIN
Joel Kirschbaum
1. Introduction 1.1 History 1.2 Structure, Nomenclature, and Molecular Weight 1.3 Appearance, Color, Odor, and Precautions 1.4 Synthesis, Biosynthesis, and Commercial Production 1.5 Nutrition, Physiolog, and Medicine 1.6 Reactions of Cyanocobalamin 2. Physical Properties of Crystalline Cyanocobalamin 2.01 Single Crystal X-Ray Diffraction 2.02 X-Ray Powder Diffraction, Spectrometry, and Activation Analysis 2.03 Mass Spectrometry 2.04 Magnetic Susceptibility 2.05 Mossbauer Spectrometry 2.06 Thermal Analysis 2.07 Microscopy and Crystal Type 2.08 Surface Area 2.09 Hydration 2.10 Polymorphism 3 . Spectrometry of Cyanocobalamin in Solution 3.1 Nuclear Magnetic Resonance Spectrometry (NMR) 3.2 Electron Spin Resonance Spectrometry 3.3 Infrared Spectrometry 3.4 Raman Spectrometry 3.5 Absorption (Visible and Ultraviolet Spectrometry) 3.6 Optical Rotatory Dispersion and Circular Dichroisni Spectrometry 3.7 Fluorescence Spectrometry 4 . Bulk Solution Properties 4.1 Intrinsic Dissolution Rate 4.2 Solubilities in Aqueous and Non-Aqueous Solvents 4.3 Partition Coefficients 4.4 Ionization 4.5 Hydrodynamic Properties 5. Methods of Analysis 5.1 Compositional Analysis 5.2 Identity, Spectrophotometric, and Colorimetric Methods 5.3 Chromatographic and Other Separation Analyses 5.4 Electrochemical Analyses
184 184 185 188 188 192 194 199 199 20 1 20 1 204 204 204 205 205 206 206 206 206 210 21 1 211 213 214 215 215 215 215 216 216 217 217 217 218 222 235
ANALYTICAL PROFILESOF IIALIC SUHSTANCES. 10
183
184 5.5 Radioassays 5.6 Microbiological Assays 5.7 Enzymatic Assays 5.8 Mass Fragmentography 5.9 Comparison of Methods Stability 6.1 Inactivation 6.2 Stabilization Metabolism Acknowledgments References
JOEL KIRSCHBAUM 237 242 247 248 249 250 250 253 254 254 255
6.
7. 8. 9.
1.
Introduction
1.1
History
In 1855, Thomas Addison' of Guy's Hospital, London, described a form of "idiopathic anaemia". About 50 years later clinicians found that deficiencies of dietary factors could lead to some diseases in humans. Casimir Funk, in 1911, called these essential compounds r'vitamins" (Latin vita, life, + amine) because many contained amine functions. G.H.Whipple2, in 1922, reported that beef liver, beef heart and various other food enhanced hemoglobin formation in patients suffering from pernicious anemia, so-called because it usually lead to death.
G.R. Minot and W.P. Murphy3 reported clinical improvement in patients eating daily a special diet containing 120-240 grams of liver. Minot, Murphy andiqhipplereceived the Nobel prize in physiology and medicine in 1934 for these discoveries. 4 E.J. Cohn and his associates started fractionating5 liver using the responses of untreated human patients, each acting as his own control. Normal gastric juice was also administered orally. The food factor was called "extrinsic factor'' and the essential substance in gastric juice was called "intrinsic factor", which was unnecessary if large amounts of the food factorwereassimilated. Only slow progress was made to obtain purified material until the growth of Lactobacillus lactic
CYANOCORAI A M I N
185
Dorner6I7 was used to monitor the purification of the "anti-pernicious anemia" factor. In 1948 , this simplified analytical method enabled a group of investigators headed by K. Folkers8, at Merck & Company, to announce that they isolated from liver a red, crystalline compound, which, in microgram quantities, produced hematological remissions in patients with Addison's pernicious anemia. They named this new agent vitamin BIZ. Eight days later, E.L. Smithq'lo of Glaxo laboratories, independently reported on the purification of an anti-pernicious anemia factor in liver. A single dose of 3 to 6 ug produced hematological responses in patients with pernicious anemia. 1.2 Structure, Nomenclature and Molecular Weight
Cyanocobalamin is the United States adopted namell. The preferred chemical name12 is cobinamide cyanide phosphate 3'-ester with 5,6-dimethyl-l-aD-ribofuranosylbenzimidazole inner salt. other names include vitamin B 1 2 , 5,6-dimethylbenzimidazolyl cyanocobamide, and Co -[a-(5,6dimethylbenzimidazolyl)3 -Co -cyanocogamide. Trade names include Bevatine-12! Berubigen, Betalin-12 crystalline, a-Twelv-Oral Depinar, Dodecavite, Dodex, Endoglobin, Hepcovite, Normocytin, Poyamin, Rubramin PC, Sytobex, Vibalt, Vitron-C-Plus, ViTwel and Tulag. Cyanocobalamin 57C0 (CAS 13115-03-2 and 41559-38-0) and 6 o C o (CAS-13422-53-2) are diagnostic aids (also called Rubratope-57 and Rubratope 601, frequently used for the Schilling test; cf. section 5.5, Radioactive Methods of Analysis. The molecular weight is 1355.42 daltons; C63H88CON14014P. Vitamin B12 is given the chemical abstracts systematic number 68-19-9. Figure 1 shows the structure and numbering of cyanocobalamin and its most important analoque, coenzyme B12 [a-adenylcobamide coenzyme, a-5,6dimethyl benziminazoly1)-cobamide 5'-deoxyadenosylcobalamin and cobinamide coenzyme]. The corrin nucleus is shown darker. Corrinoid is a general term used for B12-group compounds. Bound to one
186
JOEL KIRSCHBAUM
33
Coenzyme form -CN is replaced by
Figure 1.
Cyanocobalamin (Vitamin B 1 2 ) The c o r r i n nucleus is shown darker.
CYANOCOBALAMIN
I87
carboxyl (position f, D ring) is Dg-l-amino-2propanol (isopropanolamine) which is esterified to the phosphate of 3'-mononucleotide. The sugar is D-ribofuranose, which is linked by an a glycoside rather than the usual B-linkage of nucleic acids. The 5,6-dimethylbenzimidazole moiety can be replaced by such organic bases as adenine (B12b), benzimidazole, 5-hydroxybenzimidazole, guanine, 2methyladenine (B12m), 21methylhypoxanthine and hypoxanghine, and by OH (hydroxocobalamin, B12a)r and NO2 (nitritocobalamin, B12C); cf. section 16. Reactions of Cyanocobalamin. Below is another representation of cyanocobalamin, showing the relationship of the corrin nucleus to the other groups, as modified from the frontispiece of reference 18.
188
JOEL KIRSCHBAUM
1.3
Appearance, Color, Odor and Precautions
Cyanocobalamin is a deep red, odorless, free flowing, crystalline powder. The color has been used to follow purification and to detect the compound. All corrinoids are beautifully colored compounds with colors varying from violet, red and yellow to blue, brown and green. Since the human dose is approximately 3 micrograms, care should be taken to avoid inhaling the powder, although there is no evidence that cyanocobalamin itself is toxic, even in large doses. Allergic type reactions have been reported only rarely, and it is not known whether those were related to the drug, the excipients, or neither. 1.4 Synthesis, Biosynthesis and Commercial Production
The synthesis of c anocobalamin was a joint and A. Eschenmoser 6. effort by R. B. WoodwardT4 It is shown in abbreviated form in Figure 2 . The cornerstone of the synthesis was the compound 8corrnorsterone (I) named by Woodward. It was made in a 37 step synthesis and contains 6 chiral centers. The Woodward-Hoffman rules of orbital symmetry for predicting the feasibility, stereochemistry and products of reactions originated with this work. After several other reactions, the resulting cyanobromide (TI), containing the A and D rings, was condensed with thiodextrolin (III), which contains the B and C rings, to form IV. Several steps later, the thiolactam IV was freed of sulfur via (V) concomittantly with a mixture of 8 diastereomers. High-pressure liquid chromatography was used at several steps to monitor and purify intermediates in the route to the cyclized, cobaltcontaining intermediate VI. Bridgehead structure was introduced stepwise to give VII, which, in several steps including preparative high-pressure liquid chromatography to separate two isomers, was converted to cobyric acid, VIII, containing two cyanide groups. The side chain was synthesized starting with D-ribose (IX), which, in several steps, was converted
IV
C N
%OM.
VI
Figure 2.
Selected Steps i n
tile
C i i e r r i c a l Synthesis of C y a n o c o b a l a m i r i .
190
JOEL KIRSCHBAUM
to a a-ribazole-3-phosphate (XI. Following conversion to the D-1-amino-2-propanol ester (XI), condensation with cobyric acid(VII1) yields cyanocobalamin. Details of this synthesis have been The biosynthesis of cyanocobalamin begins with 6-aminolevulinic acid (Figure 3, I)19, which is also the precursor of the porphyrins, chlorophyll, heme and the cytochromes. These pigments are metal complexes involved in oxygen, electron or redox reactions. I was enzymatically converted to porphobilinogen, 11. A deaminase condenses 4 moles of I1 to form the linear molecule bilane I11 was converted to uro'gen(III), where X=NH3+. I11 (IV). The order of assembly was investigated using NMR, 111, IV, and the deaminase partially saturated with 13C-II. Apparently, the first unit20 of I1 to bind to the enzyme became ring A, the second unit became ring B, the third C and the final one, D. Following cyclization to IV, ring D rearranged to dihydrosirohydrochlorin, V. (An alternative pathway from IV leads to protoporphyrin IX, and the porphyrins). The methyl groups are from S-adenosylmethionine. Further methylation2I gave VI, which subsequently lost the methylene group at C-2.0, as the carboxyl carbon of acetic Decarboxylaacid,22 to form cobyrinic acid (VII) tion at C-12 also occured prior to cobyrinic acid formation. Cobyrinic acid, the biological precursor of cyanocobalamin, is closely related to cobyric acid (Figure 2, UrII) which was a key intermediate in the chemical synthesis of vitamin B 1 2 .
I,-Threonine is the precursor of (R)-1-amino2-propanol, from studies using labeled amino acid. The 5,6-dimethylbenzimidazole moiety and riboflavin appear to have a similar biogenetic source. There also exist pathways in many organisms to convert one corrinoid into another by a net exchange of free and bound base.23 It has been suggested24 that vitamin B12 is an artifact and the naturally occuring, interconvertible form is the related coenzyme, adenosylcobalamin. Cyanocobalamin is produced by a large number of diverse microorganisms.25i26 The vitamin is usually adsorbed on charcoal, calcium montmorillo-
F i g u r e 3.
S e l e c t e d S t e p s i n the B i o s y n t h e s i s of Cyanocobalamin.
A1
IIA
IA
I1
192
JOEL KIRSCHBAUM
nite (Fuller's Earth), sodium montmorillonite (bentonite) or resins; eluted, purified by extraction, and precipitated as the copper- or zinccyanocobalamin complexes. Recrystallization is often from acetone. With commercial improvements of yields, the price dropped from about $800 to $18 a gram. A brief summary of patents is also included in reference 12. For formulation into animal feed and vitamin supplements, cyanocobalamin is available as low, medium or high concentrates, as crystals and adsorbed on such carriers as resins (cf. section 6.2 , Stabilization)
1.5
Nutrition, Physiology and Medicine
Occurrence in food has been summarized.27 Because plants lack corrinoids, either vitamin B12, or related compounds, must be obtained by animals from intestinal bacteria, milk, meat, fish or voluntary or involuntary coprophagy. Human daily dietary allowances28 in pg are: Infants to 1 year, 1.5; children 3 ; males 3 ; f.emales 3 , and pregnant or lactating females, 4. Much of the human requirement is met by intestinal bacterial synthesis. In lower animals, the requirement for cyanocobalamin depends on the species, and is about 2 to 20 pg per kg of diet. Once ingested by man and other mammals, dietary cyanocobalamin is bound by intrinsic factor and R binder proteins which are located in the gastric and intestinal juices.29 The intrinsic factor-cyanocobalamin complex appears to join, at neutral pH and with calcium, to a third protein in the small intestine, membrane-bound intrinsic receptor. Cyanocobalamin is transported in blood attached to a fourth protein, transcobalamin I1 and a binder, transcobalamin I. The binding capacity is determined by the number of such sites. The binding capacity measurements have been used diagnostically to differentiate between different diseases, such as leukemia and nonleukemic leukocytosis. In microorganisms, binding of cyanocobalamin initially is to the outer membrane. Transf,er into the interior of the cell requires a proton motive force and, perhaps, an additional carrier.
CYANOCOBALAMIN
193
In humans, cobalamin content of normal tissues, in ng/g are as follows,3o with the percentage of cyanocobalamin in parenthesis; liver, 1050 (0%); kidney, 134 (0%); spleen, 63 (0%); brain, 8 1 ( 0 % ) ; pituitary, 230 (0%); bone marrow, 13 ( 2 % ) , leucocytes, 4 ( 4 % ) ; plasma, 0.4 ( 2 % ) ; erythrocytes, 0.2 (6%); bile, 18 ( 7 % ) and cerebrospinal fluid, 0.02 (10%). The rest of the cobalamins are the methyladenosyl- or hydroxo- forms, as determined by chromatography and bioautography. Vegetarians, as expected, frequently show abnormally low concentrations of cobalamins in their body fluids, as do some individuals ingesting megadoses (more than 2 grams a day) of vitamin C . Other conditions and diseases producing low concentrations in human serum include malabsorption because of pregnancy, iron deficiency, primary folate deficiency, cancer (especially multiple myeloma), aplastic anemia, hereditary absence of transcobalamin I and use of oral contraceptives. In lower animals, cobalt may be deficient in the soil of the region, and lead to vitamin B 1 2 deficiencies unless supplemented. Deficiency symptoms in man31 include pernicious anemia, which results from a diminution in the reduction of ribonucleotides to deoxyribonucleotides. Large red blood cells may form which contain immature nuclei. The spinal cord may degenerate. Insufficient cyanocobalamin is often indicated by the excretion of methylmalonate into the urine, due to impaired conversion to succinyl coenzyme A ( c f . Section 1.62, Reactions, I n V i v o ) . In lower animals,32 deprivation of cyanocobalamin is first seen in the young by poor growth and then in all animals by fewer red blood cells, less plasma protein, higher non-protein nitrogen values in serum, less glucose in serum and, eventually, by higher mortality rates. Typically, patients testing below 200 ng cyanocobalamin per liter (148 pmol/L) are retested for vitamin B 1 2 and folate.33 Treatment usually includes cyanocobalamin, with or without added folic acid.
194
JOEL KIRSCHBAUM
1.6
Reactions of Cyanocobalamin
1.61
In V i t r o
The cyanide group in vitamin B can be removed by photolysis or reduction3d2to give aquocobalamin (H~O-CO:~) which, in turn, can react with various acids to form the bromide, chloride, cyanate, nitrate, sulfate and thiocyanate. A s expected, cyanide can reverse these reactions to form vitamin B12. Potassium cyanide added to aqueous cyanocobalamin displaces the nucleotide giving the purple dicyanide. The addition of acids, results in the expected hydrolysis of amide, ester and glycosylamine bonds, as well as protonation (cf. Section 4.4, Ionization). Cyanocobalamin reacts with chlorine in mild acid to form a lactone [8-hydroxy-a(5,6-dimethylbenzimidazolyl) cobamic acid a b d e g pentamide-c-lactone]. Reduction (cf. Section 5.42, Polarography) of vitamin B12 [cob(III)alaminl , by such compounds as cysteine, results in the brownorange to gray-green intermediate cob(I1)alamin or B12r. Biological reductants appear to be thiol or flavin compounds. Further reduction of Bizr gives Bizs or cob(1)alamin. B12r can disproportionate to B12s and aquo-B12. B12s in aqueous acid decomposes to cob(I1)alamin and molecular hydrogen. B 1 2 S is one of the most powerful nucleophiles known.35 It reacts with BrCN to give B12-CNI with H3CI or dimethylsulfate to form B12-CH3, with Br-CzCH to yield B12-CzCH, with HCzCH or H2C=CHBr to produce B12-CH=CH2, with ethylene oxide or C1CH2-CH20H to give BI2-CH2-CH20H, with ethyleneimine to yield B12-CH2-CH2-NH2, with H2-C=CH-C02H to give B12-CH2-CH2-CO2H and with acetyl chloride or acetic anhydride to give B ~ z - C O - C H ~ . ~ ~ Immobilized derivatives of cobalamins have been prepared37 and used to purify
CYANOCOBALAMIN
195
cobalamin-dependent enzymes and binding proteins.

1.62
I n Viuo
Vitamin B 1 2 is converted to the orangeyellow coenzyme by replacement of the cyanide group, attached to the central cobalt atom, by 5'-deoxyadenosine. Reactions involving coenzyme B I 2 can be generalized to be of the type shown below, with the exception H X X H
of the nucleotide reductase conversion. A hydrogen migrates from one carbon atom to an adjacent one with the concomitant movement of an acyl, alkyl or electronegative group(X) from the adjacent carbon atom to the one to which the hydrogen was originally bound.38 The less branched structure has the greater thermodynamic stability.39
Reactions involving methylcorrinoid as the intermediate, as shown below, appear to also require folic acid (pteroylglutamic acid) in the form of tetrahydrofolate.
TTransme t h y l a tio n
I methy 1 , N5-methyl-H4-folate+HS-CH~-CH2-CH-COzH \trans ferase homocysteine

H4-folate N5-methyl-H4-folate N5-methyl-H4-folate
H3C-S-CH2-CH-CO2H methionine methane acetate
> -
> -.
196
TOEL KIRSCHBAUM
The reaction appears to involve a Co-CH3 complex as Curiously, en~yme-bound~ [14C]-methyl-B12 ~ is less light-sensitive than free [14Cl-methyl BI2. Methane can be formed by microorganisms from methanol, carbon dioxide, formate or and formaldehyde.
Carbon SkeZeton Rearrangements
4 3 2 1 methyl \ 4 3 1 H02C-CH2-CH2-CO-SCOA H02C-CH-CO-SCOA I malonyl Succinyl CoA mutase 2AH 3 Coenzyme A (SCoA) (R)-methylmalonyl-CoA
a-methyleneglutaric acid L-glutamic acid

CI 2
a-methylene \methylitaconic acid glutarate mutase

\
lutamate\ mutase
L-threo-B-methylaspartate
The reactions catalyzed by these enzymes are similar in that a substituent group is transferred between the c1 and B position of a propionate moiety, while a hydrogen atom is moved in the opposite These studies involved 14C-labeled compounds and
D2O.
H2C-CH2-CO2
-2
,*
H3C-CH-CO2
R
In such conditions as pernicious anemia, the conversion of (R)-methylmalonyl-CoA is diminished, and relatively large concentrations of methylmalonic acid are found in the urine (which may be used as an indicator of cyanocobalamin d e f i ~ i e n c y ~ ~ )This . reaction is the only coenzyme Bi2-dependent rearrangement47 occuring in higher animals and man as well as in bacteria. It enables propionyl-CoA,
CYANOCOBALAMIN
197
originating from branched fatty acids and amino acids,to be metabolized via the citric acid cycle.48
Amino Group M i g r a t i o n s
H2N-CH2-CH2-CH2-CH-CH2-CO2H
L- O-lysine
NH 2
L- B - lysine, mutase
L-erythro-3,5-diaminohexanoic acid Ethanolamine D-a-Lysine D-Ornithine
'
ethano1amine, deaminose
acetaldehyde + NH3
D-a -1ysine, mutase - D-2,5-diaminohexanoic acid
,mutase
ornithine,
D-thre0-2~4-diaminopentanoic acid
The exchange of the amino group and hydrogen between adjacent atoms in the ethanolamine deaminase (EC 4.3.1.7), lysine mutase and ornithine mutase reactions is similar to the previously-discussed carbon skeleton rearrangements. The migrating hydrogen is transferred stereospecifically from substrate to coenzyme. Tritium is transferred from B,, coenzyme specifically labelled in the 5'methylene position of the 5'-deoxyadenosyl moiety to acetaldehyde by ethanol deaminase, to 3,5-diaminohexanoate by B-lysine mutase, and to 2,5-diaminohexanoate by a-lysine mutase. Addition of substrate to ethanolamine deaminase-B12 coenzyme gives rise to electron spin resonance signals characteristic of a radical.49 The bound enzyme was found to be 95% in the form of cob(II)alamin.50
198
JOEL KIRSCHBAUM
Net R e d u c t i o n
I OH I OH I H
ribonucleoside, -CH2-O-triphosphateT triphosphate reI ductase H
ribonucleoside triphosphate Base- -C-C-C-CH2-0-triphosphate H H I H OH deoxyribonucleoside triphosphate propanediol, propionaldehyde + l12-Propanediol T\ dehydrase water glycerol\ 'dehydrase
B-hydroxypropionaldehyde water
F J
Diol dehydrase (EC 4.2.1.28) and glycerol dehydrase (EC 4.2.1.30) catalyze the transfer of a hydrogen from the C-1 position of the substrate to the C-2 position, while the hydroxyl qroup on C-2 moves to C - 1 of the product.5 This is analogous to the preceeding amino group rearrangements. Stereochemical results and cobalt involvement in these reactions have been discussed.52 Ribonucleotide reduction to 2'-deoxyribonucleotide is required for deoxyribonucleic acid synthesis and, eventually, growth and red blood cell production. The enzyme, ribonucleotide triphosphate reductase, replaces the hydroxyl group at C2' of a ribonucleotide by hydrogen in an SN1 type of reaction. The hydrogen was found to be carried by the B12 coenzyme and cobaltcoenzyme bond cleavage occurs.53 The source of the hydrogen was reduced nicotinamide adenine dinucleotide phosphate, which was transferred to a low molecular weight dithiol protein. Electron spin resonance studies show the presence of B12r signals, together with signals ascribable to organic freeradicals.54
CYANOCOBALAMIN
199
2.
Physical Properties of Crystalline Cyanocobalamin 2.01 Single Crystal X-Ray Diffraction
The three-dimentional structure was obtained originally by Dorothy Crowfoot Hodgkin and cow o r k e r ~ ~ ~by ' means ~ ~ ' ~ of~ single crystal X-ray diffraction. Partly as a result of this work, she was awarded the Nobel prize for Chemistry, in 1964. Crystals of cyanocobalamin were grown in water: Molecular weight 1752; probable formula, C63H88N14014Co*P-22H20. The structure is shown in Figure 4. The agreement factor, R, is 26%. The structure of B12-5'-phosphateI the biosynthetic precursor, was similar58 to that of B12 found by Hodgkin and coworkers.59 However, slight differences in rotations of the a-glycosidic bonds, two acetamide groups and two water molecules were apparent. The structure of the 5,6-dimethylbenzimidazolylcobamide coenzyme wag also determined60 to a spacing limit of 0.9 A. In aqueous solution, the average orientation of the deoxyadenosyl moiety is significantly different from that found in the crystalline state.61 The corrin macro-ring is thought to exist as photostable and photolabile isomers. Isomerization depends on pH and solvents, and is reversible. In an epimer62 at C-13, the propionamide group can orient itself up or down, following different puckering of the corrin ring. Little change was found to be apparent63 in molecular packing. The ratio of neo to normal form is 3 or 4 to 1. The structure of an impurity in commercially available cyanocobalamin was studied by X-ray crystallography.64 Base was lacking in what amounts to 0.08% of the usual form. Neutrons65 can be used to determine molecular structure similarly to X-rays. However, neutrons are difficult to generate, cobalt was seen as a "light" atom in the neutron Fourier diagram because all nuclei scatter neutrons within approximately the same order of magnitude, and hydrogen
200
JOEL KIRSCHRAUM
Figure 4. Stereoview of Cyanocobalamin, upper portion, lower portion, Enlargement Showing Position of the Atoms. (From Reference 58, by permission).
CYANOCOBALAMIN
20 1
cannot be neglected in the vector diagram, thus requiring more reflections. Because of these obstacles neutron structure elucidation is rarely used. The three-dimensional X-ray and neutron analyses of cyanocobalamin gave similar results.66 2.02 X-ray Powder Diffraction, Spectrometry and Activation Analysis Fi ure 5 is the powder X-ray diffraction pattern?., as obtained using a Philips powder diffraction unit emitting CuKa radiation at 1.54 8. With the aid of a scintillation counter detector, the sample was scanned and recorded from approximately 2 to 36 degrees ( 2 8 ) . The cobalt complex in cyanocobalamin was studied by X-ray photoelectron spectroscopy68, to give peak separation and binding energies. Bombardment by 8eV 3He gave rise to X-ray emissions which can be used to detect cobalt with a 1 to 10 ng limit of detection.69 2.03 Mass Spectrometry Because conventional mass spectroscopy techniques using volatilization induce thermal strain in cyanocobalamin, a gentler mode of ionization was used. Field desorption mass spectrometry, utilizing an electrostatic field of l o 7 to lo8 V/cm to cause but one electron to be lost, is ideal for the analysis of vitamin B12. Figure 6 is the field-desorption mass s ectrum70 of cyanocobalamin using laser heatingg1 to increase ionization efficiency and sensitivity. The molecular ion was apparent at m/e 1354.567. The ion at m/e 1295.530; elemental composition C61H 3N13013 COP, may originate from the loss of acetamiae. Dehydration may produce the ion at m/e 1318.546; elemental composition C63HS4N14Ol2C0P. The base peak at m/e 914.445 agrees with an ion with the elemental composition C45H 5Nl106Co, which may arise from the splitting op the nucleotide side chain.72 Com ounds related to cyanocobalamin and depletion of 73C during the biosynthesis of vitamin B1 were studied by this technique using Mass fragmentography is but 1 ug o ! $ material. discussed in Section 5.8.
\n
Qd
t-
'
3
'h
3 _,
I"
202
Figure 5.
Powder X-ray Diffraction Pattern of Cyanocobalamin. See text or details.

w
X
0
I
f ' 8
/E
0 1
LIP:
[I].
e,Z
203
204
JOEL KIRSCHBAUM
2.04 Magnetic Susceptibility Cyanocobalamin is in solution and the solid state. The molar susand the Bohr ceptibility, xm is -750+100 x magnetron ( v ) value77 i s approximately 1.21. An impurity present in some preparations, with a concentration <0.1%, is paramagnetic, and has lead to error in some determinations.77 The related aquo- and adenosylcobalamin are also diamagnetic. The triple charge of the cobalt is neutralized by the negative charges of the cyanide ion, one of the corrin ring nitrogens and the phosphate of the nucleotide. 2.05 Mossbauer Spectrometry Mossbauer spectra are characteristic of the highly conjugated nature78 of cyanocobalamin. A doublet was visible at 77OK using57 Co-labeled vitamin B12. Emission Mossbauer spectra of cyanocobalamin, with and without the 5,6-dimethylbenzimidazole base, were studied.79 The spectra may be explained on the basis of considerable delocalization of electrons onto the corrin ring. 2.06 Thermal Analysis From 2OoC to 14OoC, water was found to be lost in a single step. Visiblego during thermal gravimetric analysis, was a sharp symmetrial peak at 135OC. Between 14OOC and 145OC an approximately 2% l o s s in mass was correlated with removal of the cyanide group using ultraviolet and infrared spectrometry (loss of 2137 cm-' absorptidn), and quantitative determination of cyanide using a ion-selective electrode (cf. section 5). Four endothermic effects were found for the black colored residue at about 23OoC, 280C, 39OOC and 520OC. At approximately 235"C, the spectrum up to 1200 cm-l indicates cleavage of the bond between 5,6-dimethylbenzimidazole and the corrinoid system. At 8OO0C, the remaining blue-violet mixture of phosphorus and cobalt oxides, if calculated as P205 and C02O3 is equal to 11.3% of the initial cyanocobalamin content (theoretical value, 11.1%). The thermogravimetric weight loss appeared to be a zero order reaction with an activation energy of 26.7 2 1.9 kcal/mole.
CYANOCOBALAMIN
205
In another studys1 using a different preparation of cyanocobalamin, an endotherm occurred at 97OC, indicating variability of the vitamin B 1 2 since this peak is also ascribed to the loss of water. Endotherms at approximately 24OOC and 25OOC were accompanied by melting and decomposition, precluding analysis of purity by differential scanning calorimetry. Following the U.S.P. procedures2 for class 1A compounds, the melting range of cyanocobalamin was determined. Decomposition started at 2 0 5 O C and appeared to end at 209OC, in good agreement with the Merck index12 value of darkening of the crystals at 21OOC.
2.07
Microscopy and Crystal Type
Microscopically,81 three sizes of particles are visible: 10 x l o p , 1 5 0 x 15011 and 500 x 50011, in a commercial preparation of cyanocobalamin. The largest particles are not agglomerated. For good crystals the refractive indices are CY = 1 . 6 1 6 , B = 1 . 6 5 2 and y = 1 . 6 4 4 . The system is orthorhombic and the crystal habit prismatic. When crystals of vitamin B 1 2 are grown in water,57 the unit cell constants are; a = 2 5 . 3 3 1, b = 2 2 . 3 2 A and c = 1 5 . 9 2 1, space group P 2 1 2 1 2 1 ; n = 4 ; density 1 . 2 9 (measured in a wet mixture of acetone and bromobenzene; calculated value 1 . 2 9 2 ) . Here, the crystals measured 0.1 to 0 . 4 mm in each dimension, with well defined faces, usually (110) and (1111. Cyanocobalamin was imaged usiilg field ion microscopy.83 The vitamin was added to platinum, which was electrodeposited on the tungsten specimen tip prior to the gradual removal of surface layers by controlled field evaporation. A few two-fold symmetrical patterns are visible.
2 . 0 8 Surface Area
A s measured by gas adsorption,81 the surface area of one commercial preparation of cyanocobalamin is 0 . 5 7 m2/g.
206
JOEL KIRSCHBAUM
2.09 Hydration The number of molecules of water per mole of water-grown crystals of cyanocobalamin is 22; 18 are present per molecule of air-dried vitamin.57 The unit cell contains water in channels, at such a high concentration that single crystal X-ray studies show that these pools of water may have some freedom of movement. Elemental and vapor phase chromatographic analyses of a commercial preparation (cf. section 5.1) gave values of approximately 5 moles of water per mole of tiitamin B12; indicating variability of the water content.
2.10 Polymorphism
Different crystal forms of cyanocobalamin are likely to exist since X-ray crystallography studies show that contacts between invidual molecules can vary, especially if the water content varies. In addition, the corrins can exist as interconvertible isomers6 1 (cf section 2.1) . 3. Spectrometry of Cyanocobalamin in Solution
3.1
Nuclear Magnetic Resonance Spectrometry (.NMR) 3.11

1
H-NMR
Figure 7 is the 360 MHz spectrum of 10 mg cyanocobalamin per mL deuterium oxide, pH 6, at 25O. The assignment^^^ for the chemical shifts (6) in ppm, relative to 2,2-dimethyl-2-silapentane sulfonic acid measured at 360 MHz, and the longitudinal relaxation rate (Ti), measured at 100 MHz, are given below. The location of the atoms is shown in Figure 1. Assignment 0.448 1.187 1.253 (doublet = 6Hz) JIH
1.380
55 144 195 132 116 203 144
c-20
c-47 methylene (1: of isopropanolamine C-35 and C-53 and B-11 B-10 and B-11
B-10
1.863 2.253 2.536 2.570
2 PPM
7.5
7.0
6.5
6.0
PPM
Figure 7.
Low Field (6.0-7.5) and High Field (0-4.5) portions of the 360 MHz IH-NMR Spectrum of Cyanocobalamin, courtesy of A. Nath.
208
JOEL, KIRSCHBAUM
Assignment 6.081 6.349 (doublet) 6.503 7.081 7.275 205 239 294 298 335 c-10 R- 1 B-4 B-7 B-2
These results agree with the 220 MHz assignm e n t ~ ~ ~ ' The ~ ~ spectrum ' ~ ~ ~ was ~ found ~ . to be dependent on pH from 2 to 9 (cf. Ionization, section 4.4).
3.12 13C-NMR
High-resolution Fourier transform NMR at 15.08 MHz was used to observe the proton-decoupled, natural-abundance 13C spectrum of 0.024 M cyanocobalamin in Figure 8. Assignments were made using splittings arising from 13C-31P coupling, chemical shift comparisons, off-resonance single frequency proton decoupling and partially-relaxed Fourier transform spectra. See Figure 1 for the locations of the carbon atoms. Assignment Corrin ring carbons 5 15 10 1 19 2 Chemical Shift 85.9 89.3 98.5 108.1 118.3 134.1 137.5 139.3 142.0 145.1 145.9
;I
13
121 17 18 a-Ribazole and isopropanolamine carbons 2 51.6 9 56.7 58.4 8 5 60.5 6 63.5 4 76.9 7 82.0
22
47
151
1 6
Figure 8.
MHz.
13C-NMR Fourier Transform Spectrum of 0.024M Cyanocobalamin at 15.08 A, Normal Spectrum of upfield portion of cyanocobalamin at 6 1 O using 4096 points in the time domain and 16,284 scans, B , partially relaxed Fourier transform spectrum, ~=1.02,to determine nonprotonated carbons of the corrin ring, and C, completely proton decoupled I 3 C spectrum. See reference 89 and text for details.
210
JOEL KIRSCHBAUM
Assignment
Chemical Shift 106.2 111.2 120.0 120.3 132.5 124.3 147.8
1' 4' 2' 3' 5' CH (doublet) CH2
13C-NMR spectra have also been obtained for related compounds.89'90'91 3.13 15N-NMR Although 15N produces sharp resonances because of its spin of 1/2, its NMR sensitivity is 1.04 x that of the proton. In addition, it is less abundant (0.37%), giving a sensitivity of that of the proton. These difficulties 3.8 x can be partially overcome by placing large sample volumes, 15-30 mL, in 2 5 - w . diameter tubes. A spectrum at 18.25 M H z , using a Bruker WH-180 Fourier-transform, superconducting spectrometer, of cyanocobalamin enriched (2.4%) in 15N showed seven resonancesg2 at 256.8 to 268.2 ppm. The external shift reference is 0.1 M DI5N03 in deuterium (D) oxide. These amide nitrogens resonate in the same region as other large biological molecules. The ring nitrogens were not observed. Perhaps the lack of signal intensity was due to coupling to cobalt, nuclear Overhauser effects and long relaxation times. 3.14 31P-NMR A Nicolet NTC 150 widebore superconducting spectrometer was operated at 60.7 MHz. The external chemical shift reference was 85% phosphoric acid (0.0 Hz). At a concentration of 0.0012MI the chemical shift was 4.79 Hzg3 with a line width of 5 . 7 Hz at pH 8.1. 3.2 Electron Spin Resonance Spectrometry
Lack of electron spin resonance (ESR) signals both in frozen solution and the crystalline solidq4 demonstrate that the central cobalt atom in cyanocobalamin is trivalent and diamagnetic. ESR has
CYANOCOBALAMIN
21 1
been applied87 to the study of vitamin B12-dependent enzymes and to derivatives of cyanocobalaminq5 (cf Section 1.42, reactions of cyanocobalamin, i n v i v o ) characteristics of these spectra are g 1 = 2.25, gl1=2.003, AllCo= 196 + 2 x 10-4 cm-l cm-l, with the hyperand A , , = 15.8 + 0.5 x fine and superhyperfine splitting sensitive to the nature of the axial ligands.96 Cob(I1)alami.n is paramagnetic and cob(I)alamin, as expected, is diamagnetic. 3.3 Infrared Spectrometry
Figure 9 shows the infrared spectrum of cyanocobalamin recorded as a KBr pellet using a Perkin-Elmer infrared spectrophotometer. Below are the interpretations of the various absorbance~. Absorption (cm-l) 3200 broad 2950 2060 1660 1570 1490 1060 broad Assignment N-H
C-N
0 -H
cEN
c
Raman Spectrometry
c=c
PO4
C=N -
c=o
Infrared spectra of cyanocobalamin and such related compounds as adenosylcobalamin and aquocobalamin were compared in another study.98 The spectra closely resemble each other. 3.4
Raman spectra of aqueous solutions of cyanocobalamin at concentrations of %lo-4 M were obtained using Ar+ and He/Ne lasers. The intensity of a strong band at 1502-1504 cm-l depends on the wavelength of the exciting radiation used,99r100 confirming that these are resonance Raman spectra, and are due to the corrin ring.lol A study of vitamin 312 and related compounds indicatedlo2 that the ~ 1 5 0 0 cm- band was due to T - V * transitions. The conformation of the corrin ring and
31dWVS
Figure 9.
33NVBIOSBV
l n
Infrared Spectrum of Zyanocobalamin. See text for details.
CYANOCOBALAMIN
213
the degree of coordination of the cobalt atom was also studied using Ar and Kr lasers.lo3 The effect on the spectra of other water-soluble vitamins was studied.lo4 Only riboflavin had a significant effect on the 1504 cm-1 signal.
3.5
Absorption (Visible and Ultraviolet Spectrometry)
The figure below shows the absorption specand 77OK(---) of cyanocabatrum at 298OK (-) lamin in ethanol. These spectra, countesy of P . S. Song,lo5 illustrate the classical a , B and y "Soret" bands characteristic of porphyrins and chlorophylls. The electronic spectra of corrins originate primarily in the 14 ?r-electron system of the conjugated corrin and secondarily in the cobalt 1igand.lo6'lo7 The cyanide group above the plane of the corrin ring perturbs the Ti-electron system.lo8 Changes in the oxidation state of the central metal are accompanied by considerable changes in the spectrum. The spectrum varies with solvent (as shown below),log pH84 and temperature*109~110
0.:
Of
0 . :
w
a m
so4
0 . :
Q
0 . 2
0. I
0
214
JOEL KIRSCHBAUM
Solvent Dimethylsulfoxide Water Me thano1 Ethanol(l:4) 3.6
Absorption (A) nm 360

5 19
Absorbance Relative to X band (a360nm) 1.00

0.29
546 360 360 518

547
0.33
518 549
1.00 0.27
0.30
1.00 0.28
0.31
Optical Rotatory Dispersion and Circular Dichroism Spectrometry.
Cyanocobalamin has 1 5 asymmetric carbons in addition to asymmetry around the cobalt atom. The figure below shows the optical rotatory spectrum of 0 . 0 5 % aqueous vitamin B12, modified from reference 111.
400
500
WAVELENGTH. IN
600
700
mp
Such a major change as substitution of cyanide by water coordinating to the cobalt atom results in only minor spectral differences, indicating a limitation of this technique. Spectral differences exist between the Co(II1) and the Co(I1) forms of cyanocobalamin. Circular dichroism studies show a marked solvent effect,l09 as shown on the next page.
CYANOCOBALAMIN
215
Solvent Dimethylsulfoxide Water MethanolEthanol (1:4)
Wavelength ( A ) nm 310 519 360 518 360 518
Dichroism ( A F ) -6.0 +11.5 -8.6 +15.1 -8.9 +13.3
The spectrum obtained at room temperature is inverted at the approximately -180O temperature of liquid nitrogen, indicating a change in conformation ( A H = %2-3 Kcal). 3.7 Fluorescence Spectrometry
Although c anocobalamin has little natural fluorescence, I Y fluorescence can be induced by photolysis or cyanolysis of the carbon-cobalt bond (cryptofluorescence). Such a technique, coupled with sensitive, low-noise microprocessor circuits should be useful in developing assays for cyanocobalamin. 4. Bulk Solution Properties 4.1 Intrinsic Dissolution Rate
The intrinsic dissolution rate was determined115 after compressing powder under 1500 PSIG using 3/8" diameter, disc-shaped dies. The surface area was 0.713 cm2. In one liter of water at room temperature, agitated at a rate of 50 rpm, the intrinsic dissolution rate of cyanocabalamin is 0.275 mg m i n . ' l cm2, using ultraviolet spectrometry at 361 nm (cf. section 5.2, spectrometry). 4.2 Solubilities in Aqueous and Non-Aqueous Solvents
Solubilities of cyanocobalamin were determined116 in various solvents at room temperature with about one minute of mixing. Results are reported using the U.S.P. definitions.l17
216
JOEL KIRSCHBAUM
Solvent Water Hydrochloric acid, 0.1M Sodium hydroxide, 0.1M Ace tone Ace tonitr i1 e Acetonitrile-water ( 1 :1 Chloroform Dimethylsulfoxide Ethanol Ethyl ether Hexanes Met hano1 Methanol-water (1 :1) n-Oc t anol Propylene glycol 4.3
Solubilitv Slightly soluble Slightly soluble Very Slightly soluble Very Slightly soluble Very Slightly soluble Slightly soluble Practically insoluble Slightly soluble Slightly soluble Slightly soluble Very Slightly soluble Slightly soluble Slightly soluble Practically insoluble Practically insoluble
Partition Coefficients
Cyanocabalamin was partitioned118 between hexanes and distilled water and between chloroform and distilled water at 2 2 ' . After mixing, the vitamin content of both phases was determined by spectrophotometry at the 361 nm maximum (cf. Section 5 . 2 , spectrophotometry). Blanks consisted of the solvent saturated with the other phase. The partition coefficient of hexanes/water was found to be 0.087 and for chloroform/water it was 0.023. At 210, partition coefficients119 for phenol/water (pH 6.2) was 0.055, and at 22', for butanol/water (pH 6.0) it was 0.045, using the maximum at 361 nm . Cyanocabalamin and hydroxocobalamin were determined120 on the basis of partitioning in benzyl alcohol and water ( l : l ) ,again using spectrophotometry at 361 nm. The partition coefficient in this system was 1 . 2 . Other cobalamins can be separated by counter-current distribution using the system.121 4.4 Ionization
Using the dependance of the proton-NMR chemical shift88 on pH, base atom B-2 (cf Section 1 . 2 for location of the atom) gave a pK of 3.28 + 0.04. This value is in excellent agreement with the previously-reported value122 of 3.3. pK values for cobalamins and cobinamides have been
CYANOCOBALAMIN
217
discussed.123 The limiting conductance of the cyanocobalamin ion is 33 mhos.124 4.5 Hydrodynamic Properties
The partial specific volume125 of cyanocobalamin at 25' was found to be 0.662 and the diffusion constant was 2.9 x 10-6 cm2 sec.-l Using these values, the calculated molecular weight is 1380, in excellent agreement, considering the errors of these techniques, with the theoretical M.W. of 1355 daltons. Using ultracentrifugation,lZ6 the diffusion constant was found to be 2.7 x 10-6 cm2 sec.-l and the sedi, ~ 0.54 x 10-13 s e e . mentation coefficient, ~ 2 0 was at 0.01%. At a concentration of 1%, S ~ O =, 0.6 ~ ~ x 10-13 sec. 5. Methods of Analysis 5.1 Compositional. Analysis 5.11 Elemental Analysis
The elemental analysislZ7 of cyanocobalamin is as follows:

E 1ement
Found 52.40 6.83 13.62 2.1
Theoretical 52.51 6.83 13.66 2.28
Carbon Hydrogen Nitrogen Phosphorus 5.12
Water Content
Based on an apparent molecular weight of 1443.95-1355.42 (actual) = 88.53 daltons, 4.9 molecules of water or 6.1% moisture were present in a commercial preparation. Vapor phase chromatography128 was used to analyze for water in a commercial preparation of cyanocobalamin. The vitamin was dissolved in pyridine and, after retention on a precolumn, water content of 5.7% was determined by comparison with external standards.
218
JOEL KIRSCHBAUM
5.13
Emission Analysis
Emission spectrochemical analysis was performed.129 A commercial preparation of cyanocobalamin was found to contain 3.9% cobalt, which, after correction for water, is 4.35% (4.39%, theoretical content). Phosphorus content is 2.3%, in excellent agreement with the results found by elemental analysis. Metallic impurities in pg/g were: zinc, 38; iron, <5; aluminum, <5; magnesium, <5, and calcium, 4. Emission spectroscopy has been used to determine the purity of vitamin B 1 2 at the 3435.5a cobalt analytical line.130 5.14 Atomic Absorption
More recently, atomic absorption methods have been used to determine cyanocobalamin in bulk131,in dosage f ~ r m s ~ and ~ ~in - dry ~ ~ ~ , feeds.136 Inorganic cobalt is usually the standard.136 Interferences by phosphate and other ions are minimized by nebulising the cobalt into the flame in a solution of 8hydroxyquinoline.137 The determination of cobalt in various forms in blood has been reviewed138and a standard method given. Blood and serum are oxidized with a mixture of nitric, perchloric and sulfuric acids. Cobalt is extracted from the aqueous ash using 1-nitroso-2-naphthol in chloroform prior to atomic ab~orptioq~analysis.The g. Recovery limit of detection is 10 of 5 7 C 0 is 99.8+0.1%. Prior to the assay of cyanocobalamin in tissues, enzymatic hydrolysis (proteolysis) is often useful. 5.15 Titration
An iodometric titration for cobalt in ashed cyanocobalamin is claimed to have an error of +0.5%.l4O A perchloric acid titration of cyanocobalamin in glacial acetic acid indicated at least six weakly basic groups.141
5.2
Identity, Spectrophotometric and Colorimetric Methods
CYANOCOBALAMIN
219
5.21
Identity Tests
Compendia1 methods14* involve (1) fusion of cyanocobalamin with potassium pyrosulfate and reaction with nitroso R salt solution, (2) digestion and acetous ammonium cyanate addition and (3) reaction with hypophosphorous acid, distillation and the addition of ferrous ammonium sulfate solution, sodium fluoride and acid. All of these methods yield strong colors. 1 4 3 A spectrophotometric identification test144 is based on the ratio of absorbance at 361 nm to 278 nm being between 1.70 and 1 . 9 0 , and the ratio A361/A550 being between 3.15 and 3.40. The maxima should be +1 nm at 361 and 278 nm, and +2 nm at 550 nm. 5.22 Spectrophotometric Methods
Aqueous solutions exhibit absorption maxima144 in the ultraviolet and visible regions (cf. Section 3.5). Using traditional nomenclature, at 278 nm & 1 nm, E (l%, 1 cm) = 115, at 361 nm ? 1 nm, E (l%, 1 cm) = 207, and at 548 nm k 3 nm, E (l%, 1 cm) = 63. A compendia1 assay145 is based on the comparison of the sample in water with the absorbance of an authentic standard at the peak maximum of 361 nm. Subdued light should be used since aqueous cyanocobalamin is converted by light to hydroxocobalamin, which has a lower absorptivity at 361 nm.144 Alternatively, the isosbestic for the two compounds or absolute ethanol can be used. Spectrophotometric d e t e r m i n a t i o n ~ lmust ~~ be compensated for variable hydration of various lots of cyanocobalamin. Vitamin B 1 2 has been determined in the presence of light-scattering c o m p ~ n e n t s l ~ ~ , hydroxocobalamin148~14q, related corrinid^^^^-^^^, other ~ i t a m i n ~ ~ and ~ ~ r ~ ~ ~ biological constituents.159 Cyanocobalamin has also been extracted with 0.025% sodium nitrite prior to spectroscopy160,or with benzyl alcohol prior to determination as the dicyanide.161 A differential assay in
220
JOEL KIRSCHBAUM
acidic and basic solution was developed.162 The infrared absorption band of the cyano-group at 2137 cm has been used to quantitate cyanocobalamin. Using a KBr pellet163,the error is * 5 % . In benzyl alcoho1164 the error is 52%. The fluorescence of the 5,6-dimethylbenzimidazole moiety, after acid hydrolysis and extraction into organic solvents, has been used to determine the c anocobalamin content of pure solutions.1 6 Y , 1 6 6 , 1 6 7 Excitation at 275 nm and emission at 305 nm has been used168 to quantitate the vitamin at concentrations of 0.1 pg/ml. 5.23 Analysis by Chemical Reaction of a Functional Grcup 5.231 Cobalt
Tabulated below are selected methods for quantitating cyanocobalamin based on cobalt content. Reference 169 170 171 172 173 174
175
Principle and Comments Colorimetry, linearity 2 to 20 vg Co/ml N-Bromosuccinimide, titration, 99.2% Accuracy Colorimetry, selective EDTA complex, 412 and 418 nm maxima Colorimetry, selective Complex, gas-liquid chromatography Complex, spectrophotometry Chemiluminescence, detection to 2x10 Chemiluminescence, 0.07 ug Co/mL Complex, colorimetry, many interferences
-9
176 177
178
CYANOCOBALAMIN
221 pg
179
180
Complex, linearity 0.23-4.5 Complex, poor sensitivity
Co/mL
181 182 183 184 ,185

186
{
Colorimetry nitroso-R salt, 100 to 600 ug B I 2 quantitated Colorimetry, kinetic quantitation Ozonization, complex with EDTA Ozonization, 13% error Complex, 5% error Complex, linearity 1-15 pg/mL Complex, linear response at 570 nm 5.232 Cyanide
187 188 189
Cyanocobalamin is 1.92% cyanide. Summarized below are selected assays based o n cyanide content. Reference Principle and Comments 190 Complex, relative standard deviation of
2%
191 192,193 194 195 196

197
Irradiation, complex, and colorimetry Automated, recovery of 94-103% Complex, error of 4% Complex with 2 agents, Recovery of Microdiffusion and colorimetry Complex, spot test 5.233 Other Functional Groups
%loo%
The Kuhn-Roth method for oxidizing C-methyl groups with chromic acid in sulfuric acid, reducing excess reagent with hydrazine
222
JOEL KIRSCHBAUM
and adjusting the pH to liberate acetic acid has been used198 to analyze for cyanocobalamin. The approximately 50 pg of acetic acid formed was determined by chromatography rather than the usual titration. Vitamin B 1 2 has been quantitated based on the phosphate content after decomposition of the vitamin as phosphomolybdate.199 The molybdate can be determined either colorimetrically as the thiocyanate (sensitivity: 0.01 pg P/mL or 0.43 vg cyanocobalamin/mL) or polarographically in nitric acid-ammonium nitrate solution (sensitivity 0.01-0.02 vg P/ml). The hydrolysis of vitamin B 1 2 in hydrochloric acid gives 5,6-dimethylbenzimidazole. This compound has been determined colorimetrically in a multistep reaction via 4,5-dimethyldibenzoyl-o-phenylenediamine (11) with acetylacetone.200 Alternatively, I1 can be reacted with alloxan to form 6,7-dimethylalloxazine, which fluoresces. Reproducibility of both methods is approximately ?2%. 5.3 Chromatographic and Other Separation Analyses
5.31
High-pressure Liquid Chromatography

(HPLC)
To this author, the preferred methods are chromatographic since retention time usuaPly depends on the interactions of the molecule, via weak bonding forces, with the mobile and stationary phases. HPLC was used by Woodward and coworkers in 1971 to purify intermediates in the synthesis of cyanocobalamin and to determine the purity of synthetic vitamin B 1 2 using normal phase chromatography201 (cf. Introduction). The preparative column was 240 x 2.3 cm and packed with 37-80 v silica gel particles. The mobile phase was hexaneisopropanol-methanol ( 5 : 2 : 1 ) flowing at 34 mL/min. The size of the injected sample was 5 9. Smaller samples were analyzed on a column 180 by 0.2 cm, inner diameter, containing 37 p silica. Better
CYANOCOBALAMIN
223
resolution of the various components can be achieved presently using 5 or 10 LI columns. HPLC procedures are summarized below. Analyte or Matrix Bulk
Other
Reference 202
Parameters and Comments Octadecylsilane (ODS) column, aqueous 12% acetonitrile (0.7 mL/min.), W 5 1-1 O D s , 3 7 % methanol-0.04 M tartarate-disodium hydrogen phosphate buffer, pH 3 . 0 ( 0 . 8 3 mL/min.), 254 nm
O D s ; gradient, 0.05 M
203
Ana1ogues
204
Cobalamins
sodium acetate (pH 4.0) or 0.05 M sodium dihydrogen phosphate and methanol (1.8 mL/min) 254 nm. Octylsilane, 3 0 % acetonitrile-70% water (2 mL/ min) or gradient; 0 . 0 8 3 M phosphoric acid (pH 3 . 3 ) with triethanolamine and acetonitrile from 10 to 25% in 10 min. Ethylsilane, 40% (1% acetic acid in water)-60% methanol (1 mL/min), 360 nm .
O D s , 90% methanol-0.2% aqueous ammonia (2.6 mL/ min)
205
Cobalamins
206
Analogues
206
Analogues
207
Analogues
Cyanocobalamin is not resolved from methyl cobalamin, 360 nm.

ODs, gradient; 0.001 to
208
Vitamins
0.5 M potassium dihydrogen phosphate solution.
224
JOEL KIHSCHRAUM
209
B vitamins
ODS, 20% (1% citrate in water)-80% methanol (pH 7.3).
209
B vitamins
B vitamins
Amino column, 20% water80% methanol. Amino column, 95% methanol-5% water (1 mL/min. 266 nm. Amino; gradient, 0.005 iz! potassium dihydrogen phosphate-86.5 to 63% acetonitrile in 8.7 minutes. Octylsilane column, 0.005 M ammonium acetate-acetonitrile (85:15).
M ammonium chloride (pH 4) ,
)
210
211
Vitamins
211
F o l k acid and B 1 2
B vitamins
212
Cation exchange column, 2
1 mL/min.
213
Vitamins
O D s , 1% aqueous acetic acid-acetonitrile (9O:lO). 280 nm.
213
Vitamins
Extract into metabisulfite buffer, ODS column acetonitrile-0.005 M oxalic acid, pH 4.1 (15:85) 546 nm
214
Vitamins and Cobalamins
Amino column at 40, 0.005 M phosphate buffer (pH 4.35), acetonitrile (237 7 % , 4 mL/min, 220 nm. Low error of 1.2%, Hydroxocobalamin is not resolved. Reverse-phase column (no details given - ODS assumed), 0.01 M potassium dihydrogen phosphatemethanol (3:1), 0.5 mL/ min., 550 nm.
215
Sludge
CYANOCOBALAMIN
225
216
Veterinary formulation
Octylsilane; gradient, 1545% methanol-0.05% sulfuric acid, 1.3 mL/min., 350 nm. ODs; 50 mM sodium dihydrogen phosphate and methanol in combination of gradient and isocratic system. Extraction is necessary. 254 nm. Electrochemical detector (ECD). Disadvantage is inability to detect resolved compounds in the same oxidation state as that produced by the detector. Advantage is that the more sensitive ECD may be used to analyze cyanocobalamins in tissues and body fluids.
217
Corrins in vivo
218
Various
Figure 10 shows liquid chromatograms of cyanocobalamin separated from a mixture of vitamins (A) and from f o l k acid (B), taken from reference 211. The figure exemplifies the use of microprocessor controlled HPLC equipment. Sample and standard solutions, in vials, can be automatically injected into a chromatographic system using a mobile phase that can separate multiple components. The assay results can be displayed with parameters indicating the validity of the assay.
Sample size
10 ! I
Sample size 10 pI Column 25cm x 4 6 r n m i d LiChrosorb NH2, l O p m Mobile phase A 0 005 M KH2 PO4 B acetonitrile
time
'7'
ir*pT
I..
Niacinamide
! . .-0
8,
4 . :4
8,
5.5
Niacin
Pantothenicacid.
I .' L
1.
L.
' 0 ' 2
4.74
5.53
5.19
-.-I
..
E .
5L -0
..I
.*a
L:
1.0080 t *
Figure 10. High-pressure Chromatography of Cyanocobalamint A. Gradient analysis of water-soluble vitamins (standard solution) .
-. B
FLl7ld
5$ 1 1 L1 . NN P MA.( P MIIl F
--TEMP H c-TEMP 8 O'JEN T E N P
':I4
2.88 15.8
1.98
14.5 54
41
488 8 48 "5 48
Sample size 10 pI Column 2 5 c m x 4 6 mm I d
LiChrosorb' AP-8
10 pm
25 48
:
688
;GNL
278
IJAVL S:P SPD IEPrJ ATTM 27 APEA PEJ ELP SEN?

CHT
8.58
18.8 3
Mobile phase 0 005 M amonium acetate buffer/ acetonitrile (85115 )
3808
8.28
S:P
a) Standard Solution b) Solution of Tablet

8
4.BE
IJAVL
212 :
L3: rc .,
.14
Fdlc u l d
P=====4 . 7-6
a,,
'5 T 1 3F
hr
1038 B 15
BTL:
Figure 10. B. Isocratic analysis of folic acid and Courtesy of Hewlett-Packard Corp. vitamine B 1 2
228
JOEL KIRSCHBAUM
5.32
Thin-Layer Chromatography (TLC)
TLC can be used to separate and estimate or quantitate cyanocobalamin in various materials. The precision and accuracy of the method depend on the concentration, amount of "bound" vitamin B 1 2 , method of detection, and skill of the analyst to spot or streak, develop and assay the regions on the plate. For quantitation, scraping the plate and eluting the vitamin, followed by detection yield the best results. Table 1 summarizes TLC methods for cyanocobalamin. Toluene should be substituted for benzene for health reasons.
5.33
Paper Chromatography
Paper chromatography is presently used less frequently because of the superior resolving power, and ease of HPLC and TLC. In the past, it was used to determine the purity of cyanocobalamin256-261, to separate cyanocobalamin from related corrins262-268, and to separate vitamin B 1 2 from other vitamins269 and from such other substances as ~ o b a l t ~ m ~e ~ th -i ~ ~n ~i ~ ne ,~ n~ u~ ~, leotides~~~ and penicillin, using in this instance, a single thread of cotton.274 The chromatographic system must be protected from light to prevent cobaltcyanide cleavage.
5.34
Ion-Exchange Chromatography
Ion-exchange chromatography, generally using a column open at the top and filled with a resin, has been used to purify or determine the purity of c y a n ~ c o b a l a r n i n ~ ~to ~ -separate ~~~, cyanocobalamin from other corrins287-294,and to separate vitamin B 1 ? from other ~ i t a m i n s ~ ~ ~ and - ~other O ~ , constituents in 1iver3O7,3O8r309, serum310, neomycin311, feed rnixtures3l2,orange juice313, nucle~tides~ and ~ ~syrups316,317. r~~~ HPLC and TLC are generally faster and more convenient, precise and accurate.
5.35
Size-Exclusion Chromatography
Exclusion gels reject larger molecules and compounds from their interiors, while interacting and retarding the elution of smaller species.
Table 1: Support
Thin-Layer Chromatography of Cyanocobalamin Mobile Phase Detect ion Reference
CyanocobaZamin
Silica gel Silica gel with acetate Dextran gels and polyethylene micro particles Silica gel, cellulose Sodium silicate on Aluminum foil Glacial acetic acid-acetone-methanol -benzene (5:5: 20: 7 0 ) , or water Methanol-water (19:1) Visible, radioactive
360 nm
219 220 221
Methanol-water (19:l) Acetic acid-methanol (3:l)
Visible, o-toluidine
222 223
Table 1
TLC, continued
Support
C o r r in s
Mobile Phase
Detection
Reference
Silica gel Silica gel
Butanol-acetic acid-0.066 M KH2P04-methanol (4:2: 4: 1)

0.4% Pyridine, 3% phenol, 0.01% NaCN and 10% acetic acid, thymol saturated
361 nm Visible
224 225
f3
Alumina G Silica gel Silica gel or aluminum oxide Cellulose Alumina Cellulose
Acetic acid-H20-methanol-CHC13butanol (2:9:10:20: 5 0 ) Anhyd. acetic acid-H20-methanolCHC13-butanol(9:ll:5:10:25)
Bioautography Bioautography Visible
226 226 227 228 229,230 231
Methanol-2% aqueous KCN (19:l) 2-BuOH-0.1 M acetate buffer, p H 3.5-methanol (4:12:1), lower layer 2-BuOH-2-propanol-water (1:l:l) See-butyl alcohol-water (19:8)
Visible Visual
Table 1
YLC, continued
Support Cellulose Cellulose Alumina
Mobile Phase See-Butyl alcohol-acetic acid-water (1OO:l: 50) Butanol-acetic acid-water upper layer (9:1:15)
Detection Visual Visual 360 nm
Reference 231 231 232 233 234
Isobutyl alcohol-isopropyl alcoholwater (1-1.5 :1 :1) Butanol-methanol-water See-Butanol-NH3-water (30:30:15) (190:5:55)
2
N
silica gel Silica gel or Cellulose Silica gel Silica gel Silica gel
Visual Bioautography
Ethanol-water
(70:30) (48:48:4)
361,545 nm Radioactivity
235 236 237
Butanol-acetone-water
2-Butanol-2-propanol-waterconc. NH40H (50:50:50:1)
Table 1
TLC, continued Mobile Phase Acetic acid-methanol-water (1:6 0 : 140) n-Butanol-ethanol-water (10:3:7) plus 0.5% N H 4 0 H Detection Reference 237
238
Support Silica gel, s ilanized Microcrystalline cellulose
Radioactivity Visual
S e p a r a t i o n from o t h e r B v i t a m i n s a n d v i t a m i n C
c3
Silica gel Cellulose
w c3
Acetone-isopropyl alcohol12% aqueous ammonia Propanol-water (6:4) or propanol-ethyl acetateN H b O H (5:3:2) Methanol-water ( 9 5 : 5 ) Butanol-acetic acid-water (4:1:5) Propanol-ethyl acetate-water (2:1:4)
Colorimetry
239
254 nm
240
Silica gel Silica gel silica gel
241 242 242
Table 1
TLC, continued Mobile Phase Butanol-water-acetic acid (120:60:24)

Sec-Butanol-ethanol-water32% NH3 (50:30:19:1)
Support Silica gel Silica gel Silica gel
Detection Visual Visual Colorimetry
Reference 243 243 244
Ethanol-water (2:1)
f3
Cyanocobalamin i n Multivitamin FormuZations
Alumina Silica gel Silica gel
Acetate-acetic acid-methanolbenzene (1:1:4:14) Water-96% ethanol-2 (45:48: 0.2)

M
o-toluidine Visual Mu1tiple colorimetric reactions Visible C1-o-toluidine
245 246 247
HC1
Benzene-petroleum ether-acetic acid (35:65:1) Acetone-acetic acid-benzene1:14 :4) methanol (1: Water
Silica gel Silica gel
248 249
Table 1
TLC, continued
Support Silica gel
Mobile Phase Benzene-light petroleum-acetic acid (35:65:1)

6 solvent systems
Detect ion 5% ethanolic molybdophosphoric acid
Reference 250
Silica gel
B I Z in l i v e r
251
Silica gel
50% aqueous ethanol
0.1% KCN, acetic acid, 550, 525 nm
252
B,, in plasma
Cellulose and silica gel (3:l) Silica gel See-Butanol-ammonia water (75:2:25) BioautoWaPhY B ioauto graphy 253 254 255
2-Butanol-2-propanol-waterammonia (30:45 :25: 2)
CYANOCOBALAMIN
235
Sephadex resins have been used to separate cyanocobalamin from other c ~ r r i n s ~ ~ * from , ~ sea ~ ~ , water320, urine, plasma and hemodialysis plasma322, gastric juice323 and 1iver324,325. As with all chromatographic techniques, interference by other substances is possible; for example, sulfitocobalamin interferes326 with cyanocobalamin in an exclusion separation utilizing Sephadex C-25. Vitamin B 1 2 has been used as a marker to determine the elution volume corresponding to its molecular weight. 5.36 Other Methods of Separation, Including Adsorption, Ultrafiltration and Partitioning
Adsorption on charcoal has been used to purify cyanocobalamin from ~ e w e r a q e ~ * to ~, concentrate it from urine329, and, when coated on a molecular sieve, to separate vitamin B I 2 from (cf. Radioassays) serum3
Membrane ultrafiltration 3 3 1 , 3 3 2 r 3 3 and 3 dialysis tubing334r335 have been used to separate free from bound forms of cyanocobalamin. Cyanocobalamin has been separated from related corrins by simple (one-pass) partitioning.336r337 Multiple partitioning and counter-current distribufrom tion has been used to separate vitamin B , and rat erythrocytes 8 , other corrinsl from bacterial cells.339 E l e c t r o f l ~ t a t i o nhas ~~~ been used to purify cyanocobalamin. Benzoquinone and naphthoquinone, which combine with nitrogeneous compounds, have been used to complex341 vitamin The complex can be removed by carbon and the B1?. quinone regenerated. 5.4 Electrochemical Analysis 5.41 Electrophoresis
Paper electrophoresis was used to separate cyanocobalamin from related compounds342 in 2 M acetic acid for 16 hours at 270 V, using bioautoThe duration was qraphy to detect activit shortened to 1.5 hours26 T by using barbital buffers, p H 8.0 and 9,O.or to 3 hours using phosphate
236
JOEL KIRSCHBAUhl
buffer at pH 7. Various corrins344 in sheep rumen were released from their bound forms by hydrolysis and then separated from one another by highvolta e paper electrophoresis in dilute acetic acid.9 4 5 Sodium acetate, sodium borate and ammonium chloride electrolyte were used. to separate and identify 1 2 water-soluble vitamins.346 An isoelectric focusing347 separation has been described. Unfortunately, complexes may have formed between the vitamin and the ampholyte. The isoelectric point of cyanocobalamin is 1.5, as found by electrophoresis.348 5.42 Polarography and Related Techniques
Redox processes for cyanocobalamin have been investigated using p o l a r ~ q r a p h y ~ ~ coulo~"~~~, metry362,363,364, cyclic voltammetry365-368 and chronopotentiometry.369 Oscillopolarography has been used to identify vitamin B 1 2 in aqueous solution.370 Cyanocob(II1)alami.n is reduced to B12rr cob(II)alamin, with a half-wave potential of -0.63V. Further reduction gives B12SI cob(1)alamin (cf. section 1.6). The B12r-B12Scouple depends on the kinetics of the base-off/base-on as studied by cyclic voltammetry at various pH's. Vitamin BI2 may be regenerated by oxidation. Assays for cyanocobalamin and related compounds have been developed using alternating current polarography in dirnethylf~rmamide~~~ at concentrations of 0 . 1 to 0.5 mM. Cf. section 5.31 for a description of an HPLC separation using an electrochemical detector. 5.43 Determination by an Ion-Selective ) Electrode (Cyano
Bulk cyanocobalamin, B 1 2 in a multivitamin capsule and B 1 2 in a liver hydrolysate can be reduced with either ascorbic acid, tin(I1) chloride in hydrochloric acid, or calcium hypophosphite to quantitatively release hydrocyanic acid. Alternatively, hydrocyanic acid can be released photo500 watt lamps. The hydrocyanic chemically, acid is d e t e E E B : 9 7 at microgram concentrations using a cyanide-selective membrane electrode, with an error of 2 5 % . Cyanide can also be determined by titration with silver nitrate at the electrode.
CYANOCOBALAMIN
237
In a simpler procedure374, cyanocobalamin is irradiated at pH 3-4, and the released cyanide is quantitated with a cyanide-selective electrode at a pH of 12-13. Recoveries are 98-101% in the presence of other corrins, indicating specificity of the method. Similarly, vitamin B 1 ? can be determined in the presence of cobinamide and hydroxocobalamin.375 5.5 Radioassays 5.51 Introduction and Example Assays
Radioassays are rapid convenient, cheap and generally unaffected by the presence of antibiotics, antimetabolites and tran uilizers. Cyanocobalamin can be labeled with 52C0, 57Co, 58C0 and 6oCo by adding labeled cobalt chloride to the fermentation medium of microorganisms producing Countin efficiencies are ood to vitamin B 1 2 . very good for 56C0, 57C0 and 6oCo. For 8 7 C 0 , the counting efficiency varies with the type of counter used, but is very good for the scintillation type. The total liver absorbed in rad/u Curies are: 56C0, 1.2; 57C0, 0.3; 58C0, 0.6 and 6oCo, 8.3, indicating one reason for the popularity of 5 7 C 0 and the paucity of uses, in vivo, for 6 oco
The classic and most reliable radioassay is the Schilling t e ~ t ~ ~ which ~ , is ~ used ~ ~with , ~ borderline cases of B 1 2 deficiency; i.e. 0.2 ug B 1 2 / 1 0 0 ml plasma.380 The Schilling test is an assay in vivo which measures the extent to which cyanocobalamin can be absorbed through the terminal ilium by examining the absorption of a small quantity of radiolabeled vitamin B 1 2 competing with a large amount of nonradioactive cyanocobalamin for receptor. It is more a diagnostic technique measuring the ability of the body to absorb B 1 2 than an assay for cyanocobalamin. The determination of the tissue381 or body fluid content of cyanocobalamin requires the complete separation of B 1 2 from various binders, usually by hydrolysis of homogenized tissue in 0.5 M HC1 for 15 min. at 100' or by boiling in acetatecyanide buffer. In this binding type of assay,
238
JOEL KIRSCHRAUM
the cyanide converts other forms of B12 into cyancobalamin. A known quantity of labeled vitamin B 1 2 is added, the homogenate shaken or boiled, and a binder or "carrier" capable of binding 2/3 to 3/4 of the labeled cyanocobalamin is added. From the pool of mixed labeled and unlabeled vitamin B 12, the carrier will remove a fraction equal to the binding capacity of the factor.382 Bound cyanocobalamin is separated from free B 1 2 by various methods (cf. Section 5.53, Technical Aspects), including chromatography and coated charcoal. The charcoal is coated with a high molecular weight compound like hemoglobin, dextran or albumin. The coating insures that only small molecules will be adsorbed. (Hemoglobin-coated charcoal is prepared by suspending 5g of a pharmaceutical grade of charcoal in 100 ml of distilled water. Human red blood cells are washed with 0.9% sodium chloride solution, then hemolyzed with twice the volume of distilled water, and then 0.5 volume of toluene is added. After shaking and centrifuging, the bottom layer is collected and filtered. Hemoglobin solution equivalent to 0.25 g in 1 0 0 m L is added to the charcoal suspension and shaken. The capacity of the binder is determined and portions frozen.) The binder, a known amount of labeled cyanocobalamin and the unknown quantity of labeled B12, are allowed to react at room temperature for 30 min. After adding 2 mL of coated charcoal suspension, mixing, and centrifugation (or other separation, cf. Technical Aspects section), the supernatant radioactivity is determined. Pg B12 per ml
- x n
pg
' C O - B ~ ~(B g , - 1)
Where n = mL of serum assayed, B = net counts per minute of binder-concentrate control tube and B ' = net counts per minute of the tube with unknown serum. Simplified methods involve commercial kits.383 These convenient tests have reduced the reagent cost per assay of cyanocobalamin in body fluids to less than a dollar and the time to less than an hour. The general basis requires labeled cyanocobalamin to be mixed with the substrate, usually
CYANOCOBALAMIN
239
serum. Once labelled and unlabeled vitamin B 1 2 have equilibrated, an equal fraction of each form should be bound to a matrix selective for cyanocobalamin. (At least three distinct types of protein have an extremely high affinity for cyanocobalamin.384 Intrinsic factor (cf. Introduction,) a protein secreted bx the stomach, is extremely specific ( K = 6 x 1 0 M-). Transcobalamin 11, a polypeptide in plasma, binds cyanocobalamin (K % 3x1Ol1 M-I) and some analogues. The glycoprotein, R. protein, binds analogues385 as well as vitamin B I Z , and cannot be used in radiodilution assays because of the resulting erroneous values. Some analogues are inert, some may have cyanocobalamin After activity and some may inhibit vitamin B 1 2 . ) separation, the ratio of labeled to unlabeled cyanocobalamin is determined by some radioactive counting technique. From the initial concentration of labeled vitamin B 1 2 , the content of unknown cyanocobalamin is calculated. Unfortunately, the kit assa fails in 10% to 20% of the patients tested37y. The problem is that usually these patients either have very low B 1 2 levels or the B 1 2 is bound and is not released, requiring further testing using either microbiological assays or the Schilling radioassay, as previously mentioned.
A newer method is the competitive protein binding (CPB or radioimmunoassay) radioassay. It involves preparing a stable derivative of cyano87 13 88 ~ o b a l a m i n 3~ ~ ~ , suitable for eliciting a high-titer antiserum specific for vitamin B I 2 . Bound to a surface389 to increase the surface area and facilitate separation of free from bound cyanocobalamin, 1 ml of preparation can coat 20,000 polypropylene tubes. Sensitivity is 1 . 5 picoof cyanocobalamin per tube, with an grams390,388 interassay precision of 15% to 20%.
Other reviews of radioassay techniques are a~ailable~~l-~~~.

5.52
Radioassays for Cyanocobalamin in Various Materials
A selected list of radioassays in various matrices is tabulated below.
240
JOEL KIRSCHBAUM
Matrix Blood Blood Cells Cerebrospinal fluid Feces Feces Feces Feeds Food Food Food Liver Sea Water Serum Serum Serum Serum Serum Serum Serum Tissue Urine Urine Urine Vitamins Vitamins Total body Total body Total body
Reference 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425
Comment Sephadex-intrinsic factor binders Receptor-coated Red blood cells High sensitivity Double-tracer technique Also whole-body Also uses 51Cr 20 Assays/hr. Rigorous extractions 14c02 from B~~ Kit Recovery of 0.5 ug 2 5% Na2I4C03 uptake in diatoms
'
'
Kits, plasma values less than serum Plasma = serum values 584 Normal humans Microbiological I4CO2 Titration Parameters examined Spleen, brain & kidney Test renal function and plasma binding Also feces and liver Sensitive and specific ~ O C O - B ~ is ~ tracer And liver Used 2 counters Discusses body compartments
CYANOCOBALAMIN
24 1
5.53
Technical Aspects
The most crucial parameter of most radioassays using labeled cyanocobalamin to quantitate unknown vitamin B 1 2 is the agent used to bind426 cyanocobalamin. The National Committee for Clinical Laboratory Standards recently announced proposals based on recommendations by the Clinical Chemistry and Hematology advisory panels of the United States Food and Drug Administration (February, 1979). Manufacturers of B 1 2 kits must substantiate purity; for example, tests should be developed for such non-specific binders as R protein (that combine with analogues of cyanocobalamin and mask B 1 2 deficiencies and thus give falsely normal results).427 (The metabolic role of these analogues is unknown at this time, and may have physiological significance. In the future, these may have to be quantitated.) Intrinsic factor must be assayed for purity by such methods as insensitivity to ~ o b i n a m i d e up ~~ to ~ concentrations of 1 0 ng/ml serum, inhibition by anti-intrinsic factor blocking antibody or the intrinsic factor-cyanocobalamin complex should be greater than 95% precipitated by specific intrinsic factor precipitating antibody. The use of intrinsic factor427,429 as binding agent was discussed in the introductory radioassay section. Intrinsic factor has been immobilized430 on d e ~ t r a n ~ ~ l p01ysaccharide.s~~~ r ~ ~ ~ , and Sephad e ~ and ~ conjugated ~ ~ , to fluorescein.435 Some other binding agents are egg yolk436, magnetic particles containing fish saliva438, chronic myelogenous leukemia serum439, chicken serum44o, human serum441, oyster serum442, rabbit serum421, trout serum443, chicken serum on magnetized particles444, transcobalamin 1445, transcobalamin on S e p h a d e ~ and ~ ~a ~ cell wall protein.446 Methods of separating "free" from "bound" cyanocobalamin include affinity ~ h r o m a t o g r a p h y ~ ~ ~ , Section 5.51) and bentonite448, charcoal449 ( c charcoal coated with dextranl; or hemoglobin451,452, column c h r ~ m a t o g r a p h y ~ dialysis454, ~~, (DEAE)filtrati~n~~ diethylaminoethyl ~,~~~, c e l l u l o ~ e S ~e ~ p~h ~ a~ de ~~ and ~ ~,~ Sepha~ rOse.4601461
242
JOEL KIHSCHBAUM
Also investigated has been the effect on radioassays of a n t i ~ o a g u l a n t s ~ a ~s ~, c ~ r b a t e ~ ~ ~ , ~ autoclaving to destroy Bx2 binding proteins465, bile450, contaminants of other radioisotopes466 and p r ~ t e i n s ~ ~ ~ ~ ,' y +a ~n ~, i d e ~ ~ ~ pep~ ~ q ~ a ~ s ~ tides471 and tissue preparation.
Various radioassays have been compared with each other; for some methods there were negligible difference^^^^,^^'+^'+^^, however, for several other assays there were significant discrepancies.4761477 Combination radioassays for c anocobalamin and folate are a ~ a i l a b l eET80 . ~ Automated ~ ~ ~ ~ ~ and ~ ~ semi-automated systems have been d e ~ c r i b e d ~ ~ ~ , ~ ~ as have various methods of calculating results.483, 4 8 ' + 1 ' + ~ ~ Counting techniques have been investigated.486f487
5.6
Microbiological Assays
5.61
Introduction and Example Assay
The microbiological assay for cyanocobalamin is relatively simple, selective and sensitive but, depending on the organism, vary in specificity. This variation in selectivi;GxE8 is because the organisms contain enzymes to catalyze various transformations between analogs or release "bound" vitamin B12. Despite such other handicaps as the need for meticulously cleaned glassware, growth in blanks and non-linear responses, possibility of contamination, and poor response when some antibiotics and other drugs have been administered, the utility of the microbiological method makes it first choice for many applications. The compendia1 turbidimetric procedure489 has been modified to an automated assay. Such an automated assay as the AutoTurb ~ y ~ t e m ~ ~ limits many variables and has been successfully used for thousands of analyses. As described below, it serves both as an example of a rugged method and an aid in understanding the references that follow to related microbiological procedures. This assay measures cyanocobalamin by its effect in stimulating the rate and amount of growth of a vitamin B12-requiring LactobaciZZus Z e i c h m a n ~ ~ 4 9 2 in a turbidimetric assay system. It is applicable
CYANOCOBA1,AMIN
243
to bulk cyanocobalamin in various forms and pharmaceutical products. Grind or blend 1 to 10 tablets for a few seconds to a uniform powder. Autoclave one tablet weight (amount depends on potency) or 0.5 to 10 mg of bulk vitamin, or equivalent, for 5 min. at 121O in 200 ml of bisulfite buffer. Cool, dilute with bisulfite buffer to 4 ng/ml, filter and dilute 10fold with distilled water. Fill the tubes for the automatic system with samples and medium containing amino acids, vitamins and inorganic solids, or equivalent). Sterilize in 10 ml volumes for 20 min. at 1 2 1 ' . Cooled, medium-filled, tubes are inoculated with one drop of working inoculum. (The master culture is L. Zeichrnanii A.T.C.C. 7830.) A frozen working culture is prepared by inoculating medium with about 10% master culture 7 ' . and incubating it for about 16 hours at 3 Adjust the pH of the culture to 6.7-7 with sterile 2 M sodium hydroxide and dilute it to 7.5% with sterile glycerol. Two ml portions are frozen with dry ice and acetone, and stored at -75' until needed. When needed, 20 ml of inoculum medium are added at 37O to the frozen culture, the tube is thawed with gentle agitation and incubated at 3 7 ' for 24 hours. This working culture is diluted 1 to 100 with sterile saline prior to one drop inoculations of each Bl2 assay tube. Samples are analyzed in quadruplicate. Standards consist of 170-175 ng of U . S . P . reference standard, or equivalent (stored over silica gel at - 2 O O ) per ml of distilled water. Working solutions are dilutions of 1 : 1 0 0 0 , 1:500, 1:330, 1:250 and 1 : 2 0 0 in a 9 to 1 mixture of distilled water and bisulfite buffer. Quadruplicate assays are made also of the standards. Inoculated tubes are incubated at 37O for 1 8 to 36 hours, mixed and the turbidity of each rack of tubes is measured at 600 nm using an automatic reader. Values more than twice the relative standard deviation for the set are discarded, leaving three responses. If any remaining values exceed the statistical limit, all responses for that sample are discarded.
244
JOEL KIRSCHBAUM
The best linear fit of the standard responses is made; such as concentration v s . % transmission, or log %T, or l o g concentration u s . %T. One standard data point may be eliminated to achieve Sample correlation coefficients above -0.991. potencies are calculated using this standard plot and multiplied by dilution of the sample using a computer. Errors are less than k 5 % . Many other laboratories analyzing large numbers of samples for cyanocobalamin use related or automated methods.494 LactobaciZZus Z e i c h m a n n i i presently is the preferred organism.
5.62
Microbiological Assays for Cyanocobalamin in Various Materials
Selected representative methods for determining vitamin B 1 2 in various matrices are tabulated below. Matrix Amino acid Antibiotic Antibiotic Bile Blood Blood Blood Blood Blood Body fluids Bulk Corrins Corrins Feed Feed Feed Feed Reference
495 496 497
4 98
Comment with methionine with dihydrostreptomycin with tetracycline plus chromatography erythrocytes content in B12 deficiency micro method acidity for free B 1 2 using several organisms micro method plus chromatography plus chromatography with preservative with vitamin B6 in clover multiple constituents
499 500 5 01 502 503 504 505 506 507 508 509 510 511
CYANOCOBALAMIN
245
Matrix Feed Feed Feed Food Food Food Food Food Food Food Food Food Intrinsic factor Microbes Pharmaceuticals Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Tissue Tissue
Reference
512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 54 0 541 542
Comment plus other vitamins meat products comparison of methods sterilized in rice, etc. in fermented milk plus other vitamins obviated interferences in 86 heated foods various foods eggs milk binding to B 1 2 extract B 1 2 miscellaneous miscellaneous normal range many samples Pg B 1 2 total B 1 2 0.05-0.2 ml in anemia many assays total B 1 2 in anemia proteins precipitated proteins precipitated proteins precipitated liver extract
1 0 0 Pg B12/ml
246
JOEL KIRSCHBAUM
Matrix Tissue Tissue Tissue Urine Water Water Water Water Water Water Vitamins Vitamins Vitamins 5.63
Reference 543 544 545 546 547 548 549 550 551 552 490 553 554
Comment liver, + cyanide liver, + cyanide various species concentrated in ocean 0 . 1 - 1 pg BI2/ml fresh water 1 month assay many methods plus other vitamins multivitamins + minerals B vitamins C non-interference
Technical aspects
Automation has been used to achieve high rates of sample in the presence of 561 tetracycline559 and other Requirements for a successful automated system have been discussed.562 Autodilutors have been used to increase the number of samples analyzed.563 Data handling has been simplified by computerization.564 Computations used to evaluate the, microbiological results have been d i s cussed.565~566~567 The dose response curve has been studied.568-571 Cyanocobalamin has been assayed using the method of standard addition.572 of
growth inhibitors578, inoculum preservatives580, reducing salts582 and spectrophotometer sensitivity.583 Use of various types of plates has been studied.584r585,586 Methionine interferes587, giving lower results compared to turbidimetry.
antibiotic^^^^,^^^,^^^, cleanliness576, flask
Also studied has been the effect on the assay
CYANOCOBALAMIN
247
Other strains have been studied, including Azotobacter S U ~ S Escherichia ~ ~ ~ , coZi (50-1500 )589~590f59 1, Euglena gracilis (0.25-50 p )5925 y 8 I Ochromonas maZhamensis (50-800 pg) 599,700 and Poteriochromonas stipitata601, and their variants. The number in parentheses is the assay range. LactobaciZZus Zeichmannii (10-200 pg) is preferred489,490~602~6 especially 03 because of the short incubation times604 and its nutritional specificity.6051606 Comparative studies between strains include Tetrahymena us. Ochromonas607, LactobaciZZus and BaciZZus coZi us. Ochromonas608, and E. c o Z i , L. Zeichmannii and E. graciZis v s . 0. maZhamensis.609f
610
Turbidimetric methods have been investigated. Growth has been measured using a Coulter counter.614
6111612t613
5.7
Enzymatic Assays
Biological reactions are usually catalyzed by cyanocobalamin in the form of the coenzyme, where the cyanide group is replaced by 5-deoxyadenosine. Cf. the introduction. Methionine can be determined microbiolo ical1y6l5t6I6 or using [14C]methylmethionine617 using purified B12 methyltransferase containing 12.5 mumoles of cobalamin per mg of protein. Cyanocobalamin can catalyze this reaction converting homocysteine to methionine. Carbon skeleton rearrangements catalyzed by methylrnalonyl-C~A~~~ (E.C. 5.4.99.2) and amethyleneglutarate620 (E.C. 5.4.99.x) mutases can be used to assay for coenzyme BI2. Glutarate mutase (E.C. 5.4.99.11, however, requires a Co-5deoxyadenosyl derivative621, greatly increasing the difficulty of the assay. Ethylamine deaminase from CZostridium requires a-(adenyl)-Co-5-deoxyadenosylcobamide6z2 as coRelated
enzyme to catalyze amino group migrations. corrins can inhibit the enzyme.
248
JOEL KIRSCHBAUM
Lysine mutase (E.C. 5.4.3.3,4) requires coL-B-Lysine ~~, mutase has enzyme B 1 2 as c ~ f a c t o r ~ been assayed by determining the amino acid content of the reaction mixture by thin-layer chromatog r a p h ~ ninhydrin ~ ~ ~ reagent625 or a coupled enzyme reaction.626 Diol hydrase (E.C. 4.2,1.28) is used to assay coenzyme B 1 2 . Enzyme from A e r o b a c t e r a e r o g e n e s , grown in the absence of vitamin B 1 2 , is added to 1,2-propanediol. The amount of propionaldehyde formed627 is determined colorimetrically with benzothiazol-2-one hydrazine. Propionaldehyde can also be determined in nano ram quantities using 2 ,4-dinitrophenylhydra~ine~~ I or , via a coupled reaction, by the alcohol dehydrogenase reaction.630 Purified enzyme enables as little as 0.01 pmole of coenzyme B 1 2 to be measured using colorimetry.631 Vitamin B 1 2 , coenzyme B I 2 and related corrins were determined in picogram quantities using The glycerol dehydratase (E.C. 4.2.1.30).632 product a-hydroxypropionaldehyde was determined c o l o r i m e t r i ~ a l l ywith ~ ~ ~ a 10% error at concentrations of 5.25 picomoles. The apo-protein form of glycerol dehydratase binds strongly, to its active center, non-coenzymatic cobamides in a stoichiometric relationship. Since this complex is inactive, the amount of cyanocobalamin is proportional to the inactivation.634 This method has been automated for routine assays of cyano- and hydroxocobalamin.635
.5.8
Mass Fragmentography
Coenzyme cyanocobalamin has been quantitated using mass fragmentography.636 This increasingly useful technique can rapidly and selectively determine low concentrations of compounds in biological materials. The major problem with this procedure is its high initial cost. Picomole quantities of 5 -deoxyadenosylcorrinoids were assayed in biological material using the deuterated compound as internal standard. Serum samples containing 2 to 20 picomole(s) of cobalamin(s) were extracted with 10% trichloroacetic acid and centrifuged. After extraction and partial purification
CYANOCOBALAMIN
249
using an Amberlite XAD-2 ion-exchange column, the corrinoids were eluted with t-butanol in 0 . 5 % acetic acid and dried. The coenzyme was split with light, reduced with lithium borohydride and then alkylated with dimethylsulfate. After desalting, and reaction with sulfuric acid and mercuric sulfate, the samples were extracted into benzene (CAUTION). Methylated cobalamins were determined by examining the intensities of typical frag8,5-cycli~-5-deoxyadenosine(TMS)3 m e n t of ~ ~ ~ ~ and 5,5-d2-8,5-cyclic-5-deoxyadenosine(TMS)3.
5.9
Comparison of Methods
Greatest attention has been given to comparing radioassays with microbiological methods for the diagnosis of cyanocobalamin deficiency. Although some investi ators found radioassays to be superior6 8-6 4?! or equal6 3-6 8 , the general consensus (cf. Radioassay Sections) is that the microbiological assay649r650 gives a clearer distinction than the radioactive method between normal subjects and patients with untreated pernicious anemia. In addition, many post-gastrectomy and folate-deficient patients yielded subnormal results for cyanocobalamin concentrations in serum by microbiological methods but normal values by radioisotopic assay. In practice, the rapid, simple and inexpensive kit radioassays are used to screen for the normal, borderline and subnormal. The borderline and questionable cases are subsequently retested for vitamin B 1 2 content using lengthier and more costly, but more exact, procedures. A chemical method gave similar results to a radioassay.651 For determining cyanocobalamin in a ternary mixture of three vitamins, a direct spectrophotometric method was found to te superior to a column chromatographic procedure652 utilizing spectrophotometry for the final quantitation. Cyanocobalamin appeared to be retained on the column. Spectrophotometric and microbiological methods were found to give similar results.653 A high-pressure liquid chromatographic procedure was found to be superior to a radioisotopic, competitive intrinsic factor binding assay for
250
JOEL KIRSCHBAUM
cyanocobalamin in natural water.654 Sensitivity is from 10 to 190 picograms per mL. The enzymatic assay using glycerol dehydratase gives similar results to spectrophotometric, microbiological ( L a c t o b a c i Z Z u s Z e i c h r n a n n i i ) and cobalt-neutron activation methods.655 Many references cited elsewhere in this review give comparative data validating the procedure. To this investigator, the only assay that should be used is one that has been previously validated for the compound in its matrix, if any. Choices, if available, must be between validated methods. Decisions could be made on the basis of simplicity, convenience, precision, accuracy, time needed per assay, cost, toxicity, stability and availability of reagents. These criteria virtually eliminate such previously-used, time consuming and tedious methods as measurement of growth in weanling rats and chickens. In general, for simplicity and accuracy, high-pressure liquid chromatographic methods are preferred for bulk vitamin B12 and for vitamin B 1 2 in formulations and other, easily manipulated matrices. For sensitivity, low cost and ease, radioassay kits are preferred where great accuracy is unnecessary, or for screening many samples of, for example, human serum for cyanocobalamin deficiencies. For sensitivity without lengthy extraction or concentration steps, microbiological, some radioassay and enzymatic methods are favored. Future assays will probably include mass fragmentographic and fluorescent immunological assays.
6.
Stability
6.1
Inactivation
added to cyanocobalamin causes hydrolysis of the amide groups (in the order e > > b , d > acetamide groups, c > a , g ) and the nucleotide, and the release of 1-amino-2-propanol. In dilute acid the 5,6-dimethylbenzimidazole base was found to be prot6nated and dissociated from the cobalt.61 This form of cyanocobalamin is called "base-off". Vigorous acid hydrolysis yields the hexa- and heptacarboxylic acids and a lactone.657
CYANOCOBALAMIN
25 I
The addition of base, 0.1 M at 100' for 10 min. in air, results in the l o s s of biological activity658, although many physical properties remain unchanged. This inactive vitamin is called "dehydrovitamin B 1 2 " , cyano [8-amino-a-(5,6-dimethylbenzimidazolyl)cobamic acid-a,b, d , e , gpentaamide-c-lactam]. With 30% sodium hydroxide at 150, a mixture of penta- and h xacarboxylic w h i l e the acids and a y-lactam were formed"' nucleotide was cleaved. Barium hydroxide hydrolysis at 1 0 0 ' frees 5 moles of ammonia. Spectrophotometric properties of cyanocobalamin in acidic, basic and neutral solutions have been studied.660 No significant spectral changes are visible661 in methanol or water for up to 24 hours, but are seen in 0.1 M hydrochloric acid and 0.1 M sodium hydroxide. Potassium cyanide added to a red solution of cyanocobalamin gives a purple color. The nucleotide is displaced by cyanide in an SN 2 reaction. The dicyanide is unstable.662 Cyanocobalamin loses activity in the presence of c h l ~ r t e t r a c y c l i n e ~ m~ e ~ t ,h ~ l p a r a b e nand ~~~ sodium phenyldimethylp razolonemeth laminomethane sulfonate (novalgin).6x5 Flavors66x induce instability. Although crystalline cyanocobalamin is stable up to 100' for moderate periods of time667, autoclavinq solutions at 1 2 0 ' for 20 min. or l o o o for 75 min. causes loss of activity. The stability of buffered and unbuffered solutions, from p H 4.7 to 7, protected from light, was found to be similar within the experimental error of the Lactobacillus Zeichmanii 3 1 3 assay.668 As mentioned previously, cyanocobalamins are photosensitive.669 Light splits the orqanometallic bond giving a 5'-deoxyadenosyl radical, which was found to be capable of further reaction, and cob(II)alamin.670 The latter was stable in the absence of oxygen. With oxygen present, hydroxycobalamin is formed, which was used as the basis of an assay for the stability of cyanocobalamin in formulations.671 The rate of anaerobic photolysis
252
JOEL KIRSCHBAUM
is increased by quinones, thiols and some alcohols. 6 5 6 Oxygen induces a l o s s of 8 8 % cyanocobalamin activity in a liver preparation.672 3,3-Dimethyl2,5-dioxopyrrolidine-4-propionamide was one oxidation product.673 Ozone has been used to liberate cobalt from cyanocobalamin preparatory to assay (cf. section 5 . 2 2 1 ) . Permanganate yields various acids. Vitamin B12 was claimed675 to be stable in plasma stored at - 6 O , 5 O and 22' for 14 weeks using radioassays. Presumably, the sum of cyanocobalamin in the plasma and B12 incorporated into whatever grows at room temperature is constant. Radiation degrades cyanocobalamin in soluti01-1 proportional ~~~ to the dose, but freezing stabilizes irradiated solutions677, as determined using 6oCo-labeled cyanocobalamin.678 (This decomposition induced by radiation can be reduced by Frozen solutions adding 0.9% benzyl of cyanocobalamin and sodium chloride are less stable to irradiation than control solutions lacking saline.680 Vitamin formulations have been extensively studied for stability. Thiamine inactivates cyanobalamin.681,682r683 Stability in solution depends on pH.684r685 Ascorbic a ~ i d de~ ~ stabilizes vitamin B12, especially if such metals as copper, manganese and molybdenum are pres; ent688,689. Two papers, published 2 8 years apart,690r691gave data showing that the apparent l o s s in BI2 activity due to vitamin C was restored by cyanide. This observation may explain some conflicting data on the stability of cyanocobalamin with ascorbate,692,693 since some assay methods utilize added cyanide and some do not. Vitamin B12 was found to be unstable in the presence of thiamine and niacinamide.694r695 Significant losses have been found in various capsules, tablets and liquids after one year at room temperature.696r697 The rate of thermal degradation has been studied and extrapolated to predict shelf-life.698 Methanol vapors caused the l o s s of
CYANOCOBALAMIN
253
protected cyanocobalamin in multivitamin tablets after one month of due to the presence of ascorbic acid and niacinamide. The effect of storage on solutions of vitamin B 1 2 and other vitamins was studied.7001701 The presence of reducing agents, even in trace quantities (which may be introduced in the form of excipients in the formulation), can significantly reduce potency as a result of decomposition of the reduced form of cyanocobalamin to irreversible products.702
6.2
Stabilization
Solutions of cyanocobalamin have been stabilized by the pyrimidine moiety of thiamine703, antioxidants and chelating agent^^^^^ citric a ~ i ~d y s~ t e~ in~ e~ diisopro ~~, ylammonium dichloro acetate707, iron salts7081 7 0 9 1 T 1 0 , galactolactate, gl~conate~ lactate711, ~~, molybdate712 (which was also claimed to degrade vitamin B 1 2 in a formulation688), phosphate buffer (pH 4.6) containing 0.8% sodium chloride713, polyhydroxy compounds like ~ o r b i t o l ~ potassium ~~, sodium ~ a 1 t and ~ stomach ~ ~ extract.718 ~ , ~ 6oCo~ ~ ~ labeled cyanocobalamin is stabilized against radiation-induced decomposition by adding 0.9% benzyl Liver extracts have been stabilized by bisulfite719 and by potassium cyanide.720 Cyanocobalamin has been stabilized in a rectal suppository by coating with either crystalline mannitol, or sorbitol and talc.721 Vitamin B12 is currently stabilized prior to formulation in vitamin-mineral products. The best method722 appears to be adsorption on Amberlite IRP-64, methacrylic acid-divinylbenzene resin.723 The 0.1% of cyanocobalamin on resin was found to be unaffected by the acid pH of the stomach, but it was eluted in the mildly alkaline small intestine, where it was absorbed. After 24 months at 2501 coated multivitamin-mineral formulations retain 9 4 % of their potency724, using a spectrophotometric assay. Tests in vivo show similar absorption of free and resin-adsorbed cyanocobala-
254
JOEL KIRSCHBAUM
min, using 6oCo-labeled vitamin B 1 2 . Serum concentrations of B 1 2 and growth rates in test animals are higher using resin-adsorbed vitamin. Other vendors725 of stabilized cyanocobalamin currently use gelatin, mannitol or dicalcium phosphate. 7. Metabolism
Of the 0.5 ug of 6oCo-cyanobalaminadministered orally to normal control subjects, 0% to 42% of the radioactivity appeared in the feces.726 At 5.5 u ( , . fecal excretion ranged from 12% to 7 1 % , indicating a dependence on concentration. Patients with pernicious anemia excrete 72% to 96%727 c f . Radioassay section. Subjects with infections excrete 45% to 6 0 % , indicating that infections interfere with absorption. Antibiotics also inhibit absorption of vitamin B12.728 In normal subjects, little or no radioactivity appears in urine after the administration of labeled cyanoRadioassays) ~ o b a l a m i n ~(cf. ~
I
After oral dosing of 6oCo-labeled cyanocobalamin, considerable radioactivity accumulated in the liver730 in inverse relationship to the dose. No radioactivity was found in plasma until 4 hours had elapsed. Peak radioactivity in plasma is found in the 8-12 hour period, with a slow decline in content with time. Cyanocobalamin appears to be temporarily stored in the gastrointestinal tract.731 The most important organ for permanent storage is the Liver, where it has a biological half-life of approximately one year.732 Aquo-, hydroxo-, methyl-, cyanocobalamin and 5'-deoxyadenosyl cobalamin (coenzyme B12) appear to be interconvertible in v i v o . Oxidation and ring-opening leads to the formation and excretion of degradation products similar to the bile pigments.733
8.
Acknowledgements
The author gratefully acknowledges the assistance of the many contributors cited as "personal communication." In many instances, the information was especially obtained for this sum-
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mary. Special thanks go to Dr. Sandu Goldstein of ICN International Montreal, for a preprint of his paper on the thermal stability of cyanocobalamin, and other reprints. Dr. Amar Nath of Drexel University, Dr. Adam Allerhand of Indiana University, The Royal Society of London on behalf of Dorothy Crowfoot Hodgkin, Dr. Hans-Rolf Schulten of the University of Bonn, and Dr. Pill-Soon Song of Texas Tech University either gave permission to use figures or, very generously, supplied copies of the glossies. The other figures utilized the illustrative skills of Sal Meloni and the photographic talents of Charlotte Raymond. The preparation of camera-ready copy was by the Word Processing Center, Squibb, Lawrence Township, N.J. Dr. John Wang helped with translations from the Japanese. The author appreciates the critical reading and thoughtful comments of Raymond Poet, Glenn Brewer, Eileen Nickoloff, and Solomon Perlman of Squibb, and Dr. Amar Nath. Credit for obtaining many of the references belongs to Susan Campos, Muriel George, Phyllis Gottstine and Mary Klein. In addition to the references cited in the text, other useful ones not specifically cited are listed below.734-742 Literature was surveyed to March, 1981. 9. References
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and Pathophysioloqy", Ed B.M. Babior, John Wiley, New York, 1 9 7 5 , p. 2 1 . D.E. Wolf, W.H. Jones, J. Valiant and K. Folkers, J . Amer. Chem. S o c . , 72, 2 8 2 0 ( 1 9 5 0 ) . R. Bonnett, J.R. Cannon, A.W. Johnson and A.R. Todd, J . Chem. S o c . , ( 1 9 5 7 ) 1 1 4 8 . J.B. Armitage, J.R. Cannon, A.W. Johnson, L.F. J. Parker, E.L. Smith, W.H. Stafford and A.R. Todd, 5 . Chem. S o e . , ( 1 9 5 3 ) 3 8 4 9 . G. Bellomonte and E. Cingolani, Rend I s t . S u p e r . S a n i t a , 26, 1 0 5 0 ( 1 9 6 3 ) . B. Pate1 and E. Sadowski, Personal communication.
CYANOCOBALAMIN
285
6 6 2 . W.W. R e e n s t r a , D i s s . A b s t r . I n t . B , 3 8 , 5 9 1 3 ( 1 9 7 8 ) ; C A , 8 9 , 1 2 9 6 7 9 ~( 1 9 7 8 ) . 6 6 3 . Z.K. A s h k i n u z i , F e r m e n t . , S p i r t . Prom., 3 6 , 1 7 ( 1 9 7 0 ) ; C A , 7 2 , 1 2 0 2 3 5 ~( 1 9 7 0 ) . 6 6 4 . S . Yazdany a n d F . B a d i i , J . Pharm. S c i . , 6 5 , 745 ( 1 9 7 6 ) . 665. I. E n v e r , T u r k . I j i y e n T e c r u b i B i o Z. D e r g i s i , 2 7 , 1 4 ( 1 9 6 7 ) ; C A , 6 7 , 84799m ( 1 9 6 7 ) . 6 6 6 . B.M. T r i v e d i a n d P.R. Kapadia, I n d i a n J . Pharm. 3 1 , 60 ( 1 9 6 9 ) ; C A , 7 1 , 1 2 8 6 5 1 ~( 1 9 6 9 ) . 6 6 7 . E . H o f f - J g k g e n s e n , K . I l v e r , 0.1. J o h a n s e n a n d F . R e i m e r s , Dansk r i d s . Pharm., 2 7 , 1 1 7 ( 1 9 5 3 ) ; CA, 4 7 , 8321f ( 1 9 5 3 ) . 668. R . P o n c i , IZ Farmaco E d . S c i . , 9 , 467 ( 1 9 5 4 ) ; CA, 4 9 , 567h ( 1 9 5 5 ) . 669. A . V o g l e r , R . H i r s c h m a n n , H . O t t o a n d H . K u n k e l y , B e r . B u n s e n g e s . P h y s . Chem., 8 0 , 430 ( 1 9 7 6 ) ; C A , 85, 13411233 ( 1 9 7 6 ) . 670. H.P.C. Hogenkamp, H.A. B a r k e r a n d H . S . Mason, Arch. Biochem. B i o p h y s . , 1 0 0 , 353 ( 1 9 6 3 ) . 671. P . D e W i t t a n d S . Muck, B o l l . Chim. Farm., 1 1 2 , 422 ( 1 9 7 3 ) . 672. E.M. S t a p e r t , E . B . F e r r e r a n d L . S t u b b e r f i e l d , J . Amer. Pharm. A s s o c . , 4 1 , 5 8 7 ( 1 9 5 2 ) . 6 7 3 . F.A. K u e h l , C . H . S h u n k , M.V. Moore a n d K . F o l k e r s , J . A m e r . Chem. S o c . , 7 7 , 4418 ( 1 9 5 5 ) . 674. H. S c h m i d , A . E b n o t h e r a n d P . Karer, H e l v . Chim. A c t a , 3 6 , 6 5 ( 1 9 5 3 ) . 6 7 5 . N.P. K u b a s i k , M. Graham a n d H . S i n e , C Z i n . Chim. A c t a , 9 5 , 1 4 7 ( 1 9 7 9 ) . 676. J . W i l s k a - J e s z k a , NukZeonika, 2 7 , 1 0 1 1 ( 1 9 7 7 ) ; C A , 88, 1 1 7 1 1 1 ~( 1 9 7 8 ) . 6 7 6 . E . V . B a r e l k o , G.S. B a b a k i n a , I . V . B e r e z o v s k a y a , V.S. D e g i l o v a , L.A. P i r u z y a n , N . V . P o m o r t s e v a , V.V. S i k h a n o v a n d V.L. T a l ' r o z e , Khim. Farm. Z h . , 1 2 , 94 ( 1 9 7 7 ) ; C A , 8 7 , 1 5 7 1 1 3 h ( 1 9 7 7 ) . 678. G.H. B a r l o w a n d N.D. S a n d e r s o n , B i o ch i m . B i o p h y s . A c t a , 4 1 , 146 ( 1 9 6 0 ) . 679. R . J . B a y l y a n d E . A . E v a n s , J . L a b e l l e d Cornp., 21, 1 (1966). 680. E.V. B a r e l k o , G.S. B a b a k i n a , I . P . S o l y a n i n a , V . L. T a l ' r o z e , V . I . T r o f i m o v a n d 1.1. C h k h e i d z e , DokZ. A k a d . Nauk SSSR, 2 4 6 , ( 1 9 7 9 ) ; C A , 9 1 , 202104b ( 1 9 7 9 ) . 681. R. P o n c i , I1 Farmaco, E d . S c i . , 1 0 , 9 9 7 , 1 0 0 3 ( 1 9 5 5 ) ; C A , 50, 5991g ( 1 9 5 6 ) . 682. J . Dony a n d J . C o n t e r , J . Pharm. B e Z g . , 1 2 , 1 8 6 ( 1 9 5 6 ) ; C A , 5 2 , 2 1 3 8 ~( 1 9 5 7 ) .
286
JOEL KIRSCHBAUM
6 8 3 . R.F. Doerge, L.J. Ravin and H.C. Caldwell, J . Pharm. S c i . , 5 4 , 1 0 3 8 ( 1 9 6 5 ) . 6 8 4 . J. Ostet-Conter, An. Farm. Hosp., 2 0 , 9 5 ( 1 9 6 7 ) . 6 8 5 . I b i d . , A r c h . Pharm. C h e m i , 6 8 , 5 2 9 ( 1 9 6 1 ) ; C A , 5 5 , 2148633 ( 1 9 6 1 ) . 6 8 6 . E . Block, A. Akerkar and M. Hoholick, C Z i n . Chem., 2 4 , 1 7 6 ( 1 9 7 8 ) . 6 8 7 . T.J. Macek, Amer. J . P h a r m . , 1 3 2 , 4 3 3 ( 1 9 6 0 ) . 6 8 8 . E.M. Stapert, E.B. Fevrer and L . Stubberfield, J . A m e r . Pharm. A s s . , S c i . E d . , 4 3 , 8 7 ( 1 9 5 4 ) . 6 8 9 . A.J. Rosenberg, J . B i o Z . Chem., 2 2 9 , 9 5 1 ( 1 9 5 6 ) . 6 9 0 . D.V. Frost, M. Lapidus, K.A. Plaut, E. Scherfling and H.H. Fricke, S c i e n c e , 1 1 6 , 1 1 9 (1952). 6 9 1 . M. Marcus, M. Prabhudesai and S. Wassef, h e r . J . CZin. N u t r i . , 3 3 , 1 3 7 ( 1 9 8 0 ) . 6 9 2 . H . L . Newmark, J. Scheiner, M. Marcus and M. Prubhudesia, I b i d . , 2 9 , 6 4 5 ( 1 9 7 6 ) . 6 9 3 . V. Herberg and S.E. Jacob, J . A m e r . Med. A s s o c . 230, 241 (1974). 6 9 4 . M. Blitz, E. Eigen and E. Gunsberg, J . h e r . Pharm. A s s o e . , 4 3 , 6 5 1 ( 1 9 5 4 ) . 695. I b i d . , 4 5 , 8 0 3 ( 1 9 5 6 ) . 6 9 6 . J.A. Campbell and H.A. McLeod, J . Arner. Pharrn. Assoc., 4 4 , 263 ( 1 9 5 5 ) . 6 9 7 . I b i d . , C a n . Pharm. J . , 8 8 , 3 7 6 ( 1 9 5 5 ) ; C A , 4 9 , 1227433 ( 1 9 5 5 ) . 6 9 8 . E . R . Garrett, J . A m e r . Pharm. A s s o c . , 4 5 , 1 7 1 , 470 (1956). 6 9 9 . J.T. Jacob, R.J. Nessel and J. Blodinger, J . Pharm. S c i . , 5 7 , 1 8 5 4 ( 1 9 6 8 ) . 7 0 0 . T.J. Macek and B.A. Feller, J . Amer. Pharrn. A s s o c . , 4 4 , 254 ( 1 9 5 5 ) . 7 0 1 . F . Ebel and 0. Mastilovic, T e h n i k a , 2 6 , 1 4 4 (1971). 7 0 2 . T.J. Macek, A m e r . J . Pharm., 1 3 2 , 4 3 3 ( 1 9 6 0 ) . 7 0 3 . F. Gstirner and S.K. Baveja, A r c h . Pharm., 2 9 8 , 1 3 4 ( 1 9 6 5 ) ; CA, 6 2 , 1 1 6 3 7 e ( 1 9 6 5 ) . 7 0 4 . G . S . D . Gupta and M . K . V . Rao, R e s . I n d . , 2 5 , 1 1 1 ( 1 9 7 0 ) ; C A , 75, 2 5 3 2 0 r ( 1 9 7 1 ) . 705. S o c i e t e C i v i Z e d e Recherches S c i e n t i f i q u e s e t I n d u s t r i e Z Z e s S o d e r s i F r . Patent, 1 , 3 7 2 , 4 0 8 August 10, 1 9 6 4 ; C A , 6 2 , 3 8 9 2 e ( 1 9 6 5 ) . 7 0 6 . G . Benedikt, Ger. Patent 2 , 5 2 2 , 1 8 7 , Oct 2 8 , 1976; CA, 8 6 , 95996s ( 1 9 7 7 ) . 7 0 7 . A. Giulio, R. Ruggero and L. Conti, G i o r n . Med. M i l . , 2 2 4 , 1 7 3 ( 1 9 6 4 ) ; C A , 61, 1 1 8 5 5 9 ( 1 9 6 4 ) . 7 0 8 . S . L . Mukherjee and S.P. Sen, J . Pharrn. Pharm., 9, 759 (1957); CA, 52, 3061a (1958).
CYANOCOBALAMIN
287
7 0 9 . I b i d . , 1 2 , 2 6 ( 1 9 5 5 ) ; CA, 53, 8 5 3 6 h ( 1 9 5 9 ) . 7 1 0 . H.L. Newmark, U . S . Patent 2 , 8 2 3 , 1 6 7 , Feb. 11, 1 9 5 8 ; CA, 5 2 , 10509i ( 1 9 5 8 ) . 7 1 1 . R.G-A del Valle, Span. Patent 2 4 7 , 5 2 2 , April 1 5 , 1 9 5 9 ; CA, 5 4 , 2 3 2 1 0 ( 1 9 6 0 ) . 7 1 2 . H.J. Buehler, U.S. Patent 2 , 9 2 3 , 6 6 3 , Feb. 2 , 1960; CA, 5 4 , 13560d ( 1 9 6 1 ) . 7 1 3 . R. Kojer, A c t a P o l o n . Pharm., 1 5 , 3 5 9 ( 1 9 5 7 ) ; CA, 5 2 , 1 2 3 2 6 a ( 1 9 5 8 ) . 7 1 4 . A. David, P. Szentmiklosi and G. Horvath, Hung. Patent 1 5 0 , 8 8 5 , Oct. 2 3 , 1 9 6 3 ; CA, 60, 7 8 7 9 a (1964). 7 1 5 . F. Gstirner and S.K. Baveja, Pharm. Z t g . , 1 0 9 , 1 7 8 0 ( 1 9 5 3 ) ; CA, 62, 159951.1 ( 1 9 6 5 ) . 7 1 6 . E . Noda, K. Aoki, 0. Horizaki and Y. Asakura, Japan Patent 7 4 7 4 , April 2 2 , 1 9 6 6 ; CA, 65, 8688f (1966). 7 1 7 . I . Y . Gurevick, O.A. Ogorodnova, 0.R Zherbina and P.E. Rosentsveig, A p t e c h n D e l o , 1 5 , 2 9 ( 1 9 6 6 ) ; CA, 6 4 , 1 7 3 6 2 ( 1 9 6 6 ) . 7 1 8 . C.E. Meyer, U . S . Patent 3 , 1 6 8 , 4 4 0 , Feb. 2 , 1 9 6 5 ; CA, 62, 10301~( 1 9 6 5 ) . 7 1 9 . H.W. Loy, Jr., J.F. Haggerty and O.L. Kline, J . Assoc. O f f i e . A g r . Chemists, 35, 169 ( 1 9 5 2 ) ; CA, 4 6 , 115851.1 ( 1 9 5 2 ) . 7 2 0 . W. Pawelec, Farm. P o l s k a , 1 0 , 1 3 0 ( 1 9 5 4 ) ; C A , 48, 1 4 1 2 1 e ( 1 9 5 4 ) . 7 2 1 . L. Grippa, F a r m a c i s t a , 20, 1 8 9 ( 1 9 6 8 ) ; CA, 71, 33369w ( 1 9 6 9 ) . 7 2 2 . M. Schwartz, Personal communication. 7 2 3 . Pfizer, Data Sheet 6 7 3 , 1 9 7 5 . 7 2 4 . I b i d . , Personal communication. 7 2 5 . Hoffman-LaRoche, Merck and Reisman. 7 2 6 . R.W. Heinle and A.D. Welch, J . Amer. Med. A s s . , 133, 739 ( 1 9 4 7 ) . 7 2 7 . S . T . Collender, A. Turnbull and G. Wakisaka, B r i t . Med. J . , 1 , 1 0 ( 1 9 5 4 ) . 7 2 8 . E.D. Jacobson and W.W. Falcon, J . A m e r . Med. Assoe., 175, 1 8 7 ( 1 9 6 1 ) . 7 2 9 . A. Doscherholmen, "Metabolism of Vitamin B12I1,
University of Minnesota Press, Minneapolis, 1 9 6 5 , p. 2 6 . 7 3 0 . G.B.J. Glass and L. Stephanson, A r c h . B i o c h e m . ,
52, 251 (1954). 7 3 1 . C.C. Booth and D.L. Mollin, B r i t . J . H a e m a t . , 2, 223 ( 1 9 5 6 ) . 7 3 2 . G.B.J. Glass, G a s t r o e n t e r o l o g y , 36, 1 8 0 ( 1 9 5 9 ) .
288
JOEL KIRSCHBAUM
7 3 3 . R. Schmid and A.F. McDonagh, in "The Porphyr-
ins," Ed. D. Dolphin, AcademicPress, New York, 1 9 7 9 , Volume VI, p. 2 5 7 . 7 3 4 . D. Dolphin, Ed. "The Porphyrins , Academic Press, New York, 1 9 7 9 . 7 3 5 . W. Friedrich, Chem. R u n d s c h . , 32, 1 ( 1 9 7 9 ) . 7 3 6 . S . Fukui and S. Shimizu, Method C h i m . 11, 1 0 1
I'
(1977). 7 3 7 . M.-U.-H Hashmi, "Assay of Vitamins in Pharmaceutical Preparations", Wiley, New York, 1 9 7 3 . 7 3 8 . A. Johnson, Chem. I n d . (London), 1 , 2 7 ( 1 9 7 8 ) . 7 3 9 . Ibid., Chem. S o e . ( L o n d o n ) Rev., 9 , 1 2 5 ( 1 9 8 0 ) . 7 4 0 . G.N. Schrauzer, Angew. Chem. I n t . E d . EngZ., 1 5 , 417 ( 1 9 7 6 ) . 7 4 1 . 1.1. Shahied, "Biochemistry of the Foods and Biocatalysts," Vantage Press, New York, 1 9 7 7 . 7 4 2 . R. Strohecker and H.M. Henning, "Vitamin Assay, Tested Methods," CRC Press, West Palm Beach, FL, 1966.
EMETINE HYDROCHLORIDE
L. Valentin Feyns and Lee T. Grady
1, Introduction-History 2. Description 2.1 Name, Formula, Structure, Molecular Weight 2.2 Appearance, Color, Odor, Taste 3. Production 3.1 Extraction from Ipecac 3.2 Methylation of Cephaeline 3.3 Total Synthesis 4. Physical Properties 4.1 Spectra 4.2 Melting Range 4.3 Solubility-Partition 4.4 Dissociation Constants 4.5 Optical Rotation 5. Methods of Analysis 5.1 Identity and Color Tests 5.2 Elemental Analysis 5.3 Chromatographic Methods 5.4 Titration 5.5 Colorimetric and Spectrophotometric Methods 5.6 Spectrofluorometric Methods 5.7 Polarographic Methods 5.8 Thermogravimetric Analysis 6 . Determination in Biological Fluids and Tissues 7 . Determination in Pharmaceutical Preparations 8. Stability-Degradation 9. Toxicity-Pharmacokinetics 10. References
290 292 292 292 292 292 293 295 295 305 305 305 306 306 306 307 307 314 314 315 316 316 316 317 319 320 322
ANALYTICALPROFILES OF DHUC SUBSTANCES, 10
289
Copyright CC, 1981 by Academic Preaa, Inc. All rights of reproduction in any form rcrervrd ISBN 0~12~2fiOX1Il~O
290
L. VALENTIN FEYNS AND LEE T. GRADY
1.
Introduction - History
In 1570, a Portuguese monk living in Brazil learned from the natives about the use of the root of Ipecacuanha ("little wayside plant that causes vomiting") as a remedy in diarrhea and bleeding.' Reports of its use in Europe date as early as the seventeenth century. The son of Louis XIV was successfully treated by Helvetius with his "Brazilian root".2 For more than two centuries, ipecac was one of the very few specific chemotherapeutic agents available to pharmacists. A description of the root of Ipecacuanha appears in "The Pharmacopoeia of the Royal College of Physicians at Edinburgh", printed in London in 1748 "for John Nourse, at the Lamb, opposite Katherine-Street in the Strand".3 In the 1830 edition of "The Pharmacopoeia of the United States of America", the emetic properties of the root are described as depending "on the presence of a peculiar principle denominated emetine". The first monograph dedicated to Emetine Hydrochloride, appears in the 1916 revision. 5 Emetine is the major active constituent of the rhizome and root of Cephaelis ipecacuanha and Cephaelis acuminata Karsten. I t was first isolated in a crude form by Pelletier in 1817 and recognized as an alkaloid in 1823, but the purified alkaloid was prepared only in 1875 (1894 according to another author),6 and it was not obtained in crystalline form until 1953.7 The first correct empirical formula was reported in 1914,8 but the stereochemistry of the four assymetry centers was elucidated only in 1959, a ter decades 5 i of brilliant degradation and synthetic research. In 1964, Pakrashi isolated emetine and other related alkaloids from the seed kernels and root bark of the Indian plant Alangium lamarckii Thwaites, the first reported occurrence of emetine outside the family of Rubiaceae. 10,ll Extensive use of emetine in the treatment of amebiasis started in the first quarter of the century. The antiamebic activity and the inhibition of protein synthesis are highly stereospecific and restricted to the natural levorotatory isomer (Fig. 1 ) . Emetine salts and ipecac (the dried rhizome and roots) are listed in all major national and international Pharmacopeias.l2 Several reviews covering the chem ca biological properties of emetine have been written. If-i?Yd
i,
k
291
Stereo-formula of emetine. N a t o m s . ' )
The shaded circles represent
Emetine R
=
OCH,
Cephaeline R
=
OH
Psychotrineb) R
O-methylpsychotrineb) R
N & m l;
Emetamine R
N & H ,
=
OCHl
Fig. 1.
Emetine and related alkaloids from ipecac.
a)From ref. 13. Reproduced with permission of the copyright owner (Van Nostrand b i n h o l d CO.). b)See Section 8 for a discussion on 0-methylpsychotrine structure.
292
L. VALENTIN FEYNS AND LEE T. GRADE'
2.
Description
2.1
Name, Formula, Structure, Molecular Weight
Chemical Abstracts Name Emetan, 6',7'10,11-tetramethoxy, dihydrochloride. Other Emetine Dihydrochloride, Emetinum Hydrochloridum, Emetini Hydrochloridum, Emetini Chloridum, Ipecine Hydrochloride, Methylcephaeline Hydrochloride, Cloridrato de Emet ina
United States Pharmacopeia definition18 the hydrochloride of an alkaloid obtained from Ipecac, or prepared by methylation of cephaeline, or prepared synthetically. Chemical Abstracts Registry Numbers Emetine dihydrochloride Emet ine Empirical formula Mo 1ecula r we igh t Structural formula 2.2 C2gH40N204.2 HC1 553.57 See Fig. 1 316-42-7 483-18-1
Appearance, Color, Odor, Taste
White, odorless, crystalline powder with a bitter taste. Colorless needles of the heptahydrate (see 5.8) from hot aqueous solution, prisms from concentrated cold solutions. 3. Production 3.1 Extraction from ipecac
Warning against falsifications, the following data were reported in 1945 for the "true" ipecac: total alkaloids 2.00-2.70%, emetine 1.35%, cephaeline 0.25%, psychotrine 0.04%, 0-me hylpsychotrine 0.015-0.033%, emetamine 0.002-0.006%.~9 Other sources indicate emetine as 50-70% of the total alkaloid content2 and the ratio emetine:cephaeline as 2-3:1 in Cephaelis ipecacuanha and 1:l in Cephaelis acuminata.20 The USP XX-NF XV definition of ipecac gives the content of cephaeline as varying "from an amount equal to, to an amount not more than twice, the
293
content of emetine. ..2 1 Extraction procedures6 9 2 2 ,23 -The alkaloids are extracted with 70% ethanol or methanol (alone or 5 0 % ) , the concentrated extract is dissolved in water, the solution is made strongly basic with ammonia and extracted with diisopropyl ether. The organic extract is treated with 10-15% aq. KOH to remove cephaeline and evaporated to give emetine, which is purified via the dihydrobromide or dihydroiodide; the halides are converted to the hydrochloride by neutralizing the regenerated free base. -The Ipecac powder is treated with ammonia, and ether; the alkaloids are extracted from the ether, with diluted H2S04, the latter is nearly neutralized and washed with ether, then made strongly alkaline and treated with ether (cephaeline remains in the aqueous phase); the residue from the ether solution is taken in methanol and treated with a methanolic solution of HBr to yield emetine hydrobromide. 3.2 Methylation of cephaeline
Cephaeline, which is extracted concomitantly from ipecac, can be converted to emetine with several methylating agents such as: diazomethane, phenyltrimethylammonium hydroxide, dimethyl su fa e sodium methyl sulfate or nitrosomethylurethane. *25,23 Methylation of the N-atoms by some agents, such as dimethyl sulfate, lowers the yield of ernet ine. 3.3 Total synthesis
Total synthesis of emetine was undertaken initially to obtain confirmation of the structure and sterzyhemistry. The first synthesis was completed in 1950 ang-although more than a dozen routes were l 5 using various starting materials and reported combinations of stereospecific reactions and resolution of racemic mixtures, the research continues, stimulated by the isolation of new structurally related alkaloids and search for biologically active synthetic analogs. 2 5-86 the Fig. 2 summarizes a procedure developed by Openshaw in 196330 and reportedly used for commercial production by Burroughs-Wellcome.
n U
3 :
V 0
3 :
n U
F i g . 2.
Openshaw p r o c e d u r e f o r the total synthesis of erne t ine 30
u
0
I-i
i l
sl
v)
$\
$4
a, C
295
The condensation of 6.7-dimethoxy-3,4dihydroisoquinoline (I) with the Mannich base (11) gives the aminoketone 111 in almost quantitative yield. A very elegant resolution of racemic intermediate 111 is obtained by refluxing it in the presence of (-) camphor-10-sulfonic acid, when the desired levo-enantiomer precipitates as a salt and the dextro-enantiomer is racemized by reversible opening of ring C (with continuous precipitation of levo111, the result is an almost total conversion). The condensation of I11 with the Wittig reagent, e t h o x y c a r b o n y l m e t h y l e n e t r i p h e n y l p h o s p h o r a n e is stereocon-
servative. Condensation of IV with homoveratrylamine succeeded by Bischler-Napielarski cyclization affords 0methylpsychotrine which is hydrogenated to a mixture of emetine and isoemetine. The yield in emetine is increased by reconversion of inactive isoemetine into O-methylpsychotrine by N-chlorination and treatment with base.
4.
Physical Properties 4.1 Spectra 4.11 Ultraviolet
The values for the absorption maxima and the absorbances of a 1% solution in a 1.0 cm cell reported in the literature are presented in Table I.
A spectrum of USP Emetine Hydrochloride Reference Standard in water is shown in Fig. 3.
The effect of substituents on the UV spectra 85 isoquinolinic drugs (including emetine) has been studied. 4.12 Fluorescence
The spectrum of natural visible fluorescence of emetine in aqueous solutions was found to be analogous to that of other compounds containing one o r two o-dimethoxyphenyl groups. Both at pH 1.23 and 4.65-4.70 excitation wavelength ximum is at 360 nm and emission Y maximum is at 460 nm. 4 Other authors reported a fluorescence maximum at 318 nm with an excitation maximum at 284 nm (see ) . 5.6 and 6
296
Table I
UV Data for Emetine and Emetine Hydrochloride a)
Emetine Solvent Chloroform Xmax,nm 285 290(infl) 290 0.1 N H2SO4 285 281 5 285(infl) 4N - methanolic ammonia 285 Ethanol 235(sh) 285 360(sh) 283.5 349.5 Absolute ethanol b, Emetine Hydrochloride Water b) 229 283 279 230 283 228 281 265 122 85 (250)(238) (109)(103) 36 37 38-40 41 226 283 El% 1cm 163 148 210 208 227 208 161.5 (329) (131) weak (120) weak Ref. 31 32 32 33 34 35
Ethanol b,
0 . 1 M H2SO4
a)
b)
The values in parenthesis are calculated from reported log E values. Minimum at 256 nm.
297
ABS
0.6
0.4
0.2
0
2 50
Fig. 3. Ultraviolet Spectrum of Emetine Hydrochloride in Water. A-5.5 mg/lOO ml. B-2.2 mg/100 ml. Instrument -Beckman w 5 2 6 0 .
298
L.VALENTIN FEYNS AND LEE T.GRADY
4.13
Infrared spectra
The principal peaks in the spectrum of metine (KBr disk) are at 1514, 1256, 1228 and 1463 cm-1 33
The presence in the IR spectrum of 2'-benzoylemetine of Bohlmann bands around 2740 c m ' confirmed the chemical evidence for a trans-configuration of the quinolizidine moiety (trans-diaxial relationshi between the pair of electrons on N-5 and the H atom at 1 4 ) . E4
A spectrum of USP Emetine Hydrochloride RS in a KBr disk is presented in Fig. 4 . It shows a trong band at ZO N ' H ) . 2.93 p (H20) and a wide band at 3.83 p (
4.14
Nuclear Magnetic Resonance
An 80 MHz proton magnetic resonance spectrum of emetine in CDC13 containing TMS as an internal reference is presented in Fig. 5 . I t is identical with a previously published spectrum (no assignments).45 The signals from aromatic protons in the 6.4-7.2 ppm range and those from the etheric methyl groups around 3.8 ppm are in the expected regions, but the complexity of multiplets from the rest of the non-aromatic protons precludes any practical analytical use. The 20 M H z proton-noise decoupled I3C spectrum of the same sample is shown in Fig. 6 and the assignments of the chemical shifts, as reported in the iterature based upon comparison with related alkaloids , 4 ' are tabulated in Table 11. Both spectra were obtained on a Varian FT-80A instrument. 4.15 Mass spectra
Th mass spectrum and fragmentation pattern for emet ine11,47,28 are presented in Fig. 7 and 8. The mass spectrum in Fig. 7 was obtained by direct-probe introduction of the sample into the ion source of a DuPont 21-492B mass spectrometer operated under data system control (VG 2040, VG Data Systems, Altrincham, Cheshire, England). Operating conditions: resolution, 1000 (10% valley); ion source, 265"; electron energy, 75eV; ionizing current, 250 Damp; scan speed, 2 secldecade.
t-4
c V
300
0
r
0
N
1
0 d
51
30 1
302
I,. VALENTIN FEYNS AND LEE T. GRADY
T a b l e I1
I 3 C Chemical S h i f t s Assignments f o r Emetine
Chemical S h i f t a )
Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
C atom no.
lit46
Found 11.2 23.6 29.4 29.5 37 .O 37.0 40.3 40.8 41.8 52.0 52.4 55.9 56.1 56.1 56.4 61.5 62.5 108.9 109.5 111.8 112.1 127.1 127.1 130.4 132.3 147.3 147.4 147.4 147.7
17 16 7 4' 1 2 15 3' 3 6 1'

0-CH3
0-CH3
0-CH3 0-CH3
4 14 12 8' 9 5' 8 10' 13 9' 10

11
6'
7'
10.9 23.3 29.1 29.1 36.7 36.7 60.0 40.6 41.6 51.7 52.1 55.7 55.7 55.7 55.7 61.2 62.2 108.6 109.1 111.5 111.5 126.7 126.7 130.1 132.0 147.1 147.1 147.1 147.1
a ) I n CDC13 s o l u t i o n ; i n ppm, d o w n f i e l d from tetramethylsilane.
i
30
88 60
21
40
20
304
m/e 258
m/e 244
m/e 465
mle 451
>
%?.
mle 480
CHaO c n o 'Q Q ;
7
m / e 246
m/e 288
Fig. 8
Fragmentation pattern or emetine
305
4.2
Melting range
Emetine Hydrochloride contains water of crystallization ranging from 3 to 8 H epending mainly on ' : 4 g After drying at the solvent used for crystallization.3 105", most references indicakg a melting range between 235" and 255" with decomposition. One reference uotes a melting point of 269-270" with decomposition.39 The melting point of the amorpho s free base is 74"; crystalline emetine melts at 104-105".
I n a study of isotonic solutions the freezingpoint lowering of a 1% solution of emetine in water was found to be 0.082". 50
4.3
Solubility-Partition
Emetine: Water - O.O02g/lOO m151 Conc. NaOH, KOH - insolublez2 Methanol, ethanol, ether, acetone, acetic acid - very ~ o l u b l e ~ ~ , ~ ~ Chloroform - soluble51 Benzene - sparingly soluble22 Emetine Hydrochloride: Water - 13.1 g/100 ml (18")22352 Diluted HC1 - spa5lngly soluble22 Ethanol - 1 in 12 Chloroform - 1 in 433 Ether - insoluble33 The effect of partition coefficient of partition coefficient of chloroform and a mixtu buffer 1:l was 1252.0. pH and organic acids 8 ' 5 b h e T h e emetine was studied.5 emetine hydrochloride between water: pH 7.0 0.05 M phosphate
55
4.4
water:
Dissociation Constants The following pKb values have been reported for -at 15" 5.77 and 6.6422,60 -at 40" 5.47 and 6.3461 Other values reported: 5.73 and 6.74 (no
306
indication on the temperature) 59 4.5 Emetine Optical Rotation Solvent 50% EtOH g/100 ml 1.8 4.1 Chloroform 2 2.8 3 0.9 5.0 8.1 Chloroform
[aID
Ref. 7 6,16,22 6,22 35 62 22 6.22 79 6,22
-24.4" -25.8" -32.7" -50" -49.2" -49.7" +11.2" +17.7" +17.8" +20.9" +50.5" +53" +25.7" +51" +17" +83 "
Eme t ine Water Hydrochloride
40 16,22
22 6 6 6
5% HC1 Butanol Benzyl alcohol Bromoform
The study of the optical rotatory dispersion curves of emetine and its salts played a major r le in determining the stereochemistry of the molecule.' Since emetine hydrobromide showed no rotational change in the 300700 nm range, the benzylic centers of asymmetry at C-14 and C-l', the only ones in proximity to W chromophores, had to be antipodal to one another thus canceling each other's contribution. 5. Methods of analysis 5.1 Identity and Color Tests
Treated with ammonium molybdate or molybdenum oxide in sulfuric acif3 emetine gives a bright green color22 . 1 p g ) . The reaction is used in (sensitivity 0 official compendia as an identification test. 63,6Zom;i taliI test yields the following colors: addition of fuming nitric acid - pale yellow; evaporation-pale brown. addition of ethanolic KOH - yellow (sensitivity 1 v g ) . 33 An orange-color is produced when em tine is treated in an acidic medium with either H20265,65 or barium
307
peroxide.66 When heated in solid phase with ch emetine hydrochloride gives a red-yellow color.
8ramine,
Salts of emetine with arylsulfonic acids having characteristic melting points were prepared for identification purposes.68 Some of the paper and thin-layer chromatographic separations reported under 5 . 3 were also recommended as identity tests, preferably in conjunction with colors formed with specific visualization reagents. The use of the infrared identification test was suggested. The Identification Tests in the USP-NF monograph of Emetine Hydrochloride call for comparison of IR and UV spectra of the sample with those of USP Emetine Hydrochloride Reference Standard. All the official compendia require also an identification test for chloride. 5.2 Elemental Analysis
The calculated values for the elemental analysis of emetine hydroch ride are: C 62.91%; H 7.65%; C1 12.81%; N 5.06%; 0 11.56%.
is
5.3
ChromatograDhic methods
5.31
Paper chromatography
The paper chromatographic systems have been summarized in Table 111. System No. 1 was reported to separate emetine from some of its stereoisomers, but not from cephaeline. Emetine is visualized by examination under W light or spraying with iodoplatinate, bromocresol green, modified Dragendorff reagent or I/KI solutions. When the dried chromatogram is sprayed with a 10% chlorine solution in water acidified with acetic acid, emetine is oxidized to rubremetine and a strong orange-ye ow W fluorescence is obtained (detection limit - 2 us).
+i
Table I11 Paper Chromatography of Emetine No.

I 1
Support Paper
Developing solvent Ethyl methyl ketone satd. with 2 N HC1 BuOh: 0.1-N HC1 1:l BuOH:MeOH: HTO 45: 5 :50 B ~ O: H AC OH :H ; O (various ratios Bu0H:formic acid:H20 120:10:70 BuOH :AcOH :Ac OBu i-Bu0H:toluene (satd. H20) 1:l BuOH:AcOBu:Phenol:H20 BuOH:Toluene:AcOH:H20 10:10:5:2 Acetone:AcOEt :10% NH4OH 2: 20: 80 Bu0H:aq. citric acid 87:13
Ref. 62.70.71 72,73 74 72,73,75-79 72,73,80 81 74 81 82 83 33
2 3
5 6 7 8 9 10 11
__"__
--''--
--
--
12
13 14 15
Paper impregnated with 5% sodium dihydrogen citrate Paper impregn. CHC13 or trichloroethylene or with phosphate BuOH saturated with buffers or citrate buffers Paper impregn. Petr.ether or cyclohexane or CHC13 and with HCONH2 Et2NH ---CHC13 or mixtures with aromatic hydrocarbons ----"---Triple development: 1) cyclohexane: benzene 9:l 2) benzene 3) CHC13
----'I
__"--- --- --
__"__
84
85
76,86
87
Table I11 16 Paper impregn with HCONH~/HCOONH~ Paper impregn. with tributyr in Car boxyme t hylcellulose cation exchange paper
(contd.)
CHC13 :
benzene
9 :1
88,89
17
pH 7.4 Phosphate buffer aq. NaCl
33,90
18
91
w c 0 o
310
5.32
Thin-layer chromatography
The chromatographic systems investigated for the analysis of emetine on silica gel plates are presented in Table IV. The spots can be visualized by one of the following methods: -spraying with iodine-chloroform solution and heating to 60" for 10-15 minutes: emetinelemon yellow (under W365nm-7ellow), haelinelight brown (under UV365nm-light blue)
"58
-examination under W365nm (blue) 92 -spraying with 10% ninhydrin in 95% ethanol (no color at room temperature) and heating at: 80" (grey-purpigJ, 120" (red-violet) or 160" (brown-violet) -spraying with 1% chloranil in toluene (brown), heating to 105" fpb315 minutes and spraying with 2N H2SO4 (ochre) -spraying with Dragendorff reagent used also on cellulose or ion-exchange plates) 164-106 -spraying with potassium hexaiodoplatinate (IV) 33,107
(KZPt1(j)
System 12 separates potential impurities present in emetine of natural or synthetic origin: cephaeline, 0methylpsychotrine and isoemetine. System 7 was reported to separate emetine from its thermochemical and photochemical decomposition products. The procedure was developed into an assay by transferring the emetine spot to a column, eluting with 0.2 N - HC1 and determining the emetine concentration at 284 nm. For quantitative purposes, re-washing of the plates with methanol was recommended.9t
T e hromatog53phi behavior of emetine cellulose,lo A1203, 3"' ion-exchangers,105,188 and silica gel-glass powder sintered platesg9 was also investigated.
2 6
311
Tab. IV Thin-Layer Chromatography of Emetine

No.
Developing solvent Chlorofor :methanol (85:15) or (9:l) Benzene:toluene:ethyl acetate: diethy1amine:methanol (35:35:20:10:2) To1uene:ethyl acetate:85% formic acid (50:45:5) To1uene:Z-propano1:conc. ammonia (70:29:1) To1uene:ethyl acetate:2-propanol: 2 N AcOH (10:35:35:20) To1uene:dioxane:methanol: conc. ammonia (25:50 :20:10) CC1 :butanol:met ano1:ammonia ($0:30:30: 2) b? Benzene:ethyl acetate:diethylamine (7:2:1) Ch1oroform:diethylamine (9:l) Ethyl acetate:methanol:conc. ammonia (170: 20: 10) Methano1:conc. ammonia (100:1.5)
Chloroform:2-methoxyethanol: methano1:water:diethylamine
RF 0.3-0.5 0.54 0 0.26 0.05

0 . 9
Ref. 20,64,92 93,94 95 95 95 95 96 97 92,98 18,99 100 33 63

101 101 80 102
2 3
4
5
6 7 8
0.45-0.64 0.67 0.45 0.52
9 10
11 12 13 14 15 16
(100:20:5:2:0.5)
17
18
0 . 1 N MI3 in Methanol Ch1oroform:ace tone :diethylamine (5:4:1) Methyl ethyl ketone:ethanol:ammonia (5:4:1)
Cyc1ohexane:chloroform:diethylamine
Ch1oroform:acetone:methanol (5:4:1) Ch1oroform:acetone:dimethylformamide
0.31 0.52 0.65 0.70 0.70 0.40 0.06
102 92 92
19
a) b,
(5:4:1)
Cyc1ohexane:diethylamine
(9:l)
Single or double development. Single or followed by two-dimensional development with petroleum ether: Et20:EtOH:Et2NH (4:16:2:1)
312
L. VALENTIN FEYNS AND LEE T. G W Y
A study of the optimization of the dansylation reaction, TLC separation of mono-dansyl-emetine and fluorescence detection was reported by the same authors110 who later reported a similar HPLC procedure (see 5 . 3 4 ) .
Emetine and cephaeline were separated by TLC after a preliminary oxidation by Hg(OAfJP to products of characteristic color and fluorescence.
5.33
Gas chromatography
Gas chromatographic methods were described mostly for the s f toxicological extraction residues.SS~fPf'flS A s stationary phase 1-5% SE-30 on silanized Gas Chrom P (100-140 mesh) or Chromosorb W (60-80 mesh) was used, with nitrogen or helium as carrier gas; 5-6 feet stainless steel columns were operated at 170-230". It was reported that apparently emetine hydrochloride dissociated in the injection port ( 3 2 5 " ) since the same retenffpn times were obtained for the salt and the free base.
5.34
High-pressure liquid chromatography
The systems reported in the literature are summarized in Table V. Pre-column derivatization: -dansylation with dansyl chloride; normal phase chromatography (mobile phase--diisopropyl ether:isopropyl alcoho1:conc. ammonia 4 8 : 2 : 0 . 0 3 ) . 121 Post-column derivatization procedures: -fluorescence labeling with dansyl chloride line two-phase "solvent segmentation" flow (reaction time - 16 min at 5 6 " ; excitation emission cut-off filter >450 nm; detection in an onsystem 365 nm, limit 30 ng)
-air-segmented flow, ion-pairing with 9.10dimethoxyanthracene-2-sulfonate and extraction in chlorinated organic solvents. Excitation at 383 nm, emission - 446 nm. ktqearity range 40-600 ng. Limit of detection 0.2 ng. Used in conjunction with system 3 , the detection limit of capacitance-conductance detector was 500 ng of emetine. l a 6
Table V
HPLC Analysis of Emetine
Column Silica Silica Silica LiChrosorb RP 8 u-Bondapak C18 LiChrosorb DIOL Mercaptopropylbonded phase Aliphatic strong cation exchanger
Mobile Phase Ethyl ether (95% water saturated) + 0.5% diethylamine Ch1oroform:methanol or ether:methanol Ch1oroform:methanol:hexane 7:3:10 pH 3.0 0.02 M phosphate buffer:methanol 2:3 Methano1:water ( 5 6 : 4 4 ) or ( 6 0 : 4 0 ) + 0.5% AcOH and 2.5m M octane sulfonate 0.05-0.i M NaHC03:acetonitrile 1OO:O - 70:30 pH 3 . 1 0.0 M phosphate buffer Methanol:2 Mammonium hydroxide:l M ammonium nitrate (27:2:1) M ammonium hydroxide: 1 M ammonium nitrate Methanol:2 (27:2: 1)
Ref.
114
115
116 117 118
119 117 120 120
314
5.35
Electrophoresis
The electrophoretic mobility of emetine on paper in buffers from pH 2.3 to 11.4122-125 and on cellulosecoated glass plates in acidic and alkaline electrolytes126 was studied for separation and identification in alkaloid mixtures. 5.4 Titration
Potentiometric titration of emetine hydrochloride with 0 . 0 1 N NaOH avoids the difficulties of the visual determinatTon of thelgyd-point due to the buffering effect of the organic base. Some compendia1 assays63,128 consisted of extraction of an alkaline solution with ether, back extraction of emetine with H C 1 and titration of the excess of acid. It has been reported that products of photochyyical decomposition of emetine interfere with the method.
I n other official procedures,18,1 2 ' emetine hydrochloride is assayed in glacial acetic acid by titration . 1 N perchloric acid in the presence of with 0 mercuric acetate with crystal violet indicator.130-132 The end-point can be also determined pp53ntiometrically or using p-naphtholbenzein as an indicator.
Emetine was determined in a two phase chloroform. 0 1 M sodium water system by titration with 0 d ioctylsulfo s u , , i n a te using dimethylyellow-Oracet blue as the indicator. Emetine was radiopjlrically titrated by I3lIlabeled Dragendorff reagent.
5.5
Colorimetric and Spectrophotometric Methods
Most colorimetric methods for the determination of emetine involve its extraction from aqueous solution into an organic solvent by ion-pairing with a dye anion. Emetine forms a 1:2 complex with bromothymol blue which can be best extracted with chlorofor aqueous solutions buffered in the pH range of 4.0-5.8. ''ff9' Similar procedures were developed using methyl orange, bromocresol purple, bromocresol green, phenol red, Direct Acid Y : Y 3 5 " 3 ? w Blue, cresol red and bromophenol blue. 5
315
Complications related to the stepwise dissociation of diprotic acids such as bromothymol blue are avoided by using the singly charged tetrabromophenolphthalein ethyl ester. The absorbance o f the red extract in 1,2dichloro thane is measured at 5 7 0 n m (linearity range 2-10ft8-' M (1.1-5.5 pg/ml). The method is less pH dependent. The reaction of emetine with sodium 1,2naphthoquinone-4-sulfonate gives a co ound extractable in chloroform and measurable at 4 6 0 nm. 1TP Emetine is precipitated quantitatively from aqueous solutions as a reineckate, which may be di ~ acetone and determined colorimetrically at 525 m. 9 $ 5 ~ P $in The concentration ranges in which Beer's law is valid or the UV spectrophotometric determinations wa reported for 20 quinoline and isoquinoline alkaloids.f44 Emetine acting as electron donor forms a charge transfer complex with iodine whose absorbance in chloroform at 292 nm is greatly increased over that of the uncomplex and can be used for a spectrophotometric assay. ,
5f 33ka10id
The red shift ( 3 2 2 to 3 5 5 nm) accompanying the ionization of picrolonic acid in the presenc of emetine was developed in an assay sensitive to 2 ug/ml. Emetine forms a colored adduct with picric acid in acetic whose extinction can be measured photometrically.l&ed* The yellow color produced by the oxidation of emetine with ceric ammonium sulfate was measured photometftyally after stabilization with sodium acetate The colored product resulted from the reaction of emetine with benfzjuinone was extracted in CHC13 and measured at 5 4 0 nm. Emetine hydrochloride yields a highly colored condensation complex (Xmax 333 nm) when heated with malonic acid in acetic anhydride. The spectrophotometric method developed on the basi of this reaction has a detection limit of 0 . 0 3 bg/ml. 129
5 . 6
Spectrofluorometric Methods
The fluorescence of emetine has a maximum emission at 318 m with a excitation maximum at 2 8 4 nm. Concentrations in the l o ' M range can be determined (as
316
compared with l o ' M for W determinations) an the decomposition products seem not to interfere." The intensity of fluorescence increases linearly over the concentration range 0.01-1.00 pg/ml; it increases with decreasing pH being maximal in the 1-3 range and it decreases with the increase of temperature (0.5%/degree in the 15-30" range). 150 Emetine Hydrochloride treated with iodine in alcoholic solution gave a gold-colored fluorescence with Xmax at 570 and 620 nm (Xex 436 n m ) . Fluorescence intensity was lifffr with concentration in the range of 0.05-1.00 p.p.m. 5.7 Polaroeraohic Methods
Emetine yields catalytic waves over the pH range 0 . For quantitative determinations the wave at pH 3 3 to 1 has been employed over the concentration range 0.08-0.25 x M (the limiting current is in linear proport on to the concentration) and at pH 8 f o r l j p 0.01-0.1 x l o ' M ran e (calibration curve necessary). Half-wave potential EB/2 = -1.62V. 153
A polarometric titration of emetine after coupling with p-diazobenzene-sulfonic acid was reported.154
5.8
Thermogravimetric Analysis
Thermogravimetric analysis at 5"/min in a N2 atmosphere showed that emetine hydrochloride forms no stable hydrates, water l o s s takes place even at room temperature (the water content will fluctuate with the relative humidijjy) and a slow l o s s continues at temperatures above 105".
6.
Determination in Bioloeical Fluids and Tissues
In the earlier publications, gravimetric (preci i ation with silicotungstic acid; detection limit 20 ug/ml), colorimetric (methyl orange or brpygph 01 blue extraction) -138 were used. and UV spectrophotometric methods
P5l
Th first spectrofluorometric method was reported in 1961.1 5 ' After an extraction procedure adapted to each preparation (plasma, urine, tissue homogenates) a fluorescent compound (rubremetine) is produced by a dehydrogenation reaction with mercuric acetate. (Xex = 365
317
nm; Xem
470 nm).
After extraction by benzene or ether of blood or tissue homogenate samples at an alkaline pH, emetine is taken up in an aqueous acidic solution and determined by measuring the fluorescence 287-318 nm (sensitivity threshold: 0.010.02 pg/ml). 1% After extraction with dichloromethane from human plasma, emetine can be analyzed directly at levels above 500 ng/ml by ion-paired reversed-phase chromatography (see 5.34); by introducing an oxidation step with mercuric acetate between extraction and chromatography, the limit of spectrofluoromet detection is lowered to levels of 10 ng/ml of plasma.
18
Combustion and liquid scint llation counting were used in pharmacoki tics studies of C-labelled emetine in guinea pigs. 1 8 7. Determination in Pharmaceutical Preparations Aqueous titration Depending on the final stage of sample preparation, emetine (or total ether-soluble alkaloids in ipecac) is titrated with 0 . 0 1 or 0.1 N EC1 or the excess acid used in the final extraction is titrat with 0.02- .1 N NaOH. The method is used for ti s tablets,16 ipecac, ipecac powder or extracts.i239sy1g5-165 The buffering effect of the phenolic alkaloids and the yellow color of the extract tending to mask the end-point have been mentioned as disadvantages of the method when applied to ipecac. 166
Non-aqueous titration in glacial acetic acid with 0 . 1 N perchloric acid using crystal violet a ator is used for the assay of emetine formulations. s3Si:Pf A modified procedure distributes the sample over a mixture of magggsium oxide and Celite and elutes the free base with CHC13. N e p h e l ~ m e t r i c and ~ ~ phototurbidimetric168 titrations were also reported. Colorimetric and spectrophotometric methods
In 1942 an author was writing a satisfactory colorimetric method for the determination of [emetine] could not be found...The reaction with hydrogen peroxide in the
318
presence of hydrochloric acid, which produces an orange colour...is y capable of detecting gross errors in dispensing".
na4
The acid-dye technique has been widely used in assaying the alkaloi i emetin f mulations, ipecac powder and tinctures. 1 ' 8 , 1 2 1 , 1 6 6 , 1 ' o p r 2 A s an example of the technique, the complex formed between the alkaloid and methyl orange at pH 5 is extracted with chloroform and treated with 0 . 1 N NaOH to liberate the dye and extract the phenolic alkaloids. The liberated dye determined at 460 nm in the alkaline extract is a measure of the total alkaloids. The non-phenolic alkaloids are extracted from the chloroform phase with 0 . 1 NH2SO4 and their concentration in the acid extract is determined at 283 run and calculated as emetine. The procedure is reported as less time-consuming an equiring less sample than the compendia1 procedures.f6f; After separation from phenolic alkaloids, emetine was assayed in liquid extracts of Ipecacuanha and powdered root by determining the W absorbagte (292 nm) of the chargetransfer complex with iodine. The yellow color arising by the action of iodine in the presence of aqueous sodium acetate was used previously for the deter ation of emetine in ipecac and its galenical preparations.rps9 Phosphomolybdic acid was used to precipitate emetine from ipecac extract. The pref+litates were taken in acetone and assayed colorimetrically. After buffering the injection at pH 9.5, emetine was extracted in ethylene dichloride and interacted with picrolonic acid. The absorbance at 362 (anionic band of picrolonic acid) is used for the assay. 1 8 Pharmaceutical preparations were subjected to dialysis across a cellophane membrane and the amounts diffusing after a fixed time interval wefg5determined colorimetrically using Lautenschlager's method. Powdered ipecac was extracted with MeOH/HCl, the extract evaporated, mixed with basic A1203 transferred to a column and eluted with CHC13. Emetine was determined as the difference between the total alkaloid content (measured at 286 run) and the cephaeline content (determined co metrically with 2,6-dichloroquinone-chloroimide).
w-
319
Ultraviolet spectrophotometric assays of emetine and cephaeline in ipecac became possible after an elaborate four-column chromatographic syste ceous earth in different buffers) was developed. The procedure was adopted as an assay for Ipecac and its syrup fluidextract and powdered eparations in the United States Pharmacopeia'" and the Official Methods of Analysis of The Association of Official Analytical Chemists180
'"st''
Other attempts to extract, purify and separate the alkaloids in ipecac into phenolic and non-phenolic fractions rri d on Fluorisi 181 by c o l y ~ chromatography ~ were 8 ' oxycellulose~84 and A1203, 185ion-exchange resins ,'$3 1 Celite. After oxidation in aqueous solution with acidic KMn04, emetine in pharmayjgtical preparations was assayed fluorimetrically. Chromatographic procedures Thin-lay r chromatograph e arations followed by den~itometric'~~ or spot area" 9 p88 measurements were used for the quantitative determination of emetine in ipecac and its preparations. Emetine-containing syrups and capsules were subjected to derivatization with dansyl chloride. Separations carried out by TLCljf HPLC were followed by fluorometrical determinations.
8.
Stabilitv - Degradation
Solid forms and solutions of both emetine and the hydrochloride turn yellow on exposure to light and heat. The thermal and photochemical stability of s solutions 8 B " ' Y pH 3 being of emetine hydrochloride are pH dependent,1 Cysteine, aminoacetic acid, that of maximum stability. 1 8 ' thioglycolic acid, D-penicillamine, Na2S204, NaHS03, Na2S03, Pb2+ and jsg8&yy2edetate increased the stability of the solution. The following compounds were identified among the products of the photochemical and thermal decomposition of emetine:emetamine, 3,4-dihydro-6,7-dimethoxyisoquinoline, 0methyl-psychotrine, l-methyl-3,4-dihydro-6,7-dimethoxyisoquinoline, tetradehydroemetinium chloride, rubremetinium chloride, 1' , 2 '-didehydroemetine, 2-methyl-3-ethyl-
320
1,4-dihydro-9,10-dimethoxybenzo[a]-quinolizinium chloride, 3-ethyl-1,4-dihydro-9,lO-dimethoxybenzo[a]quinolizinium chloride and a benzoquinolizinium dimer. The fragment products resulted only by photochemical decomposition.? G O n
One of the compounds isolated in the above mentioned study, didehydroemetine, was synthesized in 1961 by etine with mercuric acegate and designated by oxidation of A recent publication proved the structure A . degradation and synthetic products to be identical, on the basis of their UV, IR and mass spectra and their chromatographic behaviour and assigned to didehydroemetine the structure B. The same paper assigns to O-methylpsychotrine structure C , instead of the previously reported structure B.
9.
Toxicitv
Pharmacokinetics
In mice, the following acute LDs0 values were reported: subc taneous - 35 mg/kg2 (32 mg of base/kg 195 oral - 3 0 mg/kgy3,195 and intraperitoneal - 62 mg/kg. rats, y d e r intraperitoneal administration, LDz0 is 12.1 The therapeutic dose in men is 1 mg/ g body weight mgfkg. dailyigyubcutaneously, a course of treatment lasting ten days. The toxic dose by accumulation is between 1.1 and 1 . 8 g6. 700 mg is considered to be the fatal dose in 33 humans.
Emetine is rapidly absorbed and is distributed mainly
321
in the liver (high concentrat ondrial s : f 5 6 : l % : l % ! f ? 8 $ Low concenfraction), kidney and spleen.f trations were found in the brain, in agreement with unsucces f 1 attempts to treat amebic cerebral abscess with e r n et ine f 6y
Emetine does not appear to be metabolically transformed and it is slowly excreted, which may account for the cumulative toxicity (dehydroemetine is eliminated more rapidly). In contradiction with previous findings about emetine being primarily excreted in urine, a 1965 study reported th t after intraperitoneal injections of guinea pigs with "C-labelled emetine, 95% of the injected radioactivity was recovered from the feces, while only 5% appeared in the urine. It was suggested that emetine passes into the bowel through the gastro-intestinal wall where rather high concentrations were found rather than through the bile. In humans, excretion all other routes than urine is reported to be negligible. Excretion in urine begins 20 m Utes after injection and continues for as long as 2 months. $3 Acknowledgments The authors wish to thank Dr. J. A. Kelley, Laboratory of Medicinal Chemistry and Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland for the acquisition and help in the interpretation of the mass spectral data, Vivian A. Gray for the technical assistance and Ann K. Ferguson, Barbara Bowman and Patricia Perando for processing the manuscript.
322
10.
References F. b . Flkkiger, "Pharmacographia. A History of the Principal Drugs of Vegetable Origin, met with in Great Britain and British India," Macmillan and Co., London, 1 8 7 9 , p. 3 7 0 . E . F. Elslager in "Medicinal Chemistry," Third Edition, Part I, p . 5 3 0 , A. Burger, Editor, Wiley-Interscience, New York, 1 9 7 0 . W. Lewis, "The Pharmacopoeia of the Royal College of Physicians at Edinburgh," London, 1 7 4 8 , p. 3 7 . "The Pharmacopoeia of the United States of America," Second Edition, S . Converse, New York, 1 8 3 0 , p. 4 0 . The Pharmacopoeia of the United States of America," Ninth Decennial Revision, J. B. Lippincott, Philadelphia, 1 9 1 6 , p . 133. L. Holmes, eds.), Vol. 3 , Chap. 2 4 , Academic Press, New York, 1 9 5 3 , p. 3 6 3 .
1.
2.
3.
4.
5.
6.
M. M. Janot in "The Alkaloids" (R. H. F. Manske and H.
7.
5, 8.
G. E. Foster and G . W. Norgrove, J. Pharm. Pharmacol., 480 ( 1 9 5 3 ) .
F. H. Carr and F. L . Pyman, J. Chem. SOC., 1 0 5 , 1593

(1914).
9.
a. E. E. Van Tamelen, P. E. Aldrich and J. B. Hester, J. Amer. Chem. SOC., 81, 6214 ( 1 9 5 9 ) ; b . A . R. Battersby et al. ~J. Chem. SOC. 1 9 5 9 , 2 7 0 4 ,
3474, 3512.
10.
S .
C.
Pakrashi and P . P. Ghosh-Dastidar, Indian J.

2 , 379 ( 1 9 6 4 ) .
Chem., 11.
H . Budzikiewicz, S. C. Pakrashi and H. Vorbrhggen, 2 0 , 399 ( 1 9 6 4 ) . Tetrahedron, "Martindale, The Extra Pharmacopeia," 27th Edition, A. Wade, Ed., The Pharmaceutical Press, London, 1 9 7 7 , p. 7 7 , 601.
12.
323
13.
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T. Kazunori and M. Ono, E i s e i Shikenso Hokoku, 1979 93, 54055 (1980). ( 9 7 ) , 21; C.A. -
M.
S . Habib and K.
J. H a r k i s s , P l a n t a Med.,
18, 270
(1970). 189.
C.A.
G . Bayraktar-Alpmen, E c z a c i l i k Bul., 13 ( l ) , 1 (1971); 75, 67447 (1971). -
190. V. S p r i n g e r , M. S t r u h a r , Z. Zembiakova, and M. Mandak, Farm. Obz., 45 ( 9 ) , 391 (1976); C.A. 90, 12252 (1979). 191. M . S t r u h a r , F. Kubek, V. S p r i n g e r , M. C h a l a b a l a , and M. Mandak, Acta Fac. Pharm. Univ. Comeniane, 7 90, 210008 (1979). (1977); C.A. -
31,
192.
C. Schuyt, G . M. J. B e i j e r s b e r g e n van Henegouwen, and K. W. Gerritsma, Pharm. Weekbl., 112 (43), 1125 (1977); C.A. 88, 41601 (1978). C. S c h u i j t , G . M. J. B e i j e r s b e r g e n van Henegouwen, and K. W. Gerritsma, Pharm. Weekbl., S c i . Ed. 1 ( l ) , 186 91, 57253 (1979). (1979); C.A. -
193.
194.
H. Auterhoff and W. J a c o b i , Arch. Pharm.,
294, 591
Tomich,
(1961). 195. 196.

K. J. C h i l d , B. Davis, M. G. Dodds, and E. G. J. Pharm. Pharmacol., 16, 65 (1964).
H. H. Miller and W.
R. J o n d o r f , J. Pharm. Pharmacol.,
22, 659 (1970). 197. A. Marino, Chemotherapia, 5, 56 (1962).
335
198.
D. E . Schwartz and J. Rieder, B u l l . SOC. Path. e x o t . , 54, - 38 (1961).
199.
P. Synek, Cas. Lek. Cesk., 113 (28), 82, 92862 (1975). -
856 (1974);
C.A.
For t h i s p r o f i l e , the l i t e r a t u r e has been searched through Chemical Abstracts Vol. 93 (1980).
GLIBENCLAMIDE
Pamela Girgis Takla
1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance, Odour, Colour 1.3 Therapeutic Category 1.4 Usual Dose Range 2. Physical Properties 2.1 Melting Range 2.2 Solubility 2.3 Infrared Spectrum 2.4 Nuclear Magnetic Resonance Spectrum 2.5 Ultraviolet Absorption Spectrum 2.6 Mass Spectrum 2.7 pKa 3. Synthesis 4. Stability 5. Drug Metabolism and Pharmacokinetics 6. Methods of Analysis 6.1 Polarography 6.2 Non-Aqueous Titration 6.3 Chromatography 7. Identification and Determination in Pharmaceuticals 8. Identification and Determination in Body Fluids 8.1 Extraction 8.2 Ultraviolet Spectrophotometry 8.3 Colorimetry 8.4 Fluorimetry 8.5 Thin-Layer Chromatography 8.6 Gas-Liquid Chromatography 8.7 High Performance Liquid Chromatography 8.8 Radioimmunoassay 9, References 338 338 338 338 338 339 339 339 339 339 339 343 343 343 344 346 348 348 348 348 348 349 349 349 349 350 350 350 351 35 1 352
338
PAMELA GIRGIS TAKLA
GLIBENCLAMIDE
1.
Description 1.1 Name, Formula, Molecular Weight
G1ibenclamide is 1- (4-(2-(5-chloro-2-methoxybenzamido)ethyl) benzenesulphonyl)-3-cyclohexylurea. It is also known2 as 5-chloro-N- (2- ( 4 - (( ((cyclohexy1amino)carbonyl) amino) sulphonyl)phenyl) ethyl)-2-methoxybenzamide and as 1- ((p- (2(5-chloro-o-anisamido)ethyl)phenyl) sulphonyl)-3-cyclohexylurea.
C 0 N H CH2C H 2
c1
Ooc':?
Molecular Weight
=
494.0
Synonyms: Glybenzcyclamide; Glyburide; HB419; U 26,452. Proprietary Names: Daonil; Euglucon; Diabeta; Maninil; Lisaghicon; Glidiabet; Euclamin; Gilemal. 1.2 Appearance, Odour , Colour
Glibenclamide is a white, crystalline, odourless powder; practically without taste.

1.3
Therapeutic Category Oral hypoglycaemic.
1.4
Usual Dose Range 2.5 to 20mg once daily.
GLIBENCLAMIDE
339
2.
Physical Properties 2.1 Melting Range
This has been reported as 172-17403 ; 169-1704; and 168-1705. 2.2 Solubility
Glibenclamide is virtually insoluble in water and ether; soluble in 330 parts of alcohol, in 36 parts of chloroform, and in 250 parts of methanol. It forms water-soluble salts with alkali hydroxides. 2.3 Infrared Spectrum
Fig. 1 shows the infrared spectrum of a sample of glibenclamide supplied by Hoechst Pharmaceuticals recorded from a potassium bromide disc using a Perkin-Elmer Model 357 grating spectrometer. The spectrum is in agreement with published spectra The major peaks are at 1163, 1333, 1471, 1515, 1613 and 1724cm-l. According to the findings from a study of the infrared spectra of a number of s u l phonylurea derivatives, assignments for the peaks observedfor glibenclamide can be made as follows: 3363 and 3313 cm to urea N-H stretch; 1515 cm- to urea, amide 11; 1333 cm- to -S02-N-; 1163 cm- (split peak) to -S02-. Salt formation has been reportedgto decrease the intensity of many of the absorption maxima.
2.4
The NMR spectrum (Fig. 2) for glibenclamide in dimethylsulphoxide-D6 (DMS) was obtained using a Perkin-Elmer R32 (9OMHz) spectrometer. The assignments made on the figure agree with those published by HajdG et a1.6, who show also the signal produced by the -SOz-NH proton (offset) at 10.27 ppm. The -CO-NH- proton observed in DMS as a doublet at 6.27ppm, disappears when the spectrum is determined using trifluoroacetic acid as solvent. 2.5 Ultraviolet Absorption
lo
The ultraviolet absorption spectra for glibenclamide shown in Fig. 3 were determined in 0.01M methanolic hydrochloric acid using lcm silica cells with a Pye-Unicam SP 1800 spectrophotometer. Absorbance measurements at the wavelengths of maximum absorption were made with a Pye-Unicam
a,
a
a ,
rcl
H
k c d k
c a
ri
I
ri
<
a
.rl
t-aJ
I z
n
0 " cnv
-u-uV
1 " I
I
0
aJ
a ,
E : f j
I
k
(d
a,
0 G
Figure 3.
Ultraviolet absorption spectra f o r glibenclamide in 0.01M methanolic hydrochloric acid.
GLIBENCLAMIDE
343
SP 500 Series 2 spectrophotometer. The sample of glibenclamide used showed ne ligible loss of weight on drying to constant weight at 105% ( l e s s than 0.05% of its weight). The compound shows a characteristic curve with maxima at 229.4nm (A 1%,lcm about 600), 275nm (A 1%,lcm 29.6) and 300.4nm (A 1%, lcm 63.5). In 0.1M sodium hydroxide, the spectrum lcm about 480), 274nm (A 1%, shows maxima at 226nm (A 1%, lcm 23) and 300nm (A 1%, lcm 53).
2.6
Mass Spectrum
The mass-spectral fragmentation pattern for glibenclamide has been described and discussed by HajdG et a1.6, who used a CEC 21-llOB instrument to obtain the spectrum. The principal peaks were observed at m/e 394, 368, 352, 288, 198, 169, 125, 99 and 82. 2.7
pKa
Glibenclamide is a weak acid. I t has been concluded6 that it has the same dissociation constant as tolbutamide (5.340.1), since both compounds show the same dissociation at half-neutralisation in solvent mixtures such as methyl cellosolve and water or methanol and water. The direct determination of its pKa in water is impossible owing to its low solubility. 3. Svnthesis
Various procedures have been patented for the synthesis4 1 1 - 1 4 of glibenclamide or its intermediates. The synthesis o f glibenclamide has been discussed in a review by Kantolahti and Malkonen who cite the following example: c1
t-BuOC1 OCH3 Cl OCH3
OCH3
344
PAMELA GIRCIS TAKLA
CI
OCH3
@
c1 c1 OCH3 c1
CONHCH2CH2
HOS02C1 CHC13,0-5
OCH3
=>
OCH3 c1
(@
C O N H C H 2 C H 2 0 S02NHCONH
-()
OCH3
4.
Stability
A test is specified in the British Pharmacopoeia 19801, using thin-layer chromatography on silica gel GF254 with chloroform-cyclohexane-ethanol-glacial acetic acid (9:9:1:1) as mobile phase, to limit the amounts of 4-(2-(5-chloro-2-
GLIBENCLAMIDE
345
rnethoxybenzamid0)ethyl)benzenesulphonamide (I), ethyl N-4( 2 - (5-chloro-2-methoxybenzamido) ethyl) benzenesulphony1-N-
methylcarbamate (11) or related substances which may be present as impurities in glibenclamide or glibenclamide tablets. The spots are observed under an ultraviolet lamp at about 254nm.
c1
OMe
c1
Wiseman et a1.I6 in a study of sulphamylurea hypoglycaemic agents have postulated that an initial protonation is probably the rate determining step in the hydrolysis of sulphonylureas as follows:
RSO~NH-C-NHR'
II 0
H+
RSO~NH-C-NH~R' ) -H20
0
I1
+
0% I RS02NH-G-6H2R'
j
+
OH2 I RS02NH-C
NH2R'
1
0-
346
PAMELA GIRGIS TAKLA
Thus, in the case of glibenclamide, the hydrolysis products have would be (I) and cyclohexylamine. Kuriki et a1 descr bed a procedure for the determination of glibenclamide and i s decomposition products, in which cyclohexylamine is first extracted into isoamylacetate from aqueous alkaline solution, and determined by a colorimetric procedure based on the Spingler method of assay using 2,4-dinitrofluorobenzene (DNFB). The aqueous solution is subsequently acidified and extracted into organic solvents to allow the determination of glibenclami.deby heating with DNFB to produce a yellow colour, and the determination of glibenclamide plus (I) by ultraviolet spectrophotometric measurement at 299nm. Poirier et al. have reported that (11) forms gradually from glibenclamide in methanol or chloroformmethanol (1:l) even at room temperature, and observed that the British Pharmacopoeia test for impurities should be completed immediately after the test solution (in chloroformmethanol) has been prepared. The characterization and structure of (11) formed by refluxing Flibenc1ami.de with There are no methanol has been proved by synthesis2 reports that glibenclamide shows instability under normal storage conditions. A report of l o s s of strength in tablets stored for six weeks at 20 and 75% relative humidity has however been made. 21
5.
Drug Metabolism and Pharmacokinetics
Since plasma levels of glibenclamide are general1 low, most metabolic studies have been carried out using the y4C labelled drug, although in some recent work radioimmunoassay has been used. Pharmacokinetic parameters have been estimated from a single compartment model from investigations in and in man2425. The closest similarities with man were observed in the rabbit. In man, 45% of a single oral dose of 5mg was absorbed, and peak blood concentrations of 0.044+0.004ug per ml (0.089+0.008 nanomoles per ml) were attained. Other studiesz6 2 7 however revealed a practically complete intestinal absorption, and it has been shown28-30 that the bioavailability of glibenclamide is dependent upon particle size. Fuccella et al. 3 1 reported that absorption was complete within 30 to 60 minutes after administration of a 5mg tablet containing micronized glibenclamide. Maximum plasma concentration^^^-^^' 3 1 32 are usually attained within 2 to 4 hours, and are in the range 120 to 360ng per ml after a single 5mg oral dose. KO et a1.33 found peak levels to occur within 3 to 8 hours. Dose response curves show that the decrease in blood sugar which occurs is limited, and
GLIBENCLAMIDE
347
higher doses only increase the duration of effect34. After a single intravenous injection, the initial biological halfLife period was 2 3 minutes, but the half-life under steady state conditions was 6.6 hoursz4. The increase in halflife which occurs with time makes it difficult to predict drug levels after multiple doses32. The drug is widely distributed throughout the body, and does not accumulate in the blood. Its apparent volume of distribution, owing to its lipophilic nature, is 10 to 11 litres31. It shows no substantial binding to blood cells, but is more than 99 per cent bound to serum proteinsz4. The binding of glibenclamide to plasma has been studied extensively by equilibrium dialysis3 3 5 and by a fluorescence probe technique36. Brown and Crooks have studied the effect of different salts and buffers37 and of various anionic on the binding, which was found to occur by a non-ionic mechanism. No Cotton effects are generated by glibenclamide bound to albumin39. The absorbed drug is completely metabolized, 95% of a single oral dose being excreted within 5 days in similar amounts in urine and f a e c e ~ ~ Metabolism ~ ~ ~ probably takes place in the liver32. Metabolites of glibenclamide are formed by hydroxylation of the cyclohexyl ring at positions 3 and 4 to give 4-trans-hydroxyglibenclamide (the principal metabolite) Both metabolites have been and 3-cis-hydroxyglibenclamide. identified in blood, but were without hypoglycaemic effect at the levels found. A third metabolite has been found in trace amounts in urine, but not identified24y 32. 4-Transhydroxyglibenclamide is about 5 or 6 times less effective than glibenclamide in the rat40-42, and the metabolites are eliminated rapidly with a half-life of 1 2 minutes provided renal function is Attempts have been made to fit the disposition of glibenclamide in man3 and in the dog44 into a two-compartment model with a first-order absorption rate. In another pharmacokinetic study, Balant et al. 32 have made a detailed comparison o f their findings with other published results, and found that the kinetics involved were too complicated to be resolved adequately by such a model. They suggested instead a third hypothetical slowly equilibrating deep compartment, in which the drug could accumulate during long term therapy. Further evidence in favour of this approach is cited in a later report45 for which radioimmunoassay was used to measure glibenclamide levels. Comparative studies of the metabolic parameters of various sulphonylureas including glibenclamide have been made in rabbits46 and in Happ et al? measured glibenclamide levels by radioimmunoassay in a comparative study in adult diabetics. Some reviews on glibenclamide metabolism have been p~blished~-~.
348
PAMELA GIRGIS TAKLA
6.
Methods of Analysis 6.1 Polarography
Procedures have been described by S i l v e ~ t r i ~ ~ ~ For and by T a m m i l e h t ~ ~ ~ . quantitative work, an automated system, having a flow through micro cell used with a silversilver chloride reference electrode, has been stated5 to give good reproducibility. 6.2 Non-Aqueous Titration
Tetramethylurea has been used as solvent for the titration of glibenclamide with 0.1 N lithium methoxide in 55 benzene-methanol. The end-point was determined potentiometrically or by using 0.2% azoviolet in toluene as visual indicator. Tablet excipients generally were found not to interfere with the assay. Alternatively, the assay can be performed by titration with 0.1 N potassium hydroxide in dimethylformamide solution with thymolphthalein as indicator56. 6.3 Chromatography
Several procedures65 7-62 have been proposed f o r the identification o f glibenclamide by thin-layer chromatography. Among the solvent systems described are butanolmethanol-chloroform-25% ammonia5* , propanol-cyclohexane5 and propanol-benzene-cyclohexane5
High-performance liquid chromatography has been recommended by Beyer6 for the quantitative determination of glibenclarni.de in tablets. The column packing used was 1% ethylene propylene copolymer on DuPont Zipax, with 0.01 M sodium borate containing 27.5% vfv methanol as mobile phase. Testosterone serves as internal standard. An impurity, 5-chloro-N-(p-sulphamoyl-phenethyl)-o-anasimide, was eluted as a separate peak.
7.
Identification and Determination in Pharmaceuticals
Identification tests for glibenclarni.de given in the British Pharmacopoeia depend upon: a) its infrared absorption spectrum; b) its light absorption in the range 230 to 350nm; c) the evolution of fumes having a pungent, arnine-like odour which change moistened red litmus paper to blue after boiling with 6M sodium hydroxide solution; and d) positive tests for chloride and sulphate in an aqueous
GLIBENCLAMIDE
349
extract of the residue obtained after igniting glibenclamide with anhydrous sodium carbonate and potassium carbonate. The identification tests for glibenclamide in tablets depend upon: a) light absorption measurements in the range 230 to 350nm; and b) thin-layer chromatography on silica gel GF254 with chloroform-cyclohexane-ethanol (96 per cent)glacial acetic acid (9:9:1:1 parts by volume) as mobile phase. Glibenclamide is assayed' by titration in ethanol with 0.1M sodium hydroxide using phenolphthalein solution as indicator, and protecting against exposure to atmospheric carbon dioxide. Glibenclamide tablets are assayed' by a spectrophotometric procedure which depends upon extraction of the tablets with 0.1M methanolic hydrochloric acid, and measurement of absorbance at about 300nm.
8.
Identification and Determination in Body Fluids 8.1 Extraction
Glibenclamide is extracted from aqueous acid solution or acidified plasma or serum by chloroform 6'64,65, ethyl acetate6, amyl acetate66, toluene6 and benzene6'. Alternati~ely~l, plasma can be deproteinized with acetone, the acetone evaporated to small volume and extracted with chloroform after dilution with pH 4.5 buffer solution. Balant et al. 3 2 were not successful in separating glibenclamide from its metabolites using a procedure that they had found applicable to glipizide which involved adjusting the pH of plasma to 4.3 with acetate buffer, and extracting with methylene chloride.
8.2
Ultraviolet Spectrophotometry
A procedure for glibenclami.de in serum has been described by HajdG et a1.6, but is insufficiently sensitive for normal applications. 8.3 Colorimetry
A modification6 of the colorimetric procedure reported by Spingler'' for tolbutamide in serum involves heating glibenclamide in amyl acetate with 2,4-dinitrofluorobenzene to 150' for 5 minutes. Absorbance is measured at 380nm. The method can only be used for glibenclamide when it is present in much higher concentrations than those normally encountered in serum.
350
PAMELA GIRGIS TAKLA
8.4
Fluorimetry
Glibenclami.de when excited by radiation of wavelength 290nm emits a weak fluorescence which can be measured in 0.1 M sodium hydroxide at 350nm. HajdG et a1.6 have described a procedure for serum, but the method has never been successfully applied. The detection limit for gliben. 4 1 1 per ml, and plasma clamide in aqueous alkali is about 0 blanks are likely to be high6'. Beckerg6 has reported that his fluorimetric procedure which was developed for glibornuride in plasma can be applied to glibenclamide. The lower limit of detection for glibenclamide is about 40ng per ml. The fluorescence is developed by heating an amyl acetate extract of the drug at 1 4 0 ' for 15 minutes with 7-chloro-4nitrobenzo-2-oxa-1,3-diazole (NBD chloride). The reaction depends upon the degradation of glibenclamide to give 4- (2- (5-chloro-2-methoxybenzamido) ethyl)benzenesulphonamide and cyclohexylamine. The latter compound reacts with NBD chloride present in excess to produce a fluorescent product. The reagent itself is non-fluorescent. There are no other reports of this method having been used for the determination of glibenclamide. 8.5 Thin-layer Chromatography
Balant et al.32 used silica gel plates with benzene-glacial acetic acid-ethyl acetate-acetone (65:6:12:30) for methylene chloride extracts of urine and plasma.
8.6
Gas-Liquid Chromatography
A procedure65 employing a column packed with 5% OV-17 on 80-100 mesh Chrom G-AW-DMCS has been used for the determination of glibenclamide in the plasma of healthy adults after oral administration of 5mg of the drug. The method involves derivatization of the glibenclamide by heating with 2,4-dinitrofluorobenzene in amyl acetate at 13OOC for 1 hour. A 3Ni electron-capture detector was used. The procedure was found to be specific for glibenclamide, and not subject to interference by metabolites. 4-Hydroxyglibenclamide could be determined qualitatively by a slight modification of the gas chromatograph parameters. The quantitative determination of glibenclamide was carried out using tolbutamide as internal standard. The lowest detectable amount of glibenclamide was 1OOpg. Plasma concentrations found 1, 3 and 5 hours after the administration of the drug are reported. They range from 0.05 to 134.76 ng per ml.
GLIBENCLAMIDE
351
8.7
High-Performance Liquid Chromatography
Several procedures have been reported in recent months 67,68,70 which are sufficiently sensitive for clinical 67 assays. Adams and Krueger mix canine serum with monobasic sodium phosphate solution and extract with toluene containing butyl-p-hydroxybenzoate as internal standard. The extract is evaporated to dryness, and the residue is dissolved in the h.p.1.c. mobile phase which is 50mM-NH H PO -acetonitrile 4 2 4 (1:l). Chromatography is carried out on a reversed-phase column of Lichrosorb RP-8. Detection is at 228nm. The lower glibenclamide detection limit wasabout20ng per ml of serum extracted. The major metabolites in the dog, 3-cis(1- [ (4- (2-(2-methoxy-5-chlorobenzamido) ethyl) phenyl) sulphonyl) ureido)cyclohexanol, 1- ( (4-carboxyphenyl)sulphonyl)-3cyclohexylurea, and 2-methoxy-5-chlorobenzamide did not interfere. The main metabolites of glibenclamide in human serum are also stated not to interfere. Another procedure6 developed for glipizide which has been found applicable also to glibenclamide uses a VBondapak c18 column. Glibenclamide was extracted from serum with benzene after acidification to pH3. The mobile phase was 30% 0.01 M phosphate buffer (pH 3.5) in 70% methanol. Glibornuride served as internal standard. Reinauer et a1. used an RP18 column with CH3CN-H PO4 (45:55) as mobile phase for the determination of gllbenc?amide in blood serum of diabetics. 8.8 Radioimmunoassay
A number of radioimmunoassays have been developed which have the desired sensitivity for metabolic studies. Some of t h e ~ e ~ l do - show ~ ~ cross-reaction with the two major metabolites of glibenc1ami.de. The radioimmunoassay developed by .Kawashima et al. 7 4 is however stated not to be subject to interference from these metabolites, although the closely related hypoglycaemic drug, glipizide, does show significant cross reactivity. The antiserum is produced in rabbits immunized with an antigen prepared by conjugating the diazonium salt of N-(p-amino-benzamidoethy1)-benzenesulphonylN-cyclohexylurea to bovine serum albumin through diazocoupling. Dextran coated charcoal is used to adsorb the free glibenclamide, and separate it from the bound drug. It was found possible, with this procedure, to determine as little as 2.5ng per ml of glibenclamide in plasma by using l o p 1 samples without the need for extraction. Results obtained with dog plasma samples were comparable with those obtained by the less sensitive liquid chromatography method. The paper gives also the results of plasma assay carried out in diabetic
352
PAMELA GIRGIS TAKLA
Lindner et al. 76 ,77 out in connection with this pr~cedure?~. have compared the determination of glibenclamide in the serum of diabetics by radioimunoassay and high-pressure liquid chromatography. References
1.
patients on glibenclamide treatment. A patent has been taken
"British Pharmacopoeia 1980", H.M. Stationery Office, London, 1980, 210, 773. "The Merck Index", Ninth Edition, Merck & Co. Inc. , 2. U.S.A., 1963, 4311. Aumueller, W., Bander, A,, Heerdt, R., Muth, K., 3. Pfaff, W., Schmidt, F.H., Weber, H. and Weyer, R., Arzneim.-Forsch., 1 6 , 1640 (1966) Neth. Pat. Appl. 6,603,398, Sept. 19, 1966; Ger. Appl. 4. March 16 and Aug. 26, 1965 (to C.F. Boehringer and Soehne); Chem. Abstr., 66, 65289h (1967) Weber, H., Aumueller, W., Weyer, R., Muth, K., Schmidt, 5. F.H., Ger. Pat. 1,283,837, Nov. 28, 1968 (to Farbwerke Hoechst A.-G.); Chem. Abstr., 70, 47140f (1969) HajdG, P., Kohler, K.F., Schmidt, F.H. and Spingler, H., 6. Arzneim.-Forsch., 19, 1381 (1969) "The Pharmaceutica~CodeX",Eleventh Edition, The 7. Pharmaceutical Press, London, 1979 Abdel-Wahab, M.F., El-Kinawy, S.A., Farid, N.A. and 8. El-Shinnawy, A.M., Anal. Chem., 38, 508 (1966) Khlapopina, L.N. , Moroz, V.V. , Kgm.-Farm. Zh. , 10. 9. 132 (1976); Chem. Abstr., 85, 68196d (1976) Kuhnert-Brandstatter, M. , Kofler, A. and Kramer, G. , LO. Sci.Pharm., 42, 150 (1974) 11. Neth. Pat. A E l . 6,610,580, Jan. 30, 1967; Ger. Appl. July 27, 1965 (to Farbwerke Hoechst A.-G.); Chem. Abstr., 68, 1273311 (1968) 1.2" Hung. Tei-fes 6063, April 28, 1973, Appl. CI-1112, May 6, 1971 (to Chinoin Gyogyszer es Vegyeszeti Termekek Gyara Rt.); Chem. Abstr., 2, 104974t (1973) 13. Vogt, B.R., Bernstein, J., Weisenborn, F.L., Fr. Demande 2,138,112, Feb. 2, 1973; U.S. Appl. 144,678, May 18, 1971 (to Squibb, E.R. and Sons, Inc.); Abstr., 2, 18452q (1973) 14. Weber, H., Aumueller, W., Weyer, R., Muth, K,, Stach, K., S . African Pat. 68 05, 127, Jan. 20, 1969; Ger. Appl. Aug. 12, 1967 (to Farbwerke Hoechst A.-G.); Chem. Abstr., 71,91085m (1969) 1.5. Kantolahti, E., Malkonen, P . J . , Suom. Kemistilehti A, 46, 75 (1973) -
e.
GLIBENCLAMIDE
16. 17.
18. 19.
353
20.
21.
22. 23. 24. 25. 26

I
27. 28.
29.
30. 31. 32. 33. 34.
35. 36. 37.
Wiseman, E.H., Chiaini, J . , Pinson, Jr. R., J. Pharm. Sci. 53. 766 (1964) Kuriki, T., Tsujiyama, T. and Suzuki, N., Bunseki Kagaku, 2, 872 ( 1 9 7 4 ) ; Chem. Abstr., 82, 35085b (1975) Spingler, H., Klin. Wschr., 1 9 5 7 , 3 5 , 533. Poirier, M.A. , Black, D.B. and Lovzing, E.G., 5 . J . Pharm. Sci., 15, 8 (1980) Chubb, F.L., Simmons, D.L., Can. J. Pharm. Sci., 7 , 28 (1972) Izgu, E. and Kafali, N., Bilim Kongr. Tip Arastirma Grubu Tebligleri, Turk. Bilimsel Tek. Arastirma Kurumu, 6th. 227 (1977) (Pub. 1 9 7 9 ) ; Chem. Abstr., -20601m(1980 j Kellner, H.M., Christ, O., Rupp, W. and Heptner, W., Arzneim.-Forsch., 19, 1388 (1969) Heptner, W., Kellner, H.M., Christ, 0 . and Weihrauch, D. , Arzneim.-Forsch., 1 9 (Suppl.) , 1 4 0 0 (1969) Christ, O.E., Heptner,W. and Rupp, W., Horm. Metab. Res. Suppl., 1_, 5 1 (1969) Rupp, W . , Christ, 0 . and Heptner, W., Arzneim.-Forsch 1 9 . 1 4 2 8 (1969) Rupp, W., Christ, 0. and Fulberth, W., Arzneim.-Forsch., 22, 4 7 1 (1972) Schmidt, H.A.E. and Petrides, Pl., Arzneim.-Forsch., 19, - 1 4 2 2 (1969) Fr. Pat. Appl. 2 , 2 0 4 , 4 2 5 , May 24, 1 9 7 4 ; Ger. Appl. P 22 5 3 3 1 8 . 2 , Oct. 3 1 , 1 9 7 2 , (to Farbwerke Hoechst. A.-G ) ; Chem. Abstr., 83, 33027f (1975) Rothe, W., Heinemann, H., Schmidt, F.H. and Betzien, G . , Ger. Offen. 2 , 3 4 8 , 3 3 4 , March 2 7 , 1 9 7 5 , Appl. P 2 3 48 3 3 4 . 3 , Sept, 2 6 , 1 9 7 3 ; Chem. Abstr., 83, 8 4 8 7 1 j ( 1 9 7 5 ) Borchert, H.H., Mueller, H. and PfeifG, S., Pharmazie, 31, 307 (1976) Fuccella, L.M., Tamassia, V, and Valzelli, G., J. Clin. Pharmacol., 13, 6 8 (1973) Balant, L., Fabre, J. and Zahnd, G.R., Europ. J. clin. Pharmacol.. 8. 6 3 (1975) KO, H., Royer, M . E , , Molony, B.A., Excerpta Med. Int. Congr. Ser. 382, 20 (1975) Bander, A., Pfaff, W., Ritter, K., Wohlfahrt, A. and Schmidt, F.H., Proc. Tegernsee Conference on the New Oral Antidiabetic Agent HB419, 27th-29th Jan. (1969) Crooks, M.J and Brown, K.F. , J. Pharm. Pharmac., 26, 304 (1974) Hsu Par-Lin, Joseph, K.H. and Luzzi, L.L., J . Pharm. Sci., - 63, 570 (1974) Brown, K.F. and Crooks, M.J., Can. J. Pharm. Sci., 9, 7 5 (1974)
L
I _
354
PAMELA GIRGIS TAKLA
38. 39. 40.
Brown, K.F.
and Crooks, M . J . ,
Biochem. Pharmacol.,
25, 1175 (1976)

M u e l l e r , W.E., W o l l e r t , U., R e s . Commun. Chem. P a t h o l . Pharmacol., 551 (1976) Samimi, H . , Loutan, L . , B a l a n t , L . , T i l l o l e s , M . , Fabre, J . , Schweiz. Med. Wochenschr., 107, 1291 (1977) Loutan, L . , Samimi, H., B a l a n t , L . , Favre, H . , F a b r e , J . , Schweiz. Med. Wochenschr., 108, 1782 (1978) B a l a n t , L . , F a b r e , J . , Loutan, L . , S a m i m i , H . , Arzneim. -Forsch., 29, 162 (1979) Schmidt, T H . , H r s t k a , V . E . , X I I e Congres I n t e r n a t i o n a l d e Therapeutique, Geneve 1973. C a s t o l d i , D . , Chinea, B . , T o f a n e t t i , O . , Farmaco, 2 7 1 (1978) Ed. P r a t . , B a l a n t , L . , Zahnd, G.R., Weber, F. and Fabre, J . , Europ. J . c l i n . Pharmacol., 11, 19 (1977) Fukuchi, H . , T s u k i a i , S . , K u G g a i , M. and K i t a u r a , T . , Hiroshima J. Med. S c i . , 2, 269 (1977) B i g l e r , F . , Rentsch, G., R i e d e r , J . , Denes, A . , J o u r n e e s Annu. D i a b e t o l . Hotel-Dieu, 333 (1973) Happ, J . , Nest, E., F r o e h l i c h , A . , S c h o e f f l i n g , K . , Beyer, J . , Verh. Dtsch. Ges. Inn. Med., 82, 776 (1976) Bander, A , , A u s t r a l i a n and New Zealand J F M e d . , 1 (Supp. 2 ) , 22 (1971) Chabria, N.L., Proc. Asia Ocean. Congr. E n d o c r i n o l . 5 th, 2 , 432 (1974) K a i s e r , D . G . , F o r i s t , A.A., Excerpta Med. I n t . Congr. S e r . 382, 31 (1975) S i l v e s t r i , S., Lucr. Conf. Nat. Chim. Anal., 3 r d , 1, 1 3 (1971) S i l v e s t r i , S . , Pharm. Acta Helv., 47, 209 (1972) Tammilehto, S . , Farm. Aikak., 82, 140 (1973) Agarwal, S.P. and Walash, M . I . , I n d i a n J . Pharm., 34, 109 (1972) Eichhorn, A. and Wagler, M., Z e n t b l . Pharm., Pharmakother. u . Lab.-diagnostik, 111, 1049 (1972) Guven, K . C . , B e r g i s a d i , N . , E c z a c i l i k Bul. 1 2 , 30 (1970) Thielemann, H., S c i . Pharm., 4 1 , 70 (1973) Surborg, K.H. and Roeder, E . , Pharmazie, 28, 485 (1973) Agarwal, S.P., Walash, M . I . , Blake, M . I . , I n d i a n J . Pharm., 35, 181 (1973) Walash, M . I . and Agarwal, S . P . , J . Drug Res., 5, 217 (1973) Schmidt, F., Dtsch. Apoth.-Ztg., 1 1 4 , 1593 (1974) Beyer, W.F., Anal. Chem., 4 4 , 1 3 1 2 1 9 7 2 ) Clarke, E.G.C., " I s o l a t i o n a n d I d e n t i f i c a t i o n o f Drugs" Volume 2 , The Pharmaceutical Press, London, 1975, p . 1045.
2,
41.
42. 43. 44. 45. 46.
33,
47.
48.
49. 50. 51. 52. 53. 54. 55. .56
_ -
57. 58 59. 60. 61. 62. 63. 64.
GLIBENCLAMIDE
355
65. 66. 67. 68. 69. 70.
Castoldi, D. and Tofanetti, O., Clin. Chim. Acta, 93, Becker, R., Arzneim.-Forsch., 27, 102 (1977) Adams, W.J. and Krueger, D.S., J . Pharm. Sci., 2, Wahlin-Boll , E. and Melander, A., J. Chromatogr., 164, 541 (1979) Chroneos, I.D., Ph.D. thesis, University of Wales, June 1979 J.. Fresenius 2 . Reinauer, H a , L ndner. G.. Oldendoem. _ , Anal. Chem., 301, 110- (1980) Glogner, P . , Burmeister, P. and Heni, N., Klin. Wochenschr., 51, 352 (1973) Royer, M.E., KO, H., Evans, J.S., Johnston, K.T., Anal. Lett., 9 , 629 (1976) Glogner, P., Heni, N., Nissen, L., Arzneim.-Forsch.,
27, 1 7 0 3 (1977) 221 (1979)
1138 (1979)
1 9 5 (1979)
71. 72.
73.
74.
75. 76.
Kawashima, K., Kuzuya, T., Matsuda, A., Diabetes, 2 8 , Kawashima, K. and Kuzuya, T., Jpn. Kokai Tokkyo Koho 79 0 5 , 9 5 0 , Jan 17th, 1 9 7 9 , Appl. 77169,048, June llth, 1 9 7 7 ; Chem. Abstr. 9 0 , 1866372 (1979) Lindner, G . , Reinauz, H. , Kontrolle Plasmaspiegel Pharmaka, Workshop Rahmen Kongr. Laboratoriumsmed. 7 5 (1979) (Pub. 1 9 8 0.) . , Ed. Rudolf Sommer. Lindner, G., Herbertz, L., Reinauer, H., Laboratoriumsmedizin. 4. 34 (1980)
77 *
Dorothy K . Wyatt and Lee T. Grady

1. Description 1.1 Heroin 1.2 Heroin Hydrochloride 2. Physical Properties 2.1 Infrared Spectra 2.2 Nuclear Magnetic Resonance Spectra 2.3 Ultraviolet Spectra 2.4 Mass Spectrum 2.5 Melting Range 2.6 Differential Scanning Calorimetry (DSC) 2.7 Solubility 2.8 Moisture Content 2.9 Specific Rotation 2.10 Crystal Properties 2.11 Polymorphism 3. Synthesis 4. Stability-Degradation 5. Metabolism 6. Pharmacokinetics 7. Methods of Analysis 7.1 Elemental Analysis 7.2 Color Tests 7.3 Microcrystalline Tests 7.4 Non-Aqueous Titrimetric Analysis 7.5 Chloride Titration 7.6 Phase Solubility Analysis 7.7 Thin-Layer Chromatography 7.8 Paper Chromatography 7.9 Gas Chromatography 7.10 High-Performance Liquid Chromatographic Analysis 8. Determination in Biological Fluids References 358 358 358 359 359 359 366 370 370 370 370 373 373 374 374 374 377 377 377 379 379 379 380 380 381 38 1 38 1 381 388 388 388 397
ANALYTICAL PROFILES OF DRUG SUBSTANCES, 10
357
358
DOROTHY K. WYATT AND LEE T. GRADY
1.
Description 1.1
1.1.1
Heroin Name. Formula. Molecular Weieht Heroin is 3,6,-diacetoxy-7,8-dehydro-4,5 3,6,-diacetoxy-7,8-dehydr.0-4 ,5 epoxy,egistry no. is 561-27-3 [l]. The CAS I registry :
-. N-methylmorphinan t.hylmorphinan [4]. N-me -.
2 ' 1H23N05 molecular weight 369.4 OCCH3 1.1.2 ADDearance. Color. Odor
White crystals which turn pink and emit an acetic odor on prolonged exposure to air [ 2 ] . 1.1.3 Synonyms [Z] Acetomorphine Diacetylmorphine Diamorphine
7,8-Didehydro-4,5a-epoxy-l7-methylmorphinan-
3,6a-diol diacetate (ester)
1.2 1.2.1
Heroin Hydrochloride Name, Formula, Molecular Weight
Heroin hydrochloride is 3,6-diacetoxy-7,8dehydro-4,5 epoxy-N-methylmorphinan hydrochloride mono4 ] . The GAS registry no. i s 1502-95-0 [l]. hydrate [
&
cl-. N20 molecular C21H23N05-HC1 weight H20423.9
CH3CO0
OCCH3
HEROIN
359
1.2.2
Appearance. Color. Odor
The hydrochloride is an almost white, crystalline powder, odorless when freshly prepared but develops an odor characteristic of acetic acid on storage [50,51]. 1.2.3 Synonyms 3,6-di-O-acetylmorphine hydrochloride monohydrate [50] Diacetylmorphine hydrochloride [SO] Diamorphine hydrochloride [ 501 7,8-Didehydro-4,5a-epoxy-l7-methylmorphinan 3,6a-diol diacetate (ester) hydrochloride monohydrate 121 2. Physical Properties 2.1 Infrared Spectra
The infrared spectra are presented in Figure 1. The spectra were obtained from potassium bromide and potassium chloride dispersions of previously dried material ( 1 0 5 ' , constant weight) using a Beckman 5260 grating infrared spectrophotometer. Principal bands are 1765, 1740, 1450, 1370, 1250, 1180 cm-I [61]. 2.2 2.2.1 Nuclear Magnetic Resonance Spectra Proton Spectrum The proton spectra are presented in Figure 2 .
.rl
0 k
e ,
a,
r; 0
.ri
c
0 k
c
w
0
a,
362
F i g . 2a.
P r o t o n NMR s p e c t r u m of heroin base.
. _ A , -
1200
Fig. 2b.
P r o t o n NMR s p e c t r u m of heroin h y d r o c h l o r i d e .
HEROIN
363
Spectral assignments are listed in Table I Table I H ' NMR Spectral Assignments for Heroin [ 4 8 ] Chemical Shift PPm ( 6 )
2.27, 2.11 2.88 3.33 3.70
Multiplicity
Characteristic of proton acetyl N-methyl 10
5.45
5.21
a
596 7 1 2
5.73
6.87 6.66 2.2.2
Carbon-13 Spectrum The carbon-13 spectra is presented in Figure 3.
Spectral assignments are listed in Table XI. Table I1 I3C NMR Spectral Assignments for Heroin 1711 Chemical Shift
119.1
Multiplicity d
Carbon Number
1
2
121.6
132.0
d
S
. t i
0 k
c
k
Q,
0 E
k c,
0
d)
co
a
m
d
0 P
r:
(d
..
m
(d
364
P
.rl
P)
c 0
k 0
2
.A
0 k
c
k
Q,
's
i
365
366
DOROTHY K. WYATT AND LEE T. CRADY
Table I1 -- Cont'd. Chemical Shift

149.1 88.5 67.9 129.2 128.2 58.7 20.4 131.5 131.2 42.6 40.4 34.9 46.3 42.8 20.4 168.2 20.4 170.2 2.3
Multiplicity
S
Carbon Number
4 5 6 7 8 9 10 11 12 13 14 15 16 NCH3
3CH3CO 3CH3CO
d d d d d
t
S S
S
d
t t 4 4
S
4
S
6CH3C0 6CH3CO
Ultraviolet spectra
The ultraviolet absorption spectra of heroin and heroin hydrochloride are shown in Figure 4 for the solvents listed in Table 111 (1 in 10,000 solutions used). Table 111 Solvent
0.1 N hydrochloric
X M a x (nm)
Heroin
4.8
Absorptivity Heroin HC1

4.3 [53]
278
[5];
acid
0.1 N sulfuric acid 279
1% =
cm
39 [41 4.3 [53]
4.8
[53];
1% =
cm
52 141 4.9 [53]
ethanol
281
5.3
[53];
El% = 54 [68] 1 cm
HEROIN
367
Fig.
4a.
U l t r a v i o l e t spectrum i n 0.1 N hydrochloric a c i d ( r e p r e s e n t a t i v e of h e r o i n o r h e r o i n hydrochloride).
368
Fig.
4b. U l t r a v i o l e t s p e c t r u m i n 0 . 1 N s u l f u r i c a c i d ( representative o f heroin o r heroin hydrochloride 1 .
HEROIN
369
--4
Fig.
4c.
U l t r a v i o l e t spectrum i n 95% ethanol ( repr e s e n t a t i v e of h e r o i n o r h e r o i n hydrochloride).
370
2.4
Mass Spectrum
The electron impact ionization spectrum is given in Figure 5, and the fragmentation pattern is presented in Table IV [6]. A Finnigan 3000 Peak Identifier mass spectrometer was used. The masslcharge (m/e) range scanned was 40 to 400 atomic mass units. The ionization potential was 70 eV. Table IV
Mass Spectrum Fragmentation Pattern of Heroin [47, 541
m/e 369 327 268 310
Species M+ COCH2 M+ - CH3CO0 and COCH2 M+ - CH3CO0 (c6 acetyl group) cleavage followed by of peripheral groups
2.5
Melting Range
The melting point of a heroin sample is about 170" 1 4 1 . I t is also given as 173C [ 2 ] . Additional melting ranges of 170"-172"C, 171"-174"C, and 172"-173C and a melting point of 173C have also been reported [24]. (See 2.11.) The melting range of a heroin hydrochloride sample is between 229" and 233" [51,4]. It is also described as 243-244" [5J
2 . 6
Differential Scanning Calorimetry (DSC)
The DSC of heroin and heroin hydrochloride are shown in Figure 6 [53J
2 . 7
Solubility
The approximate solubilities obtained at room temperature are listed in Table V.
4 . l h
-....
L ,
CO
a
E
a,
372
--I~
I .
1
F i g . 6 a . D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y o f heroin base h e a t i n g r a t e : 5 / m i n . ; 25 t o 200 C.
I
j
A,
- 1
f\
. .
I
I
F i g . 6 b . D i f f e r e n t i a l s c a n n i n g c a l o r i m e t r y of heroin h y d r o c h l o r i d e h e a t i n g r a t e : 5 / m i n . ; 2 5 t o 300 C .
HEROIN
373
Table V Solubility Data of Heroin at Room Temperature Approximate Solubility (gm) Heroin Heroin Hydrochloride
1700 [2,4] 3 1 [2,4] 100 [2,4] 1.5 [2,4] soluble [ 2 ]
1 in 1 in 1 in 1 in
Solvent water ethanol ether chloroform alkali

2.8
1 in 1.6 [51,4]; 1 in 2 [ 5 2 ] 1 in 12 [51,4]; 1 in 1 1 [52] insoluble [51,4,52] 1 in 1.6 [51,4]
Moisture Content Karl Fischer Titration
2.8.1
An accurately weighed sample of heroin or heroin hydrochloride is dissolved in methanol which has been titrated to end-point and titrated with Karl Fischer reagent using the dead stop end-point technique and a 20-second delay (heroin hydrochloride exists as the monohydrate) [ 5 3 ] .
2.8.2
Loss on Drying
Heroin has been dried at 105" to constant weight

[51.
Heroin hydrochloride has been dried at 105" to constant weight [ 5 1 ] .

2.9
Specific Rotation
The specific rotation of heroin determined in 0.015 N methanolic hydrochloric acid at a concentration o f 0.5% a t 25C is given in Table VI [5,53]. Table VI Specific Rotation data Wavelength (nm)
589 578 546 436
[a]
25"
Specific Rotation ( " ) Heroin [ 5 ] Heroin Hydrochloride [ 5 3 ]

-147 -154 -175 -303
-1 33 -139 -159 -27 5
374
The specific rotation of heroin in methanol [a]i5" is -166" (C = 1.49) [ 2 ] . The s e ific rotation of heroin hydrochloride in water at 24" [a]!56 is -156" (C = 1.044) [2]. 2.10 Crystal Properties
The crystal structure, configuration and bond distances are presented in Figures 7 , 8, and 9. Heroin crystals were formed after addition of heroin hydrochloride t o aqueous sodium acetate solution. Clear hexagonal crystals of diacetylmorphine free base were obtained. Reflections were measured with a Syntex P2 diffractometer with a 0-20 technique on a crystal 0.7 x 0.2 x 0.2 mm [49]. 2.11 PolvmorDhism Heroin can exist in two polymorphic forms. Form
melting point of 172"-173C. Form 11, consisting of spherulites, melts at about 168C and is readily converted into Form I [24].
3.
I, consisting of rods, oblique plates, and needles, has a
Svnthesis
1.
Synthesis from morphine [5].
HEROIN
Fig. 7. Arrangement of the molecules in the unit cell ( 0 oxygen). From an origin in the lower left front corner, c is to the right, b is vertical and a is into the page.
Fig. 8. Configuration drawing of diacetylmorphine with endocyclic torsion angles for rings A, B y C and D.
Fig. 9 . Bond distances ( A ) . The estimated average standard deviation in bond length is 0.014 A in diacetylmorphine.
376
DOROTHY K . WYATT AND LEE T. CRADY
2.
Synthesis from Sinomenine [ 2 9 ] .

Ti
,c1!3
catalytic
S "
reduction
sinornenine
ec;;
HEROIN
377
4.
Stability
Degradation
Heroin is rapidly hydrolyzed in alkaline solutions. is rapidly hydrolyzed in vivo after mixing with blood to 0monoacetylmorphine and then at a slower rate t o morphine. Heroin also degrades to 0 6Inonoacetylmorphine in buffered aqueous solutions (pH 7.4) at 23C. The hydrolysis is more rapid at higher pH value (pH 6 . 4 ) . No evidence of further conversion to morphine at pH 7.4 is observed in 24 hours [7]. Heroin hydrolyzes to 06-acetylmorphine in 0.5 M sodium carbonate with a half-life Tf only 4.2 min. Subsequent hydrolysis to morphine has a half-life of 55.5 min. 191. Half-life for hydrolysis in human blood is 12.6 min.; in serum, 19.8 min. [9]. In pH 4 phosphate buffer, the halflife was 415 min.; in fresh dog plasma, 8 min. [9]. Heroin stability increases with increased alcohol content in Brompton mixtures [28]. Heroin reportedly is most stable at pH 4.0-4.5 [28] and at pH 4.3 [39]. 5 . Metabolism
it
Heroin is a short-acting (2 hours) narcotic analgesic. It is rapidly hydrolyzed in vivo by serum cholinesterase [38] to 06-monoacetylmorphine and then at a slower rate to morphine-[4]. Heroin rapidly passes out of the blood [32] after conversion to 06-monoacetylmorphine and appears in the brain as 06-monoacetylmorphine where it is sLowly hydrolyzed to morphine. Heroin and 06-monoacetylmorphine have a considerably greater ability to penetrate the blood brain barrier than does morphine which is the probable explanation 6monoacetylfor the higher potency of heroin [38]. 0 morphine, morphine, and morphine 3-glucu~onideare the major metabolites of heroin excreted in the urine [9]. Minor or negligible amounts of normorphine and its glucuronide as well as morphine 6-glucuronide have been determined in urine [9]; dihydromorphinone [26], 6-acetylmorphine 3-glucuronide [26], and norcodeine [17] have also been detected in urine. Additional possible metabolic pathways of opiates in man are presented in Figure 10 [17]. 6. Pharmacokinetics
Heroin is rapidly hydrolyzed to morphine and other metabolites and is rapidly excreted. Heroin, 6-acetylmorphine, morphine, the sum of morphine and 6-acetylmorphine and total normorphine, determined 24 hours after initial IV administration of a 10 mg/70 kg dose, was found to be 0.5, 1.5, 7.2, 54 and 4%, respectively, of administered dose.
Excretion products
-free morphine morphine conjugates --free nomorphine normorphine conjugates
ii
iv
I\ICH3
16
H
iii
--free norcodeine norcodeine conjugates

4
ii
--free codeine codeine conjugates
CX30
OH
CH3O
OH
Figure 10: P o s s i b l e metabolic pathways of o p i a t e s i n man(ixP1-methylation; ii=EI-dimethylation; i i i = 0-methylation; i v = 0-demethylation)
HEROIN
379
Eighty-eight percent of free morphine and 84% of the total morphine (including morphine glucuronide) found in urine was excreted within the first eight hours [ 4 7 ] . Of the morphine found in the urine, 88% was bound as the glucuronide and 11% was free morphine [ 6 2 ] (50-60% bound and 7% free [ 2 6 ] ) . The amount of heroin detected in the urine as morphine after a single intramuscular injection of 5 mg is about one third that detected after a single intramuscular injection of 1 5 mg of morphine.
7.
Methods of Analysis
7.1
Elemental Analysis Heroin Theoretical % [ 2 ]

68.28 6.28 3.79 21.66
Heroin carbon hydrogen nitrogen oxygen chlorine

7.2
Heroin HC1 (anhydrous) Theoretical % [ 2 ]

62.14 5.96 3.45 19.71 8.74
--__
Color
Color tests Ref.

496
Agent
1.
sulfuric acidformaldehyde (Marquis) ammonium molybdate
purple (sensitivity 0.05 wg) red-purple-bluelight green (sensitivity 0.05 L i g ) faint blue-gray (sensitivity 1.0 r i g ) faint yellow & faint yellow/orange 1 8 ) (sensitivity 1.0 $ yellow green deep green purple
2.
3.
ammonium vanadate Vitali's test
4.
5.
nitric acid base (heroin HC1)
20,51
6. Mecke test
7.
6
42
cobalt thiocyanate
380
Agent
8.
Color
Ref. 51
sulfuric aciddeep blue potassium hexacyanoferrate (111)iron (111) chloride heroin HC1) nitric acidphosphoric acid 7 . 3 yellow to red brown (depending on concentration)
9.
57
Microcrystalline Tests Crystal type needles hexagon needles needles blades and needles blades and needles amorphous precipitate followed by irregular dichroic plates or blades Ref.
20 20 6,20 6,20 6,20
Agent mercuric iodide sodium acetate platinum chloride goId bromide mercuric chloridehydrochloric acid iodine-potassium iodide bromauric acid-phosphoric acid-hydrogen bromide
70 70
7.4
Non-aqueous Titrimetric Analysis
An accurately weighed sample of heroin is dissolved in glacial acetic acid and titrated to the potentiometric end-point with 0.1 N acetous perchloric acid, using a glass indicating electrodeand a calomel reference electrode filled with 0 . 0 2 N lithium chloride in glacial acetic acid. The titrant i s standardized against dried potassium biphthalate. A blank titration is run [5]. An accurately weighed sample of heroin hydrochloride is dissolved in glacial acetic acid; mercuric acetate is added. The solution is titrated to the potentiometric end-point with 0.1 N acetous perchloric acid using a glass indicating electrodeand a calomel reference electrode filled with 0.02 N lithium chloride in glacial acetic acid. The titrant i s standardized against dried potassium biphthalate. A blank titration is run [53]. Crystal violet indicator may also be used [Sl].
HEROIN
38 1
7.5
Chloride Titration
An accurately weighed sample of heroin hydrochloride is dissolved in 1.5 N sulfuric acid and titrated to the potentiometric end-point with 0.1 N silver nitrate using a silver indicating electrode and a mercurous sulfate reference electrode. The titrant is standardized with dried sodium chloride and a blank titration is run [ 5 3 ] .
7.6
Phase Solubility Analysis
The United States Pharmacopeia procedure was followed [ 5 2 ] . The heroin solvent was (3:l) hexane:dioxane (solubility 13 mg/g) [ 5 ] . Dioxane was used for heroin hydrochloride (solubility 26 mg/g) [ 5 3 ] . The solvents were commercial distilled-in-glass solvents which had been degassed prior to mixing. The bath temperature was 25O, rotation was 28 rpm.
7.7
Thin-layer Chromatography
Thin-layer chromatography has frequently been used for the analysis of heroin. Methods of detection and solvent systems are listed in Table VII.
7.8
Ascending paper chromatography was accomplished using Whatman #l paper which had been buffered by dipping into a 5% solution of sodium dihydrogen citrate, blotting, and drying at 25" for 1 hour. The solvent consisted of 4.8 g of citric acid in a mixture of 130 ml of water and 870 ml of 1-butanol. 2.5 p 1 of a 1% solution in 2 N acetic acid, 2 N hydrochloric acid, 2 N sodium hydroxide, o r ethanol were spotted on the paper. Visualization was accomplished using ultraviolet light or iodoplatinate spray (Rf = 0.33) [ 4 ] . Reversed phase ascending paper chromatography was conducted using Whatman 81 or 83 paper impregnated by dipping into a 10% solution of tributyrin in acetone and drying in air. Acetate buffer pH 4.58 was used as solvent. Samples were spotted from a 1 to 5% solution in ethanol or chloroform. Iodoplatinate spray was used for detection (Rf = 0.84) [ 4 ] . An additional reversed phase ascending paper chromatography system consisting of phosphate buffer (pH 7 . 4 ) solvent and Whatman "1 or #3 paper impregnated with a
Table VII Thin-layer Chromatography of Heroin Plate silica gel silica gel silica gel
w
Solvent methano1:aqueous ammonia (100:1.5) aqueous ammonia-benzenedioxane-ethanol (5:50:40:5) acetic acid-ethanol-water (30:60:10)
benzene-dioxane-ethanol25% aqueous ammonia (1O:g:l:l)
Method of Detection A,B,C,D,E,J B A,B,C,D A,B,C,D A,B,C,D A,B,C,D

A
Rf x 100
45, 38, 50 76, 60 35,44 ,50 , 35,35 46,76
Ref.
4 ,5 ,20 ,56
---
4 , 20 4,5,20,21, 22 5,21
silica gel F-254 silica gel F-254 cellulose silica gel silica gel
methanol
38
72 44 85
5 5
2-propanol-water-glacial acetic acid (8:l:l)

butyl ether-ethyl etherdiethylamine (45:45:10) chloroform-dioxane-ethyl acetate-aqueous ammonia (25:60:10:5) chloroform-methanol (9:l)
6
6
silica gel
A,C,J
61,--
6,56
Table VII Plate silica gel silica gel. silica gel HPTLC-silica gel F-254 HPTLC-silica gel F-254 silica gel G+ 0 . 1 M KOH silica gel G+ 0.1 M KOH silica gel G+ 0.1 M KOH silica gel+ 0.1 M NaHS04 silica gel+ 0 . 1 M NaHS04 methanol 95% ethanol Solvent chloroform saturated with ammonia-methanol (18:l) chloroform-methanol (8:2) chloroform-cyclohexanediethylamine (8:10:3) toluene-methanol-aqueous ammonia (50:50:1) 2-propanol-n-heptaneaqueous ammTnia ( 5 0 : 5 0 : 1) cyclohexane-benzenediethylamine (75: 15:10)
--
Cont'd. Rf x 100
70
Method of Detection
Ref. 6
A
E
9,67
18
60
19 19 21 21 21 21 21
10 22 39
20
-------------
24
Table VII -- Cont'd. Plate silica gel silica gel cellulos e (previously dipped in 5% sodium dihydrogen citrate and dried one hour) silica gel silica gel silica gel MN-cellulose powder 3 0 0 G MN-cellulose powder 3 0 0 G Solvent
methanol-n-butanol-benzenewater ( 6 0 y 1 5 : 1 0 : 1 5 )
Method of Detection
E
Rf x 100
35,35
Ref.
21,22 21,22
61
ethanol-pyridine-dioxanewater ( 5 0 : 2 0 : 2 5 : 5 )
37,37 43
citric acid ( 4 . 8 g) in water-n-butanol (130:870) -
A,E, K
k ? . P
alcohol-n-butyl etherwater ( 8 0 : 7 : 1 3 )
n-butanol-glacial acetic
15,15
61,61 32,32
21,22 21,22 21,22
acid-water ( 4 : 1 : 2 ) n-butanol-concentrated HC1
saturated with water ( 9 : l )

90,90 95,65 21,22 21,22
methanol-n-butanol-benzenewater ( 60y15 :10 :15)
t-amyl
alcohol-n-butylether-water ( 8 0 : 7 : 1 3 )
Table V I I
P
--
Cont'd. Rf x 100
67
Plate
Solvent
chloroform-dioxane-ethyl-
Method of Detection
E
Ref.
21
silica gel
acetate-aqueous ammonia
(25 :60: 10: 5)
silica gel
ethanol-chloroform-dioxanepetroleum ether-benzeneaqueous ammonia-ethyl acetate

(5:10:50:15:10:5:5)
73
21
silica gel
W 0 00 7
ethyl acetate-benzene-aqueous ammonia (60:35:5) ethanol-dioxane-benzeneaqueous ammonia (5:40:50:5) acetone-methanol-aqueous ammonia (50:50:1) chloroform-acetone-aqueous ammonia (50:50:1)
ethanol-dioxane-benzeneaqueous ammonia (5:40:50:5)
19
76
21
silica gel silica gel silica gel silica gel silica gel
22
58
23
23
64
76
23
ethanol-di-n- bu tyl etheraqueous ammonia (60:35:5)
11
23
Table V I I Plate silica gel Solvent chloroform-acetone (9:l)
--
Cont'd. Rf x 100 Ref.

56
Method of Detection A,C,J
--
Method of Detection
A.
B. C. D. E. F.
G.
H.
I. J. K .
shortwave ultraviolet light longwave ultraviolet light 0.5% iodine in chloroform acidified iodoplatinate followed by exposure to ammonia vapor potassium iodoplatinate Dragendorff's reagent followed by heat (120OC) for 5 minutes and spraying with 10% sulfuric acid. potassium permanganate cobalt thiocyanate bromocresol green iodine in methanol + copper chloride (Ludy-Tenger) dilute hydrochloric acid
HEROIN
387
10% solution of tributyrin in acetone was used. The samples were spotted using a 1 to 5% solution in ethanol or chloroform. Iodoplatinate spray detection was used (Rf =
0.12) [4].
Paper chromatography was also conducted using the systems in Table VIII. Table VIII Paper Chromatography of Heroin Paper + Treatment Solvent
*
Ref.
21
Detection Rfxl00
Whatman #1 impreg- chloroform nated with formaldehyde and 1% acetic acid Whatman #l impregnated with 5% zirconium phosphate Whatman #I.
-----
76
5% acetic acid
-----
33
21
l-butanolCYE,G,H,I glacial acetic acid-water

(12: 3 :5)
74
21
Whatman #I.
1-butanol-1 N CyEyG,HyI sodium acetate1 N hydrochloric acih (7:120:60) l-butanolCYEY GY H,1 glacial acetic acid-water
(12:3: 5)
89
21
Whatman #l buffered with 5% sodium dihydrogen citrate Whatman #1 buffered with 5% sodium dihydrogen citrate
32
21
1-butanol-1 N C,E,G,H,I sodium acetate1 N hydrochloric acid (7:120:60)
16
21
*Spotting solvent was
not given.
388
7 . 9
Gas Chromatography
Gas chromatographic systems used for analysis are listed in Table IX. Flame ionization detection was used unless otherwise specified.
7.10
High-Performance Liquid Chromatographic Analysis
High-Performance Liquid Chromatography has been used extensively for the analysis of heroin. The various HPJX systems used for the analysis are given in Table X8 . Determination in Biological Fluids
P1asma- Serum Organic solvent extracts were obtained with ethyl acetate-isopropanol (85:15), or benzene-butanol (85:15) from . 9 carbonate buffer. The extracts plasma mixed with pH 8 were dried and reconstituted in acetone and analyzed on a bonded phase column using a mobile phase consisting of methanol (0.1% (NH4)2C03 and 0.01 M (NH4)2HP04, pH 6.98) (55:45) and W detection (254 n m ) . Heroin, 6-0acetylmorphine, and morphine were separated [9]. Similarly, TLC was used for separation of heroin, morphine, 6-0acetylmorphine and morphine glucuronide [9] on silica gel plates using chloroform-methanol (80:20) and iodoplatinate for visualization. Blood Heroin has been analyzed by gas chromatography following the extraction scheme presented for urine below [62]. Silyl derivatives was analyzed on QF-1 or SE-30. Urine The following procedure was used for the quantitation of heroin and morphine by gas chromatography. Extraction from sodium bicarbonate solution into ethyl acetate is followed by extraction with 0 . 0 5 N hydrochloric acid. Ammonium hydroxide, sodium chloride, and sodium bicarbonate are added to the hydrochloric acid solution. Extraction with ethyl acetate, silylation and analysis on QF-1 and SE30 using flame ionization detection follows [62]. Metabolites in urine were also determined after acid hydrolysis and silylation followed by gas chromatography [26]. An alternative analysis using thin-layer
Table IX Gas ChromatoeraDhv of Heroin Column 1% SE-30 2.5% SE-30 5% SE-30

3% ov-1 3% OV-17 3% ov-1
SUDDOrt Anakrom ABS Chromosorb WAW HMDS Chrom0so rb WAW Gas Chrom Q
Mesh 100-120 80-100
Length 6ftx4mm glass 5ftx4mm glass
Temp. ( " ) 250 225
Flow (ml/min);
Carrier Gas
Ref.
80; argon
50; nitrogen
30; nitrogen
5 0 ; helium
4 4
4,21 5
60-80
100-120
5 ft x 118" 230 stainless steel 1.2 m x 4 mm glass 6ftx4mm glass 6ftx4mm glass 0.6 m x 4 mm glass
1.83 m x 4 mm glass
210 250 250 220 220-240
--------------Chromosorb GHP Gas Chrom Q
----
6
6
----
10% ov-1*
3% OV-17*
---100-120
40; 5% methane in argon

100-120; nitrogen
7 8,14
Table IX -- Cont'd.
Flow (ml/min);
Column
0.04% SDBS 0.02% FFAP 0.06% SP-525
Support glass beads Gas Chrom Q Chromosorb W H P Chromosorb W Chromosorb W Supelcoport Supelcoport Supelcoport
Mesh
70-90
Length
1 . 6 m x 3 mm glass
1 m x 6.35 mm 0.d. glass
Temp. ( " )
240
Carrier Gas
0.5 kg/cm2; nitrogen 55; nitrogen
Ref.
13
16
18
3% OV-17
3% OV-17
W CD 0
80-100
235
3%
ov-1
80-100 80-100 100-120 100-120 100-120
mm 0.d.
1.2 m x 6.35 glass
250 280 250 255 230, 245
60; nitrogen 60; nitrogen 60; helium 50 ; he1ium
20 20 55 55
3% OV-17 5% SP-2401-DB 5% SP-2401-DB
1.2 m x 6.35 mm 0.d. glass 1.2 m x 2 mm i.d. glass 1.2 m x 2 mm i.d. glass 1.2 m x 2 mm i.d. glass
3% SP-2401-DB
55
Table IX -- Cont'd. Column

3% OV-25
Support Gas Chrom Q
Mesh
80-1 00
Length
1.8 m x 3.18 mm stainless
Temp. ( " )
240
Flow (ml/min); Carrier Gas

30; nitrogen
Ref. 20
steel
6% Dexsil 400 Gas Chrom Q
80-100
1.8 m x 3.18 mm stainless steel 1.83 m x 4 mm glass 1.83 m x 3 mm glass
240
30; nitrogen
20
1% SE-30
W
Chromosorb W
-----
210, 225, 250 175, 200, 225
(argon 6-ionization)
t ion)
21
E
0.1% polyethylene glycol 9000 + 1.15% SE-30
----Chromosorb P washed with concentrated HC1 and methanolic potassium hydroxide and treated with hexamethyldisilazane
Gas Chrom P
100-120 80-100
(argon B-ioniza-
21
1% Hi EFF8B
0.92 m x 3.2 glass
220, 250 200, 250
HF I
21
3% cyclohexChromosorb W ane dimethanol succinate
5ftx4mm i.d.
60; nitrogen
61
Table IX -- Cont'd. Column Support Chromosorb W H P Chromosorb WHP Varoport 30 (OV-17) Chromosorb WAW
(SE-30)
Mesh 100-120 100-120 80-100
Length
Temp. ( " ) 218 218
Flow (ml/min); Carrier Gas
Ref.
3% SE-30**
2.7% QF-1
3% OV-175% SE-30 (1: 1)
0
5.3 ft x 2 mm i.d. 9 ft x 2 m i.d.

6 ft x 2 i.d.
UUII
95% of controller; 62 nitrogen
60% of controller; 62 nitrogen

65
co w
temperature 30 ml/min; helium program: 250 (12 min); 10/ min, 280 (12 min) 225
40 ml/min; helium
3.8% UCW-98
Chromosorb W H P
80-100
6 f t x 4 mm
67
* **Derivatized Derivatized
with heptafluorobutyric acid anhydride in acetonitrile, 5 min., with BSA.
60C.
Table X High-Performance Liquid Chromatographic Systems for Heroin Column SCX (1.0 m ) Mobile Phase
0.4-1.4 M sodium perchloratein 0.01 M pH 6.8 aqueous phosphate buffer containing 10% ethanol
Flow/ Temperature Pressure

35"
Detector ( A nm)
Ref.
5
1000 psi
UV ( 2 5 4 )
bonded phase
% w
LiChrosorb Si60 (10 vm)
methanol-(0.1% ammonium carbonate 0.01 M ( N H 4 ) HP04) pH 6.% ( 6 : 4 3 ; ( 5 5 : 4 5 )

0.2 N aqueous ammonia
ambient
2 ml/min
uv
(--)
ambient ambient
3 ml/min
1.5 ml/min
------UV ( 2 8 0 )
12 15
UBondapak c18 acetonitrile-(aqueous buffer containing 0.75 g ammonium acetate) (65:35) LiChrosorb Li 6 0 ( 5 ~ ) diethyl ether-isooctane-methanoldiethylamine ( 5 2 . 8 : 35:12:0.2)
ambient
1.5 ml/min UV ( 2 5 0 ) 175 bar pressure
18
Table X Column UBondapak c18 Mobile Phase
--
Contd. Detector (A nm) UV (235) Ref. 28
Flow/ Temperature Pressure ambient
M monobasic
acetonitrile-(0.015 potassium phosphate adjusted to pH 3.0 with 2 N phosphoric acix) (1 :3)
0 . 8 mljrnin (620 psi)
pBondapak c18 acetonitrile-(0.015 M (heroin KH2P04 adjusted to hydrochloride) pH 3.5 with 2 N phosphoric acid) (3:7) Whatman acetonitrile-water Partisil-10 with 0.1% (NH4)2C03 ODS (heroin (6:4) hydrochloride) UBondapak C18
ambient
1 ml/min
(800 psi)
UV (235)
39
ambient
2 ml/min
UV (254)
40
(50% methanol/0.05 M phosphate buffer p H 6.2)-methanol; 0-100% methanol, l%/min linear gradient
(50% methanol/0.05 M phosphate buffer p H 7.4)-methanol; 0-100% methanol, l%/min linear gradient
ambient
1.2 ml/min
UV (254)
54
UBondapak c18
ambient
1.2 rnljmin
UV (254)
54
Table X Column Zipax SCX Mobile Phase (0.2 M H3BO3 adjusted to pH 9.7 with 40% sodium hydroxide)-(0.2 H3B03acetonitrile-z-propanol (86:12: 2) adjusted to pH 9 . 8 with 40% sodium hydroxide); 0-100% linear gradient hexane-(chloroformmethanol-diethylamine (100:300: 1 ) ) gradient chloroform-methanol (9:1, 8:2, 7:3) diethylether-methanol (8:2, 7:3, 6:4)
--
Cont'd. Detector (A nm)

UV (270)
Flow/ Temperature Pressure ambient 2 ml/min
Ref. -
60
Corasil I1
ambient
600 psi
UV (254)
64
01 CD
Merckosorb Si-60 Merckosorb Si-60
20" 20"
50-250 kg/cm2 50-250 kg/cm2
UV (254)
UV (254)
68
68
396
chromatography with a 1-butanol-acetic acid-water (35:3:10) system on silica gel plates and ethyl acetate-methanolammonium hydroxide (17:2:1) on silica gel plates has also been reported [26]. Plates were visualized using iodoplatinate spray. Morphine metabolites in urine were also analyzed after incubation with acid followed by adjustment to basic pH and benzene extraction. Thin-layer chromatography was then done using ethanol-benzene-1,4dioxane-concentrated aqueous ammonia (50: 40: 5: 5) and 1,4dioxane-chloroform-ethyl acetate-concentrated aqueous ammonia (60: 25: 10: 5) systems, silica gel plates, and potassium iodoplatinate spray [17,66].
Acknowledeements The authors wish to thank the chemists of the USP Drug Research and Testing Laboratory for experimental data and Ann K . Ferguson for providing the computerized literature search, and William K. Wyatt and Barbara A. Bowman for their assistance.
HEROIN
397
References
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Klosler, G., Roder, E., Machulla, H.J., "Synthesis, Chromatography, and Tissue Distribution of Methyl 'CMorphine' and Methyl 'C-Heroin' ," J. Labelled Compounds XVI 441 (1978). and Radiopharmaceuticals Zoccolillo, L., "Use of Glass Beads in Gas Chromatographic Analysis of Drugs," J. Chrom. 178 311 (1979). Moore, J.M., Klein, M., "Identification of 0'Monoacetylmorphine in Illicit Heroin Using Gas Chromatography-Electron Capture Detection and Mass Spectrometry," J. Chrom. 154 76 (1978). Love, J.L., Pannell, L.K., "A High Performance Liquid Chromatographic Method for the Analysis of Illicit Heroin," J. Forensic Sci. 25 320 (1980). Lim, H.Y., Chow, S., "Heroin Abuse and a Gas Chromatographic Method for Determining Illicit Heroin Samples in Singapore," J. Forensic Sci. 319 (1977). Boerner, U., Roe, R.L., Becker, C.E., "Detection, Isolation and Characterization of Nomorphine and Norcodeine as Morphine Metabolites in Man," J. Pharm. Pharmac. 26 393 (1974). Huizer, A., Logtenberg, H., Sleenstra, A.J., "Heroin in the Netherlands," Bulletin on Narcotics Vol. XXIX #4 65 (1977) Gubitz, G., Wintersterger, R.,"Identification of Drugs of Abuse by High-Performance Thin-Layer Chromatography," J. Anal. Toxicol. 4 141 (1980). Manura, J.J., Chao, J.M., Saferstein, R., "The Forensic Identification of Heroin," J. Forensic Sci. 45 (1977). Zweig, G., Handbook of Chromatography, CRC Press, Ohio (1972). Stahl, E., Thin-Layer Chromatography, Springer-Verlag, Berlin (1965). Macek, K., Pharmaceutical Applications of Thin-Layer and PaDer ChromatograDhv. . . , - . Elsevier PublishinE - Co., Amsterdam (1972).
~~~
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Borka, L., "The Polymorphism of Heroin and its Forensic Aspects," Acta Pharm. Suec. 14 213 (1977). May, E., Jacobson, A.E., "Chemistry and Pharmacology of Homologs of 6-Acetyl and 3,6-Diacetylmorphine," JPharm. Sci. 66 285 (1977). Yeh, S . Y . , McQuinn, R.L., Gorodetzky, C.W., "Identification of Diacetylmorphine Metabolites in Humans," J. Pharm. Sci. 66 201 (1977). Garrett, E.R., Gurkan, T., "Pharmacokinetics of Heroin and Its Derived Metabolites in Dogs," J. Pharm. Sci. 69 1116 (1980). Poochikian, G.K., Cradock, J.C., "Stability of Brompton Mixtures: Determination of Heroin and Cocaine in Presence of Their Hydrolysis Products,'' J. Pharm. Sci. 69, 637 (1980). Iijima, I., Minamikawa, J.I., Jacobson, A.E., Brossi, A., Rise, K.C., "Studies in the (+) Morphinan Series 4 . A Markedly Improved Synthesis of (+) Morphine," J. Org. Chem. 43 1462 (1978). Moore, J.M. , "The Problem of Negative Interference Associated with Liquid-Liquid Extraction-Derivatization Techniques," Clin. Chem. 24 1081 (1978).
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34.
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Abood, L.G., Atkinson, H.G., MacNeil, M., "Stereospecific Opiate Binding in Herman Erythrocyte Membranes and Changes in Heroin Addicts," J. Neuro. Res. 2 427 (1976). -Northway, M.G., Burks, T.F., "Indirect Intestinal Stimulatory Effects of Heroin: Direct Action on Opiate Receptors," Eur. J. Pharmacol. 59 237 (1979). Ho, W.K.K., Wen, H. L., Ling, N., "Beta-Endorphin-like Immunoactivity in the Plasma of Heroin Addicts and 19 117 (1980). Normal Subjects, Neuropharmacol. "
36.
37.
38
Lockridge, O., Mottershane-Jackson, N . , Eckerson, H.W., Ladu, B.N., "Hydrolysis of Diacetylmorphine by Human Serum Cholinesterase," J. Pharmacol. Expt. Ther. 215 1 (1980) Poochikian, G.K., Cradock, J.C., "A Simple High Performance Liquid Chromatographic Method for the Separation of 3,6-Diacetyl Morphine Hydrochloride (Heroin) and Hydrolysis Products," J. Chrom. 171 371 (1979)
39.
40. Reuland, D.J., Trinler, W.A. ,"An Unequivocal Determination of Heroin in Simulated Street Drugs by a Combination of High Performance Liquid Chromatography and Infrared Spectrophotometry Using Microsampling Techniques," Forensic Sci. 1 1195 (1978).
41. Nakamura, G.R., Noguchi, T.T., "Forensic Identification of Heroin in Illicit Preparations Using Integrated Gas 44 Chromatography and Mass Spectrometry," Anal. Chem. 408 (1972). Masoud, A. , "Systematic Identification of Drugs of 64 841 (1975). Abuse I: Spot Tests," J. Pharm. Sci. Garrett, E.R., Jackson, A.J., "Pharmacokinetics of Morphine and Its Surrogates 111: Morphine and Morphine 3-Monoglucuronide Pharmacokinetics in the Dog as a Function of Dose," J. Pharm. Sci. 68 753 (1979).
42. 43.
44. Garrett, E.R., Gurkan, T., "Pharmacokinetics of Morphine and Its Surrogates I : Comparisons of
Sensitive Assays of Morphine in Biological Fluids and Application to Morphine Pharmacokinetics in the Dog," J. Pharm. Sci. 67 1512 (1978).
HEROIN
40 1
45
Yeh, S.Y., "Separation and Identification of Morphine and Its Metabolites and Congeners," J. Pharm. Sci. 62 1827 (1973).
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49. Canfield, D., Barrick, J., Glessen, B.C., "Structure of Diacetylmorphine," Acta Cryst. B35 2806 (1979).
50. Martindale, The Extra Pharmacopoeia Twenty-sixth Edition, The Pharmaceutical Press, London (1975) p. 1114. The British Pharmacopoeia, Her Majesty's Stationery Office, University Printing House, Cambridge (1980) p. 146. The United States Pharmacopeia, 20th revision, Mack Publishers, Easton, Pennsylvania (1980). The USP Drug Research and Testing Laboratory, 12601 Twinbrook Parkway, Rockville, Maryland 20852. Knox, J.H., Jurand, J., "Application of High-speed Liquid Chromatography to the Analysis of Morphine, Heroin, 6-(O-acetyl) Morphine and Methadone," J. Chrom. 87 95 (19737. "GC Separation of Heroin and Related Street Drugs," Bulletin 7348, Supelco, Inc., Bellefonte, Pennsylvania. McLinden, V.J., Stenhouse, A.M., "A Chromatography System for Drug Identification," Forensic Sci. International 13 71 (1979).
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57.
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Wu, C., Siggia, S . , Robinson, T., Waskiewicz, R.D., "Separation of Cinchona Alkaloids, Opium Alkaloids, Heroin, and Related Narcotics by Dynamic-Coating High Speed Liquid Chromatography," Anal. Clin. Acta 63 393
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Nakamura, G.R., Ukita, T., "Paper Chromatography Study of I n Vitro and In Vivo Hydrolysis of Heroin in Blood," J. Forensic Sci.56 294 (1967). Twitchett, P.J., "Analysis of Illicit Diamorphine Preparations by High-pressure Liquid Chromatography, J . Chrom. 104 205 (1975). Curry, A . S . , Patterson, D.A., "A Procedure for the Analysis of Illicit Diamorphine Samples," J. Pharm. Pharmac. 22 198 (1970). Elliott, H.W., Parker, K.D., Crim, M., Wright, J.A., Nomof, N., "Actions and Metabolism of Heroin Administered by Continuous Intravenous Infusion to Man," Clin. Pharm. Ther. 12 806 (1971). Nakamura, G.R., Ukita, T., "A Study of Hydrolysis of Heroin by Paper Chromatography," J. Forensic Sci. 7 465
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'I
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Ziegler, H.W., Beasley, T.H., Smith, D.W., "Simultaneous Assay for Six Alkaloids in Opium Using High Performance Liquid Chromatography," -JAOC 5 8 888
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Prager, M.J., Harrington, S.M., Governo, T.F., "GasLiquid Chromatographic Determination of Morphine, Heroin, and Cocaine," JAOAC 62 304 (1979). Yeh, S.Y., McQuinn, R.L., "GLC Determination of Heroin and Its Metabolites in Human Urine," J. Pharm. Sci. 64
1237 (1975).
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403
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Verpoorte, R., Svendsen, A.B., "High-speed Liquid Chromatography of Alkaloids I," J . Chrom. 100 227 (1974). Nakamura, G.R., "Rapid Estimation of Diacetylmorphine in the Presence of Acetylcodeine," -JAOAC 46 770 (1963). Fulton, C.C., "Microcrystal Tests for 03-MonoacetylO6-MonoaceTylmorphine, morphine in Comparison with Diacetylmorphine, Morphine, and Codeine," Microchemical Journal VI 51 (1962). Carroll, F.I., Moreland, C.G., Brine,.G.A., Kepler, J.A., "Carbon-13 Nuclear Magnetic Resonance Spectra of Morphine Alkaloids," J. Org. Chem. 41 996 (1976). Beyerman, H.C., Liets, M.L., Bosman, H.H., Buurman, E . , Bijsterveld, E.J.M., Sinnige, H.J.M., "A Convenient Synthesis of Codeine and Morphine," Rec. Trav. Clin. 95 24 (1976).
69. 70.
71.
72.
HYDROCHLOROTHIAZIDE
Hans Peter Deppeler
1. Description 1,1 General Information 1.2 Nomenclature 1.3 Formula and Molecular Weight 1.4 Appearance 1.5 Official Compendia 1.6 Other Compendia 2. Physical Properties 2.1 Spectra 2.2 Physical Properties of the Solid State 2.3 Solubility 2.4 Ionisation in Aqueous Solution 3. Synthesis 4. Stability and Degradation 4.1 Bulk Stability 4.2 Solid-Solid Interactions 4.3 Stability in Solution 5. Methods of Analysis 5.1 Elemental Analysis 5.2 Identification 5.3 Colorimetry 5.4 Ultraviolet Spectrophotometry 5.5 Phosphorimetry 5.6 Fluorimetry 5.7 Polarography 5.8 Titration 5.9 Chromatography 5.10 Electrophoresis 6. Interferences of Hydrochlorothiazide in Analytical Methods 7. Pharmacokinetic and Metabolic Studies 7.1 Analytical Methods Used for Biological Material 7.2 Absorption 7.3 Distribution 7.4 Basic Pharmacokinetics 7.5 Bioavailability 8. Acknowledgements 9. References 406 406 406 407 407 407 407 407 407 419 422 424 424 425 425 425 425 426 426 426 426 427 427 428 428 428 429 430 432 432 432 432 433 433 434 435 436
ANAI.Y?Ii:AI. IROFII.ES OF DRUG SURSTANCES. 10
405
406
HANS PETER DEPPELER
1. 1.1
Description General Information
Research in sulfonamide chemistry has brought a rich yield of valuable therapeutics. One of the great successes was the discovery of the benzothiadiazines as potent diuretics of low toxicity(1). In 1958 De Stevens et a1.(2) reported on the condensation product of 4-amino-6-chloro-3,5-disulfonamide and formaldehyde which was found to be identical with the hydrogenation product of chlorothiazide(3) and which soon became a widely used saluretic: Hydrochlorothiazide 1.2 Nomenclature
1.2.1 Chemical Names Hydrochlorothiazide is the recommended international nonproprietary name(4) of 6-Chloro-3,4-dihydro-7-sulfamoyl-2H-1,2,4-benzothiadiazine l,l-dioxide(5) or 6-Chloro-3,4-dihydro-2H-lf2,4-benzothiadiazine-7sulfonamide l,l-dioxide(5,6) or 6-Chloro-7-sulfamyl-3,4-dihydro-1,2,4-benzothiadiazine l,l-dioxide(5) or 2H-1,2,4-Benzothiadiazine-7-sulfonamide, 6-chloro-3,4-dihydro-,l,l-dioxide(6) CAS registry number: 58-93-5 1.2.2 Trade Names The Merck Index(5) quotes 28, and Index Nominum(7) 64 trade names not including the combinations with other active substances. Therefore, only a few examples can be listed here. Trade names including combinations in different countries: France: Adelphan-Esidrex, Esidrex, Esimil, Hydromet, Moduretic Germany(BRD1: Di-Chlotride, Diu 25, Esidrix Germany(DDR1: Disalunil, Urodiazin Great Britain: Direma, Esidrex, Hydrosaluric, Sa1upres Japan: Esidrix, Dichlotride Esidrix, Hydrodiuril, Oretic, USA : Serapes, Thiuretic.
IIYDROCHLOROTHIAZIDE
1.3
Formula and M o l e c u l a r Weight

0 0 0 0
c7H8C1N3O4S2
M o l e c u l a r Weight 297.73
1.4
Appearance
White, o r p r a c t i c a l l y w h i t e , p r a c t i c a l l y odourless, c r y s t a l l i n e powder(6). S l i g h t l y b i t t e r taste( 8 ) . 1.5 O f f i c i a l Compendia
Monographs o n h y d r o c h l o r o t h i a z i d e and hydroc h l o r o t h i a z i d e t a b l e t s are i n c l u d e d i n t h e f o l l o w i n g compendia: BP 73, DAB 7 ( D D R ) , Ph. I n t . 11, Ph. J a p . 1 9 7 1 , Ph. Nord. Add., USP X I X Monographs i n Ph. Eur. are p r o p o s e d . A USP Hydroc h l o r o t h i a z i d e Reference Standard i s a v a i l a b l e .
1.6
O t h e r Compendia
Summaries i n c l u d i n g a n a l y t i c a l and pharmaceut i c a l i n f o r m a t i o n s are g i v e n i n The P h a r m a c e u t i c a l Codex( 9 ) and i n Kirk-Othmer, E n c y c l o p e d i a of Chemical T e c h n o l o g y ( l 0 ) .

2.
P h y s i c a l Properties Spectra Infrared(l1)
2.1
2.1.1
The i n f r a r e d spectrum i s p r e s e n t e d i n F i g u r e 1. The s p e c t r u m was o b t a i n e d from a m i n e r a l o i l m u l l on a P e r k i n E l m e r Model 157 i n f a r e d s p e c t r o p h o t o m e t e r -f A s s i g n m e n t s f o r t h e i n t h e range of 4000-650 c m c h a r a c t e r i s t i c b a n d s i n t h e spectrum a r e l i s t e d i n Table I.
408
HANS PETER DEPPELEH
Table I
Infrared absorption Wavenumber cml 3 3 7 0 , 3 2 7 0 , 3170 1600, 1550, 1520 1335 1180 Assignments
NH + NH2
heterocyclic ring system s02
/ 1320 / 1 1 6 5 / 1150
2.1.2
Raman( 11)
T h e Raman spectrum of h y d r o c h l o r o t h i a z i d e powder i s shown i n F i g u r e 2 a n d Table 11. I t w a s b b t a i n e d o n a C a r y Model 8 3 spectrometer u s i n g t h e a r g o n 488 nm e x c i t a t i o n of a Lexel Model 7 5 i o n laser
Table I1
Raman spectrum Frequency c m 3380, 3080, 2960, 1600, 1335, 1165, 940, 710,
-1
~~
Assignments
NH s t r e t c h i n g aromatic CH s t r e t c h i n g CH s t r e t c h i n g C=6 stretching
3 2 8 0 , 3180 3020 2900 1525, 1460 1320 1155 900 675
SO2 asym. s t r e t c h i n g S O sym. s t r e t c h i n g S-& s t r e t c h i n g + NH deformation r i n g deformations
F i g . 1.
I n f r a r e d spectrum of h y d r o c h l o r o t h i a z i d e .
HYDROCHLOROTHIAZIDE
411
2.1.3
Ultraviolet
T h e U V spectrum of h y d r o c h l o r o t h i a z i d e i n e t h a n o l i s shown i n F i g u r e 3 (11). I n f o r m a t i o n a b o u t t h e UV a b s o r p t i o n i n o t h e r s o l v e n t s i s g i v e n i n T a b l e 111.
T a b l e I11
U1 t r a v i o1 e t ab s o r p t i o n
So lve n t
X max nm
225 269 316 226
1 min nm
log
e t h a n o l ( 11
methanol( 1 2 1
241.5 271 294 317 270 315 270 315 272 323 221 247 273 299
31 9 / 3 2 0
water( 13 1
0.01 N H C l ( 1 3 )
0 . 0 1 N NaOH( 1 3 )
0 . 1 N NaOH(14)
4.576 4.307 3.505 4.513 3.129 4.279 3.272 3.471 4.286 3.495 4.290 3.500 4.193 3.435 4.448 4.198 3.456
418
HANS PETER DEPPELEH
F i g u r e 3 . U l t r a v i o l e t s p e c t r u m of h y d r o c h l o r o thiazi.de i n ethanol
Spectrum No 6 5 7 5 1 Sample 76-104 ?93 Prod.Std.76 M o l e c u l a r ),eight 297,75 S o l v e n t *ethanol
FO 6 2
Concentra t 1 on 2 , 5.10-5H01 ./Lit C e l l p a t h 1,Ocm I n s t r u m e n t Cary 1 1 8 O p e r a t o r Dmo. D a t e 1 9 . 3 ~ 1 1 19.9
HYDROCHLOROTHIAZIDE
413
2.1.4
1 H-Nuclear Maqnetic R e s o n a n c e ( l 1 )
The 'H-NMR s p e c t r u m shown i n F i g u r e 4 was o b t a i n e d from a s o l u t i o n i n a c e t o n e - d a t a m b i e n t t e m p e r a t u r e on a V a r i a n XL-100-12 s p e g t r o m e t e r a t 1 0 0 MHz. The a s s i g n m e n t s of t h e s i g n a l s are l i s t e d i n Table I V .
Table I V
'H-NMR
I
Signal
Chem. shift ppm v s . T M S
t i p 1i c i t y M u1
Number
Of
Species
protons
I
7.16
7.04
1 broad singlet singlet broad singlet broad singlet broad singlet singlet multiplet 1 1 1
2 2
6.75 6.65 4.93 3.0 2.05
arom. proton sulfonamide N H arom. proton arom. NH

Ar-S02NH2
m et h y l e n e protons solvent solvent
S i g n a l f i s b r o a d e n e d due t o u n r e s o l v e d c o u p l i n g w i t h t h e NH p r o t o n s .
NMR spectrum no. 105523
I
C
1 +
j6
5
4
1 0
0
ppm
Figure 4. 'H-NMR
spectrum of hydrochlorothiazide
HYDROCHLOROTHIAZIDE
415
2.1.5
13C-Nuclear M a g n e t i c Resonance( 111
The 13C-NMR s p e c t r a shown i n t h e F i g u r e s 5 , 6 , 7 w e r e r e c o r d e d a t 25.2 MHz and a m b i e n t t e m p e r a t u r e w i t h a V a r i a n XL-100-15 spectrometer u s i n g a s o l u F i g u r e s 5 and 6 show d e c o u p l e d t i o n i n acetone-d spectra. The undegoupled s p e c t r u m i n F i g u r e 7 shows t h e m u l t i p l i c i t i e s of t h e s i g n a l s . The a s s i g n m e n t s of t h e s i g n a l s are l i s t e d i n Table V.
Table V
13C-NMR
~~~
M u l t i p l i c i t y (umber S p e c i e s S i g n a l Chem. If shift zarbons ppm vs. T M S

I
1
5 9
55.9 118.9 120.8 127.3 129.6 135.9 147.7 d,m ( b r o )
1 1 1
1 1 1
h-CH
/
-N - 2 \ arom. C-H arom C-S02 -NH-CH2
8 6
7
arom. C-H arom. C-C1 arom.

C-S02NH2 arom. C-NH-R
10
d : d o u b l e t ; t: t r i p l e t ;
in:
multiplet;
( b r o ) : broadened
416
0
Figure 5. 13C-NMR spectrum of hydrochlorothiazide
-I
N
I d .d 2 4J 0 k 0
.rl N
a,
c V
2
w
0
E 3
4J
0 k
V a,
4
m
m
2 l -.t
B z I
d
P;
n m
W
a, k 3
br
417
F i g u r e 7 . Undecoupled 13C-NMR chlorothiazide
r-
spectrum of hydro-
HYDHOCHLOROTHIAZIDE
419
2.1.6
Mass( 11)
S p e c t r a were r e c o r d e d o n a V a r i a n CH7 mass s p e c t r o m e t e r u s i n g t h e d i r e c t i n l e t s y s t e m , 70eV e l e c t r o n e n e r g y a n d a n i o n s o u r c e t e m p e r a t u r e of 18OoC. A t a sample t e m p e r a t u r e o f a b o u t 24OoC t h e m o l e c u l a r i o n m/e 297 c o u l d be d e t e c t e d b u t t h e s p e c t r u m w a s c o m p l i c a t e d and d i f f i c u l t t o i n t e r p r e t d u e t o p y r o l y t i c d e g r a d a t i o n . R o u t i n e mass s p e c t r o s c o p y i s c o n s i d e r e d t o be i n a d e q u a t e f o r t h e c h a r a c t e r i z a t i o n of h y d r o c h l o r o t h i a z i d e .
2.2 2.2.1
P h y s i c a l P r o p e r t i e s of t h e S o l i d S t a t e Thermal A n a l y s i s ( 1 5 ) Meltinq p o i n t
Melting p o i n t s reported i n l i t e r a t u r e (5,14,16,17,18,19,20) vary within t h e temperature r a n g e o f 263 t o 275OC. The s t r o n g dependence o f t h e m e l t i n g p o i n t on h e a t i n g c o n d i t i o n s h a s b e e n c o n f i r m e d w i t h t h e Mettler FP-2 h o t s t a g e micros c o p e as w e l l as w i t h t h e P e r k i n - E l m e r DSC-2. The e f f e c t which c a u s e s t h e anomalous m e l t i n g behaviour(20) i s not c l e a r l y understood. D i f f e r e n t i a l Scanninq Calorimetry The m e l t i n g p o i n t of h y d r o c h l o r o t h i a z i d e a c c o r d i n g t o DSC-2 m e a s u r e m e n t s , O v a r i e s from 266.OoC f o r a s c a n s p e e d of 1125 C min-' t o 273.3OC f o r a s c a n s p e e d o f 8OoC min( F i g u r e 8 1 . A s any e f f e c t (e.g. decomposition or t r a n s i t i o n i n t o another c r y s t a l modification) i s suppressed a t a h i g h s c a n speed, one can r e g a r d t h e v a l u e f o r an i n f i n i t e l y f a s t s c a n s p e e d as t h e t r u e m e l t i n g p o i n t . E x t r a p o l a t i o n of t h e c u r v e shown i n F i g u r e 8 g i v e s a m e l t i n g p o i n t o f 274.5 + 0.3OC*. T h i s a g r e e s w e l l w i t h t h e m e l t i n g p o i n t r e p o r t e d i n The Merck I n d e x ( 5 ) . However, t h e p u r i t y v a l u e s o b t a i n e d from m e l t i n g c u r v e s measured w i t h t h e DSC-2 a r e i n d e p e n d e n t of t h e s c a n s p e e d as w e l l as of t h e s u r r o u n d i n g a t m o s p h e r e . When a sample of hydro-
E r r o r of t h e mean v a l u e i n terms of c o n f i d e n c e
i n t e r v a l s on a 95 % l e v e l
420
HANS PETER DEPPELER
c h l o r o t h i a z i d e w a s t s t e d a t scan speeds of 1.25, 2 . 5 , 5 and 2OoC min -7 t h e mean v a l u e o b t a i n e d f o r t h e p u r i t y w a s 99.3 2 1 . 0 mole per c e n t . A t y p i c a l DSC m e l t i n g c u r v e is shown i n F i g u r e 9. A s l i g h t exotherm, i n d i c a t i n g decomposition i n t h e l i q u i d p h a s e , i s s e e n above t h e m e l t i n g p o i n t . Thermogravimetry
E l m e r t h e r m o b a l a n c e TGS-1.
Measurements were p e r f o r m e d w i t h a P e r k i n The r e s u l t s were: less t h a n 0 . 1 % v o l a t i l e i m p u r i t i e s up t o a t e m p e r a t u r e of 28OoC. d e c o m p o s i t i o n s t a r t s a t 307OC. Density
2.2.2
1.68 + 0 . 0 1 g ~ m ( 1 ~9,211 2.2.3

X-ray D i f f r a c t i o n
S t u d i e s by Dupont a n d D i d e b e r g ( l 9 ) o n a s i n g l e c r y s t a l m e a s u r i n g 0.2xO.4xO.l mm g a v e t h e f o l l o w i n g crystallographic data Monoclinic System : Space group: p2 1 Unit cell: Z = 2 molecules a = 7.419 + 0 . 0 0 6 8 b = 8.521 7 0 . 0 0 3 8 c = 10.003 7 0 . 0 0 2 8 6 = 111.720:v = 587.5 Calculated density: 1.672 g
L i n e a r a b s o r p t i o n c o e f f i c i e n t p = 6 . 7 1 cm (Mo K : 0 , 7 1 0 7 ) . T h e s e r e s u l t s , found on a c r y s t a l c r y s t s l l i z e d from e t h a n o l , c o r r e s p o n d w e l l w i t h t h o s e f o u n d on a sample c r y s t a l l i z e d from m e t h a n o l i n a preliminary study(22 1 . A powder d i a g r a m , c a l c u l a t e d from t h e s i n g l e c r y s t a l d a t a , a g r e e s v e r y w e l l w i t h measurements o n i n d u s t r i a l p r o d u c t i o n l o t s . The powder d i f f r a c t i o n p a t t e r n , a s shown i n T a b l e V I , was o b t a i n e d w i t h a Guinier-DeWolf N o . 2 camera w i t h CuKa ( 1.54178 8) r a d i a t i o n ( 2 3 ) .
F i g u r e 8 . M e l t i n g p o i n t of h y d r o c h l o r o t h i a z i d e as a f u n c t i o n of scan s p e e d (DSC)
275 274 273 272 271 270 269 268 267

266
265
80 2 0
2.5
4
1.25
Scan s p e e d OC v i n - 1
F i g u r e 9. DSC m e l t i n g p o i n t c u r v e of h y d r o c h l o r o t h i a zi d e
Range 2 mcal s-l Scan speed: 1-25
OC
--
rn1n-l
Sample w e l g h t 4 . 1 5 0
r n s
Heat of f u s i c - :
260
261
262
263
264
265
266
267
266
269
Temp. OC
422
HANS PETER DEPPELER
Table V I X-ray d i f f r a c t i o n p a t t e r n of h y d r o c h l o r o t h i a z i d e powder
I n t e n s it y
9.3 6.9 6.3 5.35 4.75 4.65 4.26 4.15 4.09 3.87 3.62 3.44 3.39 3.19 3.14 3.10 2.89 2.74 v e r y weak weak v e r y weak very strong strong strong very strong very strong v e r y weak moderate very strong moderate weak strong weak strong weak weak
2.71 2.67 2.62 2.50 2.45 2.40 2.38 2.35 2.29 2.25 2.21 2.19 2.16 2.12 2.07 2.06 2.03
Intensity v e r y weak
strong
medium weak moderate v e r y weak v e r y weak v e r y weak moderate v e r y weak v e r y weak v e r y weak weak weak v e r y weak v e r y weak moderate
2.3 2.3.1
Solubility S o l u b i l i t y i n Homogeneous Media
H y d r o c h l o r o t h i a z i d e i s s o l u b l e i n a q u e o u s sol u t i o n s of i n o r g a n i c b a s e s l i k e sodium h y d r o x i d e ( 6 ) o r ammonium h y d r o x i d e ( 5 ) and i n o r g a n i c b a s e s l i k e n - b u t y l a m i n e ( 6 ) . S o l u b i l i t i e s i n aqueous s o l u t i o n s a r e g i v e n i n T a b l e V I I , and i n some commonly u s e d organic solvents, i n Table V I I I . The s u r f a c e t e n s i o n of t h e s a t u r a t e d a q u e o u s . I N p e r c m by s o l u t i o n a t 23OC w a s f o u n d t o b e 724 ! L e r k and L a g a s ( 2 1 ) . The i n c r e a s e of t h e s o l u b i l i t y upon a d d i t i o n of n o n - i o n i c s u r f a c t a n t s was s t u d i e d by A b o u t a l e b e t a l . ( 2 4 1 .
HYDROCHLOROTHIAZIDE
423
Table V I I S o l u b i l i t y i n aqueous s o l u t i o n s ( 13 1
Solvent
tC p H of t h e solution
water water 0.9 % N a C l

0.1 N HC1 0.1 N acetic acid 0.1 N acetic b u f f e r pH 4.4 0.067 M p h o s p h a t e b u f f e r p H 7.4 0.05 M borate b u f f e r p H 9.0 1 M ammonia( 2 5 ) 0 . 1 N NaOH simulated gastric f l u i d p H 1.1 simulated i n t e s t i n a l f l u i d p H 7.4
Solubility g i n 100 m l solution 60.9 108 59.4 60.8 63.6 62.3 61.6 103 2.2 1.79 108 109
25 37 25 25 25 25 25 25 25 25 37 37
6.2 7.2 6.1 1.0 2.9 4.5 7.4 8.9 11.6 10.2 1.1 7.5
. . . . -3 . . 10
.
.
.
.
T a b l e VIII
r-Solvent
S o l u b i l i t y i n non aqueous s o l v e n t s
Solubility g i n 100 m l s o l u t i o n I acetone 25 13.7 (25) 25 acetic acid 0.15 (25) ace t o n i t r i l e 25 2.0 (25) ethylacetate 25 0.59 (25) chloroform 23 0.1 ( 131 ethanol (96 % 1 23 1.3-1.4 ( 131 methanol 23 3.9-4.1 ( 131 d i c h l o r o m et h a n e 23 < 0.02 ( 13 1
temp. 0C ca.
424
HANS PETER DEPPELER
2.3.2
Partition Coefficients
The p a r t i t i o n between n - o c t a n o l and a q u e o u s p h a s e s a t 25OC i s e x e m p l i f i e d by t h e f o l l o w i n g d a t a ( 13 1 0.1 N H C 1 (pH 1 . 0 6 ) : 1.94 pco r g l c a q 0.1 M glycine b u f f e r (pH 3 . 0 ) : pCorg/Caq = 0.866 0.067 M p h o s p h a t e pCorg/Caq = 0.855 b u f f e r (pH 7 . 4 ) :
2.4
I o n i s a t i o n i n Aqueous S o l u t i o n
The i o n i s a t i o n c o n s t a n t s q u o t e d i n t h e l i t e r a t u r e d i f f e r . The v a l u e s r e p o r t e d by Mollica e t a 1 . ( 2 6 ) and by S t a h l a g r e e b e s t w i t h t h e s o l u b i l i t y b e h a v i o u r ( l 3 ) . They a r e l i s t e d below i n

Table I X .
Table I X
pK-Values pK-Value pKa pKa pKa pKa PKa

1 2 1 2
i n aqueous s o l u t i o n s
Method 8.81 + 0 . 0 5 10.4 ,+ 0 . 1 8.6 9.9 8.7
photom. t i t r . photom. t i t r potent. titr. potent. titr. spectrophotom.
13 1 13 1 (26) (26) (26)

( (
3.
Synthesis
A c c o r d i n g t o Kleemann(27) t w o ways of s y n t h e s i s are u s e d
5-Chloro-2.4-disulfamoylaniline and p a r a f o r m a l d e h y d e r e a c t i n non a q u e o u s media t o g i v e hydrochlorothiazide.
HYDROCHLOROTHIAZIDE
425
b)
0 0 0 0
(29)
0
H
6-chloro-7-sulfamoyl-2H-l.2.4-benzothiadiazine1.1-dioxide reacts with formaldehyde in aqueous alkaline solution to form hydrochlorothiazide.
4. 4.1
Stability and Degradation Bulk Stability(25)
Hydrochlorothiazide stored at room temperature for five years shows no degradation and heat affectg it very slowly, e.g. treatment for 2 hours at 230 C gives a yellowish discoloration but no significant change of the physical properties. Although hydrochlorothiazide is fairly stable in normal daylight, it should not be exposed to intense light: 38 hours at 180 0 0 0 Lux (Xenotest) destroyed about 3 per cent of a sample spotted on glass fibre paper. 4.2 Solid-Solid Interactions
Bornstein and Lach(30) found that hydrochlorothiazide reacts under the influence of humidity with adjuvants containing metal compounds. The changes in UV-absorption spectra obtained by diffuse reflectance spectrometry were interpreted as the result of charge-transfer chelation. In a compatibility study with Aerosil 2000, calcium stearate and talc using diffuse reflectance spectroscopy, tlc and UV spectroscopy after extraction, no indications of degradation under usual manufacturing and storage conditions were found( 13 1. 4.3 Stability in Solution
In aqueous solutions, hydrochlorothiazide undergoes hydrolysis to give formaldehyde and
126
HANS PETER DEPPELER
6-Chloro-2.4-disulfamoylaniline. The dependence o f t h e r e a c t i o n r a t e on t e m p e r a t u r e and p H was s t u d i e d by Mollica e t a 1 . ( 2 6 , 3 1 ) and Yamana e t a l . ( 3 2 ) . Between pH 2.5 and p H 11.5 t h e rate fo llo ws a b e l l s h a p e d c u r v e w i t h a maximum a t a b o u t pH 7 . 2 . B e l o w p H 2 and above g H 1 2 t h e r e a c t i o n r a t e increases rapidly. This p H r a t e p r o f i l e and t h e l a c k of s i g n i f i c a n t b u f f e r c a t a l y s i s was e x p l a i n e d by M o l l i c a by t h e f o r m a t i o n of i n t e r m e d i a t e s of t h e i m i n e t y p e a n d , upon h y d r a t i o n , of t h e hydroxymethylamine type( 26 1.
H2
h
H
c1
N
CH20
5.
Methods of A n a l y s i s Elemental A n a l y s i s ( 3 3 ) Element

C
5.1
% calculated % found
28.24 2 . 7 1 1 1 . 9 1 1 4 . 1 2 21.49 21.54 28.21 2 . 7 2 1 2 . 1 5 14.18 2 1 . 2 6 21.45
5.2
Identification
Chemical t e s t s were d e s c r i b e d by K e r t e s z ( 3 4 ) K a l a ( 3 5 ) and P e r e z ( l 8 ) . M i c r o c h e m i c a l i d e n t i f i c a t i o n methods were r e p o r t e d by d e Z o e t e n ( 3 6 1 , Groenewegen(371, K a l a ( 3 8 ) and A u e r b a c h ( 3 9 ) . Usuall y , h y d r o c h l o r o t h i a z i d e i s i d e n t i f i e d by spectros c o p i c means, e.g. by i t s I R ( 6 , 1 4 , 4 0 ) and UV ( 6 , 1 4 , 4 0 , 4 1 ) s p e c t r a o r by o n e of t h e c h r o m a t o g r a p h i c t e c h n i q u e s c i t e d i n s e c t i o n 5.9. 5.3
Colorimetry
Hydrochlorothiazide i s r a p i d l y hydrolysed i n a c i d o r a l k a l i n e s o l u t i o n s . By d i a z o t i s a t i o n of t h e h y d r o l y s i s p r o d u c t 5-chloro-2,4-disulfamoyla n i l i n e and s u b s e q u e n t c o u p l i n g w i t h a n aromatic amine o r a p h e n o l , s t a b l e a z o d y e s are p r o d u c e d .
HYDROCHLOROTHIAZIDE
427
N-(l-naphthyll-ethylenediamine( 4 2 , 4 3 , 4 4 ) , chromot r o p i c a c i d ( 451, g u a j a c o l s u l f o n i c a c i d ( 4 6 1 , and t h y m o l ( 3 5 ) h a v e b e e n r e p o r t e d as c o u p l i n g a g e n t s . T h e s e methods are c o n s i d e r e d t o b e s u i t a b l e f o r t h e a n a l y s i s of p h a r m a c e u t i c a l s and may a l s o s e r v e as l o w cost techniques i n biopharmaceutical s t u d i e s . O t h e r methods a r e b a s e d o n c o n d e n s a t i o n r e a c t i o n s of t h e h y d r o l y s i s p r o d u c t s ( 2 6 , 4 7 ) or o n d i r e c t c o l o r r e a c t i o n s of h y d r o c h l o r o t h i a z i d e w i t h d i f f e r e n t r e a g e n t s i n t h e p r e s e n c e of c o n c e n t r a t e d s u l f u r i c acid(18,42,48). R e c e n t l y , E l s a y e d and Nwakanma(49) r e p o r t e d o n i o n p a i r e x t r a c t i o n w i t h s a f r a n i n b a s i c dye as a s i m p l e , s e l e c t i v e and s e n s i t i v e new method f o r t a b l e t analysis.
5.4 U l t r a v i o l e t Spectrophotometry
Rehm and S m i t h ( 45 1 showed t h a t UV-spectrophotometry i s n o t s u i t a b l e f o r t h e d e t e r m i n a t i o n o f hydrochlo r othiazide i n t h e presence of i t s h y d r o l y s i s p r o d u c t 5-chloro-2,4-disulfamoyla n i l i n e . I n s p i t e of t h i s l i m i t a t i o n , t h e method w a s d e s c r i b e d f o r t a b l e t a n a l y s i s by S t e i n b a c h , M o e l l e r ( 5 0 ) and Ruiz Rodriguez e t a 1 . ( 5 1 ) . F a z z a r i combined UV-Spectrophotometry w i t h a column extract i o n t e c h n i q u e ( 5 2 ) . The s p e c i f i c i t y of t h e method may a l s o b e improved by c a l c u l a t i n g t h e c o n c e n t r a t i o n s from pH-induced s p e c t r a l c h a n g e s ( 5 3 , 5 4 , 5 5 ) . An a u t o m a t e d d e t e r m i n a t i o n of h y d r o c h l o r o t h i a z i d e i n s i n g l e m u l t i c o m p o n e n t t a b l e t s was d e s c r i b e d by U r b a n y i and O ' C o n n e l l ( 5 6 ) . The d e t e r m i n a t i o n of h y d r o c h l o r o t h i a z i d e i n ( b o v i n e ) serum i s p o s s i b l e a f t e r s e p a r a t i o n by d i a l y s i s ( 5 7 ) . Q u a l i t a t i v e r e s u l t s were o b t a i n e d by UV-spect r o m e t r y of p o l y e t h y l e n e c o n t a i n e r m a t e r i a l ( 5 8 ) a n d by means of d i f f u s e r e f l e c t a n c e s p e c t r o s c o p y o n powder m i x t u r e s ( 3 0 ) . U V - s p e c t r o p h o t o n e t r y i s also applied i n d i s s o l u t i o n rate s t u d i e s , e.g. i n USP X I X ( 6 ) . 5.5 Phosphorime t r y
B o w e r and W i n e f o r d n e r ( 5 9 ) r e p o r t e d on a t e c h n i q u e f o r room t e m p e r a t u r e p h o s p h o r e s c e n c e measurements o n h y d r o c h l o r o t h i a z i d e and found i t t o be a s i m p l e and s e l e c t i v e method s u i t e d t o c e r t a i n c l i n i c a l analyses.
428
HANS PETER DEPPELER
5.6
Fluorimetry
S c h a f e r , Geissler and M u t s c h l e r ( 6 0 ) developed two methods based on f l u o r i m e t r i c measurement on t l c p l a t e s . By c o u p l i n g t h e d i a z o t i s e d h y d r o l y s i s p r o d u c t of h y d r o c h l o r o t h i a z i d e t o a f l u o r e s c e n t compound, followed by chromatographic s e p a r a t i o n , 0 . 6 ng of 6-chloro-2,4-disulfamoyl-aniline could b e d e t e c t e d . Because t h e a u t h o r s found t h e hydrol y s i s s t e p d i f f i c u l t t o reproduce q u a n t i t a t i v e l y t h e y recommend measurement of t h e f l u o r e s c e n c e of u n d e r i v a t i s e d h y d r o c h l o r o t h i a z i d e . The s e n s i t i v i t y of t h e l a t t e r method i s lower b u t still s u f f i c i e n t f o r t h e a n a l y s i s of human plasma, u r i n e and s a l i v a a f t e r o r a l a d m i n i s t r a t i o n of 25 m g hydrochlorothiazide
5.7
Polaroqraphy
Cohen e t a1.(61) and Woodson and S m i t h ( 6 2 ) r e p o r t e d on t h e p o l a r o g r a p h i c r e s p o n s e of hydroc h l o r o t h i a z i d e and r e l a t e d compounds. P r a c t i c a l a p p l i c a t i o n s were d e s c r i b e d i n USP X V I I I ( 6 3 ) f o r t a b l e t s and i n a p a p e r of Kkolos and W a l k e r ( 6 4 ) f o r multicomponent t a b l e t s . The p o l a r o g r a p h i c d e t e r m i n a t i o n i s r e p o r t e d t o be s u i t a b l e f o r s i n g l e t a b l e t a n a l y s i s w i t h o u t s e p a r a t i o n of o t h e r components.
5.8
Titration
The t i t r a t i o n of t h e p u r e compound w i t h s t r o n g b a s e s i n non aqueous s o l v e n t s h a s found widespread application(35,65,66,67,68). USP X I X ( 6 ) t i t r a t e s h y d r o c h l o r o t h i a z i d e w i t h sodium methoxide i n n-butylamine w i t h azo v i o l e t a s i n d i c a t o r . BP 7 3 ( 8 ) d e s c r i b e s a potentiometric t i t r a t i o n with t e t r a butylammoniumhydroxide i n p y r i d i n e . Other t i t r a t i o n t e c h n i q u e s a p p l i e d t o hydrochlorothiazide a c t i v e substance o r formulations a r e l i s t e d below: Complexome$$ic a f t e r p r e c i p i t a t i o n w i t h Pb++(69 1 o r w i t h H g ( 7 0 , 7 1 1 . Amperometric w i t h n i t r i t e (72). Bromatometric(73). Thermometric w i t h sodium h y d r o x i d e ( 7 4 ) . S u l f a t e d e t e r m i n a t i o n a f t e r mineral i s a t i o n ( 7 5 ) . Argentometric a f t e r m i n e r a l i s a t i o n
(76).
HYDROCHLOROTHIAZIDE
429
5.9
Chromatoqraphy
5 . 9 . 1 T h i n Layer Chromatography P a p e r c h r o m a t o g r a p h y w a s of some i m p o r t a n c e f o r t h e i d e n t i f i c a t i o n of h y d r o c h l o r o t h i a z i d e ( 1 7 , 4 0 , 7 7 , 7 8 , 7 9 , 8 0 ) b e f o r e i t w a s r e p l a c e d by t h i n l a y e r chromatography. I n t l c , s i l i c a g e l l a y e r s a r e t h e most o f t e n u s e d s o r b e n t s . They were shown t o be s u i t a b l e f o r q u a l i t a t i v e ( 7 9 - 9 1 ) and q u a n t i t a t i v e ( 5 0 , 6 0 ) a n a l y s i s of h y d r o c h l o r o t h i a z i d e i n pharmaceuticals(50,79,87) and i n b i o l o g i c a l mater i a 1 ( 6 0 , 8 8 , 9 0 , 9 1 ) . O t h e r s o r b e n t s l i k e aluminum o x i d e ( 8 0 , 8 3 , 8 7 ) and c e l l u l o s e ( 8 1 ) t h e r e f o r e h a v e n o t r e c e i v e d much a t t e n t i o n . The d e t e c t i o n o f h y d r o c h l o r o t h i a z i d e o n p a p e r ( 1 7 , 4 0 , 7 8 ) , c e l l u l o s e l a y e r s ( 8 1 ) and aluminum o x i d e ( 8 0 , 8 3 , 8 7 ) was n o t s t u d i e d i n t e n s e l y . On s i l i c a g e l , t h e q u e n c h i n g of f l u o r e s c e n c e on l a y e r s containing a fluorescence indicator ( d e t e c t i o n l i m i t 0 . 2 p g ) and c o l o u r r e a c t i o n by h y d r o l y s i s , d i a z o t i s a t i o n and c o u p l i n g w i t h sodium chromotrop a t e ( d e t e c t i o n l i m i t below 0 . 1 p g ) were found t o b e r e l i a b l e and s e n s i t i v e methods. O t h e r r e p o r t e d v i s u a l i s a t i o n techniques(81,85,87,88,92) w e r e n o t found t o be of comparable s e n s i t i v i t y .
S u c c e s s f u l l y t e s t e d s y s t e m s ( e x a m p l e s ) ( 93 1 : E t h y l acetate+chloroform+methanol ( 1 1 + 8 + 1 ) , a t a b o u t 23OC, o n s i l i c a g e l 60 F-254 ( M e r c k ) , Rf o f h y d r o c h l o r o t h i a z i d e a b o u t 0.3. Used f o r semiq u a n t i t a t i v e s t a b i l i t y t e s t s on d o s a g e forms. Two s t e p d e v e l o p m e n t s y s t e m . a ) Diethylether+chloroform+etyhl acetate+ methanol ( 1 0 + 8 + 6 + 1 . 5 ) , b) Ethyl agetate+chloroform ( 2 2 + 3 ) , a t a b o u t 23 C , on s i l i c a g e l S i l - G 25 HR UV 254 ( M a c h e r e y - N a g e l ) , R f a +Rfb) o f h y d r o c h l o r o t h i a z i d e a b o u t 0.6. Used kor p u r i t y t e s t i n g o n a c t i v e substance.
The d i r e c t q u a n t i t a t i o n o f h y d r o c h l o r o t h i a z i d e on t l c p l a t e s was r e p o r t e d by S t e i n b a c h e t a 1 . ( 5 0 ) ( d e n s i t o m e t r y a t 2 7 2 nm) and S c h a f e r e t a l . ( 6 0 ) ( f l u o r i m e t r y ) . The l a t t e r method allows t h e d e t e r m i n a t i o n of 2 ng h y d r o c h l o r o t h i a z i d e on t h e p l a t e and w a s u s e d f o r t h e a n a l y s i s of human body f l u i d s a f t e r s i n g l e d o s e a p p l i c a t i o n .
430
HANS PETER DEPPELER
5.9.2
L i q u i d Chromatoqraphy
S e p a r a t i o n of h y d r o c h l o r o t h i a z i d e from t a b l e t i n g r e d i e n t s by chromatography o n a l k a l i n e c e l i t e c o l u m n s ( 5 2 , 9 4 ) and from b a s i c compounds i n m u l t i component p h a r m a c e u t i c a l s on i o n exchange columns ( 56,951 was shown t o be p o s s i b l e u n d e r l o w p r e s s u r e conditions (gravity). Later on, h i g h p r e s s u r e l i q u i d c h r o m a t o g r a p h y r e p l a c e d t h e low p r e s s u r e methods c o m p l e t e l y . The d i v e r s i t y of r e p o r t e d HPLC methods i s i l l u s t r a t e d by t a b l e X . 5.9.3
G a s Chromatography
The d e t e r m i n a t i o n i n p l a s m a , b l o o d c o r p u s c l e s a n d u r i n e by g a s c h r o m a t o g r a p h y w a s r e p o r t e d by Lindstroem e t a1.(104,105). Hydrochlorothiazide w a s methylated with methyliodide, using t h e e x t r a c t i v e m e t h y l a t i o n p r o c e d u r e . For t h e q u a n t i t a t i v e evaluation, an i n t e r n a l standard, chlort h a l i d o n e , was u s e d . Chromatographic c o n d i t i o n s ( l 0 4 ) : 1 %oSE-30 o n Gas-Chrom Q (80-100 m e s h ) , Column : 225 C: C a r r i e r n i t r o g e n Injector: 23OoC Detectors: ECD, 3OO0C; F I D , 27OoC The method h a s been a p p l i e d e . g . by Beermann e t a 1 . ( 1 0 6 - 1 1 3 ) i n p h a r m a c o k i n e t i c and b i o a v a i l a b i l i t y s t u d i e s and by Wallace e t a 1 . ( 8 8 ) as a c o n f i r m a t o r y method t o t l c i d e n t i f i c a t i o n methods. Vandenheuvel e t a1.(114) d e v e l o p e d a method f o r t h e a n a l y s i s of b l o o d and plasma b a s e d on t h e 'on-column m e t h y l a t i o n t e c h n i q u e ' w i t h t e t r a m e t h y l a n i l i n i u m h y d r o x i d e and t h e u s e of 6-bromo-3,4dihydro-2H-1.2.4-benzothiadiazine-7-sulfonamide 1 , l - d i o x i d e as t h e i n t e r n a l s t a n d a r d . The same i n t e r n a l s t a n d a r d was u s e d by R e d a l i e u e t a 1 . ( 1 1 5 ) i n a m o d i f i c a t i o n of t h e L i n d s t r o e m method. 5.10 E l e c t r o p h o r e s i s R u g g i e r i ( 1 1 6 ) proposed e l e c t r o p h o r e t i c s e p a r a t i o n of h y d r o c h l o r o t h i a z i d e f o r q u a n t i t a t i v e analysis.
Table X
HPLC m e t h o d s
column CSP a n i o n e x c h . o n Zipax 30 pm 1 0 0 0 ~ 2 . 1mm

Corasil-C18
eluent: 0.005 Na2S04 i n p H 9.2 borate b u f f e r : methanol :
volumes 35 5
sample
H. + h y d r o l y s i s
ref. 96 97 98
product +hydralazine artificial m i x t u r e s of antihypert.
2 2 0 ~ 2 . 3 mm Corasil-phenyl 1 2 2 0 ~ 2 . 3mm
p Bondapak C18
sc r e e n i ng
300x4 mm S p h e r i s o r b ODS 10 pm, 250x3 mm L i c h r o s o r b S160 5 pm, 2 5 0 ~ 2 . 1mm
0.01 M aq. NaH2P04: methanol :
4 1
serum/urine serum (gel filtered)

tablets ( H. + r e s e r p i n e 1
99
00
water: methanol :
n-hexane : 2-propanol: chloroform: d i et h y 1 a m in e : n-hexane : ethanol :
0 . 0 1 M a q . C12H25Na04S: 2-propanol : 0 . 1 N a q . H2S04:
85 15 77 18 5 0.01 55 45 75 23 5
101
Lichrosorb 5 pm, 5 0 0 ~ 4 . 4 mm N u c l e o s i l 10-CN 1 0 pm, 2 0 0 ~ 4 . 8 mm

H.
serum
tablets
H. + r e s e r p i n e
102
1 03
+hydralazine
= hydrochlorothiazide
432
HANS PETER DEPPELER
6.
I n t e r f e r e n c e s of Hydrochlorothiazide i n A n a l y t i c a l Methods
Hydrochlorothiazide i n t e r f e r e s with t h e u r i n a r y e s t r i o l d e t e r m i n a t i o n s by g a s c h r o m a t o g r a p h y ( l l 7 ) and by c o l o r i m e t r y w i t h t h e Kober r e a c t i o n ( 118 1 .

7. 7.1
P h a r m a c o k i n e t i c and M e t a b o l i c S t u d i e s A n a l y t i c a l Methods Used f o r B i o l o g i c a l Material
B e s i d e s r a d i o m e t r i c p r o c e d u r e s used i n a b s o r p t i o n and d i s t r i b u t i o n s t u d i e s ( 7 7 , 1 0 6 , 1 1 9 , 1 2 0 1 , s e v e r a l colorimetric methods were d e v e l o p e d f o r t h e d e t e r m i n a t i o n of h y d r o c h l o r o t h i a z i d e i n plasma and u r i n e ( 4 2 - 4 4 , 1 2 1 - 1 2 4 ) . The p e r f o r m a n c e o f b i o a v a i l a b i l i t y s t u d i e s by colorimetric methods may p r o d u c e errors as c o u l d be s e e n f o r c h l o r o t h i a z i d e ( 1 2 5 ) . T h e r e f o r e g a s c h r o m a t o g r a p h i c methods were p r e d o m i n a n t l y u s e d , i . e . t h e method of Lindstroem e t a 1 . ( 1 0 4 ) i n k i n e t i c s t u d i e s o f t h e g r o u p of Beermann ( 1 0 7 - 1 1 3 ) and by J o r d o e e t a 1 . ( 1 2 6 ) , t h e method of Vandenheuvel e t a 1 . ( 1 1 4 ) u s e d by S u n d q u i s t e t a 1 . ( 1 2 7 ) and t h e p r o c e d u r e of R e d a l i e u e t a 1 . ( 1 1 5 ) . H i g h - p r e s s u r e l i q u i d chromat o g r a p h y ( 1 0 0 ) and f l u o r i m e t r i c ( 6 0 ) or s p e c t r o p h o t o m e t r i c ( l 2 8 ) d e t e r m i n a t i o n a f t e r s e p a r a t i o n by t l c may be a l s o s u i t a b l e f o r t h e d e t e r m i n a t i o n of h y d r o c h l o r o t h i a z i d e i n b i o l o g i c a l material.
7.2
Absorption
Aft?$ s i n g l e i n t r a v e n o u s o r o r a l a d m i n i s t r a t i o n of C - l a b e l l e d h y d r o c h l o r o t h i a z i d e (Doses: i . v . 1, 3 5 , 65 mg, n = 3; 2.0. 5 , SO, 65 m g ; n = 4 , n = 6) t o v o l u n t e e r s and p a t i e n t s 90-93 % a n d 53-83 % o f d o s e , r e s p e c t i v e l y were e x c r e t e d i n u r i n e . T h e r e f o r e a b s o r p t i o n of a n o r a l d r u g d o s e w a s i n t h e r a n g e o f 60-80 %. I t was found t o b e reduced i n p a t i e n t s w i t h c o n g e s t i v e h e a r t f a i l u r e o r r e n a l and h e p a t i c d i s e a s e s s i n c e o n l y a b o u t 4 0 % o f d o s e or less were e l i m i n a t e d r e n a l l y ( l 0 6 ) . H y d r o c h l o r o t h i a z i d e w a s e x c r e t e d i n u r i n e of r a t o r man almost c o m p l e t e l y as t h e i n t a c t substance( 77,106).
HYDROCHLOROTHIAZIDE
433
The peak plasma c o n c e n t r a t i o n s of t o t a l r a d i o a c t i v i t y i n p a t i e n t s ( n = 3 ) were 260, 386 and 616 ng/ml r e a c h e d w i t h i n 3-4 h o u r s , t h e corresp o n d i n g v a l u e s i n b l o o d c e l l s were a b o u t 3 times h i g h e r ( l 0 6 ) . S i m i l a r blood/plasma c o n c e n t r a t i o n r a t i o s were a l s o r e p o r t e d f o r v o l u n t e e r s ( l 0 6 , 1 1 4 , 1 1 5 ) . The n a t u r e of t h e b i n d i n g of h y d r o c h l o r o t h i a z i d e i n t h e e r y t h r o c y t e s i s s t i l l unknown. I n v i t r o experiments with bovine carboanhydrase showed no b i n d i n g ( 1 0 4 , 1 0 6 ) . 7.3 Distribution
Organ d i s t r i b u t i o n p a t t e r n i n r a t s a f t e r s i n g l e o r a l a d m i n i s t r a t i o n of t r i t i u m l a b e l l e d h y d r o c h l o r o t h i a z i d e ( d o s e : 5 mg) r e v e a l e d h i g h e s t c o n c e n t r a t i o n s of t o t a l r a d i o a c t i v i t y i n l i v e r ( 2 7 . 8 pg/g) and g a s t r o - i n t e s t i n a l t r a c t ( 3 6 . 0 pg/g) w i t h i n 1 h o u r a f t e r d o s i n g . A t t h e same t i m e t h e c o n c e n t r a t i o n i n plasma was 1.53 pg/ml, t h a t i n s p l e e n , m u s c l e and b r a i n 0.36-0.46 p g / g ( 7 7 ) . A l o w d e g r e e of h y d r o c h l o r o t h i a z i d e b i n d i n g t o b o v i n e serum a l b u m i n was o b t a i n e d w i t h o n l y o n e binding site c l a s s ( 5 7 ) .
7.4
Basic P h a r m a c o k i n e t i c s
I n v o l u n t e e r s a f t e r s i n g l e o r a l d o s e admini s t r a t i o n of h y d r o c h l o r o t h i a z i d e ( n = 8; d o s e s : 1 2 . 5 , 25, 5 0 , 75 mg) t h e peak plasma c o n c e n t r a t i o n s o f i n t a c t d r u g r e a c h e d w i t h i n 1.5-5 h o u r s a n d t h e area u n d e r t h e c o n c e n t r a t i o n c u r v e s ( A U C , 0-9 h o u r s ) were l i n e a r l y c o r r e l a t e d w i t h t h e d o s e . Peak c o n c e n t r a t i o n s i n d e p e n d e n c e o f t h e i n c r e a s i n g d o s e s were 70 + 1 9 , 1 4 2 + 50, 2 6 0 + 88 and 376 + 70 ng/ml, r e s p e c t i v e l y - ( % ,+ s 1 . Hydrochlor o t h i a z i d e w a s e l i m i n a t e d from plasfia m o s t l y i n a b i p h a s i c way w i t h t e r m i n a l h a l f - l i v e s of 5.6-14.8 h o u r s ( l 0 7 ) . I n t h e same v o l u n t e e r s u s i n g t h e same e x p e r i m e n t a l d e s i g n t h e u r i n a r y e x c r e t i o n and t h e d o s e a d m i n i s t e r e d were s i g n i f i c a n t l y c o r r e l a t e d too. A t o r a l d o s e s of 12.5, 25, 50 and 75 mg t h e u r i n a r y e x c r e t i o n ( 0 - 4 8 h o u r s ) was 8.5 + 2 . 0 , 1 7 . 9 + 4 . 2 , 33.4 + 8.6 a n d 4 8 . 9 ,+ 7 . 6 respect i v e l y . The c u m u l a t i v e u r i n a r y r e c o v e r y of t h e d r u g was 65-72 % o f d o s e f o r a l l d o s e s a d m i n i s t e r e d . R e n a l c l e a r a n c e was a l s o i n d e p e n d e n t of d o s e w i t h
ms,
434
HANS PETER DEPPELER
345 + 1 2 3 t o 319 + 86 m l / m i n ( l 0 7 ) . I n s e v e n pat i e n & w i t h c o n g e s t i v e h e a r t f a i l u r e ( d o s e : 50 mg, n = 6 ; 75 mg, n = 1) h i g h e s t c o n c e n t r a t i o n s o f i n t a c t h y d r o c h l o r o t h i a z i d e i n plasma were f o u n d w i t h i n 1.5-8 h o u r s w i t h 282-672 ng/ml. The t e r m i n a l h a l f - l i v e i n plasma w a s 8.9-28.9 h o u r s ( n = 6 ) and 3 . 1 h o u r s i n one p a t i e n t w i t h t h e h i g h e s t h e a r t f a i l u r e . Urinary e x c r e t i o n of i n t a c t drug (0-7 d a y s ) i n t h e s e p a t i e n t s may be r e d u c e d (20.8-71.6 % of d o s e ) f 1111 . I n hypertensive p a t i e n t s during repeated treatment w i t h d i f f e r e n t d o s e s of h y d r o c h l o r o t h i a z i d e ( d o s e s : 12.5, 25, 5 0 , 75 mg/day f o r . 2 c o n s e c u t i v e weeks; 75 mg f o r a d d i t i o n a l 4 w e e k s ) p r e - d o s e p l a s m a l e v e l s of i n t a c t s u b s t a n c e showed a l i n e a r r e l a t i o n s h i p t o i n c r e a s i n g d o s e s w i t h 15 + 7 , 1 7 2 8 , 2 7 2 11 and 34 f. 1 7 ng/ml, r e s p e c f i v e l y . T h i s w a s a l s o o b t a i n e d f o r plasma c o n c e n t r a t i o n s 5 h o u r s a f t e r d o s i n g . S t e a d y s t a t e plasma concent r a t i o n o f i n t a c t d r u g a f t e r 6 weeks of d a i l y t r e a t m e n t w i t h 75 mg h y d r o c h l o r o t h i a z i d e w a s f o u n d t o be 111 ng/ml. U r i n a r y e x c r e t i o n of i n t a c t hydroc h l o r o t h i a z i d e w i t h i n t h e l a s t 2 4 h o u r s of e a c h t r e a t m e n t p e r i o d w a s a b o u t 6 0 % o f d o s e and r e n a l c l e a r a n c e a c c o u n t e d f o r 317 + 1 2 0 m l / m i n ( l l 3 ) . O t h e r a u t h o r s , u s i n g a i e s s s p e c i f i c method, h a v e o b s e r v e d s t e a d y s t a t e plasma levels o f 970 2 90 ng/ml and 2250 f. 2 0 ng/ml ( x + s ; ) d u r i n g a 1 2 week and 2 0 week t r e a t m e n t p e r i o d - i n h y p e r t e n g s i v e p a t i e n t s a t d a i l y o r a l d o s e s o f 150 and 450 m hydrochlorothiazide, respectively(44). 7.5 Bioavailability
S i n c e h y d r o c h l o r o t h i a z i d e i s e x c r e t e d almost c o m p l e t e l y as t h e i n t a c t s u b s t a n c e i n man, i t s c u m u l a t i v e u r i n a r y e x c r e t i o n i s t h e best m e a s u r e of bioavailability.
As c o u l d b e s e e n above, t h e u r i n a r y r e c o v e r y g o f t h e d r u g a f t e r s i n g l e o r a l d o s e s of 12.5-75 m h y d r o c h l o r o t h i a z i d e ( c o m m . 25 m g t a b l e t s ) is indep e n d e n t of t h e d o s e w i t h 65-72 % ( 1 0 7 ) . The u r i n a r y e x c r e t i o n was i n t h e same r a n g e i n a n e x p e r i m e n t where b i o e q u i v a l e n c e o f t w o d o s a g e f o r m s f c o m m . 25 m g t a b l e t s of d i f f e r e n t o r i g i n ) w a s o b s e r v e d w i t h 70.8 + 1 4 . 9 vs. 65.2 + 1 0 . 1 % o f d o s e ( 108 1 . T h i s was a l s o o b s e r v e d i n s t u d i e s comparing
HYDROCHLOROTHI AZIDE
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several dosage forms of hydrochlorothiazide, but less specific colorimetric methods were used(121, 123,124). Enhanced bioavailability of hydrochlorothiazide was obtained in volunteers ( n = 8; dose 75 mg) when the drug was administered with food (74.2 + 6.5 vs. 63.2 2 8.0 % of dose measured in urine)-after the pretreatment of volunteers with the anticholinergicum propantheline ( n = 6; dose 75 mg; 5 6 . 9 + 4.4 vs. 49.3 + 5.3 % of dose in urine)(109,1iO) or after concomitant administration of polyvinylpyrrolidone(l28~. No influence on the bioavailability of hydrochlorothiazide was observed when sotalol, metoprolo1 or hydralazine were administered in combined dosage forms or separately to volunteers(l22, 126,127,129). A significant malabsorption, measured in terms of urinary excretion of intact hydrochlorothiazide was found in patients with congestive heart failure ( n = 7; dose: 50-75 mg) or in patients after intestinal shunt surgery ( n = 5; dose 75 mg) with an average urinary recovery of 40.7 and 30.7 8 of dose, respectively(lll,ll2). The relationship between bioavailability data and in vitro dissolution test results has been investigated repeatedly(l21,123).
8.
Acknowledgements
The author gratefully acknowledges the assistance of K.O. Alt, K. Brugger, E . Felber, H . Fuhrer, 0 . Heiber, H. Huerzeler, E . Marti, S . Moss, W. Padowetz, P.H. Stahl and R . Steiner who contributed much previously unpublished information about physical and chemical properties and especially of K.F. Feldmann who prepared the chapter about pharmacokinetic and metabolic studies.
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HYDROCHLOROTHIAZIDE
441
113. B e e r m a n n B., G r o s c h i n s k y - G r i n d M.; E u r o p . J . C l i n . P h a r m a c o l . l3, 1 9 5 - 2 0 1 ( 1 9 7 8 ) G r u b e r V.F., Walker R.W., 114. V a n d e n h e u v e l W.J.A., Wolf F . J . ; J. P h a r m . S c i . 6 4 , 1309-1312 ( 1 9 7 5 ) Wagner W.E. j r ; 115. R e d a l i e u E . , T i p n i s V.V., J . Pharm. Sci. 6 7 , 726-728 ( 1 9 7 8 ) 1 1 6 . R u g g i e r i R.; Ban. C h i m . F a r m . 99, 2 0 - 2 3 ( 1960) C l i n . Chem. 18, 1 1 7 . Rosenthal A.F., Tomson M.R.; 471-472 ( 1 9 7 2 ) 1 1 8 . Watanabe F . , N a k a h a r a M., T s u b o t a N . , T s u k i d a K . , S a i k i K . , I t o M.; C l i n . C h i m . A c t a 88, 2 1 - 2 5 ( 1 9 7 8 ) Brettell H.R., Aikawa J . K . ; 1 1 9 . A n d e r s o n K.V., Arch. I n t e r n a l Med. 107, 7 3 6 - 7 4 2 ( 1 9 6 1 ) 1 2 0 . C a l e s n i c k B., Sheppard H . , Bowen N . ; Fed. P r o c . 20, 409 ( 1 9 6 1 ) Needham T.E.; J. P h a r m . S c i . 68, 1 2 1 . Shah K . A . , 1486-1490 ( 1 9 7 9 ) 1 2 2 . C I B A P h a r m a c e u t i c a l Company, S u m m i t R N . J . , B i o a v a i l a b i l i t y Data of Apresazide , September 1 9 7 6 1 2 3 . Meyer M.C., M e l i k i a n A.P., W h y a t t P.L., S l y w k a G.W.A.; C u r r . T h e r . R e s . l7, 5 7 0 - 5 7 7 ( 1 9 7 51 1 2 4 . Cook D . ; P h a r m a c o l o g y 2 , 1 9 0 - 1 9 5 ( 1 9 7 2 ) 1 2 5 . Resetarits D.E, B a t e s T.R.; J. P h a r m . S c i . 6 8 , 1 2 6 - 1 2 7 ( 1 9 7 9 ) 1 2 6 . Jordoe L , J o h n G o n G. , L u n d b o r g F , P e r s s o n B.A., Regardh C.-G., R o e n n 0.; B r . J. C l i n . Phannac. 7 , 563-567 ( 1 9 7 9 ) 1 2 7 . S u n d q u i s t H . , A n t i l l a M., S i m o n A . , Reich J . W . ; J. C l i n . P h a r m a c o l . 2 , 557-564 ( 1 9 7 9 ) 128. Corrigan O.I., T i m o n e y R.F., Whelan M . J . ; J . P h a r m . P h a r m a c o l . 28, 7 0 3 - 7 0 6 ( 1 9 7 6 ) 1 2 9 . Wagner W.E., G i l l e r a n T., Zak S . ; C l i n . Pharmacol. Ther. 1 7 , 247 (1975)
L i t e r a t u r e (C.A.)
surveyed t h r o u g h 1 9 7 9
KETOPROFEN
Gary G. Liversidge
1. Description 1.1 Nomenclature 1.2 Formula 1.3 Molecular Weight 1.4 Appearance, Colour, Odour, and Taste 2. Physical Properties 2.1 Melting Range 2.2 Solubility 2.3 pH 2.4 Dissociation Constant 2.5 Partition Coefficient 2.6 Thermal Analysis 2.7 Crystallinity 2.8 Ultraviolet Spectrum 2.9 Optical Rotation 2.10 Mass Spectrum 2.11 Photoelectronic Spectrum 2.12 Nuclear Magnetic Resonance Spectrum 2.13 Infrared Spectrum 3. Synthesis 4. Stability and Degradation 5. Absorption, Metabolism and Excretion 6. Methods of Analysis 6.1 Elemental Analysis 6.2 Thin Layer Chromatographic Analysis 6.3 Ultraviolet Spectroscopy 6.4 Potentiometric Titration 6.5 Gas Chromatography 6.6 Enantiomer Analysis 6.7 Colorimetric Analysis 7. Analysis of Biological Samples 7.1 Colorimetric Analysis 7.2 Polarographic Analysis 7.3 Gas Chromatographic Analysis 7.4 Thin Layer Chromatographic Analysis 7.5 Gas Chromatography-Mass Spectroscopy Analysis 7.6 High Pressure Liquid Chromatographic Analysis 8. Analysis of Pharmaceutical Formulations 8.1 Potentiometric Titration 8.2 Pyrolysis-Gas Chromatography-Mass Spectrometry 9. Acknowledgements 0. References
444 444 444 444 445 445 445 445 445 445 445 446 446 446 449 449 449 453 453 456 456 456 460 460 460 462 462 462 462 463 463 463 463 465 466 467 467 468 468 469 469 469
ANALYTICAL PROFI1,ES OF DRUG SUBSTANCES, 10
443
441
GARY G . LIVERSIDGE
1.
Description
1.1 Nomenclature 1.1.1. Chemical Names

m-Benzoylhydratropic acid (1,2), a- (benzoylphenyl) propionic acid (3), a- (3-benzoylphenyl) propionic acid (4,5,6) , 2-(3-benzoylphenyl) propionic acid (1,2,7-11), 2- (benzoyl-3-phenyl) propionic acid (12,131, Benzeneaceticacid, 3-benzoyl-a-methyl. The latter name is used by Chemical Abstacts. The Chemical Abstract's registry number for (2)ketoprofen is 22071-15-4 , for the ( + ) enantiomer it is 22161-81-5 and for the ( - ) enantiomer it is 56105-81-5
1.1.2. Nonproprietary Name Ketoprofen 1.1.3. Propietary Name Profenid@ Alrheumat@, Alrheumun6,
Orudis@,
1.2 Formula
1.2.1.
Empirical
C16H1403
1.2.2. Structural
1.3 Molecular Weight 254.29
KETOPROFEN
445
1.4 Appearance, Colour, Odour and Taste A slightly coloured, odourless, tasteless powder with an irritant dust.
2.
Physical Properties 2.1 Melting Range
93-95OC(5)I 94-95OC(14)I 94OC(1,2,15,16) 96OC, 92OC ( 1 7 ) , 91OC ( 1 8 ) . 2.2 Solubility soluble ether soluble ethano1 s1ightly water soluble octanol soluble disopropyl ether soluble acetone soluble chloroform dimethylformamide soluble soluble methanol soluble ethyl acetate 2.3 pH The pH of a 3.95 x is 6.5 (5). 2.4 Dissociation Constant The pKa in : dioxan : water (2:l) is 7.2 (20), acetonitrile : water (3:l) is 5.02 ( 5 1 , methanol : water (3:l) is 5.937 (14). 2.5 Partition Coefficient
M solution in water
The partition coefficient of ketoprofen in an n-octanol/water (phosphate buffer pH 7.35 and initial ketoprofen concentration of 0.2542 mg/ml in this) is 0.105 ( 5 ) and at pH 7.4 (MacIlvaine's buffer and initial ketoprofen concentration of 0.0240 mg/ml in this) is 0.97 (20). At these pH's most of the ketoprofen is ionised (20) and thus an increase in the initial concentration of ketoprofen in the buffer will cause an alteration in the partition coefficient.
446
GARY G . LIVERSIDGE
2.6 Thermal Analysis 2.6.1. Differential Thermal Analysis

A D.T.A. thermogram of ketoprofen at a heating rate of 5OC per minute and sample size of 4 mg in a static air atmosphere shows an endotherm at 96OC which indicates melting (Fig. 1). If the melted sample is cooled to OC and then analysed again no peak corresponding to melting can be detected. The ketoprofen is in a glass like form. On storage the glass like form changes to the regular crystdllineform, conversion is complete in ten days at room temperature.
2.6.2. Thermogravimetric Analysis

A TGA thermogram of ketoprofen at a heating rate of 20C per minute and sample size of 6 mg in a static air atmosphere shows no l o s s of weight until 223% when ketoprofen decomposes (Fig.2).
2.7 Crystallinity 2.7.1. Polymorphism Ketoprofen can exist in two Polymorphs as mentioned in section 2.6.1. on differential thermal analysis. Ketoprofen forms white crystal prisms when crystallised from di-isopropyl ether ( 5 ) . 2.8 Ultraviolet Spectrum The UV spectra of ketoprofen (3.95 x 10-4m) in the following solvents are given in Figure 3 (using Varian Techtron M 165) 1. 0.1N hydrochloric acid pH 1.2 (5) 2. distilled water pH 6.5 (5) 3. 0.1N sodium hydroxide pH 12.9 (5) appears at 261 nm and corresponds toa The A m K ban$. This maximum is independent of p H but the maximum absorbance is slightly decreased with increasing pH. The Xmax in methanol has been The reported as 255 nm and log E = 4.33 (14 2 1 ) Xmax in ethanol has been reported as 255nm and
cm
640 ( 7 ) .
KETOPROFEN
447
Figure 1.
Differential Thermogram of Ketoprofen
g %
Weight
N"
Figure 2.
Thermogravimetric Curve of Ketoprofen
448
GARY G. LIVERSIDGE
Of
0;
Of
0:
u
p OL a
13
03 02
0.1
200
250
nrn
Mo
31
Figure 3
Ultraviolet Spectrum of Ketoprofen (see text for key)
'8D
.L
. a
* 40
.3
.2
2G
.I
0 A 0
fi
0; 2 %
-1
' 0
-20
-LO
-2
- 60
-80
-3
-L
200
250
Figure 4
CD Spectra of Ketoprofen's Enantiomers
KETOPROFEN
449
2.9 Optical Rotation
Both enantiomers show Cotton Effects at 223 nm (4) as demonstrated in Figure 4. The (+)-enantiomer shows a positive Cotton Effect indicating a S-absolute configuration and interacts more strongly with human serum albumin as well as with biotransformation enzymes than the (-)-enantiomer 23 (4) (+)-enantiomer [ a ] , + 57.1 (C = 0.76 in CH2C12) 23 (415) (-)-enantiomer [a], - 57.4O (C = 0.88 in CH2C12) 23 (415) (+)-enantiomer [ a ] , + 49.6O ( C = 1.15 in CH2C12) 23 (3) . ( C = 1.05 in CH3C13) (-)-enantiomer [ a ] , - 5 2 . 4 ' (3) 2.10 Mass Spectrum
( 2 )a- (3-benzoylphenyl) -propionic acid (3,4,5 ) .
Ketoprofen is a racemic mixture of
The mass spectrum of ketoprofen has not been published but the mass spectrum of the methyl ester has (8,10,13), see Figures 5 and 6 (using LKB900S gas chromatograph mass spectrometer) (13). The fragmentation pattern is reported in Figure 7. The fragmentation pattern for ketoprofen will be similar, the methyl of the ester being replaced by a hydrogen atom. 2.11 Photoelectronic Spectrum The photoelectronic spectrum on a Vacuum Generators UV G3 instrument (Figure 8 ) exhibits several b nds characteristic of the benzophenone group. Notably the partially overlapping bands at 9.07 and 9.45 eV, these bands are a result of the ionisation of the two degenerated phenyl orbitals. The energy at 10.62 eV corresponds to the ionisation of a free electron pair of the carboxylic carbonyl group. Bands have been assigned as:47r<n (a") <n0<2nCO<n0(a") keto<aonset ( 5)
150
100
105
GARY G . LIVERSIDCE
77
80
h
bp
v
209
x
U
4
60
C I u
L,
C
r(
I u
. . I
40
c,
03
20
0 0
L 100
, 1 ,1
200
7 90
300
m/z
Figure 5
Mass S p e c t r u m o f K e t o p r o f e n Methyl E s t e r a t 7 0 EV
100
209
80
h
bt
4
2 68
60
C
(u
+.
a,
105
40
.-!
U
>
a,
20
0 0
L
10 0 200
300
m/z
Figure 6
Mass Spectrum of K e t o p r o f e n Methyl E s t e r a t 2 0 EV
( D
N n
c, m
a,
w
+ I
Ipl 0
u
0
4-1
u-u
-. -. E
N
I "I
0:
E
E:
0
o=w
\ f
r "I 0-v
P
m
k
-09 .C( D
a,
7
c-
.?I
m
t n
!&
n
0
c
m \
a
M
c
w
0
a,
c,
x
w
0
0)
c,
U
5 k
a,
Lo
a
U -4
c,
rl
a,
c
PI
03
CI 0
a, 0
a,
&
-4
? & n
h
KETOPROFEN
453
2.12 Nuclear Magnetic Resonance Spectrum The NMR spectrum of ketoprofen in CDC13 on a EM-360 6MH,. NMR spectrometer is given in Figure 9. Table 1 compares the published values for the NMR spectrum. TABLE I
Instrument Used
I
Values
I 1
I
Ref
I 7.20-7.90(M, 9H), 11.50(s,lH) arian T60 & A601 1.52(d,3H,J = 7Hz), 3.76(q,lH,J = 7Hz),I (14) 7.2-7.8(m,9H), 11.8(s,lH) arian T60 & A601 1.51 (d,3H,J = 7.4Hz), 3.82(q.lH,J = 7.4Hz), 7.2-8.0(m,9H), 11.55(s,lH) 1 I I I
2.13 Infrared Spectrum

7 1 0 B instrument of ketoprofen is reported in
The infrared spectrum on a Perkin Elmer
Figure 10. The major band assignments are given in Table 11. TABLE I1
Band Position
I
Assignment
L
I
3200-2500 3020 2970,2930
;2880 1695 1655 1595,1580,1455 1440 1370 860-690 (several bands)
0-H stretching C-H stretching of aromatic C-H stretching of CH3 group (assymetric) Masked by 0-H stretching C-H stretching of CH3 group (symmetrical) masked by 0-H stretching C=O stretching of acid C=O stretching of ketone C=C stretching of aromatic ring C-H deformation of CH, (assymetrical) C-H deformation of CH3 ( symetrical)
C-H deformation of aromatic rings
Figure 9
Nuclear Magnetic Resonance Spectrum of Ketoprofen
?
I
.o U c
0
-w m
C
O U t
rD 0- R
0 2 * m O W >
-u
0
.O
0 N
.O
N 0
1s
n (Y
n w
0 0 m
0 v)
0 Q
0 N
"2
(%) 3JNVll I Y S N W Y I
455
456
GARY G . LIVERSID(;E
3.
Synthesis
Several methods for the synthesis of ketoprofen have been reported in the literature (15-20), 23-27, 43, 46-54). The synthesis starting from (3-benzoylphenyl)-acetonitrile is illustrated in Figure 11 (15,16,41). The synthesis starting from (3-carboxyl-phenyl)-2 propionitrile is illustrated in Figure 12 (17,20). The synthesis starting from 2- (4-Aminophenyl) propionic acid is illustrated in Figure 13 (15,17,19). 4. Stabilitv and Dearadation
Ketoprofen must be protected from light and moisture. It is stable at room temperature. Ketoprofen has been dissolved in ethyl acetate and stored for several weeks at 4OC with no detectable decomposition (13). If ketoprofen is heated in an acid solution pH1 at 98OC for 30 min. no decomposition is detected (28).
5.
Absorption, Metabolism and Excretion
Ketoprofen is absorbed rapidly regardless of the route of administration. It reaches a peak maximum in the first hour of administration if taken by the oral, rectal and parental routes and six hours if taken by the subcutaneous route. Peak blood levels by the rectal route are observed after 45 mins. to 60 mins. (29,30,31). Peak blood levels by the oral route are observed after 60 mins. to 90 mins. (31,32,33) and by the intramuscular route after 30 mins. (32). The half life of ketoprofen has been reported from 1.5 hrs. to 2 hrs. (6,29,30, 32-36). From 60% to 90% of ketoprofen is bound to serum protein (29). The kinetics of elimination are first order and the rate constant is 0.350 hrs., 63% of the administered dose is excreted in the urine during the first 24 hrs. and 65. in the first 48 hrs. Minimal excretion occurs in the faeces, the rat being the exception (29). The metabolism of ketoprofen is due to two major processes, a hydroxylation process, predominant in the rat, although the prefered excretory form in the rat is unchanged ketoprofen and glucuronide conjugation in other species including man. The glucuronide conjugation pathway is predominant in the rabbit and man but in man the hydroxylation is not totallyabsent
KETOPROFEN
457
CH31
Figure 11 Synthesis of Ketoprofen Starting From (3-benzoylphenyl)-acetonitrile
458
GARY G. LIVERSIDGE
HocoC
II
H3
cLcocH
H3
It
-cN
F RI C DEL C R A F 1
1)
HYDROLYSIS
I
Figure 12 Synthesis of Ketoprofen Starting From (3-carboxy-phenyl)-2 propionitrile
KETOPROFEN
459
P O T A S S l U l l E THVLXANTHATE
7-IODOEENZOIC
ACID
POLYPHOSPHOAIC A C I D
RANEY N I C K L L
Figure 13 Synthesis of Ketoprofen Starting From 2- (4-Aminophenyl) propionic acid
460
GARY G . LIVERSIDGE
see Figure 14 ( 2 9 , 3 1 ) . As mentioned in section 2.9 Optical Rotation ketoprofen can exist in two enontiomeric formsl each having a different affinity for human serum albumin and different biotransformation pathways (Hydroxylation pathways)
(4).
6.
Methods of Analvsis
6.1
Elemental Analysis C(75.58%) I H(5.55), 0(18.87%)
6.2
Thin Layer Chromatographic Analysis
Stationery phase and platecoating Cellulose 0.10 cm thick Silica gel 0.25 cm thick Silica gel 0.25 cm thick Silica gel 0.25 cm thick Silica gel 0.25 cm thick Silica Silica
Mobile phase
Rf (References)
(21)
Silica
sec-butanol: 1.35 absolute ethanol: water:ammonia 32% (50:30 19:l) v / v . ch1oroform:methanol: 1.42 ammonia 32% (120:60:0.5) v/v hexane:acetone:water 0.72 (12.15:3) v/v. iso-octano1:dioxan: 0.88 glacial acetic acid (20:20:1) v/v . butyl acetate:methyl 1.03 ethyl ketone: glacial acetic acid:water (4:4:2:1) v/v ch1oroform:methanol 0.42 (94:6) 3.05 n-hexane : dimethylketone: acetic acid (90:10:2) dichloromethane: 0.75 methapol. ammonia t98150: 3)
(21) (21) (21) (21)
*
* * *
(5) (5)
(5)
751 c u
o m o m
hell Y Z J
P I C
Y 2 c u
u 0 u
P I C
ucll
O n 3 c u 0 u
u n z c
0 0
r
I
u--u
0
1I
u-u-0
I "
3 04i 0
I
w
w
u I
'i
o=w
O=U
462
GARY G . LIVEHSIDGE
6.3 Ultra Violet Spectroscopy Quantitative determinations of ketoprofen based on the peak maximum at 261 nm in distilled water (5) or 256 nm in methanol can be performed (21) see Section 2.8 and Figure 3. 6.4 Potentiometric titration If ketoprofen is accurately weighed and dissolved in acetonitri1e:water (3:l) and titrated with 0.1 NaOH the potentiometric curves are recorded between pH 3.45 - 12.0. This method is convenient for measuring the purity of ketoprofen in the crystalline preparation and also the content in tablets (5). 6.5 Gas Chromatography Gas chromatography is an inconvenient method for purity determination, as ketoprofen is partially decomposed by the procedure. This can be overcome by using the methylester or trimethylsilylester prepared quantitatively from ketoprofen. According to the method of Populaire et a1 (37) ketoprofen and the esters can be chromatographed on a 1 m x 3 mm column of OV-17, 5% GCQ 80-100 mesh at a temperature between 230 - 27OOC and carrier gases; argon:50 ml/min, hydrogen:79 ml/min, air:150 ml/min, and a Flame Ionisation Detector. The retention times Rt are for ketoprofen 6.35 min, for the methyl ester 4.05 min and for the trimethylsilyester 2.68 mins (5).
6.6 Enantiomer Analysis
The ratio of (+)-ketoprofen to (-)-ketoprofen in a racemic mixture can be determined by reaction with a stereospecific molecule and the product is then analysed by either gas chromatography (5) or high pressure liquid chromatography (38). The ratio of peak height or peak area respectively gives the ratio of the two enantiomers.
KETOPROFEN
463
6.7 Colorimetric Analysis
Ketoprofen can be complexed with safraniii and the absorption determined in chloroform at 5 2 0 nm ( 1 4 ) .
7.
Analysis of Biological Samples

7 . 1
Colorimetric Analysis
This method is suitable for the analysis of ketoprofen in urine. The urine is made alkali by addition of NaOH then extracted with ether, the aqueous layer is then acidifiedandthe ketoprofen extracted with hexane and evaporated to dryness. The ketoprofen (via its carbonyl group) is reacted with p-nitrophenylhydrazine to give a p-nitrophenylhydrazone which gives a violet colour with trimethylbenzylammonium hydroxide. This violet complex is then assayed colorimetrically at 5 6 0 nm and 4 6 0 nm (see Figure 1 5 for spectrum and sample - ) . The full of blank experimental detail is reported by P. Populaire et a1 ( 3 7 ) . The p-nitrophenylhydrazone formed undergoes partial decomposition to give other hydrazones, but these also absorb at 5 6 0 nm and are directly proportional to the concentration of ketoprofen in total and this decomposition does not interfere with the assay. The absorption due to ketoprofen at 5 6 0 nm is determined by subtraction of the absorption by urine blanks, at 4 6 0 nm ( 4 6 0 nm Abs = 5 6 0 nm Abs for blank) from it, this is the case for humansand cats. But for rats and rabbits the absorbance at 480 nm is deducted from the absorbance at 5 6 0 nm i.e. interference is species dependent. The precision of this method 10% over the urinary concentration range of is 10-100 mg/L and the limits of detection of 2-5 mg/L in urine.
......
7.2
Polarographic Analysis
This method is suitable for the analysis of ketoprofen in urine. The same extraction procedure asin Section 7 . 1 is used as urinary substances will interfere with the polarogram. The carbonyl groupof ketoprofen is reduced at the dropping mercury electrode, in a 0 . 2 m solution of
0
Ln
Figure 15
0 0 0
0 0
Colourmetric analysis of Ketoprofan in Biological Samples

d d
CI]
0
rd
0 . 3
I d
a,
1 0
KETOPROFEN
465
tetrabutylammonium hydroxide. The half wave potential Ef is -1.36 volts, see Figure 16 and the method employs a standard addition technique. For further experimental details see P. Populaire (37). The precision of this method is flO% of urinary concentrations in the range 10 to 1 0 0 mg/litre and the limit of detection is 5 mg/litre. 7.3 Gas Chromatographic Analysis If ketoprofen is analysed directly by gas chromatography partial decomposition results. This difficult can be overcome by working with the methyl ester (5,8,13,30,31,34,35,37,39), and using (Benzoyl-4-phenyl)-2 butyric acid as the internal standard as itsmethyl ester. Neither serum or urine samples of ketoprofen are gas chromatographed directly, an extraction procedure is employed. In the case of serum the sample is acidified then extracted by an organic solvent usually ether. In the case of urine the sample is made alkali and the unwanted products extracted with an organic solvent usually ether, the aqueous phase is then acidified and extracted with ether. The etherial extracts are washed with acid and then water, dried with magnesium sulphate and then evaporated to dryness. The samples are then methylated and chromatographed (5,29,30,31,34,35, 37). In some cases further purification prior to gas chromatography is performed using thin layer chromatography (37). Table IV gives the conditions of chromatography and retention times (Rt) of the methyl esters of ketoprofen (A) and the internal standard (Benzoyl-4-phenyl)-2 butyric acid ( B ) obtained from biological media. The accuracy of this technique has been claimed as f10% and a lower limit of detection of 0.03 - 0.04 mg/litre (37)
466
GARY G. LIVERSIDGE
TABLE IV
Length and diameter internal (Reference)

I
I
Support, s t a t i o n a r ] C a r r i e r phase and Temp. g a s and flow rate
R t (A)
R t (B)
6ft x 2 m m (35)
1.5m x 2 m m (8) 2 m x 2 m m (13) 2 m x 3 m m (29,391 1.5m x 2 m m (10) 2m x 3mm (40)

lm x 3 m m
(5)
OV-17 3% GCQ 80/100 mesh 25OoC f o r - 1 0 mins t h e n 0 280 for 4 mins. I ov-1 3% GCQ 18O0C-25OOC a t z0c/min OV-1 1% G C P 100/120 mesh 25OoC OV-17 1% Chromosorb W AW D M C S 24OoC OV-17 3% GCQ 80/100 mesh 225'C OV-17 3% Chromasorb W AW DCMS 24OoC OV-17 5% GCQ 80/100 mesh H 2 0 230-270C
N 2 60ml / min
6.29
8.85
H e 2Oml /min
14
18
H e 3Oml /min
N 2 30ml /min
N2 3 0 m l /min N2 30ml /min
4.8
6.6
4.5 6.6
4.8
A r 50ml/mi H, 79m l / m i a i r 150ml/ min
4.05
7.4 Thin Layer Chromatographic Analysis A TLC method for the analysis of ketoprofen and its urinary metabolites has beendescribed (31) using a two dimensional development system. But the separation is incomplete and the system insufficiently sensitive. After extraction from biological samples as in Section 7.3 ketoprofen can be analysed by TLC, using 250 urn thick Merck 60 F254 plates, activated at 105OC for 1 hour, with a solvent system of ether-benzene-l-butanol-methanol (85:8:6:1), giving an Rf value of 0.75. The spot can be
KETOPROFEN
467
scraped off the plate, dissolved in ethanol and analysed under UV. Accuracy of + 6 . 0 1 % and a limit of detection of 11.19 is claimed by Ballerini et a1 (7). A further method involving methylation of ketoprofen has been described ( 3 7 ) .
7.5 Gas Chromatography Analysis
Mass Spectroscopy
Ketoprofen in biological extracts is converted to methyl ester with an internal standard (benzoyl-4-phenyl)-2-butyric acid before undergoing GC-MS, as ketoprofen undergoes rearrangement when subjected to gas chromatography, see Section 6.5 (8,10,13). The limits of detection by this method are 2.5 ng and an accuracy of 10% at plasma concentrations of 25 ng/ml (8).
7.6 High Pressure Liquid Chromatographic Analysis
Most methods for the analysis of ketoprofen in biological samples require the selective extraction of ketoprofen (42) similar to Section 7.3 (12,28,39-421, or by direct injection (44). Table V gives the chromatographic conditions retention times (Rt), limit of detection and accuracy for ketoprofen.
1.0
1.2
1-4
VOLTS
1.6
Figure 16
Polarogram of Ketoprofen
3 68
GARY G. LIVERSIDGE
TABLE V
Column, Packing, diameter intercal (Reference) Hewlard Packard RP8-79918 A 25Ox4mm ( 7 ) Spherisorb-5 ODS 50 x 5 mm (44) Lichrosorb S 1 60 Spm 250 x 4.7 mm (39,401 Lichrosorb RP 18 Sum 150 x 4.7 mm
Mobile phase, flow rate pressure
Rt min
Detection limit and accuracy 0.1 pg/ml 25.1% 2.5 ng f2.1% 0.1 pg/ml
Water/Methanol 85/15, 0.8 ml/min 32-38 atmos 35% aqueous methanol pH3 2 ml/min dichloromethane/ hexane 60/40 1.3 ml/min 35 bars methanol/ acetonitrile/ phosphate 15/35/45 gH3 0.83 ml/min 7 0 bars
8.3
2 :12.a
24%
8
24%
0.1 pg/ml
40 x 4.6 mn (42)
0.5 phosphate pH 7.0 16 1 6 4 % acetonitrile 2.0 ml/min 1000 psi methanol/water 45/44 1.1 ml/min 1800 p i acetonitrile/ phosphate 0.02 m pH3 45:55 1000 psi 5.3 5.2 4.2
10-23 ng i ml
0.1 pg/ml -+5.4%
300 x 4 mm (12) Lichrosorb RP 18 5 pm or Lichrosorb RP 8 5 pm. 1 0 0 x 4.6 mm ( 9 )
methyl ester of ketoprofen
8. Analysis of Pharmaceutical Formulations

8.1 Potentiometric Titration
300 mg of ground sample are dissolved in 5 mls of acetonitrile or ethanol and 15 ml of water added. The titration is performed with 0.1 N N a O H
KETOPROFEN
469
and monitored using a glass electrode and a calomel reference electrode ( 5 ) .

8.2 Pyrolysis - Gas Chromatography Spectrometry
Mass
Ground samples are dissolved in a solvent and a known amount applied to a rotating wire. After evaporation of the solvent the material is pyrolysed in a Curie point pyrolyser at 77OoC for 5 seconds and the pyrolate purged onto a Carbowax 20 M - KOH column, that is temperature programmed from 1 0 0 to 24OoC. On pyrolysis ketoprofen is rearranged to (3-benzoylphenyl)-ethane and (3-benzoylphenyl)-ethylene which have retention indices of 2.27 and 2.52 respectively, analine having a retention of 1.00. Limits of reproducible detection range from 10 ng to 10 pg. This method can also be employed in the analysis of biological materials (45,221.
9.
Acknowledgements
I wish to express my thanks to Mrs. T. Bowler for typing this manuscript. References
1. 2.
3.
4.
5.
6. 7. 8.
9.
Drugs of Today, 1973, IX (II), 468-471. Merck Index, 9th E d . , Ed. M. Windholz, Pub. Merck & Co. Inc. Rahway, N.J., U.S.A., 1976 pp. 695-696. Rendic, S., Alebic-Kolbah, T., Kajfez, F., Sunjic, V. Farmaco, Ed. Sci. 1980, 35 (I), 51-9. Rendic, S., Sunjic, V., Kajfez, F., Blazevic, N . , Alebic-Kolbah, T., Chimia 1975, 29 ( 4 ) , 170-2. Blazevic, N., Zinic, M., Kovac, T., Sunjic, V., Kajfez, F. Acta. Pharm. Jugosl. 1975, 25 ( 3 ) , 155-64. Lombardino, J.G., Otterness, I.G., Wiseman, E.H. Arzneim. Forsch. 1975, 25 ( l o ) , 1629-35. Ballerini, R., Cambi, A F D e l Soldato, P., Melani, F., Meli, A., J. Pharm. Sci 1979, 68 (3); 366-8. HeuSSe, D - , Raynaud, L., Ann. Pharm. Fr. 1978, 36 (11-121, 631-8. Bannier, A., Brazier, J.L., Quincy, C. Feuill. Biol. 1979, 106, 91-8.
4 $0
GARY G. LIVERSIDGE
10
11.
12. 13. 14. 15. 16. 17.
18.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
Stenberg, P., Joensson, T.E., Nilssen, B., wollheim, F. J. Chromatogr. 1979, 177 (1) , 145-8. Ballerini, R., Cambi, A., Del Soldato, P. J. Pharm. Sci. 1977, 66 (2), 281-2. Farinotti, R., Mahuzier, G. J. Pharm. Sci. 1979, 68 (41, 484-5. De Grave, J., Frankinet, C., Gielen, J.E. Biomed. Mass. Spetrorn. 1979, 6 (61, 249-52. Unterhalt, B. Pharm. Ztg. 1978, 123 (41), 1801-3. Farge, D., U.S. Patent 3,641,127. Soci&te Des Usines Chimiques RhEne-Poulenc Fr. Patent6.444 M. Brunet, J.P., Cometti, A. Soci6t6 Des Usines Chimiques Rhane-Poulenc Fr. Patent 2,163,875. Allais, A., Rousseau, G., Meier, J., Deraedt, R., Benzoni, J., Chifflot, L. Eur. J. Med. Chem. - Chim. Ther. 1974, 9 (4), 381-9. Socigte Des Usines Chimiaues Rhhe-Poulenc Fr. Addn. 0296 to patentaM6,444. Brunet, J.P., Cometti, A. Ger. Patent 2,258,985. 114 (6) Lotti, B. Boll. Chim. Farm. 1975, 351-4. Irwin, W.J., Slack, J.A. Biomed. Mass Spectrom. 1978, 5 (12), 654-657. F6rge et a1 S - A , Patent 68,00,524. Farqe, D. et a1 Ger. Patent 1,668,648 Farge, D. et a1 Ger. Patent 2,024,389. Farge, D. et a1 Brit. Patent 1,164,585. Farge, D. et a1 Brit. Patent amended 1,164,585 Thomas, W.O.A., Parfitt, R.T., J. Chromatogr. 1979, 162 (1) 122-4. Courpron, P., Brazier, V.L., Meunier, P., Ribon, B., Bannier, A. Lyon Med. 1978, 239 (81, 477-82. Meunier, P., Courpron, P., Brazier, J.L., Ribon, B., Bannier, A. Rev. Rhum. Mal. OsteoArticulaires 1977, 44 (7-91, 519-24. Populaire, P., Terlain, B., Pascal, S., Decouvelaere, B., Renard, A., Thomas, J.P. 31 (121, 735-49. Ann. Pharm. Fr. 1973, Sala, G. Silvestri, N., CastegNarot E . Pollini, C., Farmaco, Ed. Prat. 1978, 33 (101, 455-60.
I
KETOPROFEN
471
33* 34. 35.

36.
Brogden, R.N., Speight, T.M., Avery, G.S., Drugs, 1 9 7 4 , 8 ( 3 ) , 1 6 8 - 7 5 . Caille, G., Besner, J.G., Brodeur, J., 36, (5-6) , Vezina, M. Ann. Pharm. Fr. 1 9 7 8 , Caille, G . , Besner, J.G., Lacasse, Y., Vezina, M. Biopharm. Drug. Disp. 1 9 8 0 , 1, 1 9 5 - 2 0 1 . Ishizaki, T., Suganuma, T., Sasaki, T., Watanabe, M., Horai, Y., Hoshi, H., Ashisuke, W. Rinsho Yakuri 1 9 7 9 , 10, ( 4 1 , 5 9 1 - 2 , Chem. Ahs. 1 9 8 0 , 9 2 : 2 0 8 7 n a . Populaire, P . , TGlain, B., Pascal, S., Decouvelaire, B., Lebreton, G., Renard, A., Thomas, J.P. Ann. Pharm. Fr. 1 9 7 3 , 3 1 , (ll), McKay, S.W., Mallen, D.N.B., Shrubshall, P.R., Swann, B.P., Williamson, W.R.N. J. Chromatog. Bannier, A., Brazier, J.L., Quincy, C . Feuil. Biol. 1 9 7 9 , XX ( l O 6 ) , 9 1 - 9 8 . Bannier, A., Brazier, J.L., Ribon, B. J. Chromatogr. 1 9 7 8 , 1 5 5 ( 2 ) , 3 7 1 - 8 . Italfarmaco, S.p.A. Belg. Patent 8 3 3 , 2 6 6 . Upton, R.A., Buskin, J.N., Guentert, T.W., Williams, R.L., Riegelman, S. J. Chromatogr. Zupancie, B., Jenko, B., Aust. Patent 3 5 1 , 5 1 6 . Thomas, W.O.A., Jeffries, T.M., Parfitt, R.T. J. Pharm. Pharmacol. 1 9 7 8 , 30 (Suppl. British Pharm. Conf. 1 9 7 8 ) , 6 6 P . Slack, J.A., Irwin, W.J. Proc. Anal. Div. Chem. SOC. 1 9 7 7 , 14, ( 8 ) , 2 1 5 - 1 7 Aziende Chimiche Riunite Angelini Francesco S.p.A. Jpn. Kokai Tokyo Koho 7 9 0 9 , 2 5 1 . Chem. A b s . 1 9 7 9 , = : 1 6 8 3 0 8 p . LEK Tovarna Farmaceutskih in Kemicnih Izdelkov n.so1.0. Fr. Demande 2 , 3 6 7 , 7 2 8 . Ibid Fr. Demande 2 , 3 6 7 , 7 2 7 . Zupancic, B. Patent Ger. Offen 2,744,833. Zuparnic, B., Jenko, Patent Ger. Offen. Zoni, G. Spanish Patent 4 4 5 , 8 4 6 . Baiocchi, L. Patent Ger. Offen. 2 , 6 2 4 , 1 7 7 . Sigurta Farmaceutici S.p.A. Be1 Patent 8 3 7 , 6 2 4 . Zoni, G. Belg. Patent 8 3 9 , 6 3 4 .
2,744,834. 1 9 8 0 , 1 9 0 ( 1 1 , 119-28. 1979 679-90. 243-52.
37.
38. 39* 40. 41. 42. 43* 44 * 45. 46. 47. 48. 49. 50.
51. 52. 53. 54.
170,
(2)
482-5.
METHYLPHENIDATE HYDROCHLORIDE
Gandharva R. Padrnanabhan
1. Description 1.1 Name, Formula, Molecular Weight 1.2 Appearance 2. Physical and Chemical Properties 2.1 Infrared Absorption Spectrum 2.2 Nuclear Magnetic Resonance Spectrum 2.3 Ultraviolet Absorption Spectrum 2.4 Mass Spectrum 2.5 Optical Rotation 2.6 Melting Range 2.7 Differential Scanning Calorimetry 2.8 Thermogravirnetric Analysis 2.9 Solubility 2.10 X-Ray Diffraction 2.11 Polymorphism 2.12 Partition Coefficient 2.13 Dissociation Constant 3. Synthesis 4. Stability-Degradation 5. Drug Metabilism and Pharrnacokinetics 6. Toxicity 7. Methods of Analysis 7.1 Identification 7.2 Elemental Analysis 7.3 Nonaqueous Titration 7.4 Phase Solubility Analysis 7.5 Thin-layer Chromatography 7.6 High Pressure Liquid Chromatography 7.7 Gas Chromatography 7.8 Gas Chromatography-Mass Spectrometry (GC-MS) 7.9 Colorimetric Methods 7.10 Infrared 7.11 Reineckate Salt 8. References 9. Acknowledgment
474 474 474 474 474 474 477 479 479 48 1 48 1 48 1 48 1 483 483 483 483 485 485 486 486 486 486 486 486 487 487 489 49 1 492 493 494
495 495
497
474
GANDHARVA R. PADMANABHAN
1.
Description 1.1. Name, Formula, Molecular Weight Methylphenidate hydrochloride is methyl a-phenyl2-piperidineacetate hydrochloride, (Rfc,Rfc)- (+) .
C14H19N02.HCl 1.2 Appearance
Molecular Weight 269.71
Methylphenidate hydrochloride occurs as a white, odorless, fine, crystalline powder.

2.
Phvsical and Chemical ProDerties 2.1. Infrared Absorption Spectrum The infrared spectrum of a mineral oil suspension o f methylphenidate hydrochloride is shown in Figure 1. The spectral assignments are listed in Table 1. TABLE 1
Wavenumber, cm-l 703, 737 1602

2300
Assignment Monosubstituted benzene Aromatic Stretch Secondary Amine Salt C=O Stretch C-0 Stretch
2700
1170
1739 1150 2.2
Nuclear Magnetic Resonance SDectrum (NMR) The NMR spectrum of methylphenidate hydrochloride i s shown in Figure 2. The spectrum was determined on a Perkin-Elmer R-24B 60 MHz spectrometer at ambient temperature. The sample was dissolved in a 1:l mixture o f deuterated chloroform and deuterated dimethylsulfoxide containing tetramethylsilane as an internal standard. The spectral assignments are shown in Table 2.
E
8
0
W 0
:
8 2
7
f
;
?
I 1
I 0
a
I
N 0
>
-0 N
0 -N
7
0 .U
.W
0
.a,
U
0 0
477
TABLE 2 Chemical Shift 6 (PPd

7.1 4.0 3.6
Multiplicity Broad Singlet Doublet Singlet
No. of
Protons
Assignment Phenyl protons -CH-COOCH, -0cg3

I
7.6 4.4 3.8
2.8
- 3.6
Broad Mu1 tiplet Broad Multiplet Broad Multiplet
-N
CH/ -
' CH2-
2.4
1.0
2.7
1.9
Solvent
2.3.
Ultraviolet Absorption Spectrum The W spectrum of methylphenidate hyd,rochloride (1 mg/mL) in methanolic 0.1N HC1 exhibits maxima and minima as shown in Table 3 and Figure 3 . TABLE 3
A max, nm
264 25 7 25 2 247
A 1% 1cm 6.1
&
165 208 159 122
7.7
5 -9 4.5
A min. at
263 nm, 255 nm, 249 n m and 245 nm.
478
GANDHARVA R . PADMANABHAN
Figure 3: Ultraviolet Absorption Spectrum of Methylphenidate Hydrochloride
0.8
0.7
0.6
0.5
a,
0
g
n
0.4
0.3
0.2
0.1
0.0
Wavelength , Nanometer
479
2.4
Mass Spectrum The low resolution mass spectrum of methylphenidate hydrochloride obtained at 70 ev using a solid probe insertion is shown in Figure 4 . The spectrum was run on a Kratos MS25 spectrometer interfaced with a data handling system. Table 4 illustrates the prominent fragments and their masslcharge ratios. TABLE 4
' ; T h e spectrum is known to vary due toethermal decomposition (27).
2.5
Optical Rotation Although the methylphenidate hydrochloride molecule has two asymmetric carbon atoms, the drug exhibits no optical activity as it is a racemic mixture. The diastereoisomer of the drug, (R",Sf:) isomer, is also referred to as "erythro isomer". The conformations of methylphenidate hydrochloride and its (&:,Sf<) isomer have been documented (1-2). Due to the low efficacy of the ( R ' ; , S $ ; ) isomer (1) the amount of this diastereoisomer is controlled in the drug to a level of 1%or less (3).
480
Figure4: Low Resolution Mass Spectrum of Methylphenidate Hydrochloride
100
90
80
70
.-
C
c
>
60
v)
c > .c
a ,
a ,
50
m a , a:
40
30
20
10
I
0
160
180
200
220
24C
MassIC harge
48 1
2.6
Melting Range Methylphenidate hydrochloride melts between 224OC and 226OC when tested according t o the USP XX Class Ia procedure.
2.7
Differential Scanning Calorimetry (DSC) The DSC thermogram of methylphenidate hydrochloride shows a melt endotherm between 1 9 5 O C and 235OC with a melting point, with decomposition, o f approximately 22OOC when the thermogram was followed in a DuPont Model 900 instrument at a scan rate o f 1O0C/minute (Figure 5 ) .
2.8
Thermogravimetric Analysis (TGA) The TGA of methylphenidate hydrochloride exhibited a weight loss of 0.34% between 3OoC and 15OOC. Above 1 5 O O C a rapid weight loss due to decomposition and/or sublimation was observed.
2.9
Solubility Approximate solubilities in different solvents were determined after equilibrating 10 mg (more, if necessary, to obtain a saturated solution) of the drug at room temperature with 1 mL o f solvent.
TABLE 5
Solvent Water
0.1N - HC1
Solubi1ity (mg/mL)
> 100
Methanol Ethanol Acetonitrile Chloroform n-Hexane Ethyl Acetate Ether

95% Ethanol
> 100 > 100 > 25

5.3
> 100 < 0.01

0.08
Petroleum Ether Acetone
< 0.01 > 50 < 0.01

0.9
482
Figure!? DSC Scan of Methylphenidate Hydrochloride
400
450
500
K
Temperature O
483
2.10
X-Rav Diffraction The x-ray powder diffraction pattern obtained for mehylphenidate hydrochloride is shown in Figure 6 . The data were collected on a GE Model XRD-spectrogoniometer using Cu K (1.542AO) with a Ni filter (Y as a radiation source.
2.11
Polymorphism No polymorphism has been reported for methylphenidate hydrochloride.
2.12
Partition Coefficient The following partition coefficient data were obtained when 50 mL o f 0.1 and 1.0 mg/mL of methylphenidate hydrochloride in appropriate aqueous solutions at room temperature were partitioned individually with 50 mL of indicated organic s o l vents. Heptane-pH 7. 4 buffer data was obtained from the literature ( 4 ) .
TABLE 6
Aqueous Phase
0.1N HC1
Organic Phase Chloroform Chloroform Ether Ether Heptane
Partition Coefficient"
+ o
22.7 20.5
3 0
pH 7 Buffer
0.1N HC1
pH 7 Buffer pH 7 . 4 Buffer
1 . 7 k0. 2
0.63
7,;
= Concentration in organic phase/concentration in aqueous phase.
2.13 Dissociation Constant

A pKa value of 9.0 was obtained for the dissociation of the protonated secondary amine function by potentiometric titration method. A value of 8.9 was also obtained (5) for the pKa by the nonlogarithmic method of Benet and Goyan ( 6 ) .
484
Figure 6: X-Ray Powder Diffraction Pattern of Methylphenidate Hydrochloride
8.3
35
30
25
20
15
10
Degrees Two Theta
485
3.
Synthesis Methylphenidate hydrochloride is prepared by the following sequence of reactions. a-Phenyl-2-pyridineacetonitrile is hydrolyzed in diluted sulfuric acid to a-phenyl2-pyridineacetamide. The acetamide is isolated and then hydrogenated over a catalyst to yield racemates of diastereoisomeric mixtures of a-phenyl-2-piperidineacetamide. The diastereoisomeric piperidineacetamide racemate mixture is converted t o (R>t,R$C) racemate by heating in sodium hydroxide solution and then to a-phenyl-2-piperidineacetic acid by heating in sulfuric acid solution. The acetic acid derivative is converted to the acid chloride and then, without isolation, reacted with methanol to yield the methyl ester free base which is then converted to methylphenidate hydrochloride (7).
4.
Stability-Degradation The drug is relatively stable in acidic solutions but is degraded extensively in basic solutions. The degradation occurs via the hydrolysis of the methyl ester to the free acid, a - p h e n y l - 2 - p i p e r i d i n e a c e t i c acid (8-10). pH of Solution
1
Temperature
OC
Time (hours) 20
20 300
% Methylphenidate HC1 Remaining

100 100
1.7
2
100
3.7
2
100
3.5
2
100
100
2
95
4.9
4.1
3
20
300
84
51
100
5.7
4
100
4
20 20
20
49
0
87
8.9
9.2
100
30
'0.1N HC1 2Phthalate Buffer 3Phosphate Buffer 4Borate Buffer
The methyl ester of phenylacetic acid has been reported as one of the products when the drug substance was subjected to thermal degradation.
486
5.
Drug Metabolism and Pharmacokinetics a-Phenyl-2-piperidineacetic acid, the lactam acid and several other unidentified polar compounds have been reported as metabolites in guinea pig, dog and human urine ( 4 , 11-13). p - H y d r o x y p h e n y l - 2 - p i p e r i d i n e a c e t i c acid and its methyl ester were also reported as metabolities in dog and rat urine ( 1 4 ) . The products of metabolism involving oxidiation, hydrolysis and conjugation processes in rats and dogs have also been reported ( 3 0 ) .
6. Toxicity
A typical sample
o f methylphenidate hydrochloride active drug gave an oral LD50 value o f 350 mg/kg with deaths (9/10) at 1000 mg/kg when a 7.5% solution was administered to male rats (15).
7.
Methods o f Analysis
7.1
Identification Two identity tests are given in the LISP XX, one an infrared absorption test and the other a test for chloride.
7.2
Elemental Analysis The following elemental composition was obtained for methylphenidate hydrochloride when a 2 mg sample was employed for analysis with a PerkinElmer, Model 240 CHN Analyzer. Element Carbon Hydrogen Nitrogen Theory, % Found, %
62.35 7.57 5.12
62.33
7.47 5.19
7.3
Nonaqueous Titration
Methylphenidate hydrochloride may be titrated in glacial acetic acid containing mercuric acetate with perchloric acid in glacial acetic acid as titrant. The titration can be carried out potentiometrically or with p-naphtholbenzein as indicator.
487
Although the titration as such is not specific for the intact drug in presence of its major degradation compound, a - p h e n y l - 2 - p i p e r i d i n e a c e t i c acid, the procedure can be made specific by extracting the drug from a pH 9 buffer with chloroform and then titrating the free base with perchloric acid without the addition of mercuric acetate ( 1 6 ) .
7.4
Phase Solubility Analysis Phase solubility analysis of methylphenidate hydrochloride has been carried out using the following systems: System I A mixture of 93 mL of benzene and 7 mL of anhydrous ethanol Approximate solubility: 14 mg/mL System I1
90 mL of benzene and 1 0 mL of methanol Approximate solubility: 65 mg/mL at 3OoC
Solvent:
Solvent:
A mixture of
System I11 Solvent: n-Butanol Approximate solubility:

7.5
13 mg/mL at 3OoC
Thin-layer Chromatography
A number of thin-layer chromatographic systems have

been developed for the identification and the determination of the drug and compounds related to the drug. System I
Adsorbent: Mobile Phase : Detection System :
The following system may be employed particularly to control the (R?:, Ssc) isomer content in the drug ( 3 ) . Silica Gel G plate, 250p thickness
A mixture containing
190 mL of chloroform, 10 mL of methanol and 1 mL of concentrated ammonium hydroxide.
Dragendorff spray reagent (0.7 g of bismuth subnitrate dissolved in 40 mL of 20% glacial acetic acid and then diluted successively with 40 mL of 40% KI solution, 120 mL of glacial acetic acid and 250 mL of water).
GANDHARVA R. PADhlANABIIAh
System I1
The following system may be employed particularly when a-phenyl-2-piperidineacetic acid hydrochloride content in the drug has to be established (3). Silica Gel G plate, 250p thickness
Adsorbent : Mobile Phase : Detection System:
A mixture containing 65 mL of chloroform, 25 mL of methanol and 5 m L of acetic acid.
1. Irradiation o f the dried plate with high-intensity W for 10 minutes followed by visualization under longwave W source.
2. Heating for 10 minutes at 1 0 5 O C after spraying with 3.5% phosphomolybdic acid in isopropanol.
Other Systems: The following systems have also been employed for the analysis of methylphenidate hydrochloride. System I11 Chloroform/Methanol (9:l); Silica Gel GF; Iodine Spray and UV Detectors
(17).
System IV
Acetone/25% Ammonium Hydroxide (99:l); Silica Gel GF; Iodine spray and W detection Systems (17). Methanol/25% Ammonium Hydroxide (99:l); Silica Gel GF; Iodine Spray and W Detection Systems (17). Ethanol/Glacial Acetic Acid/Water (5:3:2); Silica Gel GF; Iodine Spray and W Detection Systems (17). Chloroform/Methanol ( 8 4 : 1 6 ) ; Silica Gel G: Chlorine/o-Tolidine Detection System (18). n-Propanol/pH 4.0 Citric AcidPhosphate Buffer (9:l); Alumina Plate; Iodoplatinic Acid Spray Detection System ( 1 9 ) .
System V
Svstem VI
System VII
System VIII
489
System IX System X
Chloroform/Methanol ( 1 : l ) ; Silica Gel GF: Iodine Spray and W Detection Systems (17).
Chloroform/Methanol/Concentrated Ammonium Hydroxide ( 9 : l : l ) ; Silica Gel GF; Acetic acid-Commercial Chlorine Bleach-Phenothiazine Spray Reagent and Shortwave W Detection Systems (20) * Chloroform/Methanol/Concentrated ammonium Hydroxide (7:5:1); Silica Gel GF; Detection Systems Same as in System
System XI
(20).
System XI1
Acetone/Methanol containing 0.5% HCl ( 1 : l ) ; Silica Gel GF: Detection Systems same as in System X ( 2 0 ) .
System XI11 - Concentrated Ammonium Hydroxide/ Ethanol/Water (16:100:12); Silica G e l GF; Detection System not reported. Svstem XIV System XVI Methanol/Formic Acid ( 9 : l ) ; Silica Gel GF: Detection System not reported. Ethyl AcetateIAcetic Acid/Water/Hydrochloric Acid (55:35:2:2); Silica Gel G; Detection System not reported.
7.6
High Pressure Liquid Chromatography The following two systems have been reported (21) for the quantitation of the (R;b,S$C) isomer in methylphenidate hydrochloride samples. System I Mobile Phase :
A mixture containing 85 mL of chloroform, 13.5 mL o f cyclohexane, 1 . 5 mL of ethanol, and 0 . 5 mL of concentrated ammonium hydroxide.
Column: Detection:
100 cm x 2.1 mm steel column drypacked with Sil-XB (Perkin-Elmer).
W (254 nm)
Temperature: Ambient
490
System I1 Mobile Phase: Column : Detection: Sample : Temperature:
A mixture containing 80 mL of chloroform, 20 mL of cyclohexane and 1.5 mL of ethanol.

50 cm x 2.1 mm (i.d.) MicroPak-SI-10 (Var ian)
UV (254 nm) Inject 20 pL of isolated free base in chloroform Ambient
The following system has been employed for the quantitation of methylphenidate in serum samples (22). System I11 Mobile Phase : Column: Flow Rate: Detection: Internal Standard :
pH 3.5 Phosphate bufferlhigh purity
acetonitrile (80:ZO). VBondapak C18 (Waters) (4 mm x 30 cm)
Temperature: 4OoC
1.6 mL/minute
UV (192 run) 4,5-Diphenylimidazole
The following system has been employed for the determination of a - p h e n y l - p i p e r i d i n e a c e t i c acid in serum samples (23). System IV Mobile Phase : Column: Temperature: Flow Rate: Detection: Inte rna1 Standard
pH 3.8 Phosphate bufferlacetonitrile (83:7)
VBondapak C18, (Waters) (4 mm x 30 cm) 4OoC

2.0 mL/minute
(192 nm)
a,a-Dimethyl-p-methylsuccinimide
49 1
7.7
Gas Chromatography The f o l l o w i n g system h a s been employed f o r t h e a n a l y s i s of t h e d r u g s u b s t a n c e i n plasma. System I Column: Temperature: Detector: Carrier: Sample :
8 f t x 4 mm i . d . w i t h 5% XE-60 on Gas Chrom Q (100 - 120 mesh)

Column - 15OoC; D e t e c t o r I n j e c t o r - 185O Flame i o n i z a t i o n N i t r o g e n 70 cc/minute I s o l a t e d f r e e b a s e d i s s o l v e d i n DMF
185';
The f o l l o w i n g system ( 1 9 ) h a s been employed f o r i d e n t i f i c a t i o n of t h e d r u g i n b l o o d and u r i n e samples. System I1 Column:
5 ft x O . D . (3mm i . d . ) g l a s s column, 2% H i E f f 3A (Applied S c i e n c e ) on Gas Chrom Q (100 - 120 mesh).

Column - 140'C; D e t e c t o r p o r t e d ; I n j e c t o r - 220OC Flame I o n i z a t i o n N i t r o g e n a t 75 mL/minute
Temperature: Detector: Carrier:
n o t re-
The f o l l o w i n g system h a s been employed f o r t h e a n a l y s i s of s e v e r a l d r u g s i n c l u d i n g methylphenidate hydrochloride. System I11 Column: Temperature:
8 f t x l/8" g l a s s column w i t h 15% XF-1112 on Chromosorb X-HMDS

I n j e c t o r - 200OC; Column - Program 60' - 180' a t 4O/minute; D e t e c t o r not reported. Flame i o n i z a t i o n N i t r o g e n 25 mL/minute
Detector: Carrier:
The f o l l o w i n g system has been employed t o q u a n t i t a t e (R",S") isomer i n m e t h y l p h e n i d a t e h y d r o c h l o r i d e samples ( 2 5 ) .
GANDHARVA R. PAUMANABHAN
Svstem IV Column:
200 cm x 2 mm i . d . glass column with 3% OV-225 on Gas Chrom Q (80 - 100 mesh).
Nitrogen 30 mL/minute Flame ionizaton detector Column - 125OC; Detector - 200OC; Injector - 200OC Isolated free base in chloroform
Carrier Gas: Detector: Temperature: Sample:
The following system was employed for the identification of the drug in a tablet formulation. System V Column: Temperature: Carrier Gas: Sample :
7.8
8' x 4 mm i.d. with 5% XE-60 on Gas Chrom S (100 - 120 mesh)

Column - 13OOC; Injector - 158OC; Flame Ionization Detector - 175OC Nitrogen 60 cclminute Isolated free base in dimethylformamide
Gas Chromatography
Mass Spectrometry (GC-MS)
Sensitive methods for the analysis of methylphenidate hydrochloride and its hydrolytic degradation compound have been reported using GC-MS with selected ion monitoring for separation and detection. The following experimental conditions were used for the analysis of the drug and its metabolite in blood and urine samples. Method I (27) Column:
6 ft x 2 mm i.d. glass column containing 3% OV-1 on Gas Chrom-Q (100 - 120 mesh)
GC-MS selected ion monitoring m/e=91
Detection:
493
Method I (27) Temperature:
(Continued) I n j e c t o r , d e t e c t o r , c a p i l l a r y res t r i c t o r , s e p a r a t o r and l i n e of s i g h t p r o b e - 225OC; Column - 165OC; M S E l e c t r o n Impact Source - 1 7 5 O C . Helium 40 mL/minute
Carrier:
M S E I Source: 70 e v Method I1 (28) Column: Detection: Temperature:

0 . 9 m x 2 mm i . d . g l a s s column w i t h 3% OV-17 on Gas Chrom Q
GC-MS S e l e c t e d I o n Monitoring m/e=180 I n j e c t o r - 250OC; Column - 170OC; Membrane S e p a r a t o r - 1 7 O o C ; T r a n s f e r l i n e - 25OOC H e l i u m 35 mL/min
C a r r i e r Gas:
M S E I Source: 70 e v Sample : Interna 1 Standard: 7.9 Derivatized with t r i f l u o r o a c e t i c anhydride E t h y l e s t e r a n a l o g of methylphenid a t e HC1.
C o l o r i m e t r i c Methods 7.9.1 P i c r a t e Method Methylphenidate h y d r o c h l o r i d e can be asayed by t h e p i c r a t e i o n - p a i r e x t r a c t i o n method ( 3 ) . The sample i s e x t r a c t e d i n t o a d i l u t e d s u l f u r i c a c i d s o l u t i o n , mixed w i t h a p i c r i c a c i d s o l u t i o n , pH a d j u s t e d t o 5 . 0 and t h e n e x t r a c t e d a s a p i c r a t e i o n - p a i r w i t h chloroform. The e x t r a c t e d drug i s q u a n t i t a t e d c o l o r i m e t r i c a l l y by measuring absorbance a t 405 nm. The method i s s p e c i f i c f o r t h e drug i n p r e sence of i t s h y d r o l y t i c d e g r a d a t i o n compound.
494
7.9.2
Bromcresol Purple Methylphenidate hydrochloride also forms a colored complex with bromcresol purple at pH 5.0. The complex can be extracted with chloroform and the content of the drug quantitated by measuring the absorption of the acidified extract at 420 nm. The hydrolytic degradation compound does not interfere.
7.9.3
Hydroxamic Acid In basic solution, methylphenidate forms This hydroxamic acid forms a red complex with ferric ion in acidic solutions which can be quantitated colorimetrically by measuring the absorbance at 500 nm. The degradation compound does not interfere with the assay. However, the method is subject to interference from excipients in drug formulations such as lactose.
a hydroxamic acid with hydroxylamine (29)
7.9.4
1,2-Naphthaquinone Sulfonic Acid An automated procedure has been reported for the determination of methylphenidate hydrochloride in tablet formulations based on the formation of a yellow-colored complex with 1,2-naphthaquinone sulfonic acid. The complex is extracted into chloroform and the drug quantitated by the measurement of absorption maximum o f the complex at 400 nm.
7.10 Infrared
Methylphenidate hydrochloride has been assayed in certain tablets by extracting the drug from the tablet matrix, by adjusting the pH to about 9.0 and then extracting immediately with chloroform. r i ' of the By measuring the IR absorption at 1720 c concentrated extract, the drug content can be quantitated. The method has also been applied to a syrup formulation assay.
495
7.11
Reineckate Salt Methylphenidate hydrochloride has been determined gravimetrically by precipitating the reineckate salt of the free base by the addition of ammonium reineckate, NH4[Cr(NH3)2(SCN)4] to the drug in solution.
8.
References
1. Rometsch, R . , US Patent, 2,838,519 (1958).
2. Shafiee, A. and Hite, G., J. Med. Chem., 1 2 , 266, 520 ( 1 9 6 9 ) ; Shafiee, A . , Marathe, S . , BhatkarTR. and 5 6 , 1689 (1967). Hite, G., J. Pharm. Sci., 3. The United States Pharmacopeia, Twentieth Revision, Mack Printing Company, Easton, PA, 1980, page 522.
4 . Faraj, B.A.,
Israili, Z.H., Perel, J.M., Jenkins, M.L., Holzman, S . G . , Cucinell, S.A. and Dayton, P.G., J. Pharniacol. Exp. Ther., __ 191, 535 (1974). Communication.
5 . Smith, J. and Piskorik, H., CIBA-GEIGY, Personal 6 . Benet, L.Z. and Goyan, J.E., J. Pharm. Sci., 1179 (1965).
54,
7 . Weisz, I. and Dudas, A., Monatsch., 9 1 , 840 (1960)
8. Portoghese, P.S. and Malspeis, L., J. Pharm. Sci., 5 0 , 494 (1961).

9 . Siegel, S . , Lachman, L. and Malspeis, L., J. Pharm
Sci. , @, 431 (1959).
10.
Rometsch, R., CIBA-GEIGY, Personal Communication.
11. Bartlett, M.F. and Egger, H.P., Fed. Proceedings,
31,
537 (1972). 12. Sheppard, H., Tsien, W.H., Rodegker, W. and Plummer, A . J . , Toxicol. Applied Pharmacol., 2, 353 (1960).
496
13.
14. 15. 16.
Dayton, P.G., Read, J.M. and Ong, V., Fed. Proceedings, 3 1 , 1822 ( 1 9 7 2 ) . Faraj, B.A. and Jenkins, M.L., Pharmacologist, 15, 155 (1973). Jeffrey, K. and Traina, V.M., CIBA-GEIGY, Personal Communication. The Pharmacopeia of the United States of America, Eighteenth Revision, Mack Printing Company, Easton, PA, 1970, page 432. Galla, M.M. and Macek, T.J., U.S.P. Reference Standards Committee Collaborative Report, September 29, 1970. Yachontov, L.N., Mashkovski, M.D., Levkoeva, E.I., Altshuler, R.A., Tubina, I.C., Turchin, K.F., Flashurian, U.D., Tulenev, A.K., Potonova, P.G., Voljina, O.H. and Gorobsetskij, L.S., Khim-Farm. Zh., 8 , 3 (1974). Schubert, B., Acta Chemica Scand., 2 4 , 433 ( 1 9 7 0 ) . Lovering, E . G . , Drug Research Laboratories (Canada) Private Communication, July 28, 1976. Padmanabhan, G.R., Fogel, G., Mollica, J.A., O'Connor, 3, 1079 J.M. and Strusz, R., J. Liquid Chromatography, (1980). Solidin, S.J., Chan, Y.-P.M., Hill, B.M. and Swanson, J.M., Clin. Chem., 25, 401 ( 1 9 7 9 ) . Soldin, S.J., Hill, B.M., Chan, Y.-P.M., Swanson, J.M. and Hill, J.G., Clin. Chem., 25, 5 1 (1979). Noirfalise, A. and Grosjean, M.H., J. Chromatog., 37, 1979 ( 1 9 6 8 ) . Blichler, W. and Senn, H., CIBA-GEIGY, Personal Comrnunication. Mollica, J.A. and Strusz, R.F., CIBA-GEIGY, Personal Communication. Milberg, R.M., Rinehart Jr., K.L., Sprague, R.L. and Sleator, E.K., Biomedical Mass Spec., 2, 2 ( 1 9 7 5 ) .
17.
18.
19. 20 *
21.
22.
23.
24. 25.
26.
27.
497
28. 29.
G a l , J . , Hodshon, B . J . , P i n t a u r o , C . , Flamm, B.L. and Cho, A . K . , J . Pharm. S c i . , 66, 866 ( 1 9 7 7 ) . Goddu, R . F . , LeBlanc, W.F. and Wright, C . M . , Anal. Chem., 2 7 , 1251 ( 1 9 5 5 ) . g r a w y a , M.S. and Ghourab, M . G . , J . Pharm. S c i . , 5 9 , 1331 (1970). E g g e r , H . , B a r t l e t t , F . , D r e y f u s s , R . and K a r l i n e r , J . , I n Press. Acknowledgment The a u t h o r e x p r e s s e s a p p r e c i a t i o n t o I n g r i d Becue, R i c h a r d Brown and J a n e Johnson f o r h e l p i n p r e p a r a t i o n of t h i s m a n u s c r i p t .
30.
9.
NABILONE
Rex W. Souter
1. 2.
Introduction Description 2.1 Nomenclature 2.2 Formulae 2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance and Odor 2.6 Isomerism Physicochemical Properties 3.1 Spectra 3.2 Melting Range 3.3 Crystallinity 3.4 Thermal Analysis 3.5 Dissociation Constant 3.6 Solubility Profile Synthesis Stability 5.1 Accelerated Degradation 5.2 Long-Term Stability Metabolism, Pharmacokinetics, and Microbiological Transformations Methods of Analysis 7.1 Raw Material 7.2 Biological Samples 7.3 Pharmaceutical Formulations Acknowledgements References
3.
4. 5.
6. 7.
8. 9.
500 500 500 500 501 501 501 501 50 1 50 1 505 505 506 506 506 509 509 509 510 510 510 510 511 511 51 1 512
ANALYTICALPROFILES OF DRUG SUBSTANCES. 10
499
Copsright & 1981 hy Academic Im\\. lrir All rightr of reproduction in an? fnrm rrwr\rd ISBN 0-12-?60RIO-0
500
REX 1 1 . ' . SOUTER
1. Introduction Nabilone, a totally synthetic 9-ketocannabinoid, is currently being evaluated to control nausea and vomiting in cancer chemotherapy patients (1-6) and as an ocular pressurereducing agent in glaucoma patients (7,8). Such antiemetic (9) effects and ocular (10,ll) effects have heen reported f o r marihuana and the isolated natural product A ' -tetrahydroNabilone arose from an endeavor to cannabinol (A9 -THC). discover drugs which would possess beneficial effects on the central nervous system while iniiiimizing THC'S disturbing side effects (12-14), especially tachycardia and dysphoria.
2. Description 2.1 Nomenclature 2.11 Chemical Name (+) trans-3-(1,l-Dimethylheptyl)-6,6a,7,8,10, lOa-hexahydro-l-~ydroxy-6,6-dimethyl-9H-dibenzo [ b ,d] pyran-9one 2.12 Non-proprietary Name Nab ilone 2.2 Formula 2.21 Empirical 2.22 Structural
C24H36O3
7'
5'
CH3
CH3 CH3
NABILONE
501
2.3 Molecular Weight 372.5 2.4 Elemental Composition Element % Theory C 77.38 H 9.74 0 12.88
2.5 Appearance and Odor Nabilone is a white, crystalline solid having no particular odor. Isomerism 2.61 Optical Isomers Nabilone exists as a 1:l mixture of 6aR, lOaR and 6aS, lOaS optical isomers and is therefore optically inactive (Cahn-Ingold-Prelog nomenclature). 2.62 Geometric Isomerism The cis- isomer of nabilone is known to exist. This stereochemistry is defined by the position of the 6a hydrogen. Physicochemical Properties 3.1 Spectra 3.11 Ultraviolet Spectrum The ultraviolet spectrum (figure 1) in methyl alcohol from 240-360 nm exhibits maxima at 275 nm and 282 nm with molar absorptivities of 1284 and 1315 (and E 1%, 1 cm values both about 35) respectively. At 208 nm and 228 nm in methyl alcohol maxima also exists with molar absorptivities of approximately 10000 and 45800 (and E 1%, 1 cm values of about 269 and 1230) respectively. Figure 1 was plotted from data acquired with a GCA McPherson UV/Visible spectrophotometer. 3.12 Infrared Spectrum The infrared spectrum of nabilone in a potassium bromide pellet (figure 2) was plotted from data taken from a Perkin-Elmer 580B instrument and the major band assignments are listed below.
Infrared Absorption, cm-'
2.6
3.
Assignment -OH, hydrogen-bonded CH asym. in CH3 CH asym. in CH2 CH sym. in CH3 CH sym. in CH carbonyl stretch phenyl C=C probably ring vibration
3280, broad 2953 2925 2870, shoulder 2859 1696 1619 and 1574 1413
1 . 161
.871
PH NEGTRAL CONDITIONS 0.5NM
RESOLUTION 1CM PATHLENGTH
CMPD
LOT#
CONC
NABILONE REFERENCE STANDARD

178.20 MCG. /ML. IN
MEOH
F i g u r e 1. U V spectrum of n a b i l o n e
5.554
4. 165
I NT.
2.777
01
w 0
1.388
.000
4000
I , , , , I , , , , l , , , , l , , , , I , , , , , , , , , , ,
V
. . . . . . . . . . . . . . . . . . . . . . .
3600
3200
2800
2400
2000
1800 1600 1 /CM
1400
1200
1000
800
600 I n , , , ) 400
PATH PELLET ISM# 1 6 MIN SCAN CONDITIONS RESOLUTION; 3.0 WAVENUMBER
CMPD
LOT#
CONC
NABILONE REFERENCE STANDARD

IN KBr
1.36 MG.
Figure 2.
IR spectrum of n a b i l o n e
504
REX W. SOUTER
Infrared Absorption, cm-1

1385, 1 3 7 1 1363, 1359 1340 1260 1135 1038 866
Assignment
CH deformation in -c(crI3) 2 ring and side chain includes -CH deformation C-0-C aromatic ether -$-0 in polysubstituted 0, phenyl -q- aliphatic ether isolated CH, aromatic outof plane deformation
3.13 Proton Magnetic Resonance Spectrum The 60MHz proton NMR spectrum of nabilone in deuterochloroform (plotted with data from a Varian T60A spectrometer) is given in figure 3. With reference to the structure in 2.22 assignments of the resonances are also given.
Description of Resonance singlet, l H , 7.75 ppm broad singlet, 2H, 6 . 4 3 ppm doublet/doublet, l H , 4.20 ppm
triplet/doublet/doublet, l H ,
Assignment
-OH
H (at 2 a r i d 4) 10 equatorial
10 a
B -CH; ( 6 ) C H 3 (1') a-CH3 (6) 7'
singlet, singlet, singlet, triplet,
3H, 1 . 5 0 6H, 1 . 2 3 3H, 1 . 1 5 3 H , 0.85

13
2.91 ppm ppm ppm ppm ppm
3.14
Carbon Magnetic Resonance Spectrum
26.9 3 6
0 154.1106.9
CH3
27.9
r ! . ; H ! 3
18.8-
<
37*4 24.6 28.7-28.7 31.8 14.1
CH3 CH3
NABILONE 13
50s
The natural abundance C NMR chemical shifts for nabilone are shown on the preceding page. All chemical shifts are in parts per million downfield from TMS in deuterochloroform. The spectrum was recorded on a JEOL PFT100 NMR spectrometer. 3.15 Mass Spectrum Figure 4 is a plotted low resolution mass spectrum of nabilone from a Hitachi-Perkin Elmer RMU-6 instrument equipped with a System Industries System 150 data system. Fragmentation of the molecular ion of nabilone (m/e 372) along the alkylside chain, with hydrogen rearrangement yields the homolgous series of peaks m/e 330, 316, 302 and 288. M/e 288 arises by fissions to the phenyl ring, a process which may often lead to intense peaks in the spectra of alkyl benzenes. The origin of the above peaks can also be fragmentations of the alicyclic ring or combinations of alicyclic ring and side chain fragmentations. In cases where the keto group is eliminated, the possibilities can be distinguished by accurate mass measurement. M/e 177 has the composition CllH1302. 3.2 Melting Range 158-16OOC Crystallinity 3.31 Crystalline Habit Nabilone may occur in at least four distinct polymorphic forms depending upon the solvent and crystallization conditions (15). The table below summarizes these forms which have been characterized by differential thermal analysis (DTA) and X-ray diffraction powder patterns. Polymorph A B C D Crystallization Solvent hexane ethanol-water(a) ethanol-water0 ) chloroform Endothermic DTA Transition Temperature 162OC 155, 162OC 132 155, 162OC 120 140, 162OC 3.3
(a) Crystallization allowed to occur from warm ethanol-water solution. (b) Crystallization forced by the addition of ethanol solution to water. 3.32 X-ray Powder Diffraction The data below describe the pattern for the most thermodynamically stable form ( A ) of nabilone where d is equal to the interplanar spacing measured in Angstroms (A) and 1/11 are intensities of the x-ray maxima based on a value of 100 for the strongest line.
506
REX LY.SOLJTER
Cu-Ni Radiation, A
9.27 8.00 6.78 6.63 6.21 5.85 5.58 5.17 4.88 4.73 4.58
1.5405
c l I / I , - d I / I 1
100
50 30 30 40 10 05 70 90 15 20 30 40 60
4.29 4.06 3.89 3.73

3.4
40
3.56 3.43 3.28 3.12 3.07 2.92 2.72 2.62 2.44 2.39 2.33 2.24 2.18 2.04 1.89
30 05 50 02 02 02 02 02 10 05 10 02 05 02 05
Thermal Analysis 3 . 4 1 Differential Thermal Analysis (DTA) A differential thermogram of nabilone at a 0 heating rate of 5 C/min. in a nitrogen atmosphere (40cclmin) shows an exotherm at approximately 165OC indicating a melt. 3 . 4 2 Thermogravimetric Analysis (TGA) A thermogram run simultaneously with the above DTA shows a weight l o s s beginning at 150C resulting in a 0.6% l o s s at 180C (residual solvent). 3 . 5 Dissociation Constant The pka of nabilone in 66% dimethylformamide/34% water is 1 3 . 5 . Solubility Profile The sample is sonicated for one minute at ambient temperature.
3.6
Solvent Water pH 1 . 2 (USP XIX) pH 4 . 5 (USP XIX) pH 7.0 (USP XIX) methyl alcohol n-octyl alcohol diethyl ether ethyl acetate chloroform benzene cyclohexane
mglml < 0.5 < 0.5

< 0.5
< 0.5
>5.0 0.5 >5.0 >10

i
>10 > 10
>
1.0
F i g u r e 3.
P r o t o n nmr s p e c t r u m of n a b i l o n e
NABILONE
509
Synthesis The resorcinol I1 may be reacted with diene I in the presence of water and stannic chloride. I may be prepared from p-methoxyacetophenone by Grignard addition to provide 2-(4-methoxyphenyl)-2-propanol, followed by Birch reduction. The reaction probably proceeds through a ketal which is hydrolyzed to the hemiketal I11 which subsequently rearranges to the &-ketone IV. Ketone IV may finally be isomerized to nabilone by conversion with A1C13 in dichloromethane at O O C ( 1 6 ) , as shown below.
4.
OH
/4
111
nCI,
420
____)
NABILONE (trans-)
IV
5.
5.1 Accelerated Degradation Nabilone is stable to refluxing 0.1N acid and 0 . 1 N base, as well as to heating in air at l l O C or one week. However, irradiation for just over two days in ethyl alcohol with a 200 W high pressure Vycor-filtered mercury arc yields primarily the cis and trans diols formed by reduction at the 9-keto position. Another observed product is the hemiketal (I11 in the synthetic diagram).
Stability
510
REX W. SOUTER
Long-Term S t a b i l i t y Samples o f n a b i l o n e s t o r e d up t o f o u r y e a r s u n d e r v a r y i n g c o n d i t i o n s of h e a t and h u m i d i t y show e s s e n t i a l l y no change i n p o t e n c y . Metabolism , P h a r m a c o k i n e t i c s and M i c r o b i o l o g i c a l Transformations Nabiloiie h a s b e e n shown t o have a plasma h a l f - l i f e of a b o u t two h o u r s i n man a f t e r i n t r a v e n o u s and o r a l a d m i n i s t r a t i o n (17). Circulating metabolites included t h e isomeric c a r b i n o l s formed by r e d u c t i o n a t t h e 9-keto p o s i t i o n .
5.2
6.
I n v e s t i g a t i o n of t h e p h a r m a c o k i n e t i c s of n a b i l o n e and i t s c a r b i n o l m e t a b o l i t e s i n t h e dog f o l l o w i n g t h e a d m i n i s t r a t i o n of s m a l l d o s e s r e q u i r e d development of a q u a n t i t a t i v e s e l e c t e d i o n m o n i t o r i n g ( S I M ) g a s chromatography mass s p e c t r o m e t r y method ( 1 8 ) . I n t h e dog, n a b i l o n e was r a p i d l y conv e r t e d t o a m i x t u r e of i s o m e r i c c a r b i n o l s whose r a t i o i n plasma was c o n s t a n t . The a p p a r e n t h a l f - l i f e of n a b i l o n e i n dogs was 1-2 h r . w h i l e t h a t of t h e m e t a b o l i t e c a r b i n o l s w a s of t h e o r d e r of 20 h r . f o l l o w i n g a s i n g l e o r a l o r I . V . d o s e . The SIM methods f o r n a b i l o n e and i t s m e t a b o l i t e s h a v e a l o w e r s e n s i t i v i t y l i m i t of a b o u t 2 pmol ml- w i t h a coe f f i c i e n t of v a r i a t i o n of less t h a n 4% (18).
A s c r e e n i n g program was u s e d t o f i n d m i c r o o r g a n i s m s c a p a b l e of m o d i f y i n g n a b i l o n e (19) and n e a r l y one-half of t h e t e s t e d m i c r o o r g a n i s m s were found e f f e c t i v e (19,ZO). P r o d u c t s from t h r e e c u l t u r e s were c h a r a c t e r i z e d s p e c t r o s c o p i c a l l y t o determine t h e i r chemical s t r u c t u r e s ( 1 9 ) .
Methods of A n a l y s i s 7 . 1 Raw M a t e r i a l 7 . 1 1 Gas Chromatography N a b i l o n e may b e a s s a y e d u s i n g a g l a s s column W DMCS-treated packed w i t h OV-17 s t a t i o n a r y p h a s e 011 A The s a m p l e i s d i s s o l v e d i n a c h l o r o f o r m chromosorb G . s o l u t i o n of t h e i n t e r n a l s t a n d a r d , m e t h y l t e s t o s t e r o n e . Samples a r e compared t o r e f e r e n c e s t a n d a r d s p r e p a r e d i n t h e same manner. The method e x h i b i t s a p r e c i s i o n of a b o u t 0 . 7 % (R. S.D. 1. 7 . 1 2 T h i n Layer Chromatography (TLC) Using a s i l i c a g e l 60 F254 t h i n l a y e r p l a t e i n a p a p e r - l i n e d chamber c o n t a i n i n g b e n z e n e / e t h y l a c e t a t e 75~25, n a b i l o n e h a s a Rf of a b o u t 0.55. I t may b e v i s u a l i z e d by e x p o s i n g t h e p l a t e t o 2 5 4 n m UV l i g h t ( s o t h a t t h e s p o t q u e n c h e s t h e f l u o r e s c e n t i n d i c a t o r i n t h e p l a t e ) o r by spraying t h e dry p l a t e with f a s t blue B spray reagent (21).
7.
NABILONE
51 1
Biological Samples Methods for assay of nabilone and its metabolites have been described (17,18). 7.3 Pharmaceutical Formulations Following some steps to extract it from its capsule excipients, nabilone may be assayed by a gas chromatographic technique similar to that used for the raw material. Simple TLC identification of nabilone in capsules requires its extraction from excipients followed by TLC on a silica gel 60 F254 plate in a paper-lined chamber containing toluene/ Nabilone has an Rf of approximately ethyl acetate 80:ZO. 0.37 in this system.
7.2
8. Acknowledgements The assistance of Mr. Michael Gleissner in obtaining the 13C nmr data is appreciated as are the efforts of Dr. A. D. Kossoy in performing and interpreting the accelerated degradation profile of nabilone. The author thanks Dr. L. G. Tensmeyer, Dr. D. E . Dorman, Mr. J . L. Occolowitz and Mr. C . D. Underbrink for their assistance in the acquisition and interpretation of the other spectral data. The constructive criticism of Dr. R. A. Archer in proofreading this manuscript is sincerely appreciated.
512
REX \V. SOUTEIR
9.
References F. J . Fox, N . Eiigl. J . Med., 301, 7 2 8 ( 1 9 7 9 ) . T . S . Herman, S . E. J o n e s , J . Dean, e t a1, Biomedicine, 331 (1977). 3. T . S . Herman, L. H. E i n h o r n , S . E . J o n e s , e t a l . N . Engl. J . Med., 3 0 0 , 1295 ( 1 9 7 9 ) . 4. C . M . Nagy, E . F. Becky, L. H . E i n h o r n , e t a1, S c i e n t i f i c P r o c . Am. Assoc. Cancer R e s . , 3, 30 (1978). 5 . N . S t e e l e , D . Braun, M . O ' H e h i r , e t a l , P r o c . Am. Assoc. Cancer R e s . , 0, 337 ( 1 9 7 9 ) . 1265 ( 1 9 8 0 ) . 6. D. L. Sweet, J . Am. Med.Assoc., 7 . R. Weisman and J . A s h e r , S c i . N e w s , 1 1 4 , 94 ( 1 9 7 8 ) . 8. F. W. N e w e l 1 and P. S t a r k , " N a b i l o n e : A S y n t h e t i c Cannabinoid Analogue i n Open Angle Glaucoma i n Man," i n Glaucoma Update, G . P . H a l b e r g , e d . , I n t e r o p t i c s , D i v i s i o n of Woodbine P u b l i s h e r s , 1978, pp. 81-83. 9 . S . E. S a l l a n , N . E. Z i n b e r g and E . F r e i , 111, N . Engl. J . Med., 293, 795 ( 1 9 7 5 ) . 10. R. S. H e p l e r and I. M. F r a n k , J . Am. Med. A S S O C . , 217, 1392 ( 1 9 7 1 ) . 11. R. S. H e p l e r , I . M. F r a n k and J . T . U n g e r l e i d e r , Am. J . Ophthalmol., 74, 1185 ( 1 9 7 2 ) . 1 2 . L. Lemberger and H. Rowe, C l i n . Pharmacol. T h e r . , 18, 720 ( 1 9 7 5 ) . 968 ( 1 9 7 0 ) . 1 3 . L. E. H o l l i s t e r , N a t u r e (London), 1 4 . J . E. Mann, G . F. K i p l i n g e r , I . F. B e n n e t t , R. B . Forney and S . E. H a i n e , C l i n . Pharmacol. T h e r . , 11, 808 ( 1 9 7 0 ) . 1 5 . A. L. T h a k k e r , C . A. H i r s c h , and J . G . P a g e , J . Pharm. Pharmac., 3, 783 ( 1 9 7 7 ) . 1 6 . R. A. A r c h e r , W. B. B l a n c h a r d , W . A . Day, D. W . J o h n s o n , E. R. Lavagnino, C . W. Ryan and J . E . Baldwin, J . Org. Chem., 42, 2277 ( 1 9 7 7 ) . 1 7 . A. Rubin, L . Lemberger, P . W a r r i c k , R . C r a b t r e e , H. S u l l i v a n , H. Rowe and B . Obermeyer, C l i n . Pharmac. and T h e r . , 85 (1977). 18. H. R. S u l l i v a n , D. L . K. Kau and P . G . Wood, Biomed. Mass S p e c . , 5, 296 ( 1 9 7 8 ) . 1 9 . R. A. A r c h e r , D. S. Fuduka, A . D . Kossoy and B . J . A b b o t t , Appl. and E n v i r o . , M i c r o b i o . , 37, 965 ( 1 9 7 9 ) . 20. B . J . A b b o t t , D. S . Fuduka and R. A. A r c h e r , E x p e r i e n t i a , 33, 718 ( 1 9 7 7 ) . 21. K. G . K r e b s , D. H e u s s e r a n d H . Wimmer i n E. S t a h l , e d . , Thin-Layer Chromatography, S p r i n g e r - V e r l a g , New York, 1969, p . 874.
1. 2.
27,
243,
227,
22,
L i t e r a t u r e r e v i e w e d t o J a n u a r y 1981
NATAMYCIN
Harry Brik
1. Description 1.1 Name, Formula, Molecular Weight 1.2 Chemical Structure and Configuration 1.3 Nomenclature 1.4 Appearance 1.5 Standards and Regulatory Status 1.6 Antimycotic Properties 2. Chemical Properties 3. Physical Properties 3.1 Crystal Properties 3.2 Ultraviolet Spectrum 3.3 Infrared Spectrum 3.4 Proton NMR Spectrum 3.5 I3C NMR Spectrum 3.6 Mass Spectrum 3.7 Optical Rotation 3.8 Optical Rotatory Dispersion 3.9 Ionization Constants 3.10 Thermal Behaviour 3.11 Counter-Current Distribution 3.12 Solubility 4. Production 4.1 Discovery 4.2 Biosynthesis 4.3 Fermentation and Isolation 5. Stability 6. Biopharmaceutics 6.1 Pharmacokinetics 6.2 Toxicity 6.3 Other 7. Analysis 7. k Identification 7.2 Spectrophotometric Analysis 7.3 Colorimetric Analysis 7.4 Titrimetric Analysis 7.5 Chromatographic Analysis 7.6 Electrophoretic Analysis 7.7 Polarographic Analysis 7.8 Elemental Analysis 7.9 Microbiological Analysis 8. Acknowledgements 9. References 514 514 515 515 517 517 517 518 518 518 520 522 524 528 531 534 534 535 535 537 538 54 1 54 1 54 1 54 1 542 544 544 544 545 545 545 547 548 549 549 553 553 555 556 556 557
ANALYTICAL PHOFII.ES OF DRUG SUBSTANCES. 10
513
514
HARRY BRIK
1 . 1.l.
Synonym
Description Name , Formul a , Mol ecul a r Weight
Generic name Trade names Chemical name
na tamyc in
p im a r i c i n Natafucin; Pimafucin; D e l v o c i d
22 - ( 3 -am in 0- 3,6 -d id eoxy -8-D -ma nno pyranosyl )oxy-l,3,26-tri hydroxy-12-methyl -10-oxo-6,11,28-trioxatri-cycl o [22.3.1.05,7] octacosa-8,14,16,18,~0-pentaene-25-carboxyl i c acid.
S t r u c t u r a l formula*
HO
a1
HOOC
'H3
27
E m p i r i c a l formula Mol ecul a r weight Code d e s i g n a t i o n
C33H47N013 665,75 CL 12 625; A n t i b i o t i c A-5283 7681-93-8

T F3-24-6 A A0 GO KVO I U OU QU SU UUTJ BQ DO M 1 CaVQ D&Q WO-BT60TJ Cy DZ EQ F 1
CAS
r e g i s t r y number
W i swesser 1 ine notation
* I n c o n t r a d i c t i o n t o t h e I U P A C chemical name t h e C-atoms i n t h e s t r u c t u r a l formula are numbered i n t h e c l a s s i c a l way t o f a c i l i t a t e comparison w i t h 1 i t e r a t u r e d a t a which r e f e r t o p a r t s o f t h e molecule.
NATAMYCIN
515
1.2.
Chemical S t r u c t u r e and C o n f i g u r a t i o n
Natamycin be1 ongs t o t h e 1arge group o f polyene a n t i f u n g a l a n t i b i o t i c s . C h a r a c t e r i s t i c f o r t h i s group i s a m a c r o c y c l i c 1a c t o n e - r i ng w i t h a number o f conjugated carbon-carbon doubl e bonds. The chemical s t r u c t u r e o f natamycin was proposed a t f i r s t b y P a t r i c k e t a1 i n 1958 (1,2). S i x years l a t e r an e x t e n s i v e r e i n v e s t i g a t i o n was presented by Ceder ( 3 ) . A m i n o r r e v i s i o n (absence o f an OH-group a t c 8 ) o f Ceder's proposed s t r u c t u r e was made b y Golding e t a1 i n 1966 ( 4 ) . T h i s l a s s t r u c t u r e ( S e c t i o n 1.1) was confirmed by Haegele e t a1 (5 u s i n g mass spectrometry o f t h e p e r - t r i m e t h y l s i l y l d e r i v a t ve and by Ceder e t a1 ( 6 ) u s i n g 13C-NMK spectroscopy o f i.e. t h e N-acetyl d e r i v a t i ve. The hemi k e t a l - s t r u c t u r e of t h e cg-C13 p a r t o f t h e l a c t o n e - r i n g was confirmed by o p t i c a l r o t a t i o n d i s p e r s i o n (7,8) and by proton-NMK spectroscopy ( 6 ) . The a b s o l u t e c o n f i g u r a t i o n a t C25 was e s t a b l i s h e d as K by i s o l a t i o n o f an o p t i c a l l y a c t i v e d e r i v a t i v e o f t h e ';24'c26 c h a i n (9,lO). The t o t a l c o n f i g u r a t i o n o f t h e major p a r t s o f t h e molecule F i g u r e 1) was e l u c i d a t e d by Ceder e t a1 ( 6 ) u s i n g p r o t o n - and f3C-NMK spectroscopy. From t h e same experiments t h e d i a s t e r e o m e t r i c a l p u r i t y o f natamycin c o u l d be deduced as we1 1. On t h e b a s i s o f u l t r a v i o l e t data t h e t e t r a e n e system has been shown t o be a l l - t r a n s (11). S t r u c t u r a l l y , natamycin i s c l o s e l y r e l a t e d ( F i g u r e 2) t o t h e t e t r a e n e s lucensomyci n (12,13) , arenomyci n B (14) t e t r a m y c i n ( 1 5 ) and t h e t e t r i n s A and B (16,17). Contrary t o most o t h e r polyenes, natamycin c o n t a i n s l i t t l e o r no congeners.
I .3.
Nomencl a t u r e
The natamyci n-produci ng Streptornyces s t r a i n was found i n a s o i l sample f r o m t h e neighbourhood o f P i e t e r m a r i t z b u r g , a town i n t h e p r o v i n c e o f N a t a l , South A f r i c a . Therefore t h e s t r a i n i n q u e s t i o n was c a l l e d S . n a t a l e n s i s . The name natamycin i s commemorative o f t h i s s t r a i n , t h e o l d name p i m a r i c i n ( s t i l l used sometimes b u t not accepted by t h e WHO s i n c e a n t i b i o t i c s which a r e produced by a Streptomyces s t r a i n should have t h e s u f f i x " m y c i n " ) i s c a l l e d a f t e r Pietermaritzburg. The synonym t e n n e c e t i n ( S e c t i o n 4.1) i s no l o n g e r used.
516
HARRY BRIK
F i g u r e 1. C o n f i g u r a t i o n o f t h e major p a r t s o f natamycin a c c o r d i n g t o Ceder ( 6 ) . mycosami ne : absol U t e c o n f i g u r a t i o n C 1 - C 1 7 fragments: r e l a t i v e c o n f i g u r a t i o n
F i g u r e 2. S t r u c t u r a l r e l a t i o n o f natamycin t o o t h e r t e t r a e n e s which d i f f e r o n l y i n K 1 - K4 t e t raene na t amyc i n 1ucensomyci n arenomycin B tetramyci n tetrin H tetrin B

K1
K2
K3
Me n-Bu n-Bu Me Me Me
K4
H H H Et Me Me
-0-0OH OH
OH OH
H H H OH
NATAMYCIN
517
1.4.
Appearance almost t a s t e l e s s and
Hatarnycin i s a w h i t e t o cream-coloured, almost o d o u r l ess , c r y s t a l 1 ine powder.
1.5.
Standards and Kegul a t o r y S t a t u s
The m i c r o b i o l o g i c a l a c t i v i t y o f natamycin i s expressed i n ug p e r rag. The FDA master standard has an assigned potency o f 922,0 ug o f anhydrous natariiycin per m i l l i g r a m (18). T h i s standard i s equal t o Gist-Brocades ndtamycin t r i h y d r a t e r e f e r e n c e standard 1o t 711-EN-78-1. The USP r e f e r e n c e standard ( l o t F , c a t a l o g riumber 4575) i s equal t o C i st-Brocades natamycin t r i h y d r a t e r e f e r e n c e standard 1O t 705-EN-84-1. Both standards were p u r i f i e d by repeated c r y s t a l 1 i z a t i on o f a s e l e c t e d 1o t o f natarnyci n.
1.6.
Antirnycotic P r o p e r t i e s
liatamycin i s e f f e c t i v e a g a i n s t a broad v a r i e t y o f f u n y i , yeasts, some protozoa and some algae. It has no a n t i b a c t e r i a l a c t i v i t y Iiatamyci n is used t o p i c a l l y dgai n s t fungal i n f e c t i o n s o f t h e s k i n and t h e mucous r,iernbranes i n t h e form o f s e v e r a l dosage forms (suspensions , creams , o i ntrlitnts and v a g i n a l t a b l e t s ) a l o n e o r i n combination w i t h neomycin and h y d r o c o r t i s o n e o r o t h e r s t e r o i d s . H summary o f t h e t h e r a p e u t i c use o f riatamycin i s g i v e n by Kaab (19).
Natamycin i s a l s o used as a f o o d a d d i t i v e , m a i n l y d s an a n t i m y c o t i c on cheese, meat products and i n wines and f r u i t j u i c e s . The a n t i m y c o t i c a c t i o n on foods i s t w o f o l d , i t prevents economic l o s s e s as w e l l as t h e f o r m a t i o n o f inycotoxins (20,21). I n wine i t can r e p l a c e s o r b i c a c i d and o t h e r a n t i f u n g a l agents and i t a l l o w s a r e d u c t i o n o f t h e amount o f s u l f u r d i o x i d e used ( 2 2 ) . Compared w i t h c l a s s i c a l a n t i f u n g a l agents natai,iycin i s a c t i v e i n v e r y l o w c o n c e n t r a t i o n s , on cheese f o r i n s t a n c e i t i s 400 t i m e s iaore a c t i v e than potassium s o r b a t e (23). A r e v i e w on t h e use o f natamycin i n foods i s g i v e n by M o r r i s (24). The a p p l i c a t i o n o f natamycin as a food a d d i t i v e besides i t s use as a drug i s p o s s i b l e due t o a number o f f a v o u r a b l e p r o p e r t i e s : i t has a very l o w o r a l t o x i c i t y , a b s o r p t i o n t h r o u y h t h e i n t e s t i n e has n o t been demonstrated, sensi t i z i ng p r o p e r t i e s have n o t been found and ( c r o s s ) r e s i s t a n c e has never been encountered (see S e c t i o n 6.1).
518
HARRY RRIK
2.
Chemical P r o p e r t i e s
The amphoteric c h a r a c t e r o f natamycin i s r e s p o n s i b l e f o r i t s l o w s o l u b i l i t y i n most s o l v e n t s . The s o l u b i l i t y i n water o r l o w e r a l c o h o l s i s increased a t l o w and h i g h pH. I n s t a b l e , c r y s t a l 1 ine s a l t s , 1 ike t h e p o t a s s i urn s a l t arid t h e s u l phate a r e known ( 2 5 ) . Iaproved s o l u b i l i t y i n water w i t h o u t s a c r i f i c i n g any s t a b i l i t y o r m i c r o b i o l o g i c a l a c t i v i t y can be achieved by complex formation w i t h b o r i c a c i d (26) o r d modified polysaccharide ( 2 7 ) . T h i s i s a l s o achieved by chemical m o d i f i c a t i o n , f o r i n s t a n c e by f o r m a t i o n o f a l k y l e s t e r s (28,29,30), amides (31) and N-glycosyl d e r i v a t i v e s ( 3 2 ) . Natamycin forms a 1:l i o n - p a i r w i t h k a t i o n t e n s i d e s ( 3 3 ) which, u n l i k e t h e parent compound, i s s o l u b l e i n l e s s - p o l a r s o l v e n t s ( S e c t i o n 6.4). The complex f o r m a t i o n o f natamyci n w i t h s t e r o l s 1ike c h o l e s t e r o l and e s p e c i a l l y e r g o s t e r o l (34) i s t h e b a s i s o f b o t h i t s f u n g i c i d a l a c t i o n and t h e a n t a g o n i s t i c p r o p e r t i e s o f t h e above s t e r o l s (35). The t e t r a e n e chromophore g i v e s t h e riiol ecul e a h i g h l y unsaturated c h a r a c t e r . It r e a c t s r e a d i l y w i t h bromine and compounds c o n t a i n i n g a c t i ve oxygen such as permanganate, p e r s u l p h a t c and peroxides. On t h e o t h e r hand natamycin c o n t a i n s weakly a c t i v e oxygen i n t h e form o f an epoxy-group. The l a t t e r l i b e r a t e s i o d i n e when natamycin i s t r e a t e d w i t h a hot solution o f iodide i n g l a c i a l acetic acid (3). The ami nosugar nycosami ne i s 1ib e r a t e d by a c i d h y d r o l y s i s , t h e l a c t o n e i s s a p o n i f i e d by a l k a l i n e h y d r o l y s i s . See a l s o S e c t i o n 5. 3. 3.1. 3.1 .I. Physical Properties Crystal Properties O p t i c a l C r y s t a l l o g r a p h i c Data
Natamycin c r y s t a l 1 i z e s from aqueous l o w e r a l c o h o l s i n t h e form o f t h e t r i h y d r a t e as t h i n r e c t a n g u l a r p l a t e s ( F i g u r e 3) showing t h e f o l l owi ny o p t i c a l c o n s t a n t s ( 3 3 ) : r e f r a c t i v e indi ces extinction o p t i c sign a x i a l angle 2V
: 1,540 and 1,6118 : parallel : positive : < 15'
No polymorphism has been r e p o r t e d f o r t h e t r i h y d r a t e .
NATAMYCIN
519
Natarnycin methanol sol v a t e c r y s t a l 1 izes from a s a t u r a t e d methanolic s o l u t i o n of the t r i h y d r a t e a s t h i n , s t r o n g l y b i r e f r i n g e n t n e e d l e s w i t h p a r a l l e l e x t i n c t i o n and p o s i t i v e el o n y a t i on. Upon c o n t a c t w i t h water the s o l v a t e r a p i d l y c o n v e r t s t o t h e t r i h y d r a t e . The l a t t e r shows t h e same X-ray d i f f r a c t i o n p a t t e r n a s the t r i h y d r a t e c r y s t a l 1 i z e d from aqueous sol vents ( 3 6 ) . The above o p t i c a l c o n s t a n t s a r e a l s o i d e n t i c a l f o r both forms. However, the t r i h y d r a t e o b t a i n e d from the s o l v a t e a s i n d i c a t e d above, has a h a b i t w h i c h i s somewhat d i f f e r e n t from the usual t r i h y d r a t e . llnl i ke the l a t t e r (merely rectanyul a r pl a t e s ) the r e c r y s t a l l i z a t i o n product c o n s i s t s predominantly of prisms t e r m i n a t e d a t one end by a pyramid, w i t h a n e g a t i v e s i g n of e l o n g a t i o n , showing t r a n s i t i o n s t o r e c t a n g u l a r pl a t e s .
Figure 3 . Natamyci n c r y s t a l s from aqueous propanol I d i v i s i o n = 10 urn
520
HARRY BRIK
3.1 2. X-ray C r y s t a l l o g r a p h i c Data Hempel e t a l . ( 3 7 ) determined t h e c r y s t a l s t r u c t u r e o f natamycin by X-ray a n a l y s i s u t i l i z i n g CuKa r a d i a t i o n . The f o l 1 owi ng r e s u l t s were obtained: : inonocl in i c : P21 : a = 0,768(1) nm b = 0,875(1) nm c = 2,690(2) ~ i t i ti angle : 92,3(Z)O : 1 , ~ 5nm3 u n i t c e l l volume molecules p e r u n i t c e l l : 2 3.2. U l t r a v i o l e t spectrum c ry s t a 1 s t r uc t ur e space group c e l l dimensions
The u l t r a v i o l e t spectrum o f natamycin (USP r e f e r e n c e standard, l o t F ) i n methanol w i t h 0 , l X a c e t i c a c i d i s shown i n F i g u r e 4. The a c e t i c a c i d a c t s as a "wavelength s t a b i l i s e r " as t r a c e s o f a l k a l i i n t r o d u c e a small r e d - s h i f t o f up t o 2 nm. T h i s p r i n c i p l e i s used i n t h e spectrophotornetric a n a l y s i s of natamycin ( S e c t i o n 7.2). The spectrum e x h i b i t s sharp maxima a t 290, 303 and 318 nrli, a shoulder a t 280 run and a broader maximum a t 220 nm. Under t h e above c o n d i t i o n s t h e fo77 owi ng s p e c t r a l constants were o b t a i ned ( 3 3 ) : Table 1 u l t r a v i 01 e t d a t a
'max ( nm )
220 280 290 303 318
Al %* 1 cm
mol
. abs. coeff.
21 300
26 63U 52 93u 83 220 7 6 23u
320 400 795 1250 1145
*calculated w i t h reference t o the d r i e d sub stance The a b s o r p t i o n bands i n t h e r e y i o n between 280 and 320 nm a r e c h a r a c t e r i s t i c f o r an a l l - t r a n s t e t r a e n e (11), t h e maximum a t 220 n m i s a t t r i b u t e d t o t h e en-one chromophore.
NATAMYCIN
521
0,5
0,4
cu U c d n
0,3 .
0 v ,
CJ
0.2 .
0.1
200
(,
350
250
h,nm
300
F i g u r e 4. U1 t r a v i o l e t spectrum o f natamycin i n methanol w i t h 0,1% 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 3,6 pg ( c a l c u l a t e d on t h e anhydrous b a s i s ) per rnl I n s t r u m e n t used: Beckman Acta C I I I
522
HARRY BRIK
Several a u t h o r s (25,38,3Y ,40) r e p o r t somewhat lower molar a b s o r p t i o n c o e f f i c i e n t s f o r t h e t e t r a e n e chromophore. The u l t r a v i o l e t spectrum o f natamycin i s s i m i l a r t o t h e s p e c t r a o f t e t r a e n e a n t i b i o t i c s which belong t o t h e same sub-group having t h e same chromophores, e s p e c i a l l y i t s C25 b u t y l -homo1 ogue 1 ucensomyci n ( 1 2 ) , f u r t h e r t h e c l o s e l y r e 1 a t e d arenomycin B ( 1 4 ) , t e t r a r n y c i n ( 1 5 ) , t e t r i n A ( 1 6 ) and t e t r i n B ( 1 7 ) . The f o u r 1a t t e r t e t r a e n e s however 1ack an epoxy-group. It i s p o s s i b l e t h a t f o r t h i s reason t h e en-one chromophore o f these t e t r a e n e s absorbs a t somewhat 1ower wave1 engths (208-212 nm)
The a b s o r p t i o n a t 220 nm d i f f e r e n t i a t e s natamycin from t h e t e t r a e n e s n y s t a t i n and arnphoteri c i n H (see S e c t i o n 7 . 1 ) .
3.3. I n f r a r e d spectrum The i n f r a r e d spectrum o f natamyci n (USP r e f e r e n c e standard l o t F) i n a potassium bromide p e l l e t ( 4 1 ) i s presented i n F i g u r e 5. H spectrum o f t h e same sample i n l i q u i d p a r a f f i n i s e s s e n t i a l l y i d e n t i c a l t o t h e one presented. An i n t e r p r e t a t i o n o f t h e spectrum i s g i v e n i n Table 2.
Table 2 I n f r a r e d s p e c t r a l assignments (42) wavenumber (cm-1) assignment
CH def. i n CH=CH C-0-C )C-OH asymrn. s t r e t c h C-0-C epoxy CH s t r e t c h CH=CH s t r e t c h ; COOC=O l a c t o n e CH2 s t r e t c h =CH s t r e t c h NH3' OH OH herni k e t a l OH - c a r b o x y l ; water
1005 1060 1110 1270 1400 1570 1715 2950 3u20 3270 350u 3600 2400
3600
When p r e p a r i n g a K B r p e l l e t i t i s i m p o r t a n t t o evacuate f o r n o t more t h a n 10 seconds t o a v o i d d i s t o r t i o n o f t h e spectrum due t o l o s s o f water o f c r y s t a l l i z a t i o n ( 4 1 ) .
FREQUENCY (C M-)
F i g u r e 5. I n f r a r e d spectrum of natamycin t r i h y d r a t e i n a K B r p e l l e t ( x = polystyrene c a l i b r a t i o n p o i n t s a t 1600 and 1583 cm-1) Instrument used: Perkin-Elmer Model 521
524
HARRY BRIE;
3.4.
P r o t o n NMK Spectrum
DeBruyn e t a l . ( 4 3 ) have recorded a 300 MHz p r o t o n NMK spectrum o f natamyci n (Gi st-Brocades r e f e r e n c e standard 705-EFI-71-1) i n d e u t e r o t r i f l u o r o e t h a n o l c o n t a i n i n g some NaOD. The assignments a r e g i v e n i n Table 3. Table 3 Assignments o f t h e peaks i n t h e 300 MHz p r o t o n NMK s p e c t r a o f n a t a m y c m
~ ~~
chemical s h i f t , pprn
proton 6B
b',
chemical s h i f t , ppm
proton
1,27 1,37 1,40 1,73 1,83 2,12 2,13

2,26 2,40 2,65 2,97 3,21
26
1OB
8 B
8A, 14B GA 10A, 12 14A 24 B 24A 3' 5 4, 4 '
3,22 3,92 4,25 4,36 4,50 4,57 4,75 5,67 5,93 6,08 + 6,25 6,47
5' 2' 11, 13
15 7 1'
25 23 16 2 17-22 3
However, t h e r e s o l u t i o n i n t h i s s o l v e n t i s r a t h e r poor (see F i g u r e 6) and b e t t e r r e s u l t s can be o b t a i n e d u s i n g t h e Pi-acetyl d e r i v a t i v e i n p y r i d i n e - d g . Ceder e t a1 ( 6 ) p u b l i s h e d a 270 MHz spectrum and assigned most o f t h e resonances and coup1 ing constants. The spectrum c o u l d be compl e t e l y a s s i gned except f o r p a r t o f t h e t e t r a e n e moiety (HI8 - H20l u s i n g more s o p h i s t i c a t e d r e s o l u t i o n enhancement techniques on a Bruker WM 250 ( 4 4 ) . See F i g u r e 7 and Tab1 e 4.
526
HARRY BRIK
Table 4 Assignments o f t h e peaks and c o u p l i n g c o n s t a n t s i n t h e 250 NHz proton-NMK spectrum o f I l - a c e t y l natamyci n (44) proton cheirii c a l shift, p p ~ proton
s h i f t , ppn
1,256 1,431 1,517 1,814 1,836 1,956 2,036 2,050 2,107 2,144 2,222 2,582 2,802 2,953 3,1G7 3,291 3,641 4,023
26 6A 6' 8 A 10H 8B acetyl

GB
14A 24A 24 B 10B 14B 12 5 4 5' 4'
4,523 4 ,583 4,658 4,838 4,958 5 ,U83 5,202 5,266 5,572 6,173 6 ,188 6,2U0 6,352 6,358 b ,49u 6,669 8,751
2' 3' 7 25 15 1' 11 13 23 22 18-20 21 17

2
1 6
3 NH
Coup1 ing c o n s t a n t s ,
Hz
3J(14AY15) 3J(14BY15) 3J (15,16) 3J ( 1 7 ,18) 3J(21,22) 3J (22,23) 3J(23,24A) 3J(23,24B) *J(24H924B) 3J(24H,25) 3J(24B,25) J (25,26)
2,2
3J(1' , 2 ' ) 3J ( 2 ' , 3 ' ) 3J(3',4') 3J(4' , 5 ' ) 3J(5' , 6 ' ) 3J ( 3 ' , N H ) 3J(2,3) 35 ( 3 , 4 ) 3J(4y5) %(5,6H) 3J ( 5 ,GB) 2J (6HY6B) 3J ( b A , 7 )
3J(68,7) 3~ (7,8H) 3J7 ,8B) 2J(8A,8B) ZJ( ~ o H , ~ u B ) 3J(10A,ll) 3J (l0B ,11) 3J(ll,l2) 3J(12,13) 3J( 13 ,14A) 3J( 13,14B) 2 J ( 14AY14B)
3,5 2Y5 10,7 14,7 12,5 10,5 4Y8 10,2 10 ,8 8Y5 ( 1 15,2
4,4
iJY1
10,5 11 15,5
9,8
6,4 13,5 10,l 3,i fJY4
i
5
A
d
W .L
c .-
a,
a
0
f
W
a,
t
C
e:
C .*Jp
C .-
$
c r
t V
z
L
3 E
t
ul
m a
3
C
a L
I N
0 M N
ra,
3 TI
528
HARRY RRIK
3.5.
13C-NMK Spectrum
Pandey e t a l . (45) obtained 13C-NMK spectra of natamycin and N-acetylnatamycin in DMSO, b u t they assigned only four of t h e resonances, which confirmed the hemi ketal structure. The 13C-NMK spectrum of natarnycin (62,89 MHz) in deuterot r i f l uoroethanoljNaOD i s shown in Figure 8 and the assi ynrnents a r e given in Table 5 (44). A s i n the proton NMK spectrum the resolution i s poor and not a l l peaks are separated. Table 5 Assignments of the peaks in the 13C-NMK spectrum o f natam-ycin (44) chemi cal s h i f t , ppm
18 ,23 20,62 41 ,16 44 ,54 47,84 56,45 57,16 60,20 61,14 68,22 68,64 69,54 71,96 73,08
C-atom
6' 26 6 ,10 ,14 24 8 4 3'
chemi cal s h i f t , ppm

74,76 75,39
C-atom
4'*
5* 15* Y 1'
2
81,58
Y9,73 100,83 126,32 130,70 133,22 134,35 134,5Y 135 ,12 137,45 145,92 168,82 182,25
16**
17**,18**,19** 20** 21** 22** 23** 3 1 27
5
12 13* 11* 7* 2 5* 2'"
assignments may be interchanged Ceder et a l . ( 6 ) assigned part of the 13C-NMH spectrum o f N-acetyl natamycin i n pyridine-dg by comparison with the spectra of the dodecahydroderivative and the hydrogenationhydrogenolysi s product. llsi ng sel e c t i ve decoupl i ng techni ques , a1 1 resonances of the I3L-NMK spectrum of N-acetyl natamyci n (62,89 MHz) could be assi gried except for part of the tetraene moiety (C18-C22) (44). See Fiyure 9 and Table 6.
*,**
NATAMYCIN
53 1
Table 6
Assignments o f t h e peaks i n t h e 13C-NMK

Spectrum o f N-acetyl natam.yci n (44) chemical s h i f t , ppm
17,55 19,45 22,17 37,3Y 38,77 40,76 44,58 46,65 53,66 55,42 58,16 58,34 65,54 65,82 66,57 69,21 70 ,00 71,47
C- a t o m
c h eini c a l s h i f t , ppm
73,60 74,77 97,22 Y7,46 124,24 127,95 128,47 131,10 131,30 131,55 133,06 135,29 136,ll 144,15 164,36 170,27 175,25
C-atom
5' 15 1' 9
2
6' 26 8' 14 10 6 24 8 4 3' 5 12 13 11 7 25 2' 4'
23 17 18* lY* 20* 16 21* 22* 3 1 7' 27
*
3.6.
assignments may be interchanged
Mass Spectrum
Dornberger e t a1 (46) and Ceder e t a1 ( 6 ) o b t a i n e d mass s p e c t r a of u n d e r i v a t i zed natamyci n u s i n g e l e c t r o n impact and f i e l d d e s o r p t i o n iiiass spectrometry. In t h i s way a m o l e c u l a r i o n i s n o t obtained, t h e h i g h e s t mass observed corresponds t o M - mycosamine - LO2 - 2 H20. Falkowski e t a1 (47) o b t a i n e d f i e l d d e s o r p t i o n mass s p e c t r a o f several natamyci n d e r i v a t ives
R y means o f mass spectrometry o f t h e p e r t r i i n e t h y l s i l y l d e r i v a t i v e (5,48) t h e m o l e c u l a r i o n a t m/e 1169 of t h e f u l l y s i l y l a t e d d e r i v a t i v e was obtained. The mass spectrum presented i n F i g u r e 10 was r u n on a V a r i a n MAT 311A by d i r e c t sample i n t r o d u c t i o n a t 180OC. The i o n i z i n g 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 2 kV (48). The f r a g m e n t a t i o n p a t t e r n , as proposed by Haegele e t a l . ( 5 ) and c o r r o b o r a t e d by d e u t e r a t e d TMS - and a c e t y l - d e r i v a t i v e s , i s shown i n F i g u r e 11.
1
8
0
1
. . _ _ .
AlISN31NI 3 A l l V l i ' i
NATAMYCIN
533
i '
e
mje 5 3 6
1
RO W
l+.
e
+
OR
Meo
RO
NHR OR
m/e 806
m/e 3 6 2
per-TMS n a t a m y c i n +
mje 1 1 6 9
'
~
NH2
m/e 2 9 0
Re O M=
OR
OR
m/e 878
:XAoR
m/c 273
OR
m j e 608
Figure 11. Fragmentation p a t t e r n o f natamyci n. K = t r i m e t h y l s i l y l (according t o r e f . 5 )
534
HARRY BRIK
3.7.
Optical r o t a t i o n
S p e c i f i c r o t a t i o n values from d i f f e r e n t sources a r e shown i n t a b l e 7. Table 7 S p e c i f i c R o t a t i o n o f natamycin Sol vent d i m e t h y l s u l phoxide d imethyl f ormaini de acetic acid acetic acid p y r i d i ne met ha no1 conc. %emp.
LalD
reference
3Y
0,5%
27C
25C 25'C 2ooc 20C
+ 180'
t
258"
t 273"
25 19
1,@h
O,5% U,08%
t 278"*
t 294O
49
4Y
25C
+ 250
38
*mean Val ue o f 7 r e f e r e n c e p r e p a r a t i o n s , c a l c u l a t e d w i t h r e f e r e n c e t o t h e d r i e d substance, e q u i v a l e n t t o +257' f o r t h e t r i hydrate. 3.8. Optical Rotary Dispersion
The o p t i c a l r o t a r y d i s p e r s i o n (OKD) curve o f natamycin ( 7 )
i s reproduced i n F i g u r e 12.
From t h e shape o f t h e OKU curve o f natarnycin and o t h e r polyenes Chong e t a l . ( 7 ) and Dornberger e t a l . ( 8 ) concluded t h a t natamycin - i n methanolic s o l u t i o n - e x i s t s as a c y c l i c hemi k e t a l r a t h e r t h a n t h e open hydroxy-ketone form.
8 7
0 .-
-m=l
4 -
-47 250
"
3 00
3 50
400
h,nm
F i g u r e 12. O p t i c a l r o t a r y d i s p e r s i o n curve o f a inethanolic s o l u t i o n o f natarnycin ( t a k e n from r e f . 7 ) .
NATAMYCIN
535
3.9.
I o n i z a t i o n Constants
By t i t r a t i o n o f riatamycin i n 50% aqueous rnethoxyethanol , apparent pKa values o f 8,35 and 4,6 were o b t a i n e d ( 3 3 ) , correspondi ny w i t h dn isoel e c t r i c p o i n t o f 6,5 [a1 so r e p o r t e d by Kaab ( 5 0 ) l . The pH o f a 1%aqueous suspension i n water i s a c c o r d i n g l y norrnally between 5,U and I ,5.
3 .lo. 3.10.1.
Thermal Behavi our r k l t i n g Kange
4s most o t h e r amphoteric pol yeties natamyci n has no sharp m e l t i n g point,. It darkens a t about 200C and " m e l t s " under v i g o r o u s decomposition a t 280 - 3UU'C. Natamycin, when heated on a L e i t z Plodcl 350 microscope h o t stage a t a r a t e o f S"C/minute and viewed i n p o l a r i z e d l i g h t , showed a t r a n s i t i o n a t 195 - 200C t o an i s o t r o p i c inodi f i c a t i on o r d e g r a d a t i o n product ( 3 3 )
3.10.2.
D i f f e r e n t i a l Thermal A n a l y s i s (DTH)
A DTA c u r v e o f natamycin t r i h y d r a t e was run on a W e t t l e r TA 2000 d i f f e r e n t i a1 thermoanalyzer between roomtemperature and 230C (51). The o p e r a t i n g c o n d i t i o n s were as f o l l o w s : 2,32 rag sample weight sampl e atmosphere n i t rogcn heating r a t e 5"C/mi n. range 100 UV f s d sensitivity 14,5 uV/mcal / sec
The thermogram, presented i n F i gure 13, shows t h r e e subsequent broad endotherms a t r e s p e c t i v e l y 60", 77" and 1 1 7 " C , which r e f l e c t t h e l o s s o f 3 moles o f water dnd one sharp exotherin a t 196C. The l a t t e r corresponds t o t h e phase t r a t i s i t i o n observed on h e a t i n g a sample on a microscope h o t stage i n p o l a r i z e d l i g h t ( S e c t i o n 3.10.1). I n a p a r a l l e l r u n t h e weight l o s s o f 5 rlig o f sample was deterriiined by weighing t h e DTH cup on a microbalance a f t e r a warming p e r i o d ( o p e r a t i n g c o n d i t i o n s as above). From 30 t o lU0"C t h e mean weight l o s s was 5,68X, and 1,93'/, from 1OU t o 130C. The sum, (7,6 + 0,lX) i s v e r y c l o s e t o t h e t h e o r e t i c a l amount o f water (7,50%). 3.10.3. Water Content
Natamycin, c r y s t a l 1 i z e d from aqueous sol vents, c o n t a i n s approximately 7,5% o f water as found by K a r l F i s c h e r t i t r a t i o n and by l o s s on d r y i n g ( o r g a n i c s o l v e n t s a r e n o t e x p e l l e d ) ( 3 3 ) .
END0
AT
EX0
50
~ 1
'
'
100
150
20 0
"C
Figure 13. DTA curve o f nataniyci n t r i h y d r a t e (51)
NATAMYCIN
537
Extremely f a r d r i e d natamycin ( d r i e d i n vacuo t o c o n s t a n t weight over phosphorus p e n t o x i de a t roointemperature), s t o r e d i n t h e dark i n a i r shows a weight i n c r e a s e o f 7,424 w i t h i n 4 hours, a f t e r t h a t t i m e t h e weight remains constant, see F i g u r e 14 (33,49). I t i s remarkable t h a t o n l y t h e hydrated form i s c h e i n i c a l l y s t a b l e i n c o n t r a s t t o t h e dehydrated substance (see S e c t i o n 5 ) .
$ 6 c .f
t
[51 .-
o J 4 3
0
Le
a J v) m
L
c ./
I
A
I I
20 h
F i g u r e 14. A b s o r p t i o n o f water by dehydrated natamycin 3.11. Counter-current D i s t r i b u t i o n
n-amylalcohol-isoamylalcohol-phosphate b u f f e r pH 6 (12:17:29)
i s 1,38 a f t e r 190 t r a n s f e r s ( 5 2 ) .
The p a r t i t i o n c o e f f i c i e n t o f natamycin i n t h e system
538
HARRY BRI h
3.12.
Solubility
Natamycin i s p r a c t i c a l l y i n s o l u b l e i n non-polar s o l v e n t s . The s o l u b i l i t y i n c e r t a i n p o l a r s o l v e n t s can be g r e a t l y enhanced b y a d d i n g w a t e r ( t o f o r i n s t a n c e acetone, l o w e r a l c o h o l s, d i m e t h y l s u l p h o x i d e ) o r compl e x i n y a g e n t s 1 ik e c a l c i u m c h l o r i d e ( t o methanol, n e t h o x y e t h a n o l ) o r t h i o c y a n a t e s ( t o f o r i n s t a n c e a c e t o n e ) . The e f f e c t o f w a t e r on t h e s o l u b i l i t y o f natamycin i n d i m e t h y l s u l p h o x i d e i s i l l u s t r a t e d i n F i g u r e 15. A t w a t e r c o n t e n t s l o w e r t h a n 10-15% a l e s s s o l u b l e n a t a m y c i n - d i i n e t h y l s u l p h o x i d e s o l v a t e ( 1 t o Z m o l e s ) is foriiied w h i c h e x p l a i n s t h e s h a r p peak a t 15% w a t e r o f t h e s o l u b i 1 it y c u r v e . A l t h o u g h n a t a m y c i n i s s o l u b l e i n a l k a l i n e and a c i d i c rnedia t h e compound i s r a p i d l y decomposed i n such s o l v e n t s . The s o l u b i l i t y d a t a , p r e s e n t e d i n T a b l e 8, show f a i r l y l a r g e d i s c r e p d n c i es between d i f f e r e n t s o u r c e s . Presumably t h i s is p a r t l y caused by t h e f o r r n a t i o n o f s o l v a t e s : f r o m s a t u r a t e d s o l u t i o n s i n s o l v e n t s l i k e methanol, dioxane o r methoxyethanol n a t a m y c i n more o r l e s s r a p i d l y c r y s t a l l i s e s on s t a n d i n g as a l e s s - s o l u b l e s o l v a t e . F o r i n s t a n c e i n methanol up t o 15 my o f n a t a m y c i n may d i s s o l v e p e r m l . However, a f t e r t h e spontaneous c r y s t a l l i z a t i o n of t h e s o l v a t e o n l y 3,3 mg p e r in1 reiriains i n s o l u t i o n . F u r t h e r t h e s o l u b i l i t y o f natamycin i s l a r g e l y dependent on i t s p u r i t y . K e c e n t b a t c h e s a r e l e s s s o l u b l e i n a v a r i e t y o f s o l v e n t s as cornpared w i t h b a t c h e s o f ZU y e a r s ago w h i c h c o n t a i n e d a much h i g h e r p e r c e n t a g e o f i i n p u r i t i e s ( 3 3 ) . A n o t h e r example o f t h e i n f l u e n c e o f s o l v a t e f o r m a t i o n on t h e s o l u b i l i t y i s g i v e n i n F i g u r e 16 w h i c h d e m o n s t r a t e s t h e e f f e c t o f an i n c r e a s i ng c o n t e n t o f c h l o r o f o r m on t h e s o l u b i 1 it y o f n a t a m y c i n i n methanol. A t c o n c e n t r a t i o n s o f l e s s t h a n 50% o f c h l o r o f o r n i d c r y s t a l 1 in e n a t a m y c i n-methanol s o l v a t e i s r a p i d l y f o r m e d w h i c h s t r o n g l y d e p r e s s e s t h e s o l u b i l i t y o f natainyci t i . A t c o n c e n t r a t i o n s o f 50% o f c h l o r o f o r m o r more no s o l v a t e i s foriaed, n o t even upon s e e d i n g w i t h t h e methanol s o l v a t e a t -2OOC. A t t h e h i g h e s t c o n c e n t r a t i o n s o f c h l oroforrn t h e very s l i y h t s o l u b i l i t y o f natamycin i n t h i s s o l v e n t predominates. O t h e r e f f e c t s o f c h l o r o f o r m , i .e. upon hydroyen-bonding, may be involved. The s o l u b i l i t y o f n a t a m y c i n i n d i m e t h y l f o r m a m i d e i s d i f f i c u l t t o estimate. A t roorntenperature natamycin d i s s o l v e s very s l o w l y , a f t e r one n i g h t s t i r r i n y 170 mg p e r m l was d i s s o l v e d , t h e s u b s t a n c e however was p a r t l y decornposed. Upon h e a t i ny t o 60C n a t a m y c i n d i s s o l v e s r a p i d l y i n d i r n e t h y l f o r f i i a m i d e t o o b t a i n a 25 w/v;'; s o l u t i o n w i t h o u t s i g n i f i c a n t d e c o m p o s i t i o n . I n t h i s s o l u t i o n however n a t a m y c i n i s n o t s t a b l e e i t h e r ( 3 3 ) .
NATAMYCIN
539
-- 4
.e2
A
x c .-
0
0
20
40
V/V o /'
60
80
100
OMS0
F i g u r e 15. S o l u b i l i t y o f natarnycin i n dirnethyl s u l f o x i d e - w a t e r a t 20C (33).
80
E m 60
E
c .c ..-
n
I 3
!A
x 40
20
0 0
20
40
60
80
100
v/v ' / ' chloroform

F i gure 16. Sol ubi 1 i t y d f natamyci n i n inethanol -
chloroform a t A 0 C ( 3 3 ) .
540
HARRY BRIK
Table 8 S o l u b i l i t y of natamycin i n mgjml
I solvent 1 water
~~
ref .53*
0,39 9,71
r e f .33**
0,03 3Y3
60
others
0,05-0,1 (38,54 2 ( 3 8 ) ; 15 (5U 15 (38)
1 methanol
, methanol
+ 2% CaC12*** methanol/chloroform ethanol e t ha no 1/water ( 4 :1) n-butanol n-butanol s a t d . with water acetone acetone/water ( 4 : l ) acetone + 2% KCNS ethyl acetate chloroform e t h y l e n e glycol propyl ene glycol -1,2 y 1yc e rol f o rmam i d e dimethyl formami de dimethyl sulphoxide met hoxye t h a no1 methoxyethanol t t & CaC12*** ri-methyl pyrrol idone-2 g l a c i a l a c e t i c acid dioxane pyri d i ne d i e t h y l ether
;ee text
0,54 0,04 0,22
1 , 2 (50,54)
0,7
(50,54)
0,05
0,6
u,12 (50,54) 1,5 (50,54)
U,073
<
0,01 0,11
0,17 0,Ol 0,01
1Y5 14 (54); 20 (38 15 (50,54) 20 (38) 50 (38,50)
0,015
0,013 > 20
< <
> >
20
20
,ee text ;ec text 1Y 9

15
140 (38,54) 120 (38,5U) 185 (50,54)
250
0,21
>
20
0,0U3
*at L I O C , c a l c u l a t e d from evaporation r e s i d u e , corrected f o r sol vent bl ank. **mu1 t i pl c c r y s t a l 1i sed natainyci t i r e f e r e n c e standard; a t 20C a f t e r e q u i l i b r a t i o n with a s o l v a t e , i f any; f i l t r a t e analyzed by d i f f e r e n t i a1 spectrophotoinetry
+C+--C
37. h - . , - h . , A - - & - . ..... .r e r . JJ. iieAarryuraLe;
, C
iei.
.>o ariu m.
>O
...-A
L A .
,.-I,--.._
UIIK~IWWII.
NATAMYCIN
541
4. 4.1.
P roduc t i on
Discovery
I n 1955 S t r u y k e t a l . i s o l a t e d a new a n t i f u n g a l a n t i b i o t i c from a c u l t u r e o f Streptomyces n a t a l e n s i s nov. sp. (38). T h i s s t r a i n was i s o l a t e d from a s o i l sample which was taken near P i e t e r m a r i t z b u r g , a town i n t h e p r o v i n c e o f N a t a l , South A f r i c a . The t y p i c a l u l t r a v i o l e t spectrum o f t h e new a n t i m y c o t i c p o i n t e d t o a r e l a t i o n s h i p w i t h a l r e a d y known polyenes l i k e n y s t a t i n , t h e f i r s t i,iernber o f t h i s group which was discovered 5 years e a r l i e r .
I n 1959 Burns e t a1 ( 5 5 ) i s o l a t e d a compound from a c u l t u r e o f Streptonyces chattanoogensis, a s t r a i n from a s o i l sample of Chattanooga, Tennessee, which was c a l l e d t e n n e c e t i n . However, w i t h i n two y e a r s t h i s compound appeared t o be i d e n t i c a l w i t h natamycin ( 3 9 ) , so t h e name t e n n e c e t i n was del eted.
A nameless t e t r a e n e , d e s c r i b e d by Backus e t a l . i n 1959 ( 5 6 ) ,

i s most p r o b a b l y i d e n t i c a l w i t h natamycin. The substance was produced by Streptomyces g i 1vosporeus ATCC 13326. 4.2. Biosynthesis
The b i o s y n t h e s i s o f t h e C-25 b u t y l homologue o f natamycin, 1ucensomyci n, has been s t u d i e d u s i n g 1%-1 abel ed p r o p i onate and a c e t a t e (57). These p r e c u r s o r s are i n c o r p o r a t e d i n t o t h e aglycone. 1 4 b l a b e l e d natamycin c o u l d be produced i n t h e same way (58). The carbon s k e l e t o n o f mycosamine i s p r o b a b l y d e r i v e d d i r e c t l y from glucose (59). 4.3. Fermentation and I s o l a t i o n
Natamycin i s produced on an i n d u s t r i a l s c a l e by f e r m e n t a t i o n u s i n g Streptornyces n a t a l e n s i s ( 6 0 ) o r Streptomyces gilvosporeus (25). A s most o f t h e a n t i m y c o t i c i s bound t o t h e mycelium i t i s i s o l a t e d e i t h e r by whole b r o t h e x t r a c t i o n o r by e x t r a c t i o n o f t h e mycelium, u s i n g l o w e r a l c o h o l s (25,bO). The crude compound i s p r e c i p i t a t e d by pH r e g u l a t i o n o r by e v a p o r a t i ve c o n c e n t r a t i o n .
542
HARRY RRIK
5.
S t a b i 1 it y
Natamycin i s a s t a b l e compound p r o v i d e d t h e powder i s p r o t e c t e d from l i g h t arid m o i s t u r e . Only a few percent l o s s o f a c t i v i t y i s observed a f t e r several y e a r s storage a t roointemperature. T h i s i s t r u e f o r t h e t r i h y d r a t e , t h e anhydrous form however i s n o t s t a b l e . T h i s form, prepared by h e a t i n g t h e tri h y d r a t e i n vacuo a t roomtemperature over phosphorus pentoxide (see a l s o S e c t i o n 3.10.3), l o s e s 15% o f a c t i v i t y when s t o r e d f o r 48 hours a t roomtemperature i n a c l o s e d b o t t l e i n t h e dark (49). Natamycin w i l l w i t h s t a n d h e a t i n g a t up t o 1 2 i ) O C ; f o r no more t h a n one hour. However, any anhydrous natarnycin produced d u r i n g h e a t i n g i s u n s t a b l P. The methanol s o l v a t e ( S e c t i o n 3.1.1) i s an u n s t a b l e substance as we1 1
N e u t r a l aqueous natamyci n suspensions a r e n e a r l y as stab1 e as t h e d r y powder. A n e u t r a l aqueous suspension can be b o i l e d f o r a s h o r t t i m e b e f o r e a r e d u c t i o n i n potency occurs. Aqueous s o l u t i o n s a r e q u i t e s t a b l e a t pH values between 5 and Y i f s t o r e d i n t h e dark ( 5 4 ) . A t extreme pH values natamycin i s r a p i d l y i n a c t i v a t e d w i t h formation o f various kinds o f decomposition products ( F i g u r e 1 7 ) . A t a low pH t h e mycosamine m o i e t y i s s p l i t o f f . The r e s u l t i n g i n s t a b l e aglycone r e a c t s w i t h e i t h e r a second molecule o f aglycone o r w i t h a s t i l l i n t a c t molecule o f natamycin. I n b o t h cases diiners w i t h a t r i e n e r a t h e r t h a n a t e t r a e n e group a r e formed. A t t h e same t i m e t h e epoxy group i s h y d r o l y s e d t o a d i o l . H e a t i n g a t l o w pH f a v o u r s d e c a r b o x y l a t i o n o f t h e aglycone (61). A t h i g h pH values, r a p i d l y a t pH 12, t h e l a c t o n e i s s a p o n i f i e d w i t h f o r m a t i o n o f t h e m i c r o b i o l o g i c a l l y i n a c t i v e natamycoic a c i d (33). Treatment w i t h s t r o n g a l k a l i r e s u l t s i n f u r t h e r d i s r u p t i o n o f t h e molecule owing t o a s e r i e s o f r e t r o a l d o l r e a c t i o n s . Among t h e r e a c t i o n products t h e f o l l o w i n g compounds c o u l d be detected: 13-hydroxy-2,4,6,8,1O-tetradecapentaene-l-a1 (1,62), acetone (4), acetaldehyde (3,4) and ammonia (1). Natamycin i s decomposed by u l t r a v i o l e t r a d i a t i o n w i t h l o s s o f Thoma ( 6 5 ) observed t h a t t h e t e t r a e n e s t r u c t u r e (33,b3,64). natamycin decomposed f a s t e r i n aqueous s o l u t i o n a t pH 4 than a t pH 8 upon r a d i a t i o n w i t h a xenon lamp. V i s i b l e l i g h t does n o t i n a c t i v a t e natamycin unless t r a n s f e r o f photo-energy by e.9. r i b o f l a v i n takes p l a c e (66). Gamma r a d i a t i o n decomposes natamycin as He1 1 , i t can t h e r e f o r e n o t be used t o s t e r i l i z e t h e substance.
NATAMYCIN
543
OH
0
HO
I HO NHZ mycosamine
I
Me
OH
: aponatarnycin
IR = R 1 )
11 : natarnycinolidediol dimer (aglycone dirner) (R E O H )
OH
natamycin
\1
H+
pH >1/<40C: mainly I ~ H ~ I / ~ Z O * mainly C: 11
OH-
I 1 Ht>12-decarboxy-analogue
HO = M e
OH
acetone,ethanal, NH,
R1
natamycoic a c i d
13-hydroxy-2,4,6,8,10-tetradecapentaen-l-al
N2
Figure 17. Decomposition o f natamyci n i n acid and al kal ine medium ( I and 11: t e n t a t i v e s t r u c t u r e )
544
HARRY BRIK
The i n a c t i v a t i o n by peroxides o r , e s p e c i a l l y a t h i g h e r temperatures, by oxygen can be prevented by a n t i o x i d a n t s l i k e c h l o r o p h y l l , a s c o r b i c a c i d (38,67,68) b u t y l a t e d hydroxyanisol e o r b u t y l ated h y d r o x y t o l uene ( 6 9 ) . O x i d a t i v e i n a c t i v a t i o n i s promoted by several metal i o n s , e s p e c i a l l y F e ( I I I ) , N i ( I 1 ) and C r ( I I 1 ) (33). This can be prevented by adding complexing agents l i k e EDTA o r polyphosphates (69). I n a c t i v a t i o n of natamycin by l i g h t , peroxides o r oxygen proceeds a t t h e f a s t e s t r a t e i n s o l u t i o n o r i n suspension, l e s s so i n t h e s o l i d form. O x i d a t i v e d e g r a d a t i o n u f natamycin p r o b a b l y l e a d s t o t h e f o r m a t i o n o f polymers o r coli1pounds formed by a d d i t i o n o f oxygen on t h e conjugated double bonds. The l a t t e r r e a c t i o n , which takes p l a c e a t one end o f t h e polyene chain, i s d e s c r i b e d f o r several polyenes. E i t h e r an epoxy-group ( f i1ip i n and 1agosi n, 70) o r a hydroperoxide ( n y s t a t i n , 71, o r l e v o r i n and mycoheptin, 72) i s formed. I n a c t i v a t i o n occurs a l s o i n t h e presence o f s u l p h i t e s o r sodi um formal dehyde sul phoxyl ate. 6. 6.1.
B i opharmaceuti cs
Pharmacokinetics
A b s o r p t i o n o f natamycin from t h e human i n t e s t i n e a f t e r o r a l a d m i n i s t r a t i o n o f doses from 125 t o 500 mg per day d u r i n g a p e r i o d o f 1 up t o 7 days has n o t been observed. The serum c o l l e c t e d d i d n o t show any a n t i f u n g a l a c t i v i t y (73). I n animals t h e same r e s u l t s have been obtained. With r a t s and mice o r a l a d m i n i s t r a t i o n o f natamycin o n l y reduced t h e y e a s t count i n t h e faeces (38,74). 6
.%. T o x i c i t y
Uatamycin has a very l o w o r a l t o x i c i t y . The o r a l ills0 i n t h e male r a t i s 2,73 g/kg, i n t h e male r a b b i t 1,4Z y/kg (75). The c h r o n i c t o x i c i t y of natamycin was s t u d i e d by t h e a d m i n i s t r a t i o n o f natamycin i n t h e food o f r a t s and dogs. Only m i n o r e f f e c t s such as a s l i g h t decrease i n t h e i n t a k e o f food and a s l i g h t i n h i b i t i o n o f growth, were observed when 1 mg p e r kg p e r day was f e d t o r a t s f o r two years. Dogs t o l e r a t e d a dose o f 0,25 mg per kg per day f o r more tharl two years, a dose o f 0,s mg per kg per day r e s u l t e d i n a s l i g h t decrease i n body weight when administered f o r two y e a r s (75). A c i d degradation p r o d u c t s ( 1 ike aponatamycin, t h e aglycone dimer and mycosami ne) and products o b t a i n e d by a1 k a l ine d e g r a d a t i o n o r UV r a d i a t i o n o f natamycin a r e even l e s s t o x i c t h a n t h e p a r e n t compound (76,77).
NATAMYCIN
545
6.3.
Other
No s e n s i t i z i n g e f f e c t has been observed a f t e r continuous exposure t o h i g h c o n c e n t r a t i o n s o f natamycin (78,79). This i s p o s s i b l y due t o t h e low a f f i n i t y o f natarnycin f o r p r o t e i n s . Kesistance t o natamycin i s not observed (80), cross r e s i s t a n c e between nataniyci n and o t h e r polyenes has not been r e p o r t e d (81,82). The haemolytic a c t i v i t y o f natamycin i s l e s s than t h a t o f t h e more l y o p h i l i c polyenes n y s t a t i n, amphoterici n B and 1 ucensomyci n (83). tkrrcoiiler (84) r e p o r t e d nausea, v o m i t i n g and d i a r r h o e a when natamycin was given o r a l l y t o a d u l t s i n doses exceeding 1000 mg p e r day.
7. 7 .l. Hnalysis Identification
T y p i c a l c o l o u r s are formed when concentrated m i n e r a l a c i d s a r e added t o natamycin. T h i s r e a c t i o n i s based O H , x o t o n a t i o n of t h e polyene chrornophore (85). I n t h i s manner natamycin may be i d e n t i f i e d among o t h e r polyenes (33). See Table 9.
A s o l u t i o n o f antimony t r i c h l o r i d e i n c h l o r o f o r m ( C a r r - P r i c e reagent) y i ves d i f f e r e n t c o l ours as we1 1 ijatarnyci n and lucensomycin g i v e d r e d c o l o u r , n y s t a t i n a r e d - v i o l e t c o l o u r , a l l t h r e e s h i f t i n g r a p i d l y t o dark-brown. F i l i p i n g i v e s a b l u e c o l o u r , t h e heptaenes l i s t e d i n Table Y c o l o u r yreen, s h i f t i n g t o bl ue-green ( 3 3 ) .
Natamycin, 1 i k e o t h e r polyenes, r e a c t s w i t h F o l i n - D e n i s r e a g e n t (molybdotungstophosphoric a c i d ) w i t h f o r m a t i on o f a b l u e c o l o u r . T h i s t e s t i s d e s c r i b e d i n several pharmacopoeias f o r n y s t a t i n (86,87,88,89). The reagent i s h i g h l y a s p e c i f i c however s i n c e i t r e a c t s w i t h a l l r e a d i l y o x i d i z a b l e conpounds.
A somewhat more s p e c i f i c t e s t i s t h e r e a c t i o n w i t h d e c o l o u r i z e d magenta ( S c h i f f r e a g e n t ) . Upon h e a t i n y w i t h several polyenes in c l u d i ng natamyci n a r e d c o l o u r i s produced. The r e a c t i o n i s based on t h e f o r m a t i o n o f aldehydes, t h e heptaenes l i s t e d i n Table 9 do n o t r e a c t . T h i s t e s t i s a l s o d e s c r i b e d f o r n y s t a t i n i n a number o f pharmacopoei as (86,87,88).
546
HARRY BRIK
Table 9 Colour r e a c t i o n o f polyenes* polyene natamyci n nystati n group tetraene tetraene conc. H C l :onc. brown brown (gray 1 brown (gray) browngreen v i ole t v i 01 e t (gray1 green (brown) green (brown) green (brown) green (brown) H2S04 red (brown) rcd-brown (brown) red-brown (brown) green (brown green) v i 01 e t brown ilue (violet) blue (bluegreen) blue (bluegreen) blue (bluegreen) blue (yreyish-blue)
amphotcrici n A tetraene 1ucensomycin filipin tetraene pentaene
brown v io le t brown v i 01e t brown viole t green brown v i 01e t brown blue b l ue blue blue
amphoteric n B heptaene candi c i d i n aromati c heptaene a roma t ic h e p t aenc aromatic hept aene aroinat ic
1e v o r i n
partricin
t r i chomyci n
*i n parentheses: c o l o u r a f t e r a few minutes

Natamycin may be i d e n t i f i e d by means o f t h i n l a y e r chrornatoyraphy ( S e c t i o n 7.5.2), t h e d i f f e r e n t i a t i o n froiil o t h e r common polyenes i s n o t very c l ear, however. Natarnycin may be i d e n t i f i e d as a t e t r a e n e by r e c o r d i n g t h e u l t r a v i o l e t spectrum. B y t h i s means i t can a l s o be d i f f e r e n t i a t e d from t h e t e t r a e n e s n y s t a t i n and a m p h o t e r i c i n A by r e c o r d i n g down t o 215 nrn (90). Natamycin shows an a b s o r p t i o n a t 220 nm (en-one), n y s t a t i n and a m p h o t e r i c i n i-i however show an a b s o r p t i o n a t 230 nm ( t r a n s , t r a n s - d i e n c ) . The b e s t methods f o r i d e n t i f i c a t i o n of natamycin a r e IK and UV spectrophotometry combined with t h e c o l o u r r e a c t i o n w i t h s t r o n g acids. By t h i s means i t can be d i f f e r e n t i a t e d from a l l o t h e r common polycncs. For p r e p a r a t i ons c o n t a i n i ng s i y n i f ic a n t q u a n t i t i e s o f e x c i p i e n t s a combi n a t i on o f t h i n-1 ayer chromatography and UV spectrophotometry i s t o be recommended.
NATAMYCIN
547
7.2.
Spectrophotometric a n a l y s i s
U l t r a v i o l e t spectrophotonietry, u s i n g methanol w i t h O , l % o f a c e t i c a c i d as t h e s o l v e n t , may be used f o r t h e assay o f natamycin and i t s dosage forms. The method i s u s e f u l f o r r o u t i n e c o n t r o l b u t n o t f o r s t a b i l i t y s t u d i e s because o f t h e n o n c o r r e l a t i o n o f m i c r o b i o l o g i c a l a c t i v i t y and t e t r a e n e c o n t e n t upon degradation. The h i g h e s t degree o f c o r r e l a t i o n i s o b t a i n e d w i t h d i f f e r e n t i a l spectrophotometry (33) , a p r i n c i p l e which i s a l s o used f o r t h e assay o f n y s t a t i n ( 9 1 ) and t h e heptaenes rnycoheptin and l e v o r i n (92). T h i s method i s based on t h e measurement o f absorbance a t t h e main maximurn a t 303 nrn and d t t h e minima on e i t h e r side, i.e. a t 295 and 311 nm. Froin t h e s e Val ucs t h e base-1 ine a b s o r p t i o n
A303
A295
' A311
2
i s calculated. As seen i n Tables 10 and 11 t h e b a s e - l i n e lilethod g i v e s more r e 1 i a b l e r e s u l t s as compared w i t h t h e m i c r o b i 01o y i c a l assay t h a n t h e 'lone p o i n t " s p e c t r o p h o t o m e t r i c riiethod u s i n g o n l y absorbance measurement a t t h e peak a t 303 nm. I n b o t h examples a 5% natarnycin suspension was degraded and analyzed a t s p e c i f i c t i m e s (33). Table 10 shows t h e r e s u l t s o f d e g r a d a t i o n a t pH 1,5. Under t h i s c o n d i t i o n m a i n l y dirners w i t h t r i e n e a b s o r p t i o n though w i t h remarkable ( f 1 a n k ) a b s o r p t i o n a t 303 nm a r e formed. A small amount o f i n a c t i v e t e t r a e n e (aglycone) i s r e s p o n s i b l e f o r t h e b a s e - l i n e method y i e l d i n g t o o h i g h r e s u l t s . Table 11 shows t h e r e s u l t s o f d e g r a d a t i o n by long-wave u l t r a v i o l e t r a d i a t i o n . I n t h i s way no i n a c t i v e t e t r a e n e i s formed so t h e r e s u l t s o f t h e b a s e - l i n e method are n e a r l y equal t o those o f t h e m i c r o b i o l o g i c a l assay. When natamycin i s degraded i n a l k a l i n e medium a compound i s forrned which s t r o n g l y i n t e r f e r e s w i t h t h e base-1 i n e method. F o r instance, a 5% aqueous s o l u t i o n o f natamycin a t pH 12 was t o t a l l y i n a c t i v a t e d w i t h i n a few hours, s p e c t r o p h o t o r n e t r i c a l l y however, u s i n g t h e base-1 i n e riiethod, no decrease i n t e t r a e n e c o n t e n t c o u l d be d e t e c t e d (33). T h i s can be r e a d i l y e x p l a i n e d by t h e f o r m a t i on o f t h e m i c r o b i o l o g i c a l l y i n a c t i v e t e t r a e n e natamycoic a c i d , forrned by simple s a p o n i f i c a t i o n o f natamyci n (see S e c t i o n 5).
548
HARRY BRIK
Table 10 Degradation o f natamycin a t pH 1 , 5
100 81 59 39 23 21
100 74 50 31 14
100 75 46 14
2
(
5
Table 11
0,5
Degradation o f natamycin by UV l i g h t *
100 83 67 46 32 8
100
78
62
40 22 6
100 79 60 39 20 5
Natamycin shows a t r a n s i e n t b l u e c o l o u r i n f a i r l y s t r o n g h y d r o c h l o r i c a c i d , owing t o t h e f o r m a t i o n o f a carbonium i o n (85). T h i s p r i n c i p l e was used by Dryon (93) t o perform a c o l o r i m e t r i c deterrni n a t i on o f natamyci n. To f o u r volumes o f a m e t h a n o l i c s o l u t i o n o f natamycin c o n t a i n i n g 30 t o 190 ug p e r m l a r e added t e n volumes o f c o n c e n t r a t e d h y d r o c h l o r i c a c i d c o n t a i n i n g 20% o f ethanol under c o o l i n g w i t h ice. A f t e r 13 - 15 minutes t h e absorbance i s measured a t 635 nm. The b l u e c o l o u r does n o t obey B e e r ' s law. A number o f a c i d and a l k a l i n e d e g r a d a t i o n products o f natamycin does n o t i n t e r f e r e i n t h i s method ( 3 3 ) .
NATAMYCIN
549
Sol vent system n-butanol /water, saturated n-butanol/ethanol/water (5:1:4) n-propanol /water (7:3 ) t r i e t h y l ami ne/formamide/water (10:3:10), upper layer
Kf value
Keference
55
0,33
38 38
u ,33**
25
*not reported, b u t separation from three other tetraenes possible **re1 a t i ve t o chromi n
550
HARRY BRIK
900
800
si=L
A
.> 700 .-4U
-4-
73
0
U
c
u
sodium l a u r y l s u l p h a t e
60C
.-
ce .a ,
U
a
v)
50 0
40C
b
10
ml of t i t r a n t
Figure 18. Conductoinetri c t i t r a t i on o f natamyci n and sodi urn 1 auryl sul phate (each 0 , l mmol ) w i t h 0,02F1 c e t y l trirnethylarnmoni urn bromide.
NATAMYCIN
55 1
7.5.2
T h i n L a y e r Chromatography
T h i n l a y e r chromatography has been used i n q u a l i t a t i v e a n a l y s i s t o d i f f e r e n t i a t e t h e a n t i b i o t i c from o t h e r polyenes o r t o t e s t i t s p u r i t y . S e v e r a l systems a r e l i s t e d i n t h e T a b l e s 13, 14 and 15. I n t e s t i n g t h e s t a b i l i t y s o l v e n t systems 10 and 13 a r e u s e f u l t o e s t i r , i a t e mycosami ne i n n a t a m y c i n and i t s p r e p a r a t i o n s . N i n h y d r i n e i s used as t h e d e t e c t a n t . A f t e r w a r d s t h e same p l a t e can be s p r a y e d w i t h a u n i v e r s a l d e t e c t a n t l i k e s u l p h u r i c a c i d t o d e t e c t aglycone-1 i k e d e g r a d a t i o n p r o d u c t s (33,61). T a b l e 13 T h i n-1 a y e r chromatography systems f o r s t a t i o n a r y phase sol vent system (Tabl e 14) method of detection (Tabl e 15)
natamyci n "f Val ue ttef.
Silicaqel G ( N C ckS ( P H 8 ) S i 1 c a g e l G (Flerck) S i 1 cayel G (Merck) S i 1 c a g e l G (Merck) S i 1 cagel G (Merck) S i 1 c a g e l C (Merck) S i l i c a g e l G (Merck) S i 1 i c a g e l C (Flerck) S i l i c a g e l G (Merck) Silicagel G (Flerck) (pH 3 ) S i 1 i c a g e l 60 F 254 (Merck) S i 1 i c a y e l 6 0 F 254 ( Mer c k ) S i l i c a g e l GF (Analtech) Polygram S i l G f o i l (M and 1.1) Sephadex G-15
1 2 2 3 4 5
G
1 1 2,3
u ,34
0,34 u,57 u,4u 0,54 U,18 u,55 0,75 0,GO
0,5Y
Y5
Y5 96
93 93
YG 96 Y6
4 4
2,3
2,3 2,3
7
8
9
2,3
96
5
0,lO 7 ,8 78
Y ,10 11
97
G1
10
11 12 13
u,4
u ,u
0,45
45
45
33 98
14
0Y7 0,7*
*relative t o benzylpenicillin
552
HARRY BRIK
Table 14 Thi n-1 a y e r chromatography systems f o r natamyci n Sol vent systems
1. ethanol/ammonia/water (8:l:l) 2. n-butanol / a c e t i c a c i d / w a t e r ( 3 : 1: 1) 3. methanol/isopropanol/acetic a c i d (90:10:1) 4. m e t h a n o l j a c e t o n e l a c e t i c a c i d (8: 1:1) 5. ethanol /ammoni a / d i oxane/water (8 :1:1:1) 6. n - b u t a n o l / p y r i d i ne/water ( 3 : 2 : 1) 7. n - b u t a n o l / p y r i d i n e / a c e t i c a c i d / w a t e r (15:10:3:i2) 8. n - b u t a n o l / a c e t i c a c i d / w a t e r / d i oxane (6:2:2: 1 )
9. n - b u t a n o l / a c e t i c a c i d / w a t e r ( 2 : l : l ) 10. c h l oroforn/methanol / a c e t i c a c i d / w a t e r ( 6 : Z : Z : 1) 11. chloroform/methanol/~,05M b o r a t e b u f f e r pH 8 , 3 (2:2:l), lower l a y e r 12. n - b u t a n o l / a c e t i c a c i d / w a t e r (4:1:5), upper l a y e r 13. n - b u t a n o l / a c e t i c a c i d / w a t e r (4:1:2) 14. 0,025M phosphate b u f f e r pH 6,0 c o n t a i n i n g 0,5M NaCl
Table 15 Thi n-1 ayer chromatography systems f o r natamyci n Methods o f d e t e c t i o n
1. 10% p o t a s s i um permanganate/0 ,2% brornophenol b l ue 2. 5% potassium permanganate 3. c o n c e n t r a t e d phosphoric acid, 5 minutes a t 100C 4. 0,2% p-dimethyl ami nobenzaldehyde i n concentraked sul p h u r i c acid containing a trace o f f e r r i c chloride 5. 1%p-dimethylami nobenzaldehyde + 20% antimony t r i c h l o r i d e i n ethanol w i t h 20 V / V % c o n c e n t r a t e d hydroc ti1 o r i c a c i d 6. c o n c e n t r a t e d s u l p h u r i c a c i d , 10 riiinutes a t 105OC 7. i o d i n e vapour 8. c o n c e n t r a t e d s u l p h u r i c a c i d / y l a c i a l a c e t i c a c i d (1:l) 9. concent r a t e d s u l p h u r i c acid/methanol (1:2) 10. n i n h y d r i ne 11. b i oautoyraphy
NATAMY CIN
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7.5.3
High Pressure L i q u i d chromatography
HPLC has been used by Frede (99) f o r t h e i d e n t i f i c a t i o n o f natamycin i n cheese-extracts. The d e t e c t i o n l i m i t was 20 ny per i n j e c t i o n a t a d e t e c t i o n wavelength o f 303 nrn. A s HPLC i s much more s e l e c t i v e t h a n t h e UV spectrophotornetric method i t i s a u s e f u l method t o assay p a r t i a l l y degraded samples, pharmaceutical dosaye forms o r b i o l o g i c a l m a t e r i a1 Several systems a r e l i s t e d i n Table 16. A chromatograrn o f t h e USP r e f e r e n c e standard i s shown i n F i g u r e 19.
Table 16 Systems f o r HPLC o f natamyci n S t a t i o n a r y phase L i c h r o s o r b KP-8 25 cni (Merck) uBondapak C18 25 cm (Waters) VBondapak C18 25 cm (Waters) Eluent IleOH - H20 (65 : 35) MeOH-H20-HOAc (48 : 32 : 1) MeOH-H20-THF ( 4 4 : 47 : 2) c o n t a i n i n g 1 wlvX o f NH4OHc
I I
10 1
7.6.
E l ectrophoretic Analysis
Ochab (102) separated natamycin froin s e v e r a l o t h e r p o l yenes by means o f e l e c t r o p h o r e s i s on Whatman no. 4 and no. 34 paper, m o b i l i t i e s i n f o u r d i f f e r e n t e l e c t r o l y t e s a r e reported.
7.7.
Pol a r o g r a p h i c A n a l y s i s
Dornberger ( 103) determi ned natamyci n and i t s C25 b u t y l homo1ogue 1ucensomyci n p o l arographi c a l l y a t t h e d r o p p i n g mercury e l e c t r o d e i n 0,ZM hosphate b u f f e r pH 7 i n a c o n c e n t r a t i o n range o f 1 0 - t t o 10-5M. The epoxy group o f natamycin i s reduced a t a half-wave p o t e n t i a l o f -O,85 V versus t h e normal calomel e l e c t r o d e . Lucensomycin g i v e s a wave a t -1,O V. D e r i v a t i v e s o r polyenes which l a c k an epoxy group r e a c t negatively.
554
HARRY BRIK
Figure 19. Hi gh-pressure 1i quid chromatogram of 4 ug of natamycin USP r e f e r e n c e standard (104)

I nstrurnent
: Spectra Physics SP 8000
Col umn Mobile phase
Detection Sensi t i v i t y Ketention time
chromatograph w i t h Schoeffel SF770 d e t e c t o r : WBondapak Cl8 3,9 x 300 mm : m e t h a n o l - d i s t i l l e d watert e t r a hyd rof u ran (440 : 470 : 20) c o n t a i n i ng 1%o f ammoni urn a c e t a t e . Kate o f flow: 2 ml/minute. 1 t r a v i o l e t absorption a t : U
303 nm : 0,04 HUFS : 13,5 minutes
NATAMYCIN
555
7.8.
Elemental H n a l y s i s
The presence o f ash, o r g a n i c i m p u r i t i e s ( e s p e c i a l l y i n e a r l i e r l o t s ) , s o l v e n t o f c r y s t a l l i z a t i o n ( i .e. 1,iethanol , w a t e r ) may b r i n y about s u b s t a n t i a l l a c k o f agreement between 01 d e r experimental data and t h e r e c e n t t h e o r e t i c a l composition. P o s s i b l y r a t h e r because o f t h e presence o f t h e above f o r e i g n c o n s t i t u e n t s t h e r e was sometimes a f a i r l y good agreement between experimental and -meanwhi 1e obsoletet h e o r e t i c a l d a t a ( 1 , 6 2 ) . Kecent experimental d a t a ( 4 9 ) , o b t a i ned w i t h natamyci n t r i h y d r a t e r e f e r e n c e standards , conform v e r y w e l l w i t h modern t h e o r e t i c a l data. Table 17 E l emental a n a l y s i s o f natamyci n t h e o r e t i c a l composi t i on i n %
c
natamycin anhydrous riatamyci n tri h y d r a t e
Ii
59,54 55,06
found i n L;
c
58,53
H
7,32
1.1
ref.
remarks ibiean v a l u e o f seventeen analyses i n one sample (1958) mean v a l u e o f t h e a n a l y s i s o f seven r e c r y s t a l 1ized sampl cs ( 1964) mean v a l u e o f t h e analysis of three s p e c i a l l y prepared r e f e r e n c e standards
2,12
57,11
7,33
2,08
62
55,ll
7,41
1,99
34,98
49
(1973-1976)
556
HARRY BRIK
7.9.
Microbiological Analysis
Natamycin i s assayed m i c r o b i o l o g i c a l l y w i t h Saccharomyces c e r e v i s i a e RTCC 9763 as t h e t e s t organism u s i n g t h e agar d i f f u s i o n method. The assay i s recommended f o r t h e d e t e r m i n a t i o n o f natamycin i n s o l u t i o n s o r e x t r a c t s o f t h e substance, i t s dosage forms o r i n b i o l o g i c a l m a t e r i a l . The s e n s i t i v i t y o f t h e agar d i f f u s i o n rnethod i s approxirnately 0,5 uy per m l o f s o l u t i o n (105). An i n t e r e s t i ng a1 t e r n a t i ve f o r t h e bioassay o f natamyci n i s based on measurement o f t h e decrease i n heat o u t p u t r a t e w i t h t i m e o f t h e r e s p i r a t i o n o f Saccharomyces c e r e v i s i a e (106). The determi n a t i on, which proceeds by f l ow m i c r o c a l oriinetry, was c a r r i e d o u t i n a c o n c e n t r a t i o n range of 1 t o 7 x 10-6PI.
8.
Acknowledgment
The a u t h o r thanks D r J . de F l i n e s , D r H.J. Kooreman, D r R.P. Morgenstern, D r 0.A.Smink and Ir J.A. van d e r S t r a a t e n f o r r e v i e w i n g t h e manuscript, D r s G.J.B. C o r t s and D r s C. van d e r V l i e s f o r t h e i r v a l u a b l e suggestions f o r improvements, t h e many c o n t r i b u t o r s c i t e d as "personal communication" and I r J.C. Monshouwer f o r h i s t e c h n i c a l a s s i s t a n c e i n p r e p a r i n g t h e manuscri p t
NATAMYCIN
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9.
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L i t e r a t u r e surveyed through February 1981.
OXYTOCIN
Friedrich Nachtmann, Kurt Krummen, Friedrich M a d , and Erich Riemer
1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Conformation 1.6 Appearance, Colour, Odour 1.7 Biological Activity 2. Physical Properties 2.1 Infrared Spectrum 2.2 Ultraviolet Absorption 2.3 Circular Dichroism 2.4 Raman Spectra 2.5 Proton NMR 2.6 W-NMR 2.7 Solubility 2.8 Optical Rotation 2.9 Isoelectric Point 3. Production 3.1 Extraction from Gland Material 3.2 Chemical Synthesis 4. Stability 5. Metabolism 6. Analysis 6.1 Identity Tests 6.2 Quantitative Physicochemical Methods 6.3 Biological methods 6.4 Determination in Biological Material 6.5 Determination in Dosage Forms 7. References 564 564 564 565 565 565 567 567 567 567 568 568 572 573 573 573 576 576 576 576 577 578 58 1 582 582 584 590 592 595 596
564
FRIEDRICH NACHTMANN et al.
1.
Description
Oxytocin i s t h e c y c l i c octapeptide') hormone released by t h e p o s t e r i o r p i t u i t a r y and having uterotonic and galactagenic activity i n mmmls and h y p t e n s i v e a c t i v i t y i n birds.
Its 20-memberd ring i s composed of f i v e amino acids cystine, tyrosine, isoleucine, glutamine and asparagine -, and t h e s i d e chain contains a f u r t h e r 3 amino acids - proline, leucine and glycinamide. A l l the o p t i c a l l y active amino acids belong t o t h e L-series.
The s t r u c t u r e of oxytocin w a s elucidated by du Vigneaud e t al., and indepndently by Tuppy i n 1953 ( 1 , 2 ) . The structure w a s confirmed by du Vigneaud e t al . by synthesis shortly afterwards ( 3 ) .
1.1 Nomenclature
1 . 1 1 Chemical names
L-Cysteinyl-L-tyrosyl-L-isoleucyl-L-glutaminylL-asparqinyl-L-cysteinyl-L-prolyl-L-leucyl-glycinamide c y c l i c ( 1 6) disulphide L-Htni-cystinyl-L-tyrosy~-L-isoleucy~-L-g~utaminylL-asparaginyl-L-hemi-cystinyl-L-prolyl-L-leucylg lyc inamide

1.12
Generic name oxytocin [50-56-61
1.13
Brand names
The following brand names are listed i n t h e Merck Index ( 4 ) : Alpha-hypphamine; Ocytocin; Endop i t u i t r i n a ; Pitocin; Syntocinon; Nobitocin S; Orasthin; Oxystin; Partocon; Synpitan; Piton+; U teracon
1.2
Formula
1.21
Amino acid sequence 1

\
6
J
Cys-~-Ile<ln-Asn-Cys-Pro-Leu-G1y-NH2
+) Oxytocin is
also known as a nonapeptide i n the l i t e r a t u r e . Instead of 1 cystine 2 cysteines can be used f o r the characterization of the amino acid sequence (see 1.1).
OXYTOCIN
565
1.22
Structural formula
OH I
y 2
CH2
CH-CH3
S-CH2-CH-NH-CO-YH-NH-CO-CH-NH I
co
I
CH2 I CO-NH2
CH2 I CH2 CO-NH2

I
CH2 CH2
iH2
/ \
CH-CO-NH-CH-CO-NH-CH2-CO-NH2
(72
1.23
CH-CH3 I CH3 Molecular formula C43H66N12012S2
1.3
Molecular wight
1,007.23
1.4
1.5
Elemental canposition
C 51.28%
H 6.61%
M 16.69%
0 19.06%
S 6.37%
Conformation
Urry and Walter =re the first to propose a conformation for oxytocin in solution (5).
A revised version w a s pblished by Gross and Meienhofer (6) according to thich the ring has an antiparallel pleated sheet conformation with intramolecular hydrogen bonds,and the
566
FRIEDRICH NACNTMANN ~t ul.
s i d e chain i s bent back t o t h e ring. Figure 1.1 i s a diagram of the proposed configuration.
Figure 1.1
Proposed conformations of oxytocin i n solution as published by Gross and Meienhofer ( 6 ) .

A: i n dimethylsulphoxide
B: a t receptor sites
OXYTOCIN
567
1.6
Appearance, colour, d o u r
The f r e e base has not been obtained i n c r y s t a l l i n e form. By freeze-drying solutions of oxytocin a c i d i f i e d w i t h acetic acid t h e acetate i s obtained, a white f l u f f y powder w i t h a f a i n t odour of a c e t i c acid. 1.7 Biological a c t i v i t y
Oxytocin i s d i f f i c u l t t o prepare i n a pure form owing t o the canplex synthesis. Different biological a c t i v i t i e s have been reported i n the l i t e r a t u r e f o r allegedly pure oxytocin, thus suggesting t h a t the preparations i n question differed i n purity. Chan and du Vigneaud reported an a c t i v i t y of 507 f 23 units/mg ( 7 ) . Maxfield and Scheraga worked with oxytocin with an a c t i v i t y of 495 f 25 units/mg ( 8 ) . A similar a c t i v i t y , i.e. approximately 500 units/mg, was reported by Glickson e t a1 (9) and by C e r l e t t i and B e r d e (10) , while Deslauriers e t a1 described a compound having an a c t i v i t y of 510 f 23 u n i t s / q (11).Boissonnas and Huguenin reported an a c t i v i t y of 450 30 units/mg (12), and Photaki an a c t i v i t y of approximately 400 units/mg (13). Bockaert e t a l . described a 3H-oxytocin w i t h an a c t i v i t y of 440 units/mg ( 1 4 ) . The National I n s t i t u t e of Standards and Control (London, UK) indicates an a c t i v i t y of 595 units/mg f o r the Fourth International oxytocin Standard, a synthetically prepared and specially purified product.
. .
Oxytocin acetate w a s p l r i f i e d by preparative column+j3xomatcgraphy on silica gel and the e l u a t e w a s freeze-dried .The product so obtained had a biological a c t i v i t y determined by the r a t uterus method (15) of 591 f 23 units/mg. I f allowance is made f o r nonpeptide impurities (4.3% water, 8.7% acetic acid and 0.5% sodium), t h e a c t i v i t y is 684 f 27 units/mg pept i d e . The compound w a s sham by thin-layer chromatography (stationary phase: silica gel; solvent systan: chloroformmethanol 7:3 + 5% 0.2 N acetic acid) t o be hanogeneous, while HPLC (see section 6.27) shmed it t o contain less than 1% of detectable and presumably peptide byproducts.
2.
Physical properties
2.1.
Infrared spectrum
und described i n section The Infrared spectrum of t h e 1.7 was recorded f r m 4000 t o 600 ?using a Beckman Acculab
+)
W e are grateful t o Mr. H. Bossert, Sandoz Ltd., f o r preparing t h e purified a c t i v e ccmpound.
568
FRIEDRICH NACHTMANN ct al.
8 apparatus. The spectrum of a K B r p e l l e t prepared with 1.5 g a c t i v e compound and 300 m g K B r i s shown i n Figure 2.1. Some regions of the spectrum d i f f e r from the catalogues of spectra (16). T h e differences are a t t r i b u t a b l e t o the varying degrees of purity of the canpounds employed.
2.2
Ultraviolet absorption
The UV spectrum (Figure 2.2) of the product described i n 1.7 was recorded f o r an aqueous solution with a concentration of 0.3 mg/rnl over the range 210 - 320 nm using a we-Unicam a x i m u mw a s a t 275 nm apparatus, Type SP 1700. The absorption m and there was a shoulder a t 281 m. After correcting f o r nonpeptide impurities which do not absorb UV l i g h t a t 275 nm, t h e value i s 14.9 and the m l a r extinction coefficient is E & : 1500. The specis i n good agreement with the l i t e r a t u r e (16), but the mlar extinction coefficient is higher than t h a t reported i n t h e l i t e r a t u r e , ming t o the greater purity of the compound (16). 2.3
Circular dichroism
CD spectra f o r oxytocin have been described i n t h e literature by various authors (15,181. Beychok and Breslow investigated the CD spectrum a t various p H values of an oxytccin preparation W i t h an a c t i v i t y of 500 units/mg (17). The spectra obtained are shown i n Figure 2.3. The c h a r a c t e r i s t i c range of wavelengths is 215-310 nm.
A t acid p H values oxytocin displays a negative band a t 280 nm, a positive shoulder a t 250 nm and a large positive band a t 225 nm. m e n the solution i s neutralised (pH = 7.5) t h e negative band declines i n i n t e n s i t y , the shoulder a t 250nm becanes a true ~ i m m and the band a t 225 nm undergoes a s h i f t t o somewhat longer wavelengths. A t p H 10.6 (ionisation of tyrosine) a s t r i k i n g change occurs: a positive plateau appears a t 280 - 290 nm and there is a large positive band a t 245 nm. The a p t i c a l a c t i v i t y a t 280 nm i s contributed by tyrosine and t h e disulphide b n d , wlnile t h e optical a c t i v i t y i n t h e region of 225 nm i s a t t r i b u t a b l e to t r a n s i t i o n of tyrosine t o the ionised state.
WAVELENGTH
2.5
IN MICRONS
6
I
3
I
3.5
I
4
I
4.5
I
5
1
5.5
I
65
1
7
1
75
1
8
,
1 0
11
t2
11
1 6
loo 90
l-7
-70
-60
- 5 0
-40
I
I I I
I
30
- M
1 0
,
800
! o
600
4000
3000
2000
1800
lboo
1400
1200
1000
WAVENUMBER CM-'
Figure 2.1
KBr pellet; spectrometer: Beckman Acculab 8
Infra-red spectrum of oxytocin, activity 591
23 units/mg.
Wavelength [nrn]
_+
Fiaure 2.2
UV spectrum of oxytocin, a c t i v i t y 591 Concentration, 0.3 mg/ml H 0 Spectrometer: Pye Unicam S8 1700
23 units/mg
570
2000
1500
2000
1500
1000
500
ri
1000 500
0
E -500 0 .- 1 d, % 2000 a ,
U
U
a , -
-500
l I l 1 1 I
I I
D -
2000
1500
1000
cn
500
0
E
....
220 240 260 280
-10.000
1500
1000
-500
-1000
300
I 220
I-1000
300
240
260
280
Figure 2.3
x (mp) x (mp) CD spectra of oxytocln (activity 500 unlts/mgl and oxytccln analogues at various pH values; published by Beychok and Breslow (17) A Oxytocin -pH 2, pH 7.5, ----- pH 11.5 B 2,Isoleucine-oxytocin pH 2, PH 7 C De~nOQxytOCin pH 2, pH 7.5, ----- pH 11.5 D Deamino-2-isoleucine-oxytocin, pH 2
.....
.....
.....
572
FRIEDRICH NACHTMANN ef al.
2.4
Raman spectra
Investigations of the Raman spectra of oxytocin have been described i n t h e literature (8,19). Raman spectra are shown i n a s the 514.5 nm Figures 2.4 and 2.5. The incident radiation w l i n e of an argon ion laser (Spectrophysics, Model SP-1641, and the spectra were recorded with a Spex Ramalog 5.
OXYTOCIN
AMIDE I
CH,
AMIDE 111
Tyr
8%
SOLID
I I.. I I LJ U L 1600 I400 1200 1000 800 600
Figure 2.4 Laser-Raman spectrum of oxytocin i n the s o l i d state Incident l i g h t : 150 mW, r e d u t i o n : 5 integration t i m e 2 s Scan rate: 6 cm-'/min; published by Tu e t al. (13).
1600
I 4 0 0
1200
1000
800
600
F i v e 2.5 Laser Raman s p c t r u m of oxytocin i n aqueous solution and i n DqO L Incident l i g h t : 500 mW, other conditions as i n Figure 2.4; published by Tu e t a1 (19)
OXYTOCIN
573
2.5
Proton NMR
The spectrum of t h e ccmpound described i n 1.7 w a s recorded a t 360 PlRz w i t h a Bruker WH-360 spectraneter (Figure 2.6)'). 5 mg oxytocin was dissolved i n 400 p l d DMSO and one drop of mixture of C E 1 3 and lBlS w a s added to I &solution. This accounts for the small signal for CHC13 a t 8.31 p p . 1F6, 6=0 p p , w a s used as t h e i n t e r n a l standard. The spectrum i s i n good agreement with t h e data i n the literature (9,20). The IH-NMR spectrum of oxytocin has k e n discussed i n detail by Glickson e t al. ( 9 ) . A l l the amino acids were unoquivocally assigned to the speeral features. 2.6
13C-NMR
The spectrum (Figure 2.7) was recorded a t 90.5 M H z with a Bruker WH-360 spectraneter.') 100 mg of the ccrnpound described i n 1.7 w a s dissolved i n 2.5 ml D20 and the pD w a s adjusted t o 3.6 w i t h CH COOH. Diaxane, 8 = 67.8 p p , w a s used as the i n t e r n a l standard. The spectrum i s i n very good agreement with spectra published i n t h e l i t e r a t u r e . For t h e assignment of t h e s i g n a l s and i n t e r p r e t a t i o n of spectrum, t h e reader is referred t o papers by a number of authors (11,21-25). A l l t h e amino acids were unequivocally assigned t o the spectral features. 2.7 Solubility
The s o l u b i l i t y of the ccmpound described i n 1.7 (freezedried oxytocin as t h e acetate) w a s determined i n three solvents. T h e s a t u r a t e d s o l u t i o n s were assayed by HPUJ (see 6.27), and t h e r e s u l t s are shown i n T a b l e 2.1.
T a b l e 2.1
Solubility of oxytocin (as the acetate)
Solvent
Methanol
Unit s / m l
86 400
')
W e are g r a t e f u l t o M r . M. L o o s l i , Sandoz L t d . , recording the spectrum.
for
Figure 2.6
'H-NMR-Spectrum of oxytccin, activity 591 t 23 units/mg, in d6-DMS0 Apparatus: Bruker WH-360
L
L
180 170 160 150 140
70
--r----'-------
60
Figure 2.7
13C-NMR-spectm
.-
50
l ' ' -
40
r-v-
-------f
* c
r--'
, . r 7 , 7 T
30
of oxytocin, a c t i v i t y 531
23 units/mg, D 0 ($
2
IL
ijo
120
20
77-1
b
T -
7 - 7 - -
10
= 3.6)
Apparatus: Bruker '41-360
576
2.8
Optical r o t a t i o n
Optical r o t a t i o n values for oxytccin f r a n t h e l i t e r a t u r e are given i n T a b l e 2.2. Table 2.2
@tical r o t a t i o n of oxytocin
Value -26.1 -26.2' -24.0'
f
Conditions
Reference 26 26
l,Oo
+)
c = 0,53; water c = 0.53; water c = 0.5; 1 N acetic acid c = 0.51;l N acetic acid
13
-23.1'
27
-23'
28
+) n a t u r a l oxytocin
2.9
Isoelectric p o i n t
Oxytocin i s an amphoteric canpound. Accordingly, the isoelectric p i n t reported i n the l i t e r a t u r e is a t p H 7.7 (26,28, 2 9 ) , c o n s i s t e n t w i t h the presence i n the mlecule of a free amino group and a free phenol group.
3.
Production 3.1 Extraction f r a n gland material
Today the e x t r a c t i o n of cocytocin fran p o s t e r i o r p i t u i t a r y gland mterial is of l i t t l e p r a c t i c a l importance and l a r g e l y of h i s t o r i c a l i n t e r e s t . The first experiments w i t h hypphyseal extracts were carried out by Oliver and Schafer and date back t o 1895 (30). A t that tine the extracts contained t h e pressor p r i n c i p l e vasopressin as w e l l as oxytocin. Preparations having both oxytocic and pressor activity were k n m as p i t u i t r i n and wre
OXYTOCIN
577
employed i n medicine.
Kamm (31) described a method of preparing hypphyseal extracts without pressor a c t i v i t y : the gland material is dried w i t h acetone and extracted with hot 0.25% acetic acid, and a crude product i s s a l t e d out f r a n t h e concentrated extracts w i t h m n i u m sulphate. This product i s extracted w i t h acetic acid, and t h e a c t i v e material consisting of equal parts of oxytocin and vasopressin is precipitated by addition of a mixture ether/petroleum ether. The two canponents are separated by exploiting their d i f f e r e n t solubilities i n organic solvents. Ether i s added t o an acetic acid solution of the active material t o p r e c i p i t a t e the vasopressin which i s f i l t e r e d o f f . Oxytocin i s abtained a s a s o l i d substance w i t h a rubbery consistency by addition of a l i t t l e water and petroleum ether. The pressor e f f e c t of the h o m n e so obtained i s only 3 - 4% of its oxytocic a c t i v i t y .
3.2
Chenical synthesis
The f i r s t synthesis by du Vigneaud e t a l . (3,32) was followed by f u r t h e r syntheses within the next years (33-36) d i f f e r i n g i n the protective groups used, the peptide linkage methods employed and t h e plan followed i n building up t h e mlecule. The protective groups which h m e been mainly employed i n the large-scale production of oxytocin are t o s y l , carbobenzoxy and t-butyloxycarbonyl residues f o r the amino group and t h e benzyl residue f o r the mercapto group. The methods which have proved of value f o r effecting peptide linkage are the mixed anhydrides method, the active esters method using p-nitrophenol, hydroxysuccinimide o r hydroxybenztriazole i n ccmbinat i o n w i t h dicyclohexylcarbdiimide, and the a i d e method. The o p n chain N- and S-protected nonapeptide which i s s t h e precursor of oxytocin may be constructed on the 6 + 3 or the (5 + 2) + 2 plan, these intermediates being synthesised s t e p by step. In the method employing tosyl and carbobenzoxy residues, t h e last stages consist i n cleavage of t h e protective groups w i t h sodium i n liquid ammonia followed by Oxidation with a i r t o close the ring, yielding oxytocin. As an example of a 3 + 6 plan, the synthesis according t o Boissonnas (33) i s outlined i n the following scheme.
578
FRIEDRICH NACHTMANN cf nl.
le-N3 Z-Cys(BZL) v I
I
H-Gln-Asnqs (BZL) -Pro-Leu-Gl y-NH2

I
Tripeptide
\i
1. Na/NH3 2.
'i
Hexapeptide
(BZL) -Pro-Leu-Gly-NH2
Z - C y s (BZL) -TJr-Ile-Gln-Asn-Cys
Nonapeptide
O2 ( a i r )
OXrrCCIN Stability Freeze-dried oxytocin acetate m y be kept i n a r e f r i g e r a t o r (2 6OC) without special precautions f o r several years w i t h no s i g n i f i c a n t loss of oxytocic a c t i v i t y (37). However, R e s s l e r and popence (28) mention that inactivation nay occur by disulphi.de interchange. The shelf l i f e of aqueous solutions is g r e a t l y dependent on the pH (Figure 4.1).
4.
20
f
%
5
C
15
E"
0
1 0
3
I -
_ _ _ - _ _ %-----lot 2
lot 1 P H
Figure 4 . 1 Loss of a c t i v i t y as a function of p H of oxytocin solutions containing 200 units/ml which had been boiled f o r 30 minutes.
OXYTOCIN
579
Figure 4 . 1 shows the residual content of oxytocin, assayed by

HPLC, i n solutions a t d i f f e r e n t p H values, which had been
boiled f o r 30 minutes (38). The apthum p H range i s 3 - 5. I n strongly acid solutions t h e peptide linkages undergo hydrolysis. Under neutral and w a k l y a l k a l i n e conditions, dimeric and polymeric compounds are formed, especially i n concentrated solutions, by conversion of t h e intrarnolecular disulphide bridges of two o r mre oxytocin m n m r s t o intermolecular bridges (disulphide interchange) ( 2 8 ) .
A sterile aqueous concentrate of oxytocin a t optimum pH, containing a preservative, w i l l keep f o r several years i n a refrigerator. Figure 4.2 shows the oxytocic a c t i v i t y (rat uterus) of axytocin concentrate which had been stored a t various tanperatures.
The concentrate contained 200 u n i t s oxytocin/ml i n a sterile solution a t p H 3.5 , containing trichlorobutanol (39) . Concentrates which had been kept i n t h e r e f r i g e r a t o r showed no loss of oxytocin a c t i v i t y . Concentrates kept a t 21OC showed a s l i g h t loss of a c t i v i t y (approx. 1.5% per year) , whereas concentrates kept a t 3OoC showed a marked loss of a c t i v i t y (approx. 10% per year). This indicates that t h e concentrate has a shelf l i f e of a t l e a s t 3 years even a t 2 l o C , but should not be exposed t o higher tenperatures, Similar r e s u l t s were abtained w i t h d i l u t e injections of oxytocin (37). In l i n e with these findings, sane pharmacopoeias') specify the following shelf lives f o r oxytccin injection:
Eur. P.:
73: Ccmpendium Medicamentom:
B.P.
2 years a t 2 5 O C a t least 3 years a t 2
10C
5 years a t 15OC
+) Abbreviations of t h e p h a n n a c o p i a s
as i n Martindale,
The Extra Pharmacopoeia, 27th Edition
580
FRIEDRICH NACHTMANN et 01
IlY *.*
1 0 0
80
I+
0
24
Months re
20-22%
e4 j
0
24
Months
48
110
'\
\
\
3OoC
Figure 4.2
Shelf life of axytocin concentrates
OXYTOCIN
581
5.
Metabolism
The natural concentration of oxytocin i n human plasma i s low. According t o Chard e t a l . the level i s less than 0.75 p u n i t s / m l i n healthy men and women ( 4 0 ) , while Leake and Weitzmann published values of 1.4 - 1.7 punits/ml ( 4 1 ) . During labour t h e r e i s a marked rise i n oxytocin level: Kumaresan e t a l . found a concentration of 82 punits/ml (42) and Leake and Weitzrrann published a value of 6 punits/ml (41). The h a l f - l i f e of oxytocin i n the blood i s only a few minutes (43,44), t h e reason being t h a t it i s rapidly degraded mainly by the liver and kidneys. Tbm main e n z p s y s t m s are respnsible f o r t h e inactivation of axytocin. The removal of glycinamide fran t h e C - t e r m i n a l end of the oxytocin mlecule has been demonstrated i n a l l animal species investigated and i n man, b u t t h e removal of Leu-Gly-NH2 i s confined t o a few species (44,45,46). F u r t h e m r e , numerous other organs display peptidase activity and are able t o s p l i t t h e oxytocin molecule. &tracts of r a t brain i n a c t i v a t e oxytocin by uns p e c i f i c peptidases (47) and similarly microsanal and soluble f r a c t i o n s of uterus and pancreas (48,49) and testis (50) inactivate oxytocin. Hcmogenised testis i n a c t i v a t e s oxytocin by reduction of the disulphide bridge and cleavage of the ensueing cysteine-tyrosine peptide sequence. Plasma-oxytocinase is formed i n t h e uterus of pregnant women and released i n t o t h e plasma. This aminopeptidase hydrol y s e s t h e hemicystinyl-tyrosine peptide bond t o y i e l d an acyclic ccmpound (51). The biological sites of inactivation of oxytocin according t o P l i s k a and Rudinger (52) are Shawn i n t h e following diagram: 6 5 4 b b b Cys-Tyr-11e-Gln-Asn-Cys-Pro-LeuGly-NH2 I I
,r
la
1 a: SS-SH transhydrogenase 1 b: aminopeptidase, s p l i t s t h e m l e c u l e between t h e h m i cystinyl-tyrosine residues

2 3 4
J
C I
: serum oxytocinase : tyrosinase : carboxamidopeptidase
f : endopeptidases
582
FRIEDRICH NACHTMANN e t a / .
6.
Analysis 6.1 Identity tests
6.11 General tests The mthds of detection described i n section 6.122 (thinlayer chrmatqraphy) which produce various colour reactions or fluorescence, m y be regarded as general, unspecific identif i c a t i o n tests f o r peptides. 6.12 Specific i d e n t i f i c a t i o n tests 6.121 Infrared spectrum See section 2.1
6.122 Thin-layer c h r m t c g r a p h y The Rf value and visualisation by various detection procedures i s a s p e c i f i c c r i t e r i o n of identity.
A nunher of solvent systans, mainly based on butanolacetic acid-water mixtures, are enployed t o develop silica gel thin-layer plates.
The solvent s y s t m kutanol-acetic acid-water (4:l:l p a r t s by volume) i s r e c m n d e d i n t h e l i t e r a t u r e f o r use with silica gel p l a t e s (53-55). According t o Hase and Walter (54) who used Pauly reagent o r chlorine-o-toluidine reagent t o detect t h e spts oxytocin has a n Rf value of 0.45. Another butanol-acetic acid-water systan ( 4 :1:5) (upper phase) i s stated by a number of authors (56-60) t o be a s u i t a b l e mobile phase. Khan and Sivanandaiah (58) , who used kutanol - 0 . 1 N acetic acid-pyridine (5:11:3, upper phase) and s i l i c a gel G p l a t e s , reported an Rf value of 0.68 f o r oxytocin.
A solvent system canprising butanol-acetic acidpyridine-water (15:3:10:12) f o r use With silica gel G p l a t e s has also been described (57,59,60). The last-mntioned author employed t h e ninhydrin o r chlorine-o-toluidine reaction t o visualise the spots.
Flouret e t 61. (61) barked With Eastrnan Chrcmagram s i l i c a gel thin-layer sheets, developing the chromatograms with methanol-chlorofonn-acetic acid-water (38:62:2:2) and using the Pauly and/or the chlorine-o-toluidine colour reaction t o detect t h e spots (Rf = 0.4).
A solvent s y s t m derived from t h e above and modified as follows may be used t o distinguish oxytocin f r m other nona-
OXYTOCIN
583
peptides such as [8-lysine]vasopressin, [ 8-ornithine]vasopressin, 2-phenylalanineI 8-lysinel vasopressin, [des- 1-amino] oxytocin ( 6 2 ) .
Mobile phase: methanol-chlorofonn-acetic acid-water 30:70:1:6
Thin-layer plate: Canmrcial silica gel p l a t e s 60 F254, MERcK, D m s t a d t , Federal Republic of Germany, thickness 0.25 m Suitable methcds t o d e t e c t t h e spots are the FolinCiocalteau's reagent (MERCK), fluorescarnine (63) and t h e usual ninhydrin and chlorine-o-toluidine reactions. Samejima e t a l. (64) have developed a highly sensitive fluorescence spray reagent (phenylacetaldehyde-ninhydrin)
N a k a m u r a and Pisano (65) have described TIC systems f o r separating several peptides, including mytocin, derivatised with fluorescamine. The canpound i s dissolved i n h f f e r solution, s p t t e d on Merck c m r c i a l silica gel 60 p l a t e s and derivatised a t t h e start l i n e by d e v e l o p n t with o r immersion i n an acetone-hexane solution of fluorescamine. The Rf values were 0.46 with chlorofom-isopropanol-water (2: 8: 1) and 0.89 with acetone-ethyl acetate-mthanol-water (3:2:1:1) a s the m b i l e phase.
6.123 Electrophoresis
Electrophoresis w a s carried out as described by Miihlemann e t 61. (53) i n a mist chamber apparatus (CAM?G, Muttenz, Switzerland) using cel lulose-coated p l a t e s with a thickness of 0 . 1 m (MACHERY-NAGEL, Diiren, Federal R e p b l i c of Gemany) and applying a potential gradient of approximately 23 V/cm f o r 45-60 minutes. Fyridine-acetic acid-water 1:10:90, p H 3.6 w a s enployed as the electrolyte. The m b i l i t y of mytocin (relative t o arginine) w a s reported t o be m * 0.29. Arg. Flouret e t 61. (61) described the thin-layer electrophoresis of oxytocin on Eastman chranagram s i l i c a gel t h i n layer sheets i n a Brinkmann-Desaga apparatus (400 V, 2 hours; H 5.6). using 0.1 N pyridine-acetic acid h f f e r p 6.124 Amino acid analysis
Detection of the amino acids a f t e r hydrolysis of the peptide may be regarded as an i n d i r e c t methcd of identification. Oxytocin i s normally hydrolysed w i t h 6 N hydrochloric acid a t 115OC f o r 1 6 hours i n a sealed tube. The amino acids formed are separated by ion exchange chranatography, assayed a f t e r dericolorimetrical l y (ninhydrin) or f luorimetrivatisation e c a l l y (Fluram , o-phthalaldehyde) and i d e n t i f i e d by canparing t h e retention times with those of a given amount of a test
584
mixture. This method m y be used t o determine t h e amino acid ratio and the peptide content (see section 6.25). 6.125 R a t uterus roethd
- see section 6.31 Under t h e conditions described i n t h e sections on assay methods i n various pharmacopoeias, a solution of mytocin induces cont r a c t i o n s i n uterine muscle.
6.2
m a n t i t a t i v e physico-chemical mthcds 6.21 Ultraviolet spectranetry
see section 2.2

F l u o r h e t r i c mthcds
6.22
see section 6.27 Colorimetric analysis
6.23
The peptide has keen assayed using t h e well-knawn colour reactions, e.g. t h e ninhydrin reaction (66) and t h e Folin-Lany reactions ( 6 7 ) . The rnethod of Ellmann (68) has been used t o determine t h e sulfhydryl content.
6.24
Determination of nitrogen (Kjehldahl)
The organic nitrogen of t h e peptide i s converted t o m n i u m sulphate by the Kjehldahl mthcd using concentrated sulphuric acid and a suitable catalyst. The solution is rendered a l k a l i n e and t h e m n i a i s steam d i s t i l l e d i n t o a receiver containing boric acid. The boric acid i s then titrated potentianetrically with hydrochloric acid and the nitrogen, o r t h e peptide content, calculated fran t h e result. 6.25 Amino acid analvsis 6.124
- see section
Amino acid analysis m y be used to determine t h e amino acid ratio as w e l l as t h e peptide content.
Analysis of mytocin reveals t h e presence of aspartic acid, glutamic acid, proline, glycine, isoleucine and leucine i n equimolecular proportions. Cystine and tyrosine must a l s o be detectable, but since they undergo p a r t i a l deccmposition during hydrolysis, the quantity of these two amino acids i s only about
OXYTOCIN
585
70% of theory.
Further amino a c i d s are n o t d e t e c t a b l e i n s y n t h e t i c oxytocin. Hcwever, the presence of f o r e i g n amino acids, such as arginine, l y s i n e , and phenylalanine, m y serve as a crit e r i o n of p u r i t y for oxytocin of natural o r i g i n (see s e c t i o n 6.34). The peptide content i s c a l c u l a t e d from t h e y i e l d of i n t a c t amino acids present.
6.26
Gel f i l t r a t i o n
Various authors have described gel f i l t r a t i o n nrethds using Sephadex 6 1 5 and G-25 (54,56,57,61). This method which separates o f f dimers and p l p r s is mainly employed as a means of p u r i f i c a t i o n .
6.27 High performance l i q u i d chromatography (HPLC)
Since t h e introduction of chanically mdified silica g e l s as the s t a t i o n a r y phase, H E W o n reversed phase C8 or C18 columns has cane t o the fore as the m t h d of choice for t h e assay of oxytocin. Krummn and F r e i estimated the oxytccin content of i n j e c t i o n s , tablets and oxytocin concentrate (69). Isocratic e l u t i o n and short wavelength UV d e t e c t i o n a t 200-220 nm were adequate for the purpose. Figure 6 . 1 shows a t y p i c a l chromatogram (70) Since t h e mbile phase (acetonitrile/phosp h a t e Ixlffer) r e a d i l y transmits UV l i g h t a t 200-220 nm, the lower l i m i t of d e t e c t i o n is approximately 30 ng (aplprox. 30 pmol) per i n j e c t i o n (69). The HPLC results correlate very w e l l w i t h those chtained by bioassay. Figure 6.2 illustrates the gocd agreement between HPIC v a l u e s and t h e results obt a i n e d i n t h e rat u t e r u s test for 38 d i f f e r e n t batches of oxytocin (70). The c o r r e l a t i o n c o e f f i c i e n t s and critical values are s h a m i n T a b l e 6.1.
T a b l e 6.1 Correlation c o e f f i c i e n t s and critical values for t h e
r e s u l t s i n Figure 6.2, p b l i s h e d by K r m n e t al. (70). Samples Liquid Solid

-~
I EEZ~E~ I
25
Correlation Coefficient 0.9962 0.9995 0.9969
Critic?) Value
0.618 0.801 0.513
Liquid + Solid
+)
For n-2 degrees of freedan a t t h e 0.1% level.
HPLC i s so highly selective t h a t it W i l l also separate canpounds closely releated i n s t r u c t u r e . Figure 6.3 shows by way of example the separation of oxytocin and 3 Stere0i-s having 1 or 2 amino acids w i t h the D-configuration (70).
TR ICHLOROBUTANOL
OWOCIN
BY-PRODUCT
SOLVENT
Figure 6 . 1
olranatogran of 50 p l of oxytocin concentrate (200 units/rnl) Conditions were as follows: Column RP 18, 1 0 pn, 250 x 4 . 6 mm I D , isocratic e l u t i o n w i t h 18%a c e t o n i t r i l e i n phosphate buffer s o l u t i o n (1/15 ml) p H 7, roan tenperatwe, f l a w rate 2.0 rnl/min, pressure a t column i n l e t 150 bars, W monitor a t 210 m.
OXYTOCIN
587
RAT UTERUS I.U./ML OR I.U./MG

300
200
100
100
200
300
HPLC 1. U. /ML
OR
I.U./MG
Figure 6.2
Correlation between the results of r a t uterus and HPLC assays of oxytocin Correlation coefficients and critical values for the results i n Table 2.1
588
2 1
20
16
12
TIME (MINI
Figure 6.3
L
I
Qlrcmatqram of 50 p l of a mixture of oxytccin and diastereoisomers (= 100 pg/rnl each).

Conditions as f o r Figure 6.1 Larsen e t al. (71,59) separated oxytocin f r m 7 of its stereoisomers (cf. T a b l e 6.2) on a p Bondapak C18 column. Mixtures of 10% tetrahydrofuran or 18%a c e t o n i t r i l e or 16% dioxane i n acetate buffer =re used as the mbile phase. T h e nature of t h e organic solvent and t h e p H a f f e c t t h e separation. H E W w a s found t o be much superior to the classical separation on Sephadex 6 2 5 .
OXYTOCIN
589
Table 6.2 The e f f e c t of t h e solvent on the separation of oxytocin and its diastereaners by reversed-phase HPK; published by Larsen e t al. (59)
16%Dioxane LO% THF 1 8 W a stereoisaners 30%0.05M NH40Ac 82%0. O l M NH40Ac 84%0.05M NH40Ac IH 4 . 0 p H 4.0 p H 4.0 L.6 ml/min 2 . 0 rnl/min 1 . 5 ml/min k' c1 k ' a k' a 7.73 1.00 7.31 1.00 7.70 1.00 9.12 1.18 8.33 1.14 10.4 1.42 9.12 1.18 9.66 1.25 9.43 1.29 11.1 1.44 9.28 1.27 14.6 2.00 13.1 1.69 13.3 1.72 13.7 1.77 12.0 1.64 17.7 2.29 12.7 1.67 10.7 1.46
Oxytocin m y also be separated fran other peptide hormones, e. g r8-lysinel or (8-arginine] vasopressin by reversed-phase HPIC. The separation m y be effected i s o c r a t i c a l l y (72) or by gradient e l u t i o n (73) with mixtures of aqueous buffers and a c e t o n i t r i l e , methanol, dioxane or tetrahydrofuran. The mobile phase m u s t contain a minimum concentration of salt, since otherwise t h e separation efficiency i s low. The chranatographic separation is influenced by the nature of the organic solvent H and salt concentration, but these e f f e c t s and also by the p are smaller f o r oxytocin than f o r other similar peptides (69).
Nachtrnann employed an isocratic mthcd and short wavelenght W detection (74) to test t h e p r i t y of intermediates used i n t h e synthesis of corytocin. The lower l i m i t of dea s i n the ng range b t h f o r f r e e and f o r protected tection w peptides. The p r h r y m i n e group of t h e N-tenninal,hemicystine of oxytocin may be derivatised with FLURAMR and assayed fluorimetrically. Gruber e t al. (75) carried out the derivatisation i n phosphate buffer a t p H 7 before chranatographic separation on P a r t i s i l ODs with a l i n e a r gradient of 15 t o 50% acetone i n 0.03% m n i u m formate and 0.01% thiodiglycol. 15 pmol of the oxytocin derivative gave an e a s i l y detectable peak with a signal t o noise ratio of 15:l. This methcd w a s used by Live e t a l . t o test t h e p u r i t y of synthetic oxytocin (76,601. I t was possible i n t h i s way t o separate oxytocin frcm 16 similar peptides. Radhakrishnan e t al. separated oxytocin fran other polypeptides, such as [8-arginine]vasopressin, on P a r t i s i l SCX cation exchangers using volatile pyridine acetate buffers (77). An autanated fluorescamine column m n i t o r i n g system was used f o r detection.
590
Postcolumn d e r i v a t i s a t i o n with FLURAMR was employed by F r e i e t al. (78,79) and K r m n e t 61. (70). The lower l i m i t of detection w a s 5 - 1 0 ng (5 - 1 0 p l ) per injection. The ccmpound w a s chranatographed on reversed-phase C 8 o r C 18. Since the sample solutions are concentrated i n t h e column, very d i l u t e solutions can s t i l l be determined with great precision.
6.3
Biological mthods
The a c ~ v i t y of a smple of axytocin is determined by canparing it with t h e International Standard Preparation o r with a preparation which has been standardised against t h e International Standard.
A t t h e present t i m e the standard preparation f o r determination of axytocin a c t i v i t y i s t h e Fourth International Standard f o r Oxytocin f o r Bioassay (80). The standard preparat i o n f o r t h e determination of vasopressor a c t i v i t y i s the F i r s t International Standard f o r Lysine Vasopressin ( 8 0 ) . Both standards (highly p r i f i e d peptides of synthetic origin) supersede t h e Third International Standard for oxytocin and Vasopressin, Bovine, f o r Bioassay, which w a s an acetone-dried extract of posterior lobes.
Numerous biological assays have been h o w n f o r mny years and have been t h e subject of detailed reviews (81-87). For t h e expximental procedures t h e reader i s referred to these papers and t o t h e mthcds described i n t h e various phannacopoeias. Only methods included i n t h e mst important pharmacopoeias will l x discussed here. Oxytocin as an active principle i s not described i n any of t h e pharmacopoeias. The stipulated contents as a percentage of t h e declared canposition relate t o oxytocin injections.
6.31 R a t uterus m t h d
The OOntraCtiOnS induced i n isolated rat uterus by t h e oxytocin sample are canpared with those induced by a standard preparation and evaluated as described i n t h e pharmacopoeias. Pharmacopoeia +)
B.P. Eur.P.
S t i p l a t e d potency of injections (Confidence limits, P = 0.95)
90
- 111%
(80
- 125%)
Swiss Nord
+)
Abbreviations of the pharmacopoeias as i n Martindale, The Ektra Pharmacopoeia, 27th Edition
OXYTOCIN
59 1
6.32 Chicken b l o d pressure method

The depressor e f f e c t s of t h e sample and standard on the chicken blood pressure are masured and evaluated i n accordance with d e t a i l e d instructions given i n t h e following pharmacopoeias. Pharmacopoeia +) S t i p l a t e d potency of injections (Confidence limits, P = 0.95/L = confidence i n t e r v a l )
B.P. W.P.
90
- 111%
120% 120%
(80
- 125%)
. U . S . Jap .
6.33 Milk e j e c t i o n assay
Swiss Nord
85 85
(L K0.20) (L G0.15)
This method i s based on measurement of the milk e j e c t i o n pressure i n a l a c t a t i n g rat and i s proposed i n t h e 1978 addendum t o the 1973 B r i t i s h Pharmacopoeia as an a l t e r n a t i v e t o the other two methods (rat uterus and chicken blood pressure).
6.34 R a t blood pressure assay

This mthcd masures the pressor e f f e c t of oxytocin samples. It sets a l i m i t t o t h e content of vasopressin which may occur as a impurity i n a y t o c i n of natural origin. This test is not required when the cocytocin i s prepared by synt h e s i s and t h e absence of foreign amino acids i s demonstrated by amino acid analysis (88,891. Hcwever, synthetic cocytocin i t s e l f has a mall i n t r i n s i c pressor e f f e c t i n nmunals (88,90,91). The synthetic highly a s shown p u r i f i e d oxytocin described i n section 1.7, which w of peptide impurities, had a by HPLC t o contain less than 1% vasopressor a c t i v i t y amounting t o 0.9% and 1.09% of its oxyt o c i n a c t i v i t y , according t o the r e s u l t s obtained i n 4x0 independent laboratories (92).
Belcw are shcwn requirements l i s t e d i n the mst important pharmacopoeias. The USP limit i s inconsistent with t h e abovementioned i n t r i n s i c vasopressor a c t i v i t y of pure oxytocin.
+) Abbreviations of t h e pharmacopeias
The Extra Pharmacopoeia, 27th Edition
as in Martindale,
592
FRIEDRICH NACHTMANN vt al.
Pharmacopoeia +)
S t i p l a t d ximu mum vasopressor a c t i v i t y as a percentage of oxytccic a c t i v i t y
B.P.
Eur.P. Swiss Nord U . S . Jap
. .
s 5% 6 5% s 1% < 5%
Determination i n biological mterial
6 5% 6 2.5%
6.4
6.41 Bioassays The mst c m n l y enployed bioassays f o r oxytccin i n biol q i c a l f l u i d s are based on the c o n t r a c t i l e e f f e c t on t h e e a myonetrim or mnmry myoepithelium i n vitro o r i n vivo. M surement of t h e depressor e f f e c t i n t h e chicken is less sensitive. The various m e t h d s which d i f f e r i n t h e i r s e l e c t i v i t y , sensitivity and precision have been evaluated by Munsick as shown i n T a b l e 6.3 (87). The d e t a i l s m y be found i n t h e numerous reviews (83, 87,93,94). The rat milk ejection test is t h e mst s e n s i t i v e methcd, the lower l i m i t of detection being 2.5 - 5 Vunits/rnl (87,94). Despite t h e r e l a t i v e l y high s p e c i f i c i t y of t h e bioassay m e t h a , accanpanying substances frequently i n t e r f e r e so t h a t p r i o r extraction procedures are necessary and these are usually time consuming and not p a r t i c u l a r l y reproducible. Affinity chranatography on agarose-bound neurophysin m y be a s u i t a b l e alternative (see 6.42).
+) Abbreviations
of t h e pharmacopoeias as i n Martindale, The Extra Pharmacopoeia, 2 7 t h Edition
T a b l e 6.3
A q u a l i t a t i v e evaluation of several mthcds 3 + mst) ; p b l i s h d by Munsick (87)
used t o assay oxytocin (1 + least;
SpeciSpeciYcity t o ' i c i t y t o k y t o c i n txytocin
vs Other Jeuro'ypo)hysial 'eptides
vs. other
sub-
Expense Rapid creening Assay for gtocin

Of
extraneous
stances
Isolated r a t
bnsitivity to )xytocin
. -
Equip m n t for 'recision ExperiExpof mntal j rience Required setup Assay

I
S e t up
Time per
Assay
Prepa-
ration
uterus
0 0.5 mM Mg2+ Rabbit, guinea
3+ 2+ 2+
1+ 1+
2-3+
1-2+ 1-2+ 2+
3+ 3+ 2+
2+ 2+ 2-3+ 2+ 2-3+
1-2+ 1-2+ 3+ 3+ 2+
1
~
1-2+ 1-2+ 2+ 3+
1-2+ 1-2+ 2+ 3+ 2+
pig o r rat milk-ejection, i i R a t or guinea pig milk-eject i o n , i a. Avian vasodepressor
i
I
2+ 2+
2+ 2-3+
3+
1+
2+
i Ii
1
1+
1+
594
6.42 Radioimmunoassays (RIA)
Owing to its low m l e c u l a r w i g h t a y t o c i n i s not a good antigen and i n i t i a l l y t h e production of antibodies w i t h a high t i t r e presented d i f f i c u l t i e s . Gilliland and Prout shaved t h a t antibodies could be produced by administering natural unconjugated oxytocin with Freund's adjuvant t o rabbits (95). Hmver, t h e antibody t i t r e w a s low. Despite t h i s handicap, a radioimmunoassay w a s developed using t h i s technique, b u t it was no mre sensitive than t h e best bioassay methods (96,97).
Better r e s u l t s e r e obtained w i t h oxy-tocin adsorbed on carbon microparticles (40,98) and w i t h oxytocin conjugated with bovine serum albumin (99). The latter methcd has been taken up by several authors (100,101). Rabbits are used t o produce the antibodies. Nevertheless, only i n a few laboratories has it proved possible t o prepare usable antibodies (1021, and t h i s has so f a r prevented t h e application of RIA for oxytocin on a wider scale (103). Ncw, however, oxytocin a n t i s e m i s available c m r e r c i a l l y (104). The h o m n labelling is usually accanplished by iodination w i t h f251 using t h e chloramine-T method (103) Dawood e t a1 labelled oxytocin by the lactoperoxidase method (105).
For d e t a i l s of RIA methodology, t h e reader i s referred t o t h e original papers cited and t o t h e various reviews (88, 93,103,106). Problems are also encountered i n extracting oxytocin f r a n blood serum. Qlard e t 61. have p b l i s h d a method i n which oxytocin is adsorbed fran plasma on F u l l e r ' s e a r t h and eluted with aqueous acetone ( 4 0 ) . However, recovery i s not particul a r l y good (50 - 60%) and t h e reproducibility leaves sanething t o be desired (101). Sane authors therefore m i t t h e extraction s t e p and use blood serum d i r e c t l y f o r t h e RIA (100,104).
A n extract based on a f f i n i t y chranatography on agarosebound neurophysin has recently been described (107). Oxytocin can be extracted frm plasma, urine and cerebrospinal f l u i d w i t h high recovery and high s p e c i f i c i t y by this methcd.
The lower limit of detection of RIA has been variously reported as 2.0 punits/ml (104) and 0.05 punits/ml (103). 6.43 High performance liquid chranatcgraphy (HPLC)
G r u b e r e t 61. (75) determined t h e oxytccin content of r a t p o s t e r i o r p i t u i t a r i e s using the methcd described i n 6.27. These authors p i n t out t h a t the methcd can be improved by using a
OXYTOCIN
595
new fluorescence reagent.

HPLC may be expected t o play an important part i n future i n t h e assay of oxytocin i n biological material.
6.5
Determination i n dosage forms
Before HPIC becme available, oxytocin i n pharmaceutical dosage fonns w a s usually assayed by t h e chicken blood pressure method or rat uterus mthod, which were adopted by most pharnacopeias (cf 6.3).
HPLC has recently been developed and is now the mthod of choice. It i s simpler as regards t h e apparatus needed, cheaper and mre rapid than t h e bioassays and it i s appreciably more accurate (70,108). It is also s u f f i c i e n t l y s p e c i f i c t o separ a t e oxytocin frun byproducts of synthesis (e.g. stereoisaners of t h e active canpound) o r related peptides (e.g. 8-lysine vasopressin) (cf. 6.27). This mthod m y also be used f o r s t a b i l i t y tests on t h e various oxytocin dosage forms (69,70).
596
FRIEDRICH NACHTMANN ef a1
References
205, 949 (1953) 2. G p y H., B l o c h e n . B i a p h y s . A c t a 11, 449 (1953) 3 . du V i g n e a u d V. , R e s s l e r Ch. , Swan?.M. , R o b e r t s C.W. , Katsoyannis P.G. and Gordon S., J. Am. C h m . SOC. 75, 4879 (1953) 4 . The M e r c k Index, 9th ed., Merck u. Co. Inc., Rahway, W, 1976, p. 904 5 . U r r y D.W., Walter R., Proc. N a t . Acad. Sci. USA, 68, 956 (1971) 6. G r o s s E., Meienhofer J., The Peptides, V o l 1, A c a d . Press. Inc. , 1979, p. 5 7. C3anW.Y. and du V i g n e a u d V., E n d o c r i n o l o g y 71, 977 (1962) 8 . Maxfield F.R. and Scheraga H.A., Biochanisw-16, - 4443 (1977) 9. G l i c k s o n J.D., R m a n R., P i t n e r T.P., D a d o k J., bthnerBy A.A. and Walter R., B i o c h a n i s t r y 15, 1 1 1 1 (1976) 10. C e r l e t t i A. and Berde B., C1. T e r a p . 15, 313 (1958) 11. Deslauriers R., Walter R. and S m i t h I 7 . P . , Proc. N a t . A c a d . Sci. USA 71, 265 (1974) 12. Boissonnas R . A . x d H u g u e n i n R.L. , H e l v . C h h . A c t a 43, 182 (1960) h m . SOC. 88, 2292 (1966) 13. Photaki I., J. Am. C 14. Bockaert J. , Imbert PI. , Jard SFand M o r e l F. , Mol P h m a col. 8, 230 (1972) 15. B r i t i z h Pharmacopeia, L o n d o n H e r Majesty's Stationery O f f i c e 1973, p. 339 16. D i h b e m W.H. , UV and I R Spectra of Sane Important D r u g s , Editio C a n t o r A u l e n d o r f 1978 17. Beychok S. and B r e s l m E. , J. B i o l . Chm. 243, 151 (1968) 6, m 3 (1968) 18. Goodman M. and T o n i o l o C., B i o p o l y m r s 19. Tu A.T., B j a r n a s o n J.B. and H r u b y V.J., B i o c h h n . e t Biophys. A c t a 533, 530 (1978) 2 0 . Feeney J., Roberts G.C.K., Rockey J.H. and B u r g e n A.S.V. N a t u r e New B i o l o g y 232, 108 (1971) 21. Deslauriers R., Walter R. and S m i t h I . C . P . , Biochern. B i o phys. Res. C m . 48, 854 (1972) 22. B r e w s t e r A.I.R. , W-y V. J. , Spatola A.F. and Bovey F.A. , B i o c h e m i s t r y 1 2 , 1643 (1972) 23. Lyerla J . R . J r 7 a n d Freedman M.H. , J. B i o l . Chern. 247, 8183 (1972) 24. Walter R., P r a s d K.U.M., Deslauriers R. and S m i t h I.C.P., Proc. N a t . A c a d . Sci. USA 70, 2086 (1973) 25. Hruby V.J. , Deb K.K. , Spatza A.F. , Upson D.A. and Yamamot0 D., J. Am. C h m . Soc. 101, 202 (1979) 26. du V i g n e a u d V., H a r v e y Lett-s 50, 1 (1954-55)
1. du V i g n e a u d V.
, Ressler
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OXYTOCIN
597
27. R e s s l e r Ch. and du V i g n e a u d V., J. Am. Chem. Soc. 79, 4511 (1957) 28. R e s s l e r Ch. and popnoe E.A., Methods Invest. Diagn. E n d o c r i n o l . 1973, 2A (Pept.Horm.) , 681 . and P a d r Z., Chenie und B i o l o g i e der Hormone, VEB 29. H a n c 0 Gustav Fischer V e r l a g , Jena 1979, p. 221 30. O l i v e r G. and Schafer E.A., J. Physiol. 18, 277 (1895) 50, 573 (1928r 31. Kamm O., J. Am. Chem. Soc. 32. du V i g n e a u d V., R e s s l e r Ch., Swan J.M., Roberts C.W. and Katsoyannis P.G., J. Am. Chem. Soc. 76, 3115 (1954) 33. Boissonnas R.A., Guttmann St., JaqueGud P.-A. and Waller J.-P., H e l v . C h b . A c t a 38, 1491 (1955) 34. V e l l u z L., B u l l . Soc. France 1 4 m (1956) 3 5 . B e y e r m a n n H.C., Bontekoe J.S. and Koch A.C., Rec. Trav. Chim. 78, 935 (1959) 3 6 . BodansG M. and du V i g n e a u d V., J. Am. Chem. Soc. 81, 183, 1324 (19g) 2504 (1959), 81, 5688 (1959), N a t u r e 3 7 . Sandoz AG B a s x , unpublished results 38. Nachtmann F., unpublished results 39. Sanabo Ges.m.b.H., W i e n , unpublished r e s u l t s 40. Chard T. , Ebyd N.R.H. , Forsling M.L. , M c N e i l 1 A.S. and Landon J., J. E n d o c r . 48, 223 (1970) 6, 41. Leake R.D. and WeitzmG R.E., C l i n i c s i n Pharmacology 65 (1979) 42. Kumaresan P., Han G.S., Anandarangam P.B. and V a s i c k a A., O b s t . Gynecol. 46, 272 (1375) 19, 189 (1959) 43. Chaudhury R.R. and Walker J.M., J. E n d o c r i n . 44. Walter R., B i c p h y s . A c t a 422, 138 (1976) 45. Walter R. and Bawman R.H., Endocrinology 92, 189 (1973) 46. Walter R. and Shlank H., Endocrinology 897990 (1971) 47. Marks N., A b r a s h L. and Walter R., P r o c y S o c . Exp. B i o l . Med. 142, 455 (1973) 48. B r z e z i n s k a - S l e b d z i n s k a E., J. E n d o c r . 63, 361 (1974) 49. B r z e z i n s k a - Slebdzinska E., Endocr. J2xp710, - 259 (1976) 50. W o j c i k K. and B r z e z i n s k a - Slebdzinska E., B u l l . Acad. pol. Sci., Ser. Sci. B i o l . 23, 577 (1975) 51. Ferrier B .M., H e n d r i e J . M . and B r a n d a L. A., Can. J. B i o chen. 52, 60 11974) 52. P l i s k a T : a n d R u d i & e r J., C l i n . E n d o c r . 5 Suppl., 73s (1976) 53. Mtihlemann M., T i t o v M.I., Schwyzer R. and R u d i n g e r J., H e l v . Chim. A c t a 55, 2854 (1972) . and Walterk., Int. J. P e p t i d e Protein Res. 5, 54. H a s e S 283 (1973) 55. P e t t i t G.P., Synthetic Peptides, V o l m e 4, E l s e v i e r , Amsterdam 1976 87, 56. Manning M. and du V i g n e a u d V., J. Am. Chem. Soc. 3978 (1965)
598
FRIEDRICH NACHTMANN cl a / .
57. S p a t o l a A., Cornelius D. and Hruby V., J. Org. C h m . 391 15, 2207 (1974) 58. Khan S.A. and Sivanandaiah K.M., I n t . J. P e p t i d e P r o t e i n Res. 12, 164 (1978) 59. Larsen'B. , Fox B.L., Burke M.F. and Hruby V. , I n t . J. P e p t i d e P r o t e i n Res. 13, 1 2 (1979) 60. Live D.H. , Agosta W.CTand C m h r n D., J. Org. Chm. 42, 22, 3556 (1977) 61. Flouret G., Terada S., Kato F., Gualtieri R. and L i p k o w s k i A., I n t . J. P e p t i d e P r o t e i n Res. 13, 137 (1979) 62. SANDOZ AG, Q u a l i t y C o n t r o l , Basle Switzerlas, unpublished 63. E'ujiki H. and Zurek G., J. Chranatogr. 140, 129 (1977) 64. Samejima K. , Dairman W., S t o n e J. and U m r i e n d S. , Anal. Biochem. 42, 237 (1971) 65. N a k a m u r a H. and-bano J.J., J. Chrmatogr. 121, 33 (1976) 66. Moore S. and S t e i n W.H., J. B i o l . Chan. 1 7 6 , 3 6 7 (1948) 67. Lowry O.H. , Rosebrough N.J. , F a r r A.L. a x a n d a l l R.J. J. B i o l . Chem. 193, 265 (1951) 68. Ellman G.L., A r z Biochen. Biophys. 82, 70 (1959) 69. K r m n K. and F r e i R.W. , J. C h m a t s . 132, 429 (1977) 70. K r m n K., Max1 F. and Nachtmann F., Pharmaceutical Technology, Sept. 1979, 77, (1979) 71. Larsen B. , Viswanatha V., Chang S.Y. and Hruby V. J., J. Chranatogr. S c i . 16, 207 (1979) 72. K r m n K. and F r e i KW., J. Chrcmatogr. 132, 27 (1977) 73. O ' H a r e M.J. and Nice E.C., J. Chrmatogr.171, 209 (1979) 74. Nachtmann F., J. Chranatogr. 176, 391 ( 1 9 7 r 75. Gruber K.A., S t e i n S. , Brink L . ,Radhakrishnan A.N. a n d Udenfriend S. , Proc. N a t . A c a d . S c i (USA) 73, 1314 (1976) (Goodman M. 76. Live D.H., Pept., Proc. Am. Pept. Symp., 5=., and Meienhofer J., Ed.), Wiley N.J. 1977, p. 44 77. Radhakrishnan A.N., S t e i n S., Licht A., G r u b e r K.A. and Udenfriend S., J. C h r m a t o g r . 132, 552 (1977) 78. F r e i R.W. , Michel L. and S a n t i x , J. Chmatogr. 126, 665 (1976) 79. F r e i 'R.W., Michel L. and S a n t i W. , J. Chranatogr. 142, 261 (1977) 80. WHO ECBS 30th R e p r t 1 9 7 9 , Techn. Rep. Ser. N o 638, 24 81. Sawyer W.H. i n Harris G. W. and Donovan B .T. (ed. ) , The P i t u i t a r y Gland, V o l . 3, B u t t e m r t h s , London 1966, p. 288 82. Walker J . M . i n Gray C.H. and Bachxach A.L. (ed.) , Hormones i n B l o o d , V o l . 1, Acadanic P r e s s , New York 1967, p . 145 83. Stiirmer E. i n Berde B. (ed.), Handboak of Experimental Phamacology, V o l . 23, S p r i n g e r , New York 1968, p. 130 84. F i t z p a t r i c k R.J. and B e n t l e y P.J. i n Ekrde B. (ed.), H a n d b o c k of Experimental Pharmacology, V o l . 23, S p r i n g e r , New York 1968, p. 190
OXYTOCIN
599
85. Smith M.W. i n Heller H. and Pickering B.T. (ed.) , I n t e r n a t i o n a l Encyclopaedia of Pharmacology and Therapeutics, Sect. 41, V o l . 1, P e r g m n Press, Oxford 1970, p. 173 86. Chard T. and Forsling M.L. i n Antoniades H.N. (ed.) , Hormones i n Human Blood, Harvard Univ. Press, Cambridge 1976, p. 488 87. Munsick R.A. i n JosimoVich J.B. (ed.), Uterine Contraction Side E f f e c t s of S t e r o i d a l Contraceptives (Problems of H m n Reproduction, V o l . I ) , J. Wiley, New York 1973 88. Mhme H. and Hartke K., EuropZisches Arzneibuch, Band 3 , Kcmrentar, Wissenschaftliche Verlagsgesellschaft, S t u t t g a r t , Germany (1979) 89. Calm D.H., Proc. Analyt. Div. man. SOC. 5, 130 (1976) 90. Boissonnas R.A., Guttrnann S t . , Berde B. and Konzett H., E k p r i e n t i a V o l . M I , 9, p. 377 (1961) 91. Hruby V.J., Flouret G. and du Vigneaud V., J. B i o l . Chan. 244, 1 4 , 3890 (1969) 92. 2 AG, Quality Control, Basle, Switzerland, unpublished data 93. Berde B., i n Van Cawnberge H. and F r a n c h h n t P. ( e d . ) , Assay of P r o t e i n and Polypeptide H o m n e s V o l . 33., Pergamon P r e s s , Oxford 1970, p. 128 94. F i t z p a t r i c k R.J., Methods Invest. Diagn. Endocrinol. 1973 2A (Pept. Horn.), 694 95. G i l l i l a n d P. and Prout T., Metabolism 1 4 , 912 (1965) and 14, 918 (1965) 96. zeeler M. , Kogan A. and Glick S.M., Clin. Res. 14, 479 (1966) 97. Glick S., hheeler M., Kogan A. and Kumaresan P., S u m e r i e s , I n t e r n a t i o n a l Symposium on the Pharmacology of H o m n a l Polypeptides and Proteins, Milan 1967, p. 48 98. Chard T.M., Kitau J. and Landon J., J. Endocrinol. 46, 269 (1970) 99. Glick S.M. , i n J o s h v i c h J.B. (ed.), Uterine Contraction - Side E f f e c t s of S t e r o i d a l Contraceptives (Problems of Human Reproduction, V o l . I ) , J. Wiley, N.Y. 1973 100. Bossuyt-Piron C. and Bossuyt A., I n t . J. Nucl. Ekd. B i o l . 5, 1 4 4 (1978) 101. k g h a v a n K.S. , Jogender Singh and Chabra J . K . , Indian J. Med. Res. 66, 787 (1977) 102. Piron-Bossu-yt C., Bossuyt A , , Braunmann H. and Van Den Driessche R., Annales d'Endocrinologie (Paris) 27, 389 (1976) 103. Kagan A. and Glick S.M., i n Jaffee/Bennann (ed.), M e t h c d s of Hormone Radioimmunoassay, 2nd Ed., A c a d g S i c Press 1979
13,
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104. Gorewit R.C., Proc. Soc. exp. B i o l . (N.Y.) 160, 80 (1979) 105. Dam& M.Y., Faghavan K.S. and Pociask C. , J. Endocrinol. 76, 2 6 1 (1978) 106. E n Cauwenberge H., Legros J.J. and F r a n c h b n t P. i n Van Cauwenberge and F r a n c h h n t P. (ed.) , Assay of P r o t e i n and Polypeptide Hormones, V o l . 33, P e r g m n Press, Oxford 1970, p. 136 107. Robinson I.C.A.F. and Walker J . M . , J. Endocrinol. 80, 1 9 1 (1979) 108. M a x 1 F. and Krunmn K., P h m . Acta Helv. 53, 207 (1978)
PENICILLAMINE
Ching Ching Chiu and Lee T . Grady
1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Appearance, Color, Odor, Taste 2. Physical Properties 2.1 Crystal Properties 2.2 Infrared Spectrum 2.3 Raman Spectrum 2.4 Nuclear Magnetic Resonance Spectra 2.5 Mass Spectra 2.6 Optical Rotation 2.7 Melting Range 2.8 Differential Thermal Analysis 2.9 Solubility 2.10 Acid-Base Properties 2.11 Polarography 3 . Preparation 4. Synthesis 5. Stability-Degradation 6. Chemical Reactions 7. Radiolysis 8. Methods of Analysis 8.1 Elemental Analysis 8.2 Identification Tests 8.3 Color Reactions 8.4 Colorimetric Analysis 8.5 Chromatographic Methods 8.6 Automated Analysis 8.7 Polarographic Analysis 8.8 Coulometric Method 8.9 Titration Method 8.10 Optical Purity Analysis 9. Metal Complex Formation 10. Metallic Salt Formation 11. Pharmacokinetics and Drug Metabolism 12. Toxicity and Side Effects 13. References 14. Acknowledgement
602 602 602 602 602 603 603 603 604 604 610 61 1 611 612 612 612 612 612 613 613 613 615 616 616 616 617 617 617 623 623 624 624 624 625 626 626 627 629 637
602
CHING CHING CHIU AND LEE T. GRADY
1.
Description
Penicillamine is the characteristic degradation product I t was first isolated and 1 ) . It is characterized by Abraham and co-workers in 1943 ( used medically as treatment of medical problems such as Wilsons disease (too much copper in the body), rheumatoid arthritis, cystinuria, and lead poisoning ( 2 ) .
of penicillin type antibiotics.
1.1.
Nomenclature 1.1.1. Chemical Names:
D-3,3-dimethylcysteineY D-3-mercaptovalineY 0mercaptovaline, By$-dimethylcysteine, D-B-thiovaline, aamino-f3-rnethyl-P-mercaptobutyric acid, and D-valineY3mercap t0. 1.1.2. Generic Name:
Penicillamine is the United States Adopted Name (3) for D-penicillamine. 1.1.3. Trade Names:
Cuprimine (4) (Merck-Sharp and Dohme) , Cuprenil ( 4 ) , Depamine (4), Trolovol ( 4 ) , DMC ( 4 ) , Depen (3) and Distamine (4) (Wallace). 1.2. Formula 1.2.1. Empirical gH1 1N02S 1.2.2. HS 1.3. Structural
CH3 H C - C - CO2H CH3 NH2
Molecular Weight MW
=
149.21
1.4.
Appearance, Color , Odor , Taste Penicillamine is a fine white or practically white
PENICILLAMINE
603
crystalline powder having a slight characteristic odor and a slightly bitter taste. 2. 2.1. Physical Properties Crystal Properties 2.1.1. PolvmorDhism
Evidence for the existence of polymorphism for D-penicillamine is from infrared spectroscopy and X-ray crystallography. I t was reported that pol morph I has a minimum between peaks at 1078 and 1101 cm-I that are less intense than the adjacent peaks at about 1050 and 116 c m ' , while polymorph I1 has an absorption peak at 1092 c m ' which is more intense than the adjacent peaks at about 1050 and 1160 cm-I ( 5 ) . Under the polarizing microscope (6), both polymorphs were seen as anisotropic crystals, I as needles and I1 as plates. X-ray crystallography also confirmed the occurrence of two polymorphs of D-penicillamine ( 6 ) . 2.1.2. X-Ray Crystallography
X-ray crystallographic data on D-penicillamine.HC1.H20 were reported by Crowfoot ( 7 ) . The unit cell characteristics listed for D-penicillamine.HC1.H20 are: a = 6.85, b = 6.08, c = 12.20, 6 = 103.6", p = 1.360; space . Morphoplogy: laths {loll elongated group = P2 , N = 2 along I O I O ~ . mine.HC1.H20 2 . 2 . X-ray crystallographic data on L-penicilla8 ) . also were reported (
Infrared spectrum
The infrared spectrum of D-penicillamine as Nujol mull 9 ) . was reported ( The infrared spectrum of DL-penicillamine was also described. The spectrum was taken as Nujol mull on cesium iodide windows and recorded on a Perkin-Elmer 180 Spectrophotometer ( 1 0 ) . The infrared spectrum of USP Reference Standard Penicillamine (Lot G) obtained as Nujol mull, using a
604
Beckman IR 4250 is shown in Figure 1. Table I lists the characteristic frequencies of penicillamine.
Table I
IR Spectral Assignment for Penicillamine
Frequency (cm-l)
Assignment
3170 2920 2600
-NH3 -NH3
-SH
+ +
1615
1280
-CO2
-c o2-
2.3.
Raman Spectrum
The Raman spectrum of DL-penicillamine was reported ( 1 0 ) . I t was obtained for powders by use of a modified rotating cell on a Jarrell-Ash 25-100 Spectrometer equipped with argon-ion (5145 a ) excitation. Some characteristic Raman bands for DL-penicillamine are listed in Table 11.
2.4.
Nuclear Magnetic Resonance Spectra
Proton magnetic resonance and 13C magnetic resonance 1 0 ) . Proton and fljectra were reported for DL-penicillamine ( C nuclear magnetic resonance spectra for penicillamine (Figures 2 and 3) were obtained on a Varian FT-80A NMR Spectrometer. The sample (about 200 mg) was dissolved in 3 ml of D20 with DSS (sodium 2,2-dimethyl-2-silapentane-5s lfonate) added as an internal standard. The proton and " C chemical shifts obtained for D-penicillamine are summarized in Tables I11 and IV, respectively.
Figure 1.
Infrared Spectrum (Nujol mull) of Penicillamine. Instrument: Beckman IR 4 2 5 0
606
CHINC CHING CHIU AND LEE T. GRADY
Table I1 Characteristic Raman Bands for DL-Penicillamine
Band Frequency (cm-l) 2569 1659 1510 1597 1399
Characteristics vs* w,* br* w,* br* w,* br* m* w*
Assignment
- SH
-NH3 -NH3
-co2
-CO2-
+
576 552
S*
-NH3 -CH2-
S*
*vs
very sharp, w = weak, br
broad, mw
medium weak
C C K SIGNAL SPIN RATE
rps
TEMP
C
nz
lec
x
ACQUISITION
SPECTRAL WIDTH I S W I YO OF TRANSIENTS INT! K O U I S I T I O N TIME (AT) P U k E WIDTH (PW) PULSE DELAY IPDi 3ATA POINTS IDPl TRANSMITTER OFFSET [TO) HIGH FIELD LOW FIELD RECEIVER GAIN iRGl
re
lec
DISPLAY
SEN5 ENHANCEMENT iSE1 WIDTH OF PLOT I W P l END OF PLOT IEP! WIDTH OF CHART IWCI END OF CHART IEC! VERTICAL SCALE IVSI REFERENCE LINE (RLI
S K
nz
HI HI HZ
I
8
6
P pm L
Figure 2.
Nuclear Magnetic Resonance Spectrum of Penicillamine.
LOCK SIGNAL SPIN RATE
rpz
TEMP
'C
ACOUlSlTlON
SPE~TRAL WIDTH (SWI NO OF TRANSIENTS INTI ACQUISITION TIME IATl P l l l SE WIDTH IPWI PULSE DELAY lPDl DATA POINTS (DPI TRANSMITTER OFFSET IT01 HIGH FIELD LOW FIELD RECEIVER GAIN :RGI DECOUPLER MODE IDM) DECOUPLER OFFSET ID01 NOISE BANDWIDTH I N B ) HI
iec
.rec
<ec
kHz
DISPLAY
SENS ENHANCEMENT ISEI WIDTH OF PLOT I W P I END OF PLOT IEPI WIDTH OF CHART IWCI END OF CHART (EC: VERTICAL SCALE l V S i REFERENCE LINE IRL)
Sec
nz
nz nz nz
200
150
100
ppm
50
Figure 3.
13C Nuclear Magnetic Resonance Spectrum of Penicillamine
PENICILLAMINE
609
Table 111
' H NMR Spectral Assignments for Penicillamine

FH3 B f: - C02H HS - C bH3 NH2
Proton
Chemical Shift (ppm) (relative t o -CH3 of DDS) 1.48, 1.56 3.69 ppm from H20 Table IV
Multiplicity
-CH3 -CH
singlet singlet
*4.71
13c NMR Spectral Assignments for Penicillamine
Carbon
Chemical Shift (pprn) (relative to -CH3 of DSS) 32.90, 30.39 67.28
46.46
173.94
610
2.5.
Mass Spectra Ultramicrodetermination and selective identification
of penicillamine have been achieved by the combined gas
chromatography-mass spectrometry technique (11-13). Penicillamine was derivatized with N,O-bis-(trimethylsily1)trifluoroacetamide (BSTFA)-1% trimethylchlorosilane in pyridine (11) or N-trifluoroacetamide-L-prolyl chloride (12). The derivatives were analyzed by gas chromatography. Two glass columns, 50 cm x 3 mm i.d. packed with 1.5% OV-101 on 60-80 mesh Diatoport S and 1 m x 3 mn i.d. packed with modified OV-101 on 100-120 mesh Chromosorb W (AW) were employed. Column temperature was programmed from 140" to 170" at the rate of 5" per minute. A Hitachi RMU-6MG mass spectrometer combined with an 002 Datalizer using Hitac-10 computer for an on-line data processing was used. Ionization energy and accelerating voltage were 70 eV and 3000 V, respectively. The ion source temperature was 210" The limit of detection of penicillamine was about l o ' ' g level. The mass spectra of the fragment ions of Nester of TFA-L-prolyl-n-butyl ester and N-TMS-O-TMS penicillamine are summarized in Tables V (11) and VI (12), respectively. Table V Fragment Ions of N-Trimethylsilyl-0-trimethylsilyl Derivative of Penicillamine Ester
mle
147
Relative Intensity 21.4 3.8 5.0

9.4 8.4
128 100 75 74 73
59
45
100.0 6.4 17.7
PENICILLAMINE
611
Table VI Fragment I o n s of N-TFA-L-Prolyl-n-Butyl Ester of Penicillamine
mle
324 250 167 166 73 70 55 41 29 28
Relative Intensity
17 .O 42.4 24.0 17.8 13.3 15.9 15.0 33.0 35.5 29.5
2.6.
Optical Rotation
The reported optical rotation values or Dpenicillamine are:

[a~g50 = -63"
C = 0.1 in pyridine ( 4 ) C = 2.5 in 1.0 N NaOH ( 9 )
[a]i6"
-61.3"
The specific rotation range required by official compendia (14, 15) is between -58" and -68" determined in a 5% solution in 1 . 0N sodium hydroxide at 25C.
2.7.
Melting Range
The melting point temperatures reported or penicillamine are: m.p. = 198C (1) m.p. = 198.5"C ( 4 ) m.p. = 212C ( 9 )
612
2.8.
Differential Thermal Analysis
The only thermal event in the differential thermal analysis curve of D-penicillamine is the melting endotherm 6 ) . Either polymorph of D-penicillamine gives same at 185" ( endotherm.
2.9
Solubility
Penicillamine is soluble in 9 parts of water, soluble in 530 parts of ethanol (95%), slightly soluble in other alcohols, practically insoluble in chloroform and ether at
20C (14). 2.10.
Acid-Base Properties
revealed the presence of three ionizable groups with pKa values of 1.8, 7.9, and 10.5, which correspond to the carboxyl, a-amino and 8-thiol group.
A titration curve of penicillamine hydrochloride at 25C was first presented by Abraham (1),(16). The curve
Recently, the ionization constants for acid functions of D-penicillamine were verified by pH titration at 37C and 0.15 M ionic strength (17, 18). These results correspond to that previously obtained by other workers (19-23).
2.11.
Polarography
The polarographic behavior of penicillamine was studied by differential pulse polarography (24). The anodic polarographic wave was shown to be diffusion-controlled. A rectilinear calibration plot was obtained in citratephosphate buffer at pH 2.5 over the range 0.1-5.0 x M. The peak potential varied rectilinearly over the pH range 2-8 [Ep(V) = -0.030-0.059 pH]. The peak current is highly dependent on pH. It has maximum values at pH 3 and 10 (4.7 and 3.8 I . ~ Arespectively) with a minimum value (3.2 PA) at pH 7.
3.
Preparation
It is prepared by acid hydrolysis of penicillin (16) followed by precipitation from the hydrolysis mixture as the mercuric salt which is then collected, suspended in water and treated with hydrogen sulfide to liberate the free acid. Purification involves only recrystallization from water (16),(25). Several modifications of the preparation
PENICILLAMINE
613
procedure have been made to either increase the yield or simplify the process (26-28).
4.
Synthesis
Penicillamine was first synthesized independently by several groups utilizing a series of reactions involving benzyl mercaptan and 2-phenyl-4-isopropylidene oxazolone (29, 30), (Figure 4 ) . This involves the addition of benzyl mercaptan to 4-isopropylidene-2-phenyl-5(4)-oxazolone (I) using sodium methoxide as catalyst. Mild hydrolysis of the addition compound (11) gives (111), from which the benzoyl group was removed by strong acid hydrolysis. The S-benzyl amino acid (IV) was reduced with sodium and liquid ammonia to give the racemic penicillamine hydrochloride (V). Alternately, IV is first resolved by crystallization of the brucine salts of the N-formyl derivatives (VI) into the components D-and L-S-benzyl penicillamines which in turn were reduced to the corresponding D-and L-penicillamines. Several other synthetic routes for the synthesis of penicillamine were also reported (31).
5.
Stability-Degradation
Penicillamine is relatively stable in both light and air. Aqueous solutions of D-penicillamine are comparatively stable at pH 2-4 (14). In aqueous solution, penicillamine degrades slowly by first order or pseudo-first order kinetics. A 3% solution of penicillamine hydrochloride stored under nitrogen in a sealed container at 20" decomposed to the extent of about 10% per year (32).
6.
Chemical Reactions
1. On treating penicillamine with bromine water, the thiol group is oxidized to a sulfonic acid group and a crystalline compound called penicillaminic acid is obtained (33).
2. The nitrogen of the a-amino group of penicillamine reacts as a-amino nitrogen in the Van-Slyke procedure (33).
3.
Penicillamine reduces ammoniacal silver nitrate
(25)
614
CHING CHINC CHIU AND LEE T. GHADY
I
4-isopropylidene-2-phenyl-5(4)-oxazolone
CH3 CH3-I-FH-COZH C6H5CH2-S NH2 IV Acid Hydrolysis
I
0
Hiirolysis
CH3-q-qH-C 0 2 H C6H5CH2S NH-G-CsH5

0
P3
111
S-benzyl-DL-penicillamine
CH3-7-FH-CO2H HS NH2(HX) V (Racemic)
Na/NH3
(liquid)
F 3
N-benzoyl-S-benzylDL-penicillamine
FH3 CH3-$-7H-C02R1 C6H5CH2-S NH-5-H
VI
FH3 CH3-F-VH-CO2H H-S NHz D, L - i s o m e r s
Na/NH3
CH3-$-$H-C02H C H CH S NH2 VII D, L - i s o m e r s
b r u c i n e s a l t s of Nf o r m y l derivative of p e n i c i l l a m i n e
F 3
Figure 4 .
S y n t h e s i s of P e n i c i l l a m i n e
PENICILLAMINE
615
4 . On heating a solution of penicillamine and pnitrophenylhydrazine in 1 N hydrochloric acid at l o p , glyoxal-p-nitro-phenylosazone is produced (25).
5. The kinetics and mechanism of the redox reaction of penicillamine in solution by chromium (VI) were investigated by stopped-flow technique. Three moles of penicillamine are required to reduce chromium (VI) to chromium (111). Several chromium (111) products have been identified by ion-exchange technique (34, 35).
7. Radiolysis of penicillamine has been studied extensively spin resonance spectroscopy at room 7 7 ' K (36, 37) and more recently at 4 . 2 ' K by These experiments sought insights into the penicillamine as radiation protective agent.
Radiolysis using electron temperature at Box (38, 3 9 ) . application of
Solid state studies on crystalline penicillamine at 4 . 2 ' K have shown that electron addition to the carboxyl group is the major electron-capture route. At higher temperatures, dissociative electron capture to give MI3 has also been observed (38,39). The effects of radiation dose and exposure time on the number of detectable paramagnetic reasonance centers were investigated ( 4 0 ) . Recently penicillamine has been exposed to 6oCo gamma rays at 77"K, both in pure state and in dilute methanolic solution or aqueous glass (41). In methanol, the major step seems to be.loss of the thiol group (.SH) to give the radical Me2C-CH(NH2)C0i which is readily characterized by its esr spectrum. Loss of thiol (.SH) clearly dominates in the protic media. Mechanistic aspects of the radiolysis of penicillamine in N20 saturated solution at pH 5 was investigated (42). Penicillamine disulfide, penicillamine trisulfide and valine were formed. The formation o f the latter two products was attributed to secondary reaction of radical I. Other minor products like ammonia and compound I1 were also found. These radiolysis results are in agreement with earlier studies (43-46).
616
CHING CHINC, CHIU AND LEE T. GRADY
+
(CH3)2-?-CH(NH2)CO*H
S
CH2=C(CH3)-FH-(NH3) COqL
I1
Frozen aqueous mixtures containing a macromolecule and penicillamine (Sephadex or Thiogel and the reduced or oxidized form of penicillamine) were irradiated in vacuo with X-ray. In frozen aqueous mixture, radiation-induced unpaired spin may be transferred from a macromolecule to the penicillamine, whereas the intermolecular transfer of spins between solutes appeared to occur primarily during heat treatment. Electron spin resonance measurements were made at 7 7 ' K after heat treatment at 2 2 3 ' K (47).
8.
Methods of Analysis Elemental Analysis The elemental composition of penicillamine is: E1ement
% Theoretical
8.1.
40.25% 7.43% 21.45% 9.39% 21.49% 8.2. Identification Tests
The following identification tests are listed in the compendia:

1 . Addition of phosphotungstic acid solution to a penicillamine solution gives a deep blue coloration (14,15).
2 . Addition of acetone-hydrochloric acid solution to a penicillamine solution gives a white precipitate (14,15).
PENICILLAMINE
617
3 . Addition of ninhydrin test solution to a penicillamine solution gives a blue or blue-violet coloration (14,15).
4. Comparison of the infrared spectrum of the penicillamine sample with that of a reference standard (15).
8.3.
Color Reactions
1. Penicillamine gives a deep blue coloration with ferric chloride (25).
2. Penicillamine gives a green color with Fehlings solution (25).

3 . Penicillamine gives a strong purple coloration with alkaline sodium nitroprusside ( 3 3 ) .
8.4.
Colorimetric Analysis
Colorimetric methods of analysis were reported for penicillamine. These were based on the measurement of the absorption maximum at 645 nm of the blue coloration formed when penicillamine is reacted with tetrazolium salts in low concentration (48) (after 1 hr.) or with ferric chloride-potassium cyanide (49). (after 5 min.) at 65C. Quantitative determination of penicillamine during production process was based on orange color produced by penicillamine with Nessler reagent (50). The sensitivity is 3 x g of penicillamine. Colorimetric analysis based on copper (11) complex formation was also used (51). The absorption maximum is measured at 522 nm (after 30 min.). The method is selective for the unaltered drug in partially degraded solution. Recently, a simple colorimetric method based on the use of Ellmans reagent, 5,5dithiobis-2(nitro-benzoic acid) was used for plasma penicillamine concentration (52). 8.5. Chromatographic Methods 8.5.1. Pauer Chrornatoerauhv Table V I I gives various paper chromatographic
618
systems used for the detection of penicillamine (53, 5 4 ) . Ninhydrin spray reagent'hasbeen used. Table VII Paper Chromatographic Systems for Penicillamine Analysis
Developing Solvent 1-butanol-glacial acetic acid-water (120:30: 50) phenol-water (160 g:40 ml) (solvent A) solvent A-conc. ammonium hydroxide (200: 1) solvent A-ethanol-water (150:40:10) ethanol-water-conc. ammonium hydroxide (180: 10: 10)
2-methyl-2-propanol-water-methyl
Rf x 100 Reference 19
50 55 33 11 22
ethyl ketone-diethylamine (80:80:40:8) methanol-water-pyridine (160:40:8)

1-butanol-pyridine-water
42 25 81
73
(60:60:60)
pyridine-acetone-ammonia-water (45:30:5:20)
2-propanol-formic acid-water (75:12.5: 12.5) 8.5.2. Thin-Layer Chromatography
Thin-layer chromatography was used mainly t o detect the presence of penicillamine as impurity in penicillin type antibiotics or as conversion product from these
PENICILLAMINE
619
antibiotics. Table V I I I summarizes the thin-layer chromatography systems for penicillamine analysis.
8.5.3.
Gas Chromatography
Penicillamine is a polar nonvolatile molecule, derivatization is a requirement for analysis by gas chromatography. Jellum (60) reported the separation of amino thiols and diamino disulfides by reacting with pivalaldehyde at room temperature and neutral pH to form volatile neopentylidene derivatives which can be separated on a column of 5% SE-30 (on Aeropack 30) (5 ft x 118 in.) with a helium carrier-gas flow of 35 ml per minute. The temperature was programmed from 80" to 250" at a rate of 10" per min. The structures of the derivatives were confirmed by mass spectrometry. Recently, penicillamine was analyzed as a cyclic oxazolidine derivative (61) by reacting with formaldehyde followed by 1,3-dichlorotetrafluoroacetone. The separation was done on a 3% OV-17 or SP-2250 (2 m x 2 mm) on 80-100 mesh support or 3% SE-30 (1 m x 2 mm) on 45-60 mesh Chromosorb WAW DMCS column using a flame ionization detector. The nitrogen flow rate was 30 ml per minute. Detector and injector temperatures were 250 and 200C respectively. Column temperature was linearly programmed from 120 to 240" for both columns, at 8C per minute. Glass as the column material and silanization of the chromatographic support are basic requirements, without which the analysis fails.
8.5.4.
Pressurized Liquid Chromatography
Kenyhercz (62) developed an assay for penicillamine involving pressurized liquid chromatography with an electrochemical detector. A 50 cm x 2 nun glass column dry packed with a pellicular cation-exchange resin and aqueous mobile phase consisting of 0.018MH2S04 and either 0 . 1 M or 0.2 M Na2S04 were used.
Table VIII Thin-Layer Chromatographic Systems for Penicillamine Analysis Stationary Phase Developing Solvent Cellulose powder MN-300 2-propanol-butanone-1 (60:15:25) Visualization Reference (55) (55)
2 HC1
Ninhydrin-cadmium acetate reagent
2-methyl propanol-2-butanonepropanone-methanol-water-
conc. ammonia (40:20:20:1:14:5) Silica Gel G butyl acetate - methanol butanol - H20 - acetic acid (75:45:45:30:7.5) 2-isopropanol - H20 (85:15) 1-butanol - acetic acid - H20 (72:18:18) Methanol - H20 (64:36)
2% ninhydrin in ethanol (w/v)

Ninhydrin spray
(14)
Silica Gel
(57)
Table VIII
Cont'd.
Stationary Phase Silica G e l G
Developing Solvent 1-butanol - H20 - ethanol acetic acid (5:2:1.5:1.5) 1-butanol
Visualization
Reference (58)
- H20 - acetic acid (4:1:1) acetone - acetic acid (95:5)

85% aqueous acetone Silica Gel G/F barbital acetate buffer acetone (94:6)
(1) Ninhydrin spray (2) Spray consecutively with the following reagents (a) 2 N NaOH (b) Iodine-azide (c) 1% starch solution
Bioassay with Sarcina Lutea (ATCC 9431)
(59)
622
Recently, a method for the determination of the reduced and total penicillamines in biological fluids was reported (63). The separations were performed on a 30 cm x 0.2 cm column of Zipax SCX strong cation-exchange resin with a 3 cm x 0.2 cm precolumn of Zipax SCX. A pH 3.0 phosphate-citrate buffer of 0 . 0 4 M ionic strength, deaerated with oxygenscrubbed nitrogen, was used as eluent with a flow rate of 0 . 6 ml per minute. Separations were detected with a mercury-based electrochemical detector. Electrochemical detectors were also applied in monitoring the separations o f penicillamine (64, 65). Blaha (66) reported the analysis of penicillamine as degradation product of penicillin G potassium by HPLC. The liquid chromatograph used was equipped with a W detector operating at 254 nm and a column packed with Bondapak AX/Corasil (0.61 m x 2.3 m m ) . The mobile phase was a 0.1 M citric acid-0.2 M disodium phosphate buffer solution elutiyg at a flow rate o f 0.7 ml per minute. Penicillamine was eluted with a retention time of 4.5 min. Recently, a simple method for the determination of penicillamine in serum at therapeutic levels was described (67), 50-300 mg of penicillamine can be determined. The analysis was based on a fluorescence derivatization of the sulfhydryl group with 5-dimethylaminonaphthaline-lsulfonylaziridine combined with pressurized liquid chromatographic separation and fluorescence detection. Separation was achieved with a LiChrosorb RP-18 column (25 cm x 4 . 6 mm) with a mobile phase consisting of acetonitrile(pH 8.2, 0.0033 M) phosphate buffer (1:2) + 0.05% ethylenediamine maintained at a flow rate of 1 ml per minute. The retention time for the derivatized penicillamine was 8 minutes. Chromatographic separation of penicillamine and other sulfur containing amino acids (68) was performed on 60 cm x 0 . 6 cm column of Amberlite CG-4B anion-exchange resin (in the chloride form) operated at room temperature and 200 psi pressure. The column was eluted with 0.1 M HC1 and maintained at a flow rate of 1.0 ml per miyute. Iodoplatinate reagent (chloroplatinic acid-potassium iodide) was used for detection. 8.5.5. Ion-Exchange Chromatography Penicillamine present in urine and plasma was
PENICILLAMINE
623
determined by conversion to penicillaminic acid and then chromatographed on a Dowex 1-X 8 column. The mobile phase consisted of 0.25 M formic acid, 0.2 M NaOH plus 5 ml of Brij 35 per liter TpH 4 . 2 ) . The effluent was treated with ninhydrin ( 6 9 ) . Earlier, Frimpter (70, 71) reported the separation of penicillamine with sulfonated polystyrene cation-exchange resin column.
A column of 16 mm diameter containing 18 g of polyamide covered with 3.6 ml of tributyltin chloride was used to separate penicillamine. Tributyltin chloride has the properties of a liquid anion exchanger possessing special affinity for mercapto groups (72, 7 3 ) . The column was eluted with buffer solutions of pH decreasing from 8 to 4 with gradient elution by addition of solvent A ( 0 . 1 M citric acid + 0.2 M boric acid + 0.2 M monosodium phosphate7 to 50 ml of a solution of four partssolvent B (0.3 M disodium phosphate + 0 . 1 M trisodium phosphate + 0.5 g disodium EDTA) and one part solvent A.
8 . 6 .
Automated Analysis
Automation for analysis of penicillamine has been described. Chromatographic separation of penicillamine was achieved on a dual column packed with ion-exchange resin with automated system (74-76). Friedman (77) described the use of Durrum DC-4A column (48 cm x 1.75 mm) eluted with citrate buffer at gradient pH of 3.25, 4.25, and 7.9 and detected with ninhydrin at 590 nm. Norleucine was used as internal standard and eluted at 120 minutes. Automated injection system was used. Purdie (78) has successfully used a Technicon, Model NC-1 amino acid analyzer to separate penicillamine on a column of Chromobeads (cation-exchange resin) (150 cm x 0.60 cm) at 60" and it was determined with ninhydrin. A buffer eluent flow rate of 35 ml per hour was used. A rapid and sensitive automated analysis of penicillamine on micro sample in the 1-10 nmole range in physiological fluids was reported ( 7 9 ) . Bonnot also achieved a similar separation on a different size column (140 cm x 0.3 cm) with citrate 8 0 ) . buffer ( 8.7. Polarographic Analysis Polarography has been used for the determination of
624
penicillamine present in biological fluids or as conversion product from penicillins (81, 82).
8.8.
Coulometric Method
The use of coulometrically generated mercury (11) for the titration of penicillamine was reported. When pure penicillamine was titrated two equivalence points were obtained ( 8 3 ) . The potential at the first equivalence point lies around +80 mV. Titration took place in a 0.4 M acetate buffer solution at a generating current of 5 mA (3 before the second equivalence point).
8.9. Titration Method

Billabert (84, 85) used mercury (11) acetate for a titration at pH 6, two equivalence points were found, the first corresponding to formation of a sulfide and the second to formation of a chelate. A 1:l stoichiometry exists between Hg (11) and penicillamine during titration. Other titrimetric methods, such as titrating with silver (I) ions (86), lead (11) ions (87, 8 8 ) , alkaline solution after proton displacement reaction (89) and other metallic salts (90) are also reported. 8.10. Optical Purity Analysis
The enantiomers of this drug differ in their efficacy and activity. D-Penicillamine is the enantiomer required for pharmaceutical preparations. The L-enantiomer is toxic and it is also absorbed by the human body more than the Dform (91). Much of the allergic toxicity can be attributed to the use of L-form. It is known that L-penicillamine exerts an anti-vitamin B6 effect in rats. Since this drug is administered often in doses of up to one gram per day (92), an impurity even of a fraction of a percent of Lisomer may be hazardous. A method for the analysis of the optical purity is necessary. Cockerill (93) developed a NMR method for assaying the enantiomeric composition of penicillamine. The NMR spectrum of the thiazolidene derivative of penicillamine was taken in the presence of the optically active europium shift reagent, tris-(3-heptafluorobutyryl-d-camphorato)europium. The derivative was prepared from the mixture by successive treatment with acetone-HC1 and ethereal diazomethane. The L-enantiomer can be detected at levels of 0.4-0.5% in DL mixture. The shift reagent causes significant separation of
PENICILLAMINE
625
equivalent peaks due to the Jl- and L- forms of penicillamine e.g. A6 = 1.84 for the methine C-H protons and A 6 = 1.82 and 0.65 for the methyl groups of penicillamine. Gas chromatography-mass spectrometry (94) were used to determine the optical purity of penicillamines. The mixture of penicillamines was desulfurized with Raney nickel, derivatized with pentafluoropropionic anhydride and operated by a gas chromatograph fitted with glass capillaries (2.0 m x 0.3 mm) coated with Chirasil-Val (a novel chiral polysiloxane type stationary phase). The enantiomers were separated isothermally at 110" with a resolution factor a L / D of 1.134. The reliability of the assay was further monitored by mass spectrometry. The mass spectrometer is set to a resolution of approximately 1000 with ionizing potential of 70 eV ionizing current of 0.8 mA, interface temperature of 220" and ion source temperature of 220". 9. Metal Complex Formation
The ability of penicillamine to act as chelating agent in therapeutic treatment of Wilson's disease and mercury poison has prompted the extensive investigations by a number of laboratories on the structural chemistry of metal complexes of penicillamine, particularly that of the copper complex (95-101).
A complex was assigned to be Cu (I)/Cu (11) mixedvalence complex based on X-ray diffraction data. Six out of the fourteen copper ions are present in square, planar 1 1 ) . The high absorption environment commonly found for Cu ( in the blue region allowed the proposal of some similarity to copper chromophores.
Investigation of complex formation of penicillamine with non-transition and transition metal ions was undertaken by means of analytical potentiometric and spectroscopic techniques such as nuclear magnetic resonance, electron magnetic resonance and circular dichroism. Among the metallic ions examined include nickel (TI) (17), zinc (11) (102), lead (11) (102), antimony (111) (103), cobalt (111) (104, l05), indium (111) (106), mercury (11) (go), (107), gold (I) (108-110), and cadmium (11) (111). Stability constants (112) for metal complexes of Dpenicillamine with bivalent metallic ions such as magnesium
626
CHING CHING CHIU AND LEE T. GRAD1
(11), calcium (II), manganese (II), iron (II), cobalt (11), nickel (11), copper (11), zinc ( I I ) , lead (11), and mercury (TI) were reported. These constants were calculated from pH values using known mathematical relations and computer programming.
10. Metallic Salt Formation

The preparation of calcium chloride salt of Dpenicillamine (m.p. 30OoC) was described. This salt is used as a chelating agent in tablets or capsules (113). 11. Pharmacokinetics and Drug Metabolism
D-Penicillamine is the accepted therapeutic agent for the treatment of Wilson's disease (114, 115), cystinuria (116), lead poisoning (117-119), rheumatoid arthritis (120), and a possible protective agent against radiation (121). Overall, the biochemical actions of D-penicillamine are attributed to its aminothiol properties, i.e., chelating of metals, reaction with carbonyl groups and interference with sulfhydryl disulfide exchange reactions. The therapeutic value in Wilson's disease as well as the treatment of heavy metal poisoning results from its strong in vivo metal chelating properties. Theories have been advanced for the mechanism of action of penicillamine in rheumatic disease (122, 123). A striking similarity between side effects in rheumatoid arthritis patient caused by gold treatment and penicillamine therapy has been observed. The mode of action of penicillamine in cystinuria is well understood, the mixed disulfide formed with cysteine is more soluble than cystine (124). Stability constants were determined for penicillamine at simulated biological condition with calcium (II), iron (11), and gold (11). Only iron (111) formed both 1:l and 1:2 chelates with D-penicillamine (125). Based on electron spin resonance and chemical studies of penicillamine and copper (11), a mechanism for the action of penicillamine in the treatment of Wilson's disease was proposed by Peisach (125). ESR data indicated that penicillamine initially produces a complex with copper (11) through a microscopically reversible addition of a single ligand atom, i.e., nitrogen to the metal ion. The addition of the second ligand atom - sulfur is quickly accompanied by an electron transfer reaction whereby the metal i o n is
PENICILLAMINE
627
reduced to copper (I) and the ligand sulfur is oxidized to a sulfur free radical. In spite of its wide use in medicine, the pharmacology of D-penicillamine in man is little understood. Gibbs and Walshe (126) studied the fate of orally administered [35S] DL-penicillamine in six cases of Wilson's disease. Wass and Evered (127) have studied the intestinal absorption of L and D-penicillamine in the rat. Recently, a study on D-penicillamine metabolism in cystinuria, Wilson's disease and rheumatoid arthritis was undertaken. For subjects undergoing treatment for Wilson's disease, cystinuria and rheumatoid arthritis, the total percentage of the D-penicillamine dose excreted was 34, 40, and 3 4 percent, respectively. D-Penicillamine caused a 32 percent reduction in the urinary excretion of cysteine residues in cystinuria, but 400 percent increase in their excretion in rheumatoid arthritis. The metabolites of D-penicillamine known to occur in man are D-penicillamine disulfide, the mixed D-penicillamine-cysteine disulfide and S-methyl-D-penicillamine (128). In vivo, the free thiol has also been found in plasma and urine of patients with rheumatoid arthritis after treatment with the drug. It was also found the stable copper-D-penicillamine complex shows a high superoxide reactivity which may be responsible for the therapeutic effects in rheumatoid arthritis. When incubated with the contents of the stomach and duodenum of the chick, penicillamine was either destroyed or chemically bound and was no longer detected on the chromatograph (129). D-Penicillamine is slowly oxidized by D-amino acid-oxidase. Both enantiomers of penicillamine is desulfhydrated by L-cysteine desulfhydrase, only the Lisomer inhibits the action of this enzyme (130). In vitro effects reported in the literature include inhibition of DNA and protein synthesis (131), selective inhibition of polio virus growth (132) and prevention of collagen cross linking (133, 134). 12. Toxicity and Side Effects
When administered to mice, penicillamine was tolerated up to 1750 mg/kg intravenously, >5 g/kg orally and exhibited a LD50 value of 2300 mg/kg. When administered orally at 50 mg/kg daily for a month, it did not affect the composition
628
of blood or the functioning of kidneys and liver in rabbits or gastric peristalsis in dogs ( 1 3 5 ) . Acute sensitivity reactions manifested by fever, rashes (pruritic, morbilli form and urticarial), leukopenia, eosinophilia and thrombocytopenia have been encountered early in the course of therapy. Infrequently, anorexia, nausea and vomiting occurs. Loss of taste for salts and sweets have been observed. Rarely nephrotoxicity has been reported. Extravasation of blood into the skin over pressure joints occurs in some patients after prolonged administration of high doses of penicillamine (136).
PENICILLAMINE References
629
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E. P. Abraham, e t a l . The Chemistry of P e n i c i l l i n , p. 26, H. T. Clarke, J. R. Robinson, and R. Robinson, Eds., P r i n c e t o n U n i v e r s i t y , P r i n c e t o n , N J (1949).
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U n i t e d S t a t e s Pharmacopeia D i s p e n s i n g I n f o r m a t i o n , p. 354, Mack P r i n t i n g Co., E a s t o n , P A (1980). USAN and t h e USP D i c t i o n a r y of Drug Names, p. 244, M. G r i f f i t h s , Ed., Mack P r i n t i n g Co., E a s t o n , PA (1980). Merck I n d e x , 9 t h Ed. p. 918, No. 6880, M. Windholz, Ed., Merck and Co., I n c . , Rahway, N J (1976).
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D. Crowfoot, C. W. Bunn, B. W. Rogers-Low, and A. Turner-Jones, The Chemistry of P e n i c i l l i n , p. 319, H. T. C l a r k e , J. R. Robinson, and R. Robinson, P r i n c e t o n U n i v e r s i t y P r e s s , P r i n c e t o n , N J (1949). S . N. Rao, R. P a t h a s a r a t h y and F. E. C o l e , Acta C r y s t . B 29, 2373 (1973)
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A l d r i c h L i b r a r y of I n f r a r e d S p e c t r a , 2nd Ed., p. 313, C. J. P o u c h e r t , Ed., A l d r i c h Chemical Co., L t ' d . , M i d d l e s e x , N J (1978).

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25, 285 -
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25.
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2,
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28.
29.
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5,
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PENICILLAMINE
631
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633
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a,
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L a u r i e , J. Chem. SOC., (Dalton) 1389 (1975).
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635
97. 98.
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L a u r i e , and B.
S a r k a r , J. Chem. SOC., 1822 (1977).
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102.
A. M. C o r r i e , M. L. D. Tonche, and D. R. W i l l i a m s , J. Chem. SOC. ( D a l t o n ) , 2561 (1973).

S.
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104.
Wandiga, J. Chem. SOC. ( D a l t o n ) , 1894 (1975).
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T. F a i r b u r s t , J. Am. Chem.
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J. C. Crawhall, E. F. Scowen, and R. W. Watts, Br. Med. J., 1 , 588 (1953). J. E. Boulding and R . A. Baker, lan c e t , 2, 985 (1957). S.
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637
132.
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(1965). R. A. Veis, I. A. Storozhev, L. E. Mogilevchik, Y . 0. Sazykin, R. M. Voronina, and V. B. Korchagin, Antibiotiki, 1 4 , 839 (1969).
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(1948).
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L i t e r a t u r e reviewed t o October, 1980.
Acknowledgement The a u t h o r s wish t o thank M r . R. Salkot of FDA, Bureau of Drugs Medical L i b r a r y ( R o c k v i l l e , Maryland) f o r l i t e r a t u r e r e t r i e v a l work, D r . L. V. Feyn f o r obtahning t h e NMR s p e c t r a , M r . T. J. Nowak f o r o b t a i n i n g t h e I R spectrum and F r e d e r i c k Cancer Research Center ( F r e d e r i c k , Maryland) f o r t h e l i b r a r y f a c i l i t i e s . Ching Ching Chiu wishes s p e c i a l l y t o thank D r . Sy-rong Sun f o r h i s support and c o o p e r a t i o n d u r i n g t h e p r e p a r a t i o n of t h i s manuscript.
PROBENECID
Abdullah A . Al-Badr and H . A . El-Obeid
1. Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance 2. Physical Properties 2.1 Melting Point 2.2 Solubility 2.3 Identification 2.4 Spectral Properties 3. Synthesis 4. Absorption, Metabolism, and Excretion 5. Methods of Analysis 5.1 Titrimetric Method 5.2 Spectrophotometric Methods 5.3 Chromatographic Methods Acknowledgements References 640 640 640 640 640 640 64 1 641 641 64 1 641 646 652 655 655 655 658 661 662
Copyright t, i9Xi hg AradcoricPre\a. Inc. All right, of reproduction i n any form rewned ISBN 0-12-2608113-0
640
ABDULLAH A. AL-BADR AND H . A. EI,-OREID
1. Description
1.1
Nomenclature 1.1 1 Chemical Names
4-[ (Dipropylamino) -sulfonyl]-benzoic acid p-(Dipropylaminosulfony1)-benzoio acid. p- (Dipropylsulfamyl)-benzoic acid. 4-(Dipropylsulfamoyl)-benzoic acid. 4-(Dipropylsulfamy1)-benzoic acid. 1.1 2 Generic Name
Probenecid 1.1 3 Trade Names Benacen, Benemid, Benemide, Benn, Probalan, Probecid, Proben, Probenid, Robenecid, Uricocid. It is also an ingredient of Amcil-GC, Benn-C, Colbenemid, Polycillin-PRO, Probampacin, Prolongine, Principen with Probenecil, Robenecol and Robenecid with Colchicine. 1.2 ___Formulae 1.2 1 EmDirical
1.2 2 Structural
0
tI0 - C
It
0
II
CH CH CH
2 2
1.2 3 Wiswesser Line Notation QVR DSWN3&3 1.3 Molecular Weight 285.36 1.4 Elemental Composition C 54.72%, S 11.23%. (1)
H 6.71%,
N 4.91%,
0 22.43%,
1.5 Appearance White o r almost white crystalline powder.
PROBENECID
641
2.
Physical ProDerties 2.1 Melting Point Melts between 1 9 8 ' 2.2 Solubility Practically insoluble in water, soluble in 25 parts of alcohol (95%), and 12 parts of acetone; also soluble in chloroform and in dilute solutions of alkali hydroxides and of sodium hydrogen carbonate; insoluble in dilute mineral acids (2,4). 2.3 Identification 2.3 1 Infrared Spectroscopic Test B.P. 1973 (2) and U . S . P . XIX (5) cite the use of infrared absorption spectrum of probenecid as a mean of identification comparing some characteristic absorption bands of the drug. This will be discussed in the infrared spectral properties of the drug. 2.3 2 Ultraviolet Spectroscopic Test B.P. 1973 (2) and U.S.P. XIX (5) cite the use of ultraviolet absorption spectrum of probenecid in alcohol (95%) as a mean of identification comparing some characteristic absorption maxima of the drug. This will be discussed in the ultraviolet spectral properties of the drug. 2.3 3 Thin Layer Chromatographic Test B.P. 1973 (2) describes a thin layer chromatographic method or the identification of probenecid comparing the principal spot in the chromatogram of the substance being examined with that of probenecid obtained under identical conditions. 2.3 4 Melting Point Test B.P. 1973 (2) also uses the melting point of the drug as a mean of identification. 2.4 Suectral ProDerties 2.4 1 Ultraviolet Spectrum The ultraviolet spectrum of probenecid in neutral methanol solution in the region of 200 t o 350 nm exhibits two maxima at 224 nm and246 nm and two minima at 214 and 233 nm The spectrum is shown in Figure 1. According to B.P. 1973 the spectrum of and 2 0 0 ' (2-4).
642
ABDULLAH A. AL-BADR AND H. A. EL-OBEID
FIGURE 1.
U l t r a v i o l e t spectrum of probenecid i n n e u t r a l methanol.
PROBENECID
643
probenecid in ethanol shows maxima at 225 nm and 248 nm ( E 1%, lcm 330). The ultraviolet absorption spectrum of the drug is used as a mean of identification of probenecid in B.P. 1973 and I1.S.P. XIX. According to the B.P. a 2cm layer of 0.0015% w/v solution in alcohol (95%) exhibits two maxima, at 225 nm and 248 nm; extinction at 248 nm, about 0.99. 2.4 2 Infrared Spectrum The infrared spectrum o f probenecid is shown in Figure 2. The spectrum was obtained from nujol mull. The structural assignments have been correlated with the following band frequencies: -1 Frequency (cm ) Assignments 1705 1610, 1583 1295, 1315
1160 0 Vibration C Stretch of ayomatic ring. Asymmetric-sO -Stretch 2 Symmetric-S02-Stretch
= =
C C
770, 780
CH deformation
The infrared absorption spectrum of the drug is used as a mean of identification of probenecid in both B.P. 1973 a . n d U . S . P . XIX. 2.4 3 Proton Nuclear Magnetic Resonance Spectrum(PMR) A typical PMR spectrum of probenecid is shown in Figure 3. The sample was dissolved in acetone-d6. The spectrum was determined on a Varian-T6BA NMR spectrometer with TMS as the internal standard. The following structural assignments have been made for Figure 3: Chemical shift (6) 8.07 3.17 1.62 0.87 Assignments Aromatic -CH2CH2CH3
- CH2CH2CH3
- CH2 CH2CH,
2.4 4 13C Nuclear Magnetic Resonance Spectrum (13CNMR) The I3C NMR spectrum of probenecid in acetone-d6 using tetramethylsilane as an internal reference is obtained on a
644
.
I
' . l ' . . . , . - . . l . . . . , . . . .
. . _ .. _ . . ,
I
480
i.0
(b
FIGURE 3.
PMR Spectrum o f p r o b e n e c i d i n a c e t o n e - d
as i n t e r n a l s t a n d a r d .
w i t h TMS
646
ABDULLAH A. AL-BADR AND H. A . EL-OBEII)
Jeol FX100, 100 MHz instrument at an ambient temperature and using 10 mm sample tube. The spectrum is shown in Figure 4 and the carbon chemical shift values, shown in Table 1 , are derived from the off-resonance spectrum. Table
1
I3C NMR Characteristics of Probenecid

Carbon No. Chemical Shift (ppm) 50.87 22.75 11.30 50.87 22.75 11.30 145.46 Carbon No.
8 9
Chemical Shift (ppm) 131.03 127.91 134.73 127.91 131.03 166.31
1
2 3
10
4
5
11
12 13
2.4 5 Mass Spectrum and Fragmentometry The mass spectrum of probenecid (Figure S ) , obtained by electron impact ionization, using Nermag GC-Mass spectrometer model R 1010 shows, a molecular ion M+ at m/e 285 (relative intensity 4.1%) and a base peak at m/e 256. Table 2 shows the proposed fragmentation of probenecid. 3. Synthesis Probenecid can be synthesized by one of the following methods : a) Di-n-propylamine is condensed with p-cyanobenzenesulfonyl chloride followed by hydrolysis of the nitrile to the carboxylic acid ( 6 ) . CH CH CH / 2 2 3 N C e S O 2 C l
+
HN'CH2CH2CH3
TMS
I
PPM ( 6 )
F I G U R E 4.
l3C NMR Spectrum o f p r o b e n e c i d i n acetone-d6 w i t h TMS i n t e r n a l reference.
Figure 5.
Mass Spectrum of probenecid (EI)
PROBENECID
649
Table 2 Proposed fragmentation of Probenecid
(El)
ion
m/e
287 286 285 268
Relative Intensity%
2.0 10.2 4.1

10.9
M+2
M+ 1 M+
OC
CH CH CH / 2 2 3 N\CH CH CM 2 2 3
1 '
256
100 O S @ C O O H
N / CH2
\C H ~ C H ~ C H ~
2 14
4.7
HOCC @O2N-CH2
I+
185
25.1
HOOC-@-
SO2 l+
121
39.2
HOG@
'?
650
NC
0'
SO2-
I I C I I CH / C 2 2 3 CH2CH2CH3
L -
HOOC
a
0
~
1-N
'
CH2C112CH3
CH2CH2CH3
b)
Di-n-propylamine is condensed with p-carboxybenzenesulfonyl chloride (6)
/ CH2CH2CH3
S-N
0
___)
HOOC
'
CH2CH2CH15
c)
Toluene is treated with chlorosulfonic acid, and the resulting p-toluenesulfonyl chloride is hydrolysed to p-toluenesulfonic acid. Oxidation of the methyl group with potassium permanganate produces a p-carboxybenzenesulfonic acid. This acid is then converted into the corresponding sulfonyl chloride by treatment with chlorosulfonic acid. The resulting sulfonyl chloride is then reacted with di-n-propylamine. Crude probenecid is precipitated by pouring the reaction mixture into cold water. The precipitate is collected, washed and purified by recrystalli4 ) . zation from a suitable solvent such as alcohol (
PROBENECID
651
CH3
-@)+
HOOC
s03
KMn04
HOOC
-@-SO3
C1S03H
-@- s O z c 1
NH(CH2CH2CH3)
t
HOOC
1
0
0
CH2CH2CH3
\ ~ H ~ C H ~ C H ~
d)
p-Toluenesulfonyl chloride yields p- (chlorosulfonyl) benzoic acid. The latter added in portions to a cooled stirred solution of di-n-propylamine in dry acetone, stirring continued for 30 minutes, the suspension concentrated on steam bath to one-third volume, the residue poured into cold water, the solution acidified with dilute H C 1 , the crude product filtered, decolorized in dilute sodium bicarbonate solution, filtered and the filtrate acidified with an excess of dilute H C 1 to give probenecid ( 7 )
HOOC
0
O C
SO C1 2
NH(CH2CH2CH3)
/ C H
CH CH CH 2 2 3 2 CH 2 CH 3
e 0 I! N I
652
4.
Absorption, Metabolism and E x c r e t i o n Probenecid i s r e a d i l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t and i s e x t e n s i v e l y bound t o plasma p r o t e i n s . The drug i s s l o w l y m e t a b o l i z e d and e x c r e t e d i n u r i n e ( 8 ) . The metabolism of t h e drug h a s been e x t e n s i v e l y s t u d i e d i n s e v e r a l s p e c i e s i n c l u d i n g humans. Dayton e t .a1 (9) s t u d i e d t h e p h y s i o l o g i c a l d i s p o s i t i d i o f t h e drug i n c l u d i n g r e n a l c l e a r a n c e i n man. The d r u g f w a s found t o be r a p i d l y and almost completely absorbed from t h e human d i g e s t i v e t r a c t . The h a l f - l i f e , as e s t i m a t e d by r a t e o f plasma level d e c l i n e of t h e d r u g , was t h e same, whether i t was given o r a l l y o r i n t r a v e n o u s l y ; among 9 s u b j e c t s given 2 gm., t h e h a l f - l i f e ranged from 4 t o 17 h o u r s , l e s s t h a n 5% of t h e dose was e x c r e t e d i n t h e u r i n e a s unchanged form of t h e drug i n 24 h o u r s . The remainder must have been t r a n s f e r r e d t o unknown m e t a b o l i t e s o r conjugates. Other s t u d i e s a r e p u b l i s h e d by P e r e l e t . a l (10) f o r t h e i d e n t i f i c a t i o n and r e n a l e x c r e t i o n o f probenecid [I] metabol i s m i n man. The drug g i v e n o r a l l y t o man was e x c r e t e d i n t h e u r i n e m o s t l y as t h e probenecid monacylglucuronide [V] (40%) and o t h e r m e t a b o l i t e s ( F i g u r e 6 ) . The m e t a b o l i t e s were e x c r e t e d mostly i n t h e free form [ I ] , a small amount (3.4%) of t h e secondary hydroxy [11] and N-depropylated [ IV] m e t a b o l i t e b e i n g p r e s e n t p r o b a b l y as B-acylglucuronides. M e l e t h i l and Conway (11) have used a gas l i q u i d chromatog r a p h i c a s s a y t o s t u d y t h e e x c r e t i o n of p r o b e n e c i d and i t s m e t a b o l i t e s i n t h e u r i n e of human s u b j e c t s . The major m e t a b o l i t e , probenecid a c y l g l u c u r o n i d e [V] accounted f o r 34-47% o f t h e dose g i v e n . Approximately e q u a l amounts (10-15%) o f t h e mono-N-propyl [ I V ] , secondary a l c o h o l [ 111 and c a r b o x y l i c a c i d [111] m e t a b o l i t e s were e x c r e t e d i n t h e unconjugated forms with o n l y t r a c e s i n t h e c o n j u g a t e d forms. The u r i n a r y e x c r e t i o n o f t h e unchanged drug i s dependant on b o t h t h e pH and f l o w r a t e of u r i n e . P e r e l e t . a l (12) a l s o p u b l i s h e d a s t u d y on r e n a l e x c r e t i o n o f probenecid a c y l g l u c u r o n i d e [V] i n man. Following a s i n g l e o r a l dose t o both normal and gouty s u b j e c t s , about 25% of t h e probenecid was c o n v e r t e d t o i t s a c y l g l u c u r o n i d e and o n l y a small amount o f t h e drug was e x c r e t e d unchanged. Dayton and P e r e l (13) have s t u d i e d t h e metabolism o f probenecid i n man. Q u a n t i t a t i v e TLC showed t h a t t h e d r u g ' s metabolism i n man y i e l d e d monohydroxylated d e r i v a t i v e a t the secondary [II] ( 7 . 2 - 12.5%) and t e r m i n a l [ V I ] (1.6-3.7%) p o s i t i o n s o f p r o p y l s i d e c h a i n and carboxy [111] ( 6 . 3 9 . 2 % ) and N-depropylated [IV] ( 4 . 6 - 8 . 0 % ) compounds.
PROBENECID
653
HO
CH,CH,CH,
CH2CH2CH3 HO-C
I11
! G
CH2CH2COOH CH2CH2CH3
rv
!!
V
0 [I - N < H
Probenecid monoacylglucuronide
VI
Figure 6. Metabolites
of
Probenecid.
654
These metabolites were excreted mainly in the free forms with 3.4% of secondary hydroxy and N-depropylated derivatives probably present as B-acylglucuronides. No phenolic metabolites were formed probably due to electron deactivation of the ring. Guarino and Schanker (14) studied the billiary excretion of probenecid and its glucuronides. In rat, the drug administered with ligated renal pedicles, readily appeared in the bile as the unchanged drug and as the glucuronide of a metabolite of the drug. Gigon and Guarino (15) investigated the uptake of probenecid by rat liver slices. The uptake of p r ~ b e n e c i d - ~ by ~C rat liver was studied by standard tissue slice method. No metabolites of this transport inhibitor accumulated in the slices, but the parent drug and a single metabolite appeared in the incubation media. The metabolite was identified by GLC/MS procedure as the glucuronide of side chain-hydroxylated probenecid. Conway and Melethil ( 1 6 ) studied the excretion of probenecid and its metabolites in bile and urine of rat. Within 8 hours after i.v. administration of the drug to anesthetized rats, 63.8% of the dose was accounted for in the bile, consisting of (as % of dose) the drug (lo%), probenecid glucuronide (15.7%), glucuronideof[II](20.3%), glucuronide of [VI] (14.2%) and p- (N-propyl-N-2-carboxyethyl sulphamoyl) benzoic acid [111] (3.6%). Ligitation of the renal pedicles increased excretion of all metabolites to a total recovery of 86.6%. p-(N-Propylsulphamoyl) benzoic acid [ IV] , unconjugated N-2-hydroxy propyl [ 111 ,N-3-hydroxypropyl [VI], and N-2-carboethoxy derivative [111] of the drug. Neither the drug nor its glucuronide were found in the urine of probenecid-treated rats. Cunningham et.al (17) have studied the _ in vitro metabolism of probenecid in rat, mouse and human liver preparations and the factors affecting the site of oxidation. The metabolism was limited to the side chain, involved mono-Ndepropylation and hydroxylation in the 2 and 3 positions (3-hydroxyprobenecid was in part converted to the carboxy metabolite). Acylglucuronide conjugation was minimal. Balogh et.al (18) have reported the pharmacokinetics of probenecid during the neonatal period. The drug was reported to have eliminated from the serum more slowly in 5and 15-days old rats than in older rats. The drug was eliminated entirely in the form of metabolites in which four were detected in urine. The drug was excreted in both urine and bile. Alkalinization of the urine accelerated the drug excretion in adult rats.
PROBENECID
655
5. Methods of Analysis 5.1
Titrimetric Method
B . P . 1973 (2) and U.S.P. X I X (5) describe for the determination of probenecid powder a titrimetric assay method based on neutralizing the carboxylic acid group of the drug with standard alkali.
Spectrophotometric Methods 5.2 1 Ultraviolet Spectrophotometric Method a)
B.P. 1973 (2) uses f o r the assay of probenecid tablets a spectrophotometric method which is based on the extraction of drug with acidified alcohol and measuring the extinction of a 1-cm layer of the resulting solution at a maximum at about 248 nm. The amount of the drug is calculated taking 332 as the value of E ( I % , lcm) at the maximum at about 248 nm.
The spectrophotometric method of U.S.P XIX (5) for the determination of probenecid in tablets utilizes chloroform as the solvent for extraction and the absorbance is determined in 1-cm cells at the wavelength of maximum absorbance at about 257 nm. Tillson et.al (19, 20) described a method for the determination of probenecid in plasma and urine. According to this procedure the drug is extracted into chloroform from the acidified fluid, then re-extracted into dilute aqueous sodium hydroxide solution and determined electro photometrically in the ultraviolet region at 242.5 nm. The extinction coefficient in g/1 is 36.43. The recovery i s 95% or better. A reagent blank is run alongside and should not have an optical density greater than 0.50. As the method is not specific, blanksshould be obtained from each patient before the drug is administered . Tillson et.al (19) reported a colorimetric method for the estimation of the drug in body fluids suitable for clinical purposes as it is unaffected by most medicaments and endogenous metabolic products. In
b)
c)
5.2 2
Colorimetric Methods a)
656
t h i s method t h e drug i s e x t r a c t e d i n t o chloroform from t h e a c i d i f i e d f l u i d , and t h e s e p a r a t e d chloroform l a y e r i s shaken w i t h an aqueous s o l u t i o n o f methylene b l u e . The c o l o r e d s a l t formed i s s o l u b l e i n chloroform and t h e amount formed can be determined c o l o r i m e t r i c a l l y a t 635 nm. b) Beltagy e t . a l (21) a l s o r e p o r t e d a similar c o l o r i m e t r i c method or t h e d e t e r m i n a t i o n of p r o b e n e c i d . I t i n v o l v e s t h e t r e a t m e n t o f t h e drug w i t h a NaH2P04/Na2 HP04 b u f f e r (PH 6 . 8 ) , methylene b l u e and chloroform, t h e n measuring t h e absorbance of t h e chloroform e x t r a c t o f t h e complex. The maximum a b s o r p t i o n (Xmax) was 640 nm. T h i s method i s u s e f u l f o r small amounts o f t h e drug (> - 0 . 1 mg) . Wahbi e t . a l (22) p u b l i s h e d a c o l o r i m e t r i c method f o r t h e e s t i m a t i o n of probenecid i n t a b l e t s which i s based on t h e u s e of b a s i c f u c h s i n a s an i o n - p a i r i n g r e a g e n t f o r t h e a c i d i c d r u g . According t o t h i s method t h e a c t i v e drug was e x t r a c t e d from t a b l e t s , and t h e drug aqueous s o l u t i o n a t pH 7 t r e a t e d w i t h aqueous b a s i c - f u c h s i n . The s a l t was e x t r a c t e d i n t o chloroform and t h e absorbance measured a t 550 nm, and t h e c o n c e n t r a t i o n o f t h e drug c a l c u l a t e d from t h e c o r r e s p o n d i n g c a l i b r a t i o n graph. B e e r ' s Law i s obeyed f o r 20 t o 80 ug of p r o b e n e c i d i n 50 m l o f chloroform s o l u t i o n . The method i s a p p l i c a b l e t o t h e bulk drug or t o tablets. t d)
A t e c h n i q u e f o r i d e n t i f y i n g probenecid i n
t a b l e t dosage form by s p o t t e s t on p a p e r had been u t i l i z e d by Cooper ( 2 3 ) . The t e c h n i q u e was based on t h e r e a c t i o n o f t h e drug w i t h f e r r i c i r o n and dimethylaminobenzaldehyde t o produce c o l o r e d p r o d u c t s . The two r e a g e n t s used f o r c o l o r p r o d u c t i o n a r e , a s l i g h t l y a c i d i f i e d aqueous s o l u t i o n of f e r r i c c h l o r i d e and an a c i d i f i e d e t h a n o l i c s o l u t i o n of p-dimethylaminobenzaldehyde ( E h r l i c h ' s r e a g e n t ) . The i n t e r f e r i n g f a c t o r s and t h e c o l o r o b t a i n e d by r e a c t i o n o f t h e s e reagents w i t h probenecid a r e shown i n Table 3.
PROBENECID
657
Table 3 C o n d i t i o n s f o r Spot I d e n t i f i c a t i o n o f Probenecid Conditions Blank-Reagents d r i e d on Paper 5% KOH

10% H C 1
F e r r i c Chloride Reagent p a l e yellow ochre no c o l o r no c o l o r ochre
Ehrl i c h Reagent p a l e yellow no c o l o r no c o l o r no c o l o r primose
Excipients Probenecid + a c i d i f i e d reagent 5.2 3
Spectrophotofluorometric Method
A spectrophotofluorometric a s s a y f o r probenec i d was p r e s e n t e d by Cunningham e t . a l ( 2 4 ) . The method was based on c o n v e r s i o n o f t h e drug t o a fluorescent anthranilic acid derivative. Blood plasma o r c e r e b r o s p i n a l f l u i d was a c i d i f i e d w i t h 3 N-hydrochloric a c i d and e x t r a c t e d w i t h 1 , 2 - d i c h l o r o e t h a n e . An a l i q u o t o f t h e o r g a n i c l a y e r was e v a p o r a t e d t o d r y n e s s w i t h a i r stream a t room t e m p e r a t u r e and t h e r e s i d u e was c o n v e r t e d t o t h e a n t h r a n i l i c a c i d d e r i v a t i v e . The f l u o r e s c e n c e i s measured a t t h e emission wavelength o f 415 nm w i t h e x c i t a t i o n a t 340 nm. The f l u o r e s c e n c e i s d i r e c t l y p r o p o r t i o n a l t o t h e amount of probenecid from 4 t o 100 ug. The a s s a y was e s p e c i a l l y a p p l i c a b l e w i t h c l e a n b i o l o g i c a l f l u i d s (such as c e r e b r o s p i n a l f l u i d and blood) and o f f e r e d s e v e r a l f o l d g r e a t e r s e n s i t i v i t y than t h e commonly used u l t r a v i o l e t methods. P e n t o b a r b i t o n e , s a l i c y l a t e s and m e t a b o l i t e s of probenecid did not i n t e r f e r e .
5.2 4
Molecular Emission C a v i t v Method Al-Abachi (25) d e s c r i b e d a m o l e c u l a r emission c a v i t y a n a l y t i c a l (MECA) method f o r t h e d e t e r m i n a t i o n of s u l f u r c o n t a i n i n g d r u g s , amino a c i d s and p r o t e i n s from t h e i r S2 emission a t 384 nm. Probenecid gave tms o f 1 . 4 5 , c a l i b r a t i o n range of 5-100 ng S , l i m i t o f d e t e c t i o n o f 2 . 0 ng S and c o e f f i c i e n t v a r i a t i o n o f 3.10%.
658
ABDULLAH A. AL-BADR AND H. A. EL-OREID
5.2 5 Nuclear Magnetic Resonance S p e c t r o m e t r i c Method A n NMR procedure i s d e s c r i b e d by which probenec i d i s determined i n p u r e and t a b l e t f o r m u l a t i o n . The method i s r a p i d , a c c u r a t e and p r e c i s e (26) ( s . d . 0 . 7 3 and l . l l ) , and a l s o p r o v i d e a method o f i d e n t i f i c a t i o n o f t h e drug. The spectrum was run i n acetone-dg w i t h t h e u s e o f m a l e i c a c i d a s an i n t e r n a l s t a n d a r d u s i n g t h e a r o m a t i c p r o t o n s of probenecid a t 8.07 ppm and t h e s i n g l e t a t 6 4 2 ppm o f t h e methylene p r o t o n s o f t h e m a l e i c acid (the i n t e r n a l standard) a s t h e c r i t e r i a f o r analysis.
5 . 2 6 Mass S p e c t r o m e t r i c Method
Probenecid was assayed by s e l e c t i v e i o n monitori n g (m/e 388) u s i n g m- ( d i i s o b u t y l s u l f o n y l ) benz o i c a c i d a s an i n t e r n a l s t a n d a r d (27). The s t a n d a r d curve was l i n e a r over t h e range 4-20 1.18 p r o b e n e c i d a n d t h e p e r c e n t a g e recovery o f t h e drug was 92.7%. The method was a p p l i e d f o r d e t e r m i n a t i o n o f probenecid i n human c e r e b r o s p i n a l f l u i d and could be i n c o r p o r a t e d i n t o t h e s e l e c t i v e ion monitoring procedure used t o q u a n t i t a t e o t h e r a c i d i c and n e u t r a l compounds on such samples. 5 . 3 Chromatographic Methods 5.3 1 Thin Layer Chromatography (TLC) a)
A TLC procedure appears i n B . P . 1973 (2) f o r t h e i d e n t i f i c a t i o n of probenecid and t o t e s t f o r r e l a t e d s u b s t a n c e s . The method u t i l i z e s g e l G/UV 254 a s t h e c o a t i n g s u b s t a n c e and a mixture o f a l c o h o l and ammonia a s t h e mobile phase. A f t e r d r y i n g , t h e chromatogram i s examined under u l t r a v i o l e t lamp having a maximum o u t p u t a t about 254 nm.
Gecgil (28) r e p o r t e d a TLC method f o r t h e i d e n t i f i c a t i o n o f probenecid and r e l a t e d s u l fonamide-type d i u r e t i c s i n t a b l e t s and suppos i t o r i e s . The d r u g s a r e e x t r a c t e d from t a b l e t s and o t h e r pharmaceutical p r e p a r a t i o n s w i t h e t h a n o l o r a c e t o n e and t h e e x t r a c t s a n a l y s e d by TLC on s i l i c a g e l u s i n g e t h a n o l chloroform-heptane ( 1 :1:1) a s s o l v e n t . The
b)
PHOBENECID
659
s p o t s a r e v i s i b l e i n UV l i g h t and g i v e c o l o r r e a c t i o n s when sprayed w i t h p e n t a c y a n o n i t r o s y l f e r r a t e - 1 1 1 (Sodium n i t r o p r u s s i d e ) i n NaOH, o r w i t h t h e Van Urg r e a g e n t (dimethylaminobenzaldehyde i n HC1 and 95% e t h a n o l ) , o r Bratton-Marshall r e a g e n t (5% NaNO2 + 1 % N- (l-naphthyl)ethylenediarnine-2HCl) o r with Hg*(N02)2 ( o b t a i n e d by t r e a t i n g an e x c e s s of Hg with HN03). The Rf v a l u e o f probenecid i s 0.98. c) Another TLC method (29) i s used t o i d e n t i f y nonmercurial d i u r e t i c s on s i l i c a g e l u s i n g toluene, xylene, 1,4-dioxane, isopropyl a l c o h o l , 25% NH40H (10:10:30:30:10) a n d . p r o p y l a l c o h o l - e t h y l a c e t a t e - w a t e r - 2 5 % NH40H (50:10:30:10) a s s o l v e n t s and a 5 : l m i x t u r e o f 10% CaS04 and 2% NH40H a s t h e s p r a y i n g agent which g i v e s a f t e r h e a t i n g t o l l O e f o r 1 0 min, b l u e o r brown c o l o r r e a c t i o n s with s u b s t a n c e s c o n t a i n i n g amido groups. With t h e two s o l v e n t s probenecid had Rf values of 0 . 5 and 0.78 r e s p e c t i v e l y .
5 . 3 2 Gas -Liquid Chromatography (GLC)
a)
A method (30) i s r e p o r t e d f o r t h e determina-
t i o n of probenecid i n c e r e b r o s p i n a l f l u i d which employs g a s - l i q u i d chromatography and e l e c t r o n c a p t u r e d e t e c t i o n (GLC-ECD) The drug i s r e a c t e d with t r i f l u o r o a c e t i c anhyd r i d e and p e n t a f l u o r o p r o p a n o l t o o b t a i n pent a f l u o r o p r o p i o n a t e e s t e r . The high s e n s i t i v i t y of ECD f o r t h i s d e r i v a t i v e allows f o r t h e d e t e r m i n a t i o n of probenecid i n a s l i t t l e a s 20 ~1 o f c e r e b r o s p i n a l f l u i d .
b)
A q u a n t i t a t i v e method f o r t h e d e t e r m i n a t i o n
o f t h e drug i n b i o l o g i c a l f l u i d s i s a l s o d e s c r i b e d by Zacchei and Weidner ( 3 1 ) . The method employs t h e N-dibutyl analog o f probenecid a s t h e i n t e r n a l s t a n d a r d t o be added t o plasma o r u r i n e samples followed by a c i d i f i c a t i o n and e x t r a c t i o n i n t o benzene. The a c i d s t h u s e x t r a c t e d were converted t o methyl e s t e r s by r e a c t i o n with e t h e r e a l diazomethane and a n a l y s e d by gas chromatography. As l i t t l e a s 250 P g o f probenecid could be d e t e c t e d i n 1 m l o f plasma.
660
c) A gas-liquid chromatographic assay for probenecid and its metabolites and the utilization of this method for the examination of the disposition of the drug in rats and human subjects is developed by Melethil (32). d) A sensitive gas chromatographic (GC) method for the measurement of probenecid in biological fluids is described by Sabih ( 3 3 ) . The method involves the conversion of probenecid to its methyl ester by treatment with dimethylsulfate under basic conditions. Analysis was performed on Gas Chrom. Q coated with DC-200. The major metabolic product of probenecid, the glucuronide conjugate o f the unchanged drug, was identified by combined GC and high resolution mass spectrometry following enzymic hydrolysis. e) Conway and Melethil (34) described a gas chromatographic assay method f o r probenecid and its metabolites in biological fluids. The method is capable of monitoring concentrations of the drug and its metabolites in urine at least 3 days after oral injection of 0.5 g of probenecid. According to the method the free acids, o r the acids liberated by acid hydrolysis, were extracted from acidified urine with methylene chloride. After the addition of N, N-dibenzyl-2,5-dimethylbenzenesulfonamideas internal standard, the acids were determined by GLC on a stainless steel column (6 ftx0.125 in.) packed with 10% of OV-1 on Chromosorb W-HP (80 to 100 mesh) and operated at 250, with N as carrier gas (23 ml min-l) and a flame ionization detector. Calibration graphs were rectilinear for 25 to 700 1 . 1 8of probenecid o r metabolite. RecoveriPs of the N-monopropyl, N-carboxyethyl andN -(2-hydroxypropyl) -metabolites were in the range of 10 to 400 pg per 2 ml o f urine.
5 . 3 3 High Pressure Liquid Chromatography (HPLC)
a) An HPLC method (35) for the determination of probenecid in oral suspensions of ampicillin gave good intra- and interlaboratory reproducibility and accuracy; and interference from excipients and flavour ingredients was
PROBENECID
66 1
e l i m i n a t e d . Average r e c o v e r i e s ranged from 95.2 t o 99.1% with c o e f f i c i e n t of v a r i a t i o n from 1 . 6 3 t o 4 . 9 % . I n s t e a d of an i n t e r n a l s t a n d a r d , use of a c l o s e d - l o o p i n j e c t i o n system (or o t h e r means o f o b t a i n i n g a c o n s t a n t i n j e c t i o n volume) i s recommended. I t i s a l s o recommended t h a t t h i s method r e p l a c e s p r e s e n t e x t r a c t i o n methods f o r o r a l s u s p e n s i o n s i n t h e Code o f F e d e r a l R e g u l a t i o n s and o t h e r o f f i c i a l compendia. b)
A h i g h performance l i q u i d chromatography method (36) was a l s o used t o d e t e r m i n e prob e n e c i d i n serum. S a t i s f a c t o r y r e s u l t s were o b t a i n e d o v e r t h e normal t h e r a p e u t i c r a n g e o f drug ( 5 1 5 0 mg/ml). The s e n s i t i v i t y o f t h e p r o c e d u r e was about 1 ug/ml. N e i t h e r t h e m e t a b o l i t e s of probenecid n o r e p i c i l l i n , which i s f r e q u e n t l y co-administered w i t h t h e d r u g , i n t e r f e r e d w i t h t h e chromatographic b e h a v i o u r of p r o b e n e c i d .
ACKNOWLEDGEMENTS The a u t h o r s wish t o acknowledge t h e t e c h n i c a l a s s i s t a n c e o f Mr. K h i d i r H. Babiker, M r . Khalid N . K . Lodhi and Mr. W . Dessanayke i n running t h e s p e c t r a and M r . A l t a f H. Naqvi for t y p i n g t h e m a n u s c r i p t .
662
REFERENCES
1. CRC, Atlas of S p e c t r a l Data and Chemical C o n s t a n t s f o r Organic Compounds, 2nd Ed., p u b l i s h e d , CRC Press, I n c . Vol. 11, p . 436.
2 . B r i t i s h Pharmacopoeia, London, Her M a j e s t y ' s S t a t i o n e r y Office, (1973), p.384.

3. E.G.C. C l a r k e , " I s o l a t i o n and I d e n t i f i c a t i o n o f Drugs", The Pharmaceutical P r e s s , London, (1969), p . 512.
4 . Remington's Pharmaceutical S c i e n c e s , Mack P u b l i s h i n g Company, Easton, Pennsylvania, 1 5 t h Ed. (1975), p . 873.

5. The United S t a t e s Pharmacopoeia X I X , United S t a t e s Pharmac o p e i a l Convention, I n c . , R o c k v i l l e , Md., 20852,(1975),p.406.
6 . C.S. Miller, U.S. P a t e n t 2 , 608, 507 (1952). 7 . Sharp and Dohme, I n c . F jrit., 674, 298 (1952). 8 . M a r t i n d a l e , The E x t r a Pharmacopoeia, The Pharmaceutical Press (London), Ed. A . Wade, 27th Ed. p . 337. 9 . P . G . Dayton, T . F . Yu, W. Chen, L . Berger, L . A . West and A . B . Gutman, J . Pharmacol. E x p t l . T h e r a p . , __ 140, 278 (1963). 10. J . M. P e r e l , R . F . Cunnigham, H.M. Life Sci., 9( 2 3 ) , 1337 (1970).
11. M. M e l e t h i l and W.D. (1976).
F a l e s and P . G .
Dayton,
Conway, J . Pharm. S c i . , 6 5 ( 6 ) , 861 Gutman, Eur. J .
1 2 . J.M. P e r e l , P . G . Dayton, T . F . Yu and A . B . C l i n . Pharmacol., 3 ( 2 ) , 106 (1971).

L~
1 3 . P . G . Dayton and J . M . 399 (1971).
P e r e l , Ann. N . Y .
Acad. S c i . , 179,
1 4 . A.M. Guarino and L.S. Schanker, J . Pharmacol. E x p t l . T h e r . , 164, 387 (1968). 15. P . L . Gigon and A.M. Guarino, Biochem. Pharmacol., - 19, 2653 (1970). 1 6 . W.D. Conway and S . M e l e t h i l , J . Pharm.Sci., 6 3 , 1551 (1974).
PRORENECID
663
17. R . F . Cunningham, J . M . P e r e l , Z . H . I s r a i l i and P . G . Dayton, Drug Metab. Dispos., 5 , 205 (1977). 18. A. Balogh, H.Braeunlich, F.K. 33, 365 (1978). Pharmazie, S p l i n t e r and J . Zaumseil,
19. E . K . T i l l s o n , G.S. Schuchardt, J . K . Fishman and K . H . J . Pharmacol. E x p t l . T h e r a p . , 111, 385 (1954). 20. E . K . T i l l s o n , N . W . Pusey and K . H . E x p t l . Therap., 1 1 2 , 252 (1954).
Beyer,
Beyer, J . Pharmacol.
2 1 . Y . A . Beltagy, S.M. Rida and A. I s s a , Pharmazie, 29, 64 (1974). 2 2 . A.M. Wahbi, H. Abdine, M . A . Analyst, 103, 876 (1978).
23. P. Cooper, Pharm. J . , Korany and M.H. Abdel-Hay,
177,495
(1956).
2 4 . R . F . Cunningham, Z . H . I s r a i l i and P . G . Dayton, J . Pharm. S c i . ,67, 434 (1978). 25. M . Q . A 1 Abachi, Proc. Anal. Div. Chem. S O ~ . , (1977). 26. H . A . El-Obeid and A . A .
14,251
Al-Badr (Unpublished work). Barchas, Biomed. Mass
27. K.F. F a u l l , J . R . DoAmaral and J . D . 5 , 317 (1978). Spectrom., -
28. S . G e c g i l , E c z a c i l i k B u l t e n i , 7 , 100 (1965). 29. K . C . Guven and S. Cobanlar, E c z a c i l i k B u l t e n i , 9 , 98 1967).
30. E . Watson and S. W i l k , J . Neurochem., 2 1 , 1569 (1973 31. A . G . Zacchei and L. Weidner, J . Pharm. S c i . , 62,1972 1973).
32. S.K. M e l e t h i l , ( S t a t e Univ. N e w York, B u f f a l o , N . Y . ) . 1973. 103 pp. Avail Univ. Microfilms, Ann Arbor. Mich., Order No.74-4427. 33. K . Sabih, J.Pharm.Sci., 34. W.D.
60, 745 (1971).
Conway and S. M e l e t h i l , J . C h r o m a t o g r . , U , 2 2 2 (1975).
35. P . J . Vollmer, T.G. Alexander and C . Haneke, J . Assoc. O f f . Anal. Chem.. 61. 687 (1978). 36. R . K . Harle and T . Cowen, A n a l y s t , 103, 492 (1978). .
SALBUTAMOL
Hassan Y. Aboul-Enein ,Abdullah A. Al-Badr, and S. E . Ibrahim
1, Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance 2. Physical Properties 2.1 Crystal Properties 2.2 Optical Rotation and Circular Dichroism 2.3 Solubility 2.4 Identification 2.5 Spectral Properties 3 . Synthesis 4. Metabolism, Absorption, and Excretion 5. Methods of Analysis 5.1 Titrimetric Methods 5.2 Spectrophotometric Methods 5.3 Chromatographic Methods 5.4 Mass Fragmentography 6. References Acknowledgements
666 666 666 666 667 667 667 667 668 669 669 670 680 683 684 684 684 685 687 688 689
ANALYTICAL PROFILES OF U R U C SUBSTANCES, 10
665
Coplright (C 1981 by AcademicPresq, Inc. All right? of reproduction in any form rmeerued ISBN U-12-260RIO-0
666
HASSAN Y . ABOUL-ENEIN et a / .
1. Description 1.1 Nomenclature __ 1.11 Chemical Names

a1- [ (tert - Butylamino) methyl] -4-hydroxy-m-xylenec1
- a-diol N-tert-Butyl-2-(4-hydroxy-3-hydroxym~.tl~ylphenyl)2-hydroxylamine
2-(tert-Butylamino)-l-(4-hydroxy-3-hydroxpethylphen y 1) - ethano 1 4-Hydroxy-3-hydroxymethyl-a-[(tert-butylamino) methyl] -benzyl alcohol a- [ (1-tert .- Butylamino) methyl] -4-hydroxy-mxylene-a-u-diol 1-(4-hydroxy-3-hydroxypheny1)-2-tert -butylaminoethanol.
1.12 Generic Names Salbutamol, Albuterol, AH 3365, Sch 13949W. 1.13 Trade Names Aerolin, Broncovaleas, Sultanol, Venetlin, Vent o l in. 1.14 Registry No. 1.2 Formulae 1.21 Emperical CA 18559-94-9.
1.22 Structural CH OH
\ 2
Ho
CHOHCH2NHC (CH3)
1.3 Molecular Weight 239.31
SALBUTAMGL
667
1.4 Elemental Composition C, 65.24

1.5 Appearance
H, 8.85
N, 5.,85 0, 20.06
White crystalline powder from ethanol-ethyl acetate o r ethyl acetate-cyclohexane (1). The powder is odorless and almost tasteless. 2. Phvsical ProDerties 2.1 Crystal Properties 2.11 Crystallinity and X-ray Crystallography Beale and Stephenson (2) had determined the X-ray crystallographic structure of certain bronchodilators which included salbutamol, they reported that the drug had conformational characteristics of a 8-adrenoreceptor stimulant. Furthermore, Beale and Grainger ( 3 ) had published the X-ray analysis of salbutamol and found that it belongs to the space group Pbca with a 21.654[10] and b 8.798 [4] c 14.565 [ 7 ] A ; Z = 8; d[obsd] ~ 1 . 1 5 , d[calcd] = 1.15.
The benzene ring is inclined at 74.5[2]' to the plane of the C[7]-C[8]-N-C[9] atoms. It was reported that the tert. butyl group is the opposite side of the salbutamol molecule to the amino and the hydroxy groups. The bond distances and angles as well as the atomic position coordinates were discussed.
2.12 Melting Point The British Pharmacopoea (B.P.) 1973 (4) specifies a m.p. of salbutamol at about 1 . 5 6 ' . Other melting ranges of salbutamol are given below: Melting range, C" 151-152 157-158 Ref.
668
HASSAN Y. ABOUL-ENEIN et al.
2.2 Optical Rotation and Circular Dichroism Hartley and Middlemiss ( 5 ) had separated the two optical isomers of salbutamol by forning a benzyl ether I1 from its synthetic precursorese I followed by the precipitation of I1 with either the (+) o r ( - ) di-p-toluoyltartaric acid. In each case only one isomer formed a crystalline salt and the antipode was recovered from the mother liquor. The purified salts were neutralized and liberated to yield the required optical isomer of salbutamol 111.
COOCH3
CH2Ph LiAlH4 CHOHCH2 NC(CH3)3 I CH2Ph
fH2Ph -- o(I> 0 CHOHCH,NC (CH3)
CH20H
CH2Ph I1
CH20H Catalytic Pd/H7 debenzylat ion
HO
-@
I11
CHOHCH2NHC(CH3)3
The circular dichroism (CD) spectral studies indicated that the (-)-isomer had the R-configuration. The (-)-salbutamol showed a clear negative cotton effect at 276-280 nm. At a lower wavelength, 220-230 nm, the curves tended towards a further negative peak although this was somewhat masked by the high aromatic absorption ( 5 ) . The physical properties of the optical isomers are shown below:
SALBUTAMOL
669
m.p.C"
C.D.
R- (-) Salbutamol 144.3 acetate monomethanolate.
-36.9 (C,0.27 H 2 0 )
S ( + ) Salbutamol acetate monomethanolate
145.7
+36.9 (C, 0.23)
2.3 Solubility
Salbutamol is soluble 1 in 70 of water and 1 in 25 ethanol, slightly soluble in ether and solube in most organic solvents (4).
2.4 Identification
The following tests are cited from B.P. 1973 (4):a) The infrared absorption spectrum exhibits maxima which are only at the same wavelength as, and, have similar relative intensities to, those of the spectrum of salbutamol A . S . b) The light absorption, in the range 230-250 nm, of a 2 cm layer of 0.004% w/v solution in 0.1 N HC1 exhibits a maximum only at 276 nm; extinction at 276 nm about 0.56. c) Dissolve I 0 mg in 50 ml of 2% w/v solution of borax, add 1 ml of 3% w/v solution of 4-aminophenazone, 10 m l 2% w/v solution of potassium ferricyanide and 10 ml of chloroform, shake and allow to separate, an orange-red color develops in the chloroform layer. d) To 2 ml of 1% w/v solution, add 2 drops of FeC13 T.S., a reddish-orange color develops which does not change on the addition of sodium hydrogen carbonate solution,
670
Furthermore, salbutamol gives a pale-yellow color with Marquis-reagent (6) . Other color tests include the following:Reagent H2S04
Co 1or
Yellow Palc - yellow
-f
Sensitivity
1.0
Pg
n2s04/ncno
Ammonium Molybdate Ammonium Vanadate Vitalis test
1.0 ug
Green yellow 0 . 1 iig Blue rim brown rim 0.1 wg Pale yellow-bright 0 . 1 iig orange
-f
Salbutamol can be identified by forming irregular plates with gold-bromide solution, sensitivity 1 : 100 ( 6 ) .
2 . 5 Spectral Properties 2 . 5 1 Ultraviolet Spectrum
Salbutamol sulphate in 0.1 N hydrochloric acid 1 cm 310) and 276 nm shows maxima at 225 nm (E 1% (El%,l cm 60) (6). In 95% neutral ethanol, sa! butamol base absorbs ultraviolet radiation at 276 nm and 278 nm as shown in Figure 1. The u l traviolet spectra of salbutamol at pH 2 276 nm) and pH 12 (Xmax296 nm) were reported ( m a x by Evans et_ a1 (7) for the comparison with the metabolite isolated in man which does not show bathochromic shift as shown in Figure 2 . The bathochromic shift observed by changing the pH from acid to alkaline condition was accompanied by hyperchromic effect.
2 . 5 2 Fluorescent Proverties
The fluorescent properties of salbutamol were et_ a1 (7) using Fluorispic l O O E studied by Evans spectrophotofluorometer at pH 7. The maximum wave length of excitation and emission reported were 230 nm and 312 nm respectively.
SALBUTAMOL
67 1
WAVE1E N S M am
Fig. 1:
Ultraviolet spectnm of rrlbutuol in 951 ethanol.
0 u-l N 0
Fig. 2 .
a t pH2 a n d pH12.
T h e u l t r a v i o l e t s p e c t r u m of s a l b u t a m o l and m e t a b o l i t e
0 a 2 N 0
6, N
SALBUTAMOL
673
2.53 I n f r a r e d Spectrum The i n f r a r e d spectrum of salbutamol base i n n u j o l mull i s given i n Figure 3. Major band assignments a r e as f o l l o w s : Frequency c m
-1
Assignment. P h e n o l i c OH, a l c o h o l i c OH and NH s t r e t c h i n g bands. Aromatic r i n g C=C s t r e t c h i n g . Phenolic C - 0 s t r e t c h i n g . Aromatic CH bending.
3320, 3200, 3160 1610 1370, 1270, 1190. 1150 and lower.
Other f i n g e r p r i n t bands c h a r a c t e r i s t i c t o s a l b u t a mol (6) (determined i n K B r d i s c ) , a r e : 1038, 1075, 1263, 1228 and 1333 cm-l, as shown i n F i g u r e 4 .
2 . 5 4 Nuclear Magnetic Resonance Spectrum
The 60 MHz PMR spectrum of salbutamol b a s e i n d e u t e r a t e d d i m e t h y l s u l p h o x i d e i s shown i n Figure 5 . The spectrum was determined i n Varian T60 A NMR spectrometer with T M S as t h e i n t e r n a l s t a n d a r d . Assignments o f t h e bands a r e as f o l l o w s : Chemi c a1 sh i f t (ppm) S i n g l e t a t 1.0 Assignment -C(CH3) 3
OH
Doublet c e n t e r e d a t 2.6
- CH - CH2 -NOH
S i n g l e t (due t o SDO s i g n a l ) overlapped by a t r i p l e t centered a t 4.50*. Singlet a t 5.03
- ECH - N -
-CH OH ( b e n z y l i c
protons)
*When salbutamol b a s e was determined i n g y r i d i n e , t h e o v e r l a p p i n g due t o HDO s i g n a l was r e s o l v e d t o g i v e a t r i p l e t a t 3.97 ppm f o r -CHOH CH2-N.
Wlre'cnY micromc rcs
A 1038 or 1075 or 1263, B 1228.

Fig. 4 .
(I'
If32
F i n g e r p r i n t s b a n d s of salbutamol ( K B r d i s c ) .
U C
SALBUTAMOL
677
Multiplet between 6.667.27
Aromatic H2, H5 and
H6 o f the aromatic
ring.
Parfitt _ et _ a1 (8) have determined the optical purities of salbutamol among other substances by NMR using the chiral lanthanide shift reagent (CLSR) by applying the base line technique. The molecular conformation of several adrenergic and 6-adrenolytic substances had been studied by NMR ( 9 ) . 2.55 Mass Suectrum and Fraementometrv The mass spectrum of salbutamol base obtained by electron-impact ionization, Figure 6, shows a molecular ion M+ at m/e 239 (relative intensity 4.3) which becomes pronounced when determined by chemical ionisation (isobutane gas) as shown in Figure 7. The medium resolution EI/MS was determined by direct inlet to Ribermag -10 Mass Spectrometer. The proposed fragmentation ions given in Table 1 are consistant with the salbutamol structure. Table (1) Mass (m/e) 241 240 239 206 Relative Intensity 3.2
9.8
M+
Ions
M+ M+
4.3
10.0
M+' - H20 & HO'
135
21.1
107
86 57
14.2
100.0
55.4
1 "
L
t
0 h
0
4
In
0,
678
50
a
Fig. 7: Mass spectrum of salbutamol (CI-isobutane C4Hlo).
680
3 . Synthesis
S e v e r a l methods have been p u b l i s h e d and p a t e n t e d f o r t h e s y n t h e s i s o f s a l b u t a m o l . They a r e summarized a s f o l l o w s : a) Salbutamol has been p r e p a r e d by Lunts et a l . (10) s t a r t i n g from t h e a p p r o p r i a t e acetophenone d e r i v a t i v e I by condensation w i t h t e r t i a r y b u t y l benzylamine t o g i v e I 1 as shown i n Scheme 1. The k e t o n i c e s t e r 11 i s
I1
LiA1H4
CH20H
CH2 Ph
THF
>
HO
&CHOHCH2
I11
NC(CH3)3
'I 2 Pd/C
CHzOH
Ho
CHOHGH2NHC (CH3)
IV
Scheme 1. reduced w i t h L i A l H 4 i n THF under n i t r o g e n t o y i e l d I11 which i s s u b s e q u e n t l y d e b e n z y l a t e d w i t h hydrogen i n t h e p r e s e n c e o f Pd/C c a t a l y s t t o g i v e salbutamol IV. b) I n 1973, Lunts and Toon (11) d e s c r i b e d a n o t h e r method f o r t h e p r e p a r a t i o n o f s e v e r a l 1-phenyl-2-aminoethanol d e r i v a t i v e s i n c l u d i n g salbutamol through r e d u c t i o n o f methyl-5-(2-amino-l-hydroxyethyl)-salicylate I with L i A l H 4 t o g i v e t h e corresponding a l c o h o l I 1 which was then c o n v e r t e d t o salbutamol I11 by t h e r e a c t i o n with t e r t i a r y b u t y l c h l o r i d e a s shown i n Scheme 2 .
SALBUTAMOL
68 1
I1
ClC(CH )
t
HO
CH20H
CHOHCHzNHC (CH3) I11
Scheme 2 . c) Okumura et a1 (12) have prepared salbutamol through the reaction of 4-hydroxy-3-hydroxy methyl benzoyl chloride I with tertiary butyl isonitrile in benzene to give I1 which is reduced with LiAlH4 to give salbutamol I11 as shown in Scheme 3.
I1
Scheme 3. d) et a1 (13) have prepared several 1-(-3-hydroxy Kyotani methyl-4-hydroxyphenyl)-Z-alkylaminoethanol derivatives including salbutamol as shown in Scheme 4.
682
CH20CH2P h HO
-@
CHO
+ (CH3)3 CNC
I
AcOH Et20
\
HO
6CH2OCH213h FHCONHC ( CH3) OCOCH3

I1 111
2-B H or L i A 1 H 4 2 6
1-hydrolysis
CH OH
HO
-@
\
II
CHOHCH2NC I (CH3)
I V
Scheme 4.
SALBUTAMOL
683
Treating the 0-protected benzaldehyde I with tertiary butyl isonitrile in the presence of acetic acid to give 11. Compound I1 is hydrolysed followed by reduction with diborane o r LiAlH4 to give 111. The latter was then subjected to hydrogenolysis with Pd/C to give salbutamol IV in about 49% yield.
4. Metabolism, Absorption and Excretion

Salbutamol is readily absorbed from the gastrointestinal tract (14). Its effect occurs within 15 minutes and lasts for about 14 h o u r s . When give by inhalation, its effect occurs within 5 minutes (14, 15). The drug is excreted in urine in about 24 hours, 50% of the dose administered by mouth or 30% o f the dose by inhalation is excreted within 4 hours (15). About 80% of the tritium-labelled salbutamol given orally, intravenously or by aerosol is excreted in urine within three days. The peak-plasma concentration o f salbutamol and its metabolites is 5.1-11.7 ug% at 2 . 5 - 3 hours after an oral dose (4 mg of salbutamol)(7). E\lanset -a1 (7) reported that salbutamol was extensively metabolized to a plar metabolite in humans, which possessed spectral and chemical properties different from the parent drug. The paper chromatographic properties of salbutamol, its glucuronide and the polar metabolite will be discussed later (see section 5 . 3 1 ) . The metabolite is a conjugate which was not hydrolysed by B-glucuronidase, sulphatase, ketodase of $-glucosidase. Thus the authors reported that the metabolite formed in man was different from that formed in rat and rabbit, i.e., the metabolite is not a conjugate of glucuronic acid, sulphate or glucose. In dog's urine, 70-90% of the drug was excreted, 10% as its glucuronide metabolite (16)) while in the rabbit and rat 90 and 40% respectively was changed to the O-glucuronide. The latter possesses neither a 6-stimulant o r a B-blocking activity (14). Salbutamol does not cross the blood-brain barrier to a significant extent, but it corsses the placenta barrier. In man, about 25% of an administered dose is metabolised to the 4-0-sulphate ester (17) which is contrary to Evans et al's finding (7) The metabolic pathway of salbutamol in different spezies is shown in Scheme 5 .
684
CHqOH H O H C @ o H
C&OH
I
OH
CH2NHC (CH3)
CII OH
o-@3
I SO H
CHOHCH2NHC(CH3)
+ Polar metabolite of
undetermined structure.
CHOH CH2NHC (CH3)3 HO CH20H
40% in rat 90% in rabbit 10% in dog
Metabolic pathway of salbutamol in different species.

Scheme 5 .
5. Methods of Analysis 5.1 Titrimetric Methods

5 . 1 1 Non-Aqueous Titration
The B.P. 1973 (5) determined salbutamol and salbutamol sulphate by the non-aqueous titration with 0.1N perchloric acid using solvent blue 19 solution as an indicator. 5.2 Spectrophotometric Methods 5.21 Colorimetric Method Salbutamol sulphate tablets were determined among other 4-substituted phenols, colorimetrically, by measuring the yellow complex formed by the reaction between the phenols and sodium cobaltinitrite. The reaction is carried in aqueous acetic acid solution and the yellow complex are extracted with chloroform and measured colorimetrically (18). Another colorimetric method reported f o r the analysis of salbutamol included its oxidation with potassium ferricyanide in the presence of N,Ndimethyl-p-phenylenediamine. The colored Froduct was extracted into CHCl and the extinction determined at about 605 nm ( ? 9 ) .
SALBUTAMOL
685
HO
CHOHCH NHC (CH ) 2 3 3
5.3.
CHROMATOGRAPHIC METHODS
5.31 Paper Chromatography C l a r k e (6) d e s c r i b e d a s o l v e n t system used f o r t h e p a p e r chromatography of salbutamol c o n s i s t i n g o f c i t r i c a c i d : H20 : n-butanol ( 4 . 8 gm: 130 m l : 870 m l ) . The drug can be d e t e c t e d under u l t r a v i o l e t o r by u s i n g potassium permanganate s p r a y . Evans e t_ a1 ( 7 ) r e p o r t e d s e v e r a l s o l v e n t systems f o r t h e s e p a r a t i o n of salbutamol and i t s metabol i t e s as shown i n Table 2 . Descending p a p e r chromatography on Whatman 3 m m p a p e r [4x 55 cm] were u s e d . The Rf v a l u e s of salbutamol and m e t a b o l i t e s were determined by radiochromatogram scanning s i n c e H3-salbutamol w a s used i n Evans et al's studies. 5 . 3 2 Thin Layer Chromatography
A t h i n l a y e r chromatographic procedure f o r s a l b u -
tamol h a s been r e p o r t e d ( 6 1 , t h e s o l v e n t system c o n s i s t s of s t r o n g ammonia s o l u t i o n : methanol (1.5 : 100) which should be changed a f t e r two r u n s . S e v e r a l v i s u a l i z i n g a g e n t s can be u s e d , e . g . potassium permanganate, iodine/CClq, Dra.gend o r f f s p r a y , p-dimethylaminobenzaldehyde s p r a y as well as u l t r a v i o l e t l i g h t .
Table (2) Solvent system. Values Salbutamol Salbutamol glucuronide

Rf
: H,O
Polar Metabolite in man 0.47

0.80
1. n-Butanol
4
: Acetic acid
1
3
1.6 0.87 0.74

0.60 0.00
0.74
0.30
2. Isopropmol
7
: Ammonia (Sp. gr. 0.88) : Ammonia (Sp.gr.O.88) : H 2 0
3. n-Butanol
0.38
10
1
: n-Butanol
: Benzene : H20
4, Methanol
4
0.75
0.90
3
: H20
I
1
0.20 0.81
0.00
0.75
0.74
5. Phenol
~
6. SO% Aq.ethano1.
1
: H20
0.82
0.78 0.58
7. n-Butanol
9
: Ethanol
Oe6*
0.19
I_
SALBUTAMOL
687
5.33 Gas Chromatography et a1 (20) quantitatively determined salMartin butamol in plasma as either its trimethylsilyl or tertiary butyl dimethylsilyl ether. The derivatives were introduced for GLC at 250' on a glass column, lm x 4mm packed with 3% OV 101 on gas chromatograph (100-120 mesh).
5.4 Mass Fragmentography
Salbutamol among other substances were quantitatively determined by mass fragmentography after gas chromatography on coated capillaries. This has been achieved with a magnetic sector-type mass spectrometer with a closed loop control of the magnetic field and a digitally controlled high voltage supply. The method can detect picogram and nanogram amounts (21). Martin et a1 (20) have developed two methods for the determination of salbutamol in human plasma using the stable isotope multiple ion recording technique. The first method involved the extraction of salbutamol from plasma as its tetraphenylboron ion pair, separated from plasma cholestrol and derivatized at its trimethylsilyl ether. The drug was determined by mass spectrometry using to measure the intensity of the fragment m/e 369. Trideuterosalbutamol was used as an internal standard. The second method involved ion-pair extraction of salbutamol into heptan-3-one. The drug was derivatized to its tertiary butyl dimethylsilyl ether and determined by GC/MS using the fragment m/e 495 and 498 (fortrideuterosalbutamol tert-butyl dimethylsilyl ether derivatives). The latter method is reported to be rapid and did not require separation o f cholesterol.
688
References
1.
Merck Index, n i n t h e d i t i o n , Merck Fr Co., I n c . , Rathaway, N . J . , U.S.A., p . 30, 206, 1976.

J.P.
2.
Beale and N.C. 277, (1972).
Stephenson; J. Pharm. Pharmacol 24,
3.
J . P . Beale, and C.T. G r a i n g e r ; C r y s t . S t r u c t . Commun. ~ 1, 7 1 , (1972).
4.
5. 6.
B r i t i s h Pharmacopoeia 1973, London Her M a j e s t y ' s S t a t i o n a r y O f f i c e 1973, p . 415.

D. H a r t l e y and D. Middlemiss, J . Med. Chem., 1 4 , 895,
(1971).
E . G . C . C l a r k e , t ' I s o l a t i o n and I d e n t i f i c a t i o n o f Drugs", The Pharmaceutical P r e s s , London, p . 1095, 1975. M.E. Evans, S.R. Walker, R.T. Xanobiotica, 3, 113, (1973).
7.
8. 9.
B r i t t a i n and J.W. P a t e r s o n ;
R.T. P a r f i t t , G.H. Dewar and J . K . Kwakye; J. Pharm. Pharmacol. _30, ( s u p p l . ) , 62 P . (1978).
J . Dangoumau, Y . Barrans, and M. C o t r a i t ; J . Pharmacol. 4 , 5 (1973).
67, 10. L . H . C . Lunts, P. Toon and D.T. C o l l i n ; S. A f r i c a n 05, 591 through C.A. 71, 91066 f (1969):
11. L . H . C . Lunts and P. Toon; U.S. 3,705,233 through C.A. 78 71665s (1973).
1 2 . K . Okumura, K. Matsumoto, T . Iwasaki and M. Suzuki; Ger. Offen. 2,249,820; through C . A . 79, 18349m (1973). -
13. Y . Kyotani, S . Kabuto and N . Sawada; Japan Kokai 75 52,037; through C.A. 83, 1 1 3 9 6 2 ~(1975). 14. L.E. Martin, J . C . Hobson, J . A . Page and C. H a r r i s o n ; Eur. 1 4 , 183, (1971). - J . Pharmacol: 15. M a r t i n d a l e , t h e E x t r a Pharmacopoeia, 27th e d i t i o n p . 32, The Pharmaceutical P r e s s , 1977.
SALBUTAMOL
689
16. A . V . Cullum, J . B . Farmer, A. J a c k and G . P . Levy, B r it . J. Pharmacol . , 35, 141 (1969)
17. Drugs o f Today; 1 6 , 271 (1980). 18. A. Wahbi, H. Abdine, M. Korany and M.H. Abdel-Hay; J .- Assoc. O_ f f ._ Anal. Chem., 61, 1113 (1978). - _ _ _ 19. P r a c t i c a l Pharmaceutical Chemistry, 3rd e d . Part One, by A.H. Beckett and J . B . S t e n l a k e , p . 312, 1975, The Athlone Press of t h e U n i v e r s i t y o f London. 20. L . E . Martin; J . Rees., R . J . N . Spectrom, 3, 184 (1976). Tanner, ~Biomed. Mass
2 1 . J . Eyem; Adv. Mass Spectrom., B , 7 , 1534 (1978).
Acknowledgements The a u t h o r s wish t o thank M r . Altaf Hussain Naqvi f o r t y p i n g t h e m a n u s c r i p t . A sample o f salbutamol b a s e was k i n d l y donated by Allen G Hanhurys Ltd., Research Ware, Herts, England.
SUCCINYLCHOLINE CHLORIDE
Penelope R . B . Foss and Steven A . Benexra
1. Description 1.1 Name, Formula, Molecular Weight
1.2 Appearance, Color, Odor 2. Physical Properties 2.1 Infrared Spectrum 2.2 Nuclear Magnetic Resonance (NMR) Spectrum 2.3 Ultraviolet (UV) Spectrum 2.4 Mass Spectrum 2.5 Melting Point 2.6 Solubility 3. Synthesis 4. Stability 5 . Methods of Analysis 5.1 Elemental Analysis 5.2 Titration Analysis 5.3 Chromatography 5.4 Biological Tests 6. Metabolism and Pharmacokinetics 6.1 Metabolism 6.2 Tissue Distribution 6.3 Pharmacokinetics 7. References 692 692 692 693 693 695 698 698 699 699 699 700 700 700 700 701 702 702 702 703 703 704
ANALYTICAL PROFILES OF DRUG SUBSTANCES. 10
69 1
Copyright &, 1981 hy Academic Ircss. Inc. All rights of reproduction in any form resened. ISRN 1)-12-260810-0
692
PENELOPE R. B. FOSS AND STEVEN A. BENEZRA
1.
Description
1.1 Name, Formula, Molecular Weight

Succinylcholine chloride 2,2'-[(1,4-dioxo-1,4-butanediyl)bis(oxy)~bis[N,N,Ntrimethylethanaminiu] dichloride; bis[2-dimet.hylaminoethyl]succinate bis[methochloride]; 2-dimethylaminoethyl succinate dimethochloride; diacetylchaline dichloride; suxamethonium chloride; choline succinate dichloride; succinic acid b i s [ f + d i m e t h y l a m i n o e t h y l ] ester dimethochloride ; choline chloride succinate ( 2 : l ) ; Listenon; Anectine chloride; Scoline chloride; Lysthenon; Midarine ; Quelicin chloride; Sucostrin chloride; Ultrapal chloride; Succicuranl
CH2- CO2CH2CH2N(CH3), CH2- C02CH2CH2R (C H3)3
2cr
C14H30C12N204
Mol. wt. 3 6 1 . 3 1 397.342 (dihydrate)
Succinylcholine chloride exists as a dihydrate at room temperature. All data presented here is for the dihydrate form unless otherwise stated.
1.2 Appearance, Color, Odor
Succinylcholine chloride is characterized as having white, odorless, crystals.
693
2. Physical Properties
2 . 1 Infrared Spectrum
The infrared spectrum of succinylcholine chloride is shown in Figure 1 . It was taken as a 0.2% dispersion of succinylcholine chloride in KBr with a Nicolet Model 7199 FT-IR spectraphotometer. Table I gives the infrared assignments consistent with the structure of succinylcholine chloride. Table I Infrared Spectral Assignments for Succinylcholine Chloride Band (cm-l)
1151,1048 1738 1312 1428 1481 2930-3023 2957 2930 3426 3484
Assignment
C-0-C stretch (ester) C=O stretch (ester) CH2 wagging vibration C-H bending vibration from CH3 C-H bending vibration from CH2 and CH3 C-H stretch C-H stretch from CH3 C-H stretch from CH2
overtone of C=O stretch
0-H stretch from water o f hydration
cc, ;
0
N
c -
0
N
r-
Ln
0 0
r-
0 0 0
695
2.2 Nuclear Magnetic Resonance (NMR) Spectrum 2.21 Proton NMR Spectrum The proton NMR spectrum o f succinylcholine chloride i s shown in Figure 2. It was obtained with a Varian CFT-20 80 MHz spectrometer. Deuterated water was used as the solvent with tetramethylsilane as an internal standard. Based on the NMR spectrum, the following proton assignments are made. Proton
0
Chemical Shift (ppm) 2.69 singlet 3.13 singlet 3.65 multiplet
CH2C-0
II
(CWN+
CH2N'
0
CH2OC
I1
4 . 5 0 multiplet
2.22 Carbon-13 NMR Spectrum

The carbon-13 NMR spectrum o f succinylcholine chloride is shown in Figure 3. It was obtained with a Varian H z spectrometer equiped with a Nicolet XL-100 25 M data system. Deuterated water was used as the solvent with tetramethylsilane as an internal standard. Based on the NMR spectrum, the following carbon assignments are made. Carbon Chemical Shift (ppm) 176.36 singlet 67.19 triplet 61.26 singlet 56.52 triplet
c=o
CH2N+ CH20 CHSN
31.25 singlet
696
Figure 2 Proton NMR Spectrum of Succinylcholine Chloride

4 .
ai V
ai
697
Figure 3 Carbon-13 NMR Spectrum of Succinylcholine Chloride
698
2.3 Ultraviolet (UV) Spectrum Succinylcholine chloride does not absorb UV radiation above 220 nm. 2.4 Mass Spectrum The low resolution field desorption mass spectrum of succinylcholine chloride is shown in Figure 4. It was obtained with a Varian MAT 731 mass spectrometer coupled to a VG multispec data system. The spectrum was recorded at 20 mA. The assignment o f fragment ions is given below.6 m/z 78 m/z 325 m/z 275 molecular i o n of dimethyl sulfoxide used as a chemical marker [M-Cl]' thermal elimination o f CH3C1 followed by l o s s of C1- and acceleration of the resultant quaternary ammonium ion [M-CHzC1-Cl] result of two molecules (after thermal elimination of CH3C1 from each molecule)+ clustering and losing C1-[2M-2(CH3C1)C1]
m/z 585
100
40
20
0
m/ z
Figure 4 Mass Spectrum o f Succinylcholine Chloride
699
2.5 Melting Point Succinylcholine chloride dihydrate crystals melt at 156-163OC. The anhydrous form melts at about 190C. 2.6 Solubility Succinylcholine chloride is freely soluble in water (1 g/mL), soluble in 95% ethanol ( 0 . 4 2 g / l O O mL). It is sparingly soluble in benzene, chloroform and practically insoluble in ether. 3. Synthesis Figure 5 outlines a synthesis of succinylcholine chloride. Diethyl succinate (1) is condensed with dimethylethanolamine (2) to yield dimethylaminoethylsuccinate (3). The d i m e t h y l a m i n o e t h y l s u c c i n a t e is quaternized with methyl chloride ( 4 ) to give succinylcholine chloride ( 5 ) . 7a9b
CH,j-CO,C,H,
/CH3 + 2 HO-CH2CH N CHrC02C2H5 C H 3
-I
CH2C02CH2CH N/c H3 2 C H ,
C Hj-C O,CH,CH,N<Z
13
3
c H~ c 02cH ,
CH CH, N < ~
,,:
+CH3CI
O , C H , C C Hj- C
H , N (C H3)3
0
CHTCO~CH,C H NA H , 2 CH3
-1
2c1(C HJ3
C H,j- CO,CH,CH,N
Figure 5 Synthesis o f Succinylcholine Chloride
700
4.
Stability3 Succinylcholine chloride is stable in crystalline form. Aqueous solutions hydrolyze at an increasing rate with the increase of pH, temperature, and concentration. A five percent solution at room temperature shows very slow hydrolysis at pH 3 - 5 , moderately fast hydrolysis at pH 7 . 4 and very rapid hydrolysis at pH 10-11. A five percent solution at pH 3-5 maintains potency for more than two years when kept at 4OC, and rapidly loses potency at 100OC.
5.
Methods of Analysis
5.1
Elemental Analysis'
(Anhydrous) c1 19.63
E1ement C % calculated 4 6 . 5 4
5.2
H
8.37
N
7.75
0 17.71
Titrat.ion Analvsis
5 . 2 1 Succinylcholine chloride is dissolved in a mixture of glacial acetic acid and mercuric acetate. The solution is titrated with 0.1N perchloric acid with crystal violet as the indicator. Each mL of 0.1N perchloric acid is equivalent to 18.07 mg of Cl4II3~Cl2N2O,. 5 . 2 2 An injection sample of succinylcholine chloride is diluted with water and extracted with ether. The collected ether extracts are washed with water and discarded. The combined water washings are extracted with ether, added to the aqueous pbase from the initial extraction, and heated on a steam bath until just free of ether odor. The solution is neutralized with 0.1N sodium hydroxide using bromothymol blue as the indicator. Twenty-five milliliters of 0.1N sodium hydroxide are added to the sample solution and the blank, then refluxed for forty minutes. The cooled samples are titrated with 0.1N HC1. Each mL of 0.1N NaOH is equivalent to 18.07 mg of C,,H30C12N204. 2 5 . 2 3 Two milliters of a succinylcholine chloride injection sample are transferred to a 150-mL flask. Sodium thiosulfate (0.005N) and KI03/KI reagent are added to the flask. The flask is allowed to stand for ten minutes. A mucilage o f starch is added and
70 1
the excess sodium thiosulfate is titrated with 0.005N iodine. The result of the blank is subtracted, and the amount of succinic acid generated by hydrolysis of the succinylcholine chloride in aqueous solution is calculated.'
5.3
Chromatography
5.31
High Performance Liquid Chromatography
High performance liquid chromotography has been used to determine succinylcholine chloride in injection and Flo-pack@ sample^.^ A stainless steel column (25 cm x 3 . 9 nun i.d.), packed with PartisilB 10 was used for the separation. The mobile phase, methanol/ tetramethylammoniumchloride ( 9 : 1 ) , was adjusted to pH 3 with HC1. The LW detector was set to 214 nm. The flow rate was -0.75 mL/min which gave a sevenminute retention time for succinylcholine chloride.
5.32
Paper chromatography' was used for the determination o f succinylcholine chloride in injection samples. The paper was Whatman No. 1 and the mobile phase was n-propanol/benzyl alcohol/water ( 5 : 2 : 2 ) . The development time was 18 hr. After the chromatogram was air dried, the separated sample components were visualized with 0.05% iodine solution. The observed R value f for choline was 0 . 2 5 , and for succinylcholine was 0.105.
A second mobile phase' used for paper chromatography was n-butanol/acetic acid/water ( 4 : 1 : 5 ) . The sample components were visualized with Dragendorff's reagent. Choline gave a violet spot and succinylcholine gave an orange spot. 5 . 3 3 Thin Layer Chromatography
Thin layer chromatography" was used for the detection of succinylcholine chloride. The thin layer plate was a Merck silica G-60 with no fluorescent indicator. The mobile phase was acetone/l.ON HC1 ( 1 : l ) . The sample was dissolved in water to a concentration of 1 mg/mL and spotted in 1 pL portions. After the developed plate was air dried, the succinylcholine
702
PENELOPE R . B. FOSS AND STEVEN A. BENEZRA
chloride was visualized with Dragendorff's reagent. Succinylcholine chloride had an R o f 0 . 4 7 . f

5.4
Biological Tests
5.41
Rat Respiration Test"
A polyethylene tube was passed into the trachea o f an

anaesthetized rat. The rat's respiration amplitude was measured by the amount of water displaced in a water-filled glass flask connected to the endotrachael tube and an oxygen source. The half-height o f the maximum amplitude was recorded. Succinylcholine chloride was administered and respiration depression ensued. The time, elapsing from the moment of injection of the drug until the resumed respiration raised the level of the manometer to the previously marked half-height-,was measured and recorded as the response to a given dose. By linear regression, a curve was drawn of the time of respiratory depression versus the log of the dose of succinylcholine chloride.
5.42
Frog Muscle Response12
The amount of succinylcholine chloride in either blood serum or urine samples was determined by adding a sample to a Ringer solution (NaC1 0.6%, KC1 0 . 0 4 2 % , CaC12 0.032%, NaHC03 0.05%, dextrose 0.05%) surrounding a frog muscle. The contraction of the muscle displaced a recorder pen which traced a peak on chart paper. The height of the peak was proportional to the dose level of succinylcholine. A dose response curve was drawn and the concentration o f an unknown succinylcholine solution was determined.
6. Metabolism and Pharmacokinetics

6.1 Metabolism _______
Succinylcholine chloride, a short-acting depolarizing muscular relaxant, is a positively charged small linear molecule consisting essentially of two acetylcholine mo:Lecules.l3 Its low toxicity is due to the relatively inert nature of the drug and its rapid breakdown t o succinic acid and choline.3 Succinylcholine chloride hydrolysis in serum occurs in a two-stage reaction: one, the formation of
703
succinylmonocholine and choline, and two, the further breakdown of succinylmonocholine to choline and succinic acid. The first step3 is six times faster than the second. The two enzymes in the body concerned with the breakdown of choline esters are acetylcholinesterase and cholinesterase. Acetylcholinesterase is present in the neuromuscular junction, nervous tissue, and in the red blood cells. It does not hydrolyze succinylcholine to an appreciable extent. Cholinesterase is present in most tissues in the body: plasma, nervous tissue, cardiac muscle, intestine, and skin. Up to 90X3 (80%14) of succinylcholine is hydrolyzed by plasma cholinesterase before it reaches the neuromuscular junction. Ten percent3 o f succinylcholine is excreted unmetabolized in the urine.
6.2 Tissue Distribution

When succinylcholine is administered intravenously there is a rapid initial distribution of the drug throughout the extracellular fluid volume to the muscle mass and neuromuscular junction.l4 Whole body distribution of succinylcholine was determined in a study involving near-term macaca mulatta monkeys.l 5 The radio-labelled drug was injected into the umbilical vein of developing feti. The feti were delivered by cesarean section and examined for the whole body distribution of 14C succinyldicholine hydroiodide. The highest concentration of radioactivity was found in the intravascular space, kidney, and other highly perfused organs and cartilage. A lower concentration was found in the lung, skeletal muscle, and bone marrow. Radioactivity was absent in the brain, vitreous body, and cerebrospinal fluid. The activity in the skin, primary dentine, and in the blood vessels, and intestinal walls was especially marked. Radioactivity was rapidly taken up in the sclera, choroid layer, isis, ciliary body, and extraocular muscle.
6.3
Pharmacokinetics
In an in vitrol* study, it was found that 85% of succinylcholine is destroyed within the first half
704
minute following the mixing with blood. minutes only 5% of the drug remained.
After two
The neuromuscular blocking effect is terminated by redistribution of succinylcholine away from the neuromuscular junction and only to a limited extent by further hydrolysis in the plasma by plasma cholinesterase (pseudocholinesterase) . l 4
7.
References
1.
2. 3. 4. 5. 6. 7.
8.
9. 10.
11.
12.
13.
14. 15.
The Merck Index, Ninth Edition, p. 1148 (1976) USP M: p. 743 (1980) Gibb, D. B., Anaesth. Intens. Care, 1, 109 (1972:l Powell., H., Burroughs Wellcome, personal communication Hurlbert, B. S . , Burroughs Wellcome, personal communication Brent, D., Soltman, B., Burroughs Wellcome, pe rsona 1 communication a. Phillips, A. P., J. Amer. Chem. SOC., 71, 3264 (1949) b. Tammelin, L . E., Acta Chem. Scand., 7 , 185 (1953) Earles, M. P., Foster, G. E., Hardstone, B. L., Stewart, G. A., "The Stability of Injection of Succinylcholine Chloride" from the Wellcome Chemical Works, Dartford (1954) Rusch, D., Burroughs Wellcome, personal communication Wollman, Ch., Nagel, S . , Scheibe, E., Pharmazie, 21, 665 (1966) Salgado, A. S . , Brit. F. Anaesth., 34, 410 (1963) Kvesselgaard, N, Moya, F., Acta Anaesthesiologica Scandenavica, 5 , 1 (1961) Wingard, L . B . , Cook, D. R., Clinical Pharmacokinetics, 2 , 330 (1977) Cook, D . R,, Wingard, L. B., Taylor, F. H., Clinical Pharmacology and Therapeutics, 20, 493 (1976) Van Der Kleijn, E., Drabkova, J., Crul, J. F., British Journal of Anaesthesia, g, 1109 (1973)
3 -
TRIOXSALEN
Mahmoud M . A . Hassan and Mohammed A . Loutfy
1
Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Taste, Odor Physical Properties 2.1 Melting Point 2.2 Solubility 2.3 Identification 2.4 Spectral Properties Isolation Synthesis Metabolism Photoreactions between Trioxsalen and Nucleic Acids Method of Analysis References
2.
3. 4. 5. 6. 7.
8.
706 706 706 706 707 707 707 107 707 707 707 719 719 722 724 724 725
ANAI.YI'ICAI. t'ROFll.k~h OF DRPC SURSTANCES. 10
705
706
MAHMOUD M. A. HASSAN AND MOHAMMED A. LOUTFY
TRIOXSALEN 1. Description
1 .1
Nomenclature
1.11
Chemical Names
ab-
cd-
e-
2,5,9-Trimethyl-7H-furo(3,2-g)benzopyran7-one 2,5,9-Trimethyl-7H-furo(3,2-g) [ 1 1 henzopyran-7 -one 6-Hydroxy-3 ,2,7-trimethyl-5-benzoEuranacrylic acid 8-lactone 7H-Fur0 [ 3 ,2-g] [ 11 benzopyran-7--one, 2,5,9-trimethyl 4,5' ,8-Trimethylpsoralen.
1.12
Generic Names
I
4,5,8-Trimethylpsoralen; Trimethylpsoralen; Trioxsalen; Trioxysalen; TMP.
1.13
'Trade Names Trisoralen

;
Trioxsalen.
1.2
Formulae 1.21 Empirical C14H1 3 ' 2 1.22 Structural

CH3
I
CH3
1.3 Molecular Weight
228.25 (1,2)
;
228.24 ( 3 )
228.2 ( 4 ) .
'THIOXSALEN
1.4
E l e m e n t a l Composition
C,73.67% 1.5
H, 5.30%
0 , 21.03%.
Appearance, C o l o r , Taste, Odor C r y s t a l l i n e s o l i d or prisms, white t o off-white o r g r a y i s h , tasteless, o d o r l e s s .
2.
Physical Properties
2.1
Melting P o i n t T r i o x s a l e n m e l t s a t a b o u t 230' 234.5 - 235 ( 3 ) .
(1,2,4) or a t
2.2
Solubility Practically insoluble i n water, s l i g h t l y soluble i n a l c o h o l ( l g i n 1150 ml) , s p a r i n g l y s o l u b l e i n c h l o r o f o r m ( l g i n 84 m l ) , f a i r l y s o l u b l e i n m e t h y l e n c d i c h l o r i d e (lgm i n 4 3 ml)
2.3
Identification i) The i n f r a r e d a b s o r p t i o n spectrum of a m i n e r a l o i l d i s p e r s i o n of t r i o x s a l e n , p r e v i o u s l y d r i e d a t 105' f o r 6 h o u r s , e x h i b i t s maxima o n l y a t t h e same w a v e l e n g t h s a s t h a t of a s i m i l a r p r e p a r a t i o n of USP T r i o x s a l e n R e f e r e n c e Standard ( 2 ) .
ii) The u l t r a v i o l e t a b s o r p t i o n spectrum of a 1 i n 200,000 s o l u t i o n i n c h l o r o f o r m e x h i b i t s maxima and minima a t t h e same w a v e l e n g t h s a s t h a t of a s i m i l a r p r e p a r a t i o n of USP T r i o x s a l e n R e f e r e n c e S t a n d a r d , c o n c o m i t a n t l y measured ( 2 ) .
2.4
Spectral Properties
2.41 U l t r a v i o l e t Spectrum
The UV spectrum of t r i o x s a l e n i n methanol w a s scanned u s i n g Pye Unicam SP 8 0 0 s p e c t r o p h o t o meter; from 4 0 0 - 200 nm. Three maxima and t h r e e minima were o b s e r v e d :
708
Xmz:x
248 296
Log
Amin
225 27 2
Log
&
4.35 3.99
4.09 3.68
338
3.80
320
3.79
The spectrum i s shown i n F i g u r e 1. Other UV s p e c t r a l d a t a have been a l s o r e p o r t e d ( 3 , 5 , 6 ) .
2.42
I n f r a r e d Spectrum The I R spectrum of t r i o x s a l e n i s recorded a s a n u j o l mull on a Unicam SP 1025 spectrophotometer and i s shown i n F i g u r e 2. The assignments o r t h e c h a r a c t e r i s t i c bands i n t h e i n f r a r e d spectrum a r e l i s t e d i n Table 1.
Table 1 Frequency Cm -1 Assignment
3120
1;710
CH3
C=O (a-pyrone)
1640
c=C (a -pyrone)
1620 1600 1x90 11170
1:110
C-0-C C=C ( a r o m a t i c )
(a-pyrone;furan)
880
840
810
Furan r i n g I s o l a t e d H (Penta s u b s t i t u e d benzene)
TRIOXSALEN
709
Fig. 1 .
UV spectrum of Trioxsalen in Methanol.
LA-'
"
'
"
TRIOXSALEN
711
Other f i n g e r p r i n t bands c h a r a c t e r i s t i c of t r i o x s a l e n are : 1465, 1385, 1360, 1305, 1280, 1250, 1235, 1045, 1000, 945, 930, 860 and 760 (5-7).
2.43
2.431
P r o t o n Spectrum
The p r o t o n magnetic r e s o n a n c e s p e c t r a of t r i o x s a l e n and o t h e r p s o r a l e n s have A typical P M R been r e p o r t e d (8,9). spectrum of t r i o x s a l e n i s shown i n F i g u r e 3. The sample w a s d i s s o l v e d i n d e u t e r a t e d chloroform and t h e spectrum w a s r e c o r d e d and t h e spectrum w a s recorded on a Varian XL-200, 200 M H z NMR s p e c t r o m e t e r u s i n g tetramethylsilane as a reference standard. The PMR s p e c t r a l assignments of t r i o x s a l e n a r e g i v e n i n Table 2. Table 2: P M R C h a r a c t e r i s t i c s of T r i o x s a l e n i n CDC13
H3
CH3
Chemical s h i f t s (6)
4-CH3
d
8-CH3
S
5kH3
d
3-H d
5-H
S
4 LH
2.47 2.46
2.55
2.50 2.49
6.22 6.21
7.49
6.41 6.40
S = singlet
; d = doublet
i,
I-
TRIOXSALEN
713
2.432
13C-NMR
Spectrum
The f u l l y decoupled I3C-NMR spectrum of t r i o x s a l e n i n d e u t e r a t e d chloroform i s given i n Figure 4. Proton-coupled ( o f f r e s o n a n c e ) spectrum i n d e u t e r a t e d c h l o r o f o r m i s a l s o shown i n F i g u r e 5 . These s p e c t r a were o b t a i n e d w i t h 50.3 MHz on a V a r i a n XL-200 -200 MHz NMR s p e c t r o m e t e r a t ambient t e m p e r a t u r e u s i n g a broad band 10 mm p r o b e . The sample w a s r u n a t c o n c e n t r a t i o n s c a 1-2 M i n d e u t e r a t e d c h l o r o f o r m w i t h tetramethylsilane a s an i n t e r n a l s t a n d a r d . The p r o t o n - c o u p l e d spectrum w a s r e c o r d e d under g a t e d d e c o u p l i n g c o n d i t i o n . The a s s i g n ment of t h e i n d i v i d u a l s i g n a l s based on t h e p r o t o n - decoupled spectrum i s g i v e n i n T a b l e 3. T a b l e 3 : 13C-Chemical S h i f t s of T r i o x s a l e n
5' H3c Carbon Chemical S h i f t PPm 161.5 112.7 155.4 109.0

112.1
Carbon
Chemical S h i f t PPm 116.0 148.9 157.3 102.6
c-2
c-3 c-4 C-4a c-5 C-6 c-7
C-8
C-8a c-4 c-5
I
'
3
3
4-CH
8-CH
1
14.2 8.5 19.2
125.4 153.2
5-CH3
714
715
7 16
Other 13C-NMR chemical s h i f t d a t a of o t h e r p s o r a l e n s have been a l s o r e p o r t e d (10-13) 2.44

Mass Spectrum
The mass spectrum of t r i o x s a l e n o b t a i n e d by c o n v e n t i o n a l e l e c t r o n impact i o n i z a t i o n a t 70 eV+ shows a m o l e c u l a r i o n M a t m / e 2 2 8 . The M i o n peak i s t h e b a s e peak ( F i g u r e 6 ) . The m / e f o r t h e most prominent fragments and t h e corresponding i o n s a r e p r o p o s e d i n T a b l e 4 . Table 4 : Prominent Fragments and Corresponding I o n s of T r i o x s a l e n
Relative Intensity
Ion
77
5.3
115
7.9
1 ' '
127
4.8
CH
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 221 230 240
PT-
Fig. 6.
Mass spectrum of Trioxsalen.
718
MAHMOUD M. A . HASSAN A N D MOHAMMED A . LOUTF'Y
128
16.8
H2c
r3'
1
+.
129
4.8
171
3. 6
CH3
185
10.6
CH3
199
55.6
c@3
CH3
H3C
l+'
TRIOXSALEN
719
200
56.0
CH3
228
3.
Isolation The isolation of trioxsalen from celery diseased with the fungus sclerotinia sclerotiorum has been reported ( 1 4 ) .
4. Synthesis
The synthesis of trioxsalen is based on the conversion of 7-allyloxy -4,8-dimethylcoumarin intermediate (111) into 6-allyl-7-hydroxy-4,8-dimethylcoumarin (IV) by Claisen rearrangement (1,6,9,15,17). This procedure involves the cyclisation of the starting material 2-methylresorcinol (I) with ethylacetoacetate, with the aid of sulfuric acid. The reaction is a Pechmann type candensation and gives 7-hydroxy-4,8-dimethylcoumarin (11). The latter is treated with ally1 bromide and potassium carbonate in acetone to give 7-allyloxy-4,8dimethylcoumarin (111) which, on reacting with acetic anhydride in the presence of N,N-diethylaniline and anhydrous sodium acetate, rearranges, and esterifies to give 7-acetoxy-6-allyl-4,8-dimethylcoumarin ( V ) . Bromination of V followed by reaction with sodium methoxide yields trioxsalen (VII) (Scheme A ) . The 7-hydroxy group of IV is acetylated to minimise the possibility of ring bromination during the addition of one
720
MAHMOUD M. A. HASSAN A N D MOHAMMED A. LOUTFY
Scheme A.
CH COCH2COOC2H5 HO @o:H2s04 CH3
q j
CH3
0
CH2=CH-CH
K
(1) Br
co
lacetone
,
CH3 (111)
n e s: : : : Rearrangement
0
CR =CHCH20 2
Acetylation
0
HO
TRIOXSALEN
721
Scheme B.
CH =CH-CH 2
HO
CH
1
(VII)
ehydrogenation
CH3
722
e q u i v a l e n t of bromine t o t h e a l l y l i c double bond.

An improved s y n t h e s i s of t r i o x s a l e n has been d e v i s e d by Parekh and T r i v e d i (18). I n t h i s approach, t h e a d d i t i o n of f u r a n r i n g t o t h e 6 - a l l y 1 u m b e l l i f e r o n e i n t e r m e d i a t e (IV) h a s been achieved by mere t r i t u r a t i o n w i t h c o n c e n t r a t e d s u l f u r i c a c i d , a t room t e m p e r a t u r e , t o g i v e 4' ,5'-dihydropsoralen d e r i v a t i v e (VI) The l a t t e r , on dehydrogenation a f f o r d s t r i o x s a l e n (VII) (Scheme B ) .
5.
Metabolism The k i n e t i c s of a b s o r p t i o n , metabolism and e x c r e t i o n of t r i o x s a l e n w e r e s t u d i e d i n mice an3 human volunteers (19). Groups of mice r e c e i v e d H - t r i o x s a l e n e i t h e r o r a l l y o r i n t r a p e r i t o n e a l l y . Urine, blood, f a e c e s , s k i n and v i s c e r a were o b t a i n e d a t d i f f e r e n t t i m e i n t e r v a l s . T r i o x s a l e n o r i t s m e t a b o l i t e s were e x t r a c t e d and analysed f o r r a d i o a c t i v i t y . Over 88% o f t r i o x s a l e n , a f t e r o r a l o r i n t r a p e r i t o n e a l a d m i n i s t r a t i o n were e x c r e t e d i n t h e u r i n e w i t h i n 8 hours and over 90% w i t h i n 1 2 hours. D i s t r i b u t i o n p a t t e r n s of t r i o x s a l e n r a d i o a c t i v i t y a t d i f f e r e n t t i m e i n t e r v a l s i n blood and v a r i o u s organs ( l i v e r , skin, heart, lung, brain, i n t e s t i n e s , kidney, and s p l e e n ) r e v e a l e d t h a t t r i o x s a l e n was s e l e c t i v e l y p r e s e n t i n l i v e r , s k i n , and blood alid was b a r e l y d e t e c t a b l e i n o t h e r organs. Highest v a l u e s were o b t a i n e d between 2 and 6 hours and diminished r a p i d l y t h e r e a f t e r . T r i o x s a l e n w a s m e t a b o l i s e d i n l i v e r and e x c r e t e d i n u r i n e as s e v e r a l d i s t i n c t f l u o r e s c e n t m e t a b o l i t e s , one of which appeared t o be hydroxylated t r i o x s a l e n (11) ( h y d r o x y l a t i o n a t 3 p o s i t i o n ) . I n men r e c e i v i n g 40 m g u n l a b e l l e d t r i o x s a l e n , 80% of t h e a d m i n i s t e r e d d o s e was e x c r e t e d i n u r i n e w i t h i n 8 hours as hydroxylated o r glucuronide (111) d e r i v a t i v e s . e t_ a1 (20) have r e p o r t e d t h a t t r i o x s a l e n g i v e s Pathak as a p r i n c i p a l m e t a b o l i t e 5'-carboxy-4,8-dimethylpsoralen ( I ) . T h i s compound i s i n a c t i v e as s k i n p h o t o s e n s i t i z e r and t o t h i s t r a n s f o r m a t i o n h a s been a t t r i b u t e d t h e d i f f e r e n c e i n t h e p h o t o r e a c t i v i t y of t r i o x s a l e n when t o p i c a l l y a p p l i e d o r s y s t e m a t i c a l l y a d m i n i s t e r e d (21,22).
It h a s been a l s o r e p o r t e d (23) t h a t one of t h e major m e t a b o l i t e s of t r i o x s a l e n c r y s t a l l i s e d from u r i n e of mice and men showed f l u o r e s c e n c e a c t i v a t i o n and emission wave l e n g t h s of 355 and 430 nm. I n f r a r e d spectrum of t r i o x s a l e n m e t a b o l i t e r e v e a l e d i n t a c t methyl groups, absence of OH bond a t 3300 cm-l and a new peak
724
a t 1225 cm-'. NMR spectrum r e v e a l e d opening of t h e l a c t o n e r i n g b u t no d e m e t h y l a t i o n (IV). T h e r e f o r e , metabolism of t r i o x s a l e n i n v o l v e s ( a ) h y d r o x y l a t i o n , (b) g l u c u r o n i d a t i o n , ( c ) opening of l a c t o n e r i n g w i t h p o s s i b l e formation of f l u o r e s c e n t c a r b o x y l a t e d moeity, and ( d ) o x i d a t i o n of t h e 5'-methyl group t o y i e l d t h e 5'-carboxy d e r i v a t i v e (Scheme C)
6.
P h o t o r e a c t i o n s between T r i o x s a l e n and Nucleic Acids The photochemical a d d i t i o n of t r i o x s a l e n t o t h e pyrimidine b a s e s of DNA i s c o n s i d e r e d t h e molecular b a s i s f o r explaining i t s photobiological e f f e c t s . S e v e r a l s t u d i e s (24-35) have been c a r r i e d o u t concerning t h e format:ion of molecular complexes between t r i o x s a l e n ( o r o t h e r p s o r a l e n s ) and DNA, t h e mechanism of t h e p h o t o a d d i t i o n , t h e K i n e t i c s of formation of t h e v a r i o u s photoadducts, and t h e s p e c i f i c r e c e p t o r s i t e s of DNA.
7.
Methods of A n a l y s i s
7.1
Spectrophotometry The o f f i c i a l method adopted by t h e U.S.P. X I X (1975) i s a s p e c t r o p h o t o m e t r i c ( 2 ) . The procedure i s a s follows: T r a n s f e r about 50 mg of T r i o x s a l e n , a c c u r a t e l y weighed, t o a 100 m l v o l u m e t r i c f l a s k , add c h l o r o form t o volume, and mix. T r a n s f e r 1 m l of t h i s s o l u t i o n t o a 100 ml v o l u m e t r i c f l a s k , add chloroform t o volume, and mix. D i s s o l v e an a c c u r a t e l y weighed q u a n t i t y of USP T r i o x s a l e n Ref Crence Standard i n chloroform, and d i l u t e q u a n t i t a t i v e l y and s t e p w i s e w i t h chloroform t o o b t a i n a s t a n d a r d s o l u t i o n having a known c o n c e n t r a t i o n of about 5 i-lg p e r m l . Concomitantly determine t h e absorbances of b o t h s o l u t i o n s i n 1-cm c e l l s a t t h e wavelength of maximum absorbance a t about 252 nm, w i t h a s u i t a b l e spectrophotometer , u s i n g c h l o r o form as t h e blank. C a l c u l a t e t h e q u a n t i t y , i n mg, of C H 0 i n t h e T r i o x s a l e n t a k e n by t h e formula 14 1 2 3 ~OC(AU/AS),i n which C i s t h e c o n c e n t r a t i o n , i n p g p e r m l , of USP T r i o x s a l e n Reference Standard i n t h e Standard s o l u t i o n , and A u and A s are t h e a b s o r bances of t h e s o l u t i o n of T r i o x s a l e n and t h e Standard s o l u t i o n , r e s p e c t i v e l y .
TRIOXSALEN
725
References "Remington's P h a r m a c e u t i c a l S c i e n c e s " , 1 5 t h ed. , Mack P u b l i s h i n g Co., E a s t o n , P e n n s y l v a n i a , P. 728 (1975). The United S t a t e s Pharmacopeia, 1 9 t h e d . Co., E a s t o n , P a . , P. 525 (1975).
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, Mack
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The Merck I n d e x , 9 t h e d . , Merck P u b l i s h i n g Co., I n c . , P. 1249 (1976). M a r t i n d a l e , The E x t r a Pharmacopeia, 2 7 t h e d . , The P h a r m a c e u t i c a l P r e s s , P.459 (1977).
A. M u s t a f a , "Furopyrans and Furopyrones". I n t e r s c i e n c e P u b l i s h e r s , John Wiley and Sons, London, P.14 ( 1 9 6 7 ) .
K.D.
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K. L e e and T.O.
K.D. N.C. K.D.
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Kaufman, L.R. Worden, E . T . Lode, M.K. S t r o n g & R e i t z , J. Org. Chem. , 35, 157 (1970). Karfman, U.S.P P a t e n t 3 , 201, 421 ( 1 9 6 5 ) .
9.
10,. E. Wenkert, B.L. Buckwater, I . R . B u r f i t t , M . J . G a s i c , H.E. G o t t l i e b , E.W. Hagaman, F.M. S c h e l l , and P. M. Wovkulich, "Topics i n Carbon-13 NMR S p e c t r o s c o p y , " G . C . Levy, ed. , Wiley I n t e r s c i e n c e , New York, V o l . 2 , P. 110 (1976).
11. D. B e r g e n t h a l , K. S z e n d r e i , and J . R e i s c h , J . Arch. Pharm.

310, 390 (1977).
12. M.H.A. Elgamal, N.H. E l e w a , E.A.M. E l k h r i s y , and Helmut Doddeck, P h y t o c h e m i s t r y , 139 (1979).
18,
13. D. B e r g e n t h a l , 2 . Rozsa, I. Mester, and J . R e i s c h , Arch. Pharm. (197 8)
14. L.D. S c h e e l , V.B. Perone, R.L. L a r k i n , and R.E. B i o c h e m i s t r y , 2, 1 1 2 7 (1963). 15, S . Rangaswami and R. T. S e s h a d r i , S c i . , 7 A, 8 (1938). 16. W. Baker and O.M.
Kuppl;
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L o t h i a n , J. Chem. SOC.,
628 (1935).
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B. Krishnaswamy and T . R . 13 A,43 ( 1 9 4 1 ) . Sci., M.G.
S e s h a d r i , P r o c . I n d i a n Acad.
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Parekh and K.N. T r i v e d i , C u r r . S c i . , (1970) ; Chem. A b s t r . , 73,87816 ( 1 9 7 0 ) .
2, 3 4 9
19.
M.A. P a t h a k , F. D a l l ' Acqua, G . R o d i g h i e r o , and J . A . P a r r i s h , J . I n v e s t i g . Dermatol. , 62, 347 ( 1 9 7 4 ) . M.A. P a t h a k , B.B. Mandula, J . A . P a r r i s h and T . B . F i t z p a t r i c k , Commun. P r e s e n t e d a t V I I I n t . Congress on P h o t o b i o l o g y , Rome (1 976)
20.
21.
G . R o d i g h i e r o and F. D a l l ' Acqua, P h o t o c h e m i s t r y and P h o t o b i o l o g y , 23 ( 1 9 7 6 ) .
22. 23.
B.B.
Mandula and M.A.
Pathak, S c i .
, 193, 1 1 3 1
(1976).
M.A. T.B.
P a t h a k , B. Mandula, Y . Nakayama, J . A . F i t z p a t r i c k , J . I n v e s t i g . Dermatol.,
9, 279
Parrish, & (1975).
24.
L. Musajo and G. R o d i g h i e r o , "Photophysiology", ed. A . C . Giese, Academic P r e s s (New York) , Vol. V I I , P . 1 1 5 ( 1 9 7 2 ) .
25.
F. D a l l ' Acqua, S. M a r c i a n i , D. V e d a l d i , and G. R o d i g h i e r o , 13iochem. Biophys. Acta, 3 5 3 , 267 ( 1 9 7 4 ) .
26.
L. Musajo, G.. R o d i g h i e r o , G . Colombo, V . T o r l o n e , and F. D a l l ' Acqua, E x p e r i e n t i a , 21, 22 ( 1 9 6 5 ) .

J . F . Walter, J . J . Voorhees, W.H. K e l s e y , and E.A. Arch Dermatol. , 107,8 6 1 ( 1 9 7 3 ) . Duell,
27.
28.
P. Chandra, S. M a r c i a n i , F. D a l l ' Acqua, D. V e d a l d i , G . R o d i g h i e r o , and R.K. B i s w a s , FEBS Letters, 35, 2 4 3 ( 1 9 7 3 ) .

L. Musajo and G . R o d i g h i e r o , Photochem. P h o t o b i o l . ,
29.
27 ( 1 9 7 0 ) . 30.
K.H.
a,
Kaidbey and A.M. (1974).
Kligman, Arch Dermatol.,
109, 674
31.
32.
L. Musajo, Ann. 1st. Super. S a n i t a ' , 5 , 376 (1969).
F. D a l l ' Acqua, S. M a r c i a n i , L . C i a v a t t a , and G . R o d i g h i e r o , Z. N a t u r f o r s c h . , 3,561 ( 1 9 7 1 ) . P. Chandra, "Research P r o g r e s s i n O r g a n i c , B i o l o g i c a l and M e d i c i n a l Chemistry", North-Holland P u b l i s h i n g Co., Amsterdam-London, Vo1.3., P.232 ( 1 9 7 2 ) .
33 *
TRIOXSALEN
7 27
34.
35.
L. Musajo and G. R o d i g h i e r o , Herba H u n g a r i c a , (1971).
10,79
G. R o d i g h i e r o and F. D a l l ' Acqua, " P h o t o c h e m i s t r y and P h o t o b i o l o g y " , Pergamon P r e s s , Vo1.23 (1976).
ERRATA FOR VOLUME 9
CEFAMANDOLE NAFATE
Rrfik H . Bishai-u cind Eugene C . Rickard
Page 145, Section 6.3, line 3 Reference (45) should be (44) Page 145, Section 6.4, line 2 Reference (44) should be (45) Page 146, Section 6.5.3, bottom line Reference (45) should b e (44) Page 147, Section 6.6.2, last reference Reference (44) should be (45)
730
ERRATA FOR VOLUME 9
FLWHENAZINE DECANOATE
F i g u r e 1. I n f r a r e d spectrum of F l u p h e n a z i n e d e c a n o a t e as a t h i n f i l m . I n s t r u m e n t : Unicam SP 1 0 0 0 .
F i g u r e 3. N u c l e a r m a g n e t i c r e s o n a n c e spectrum of F l u p h e n a z i n e d e c a n o a t e i n DMSO-d6. I n s t r u m e n t : Thompson Packard.
ERRATA FOR VOLUME 9
731
GENTAMICIN SULFATE
Bernard E. Rosenkrantz, Joseph R. Greco, John G. Hoogerheide, and Edwin M . Oden
L i
F i g u r e 4 . Carbon-13 NMR s p e c t r u m of Gentamicin s u l f a t e USP R e f e r e n c e S t a n d a r d .
100
200
300
400
500
600
700
TEMPERATURE, "C
F i g u r e 6. T h e r m o g r a v i m e t r i c a n a l y s i s c u r v e (TGA) of Gentamicin s u l f h t e U S P Reference Standard.
732
ERRATA FOR VOLUME 9
NADOLOL
Lidia Slusarek and Klaus Florey
F i g u r e 5. C-13 NMR s p e c t r u m of N a d o l o l i n DMSO-d6. I n s t r u m e n t : V a r i a n XL-100-15 o p e r a t e d a t 2 5 . 2 MHz.
F i g u r e 6. C-13 NMR s p e c t r u m of T e t r a b e n z o a t e d e r i v a t i v e of N a d o l o l i n CDCl3. I n s t r u m e n t : V a r i a n XL-1001 5 , o p e r a t e d a t 2 5 . 2 MHz.
CUMULATIVE INDEX
Italic numerals refer to volume numbers Acetaminophen, 3, 1 Acetohexamide,l, 1; 2, 573 Allopurinol, 7, 1 Alpha-tocopheryl acetate, 3, 111 Aminosalicylic acid, 10, 1 Amitriptyline hydrochloride, 3, 127 Amoxicillin, 7, 19 Amphotericin B, 6, 1; 7, 502 Ampicillin, 2, 1; 4, 517 Aspirin, 8, 1 Azathioprine, 10, 29 Bacitracin, 9, 1 Bendroflumethiazide, 5 , 1; 6, 597 Benzyl benzoate, 10, 55 Betamethasone dipropionate, 6, 43 Bretylium tosylate, 9, 71 Bromocriptine methanesulfonate, 8, 47 Calcitriol, 8, 83 Carbamazepine, 9, 87 Cefaclor, 9, 107 Cefamandole nafate, 9, 125; 10, 729 Cefazolin, 4, 1 Cephalexin, 4 , 21 Cephalothin sodium, 1, 319 Cephradine, 5, 21 Chloral hydrate, 2, 85 Chloramphenicol, 4, 47, 517 Chlordiazepoxide, 1, 15 Chlordwqxmde hydrochloride, 1, 39; 4, 517 Chloroquine phosphate, 5, 61 Chlorpheniramine maleate, 7, 43 Chloroprothixene, 2, 63 Chlortetracycline hydrochloride, 8, 101 Clidinium bromide, 2, 145 Clindamycin hydrochloride, 10, 75 Clonazepam, 6, 61 Clorazepate dipotassium, 4, 91 Cloxacillin sodium, 4, 113 Codeine phosphate, 10, 93 Colchicine, 10, 139 Cyanocobalamin, 10, 183 Cyclizine, 6, 83; 7, 502 Cycloserine, 1, 53 Cyclothiazide, 1, 66 Cyproheptadine, 9, 155 Dapsone, 5 , 87 Dexamethasone, 2, 163; 4, 518 Diatrizoic acid, 4, 137; 5 , 556 Diazepam, 1, 79; 4 , 517 Dibenzepin hydrochloride, 9, 181 Digitoxin, 3, 149 Digoxin, 9, 207 Dihydroergotoxine methanesulfonate, 7, 81 Dioctyl sodium sulfosuccinate, 2, 199 Diperodon, 6, 99 Diphenhydramine hydrochloride, 3, 173 Diphenoxylate hydrochloride, 7, 149 Disdfiram, 4, 168 Dobutamine hydrochloride, 8, 139 Doxorubicine, 9, 245 Droperidol, 7, 171 Echothiophate iodide, 3, 233 Emetine hydrochloride, 10, 289 Epinephrine, 7, 193 Ergotamine tartrate, 6, 113 Erythromycin, 8, 139 Erythromycin estolate, 1 , 101; 2, 573 Estradiol valerate, 4, 192 Ethambutol hydrochloride, 7, 231 Ethynodiol diacetate, 3, 253 Fenoprofen calcium, 6, 161 Flucytosine, 5, 115 Fludrocortisone acetate, 3, 281 Fluorouracil, 2, 221 Fluoxymesterone, 7, 251 Fluphenazine decanoate, 9, 275; 10, 730 733
734 Fluphenazineenanthate, 2,245; 4,523 Fluphenazine hydrochloride, 2, 263; 4, 518 Flurazepam hypochloride, 3, 307 Gentamicin sulfate, 9, 295; 10, 731 Glibenclamide, 10, 337 Gluthethimide, 5, 139 Gramicidin, 8, 179 Griseofulvin, 8, 219, 9, 583 Halcinonide, 8, 251 Haloperidol, 9, 341 Halothane, 1, 119; 2, 573 Heroin, 10, 357 Hexetidine, 7, 277 Hydralazine hydrochloride, 8, 283 Hydrochlorothiazide, 10, 405 Hydroflumethiazide, 7, 297 Hydroxyprogesterone caproate, 4, 209 Hydroxyzine dihydrochloride, 7, 319 Iodipamide, 3, 333 Isocarboxazid, 2, 295 Isoniazide, 6, 183 Isoprppamide, 2, 315 Isosorbide dinitrate, 4, 5!25; 5, 556 Kanamycin sulfate, 6, 259 Ketamine, 6, 297 Ketoprofen, 10, 443 Khellin, 9, 371 Leucovorin calcium, 8, 315 Levarterenol bitartrate, 1 , 49; 2, 573 Levallorphan tartrate, 2, 339 Levodopa, 5, 189 Levothyroxine sodium, 5 , 225 Lorazepam, 9, 397 Meperidine hydrochloride, 1, 175 Meprobamate, 1, 209; 4, 519 6-Mercaptopurine, 7, 343 Methadone hydrochloride, 3, 365; 4, 519; 9 601 Methaqualone, 4, 245, 519 Methimazole, 8, 351 Methotrexate, 5, 283 Methoxsalen, 9, 427 Methyclothiazide, 5, 307 Methylphenidate hydrochloride, 10, 473 Methyprylon, 2, 363 Metronidazole, 5, 327 Minocycline, 6, 323 Nabilone, 10, 499 Nadolol, 9, 455; 10, 732 Nalidixic acid, 8, 371
CUMULATIVE INDEX Natamycin, 10, 513 Neomycin, 8, 399 Nitrazepam, 9, 487 Nitrofurantoin, 5, 345 Nitroglycerin, 9, 519 Norethindrone, 4, 268 Norgestrel, 4, 294 Nortriptyline hydrochloride, I , 233; 2, 573 Nystatin, 6, 241 Oxazepam, 3, 441 Oxytocin, 10, 563 Penicillamine, 10, 601 Phenazopyridine hydrochloride, 3, 465 Phenelzine sulfate, 2, 383 Phenformin hydrochloride, 4, 319; 5, 429 Phenobarbital, 7 , 359 Phenoxymethyl penicillin potassium, 1 , 249 Phenylephrine hydrochloride, 3, 483 Piperazine estrone sulfate, 5, 375 Primidone, 2, 409 Probenecid, 10, 639 Procainamide hydrochloride, 4 , 333 Procarbazine hydrochloride, 5, 403 Promethazine hydrochloride, 5, 429 Proparacaine hydrochloride, 6, 423 Propiomazine hydrochloride, 2, 439 Propoxyphene hydrochloride, 1, 301; 4, 519; 6, 598 Propylthiouracil, 6, 457 Pseudoephedrine hydrochloride, 8, 489 Reserpine, 4, 384; 5, 557 Rifampin, 5 , 467 Salbutamol, 10, 665 Secobarbital sodium, 1, 343 Spironolactone, 4, 431 Sodium nitroprusside, 6, 487 Succinylcholine chloride, 10, 691 Sulphamerazine, 6, 515 Sulfamethazine, 7, 401 Sulfamethoxazole, 2, 467; 4, 520 Sulfasalazine, 5, 515 Sulfisoxazole, 2, 487 Testolactone, 5, 533 Testosterone enanthate, 4, 452 Theophylline, 4, 466 Thiostrepton, 7, 423 Tolbutamide, 3, 513; 5, 557 Triamcinolone, 1, 367; 2, 571; 4, 520, 523 Triamcinolone acetonide, 1, 397, 416; 2, 571; 4,520; 7,501
CUMULATIVE INDEX Triamcinolone diacetate, 1, 423 Triamcinolone hexacetonide, 6, 579 Triclobisonium chloride, 2, 507 Trifluoperazine hydrochloride, 9, 543 Triflupromazine hydrochloride, 2, 523; 4, 520; 5, 557 Trimethaphan camsylate, 3, 545 Trimethobenzamide hydrochloride, 2, 551 Trimethoprim, 7, 445 Trioxsalen, 10, 705 Triprolidine hydrochloride, 8, 509 Tropicamide, 3, 565 Tubocurarine chloride, 7, 477 Tybamate, 4 , 494 Valproate sodium and valproic acid, 8, 529 Vinblastine sulfate, I, 443 Vincristine sulfate, I , 463
735

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Analytical Profiles of Drug Substances Volume 10

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Analytical Profiles of Drug Substances

A Subsidiary of Harcourt Brace Jovanovich, Publishers

New York London Sydney Toronto San Francisco

Academic Press Rapid Manuscript Reproduction

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD.

PRINTED I N THE UNITED STATES OF AMERICA

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

AFFILIATIONS OF EDITORS, CONTRIBUTORS, AND REVIEWERS

1. DESCRIPTION 1.1 Nomenclature 1.1 1 Chemical Names a. 4-Amino-2-hydroxybenzoic acid.

c7 H7 N03 1.2 2 Structural

1.2 3 Wiswesser Line Notation ZR CQ DVQ

MAHMOUD M. A. HASSAN etal.

Table 1 Bond l e n g t h s (A) i n p - a m i n o s a l i c y l i c a c i d ( l ) , w i t h s t a n d a r d d e v i a t i o n s i n p a r e n t h e s e s . I n t r a m o l e c u l a r cont a c t s i n v o l v i n g t h e 0 ( 1 ) - H ( 2 1 ) . . 0 ( 2 ) hydrogen bond a r e included.

Bond a n g l e s (") i n p - a m i n o s a l i c y l i c a c i d ( 1 ) , w i t h e s t i m a t e d s t a n d a r d d e v i a t i o n s i n p a r e n t h e s e s . Angles i n v o l v i n g t h e O(1)-H(21). . 0 ( 2 ) hydrogen bond a r e i n c l u d e d .

0 (1)-C (2) -C (3)

4 3 F i n a l atomic p o s i t i o n s (x10 ; f o r H x 10 ) f o r p-aminos a l i c y l i c acid ( I ) , with standard deviations i n parentheses.

isolated C-H out of plane deformation. C-H out of plane deformation.

820 800 770

UV spectrum of PAS in ethanol was scanned using

Chemical shifts (6)

6.13 7.50 6.20 7.56

(s) = singlet, (d) = doublet.

F i g . 4 : PMR Spectrum of p - h i n o s a l i c y l i c a c i d i n DMSO-d a n d 6 TMS .

Fig. 5 : PMR Spectrum of p-Aminosalicylic acid in Acetone-d

F i g . 6 : 200 Mttz PMR S p e c t r u m of p-Aminosalicylic acid in L)i.!SO-d

Spectrum of p-Aminosalicylic acid in DMSO-d6.

F i g . 8 : I3C NMR o f f Resonance Decoupled Spectrum o f p - h i n o s a l i c y l i c a c i d i n DMSO-d6.

m/e 52 79 107 135 136 153 3. Svnthesis

Relative Intensitv 14.3 14.3 24.6 100.0 15.1 62.1

MAHMOUD M . A. HASSAN et al.

NHCOCH Estere?ucuronide COOH

F. Bertinotti, G. Giacomello and A.M. Liquori, Acta Cryst., - 7, 808 (1945).

W. Seaman, W. Allen, R.L. Pasternak and A. Pollara,

MAHMOUD M. A. HASSAN et al.

Hassan and M. Uppal Z u b a i r , unpublished d a t a .

J . T . Sheehan, J . Am. Chem. SOC. 70, 1665, (1948).

G . F . Felemons and R . A . Aug 26 (1953). R . P . P a r k e r and J . M . June 30 (1953).

Wilkinson, B r i t . Pat.696, 132,

Smith. Jr. U.S. P a t . 2 , 644, 011,

P a t . 693, 386, J u l y 1 (1953). Roth, J . Am. Pharm. Assoc.

I . H i r a o , Y . Kosugi, T . Matsuura, Y . Hironaka and Y. Gosei, Kagaku Kyokai S h i , 25 ( 5 ) , 417 (1967).

A . Venkataraman, P . R . Venkataraman and H . B . J . B i o l . Chem., 173, 641 (1948).

F . Z i n i , Riv. C r i t . C l i n . Med. 5 3 , 308 (1953).

E . L . Way, G . Peng, N . Allawala and T . C . D a n i e l s , J . Am. Pharm. Assoc., 4 4 , 65 (1955).

50 (3), 169 (1969). -

S.H. Wan, P . J . P e n t i k a i n e n and D . L . Azarnoff, J.Pharm. 63, 708 (1974).

J . Kawamata and J . Kashiwagi, Med. J . Osaka Uni., 6 , 119 (1955).

J . Am. Pharm. Ass.

The Pharmacopeia of t h e United S t a t e s of America, p . 3 6 , 1 8 t h r e v . , Mack Pub.Co.Easton, Pa,USA, p.36 (1970).

Brews, Biochem. J . , 45, 508 ( 1 9 4 9 ) .

M. Pesez, B u l l . SOC. Chim. F r . , 30, 918 (1949).

M.I. Blake, K . Makris and J . Hunt, J. Pharm. S c i . , 60 ( l l ) , 1695 (1971).

H . P . R i e d e r , Kiln Wochscher, 39 ( 1 5 ) , (1961).

105 ( 6 ) , 365 (1966)

A. Moussa, Pharmzie, 33 ( 7 ) , 460 (1978).

ANALYTICAL PROFILES OF DRUG SUBSTANCES, 10

WENDY P. WILSON AND STEVEN A. BENEZRA

Name, Formula, Molecular Weight

1.2 Appearance, Color, Odor Azathioprine is a pale yellow, odorless powder.

921 and 857 831 and 637

WENDY P. WILSON AND STEVEN A. BENEZRA

2810 2976 3109 3191