CEFOTAXIME

Farid J . Muhtudi and Mahmoud M . A. Hassan

1 Description 140
1.1 Nomenclature 140
1.2 Formulae 140
1.3 Molecular Weight 142
1.4 ElementalComposition 142
1.5 Appearance, Color, Odor, and Taste 142
2. Physical Properties 142
2.1 Solubility 142
2.2 Moisture Content 142
2.3 pH Range 142
2.4 Optical Rotation 142
2.5 Spectral Properties 142
3. Synthesis of Cefotaxime 152
4. Metabolism 152
5. Pharmacokinetics 156
6. Microbiological Activity 156
7. Methods of Analysis 159
7.1 Identification Tests 159
7.2 Non-Aqueous Titration 159
7.3 Chromatography 159
7.4 Spectrophotometry 165
7.5 Microbiological Assay 166
8. References 167

Analytical Profiles of Drug Substances Copyright 0 1982 by The American

Volume I I 139 Pharmaceutical Association
ISBN &12-260811-9
140 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

1. Description

1.1. Nomenclature

1.1.1 Chemical Names

(a) Sodium 7-[2-(2-amino-4-thiazolyl)-2-

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

1.1.2 Generic Name

Cefotaxime sodium; HR 756; RU-24,662;

RU-24,756

1.1.3 P r o p r i e t a r y Names

C l a f o r a n ; Primafen; Z a r i v i z ; T a r i v i d .

1.2. Formula

1 . 2 . 1 Empirical C16H16N507S2Na
1.2.2 Structural
m
?2
V
I
0 =. v
I
0
I
hl
X
V
142 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

1 . 2 . 3 CAS no.

[ 611846-23-31 (1) C16H16N507S2Na

[ 60846-21-11 (1) C H N 0 S (as f r e e acid)
16 1 7 5 7 2
[63527-52-61 (2)

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

[ a~D240 + 59.30.
CEFOTAXIME 143

2.5. Spectral Properties

2.5.1 Ultraviolet Spectra
The UV s p e c t r a o f c e f o t a x h e i n a n aqueous
s o l u t i o n and i n 0 . 1 N H C 1 a r e p r e s e n t e d i n
Fig. 1 and F i g . 2 r e s p e c t i v e l y . These were
scanned from 200 t o 400 nm u s i n g a Pye-
UnicumSP 8-100 U l t r a v i o l e t s p e c t r o -
photometer. The f o l l o w i n g t a b l e 1 shows
t h e UV d a t a .
Table 1. UV c h a r a c t e r i s t i c s of cefotaxime

solvent c onc en t r a t i o n max nm. E (lX, lcm)

water 0.5 mg/ml 234

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

2.5.2 Infrared Spectra

The I R s p e c t r a of c e f o t a x i m e a s KBr-disc
and a s n u j o l mull a r e shown i n F i g . 3 and
Fig. 4 r e s p e c t i v e l y . The KBr-disc w a s
recorded on a P e r k i n E l m e r 58OB i n f r a r e d
s p e c t r o m e t e r . The s t r u c t u r a l a s s i g n m e n t s
have been c o r r e l a t e d w i t h t h e f o l l o w i n g band
f r e q u e n c i e s (Table 2).
Table 2. I R c h a r a c t e r i s t i c s of c e f o t a x i m e
-1 Assignment
Frequency Cm

3420 -NH2
3340 (broad) -NH, -NH2
2940 -S-CH2

1 7 60 -C=O lactam 0
II
1730 -C=O e a r b o x y l i c , O-C-CH3
0
11
1650 -C-NH
0
1620 -C-NH,-C=N-,-C=C-
0
1540 -C-N-
1385-13 55 -0-CO-CH3

1180 C=O i n e s t e r
1050 C-0 stretching
 230
240
250

304

350

400

Fig.1 The UV spectrum of Cefotaxtme in water

200
210

250
2 60
270

300

350

400

Fig.2 The UV spectrum o f C e f o t a x i m e in O . I N H C I .

144
 3.0 40 5.0 MICRONS 6.0 70 8.0 9.0 10 12 14

I
4000 3500 3000 2500 2000 1800 1600 1400 1200 1000 1)oo
8
CD
0
0
a0
0
0
0
4
0
0
2
0
0
$
0
0
'0,
0
0
2
0
0
0
cu
0
0
v)
cy
a
0
0
c3
0
0
In
rn
CEFOTAXIME 147

2.5.3 Nuclear Magnetic Resonance S p e c t r a

2.5.3.1 Proton SDectra

The PMR s p e c t r a of c e f o t a x i m e i n
d e u t e r a t e d d i m e t h y l s u l f o x i d e (DMSOD6) and
d e u t e r i u m o x i d e w e r e r e c o r d e d on a v a r i a n
T-60A, 60-MHZ NMR s p e c t r o m e t e r u s i n g sodium -2,
2-dimethyl-2-silapentane-5-sulfonate (DSS) a s a n
i n t e r n a l r e f e r e n c e . These a r e shown i n F i g . 5
and F i g . 6 r e s p e c t i v e l y .
The f o l l o w i n g s t r u c t u r e a s s i g n m e n t s h a v e been
made ( T a b l e 3 ) ( 6 ) .

T a b l e 3. PMR c h a r a c t e r i s t i c s of c e f o t a x i m e

Group Chemical Sh Et ( P P )
DMSQD6
D2°

-0co CH, 2.00(s) 2.12(s)

2-G2 3 . 3 3 (9) 3 . 5 3 (9)
N - E 3 3 . 8 3 (s) 4.00(s)
0-CH
-3
AC . 4.97(9) 4.83 ( d )
6-H 4.97(q) 5.20(d)
7-H 5.60(2d) 5.82 ( d )
5-H 6.70(s) 6.97 (s)
2-Nl12 7.22 (bs) -
CONH
- 9.47 ( d )

s = s i n g l e t , d=doublet, q = q u a r t e t , 2d=doublet
of d o u b l e t s , bs=broad s i n g l e t .
13
2.5.3.2 C-NMR
13
C-NMR c o m p l e t e l y d e c o u p l e d and o f f r e s o n a n c e
s p e c t r a a r e p r e s e n t e d i n F i g . 7 and F i g . 8
respectively. Both were r e c o r d e d o v e r 5000 H,
r a n g e i n d e u t e r i u m o x i d e ( c o n c . 100 m g / l m l D20)
148
0
149
150 FARID J. MUHTADI AND MAHMOUD M . A. HASSAN

4 a
1
I

-TTJ_LLl- -J ' I ' -I

'_J _ ' ' '-11 I rI ' I '

F i g . 7. The 1 3 C NMR of Cefotaxime, completely decoupled

spectrum .

'I
I I II

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

coupled) spectrum
CEFOTAXIME 151

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

t u b e and d i o x a n e a s a r e f e r e n c e s t a n d a r d a t 2OoC.
T h e c a r b o n chemical s h i f t a r e a s s i g n e d on t h e b a s i s
of t h e a d d i t i v i t y p r i n c i p a l s and t h e o f f r e s o n a n c e
s p l i t t i n g p a t t e r n ( T a b l e 4) ( 6 ) .

12
NOCH3

H2N !?1 6
OCCH3
15

COONa
13

T a b l e 4. Carbon Chemical s h i f t s o f c e f o t a x i m e

Carbon no. Chemical s h i f t Carbon Chemical s h i f t

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

s=singlet, d=doublet, t = t r i p l e t , q=quartet.

152 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

3. S y n t h e s i s of Cefotaxime

Cefotaxime i s a s e m i - s y n t h e t i c c e p h a l o s p o r i n . The
s t a r t i n g material f o r s u c h c e p h a l o s p o r i n s i s 7-aminoceph-
a l o s p o r a n i c a c i d (7-ACA) which o b t a i n e d e i t h e r by mild
a c i d o r enzymatic h y d r o l y s i s of c e p h a l o s p o r i n C . ( 7 , 8 , 9 , 1 0 )

P r e p a r a t i o n of t h e S i d e Chain: (5)

The s t a r t i n g m a t e r i a l e t h y l a c e t o a c e t a t e [ l ] w a s o x i -
mated t o produce oximinoethylacetoacetate [ 21. Methyla-
t i o n of [ 21 followed by bromination y i e l d e d syn-methoxy-
iminobromoketone [ 3 ] . Condensation of [ 3 ] w i t h t h i o u r e a
[ 4 ] i n aqueous medium and a t a low t e m p e r a t u r e gave syn-
a m i n o t h i a z o l y l d e r i v a t i v e [ 51. The a m i n o t h i a z o l y l r i n g
w a s p r o t e c t e d by t r i t y l a t i o n t o g i v e t h e N - t r i t y l d e r i v a -
t i v e [6]. S a p o n i f i c a t i o n of t h e l a t t e r by b o i l i n g w i t h
NaOH s o l u t i o n a f f o r d e d t h e c o r r e s p o n d i n g a c i d [ 7 ] . Acy-
l a t i o n of t h e amino group of 7-ACA w i t h t h e r e s u l t i n g
a c i d [ 7 ] proved d i f f i c u l t . T h i s h a s been overcome by t h e
u s e of symmetrical a n h y d r i d e [ 8 ] , which w a s o b t a i n e d by
condensing two m o l e c u l e s of [ 7 ] i n t h e p r e s e n c e of d i c y -
c l o h e x y l c a r b o d i i m i d e [ a ] . 7-ACA w a s a c y l a t e d by compound
[8] t o g i v e [ 91. Removal of t h e t r i t y l r e s i d u e under
a c i d i c c o n d i t i o n gave t h e f r e e a c i d form (R=H) of c e f o -
taxime [ l o ] .

P r e p a r a t i o n of a S t a b l e Sodium S a l t :

T h i s w a s prepared by adding t h e s o l i d f r e e a c i d t o
a n aqueous a l c o h o l i c s o l u t i o n of a n o r g a n i c sodium s a l t
t o g i v e t h e s t a b l e a c t i v e D-form. (5)

The s y n t h e s i s of c e f o t a x i m e i s p r e s e n t e d i n Scheme 1.

I t i s i n t e r e s t i n g t o n o t e t h a t t h e syn-isomer of
cefotaxime i s u p t o 100 times more a c t i v e a g a i n s t c e r t a i n
organisms t h a n t h e a n t i - i s o m e r .

4. Metabolism

The metabolism of c e f o t a x i m e i n r a t , dog and man

u s i n g 14C-labelled c e f o t a x i m e h a s been s t u d i e d by
Chamberlain e t a l . (11). Cefotaxime i s w e l l absorbed i n
t h e t h r e e species a f t e r intramuscular administration. It
i s e l i m i n a t e d mainly v i a t h e u r i n e . The major m e t a b o l i t e
being d e s a c e t y l c e f o t a x i m e . The amount of unchanged c e f o -
CEFOTAXIME 153

OH
Scheme 1: S y n t h e s i s of Cefotaxime N’

methylation

bromination

then I
N,0CH3

H2N<NH2 + ‘OC2H5

S CH*
/
[41 Br [31

condensation

i
H2N -(syl:*5
s

[ 51

tr i t y l a t ion
154 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

condensation
C 6H 1 1 N=C=NC6H1

[a1

/
N
H C-0
3
+ symmetrical anhydride
[81

COOH
7- A M

1 Acylation
 155
CEFOTAXIME

Tr,n

H /
<N),/)[;'j
S
0-CH
3

0'
(4 CH 20Ac

C02H

1
acidification

0 -CH
3
N'

H 2N ~R=H acid
C02R ~
cH20Ac

R=Na Cefotaxime
[lo1
156 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

taxime and t h e major m e t a b o l i t e e l i m i n a t e d i n t h e u r i n e

i s s i m i l a r f o r each s p e c i e s . Two f u r t h e r m e t a b o l i t e s ,
UP1 and UP2 a r e d e t e c t e d i n dog and human u r i n e b u t n o t
i n r a t urine.

The f o r m a t i o n of b o t h UP m e t a b o l i t e s i s r e l a t e d t o
t h e r a t e of r e n a l e l i m i n a t i o n of d e s a c e t y l c e f o t a x i m e
( 11) *

The m e t a b o l i c pathway f o l l o w s t h e r o u t e i n dog and

human which may occur i n t h e l i v e r i s p r e s e n t e d i n Scheme
?
L.

5. P harmacokine t i c s

The p h a r m a c o k i n e t i c s of c e f o t a x i m e w a s r e p o r t e d t o
be d o s e independent f o r d o s e s up t o 2.0 g ( 1 2 ) . Following
t h e i n t r a m u s c u l a r (i.m.) i n j e c t i o n of a 0 . 5 g (13) and
1 . 0 g d o s e s ( 1 2 ) , t h e mean peak serum l e v e l of 11.7 \ig/ml
and 22 ug/ml a t 0.5 h were o b t a i n e d r e s p e c t i v e l y . A f t e r
i n t r a v e n o u s ( i . v . ) p o l u s i n j e c t i o n of 1 . 0 g c e f o t a x i m e ,
t h e mean peak l e v e l w a s 105 ug/ml and an a p p a r e n t volume
of d i s t r i b u t i o n of 25 l i t r e s w a s e s t i m a t e d ( 1 2 ) . Values
of 32 and 37 l i t r e s f o r t h e volume of d i s t r i b u t i o n were
a l s o r e p o r t e d ( 1 3 ) . Cefotaxime i s mainly e l i m i n a t e d by
r e n a l e x c r e t i o n and s e r u m - h a l f - l i f e ranged from 0.92 t o
1.35 h a f t e r 1 g -i.m. i n j e c t i o n , and from 0.84 t o 1 . 2 5 h
a f t e r i . v . a d m i n i s t r a t i o n ( 1 2 ) . The m a j o r i t y of t h e d o s e
being e x c r e t e d unchanged i n t h e u r i n e (-51% w i t h i n 8 h)
(12). I n a n o t h e r s t u d y (13) t h e serum c l e a r a n c e of 341
mlfmin. per 1.73 m2 and r e n a l c l e a r a n c e of 130 mlfmin p e r
1.73 m2 were o b t a i n e d . P r o t e i n b i n d i n g of c e f o t a x i m e
ranged from 35 t o 45%.

6. Microbiological Activity

Cefotaxime p o s s e s s e s a n e x t r e m e l y broad a n t i b a c t e r i a l
spectrum and i s v e r y a c t i v e a g a i n s t C positive bacteria
e s p e c i a l l y on E n t e r o b a c t e r i a c e a e , Haemophilus i n f l u e n z a e
o r Neisseria gonorrhoeae. I t is a l s o a c t i v e on Bacteroides
f r a g i l i s and Pseudomonas a e r a g i n o s a ( 1 4 ) . Cefotaxime
p e n e t r a t e s w e l l through t h e c e l l w a l l s , showing h i g h 6-
l a c t a m a s e s t a b i l i t y and s t r o n g a f f i n i t y t o t h e i m p o r t a n t
t a r g e t enzymes ( 1 4 ) .
CEFOTAXIME 157

Scheme 2 : Metabolism of Cefotaxime.

OCH3
N'

co-
Cefotaxime

Deacetylation

OCH3
'N

2
-<: D;k"oE(A
Desacetyl
NH

cefotaxime
co- CH OH

Desacetyl
cef o t a x ime l a c t o n e
ELo
09 0
158 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

OCH3
N’ H H

UP 2
CEFOTAXIME 159

7. Methods of A n a l y s i s

7 . 1 . I d e n t i f i c a t i o n Tests

The f o l l o w i n g i d e n t i f i c a t i o n t e s t s are
mentioned under c e p h a l e x i n i n t h e B.P. 1980
(15) :-

A) The i n f r a - r e d a b s o r p t i o n spectrum determined

i n t h e s o l i d s t a t e , i s concordant w i t h t h e
r e f e r e n c e spectrum of c e f o t a x i m e .

B) Mix 20 mg w i t h a few d r o p s of an 8 0 % V/V

s o l u t i o n of s u l f u r i c a c i d c o n t a i n i n g 1 % V/V
of n i t r i c a c i d ; a yellow c o l o r is produced.

C) Mix 20 mg w i t h 5 d r o p s of a 1 % V/V s o l u t i o n of
g l a c i a l a c e t i c a c i d and add 2 d r o p s of a 1 % W/V
s o l u t i o n of copper s u l f a t e and d r o p of 2M sodium
hydroxide; an o l i v e - g r e e n c o l o r i s produced.

7.2. Non-Aqueous T i t r a t i o n Assay

Weigh a c c u r a t e l y a b o u t 0.15 g of c e f o t a x i m e and

d i s s o l v e i n 50 m l g l a c i a l a c e t i c a c i d . Assay
potentiometrically with 0.1 N perchloric acid.

1 m l of 0.1 N p e r c h l o r i c a c i d c o r r e s p o n d s t o
23.87 mg of c e f o t a x i m e ( 3 ) .
7.3. Chromatography

7.3.1 Thin Layer Chromatography (TIX)

TLC of c e f o t a x i m e i s perforlned on s i l i c a
g e l c o a t e d , s e l f - i n d i c a t i n g chroma t o g r a p h i c
p l a t e s (60 F 2 5 4 , Merck).

The developing s o l v e n t system is:-

Ethylacetate/Ethanol/Water/Formic a c i d
(60 : 25 : 1 5 : 1) by volume.
Development i s c a r r i e d o u t i n a l i n e d t a n k ,
and t h e s o l v e n t i s allowed t o m i g r a t e a b o u t
1 5 cm from t h e s t a r t i n g p o i n t s .
The p l a t e s are v i s u l i z e d by examining under
u l t r a v i o l e t l i g h t a t 254 nm ( 3 ) .
160 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

The above method c a n be adopted f o r t h e

d e t e r m i n a t i o n of t o t a l i m p u r i t y c o n t e n t of
cefotaxime as f o l l o w s ( 3 ) :-

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

Two s p o t s 0.01 and .0.02 m l of t h e t e s t e d

s o l u t i o n s are a p p l i e d t o t h e c h r o m a t o p l a t e
t h e s e r e p r e s e n t i n g 200 and 400 pg r e s p e c t i v e l y .

A 0.01 m l of e a c h s t a n d a r d s o l u t i o n is
a l s o applied t o t h e chromatoplate represent-
ing 2 , 4 , 8 p g r e s p e c t i v e l y .
The p l a t e i s developed a s above.
The t o t a l i m p u r i t y c o n t e n t should n o t
exceed 5 %.

Another TLC system i s recommended f o r

cefotaxime a s f o l l o w s : -

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

7.3.2 Paper Chromatography

Descending paper chromatography w a s

accomplished u s i n g Whatman no. 1 paper.
The s o l v e n t system c o n s i s t e d of i s o p r o p a n o l -
p y r i d i n e - w a t e r (65 : 5 : 30, V / V ) . The
chromatogram was s u b j e c t e d over-night
f o r development. Examination was conducted
under t h e UV l i g h t a t 254 nm.

7.3.3 Gas Liquid Chromatography (GLC)

A GLC method f o r t h e d e t e r m i n a t i o n of
cefotaxime h a s been adopted i n our l a b o r a t o r y
CEFOTAXIME 161

u s i n g a Varian GC - 3700 g a s chromatograph

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

Another GLC method h a s been d e s c r i b e d € o r

t h e d e t e r m i n a t i o n of r e s i d u a l s o l v e n t s i n
cefotaxime ( 3 ) .

Apparatus : A 1 . 5 m PORAPAK Q column programed a t 150'

with a flame i o n i z a t i o n d e t e c t o r was used.
I s o p r o p a n o l w a s used a s t h e i n t e r n a l s t a n d a r d .

Solution I : 70 mg cefotaxime s t a n d a r d (HR 756) and 0.08 mg

i s o p r o p a n o l were d i s s o l v e d i n water t o produce
1ml.

S o l u t i o n I1 : 0.05 mg methanol, 0.13 mg e t h a n o l and 0.08 mg

i s o p r o p a n o l were mixed w i t h water t o produce
1ml.

Procedure : S o l u t i o n s I and I1 w e r e i n j e c t e d and t h e

s o l v e n t l e v e l s w e r e determined from t h e
peak h e i g h t s c o r r e c t e d a g a i n s t t h e i n t e r n a l
standard.

Results : Methanol should n o t exceed 0.2 % e t h a n o l o r

any o t h e r o r g a n i c s o l v e n t should n o t exceed 1 % .
162 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

F i g 9. GLC of c e f o t a x i m e
A = cefotaxime

7.3.4 High Performance L i q u i d Chromatography (HPLC)

S e v e r a l HPLC s y s t e m s f o r t h e i d e n t i f i c a t i o n
and s e p a r a t i o n of c e f o t a x i m e and i t s metabo-
l i t e s have been r e p o r t e d . These s y s t e m s a r e
g i v e n i n T a b l e 5.

HPLC o f c e f o t a x i m e and i t s m e t a b o l i t e s
u s i n g system 1 (11) i s p r e s e n t e d i n F i g . 10.
 T a b l e 5. HPLC Systems of Cefotaxime

System Column Mobile P h a s e Retention Time Detect ion Ref.

no. (min) ( nm)

1. A n u c l e o s ill Acetonitrile- 13.0 W region

ODS column wa t e r - a c e t i c a c i d
( 8 : 9 1 : 1 by v o l )

2. A Microbondapak, wa t er-me t h a n o l
C18 corasil (39 : 7 ) c o n t a i n i n g 30 .O 254 nm
0.005 M h e p t a n e
guard column
sulfonic acid

3. Si-C18, 10 Methanol-water- - 235 nm

(1 : 5)
microns
KH2 P O 0.06 %
4
N a 2 HPO 2H20
4
t o r e a c h pH 7 . 8

4. Li ch r os o r b Phosphoric acid- 17.0 310 nm cL7 1

RP-8 methanol
 Table 5 contd.

System Column Mobile Phase R e t e n t i o n Time Detection Ref.

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

6. A 10-cm octade- 1 4 % methanol, 12.0 254 nm (19)

c y l s i l a n e column 1% acetic acid, ( s e t a t 0.005
a n d a 4-cm pre- in distilled absorbance
column c o n t a i n i n g water unit)
copellicular
oc t a d e c y l s i l a n e
CEFOTAXIME 165

A
I D

13
R e t e n t i o n time (min)

F i g . 1 0 HPLC of C e f o t a x i m e and i t s M e t a b o l i t e s
A , D e s a c e t y l c e f o t a x i m e ; B , UP1; C , UP2;
D, Desacetyl cefotaxime l a c t o n e ; E , cefotaxime.

7.4 Spectrophotometry

A PMR method f o r q u a n t i t a t i v e d e t e r m i n a t i o n
of c e f o t a x i m e and o t h e r c e p h a l o s p o r i n s i n b u l k
d r u g s and v a r i o u s d o s a g e f o r m s i s r e p o r t e d ( 2 0 ) .
The d e t e r m i n a t i o n i s based on t h e i n t e g r a t i o n
of t h e 6-H and 1 o r 7-H of t h e @-lactam r i n g
system r e l a t i v e t o t h a t of t h e n i n e p r o t o n s of
t-butanol. The method i s r a p i d , a c c u r a t e and
precise, with an average standard deviations
of 2 1 . 5 1 i n s t a n d a r d m i x t u r e s and k 1 . 1 5 i n
p h a r m a c e u t i c a l f o r m u l a t i o n s . The p r o c e d u r e
f u r n i s h e s a s p e c i f i c means o f i d e n t i f i c a t i o n
of e a c h i n d i v i d u a l c e p h a l o s p o r i n a s w e l l a s
d e t e c t i o n of t h e commonly e n c o u n t e r e d i m p u r i t i e s .

Procedure:

Weigh a c c u r a t e l y a p o r t i o n of t h e powder
e q u i v a l e n t t o 100 mg of t h e c e p h a l o s p o r i n i n t o
a g l a s s s t o p p e r e d sample t u b e . Add 1.0 m l
a c c u r a t e l y measured D20 c o n t a i n i n g a n a c c u r a t e
w e i g h t of t - b u t a n o l , s t o p p e r and s h a k e f o r 3 min.
T r a n s f e r a b o u t 0 . 5 m l of t h e r e s u l t i n g s o l u t i o n
i n t o a NMR t u b e and r e c o r d t h e s p e c t r u m , a d j u s t i n g
t h e s p i n r a t e t o r e d u c e t h e s p i n n i n g s i d e bands a s
166 FARID J . MUHTADI AND MAHMOUD M. A. HASSAN

much a s p o s s i b l e . I n t e g r a t e t h e peaks of i n t e r e s t
(The 6- o r 7-H of t h e B-lactam r i n g a p p e a r i n g a t
4.86 - 5.80 ppm and t h e 9 p r o t o n s of t - b u t a n o l
a p p e a r i n g a t 1.23 ppm) a t l e a s t t h r e e times and
determine t h e average i n t e g r a l s .

The amount of c e p h a l o s p o r i n i s t h e n c a l c u l a t e d
as follows:-

Where Ac i s t h e i n t e g r a l v a l u e of t h e c e p h a l o s p o r i n
signal, % t h a t of t h e t - b u t a n o l s f g n a l , E.Wc is t h e
m o l e c u l a r weight of c e p h a l o s p o r i n and E.W is one
b
n i n t h of t h e m o l e c u l a r weight of t - b u t a n o l (= 8 . 2 4 ) .

7.5 M i c r o b i o l o g i c a l Assay

C e f otaxime i s m i c r o b i o l o g i c a l l y assayed by t h e
a g a r w e l l d i f f u s i o n method of Grove and R a n d a l l (21)
a s modified by Chabbert and Boulinger ( 2 2 ) .

The f o l l o w i n g t a b l e 6 summarises t h e media and

t h e t e s t organisms a s r e p o r t e d .

T a b l e 6. Nedia and Test Organisms Used

Med iurn Test Organism -

Ref.

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

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

8. References

1. "Annual Drug Data Report", Ed. J . R . P r o u s , V 0 1 . 1 1 1 ,

J . R . P r o u s S . A . , B a r c e l o n a , S p a i n , p. 5 3 , ( 1 9 8 1 ) .

2. Chemical A b s t r a c t , I n d e x Guide S u p p l . , 1977-1979

c u m u l a t i v e , The American Chemical S o c i e t y , U.S.A.

3. C l a f o r a n " Q u a n t i t a t i v e and Q u a l i t a t i v e methods of

analysis" Roussel Uclaf, P a r i s , France.

4. S . Khawaja, U n i v e r s i t y of Riyadh, S a u d i A r a b i a
" P e r s o n a l Communication''.

5. R. Bucourt, D. Bormann, R. Heymes and ?I. P e r r o n n e t

6 , S u p p l . A , p. 6 3 , (1980).
J . A n t i m i c r o b i a l Chemotherapy, -

6. M.M.A. Hassan and F . J . Muhtadi, "Unpublished Data".

7. B. Loder, G.G.F. Newton and E.P. Abraham, Biochem.

J., 2,4 0 8 , (1961).
8. R.B. Morin, B . G . J a c k s o n , E . H . F l y n n , and R.W. Roeske,
J . Am. Chem. SOC., - 8 4 , 3400, ( 1 9 6 2 ) .

9. B. F e c h t i g , H. P e t e r , H. B i c k e l and E . V i s c h e r ,
Helv. Chim A c t a , 2, 1108 (1968).

10. E . H . F l y n n , Ed., C e p h a l o s p o r i n s and P e n i c i l l i n s , C h e m i s t r y ,

B i o l o g y , Academic P r e s s , New York, p. 27, ( 1 9 7 2 ) .

11. J . Chamberlain, J . D . Coombes, D. D e l l , J . M . Fromson,

R . J . I n g s , C.M. Macdonald and J . McEwen, J . A n t i -
m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 6 9 , ( 1 9 8 0 ) .

1 2 . F. Esmieu, J. G u i b e r t , H . C . R o s e n k i l d e , I. Ho and A.
L e Go, J . A n t i m i c r o b i a l Chemotherapy, - 6 , Suppl. A,
p. 83, ( 1 9 8 0 ) .

13. K. P . Fu, P . Aswapokee, I . H o , C . M a t t h i j s s e n and

H . C . Neu, J . A n t i m i c r o b . Agents Chemother, -1 6 , 592,(1979).

1 4 . E. S c h r i n n e r , M. L i m b e r t , L. P e n a s s e and A. Lutz
J. A n t i m i c r o b i a l Chemotherapy, -
6 , Suppl. A , p. 25, (1980).

15. B.P. (1980) "The B r i t i s h Pharmacopeia" Her M a j e s t y

S t a t i o n e r y O f f i c e , Cambridge ( 1 9 8 0 ) .
168 FARID J. MUHTADI AND MAHMOUD M. A. HASSAN

16. D.S. Reeves, L.O. White, H.A. H o l t , D . B a h a r i ,

M. J . Bywater and R . P . Bax, J. A n t i m i c r o b i a l Chemotherapy,
-
6, Suppl. A , p. 93, ( 1 9 8 0 ) .

1 7 . T. Bergan, R. S o l b e r g , Chemotherapy ( B a s e l ) , 27 (3),

155, (1981).

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

and H. G r o b e c k e r , Arzneim. F o r s c h . , 31 (l), 3 6 2 , ( 1 9 8 1 ) .

19. R. Wise, N . Wright and P . J . W i l l s , J . A n t i m i c r o b .

Agents and Chemother., 9, 526, ( 1 9 8 1 ) .

20. F . J . Muhtadi, M.M.A. Hassan and M.M. Tawakkol,

Spectroscop. l e t t . I n P r e s s (1982).

21. D.C. Grove and W.A. R a n d a l l , Assay Methods of

A n t i b i o t i c s , A n t i b i o t i c Monograph, Vol. 2. M e d i c a l
E n c y c l o p e d i a , N e w York, p. 3 4 , ( 1 9 5 5 ) .

22. Y . Chabbert and H. B o u l i n g e r , Rev. F r a n c . E t . C l i n .

B i o l . (-
2 ) 636, (1957).

23. N . Lambert-Zechovsky, C . A u f r a n t , E . Bingen, C. Blunn,

M.C. Proux and H . M a t h i e u , J. A n t i m i c r o b i a l Chemo-
therapy, -6 , S u p p l . A, p. 235, ( 1 9 8 0 ) .

24. H. Lode, B. Kemmerich, G . G r u h l k e , G. D z w i l l o ,

P. Koeppe and I . Wagner, J . A n t i m i c r o b i a l Chemotherapy,
6, Suppl. A, p. 193, ( 1 9 8 0 ) .
-

25. W.M. Glb'ckner, U. H o f f l e r , J. K i n d l e r , G. P e t e r s and

H.G. S i e b e r t h , J . A n t i m i c r o b i a l Chemotherapy, 5,
Suppl. A , p. 219, ( 1 9 8 0 ) .

26. J . P . F i l l a s t r e , A. L e r o y , G. Humbert and M. Godin,

J . A n t i m i c r o b i a l Chemotherapy, 6, Suppl. A , p. 1 0 3 ,
(1980).

Acknowledgment

The a u t h o r s would l i k e t o t h a n k R o u s s e l - U c l a f ,
P a r i s , France f o r kindly providing cefotaxime
sample.