CEFOXITIN, SODIUM

Gerald S. Brenner

I . Introduction 170
1.1 History 170
1.2 Name, Formula, Molecular Weight 170
1.3 Definition 170
1.4 Appearance, Color, Odor 170
2. Physical Properties 171
2.1 Infrared Spectrum 171
2.2 Magnetic Resonance Spectra 171
2.3 Ultraviolet Spectrum 179
2.4 Mass Spectrum 179
2.5 Optical Rotation 182
2.6 Solubility 182
2.7 Crystal Properties 183
2.8 Hygroscopicity 183
2.9 Acid Dissociation Constant 183
3. Synthesis 183
4. Stability-Degradation Products 185
4.1 Bulk Stability 185
4.2 Solution Stability 186
5 . Pharmacokinetics and Metabolism 187
5.1 Pharmacokinetics 187
5.2 Metabolism 187
5.3 Intramuscular Absorption and Bioavailability 187
5.4 Effect of Probenecid 188
6. Methods of Analysis 188
6.1 Identification Tests 188
6.2 Ultraviolet Spectrophotometric Analysis 189
6.3 Chromatographic Analysis 189
6.4 Perchloric Acid Titration 190
6.5 lodometric Assay 190
6.6 Microbiological Assay 190
6.7 Hydroxylamine Assay 190
7. Determination in Biological Fluids 191
8. References 192

Analytical Profiles of Drug Substances Copyriaht Q 1982 by The American

Volumc I I I69 Pharmaceutical Association
ISBN &12-260(111-9
170 GERALD S. BRENNER

1. Introduction
1.1 Historv
Sodium cefoxitin, a semi-synthetic cephamycin anti-
biotic was synthesized, tested and developed by the Merck
Sharp and Dohme Research Laboratories. Cefoxitin is active
against Gram-positive and Gram-negative bacteria ( 1 ) and is
therapeutically important because of its resistance to de-
struction by bacterial 6-lactamase (2,3,4).
1.2 Name, Formula, Molecular Weight
The Chemical Abstracts name for sodium cefoxitin
(MK-306) is 3-[: [Aminocarbonyl)oxylmethyl I-7-methoxy-8-0x0-
7~~2-thienylacetyl)aminol-5-thia-l-azabicyclo~4.2.Oloct-2-
ene-2-carboxylic acid sodium salt. The CAS registry no. is
33564-30-6.
Other names include 3-carbamoyloxymethyl-7a-methoxy-
7~-(2-thienylacetamido)-3-cephem-4-carboxylic acid sodium
salt, sodium 3-carbamoyloxymethyl-7-methoxy-7-[2-(2-thienyl~
acetamidol-3-cephem-4-carboxylate and sodium (6R,7R)-3-(hy-
droxymethyl)-7- -methoxy-8-oxo-7-[2-(2-thienyl)acetamidol-5-
thia-l-azabicyclo[4.2.Oloct-2-ene-2 carboxylate carbarnate
(ester) .

‘1 6H16N3Na07S2 Molecular Weight: 449.44

1.3 Definition
Sodium cefoxitin, unless specified otherwise, is
defined as the crystalline, non-solvated salt form of the
compound. Cefoxitin when specified refers to the free acid.
1 .4
Appearance, Color, Odor
White to off-white granules or powder having a
slight characteristic odor.
CEFOXITIN, SODIUM 171

2. Physical Properties
2.1 Infrared Spectrum
The infrared spectrum of c e f o x i t i n presented i n
Figure 1 was taken i n a KBr p e l l e t using a P e r k i n Elmer
Model 621 infrared spectrophotometer. A list of t h e
assignments made f o r some of t h e c h a r a c t e r i s t i c bands is
given i n Table I ( 5 ) .

Table I

IR Spectral Assignments f o r Cefoxitin

Frequency (ern-') Assignment

3200-3500 various N-H s t r e t c h

3000 (broad) OH s t r e t c h
1780 B-lactam C=O s t r e t c h
1715 carboxyl C=O s t r e t c h
1680 carbamate C=O s t r e t c h
1660 amide C=O stretch
1420 various -CH2 bends
1080 carbamate C-0 s t r e t c h

The infrared spectrum of sodium c e f o x i t i n taken a s

a mineral o i l m u l l is shown i n Figure 2.

2.2 Magnetic Resonance Spectra

2.21 Proton Spectrum
The proton magnetic resonance spectrum of
c e f o x i t i n presented i n Figure 3 was obtained using a Varian
A-60A spectrometer with DMSO-d6 a s t h e solvent. The
s p e c t r a l assignments a r e given i n Table I1 ( 6 ) . The proton
magnetic resonance spectrum f o r sodium c e f o x i t i n i n
DMSO-d6 is shown i n Figure 4 .

Table I1

Proton Magnetic Resonance Assignments f o r Cefoxitin

ppm ( 6 ) Relative I n t e n s i t y Multiplicity Assignment

9.38 1 Singlet 0
II
-C-NH-
7.34 1 Mu 1ti p l et thienyl-a
G -85-7.05 2 Mu1t i p l e t thienyl-B
 0
-% u
a,
0
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0 0 0 0 0 0
g c o W c t N
O/o 3 3N V l1I W S NV t l l
172
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al
H
8
0
r
I
0
0
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2
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0
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w
I73
174
 A
1m

FIGURE 4

I
A
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I . J . I .
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,

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8.0 7.0 6.0 5.0 4.0 3.0 2.0 10 0

PPM

Figure 4 . Proton Magnetic Resonance Spectrum of Sodium Cefoxitfn in DMSO-d 6 '

176 GERALD S. BRENNER

Table I1 (cont'd)
ppm (6 1 Relative Intensity Multiplicity Assignment
6.52 2 Singlet 0
I1
0-c-NH2
5.13 1 Singlet 6-H
4.49-5.0 2 Mu1tiplet 0
II
-CH20C-
0

3'83
3.08-3.80
3.40
} 6-7
Singlet
Mu1tiplet
Singlet
thien yl-CH2-C
b-CH
7-OC~;~
II

2.22 Carbon-13 Spectrum

The carbon-13 magnetic resonance spectra of
cefoxitin presented in Figures 5 and 6 were obtained using a
Varian CFT-20 spectrometer with DMSO-d as the solvent at
a concentration of 0.5 -
M. The spectra assignments are P
given in Table I11 (7).
Table I11
Carbon-13 Magnetic Resonance Assignments for Cefoxitin

ppm( 6 ) Assignment
170.4 amide C=O
162.5 carboxyl C=O
160.3 C8, lactam C=O
156.3 carbamate C=O
136.5 c21
126.5
126.4} C31 and C4l
 c a r boxy I
\

onide
C
\

c
corbomok
7 ow3
I

J1
 I c,

175 I60 PPM I25

Figure 6. C-13 WiA Spectrum of Cefoxitin in DMSO-d

6'
CEFOXITIN, SODIUM 179

Table I11 (cont'd)

ppm ( 6 ) Assignment

125.8
125.5
124.9 c j1
95.1 c7
62.8 c6
61.9 C
52.5 OEB, -I
0
II
35.9 thien yl-CH2-C-
-
25.7 c4
2.3 U l t r a v i o l e t Spectrum
The u l t r a v i o l e t absorDtion sDectrum o f c e f o x i t i n i n
pH 7.0 phosphate buffer is chabacterized by a peak a t about
2 5 nm and a shoulder a t about 262 nm (Figure 7) with
A?$m values of approximately 330 and 209 respec-
t i v e l y . The shoulder a t 262 nm is d u e t o t h e 3-cephem
chromophore and t h e maximum a t 235 nm is due to thienyl-
a c e t y l s i d e chain.

2.4 Mass Spectrum

The mass spectrum o f c e f o x i t i n was obtained u s i n g a
Finnigan 3200 mass spectrometer i n t h e e l e c t r o n impact moie
w i t h an ionizing energy of 70 eV and a probe temperature of
175OC. The output from t h e mass spectrometer was analyzed
(8) and presented i n t h e form of a bar graph shown i n Figure
8.
The ion of highest mass seen i n t h e spectrum o f
c e f o x i t i n f r e e acid (molecular weight 427) is m/e 366, which

0
corresponds t o t h e lactone formed from t h e

C H 2-CO N H OCH3 ~cr''

N N
0 CH2

0
d
Cefoxit i n 1ac tone
 \
\ \

I
250 nm 300 nm

Figure 7. UV Spectrum of Cefoxitin in pH 7 Phosphate Buffer.

Concentration: 2.75 mg/100 ml.

180
 “1
D
Ln
(3
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.d
U
*r(
x
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u
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D
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0
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N 4
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rn
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AlISN31NI 3 A l l V l 3 Y
181
182 GERALD S. BRENNER

carboxylic acid and carbamate group. The n e x t ion (m/e 322)

corresponds t o l o s s o f C02 from the l a c t o n e . Scission o f
t h e s i d e chain amide bond of t h e l a c t o n e g i v e s m/e 241,
which l o s e s CH20
J
to give m/e 210. A t lower mass one s e e s

m /e 124,
@CH=C=O +

m/e 112,

m/e 97,

2.5 Optical Rotation

The s p e c i f i c r o t a t i o n o f a 1% (w/v) sodium
c e f o x i t i n s o l u t i o n i n methanol determined a t 509 nm and
25OC i s +210° + 4' calculated on an anhydrous and
solvent f r e e b a s i s .

2.6 Solubility
The s o l u b i l i t y o f sodium c e f o x i t i n i n t h e following
s o l v e n t s a t room temperature is s t a t e d i n terms of t h e
U.S.P. d e f i n i t i o n s : v e r y s o l u b l e i n water, s p a r i n g l y
soluble i n methanol and dimethylformamide, s l i g h t l y s o l u b l e
i n ethanol and i n s o l u b l e i n e t h e r , chloroform, acetone
aromatic and a l i p h a t i c hydrocarbons.

The s o l u b i l i t y of c e f o x i t i n i n water is t y p i c a l of
an organic acid with l i m i t e d aqueous s o l u b i l i t y and
increases with increasing pH. A t pH 1 , t h e measured
s o l u b i l i t y is about 0.3 mg/ml and a t pH 4 , t h e observed
value is about 25 mg/ml ( 9 ) .
CEFOXITIN, SODIUM 183

2.7 Crvstal ProDerties

2.571 C r y s t a l l i n i t y
Sodium c e f o x i t i n exists i n s e v e r a l solvated
and i n a desolvated c r y s t a l l i n e form. An amorphous form has
a l s o been i d e n t i f i e d . X-ray powder d i f f r a c t i o n p a t t e r n s and
polarized l i g h t microscopy can d i s t i n g u i s h c r y s t a l l i n e from
amorphous forms ( 1 0 ) .

2.72 X-Ray Powder Diffraction

The x-ray powder d i f f r a c t i o n p a t t e r n o f
c r y s t a l l i n e desolvated sodium c e f o x i t i n was obtained with a
Philips-Norelco d i f f r a c t o m e t e r , using copper Kcc r a d i a t i o n .
Samples of t h i s form show broad d i f f r a c t i o n l i n e s suggesting
t h a t t h e s o l i d is m i c r o c r y s t a l l i n e and composed of extremely
small c r y s t a l l i t e s ( 1 1 ) . The amorphous form shows no
d i s t i n c t X-ray powder d i f f r a c t i o n p a t t e r n .

2.73 D i f f e r e n t i a l Scanning Calorimetry

C r v s t a l l i n e sodium c e f o x i t i n e x h i b i t s a
decomposition exotherm a t approximately 19O0-2OO0C by
d i f f e r e n t i a l scanning calorimetry (DSC). The amorphous form
does not e x h i b i t any well-defined thermal t r a n s i t i o n s by DSC
below 35OoC. Gradual decomposition of t h i s s o l i d is
observed by DSC a s temperatures increase over 15OoC ( 1 1 ) .

2.8 Hygroscopicity
The water uptake o f sodium c e f o x i t i n a s a function
of r e l a t i v e humidity was s t u d i e d a t ambient temperature
( 1 1 ) . Equilibrium water levels were approximately I-2% a t
35% RH, 4-6% a t 47% RH and 15-17% a t 76% RH.

2.9 Acid Dissociation Constant

The d i s s o c i a t i o n constant o f c e f o x i t i n derived from
aqueous t i t r a t i o n (12) and s o l u b i l i t y d a t a (9) a r e i n good
agreement and i n d i c a t e t h a t c e f o x i t i n i s a r e l a t i v e l y s t r o n g
organic acid with a pKa of approximately 2.2.

3. Synthesis
Cefoxitin has been prepared by chemical modification of
cephamycin C , a n a t u r a l l y occuring a n t i b i o t i c produced by
Streptomyces lactamdurans (13, 1 4 ) . This r o u t e i s presented
i n Figure 9. Cepharnycin C (I) is tosylated t o t h e N-tosyl
d e r i v a t i v e (11) and then e s t e r i f i e d w i t h methyl chloromethyl
ether t o y i e l d t h e methoxymethyl ester (111). The ester i s
t h e n t r e a t e d with 2-thienylacetyl c h l o r i d e t o exchange t h e
aminoadipoyl s i d e chain f o r t h i e n y l a c e t y l . The ester
protecting group i s then removed w i t h acid t o y i e l d
c e f o x i t i n (IV).
r II:
I
1 CICH20C H3

Figure 9
S y n t h e s i s of C e f o x i t i n from Cephamycin C
CEFOXITIN, SODIUM 185

C e f o x i t i n h a s a l s o been p r e p a r e d b y t o t a l s y n t h e s i s (15)
and v i a t h e a c y l a t i o n and m e t h o x y l a t i o n of 7-amino
c e p h a l o s p o r a n i c a c i d ( 1 6 , 17, 18, 19).

4. Stability-Degradation Products
4.1 Bulk S t a b i l i t v
A t room t e m p e k a t u r e sodium c e f o x i t i n i s s t a b l e for
a t l e a s t t h r e e y e a r s when p r o t e c t e d from m o i s t u r e . A t ele-
v a t e d t e m p e r a t u r e , t h e s o l i d e x h i b i t s a b i p h a s i c decomposi-
t i o n p a t t e r n t y p i f i e d by an i n i t i a l more r a p i d d e c o m p o s i t i o n
p e r i o d f o l l o w e d by a slower d e c a y p e r i o d ( 2 0 ) . T h i s phenome-
non may b e related t o d e g r a d a t i o n of c e f o x i t i n b y low l e v e l s
of w a t e r i n t h e sample. S i n c e 6 - l a c t a m c l e a v a g e is water-
consuming, t h e e x t e n t of t h i s d e g r a d a t i o n pathway is l i m i t e d
by a v a i l a b l e water i n t h e s o l i d . Amorphous sodium c e f o x i t i n
h a s been shown t o be c o n s i d e r a b l y less s t a b l e t h a n i t s
c o r r e s p o n d i n g c r y s t a l l i n e form ( 2 0 ) .

A t e m p e r a t u r e d e p e n d e n t d i s c o l o r a t i o n of t h e s o l i d
h a s been n o t e d . The d i s c o l o r a t i o n i s n e g l i b l e a t 5OC and
becomes greater a t e l e v a t e d t e m p e r a t u r e . I t h a s been shown
t h a t an i n e r t a t m o s p h e r e ( a r g o n or n i t r o g e n ) m a r k e d l y
d e c r e a s e s t h i s change. T h i s development of color is n o t
d i r e c t l y r e l a t e d t o l o s s of p o t e n c y , i . e . c o n s i d e r a b l e
d i s c o l o r a t i o n c a n o c c u r w i t h no m e a s u r a b l e loss of p o t e n c y .

S e v e r a l d e g r a d a t i o n p r o d u c t s of s o l i d sodium
c e f o x i t i n h a v e been i d e n t i f i e d . From material s t o r e d a t
6OoC for t h r e e d a y s , two compounds h a v e been i s o l a t e d and
identified.

I,p
186 GERALD S. BRENNER

4.2 Solution S t a b i l i t y
The s o l u t i o n s t a b i l i t y o f sodium c e f o x i t i n has been
studied i n aqueous b u f f e r s i n t h e pH range 3 t o 9 ( 2 0 ) . The
degradation of sodium c e f o x i t i n i n t h i s pH range follows
apparent f i r s t - o r d e r k i n e t i c s . Maximum s t a b i l i t y i n water
i s i n t h e pH range o f 5-7. Under these pH c o n d i t i o n s ,
sodium c e f o x i t i n undergoes 10% chemical l o s s i n two days a t
25OC. Ten percent l o s s a t pH 3 occurs i n about 40 hours
and a t pH 9 i n about 14 hours. TLC s t u d i e s were c a r r i e d o u t
during k i n e t i c r u n s . P a t t e r n s become complex suggesting
t h a t t h e i n i t i a l B-lactam hydrolysis product i s unstable and
s u s c e p t i b l e t o transformation t o a considerable number of
secondary products (20).

The s o l u t i o n s t a b i l i t y o f sodium c e f o x i t i n was a l s o

studied a f t e r c o n s t i t u t i o n with frequently used I . V .
infusions and admixture w i t h comnonly used I . V . and I.M.
a d d i t i v e s (21 1. S t a b i l i t y i n t h e s e systems was e s s e n t i a l l y
t h e same a s t h a t observed f o r unbuffered s o l u t i o n s . I n
t h e s e s t u d i e s , sodium c e f o x i t i n was shown t o maintain
potency i n s o l u t i o n f o r a t l e a s t 30 days a t 5OC and f o r 30
weeks when stored i n t h e frozen s t a t e .

I n an attempt t o i s o l a t e degradation products

formed i n s o l u t i o n , a 10% aqueous s o l u t i o n of sodium
c e f o x i t i n was heated f o r four days a t 8OoC and then
subjected t o preparative TLC. F r a c t i o n s i s o l a t e d were
examined by NMR, mass spectrometry and i n f r a r e d . The
following compounds were i d e n t i f i e d .

Thiophene-2-acetic acid Thiophene -2- acetamidr

CON+
N- ( 2'-mothoxyacotamido) thiophene-
2- ace t am ido
CEFOXITIN, SODIUM 187

5. Pharmacokinetics and Metabolism

The pharmacokinetics and metabolism of sodium c e f o x i t i n
i n humans following parenteral administration have been t h e
subject of a number of i n v e s t i g a t i o n s and reviews
(22,31,32).

5.1 Pharmacokinetics
Followina intravenous administration (bolus o r
i n f u s i o n ) , c e f o x i t i n i s d i s t r i b u t e d r a p i d l y between serum
and t i s s u e and e x h i b i t s a terminal serum h a l f - l i f e of 30 t o
50 m i n u t e s . Total body clearance of c e f o x i t i n ranges from
approximately 250 m l t o 350 m l / m i n while r e n a l clearance i s
approximately 200 t o 300 m l / m i n . Urine contains a t l e a s t
90% of t h e dose a s unchanged drug and less than 5% of t h e
dose i s eliminated by metabolism and b i l i a r y clearance
(23,24,25). The d i s p o s i t i o n k i n e t i c s are f i r s t o r d e r ,
showing no e f f e c t of dose (0.25 t o 3 g.) or infusion r a t e .
Multiple dose regimens i n t h i s range given every f o u r hours
do not cause accumulation i n healthy volunteers. The volume
of d i s t r i b u t i o n i n t h e vascular compartment is about 8
l i t e r s (26-34). These d a t a a r e adequately described by a
two-compartment open model w i t h elimination occurring from
t h e c e n t r a l compartment.

5.2 Metabolism
Sodium c e f o x i t i n i s not metabolized appreciably i n
man. Urine samples from s e v e r a l human s t u d i e s were sepa-
rated by HPLC and TLC techniques. These s t u d i e s show t h a t
more than 90% of t h e c e f o x i t i n administered by e i t h e r t h e
intravenous o r intramuscular route i s recovered i n t h e u r i n e
a s i n t a c t drug. A microbiologically i n a c t i v e metabolite,
descarbamoyl c e f o x i t i n , was found t o t h e extent of 1-6% i n
some i n d i v i d u a l s , 2 t o 4 hours post dosing (26,271. This
metabolite was not found i n t h e u r i n e o f a l l s u b j e c t s .

Descarbarnoyl c e f o x i t i n
188 GERALD S . BRENNER

5.3 Intramuscular Absorption and B i o a v a i l a b i l i t y

Sodium c e f o x i t i n is raDidly and completely absorbed
following intramuscular administration. Peak serum levels
a r e a t t a i n e d i n 30 m i n u t e s o r less and 85 t o 95% of t h e
i . m . dose is recovered i n u r i n e within 12 hours of
administration.

Intramuscular administration o f sodium c e f o x i t i n

with either 0.5 o r 1.0% l i d o c a i n e hydrochloride a s a d i l u e n t
has no apparent e f f e c t on t h e b i o a v a i l a b i l i t y o f t h e
an tibiotic (32,35>.

5.4 E f f e c t o f Probenecid
The concurrent o r a l o r intravenous administration
of probenecid with i . m . o r i . v . i n j e c t i o n s of c e f o x i t i n has
a l a r g e influence on t h e time course o f t h e a n t i b i o t i c i n
serum (26,36). Probenecid administered intravenously
concurrent with c e f o x i t i n i n c r e a s e s t h e serum h a l f - l i f e o f
c e f o x i t i n from approximately 40 minutes t o 80 m i n u t e s and
reduces t h e r e n a l clearance of t h e drug from 200-300 ml/min
t o less than 100 m l / m i n .

6. Methods o f Analysis
6 . 1 I d e n t i f i c a t i o n Tests
U l t r a v i o l e t spectrophotometry i s used t o i d e n t i f y
sodium c e f o x i t i n . The spectrum o f a sample dissolved i n pH
6.0 phosphate buffer scanned from 220 t o 310 nm compares
q u a l i t a t i v e l y t o t h a t of a c e f o x i t i n standard s i m i l a r l y
tested.

The i d e n t i t y o f sodium c e f o x i t i n is a l s o
established by i n f r a r e d spectroscopy. The i n f r a r e d
absorbance spectrum of a s o l i d sample prepared e i t h e r a s a
potassium bromide d i s c o r mineral o i l m u l l is compared t o a
standard sample prepared i n an i d e n t i c a l manner.

A color test has been employed f o r t h e d e t e c t i o n of

c e f o x i t i n i n s o l u t i o n (37). To t h e r e s i d u e obtained by
drying a s o l u t i o n containing 10-50 mcg of c e f o x i t i n i s added
1-2 m l of 0.01% ninhydrin i n concentrated sulfuric acid and
t h e color is allowed t o develop a t room temperature. A
vivid blue c o l o r appears i n a few m i n u t e s . Other
cephalosporin a n t i b i o t i c s do not give t h e same c o l o r
response.
CEFOXITIN, SODIUM 189

Cefoxitin and f i f t e e n other cephalosporins have

been i d e n t i f i e d by thin-layer chromatography coupled with
color r e a c t i o n s (38) and by spectroscopic methods (39).
6.2 U l t r a v i o l e t Spectrophotometric Analysis
I n t a c t sodium c e f o x i t i n e x h i b i t s a UV absorption
band near 262 nm a t t r i b u t e d t o t h e OX-N-C=C linkage i n t h e
molecule. Beta-lactam r i n g opening l e a d s t o disappearance
of t h i s absorption band. This observation is t h e b a s i s of a
q u a n t i t a t i v e assay f o r c e f o x i t i n which i s s t a b i l i t y
indicating. Calculation of i n t a c t compound i s based on t h e
net absorbance a t 262 nm of t h e sample and of t h e standard
i n pH 6.0 phosphate buffer a s determined by s u b t r a c t i n g a
base l i n e correction a t 262 nm from t h e maximum a t t h e same
wavelength. The correction i s found by extending t o 262 nm
t h e s t r a i g h t portion of t h e UV curve between 340 and 300 nm.

6.3 Chromatographic Analysis

6.31 Thin Layer Chromatography
Thin layer chromatography using t h e system
chloroform/acetone/formic acid (10:9: 1 ) w i t h 0.25 rnm s i l i c a
gel p l a t e s has been employed f o r both sodium c e f o x i t i n and
the f r e e acid. The a i r dried p l a t e is sprayed w i t h 0.2% p-
dimethylaminocinnamaldehyde i n methanol/conc. s u l f u r i c acid
( 4 : l ) and heated a t 105' f o r f i v e m i n u t e s . The Rf f o r
c e f o x i t i n i n t h i s sytem is approximately 0.45.

Cefoxitin has a l s o been chromatographed on

s i l i c a w i t h developing s o l v e n t s of n-butanol/water/acetic
acid ( 4 : l : l ) and benzene/methanol/acetic acid (50:10:6)
giving approximate Rf values of 0.7 and 0.2 r e s p e c t i v e l y .
Detection can be accomplished by fluorescence quenching or
iodine s t a i n i n g .

6.32 High Performance Liquid Chromatography

Several HPLC procedures have been developed
t o s e p a r a t e c e f o x i t i n from process i m p u r i t i e s and
degradates. A system frequently used is described below.

Mobile Phase: 20% a c e t o n i t r i l e i n water

containing 1% a c e t i c acid.

Column : Ten micron, microporous

oct,adecylsilane bonded reversed phase packing i n a 3.9 mn X
30 cm column. Column temperature and pressure a t 25OC
( o r ambient) and 1000-1500 p s i g , respectively. Flow r a t e of
I .O-l.3 ml/min.

Detection: U.V. a t 254 nm

190 GERALD S. BRENNER

Procedure: Aliquots (10 mcl) o f a sample

s o l u t i o n containing 0.25 m g h l i n 0.02 M pH 7 phosphate
buffer a r e i n j e c t e d . The r e t e n t i o n time
f o r c e f o x i t i n is
approximately 10-15 m i n u t e s .

6.4 Perchloric Acid T i t r a t i o n

Sodium c e f o x i t i n can be determined by non-aqueous
t i t r a t i o n . An accurately weighed sample of about 800 mg i s
dissolved i n about 100 m l of g l a c i a l a c e t i c acid and t i t r a -
t e d potentiometrically w i t h standardized 0.1N p e r c h l o r i c
acid i n s p e c t r a l grade dioxane. An e l e c t r o d z p a i r i s
employed c o n s i s t i n g of a calomel e l e c t r o d e which has been
r e f i l l e d w i t h 0.1N LiC104 i n a c e t i c anhydride a s t h e
i n d i c a t i n g e l e c t r o d e , and a platinum r i n g a s t h e r e f e r e n c e
electrode.

6.5 Iodometric Assay

Sodium c e f o x i t i n bulk chemical and formulations can
be determined by iodometric assay. I n t h e assay, a l i q u o t s
of t h e sample and o f a s u i t a b l e c e f o x i t i n standard s o l u t i o n
a r e hydrolyzed f o r 20 minutes with 1 N NaOH and then
a c i d i f i e d t o pH 3.0 with a c e t a t e bufTer and I N H C 1 . Then,
0.01N iodine s o l u t i o n is added and, a f t e r a 2 h i n u t e w a i t ,
t h e excess iodine is t i t r a t e d with 0.01N sodium t h i o s u l f a t e
s o l u t i o n t o a s t a r c h end p o i n t . A blank determination i s
made a t t h e same time on a l i q u o t s o f both sample and
standard s o l u t i o n s t r e a t e d only with buffer and 0.01N iodine
and allowed t o stand f o r 20 minutes.

An automated iodometric assay has been developed

based on the manual method described above w i t h t h e
exception t h a t excess iodine is measured c o l o r i m e t r i c a l l y
r a t h e r than by t h i o s u l f a t e t i t r a t i o n ( 4 0 ) .

6.6 Microbiological Assay

For bulk and formulated products an agar d i f f u s i o n
( p l a t e ) assay can be conveniently c a r r i e d out using
Staphylococcus aureua a s t h e t e s t organism ( 4 1 ) .
6.7 Hydroxylamine Assay
Sodium c e f o x i t i n can be determined by means of t h e
colored complex formed between f e r r i c ion and t h e hydroxamic
acid formed by t h e a c t i o n o f hydroxylamine on t h e beta-
lactam ( 4 2 ) . This method has been s u c c e s s f u l l y automated
CEFOXITIN, SODIUM 191

and has been specified by t h e FDA a s t h e d e f i n i t i v e assay

method f o r c e r t i f i c a t i o n of t h e a n t i b i o t i c .

7. Determination i n Biological F l u i d s
High performance l i q u i d chromatography has been u t i l i z e d
f o r t h e determination of c e f o x i t i n i n b i o l o g i c a l f l u i d s .
Cefoxitin and i t s descarbamoyl metabolite have been
q u a n t i t a t i v e l y analyzed i n human urine employing an anion-
exchange column w i t h U.V. detection ( 2 3 ) . More r e c e n t l y
Wheeler, e t a l . (43) have employed HPLC u t i l i z i n g a C-18
reversed phase packing and a solvent system of
a c e t o n i t r i l e / a c e t i c acid/O. 005M potassium dihydrogen
phosphate (25/0.5/74.5, v / v / v ) f o r the q u a n t i t a t i o n of
c e f o x i t i n i n serum and s a l i v a .

An HPLC method has been developed f o r t h e determination

of c e f o x i t i n i n serum which l e n d s i t s e l f t o automation
( 4 4 ) . The system is described below:
Mobile Phase: 11% methanol i n pH 6.86 buffer
containing 1% a c e t o n i t r i l e .

Column: Ten micron C-8 reversed phase packed i n a

3.9 mn x 25 cm column with a disposable precolumn i n l i n e
and a precolumn i n the i n j e c t i o n loop. A flow r a t e o f 3.0
m l / m i n is used.

Detection: U.V. a t 254 nm.

Procedure: Serum samples (100 1.111 a r e t r e a t e d with

100 1 of i n t e r n a l standard (aqueous cefmetazole, 15 u l / m l
and 75 u 1 10% t r i c h l o r o a c e t i c a c i d . SarnDles a r e m i x e d ,
allowed t o stand for 15 m i n u t e s and centkifuged t o remove
p r e c i p i t a t e d protein. For automated a n a l y s i s , c l e a r
supernatant is t r a n s f e r r e d t o microcentrifuge tubes. A
standard curve i s generated by spiking blank serum with
appropriate levels of c e f o x i t i n and then processing a s
described above.

Cefoxitin i n b i o l o g i c a l f l u i d s has a l s o been determined

microbiologically by t h e cup-plate diffusion-technique using
e i t h e r Staphylococcus aureus MB2876 (26,27) o r B a c i l l u s
s u b t i l i s MB36 (35) a s t h e t e s t organism.
192 GERALD S.BRENNER

8. References

1 . H. R . Onishi, D. R. Daoust, S. B. Zimnerman, D. Hendlin

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CEFOXITIN, SODIUM 193

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194 GERALD S. BRENNER

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41. Code of Federal Regulations, Sections 4 4 2 . 1 4 a ( b ) ( l ) ( i )

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L i t e r a t u r e Reviewed t o June 1981

CEFOXITIN, SODIUM 195

Acknowledgement

The author wishes t o thank Mrs. E. Moyer f o r typing t h e

manuscript, Ms. F. R. Berg and Ms. A. M. Hendrick f o r
l i t e r a t u r e r e t r i e v a l work and Mr. C. Dillman and Ms. N .
G i l b e r t f o r preparation of the f i g u r e s .