Arch. Pharm. Res.

4(1), 9~17(1981)

Determination of Ampicillin and Cloxacillin Mixture by NMR

Moon-Hee Shin, Man-Ki Park, Chang-Hwa Yu and Jung-Kap Choi
Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul 151, Korea

(Received 27 January 1981)

Abstract[ZA simple, accurate and specific NMR

procedure is described for the determination of
ampicillin and cloxacillin mixtures in injection
dosage form and capsules. The solvent was dimethylsulfoxde-d6and maleic acid was the internal
standard. 'By integrating the peak at 2.68 ppm and
4.57 ppm, cloxacillin and ampicillin could be
determined respectively. The relative proton ratio
of ampicillin trihydrate and cloxacillin were 1.038
and 0.950. The coefficentsof variation of ampicillin
trihydrate and cloxacillin in a few commercial
preparation were 1.557o (n=9), 2.6970 (n=15).
KeywordsI--1Ampicillin and cloxacillin mitxure-NMR procedure-Solvent; dimethylsulfoxide-d6Internal standard; maleic acid-Integration of
the peak at 2.68 ppm and 4.57 ppm.

producing staphylococci and most strains

of gram-negative bacteria 2~3~ was analyzed
by NMR.
The analysis of this drug involves a variety
of

analytical techniques.

Ampicillin

and

cloxacillin mixtures have been assayed by

ion exchange chromatography4~, thin layer
chromatography 5~, colorimetry e,

ultraviolet

(UV) spectrophotometry7~ and iodometry 8~.

But these methods have been found to be
time-consuming and complicated.
Ampicillin sodium (Amp. Na), ampicillin
trihydrate (Amp 9 3H20)

and

cloxaciUin

(Clox) were assayed alone and mixed form

by NMR

spectrometry. The results were

compared with those obtained by an UV

spectrophotometry which has been reported
Recently considerable efforts have been
devoted

to

the

quantitative analysis of

components in pharmaceutical mixtures by

nuclear

magnetic

resonance

(NMR)

by A. G. Davidson and J. B. Stenlake r~.

Analyzing the ampicillin and c!oxacillin
mixtures by N M R method was found to
be simple, specific and accurate.

spectrometry.
Application

of NMR

spectrometry to

EXPERIMENTAL

antibiotics has been reported by William

L. Wilson et aL 17. In this report the NMR

Reagents

spectra of various commertially available

penicillins and cephalosporins have been given.
A mixture of ampicillin and cloxacillin

Standard : Amp. 3H20 (assay: 84.22~o,

Chong Kun Dang Co.)Clox
(assay: 875 mcg/mg, Chong

which provides a broad-spectrum antibiotic

combination effective against penicillinase-

Kun Dang Co.)

Internal standard : maleic acid (Wako

M.H. SHIN, M.K. PARK, C.H. YU and J.K. CHOI

10

pure chemical industries)

Solvent

: dimethylsulfoxided~ for NMR

~ample

spectroscopy (Merck)
: Amp.Na
& Clox 500mg
injections

and

A m p . 3H20

mg Amp= IAmp
W.A~p/1
IMp1 M.W.~al/2

mg Clox= Icl~ M.W.c~o~/3

I~al x M.W.Mal/2 mg Mal RVR
where IAmv =integral value of the signal

& Clox 500mg, 250mg capsules

from

various

Mal

from Amp

commercial

Iclox =integral value of the signal

sources

from Clox

Apparatus

IMaI =integral value of the signal

2qMR spectrometer : Perkin-Elmer R32

(90 MHz) All spectra

from maleic acid

M.W.amp=molecular weight of Amp

were

M.W.c~ox=molecular weight of Clox

probe

scanned

at

temperature

M.W. Mo~= molecular

of 35~
Micro-balance : Matfler type M-5
UV spectrophotometer: Pye Unicam SP
1750
Procedure

weight

of

maleic acid
RvR

= Relative

proton

ratio

RpR of Amp= IAmp M.W.Amv/1

Mal
IMa M.W.ml/2xmg Amp

1) Standard procedure by NMR

RVR of Clox= Ioo, M.W.cIo./3 mg Mal

Iml M.W.Ma/2X mg Clox

Amp. Na, Amp. 3H20, Clox were weighed

2) Standard procedure by UV method

:about 40 mg accurately and were used alone

(1) Determination of ampicillin

.or mixed form as a sample. The sample was

The mixture was weighed correspond to

transferred to a glass-stoppered tube and

10 mg of Amp accurately and placed in 25 ml

25 mg of maleic acid (weighed accurately)

mass flask. The sample was dissolved and

was added as the

internal standard. It was

the flask was filled to 25 ml mark with pH

dissolved completely with 0.5 ml of DMSO-d6.

5 buffer solution. At the same time, the

Then 0.4 ml of this solution was transferred

mixture

was

weighed

same

amount - as

to a NMR tube and one drop of tetramethyl-

above into 25 ml flask, dissolved and filled

silane (TMS) was added. The tube was placed

the flask to 25 ml with pH 9 buffer solution.

in the NMR spectrometer and the spectrum

The absorbance of the pH 5 solution of

was obtained.

mixture was read against the pH 9 solution

All peak field positions were referred to

of the mixture at 268 nm. The concentration

TMS as 0 ppm on the delta scale. Each of

of Amp was determined by comparison with

the peak of interest was integrated at least

five times. Then the amount of Amp and

the absorbance of a standard solution of Amp.

(2) Determination of cloxacillin

Clox was calculated individually as follows:

The mixture was weighed correspond to

10 mg of Clox accurately and transferred to

Arch. Pharm. Res. Vol. 4, No. 1, 1981

Determination of AmpiciHin and Cloxacillin

11

25 ml mass flask. It was dissolved and filled

the total volume to 25 ml with p H 5 buffer
solution.

The concentration

of Clox was

obtained by measurement o f the absorbance

at 275 nm of the mixture in p H 5 buffer
solution read against a p H 5 buffer blank.
The absorbance at 275 nm of a standard
solution of A m p was also measured. The
influence of A m p

on the

absorbance

Fig. 2: The NMR spectrum of ampicill_;n. 3H20.

of

sample at 275 n m was corrected. The corrected

absorbance at 275 n m was propotional to
the concentration of Clox.

RESULTS AND DISCUSSION

N M R Spectra of Amp. and Clox.

When 30 mg of the sample was taken, the

Fig. 3: The NMR spectrum of cloxacillin.

signal to noise ratio was large enough to

analyze. Therefore, in this experiment the
sample amount was decided to 40 mg.
By N M R standard procedure, the spectra
of A m p . N a , A m p . 3HzO and Clox appeared
as Fig. 1-3.
Fig. 4-5 represents the N M R spectra o f
A m p 9 N a and Clox, A m p 9 3HzO and Clox
Fig. 4: The NMR spectrum of ampicillin 9 Na anti

mixture.

cloxaciUin mixture.

Fig. 1: The NMR spectrum of ampicillin 9 Na.

Fig. 5: The NMR spectrum of ampicillin 9 3H20 and

cloxacillin, mixture.

Arch. Pharm. Res. Vol. 4, No. 1, 1981

M.H. SHIN, M.K. PARK, C.H. YU and J.K. CHOI

19
As shown

in Fig. 4, C H peak of Clox

appeared at 2.68 p p m and C H peak o f A m p 9

N a and A m p . 3H20 at 4.57 ppm, specifically.
By integrating these two peaks, A m p . N a and
Clox

could

be

determined

easly without

a n y interference.
In the N M R

spectrum

of A m p - 3 H 2 0 ,

C!ox mixture (Fig. 5), it seem to be impossible

to determine this mixture because the water

Table I: The chemical shifts of ampieillin-Na,

ampie~llin. 3HzO and elaxaeillin.
Amp 9 Na
a
b
c
d
e
f
g

Amp 9 3H20

1.47
1.58
3.98
4.50
5.46
7.37
8.70

Clox

h 1.40
i 1.50
j 4.08
k 4.90
1 4.96
m 5.40
n 6.95
o 7.41
p,~9

p e a k and the C H peak of A m p 9 3H20 were

q
r
s
t
u

1.46
2.70
3.52
3.95
5.46
5.52
v 7.59
w 8.16

overlapped.
The chemical shifts of A m p . Na, A m p . 3H20
a n d Clox were appeared as Table I.

standard.

Selection o f the Internal Standard.

Addition Effect o f Maleic Acid as the Internal

The internal standard should not interact

with sample and overlap with other peaks.

Standard
Adding 10 mg of maleic acid in the mixture,

I t should freely dissolve in solvent and shows

the water peak

a sharp and single peak.

the integration o f maleic acid was difficult

On

this experiment,

maleic acid which

presents a sharp and single peak at 6.1-6.3

p p m found to be the most satisfied internal
(g)
(f)

~H 2 ~
_

of Clox disppeared. And

because the maleic acid peak was overlapped

with a broad p e a k (Fig. 6).
Then

the

amount

of maleic acid was

increased 15,20,25 mg in the mixture separately.

(g)
H

(e)

I N
H
_ N~ _ _ _ _ ~ s

..~.

{b)
CH~

%" *

When 25 mg o f maleic acid was added, the

maleic acid peak was separated completely

'

so that the integration of maleic acid was

0
>~r~icillin sc~i~a
(nl

possible (Fig. 7).

(c)

So in this experiment,

(P) ~m)
j H
!/

14

N
0

(i)

-3Ho 0
--"~"""'
O0 OH
[

(j}

t.

~ /

cH3 H[W)

~'

[v~

Cloxac-iilin

of

maleic acid was decided to 25 mg.

In Fig. 7, it was noticeable that the water
peak of Clox and C O O H peak of maleic
acid were disappeared and a very broad
peak was appeared at 8-10 ppm.

(u)

Interaction between Maleie Acid and Water

"f-- " ~
L___ ,14

'

amount

f;cj

ArT~icil] J n t r ~hydr~te
(r)

N/O~

the

'

T".,, (~)

"OH3 " H2oIs)

7""'" co0 N~.

Hit ~

When water was added in maleic acid

from 2 y1 to 10 /4 gradually, an interaction
was found between C O O H of maleic acid
and water (Fig. 8).

Arch. Pharm. Res. Val. 4, No. 1, 1981

Determination of Ampicillin and CloxaciUin

13

Fig. 6: The NMR spectrum of maleic acid as an

internal standard (added 10 rag).

Fig. 10: Ampicillin. 3H20 calibration curve by

NMR

l~g. 7: The NMR spectrum of maleic acid as an

internal standard (added 25mg).

Fig. 11: Cloxacillin calibration curve by NMR.

l~g. 8: Interaction of maleic acid and water.

As a result of this interaction, water peak

was shifted to the low field (water peak
ordinarily appears at 4.7 ppm). From this
fact, the broad peak which was overlapped
with maleic acid in Fig. 6 was found to be
the crystal water of Clox.
Because the maleic acid moved the water
F/g. 9: The NMR spectrum of ampicillin. 3H20
eloxacillin mixture & maleic acid.

peak,

A m p . 3H20

& Clox mixture

was

possible to determine which seemed to be

Arch. Pharm. Res. Vol.4, No. 1, 1981

14

M.H. SHIN, M.K. PARK, C.H. YU and J.K. CHOI

Table H: Determination of Amp and Clox standard mixtures by NMR.

Maleic
acid
added mg

Amp 9 Na
found
recovery
mg
~o

--added
mg

25.195

Clox
found
mg

added
mg

recovery
~o

Amp 9 3H O
found
recovery
mg
Yo

--added
mg

40.320

40.493

100.43

25.260

40.000

39.699

99.25

.
.

25.630

40.460

41.244

101.94

25.115

--

--

--

40.755

41.319

102.35

--

--

25.300

--

--

41.230

42.698

103.56

--

25.025

--

--

--

39.360

40.974

104.10

--

--

25.170

40.335

41.752

--103.51

25.035

39.960

41.247

103.22

25.030

41.445

42.668

102.95

24.970

39.575

39.738

100.41

40.755

41.829

102.64

--

--

--

25.350

40.820

40.160

98.38

39.910

40.064

100.39

--

--

--

25.160

40.100

39.740

99.10

25.085

--

41.600

41.235

99.12

--

--

39.825

40.460

101.59

39.880

39.893

100.03

--

25.310

--

--

--

39.945

40.913

102.42

40.125

40.300

100.44

25.330

--

--

--

40.320

40.601

100.70

40.120

39.721

99.00

M.V.=99.92

M.V.= 101.87

M . V . - - 101.53

c. v.= 1.16%

C.V.=1.47~o

C.V.=1.74~

M . V. = m e a n v a l u e
C. V. = coefficent of variation

impossible

(Fig.

Calibration
Amp

and

respectively

Fig.

of 30-50mg per 0.5ml (Fig. 10-11).

Good linearities were found both in Amp

9).

Curve
Clox

by

standard

NMR

method

were
in

calibrated

and in Clox calibration curve. Therefore,

the

the quantitative analysis of Amp and Clox

range

12: S t a b i l i t y o f a m p i c i l l i n N a 9 a n d c l o x a c i l l i n .
mixture.

Fig.

13. S t a b i l i t y o f a m p i c i l l i n 9 3 H 2 0 a n d C l o x a c i l l i n
mixture.

Arch. Pharm. Res. VoL 4, No. 1, 1981

Determination of Ampicillin and Cloxacillin

Table IH: Determination o f ampicillin and e l o x a c i l l i n in standard mixtures by

Amp

15

UV.

Clox

added mg

found mg

recovery%

added mg

found mg

recovery %

9.97
9.96
9.98
9.96

9.98
9.82
9.91
9.82

98.50
98.60
99.30
98.59
----98.50
98.59
99.30
99.89

----10.03
10.01
10.07
10.00
10.03
10.01
10.07
10.00

----10.08
9.97
10.00
9.92
10.08
9.97
10.00
9.92

---

--

---

9.97
9.96
9.98
9.96

---9.82
9.82
9.91
9.82

M.V.---98.75 C.V.-~0.33~
M. V. =mean value
C.V. =coefficent of variation

M.V.=99.66

100.53
99.60
99,30
99.20
100.53
99.60
99.30
99.20

C.V. =0.53~

Therefore, if the whole procedure would

was possible in this range.

Also relative proton ratio was determined
by these standard treatment. Relative proton
ratio of A m p was 1.038 and that of Clox was
0.950.

be finished within 2 hrs, the error could be

minimized.

Determination in Commercial Preparations

Two lots o f A m p 9 N a & Clox mixtures in

Determination of Standard Mixture

vials and three lots o f A m p . 3H20 & Clox

A m p . Na,

mixtures in capsules were assayed by N M R

Amp 9 3H20,

Clox

standard

alone and mixtures were assayed by N M R

method. The relative contents to the declared

method (Table R, and by UV method (Table

strength of each c o m p o u n d were calculated

Ill).

as follows.

N M R method was more accurate than UV

method but less precise than UV method.

Stability of the Sample Solution

After assaying the mixture, the stability
was observed for 18-20 hours. As shown

ASA ~g S A filled a m o u n t x 100

A m p %=-- AST ~ag
DS
CsA mg S C filled a m o u n t x 100
Clox ~/o:-- CsT mg
DS
where AsA rag=Amp sample

in Fig. 12, A m p . N a was decreased 2.3%

after 2 hrs, 50 % after 18 hrs.
In Fig. 13, A m p - 3 H 2 0

found by N M R
CSA mg=Clox sample

A m p . 3 H 2 0 and 1.6 % of Clox were decreased

after 20 hrs.

method

Asr ~ g = A m p standard a m o u n t

and Clox were

still stable after 6 hrs. and only 6.9 % o f

amount

found by N M R

method

amount

found by N M R

method

Csr ~g=Cl0x standard a m o u n t

Arch. Pharm. Res: Vol.4, No. 1, 198l

16

M.H. SHIN, M.K. PARK, C.H. YU and J.K. CHOI

Table IV: Determination of ampieillin & eloxacillin in commertial mixtrues by NM]R.

Maleic
acid
mg
25.090
25.700
25.070
25.535
25.165
25.535
25.025
25.610
25.415
25.375
25.275
25.130
25.225
26.015
25.245

Sarnaplefilled
in vial or cap.
nag
582.080
576.740
573.500
565.510
555.250
571.785
591.825
604.720
616.475
610.750
608.610
603.760
299.890
310.905
301.580

Taken

found by NMR (mg)

sample Amp 9 Na
nag
80.740
46.429
80.082
42.019
80.095 41.528
79.855 39.433
80.265 41.588
80.310
40.203
80.035
-80.500
-80.485
-79.990
-80.565
-80.295
-80.230
-80.595
-80,760
--

Amp 9 3H20
------40.445
40.640
40.479
39.074
39.663
38.960
40.033
38.598
40.591

recovery .%o
Clox

Amp 9 Na

40.394
40.853
40.046
42.645
40.695
42.341
40.432
40.511
37.956
40.334
40.649
39.713
40.871
40.035
39.142

Amp 9 3H~O

M.V.=101.21

C. V.= 1.55y,
found by NMR

The

101.92
102.96
100.36
105.70
98.53
102.52
104.29
106.51
101.75
107.78
107.48
104.51
106.94
108.11
103.02

--

-----100.40
102.85
104.45
100.51
100.94
98.69
100.82
100.32
101.95

Clox

104.16
2.69~

method

b e i m p o s s i b l e to d e t e r m i n e because C H p e a k

TA

= strength o f A m p s t a n d a r d

a n d w a t e r p e a k o f A m p . 3 H 2 0 were o v e r l a p p e d

TC

= s t r e n g t h o f Clox s t a n d a r d

But

DT

= d e c l a r e d strength

A m p . 3HzO a n d shifted the water p e a k to

result a p p e a r e d

as T a b l e t/

The content of Amp.

N a c o u l d n o t be

maleic a c i d i n t e r a c t e d with w a t e r o f

s e p a r a t e the

CH

p e a k.

Therefore,

it was

p o s s i b l e to d e t e r m i n e the A m p . 3 H 2 0 mixture.

d e t e r m i n e d b e c a u s e it was dit~cult to o b t a i n

3.

t h e s t a n d a r d o f A m p 9 N a in this experiment.

a g o o d linearity, it was sure t h a t observed

Because the c a l i b r a t i o n curve represented

amount by NMR
CONCLUSION

m e t h o d was p r o p o r t i o n a l

t o the s t a n d a r d a m o u n t .
T h e relative p r o t o n r a t i o o f A m p 9 3H20

t.

In NMR

s p e c t r u m o f A m p . N a & Clox

a n d Clox was 1.038 a n d 0.950 respectively

m i x t u r e , the p e a k at 2.58 p p m represented

4.

C l o x a n d t h a t at 4.57 p p m represented A m p

b u t the A m p 9 N a was r a t h e r unstable. The

:specifically.

whole NMR

By i n t e g r a t i n g these peaks, A m p a n d Clox

9c o u l d be d e t e r m i n e d . M a l e i c a c i d was used

The

Clox a n d A m p . 3H20 were stable

p r o c e d u r e h a d to be finished

within 2 hrs.
5.

T h e coefficients o f variation o f A m p . 3 H 2 0

as

the i n t e r n a l s t a n d a r d .

a n d Clox in v a r i o u s c o m m e r c i a l p r e p a r a t i o n s

2.

A m p 9 3 H 2 0 & Clox mixtures seemed to

were 1.55 ~ ( n = 9 ) a n d 2.69 ~ ( n = 1 5 ) .

Arch. Pharm. Res. Vol.4, No. 1, 1981

M.H. SHIN, M.K. PARK, C.H. YU and J.K. CHOI

6. Determination by N M R method was

~simple, specific than UV method, and
.accurate. It took only 15 rains to determine
sample.
This determination of Amp and Clox by
N M R could be used as a base of micro-analysis
b y using time averaging computor and drug
a

:na0nitorring

in biological material.
LITERATURE CITED

:1) Wilsion, W. L., Avodovich, H. W., and Hughes,

D. W., Applications of nuclear magnetic resonance spectroscopy to antibiotics. I. Specific identification of penicillins and cephalosporins. J.
AOAC., 57, 1300 (1974).

2) Acred, P., and Sutherland, R., Antibacterial

activities of combinations of ampicillin and cloxacillin. Antimicrob. Ag. Chemother., 53 (1966).
3) Sutherland, R., and Batcbelor, F. R., Synergistic
activity of penicillins against penicillinase-produc-

17

ing gram-negative bacteria. Nature, 201, 868

(1964).

4) Saccani, F., Neri, C., and Sudano, F., Separation

and determination of ampicillin from mixture
with dicloxacillin or cloxacillin. Bull Chim. Farm.,
108, 777 (1969).

5) Murakawa, T., Wakai, Y., Nishida, M., Fujii, R.,

Konno M., Okada k., Cioto S., and Kuwahara S.,
Chromatographic assay o f the mixed penicillins,
ampicillin and cloxacillin in body fluids. J. Antiblot., 23, 250 (1970).
6) Celletti, P., Moretti, G. P., and Petrangeli B.,
New methods for chemical determination of ampicillin and ctoxacillin alone or in combination.
Farmaco. Ed. Prat., 27, 688 (1972).

7) Davidson, A. G., and Stenlake, J. B., The specto-photometric determination of ampicillin and
cloxacillin in combined preparations. J. Pharm.
PharmacoL, 25 (suppl.), 156 (1973).
8) De Leo, S., and Pitrolo, G., Iodometric determination of ampicillin and cloxacillin in a mixture.
Bull. Chim. Farm., 112, 487 (1973).

Arch. Pharm. Res. Vol. 4, No. 1, 1981