Freseniu$' Journal of

Fresenius J Anal Chem (1991) 340:53- 56

High-performance liquid chromatographic analysis

of ampicillin and cloxacillin and its application
to an intramammary veterinary preparation

@ Springer-Verlag1991

D. Thorburn Burns 1, M. O'Callaghan 1, W. Franklin Smyth 2, ,, and C. J. Ayling 3

1 Department of Analytical Chemistry, The Queen's University of Belfast, Belfast BT9 5AG, Northern Ireland
2 Department of Pharmacy, The Queen's University of Belfast, Belfast BT9 5AG, Northern Ireland
3 Norbrook Laboratories, Station Works, Newry BT35 6JP, Northern Ireland
Received November 3, 1990
Summary. Reverse phase high-performance liquid chromatography with ultra-violet detection has been used for the
simultaneous identification and determination of ampicillin,
cloxacillin and some of their degradation products following
their extraction from an intramammary preparation used in
veterinary medicine. Extraction of the intramammary base
into petroleum ether (40-60C) and partition of the
penicillins into the mobile phase gave a mean recovery of
100.8 % for ampicillin with coefficient of variation of 1.0 and
103.4% for cloxacillin with coefficient of variation of 1.2
(n = 6). Using optimised HPLC conditions ampicillin eluted
in approximately 2 rain and cloxacillin in approximately
8 rain. The overall method was found to be stability indicating, since cloxacillin and ampicillin eluted independently of their degradation products.

Introduction
fl-Lactam antibiotics such as cloxacillin, ampicillin,
penicillins G, V, amyoxycillin etc. can be assayed in raw
materials and formulations by a variety of methods.
Microbiological methods [i, 2] reflect the end use of the
antibiotic but are slow, tedious and of limited selectivity.
Iodometric titrations [3 - 6] differentiate between molecules
having an intact penicillin nucleus and degradation-products
containing "open" fl-lactam structures, but again the methods are time consuming and of limited selectivity since
certain penicillin precursors and polymers interfere.
Spectrophotometric methods such as those based on
the imidazole mercury reagent for the determination of
cloxacillin and ampicillin [7], the ninhydrin reagent for
amoxycillin [8] and the copper[II] acetate reagent for
penicillin G [9] again have limited selectivity but are more
rapid, fl-Lactam salts are not sufficiently volatile or
thermally stable to be analysed by gas chromatography. In
addition, they are not directly electro-reducible or -oxidisible
which excludes polarographic analysis.
Increasingly in recent years, HPLC has become the
method of choice for the determination of fl-lactams and
their precursors, degradation products and polymeric products in a variety of matrices (raw materials, formulations,
*Now at Department of Applied Physical Sciences, University of
Ulster, Coleraine, Co. Londonderry BT52 1SA, Northern Ireland
Offprint requests to: D. Thorburn Burns

biological fluids, tissues etc.). The technique under optimum

operating conditions combines acceptable accuracy, precision and speed of assay with the necessary sensitivity and
selectivity required.
The first HPLC assay of ampicillin appeared in 1975 [10]
although HPLC methods had been reported previously for
the detection of ampicillin in nitrofurantoin [11] and in a
cephalosporin [12]. Ion-exchange was employed with a
mobile phase of 0.02 mol/1 NaNO3 in 0.1 mol/1 pH 9.15
borate buffer with detection at 254 rim. Penicillenic and
penicilloic acid degradation products were also detected together with penicillin G. The HPLC method was compared
with the officially recognised iodometric method and no
statistically significant difference was found. However, aged
ampiciUin trihydrate powders showed a 12.5% reduction in
ampicillin concentration by HPLC and by microbiological
assay but no loss was found when determined by iodometric
titration. Other relevant HPLC studies include the analysis
of ampicillin [13], impurities in ampicillin [14], body fluid
analysis [15], separation of ampicillin from epicillin [16] and
polymeric products of ampicillin [17]. Reverse phase HPLC
has been reported to produce better peak separation in an
aqueous medium than ion exchange HPLC [16, 1 8 - 20]. As
such, RP-HPLC has proved to be an effective method for
the analysis of biological materials by direct detection at
225 nm [15], at 210 nm [21] or by post column derivatisation
[22], for separating diastereoisomers [23] and for regulatory
control of the potency of the trihydrate and anhydrous forms
of ampicillin and its sodium salt [24].
There is significantly less information available on the
assay of cloxacillin and its degradation products, perhaps
an indication of this antibiotic's relative chemical stability.
Lauriault et al. [25] have developed such an RP method with
a buffer-methanol or -acetonitrile mobile phase to detect the
penilloic and penicilloic degradation products of cloxacillin,
as identified by their 2max values. This RP-HPLC method
was applied to the analysis of eleven capsule formulations
containing cloxacillin sodium. Excellent correlation between
this and the B. P. colorimetric method [7] (imidazole mercury
reagent) was found. The penicilloic and penilloic degradation products of cloxacillin (approximately 0.1% total) were
identified by comparing their retention times with standard
compounds.
To date no HPLC assay has been reported for a combined
formulation of ampicillin and cloxacillin such as those used
for mastitis. The B. P. Veterinary [26] details an appropriate
colorimetric method. Such an HPLC method with the added

54
facility for detection of relevant degradation products is now
reported.

Experimental

Apparatus. A Shimadzu LC-4 high-performance liquid

chromatograph was used throughout the investigation. Accessories for programmable gradient elution chromatography and on-line helium degassing were available. A
variable wavelength UV-visible detector and a 20 ~tl loop
injection valve were used. The column was a Spherisorb $5
ODS-2 (Phase Separations, Queensferry, U.K.) 12.5 em x
4.5 mm. This was used in combination with a guard column
(reversed phase pellicular material) in order to extend the
life time of the analytical column. The pH-meter used to
measure the buffer and mobile phase pH was a Pye General
Purpose pH-meter. Absorption spectra of the penicillins
were obtained with a Pye Unicam SP8-400 U.V.-Visible
spectrophotometer.

Standard solutions and reagents. AnalaR chemicals were used

throughout the study and L. C. grade solvents such as water
and methanol employed in the liquid chromatographic
separations. Petroleum ether ( 4 0 - 6 0 C) was of laboratory
reagent grade. Samples of the penicillins in the form of
their sodium salts and chloramphenicol were supplied by
Norbrook Laboratories Ltd.

Techniques
The intramammary products under investigation contained
appropriate quantities of ampicillin and cloxacillin in the
form of their sodium salts suspended in an oily base. In order
to subject the penicillins to RP-HPLC investigation they
must be extracted from this base into the mobile phase.
The extraction procedure employed in this investigation
was as follows:
The contents Of the intramammary tube (ca. 5 g) were
placed in a 100 ml separating funnel, to which 50 ml of
petroleum ether (40 - 60 C) were added. The penicillins were
extracted with 3 x 30 ml of mobile phase shaking vigorously
each time for 1 min. The aqueous phase was transferred to
a 100 ml volumetric flask and after the final extraction made
to volume with mobile phase. A 5 ml aliquot of this solution
was made to volume in a 50 ml volumetric flask first adding
1 mi of internal standard solution (approximately 50 mg
m1-1 chloramphenicol in methanol). The solution was
filtered through a 0.45 l~m filter to remove particulates prior
to injection on the column.
The RP-HPLC conditions employed in the initial study
were:
(1) Mobile phase-methanol: 0.05mol/1 potassium
dihydrogen orthophosphate pH 6.0 (50:50). This was
degassed for 15 min after filtration through a 0.45 gm filter.
(2) Flow rate 1 ml min- 1.
(3) Injection volume 20 gl.
(4) Detection wavelength 220 nm.
A standard solution was prepared by weighing accurately
cloxacillin sodium (220 mg) and ampicillin sodium (75 mg),
dissolving in the mobile phase and making up to volume in
a 100 ml flask. This standard was prepared daily. Using the
above conditions resolution of ampicillin (tr 1.8 min) and
cloxacillin (tr 4.0 rain) was achieved. Although suitable for

Table 1. Variation of retention times of ampicillin and cloxacillin

with mobile phase composition

Mobile phase ratio (v/v)

Retention time (min)

% Methanol

% Buffer

Ampicillin

Cloxacillin

30
40
45
50
60

70
60
55
50
40

3.0
2.0
2.0
1.8
1.4

41.2
13.6
7.0
4.0
2.4

quality control purposes the comparatively short elution

times were inadequate for detection of degradation products
eluting close to the parent compounds.
Results

(a) Optimisation of HPLC conditions

for the simultaneous determination of cloxacillin,
ampieillin and some of their degradation products
Ampicillin has a 2max of 204 nm and cloxacillin at 206 nm.
Maximum sensitivity of the UV-detector should therefore
be achieved by choosing a detection wavelength as close as
possible to the above figures. In practice, 220 nm was chosen
because of optimum signal-to-noise ratio.
A range of mobile phases were prepared with methanol
content 30, 40, 45, 50, and 60% by volume to the appropriate
proportion of 0.05 mol/1 phosphate buffer. The pH was
maintained at 6.0. Standard solutions of ampicillin sodium
and cloxacillin sodium were prepared as previously described
in the appropriate mobile phase. The retention times
obtained are shown in Table 1.
It can be seen that the variation in mobile phase composition has a greater effect on the retention time of cloxacillin
compared to that for ampicillin. The optimum ratio 45:55
(methanol:buffer) gave adequate chromatographic resolution in a total run time of approximately 8 min.
Variation of the mobile phase pH should alter the retention times of cloxacillin and ampicillin which contain acidic
and basic functional groups. The effect of mobile phase pH
was investigated for three mobile phase ratios 40, 45, and
50% methanol, the pH being adjusted with 0.1 mol/1 NaOH
or 0.1 mol/1 HC1 to give mobile phase solutions in the pH
range 3.0 to 7.0. Standard solutions of ampicillin and
cloxacillin were prepared in the appropriate mobile phases.
The results are summarized in Table 2.
Graphs of retention time vs. pH of the mobile phase are
concave with minima at pH 5 - 6. As the methanol content
of the mobile phases increases the curves become flatter and
the influence o f p H decreases. At pH's equal to the isoelectric
points of the amino acids cloxacillin and ampicillin minimum
retention times are obtained. At these pH's they exist as
zwitterions and in this form are most stable. The minimum
retention time for ampicillin using a 45: 55 methanol buffer
ratio is found at pH 5.0 and at pH 5.3 for cloxacillin. A
pH of 5.0 was therefore chosen since maximum chemical
stability of cloxacillin and ampicillin would exist with adequate resolution of the antibiotics and degradation products.
The following chromatographic conditions were then
adopted:

55
Table 2. Variation of retention time with mobile phase pH
Mobile phase ratio (v/v)

Mobile phase pH

Methanol

Buffer

3
4
Retention time (min)

60
55
50

Ampicillin
40
45
50
Cloxacillin
40
45
50

60
55
50

3.4
2.8

2.6
2.1
2.0

2.0
1.8
1.8

2.1
2.0
1.8

2.5
2.2
2.0

40.8
22.4

23.4
10.8
9.2

9.6
8.0
5.2

9.8
7.0
4.0

11.6
9.0
6.0

1. detection wavelength 220 nm; 2. methanol: buffer

ratio 45: 55; 3. mobile phase pH 5.0: 4. flow rate 1 ml rain- 1.

(b) Linearity of response~choice of internal standard

Linear calibration of peak height vs. concentration was
found for ampicillin in the concentration range 0.029 - 0 . 1 4 7
mg m1-1 with correlation coefficient r = 0.9992 and for
cloxacillin in the concentration range 0.088 - 0.441 mg m l - 1
with r = 0.9989. Chlorarnphenicol (tk = 4.8 rain under
optimal HPLC conditions) was found to be almost an ideal
internal standard since it gave little or no interference to the
chromatographic resolution of ampicillin, cloxacillin and
their degradation products. Plots of peak height of the
penicillin/peak height internal standard vs. the penicillin
concentration were again linear over the above concentration ranges with r = 0.9924 for ampicillin and r = 0.9969
for cloxacillin.

(c) Extraction of ampicillin and cloxacillin

from intramammary product
A bulk preparation of ampicillin sodium (0.7489 g) and
cloxacillin sodium (2.197 g) in the oily base (54.47 g) was
prepared with thorough mixing. Six portions of
approximately 5 g were removed and extracted as described
above adding the internal standard solution just prior to
HPLC. Each sample was injected three times with an injection of standard solution on either side of each extract to
ensure reproducibility.
A mean recovery of 100.8% (coefficient of variation 1.0)
was found for ampicillin and a mean recovery of 103.4%
(coefficient of variation 1.2) for cloxacillin. Both antibiotics
are therefore efficiently extracted from the intramammary
base with satisfactory coefficients of variation.

(d) Estimation of average sample tube contents

A sample pack of finished product was supplied by
Norbrook Laboratories and the contents of five intramammary sample tubes were extracted and subjected to
HPLC analysis as described in this paper. Using the internal
standard method of quantitation the average weight of
ampicillin sodium per sample tube with 95% confidence
limits was found to be 95.8 + 3.6 mg and 238.5 + 3.3 mg for
cloxacillin sodium.

(e) HPLC Study of the degradation of ampicillin

and cloxaciIlin
A number of samples of aged pharmaceutical product,
stored under adverse conditions and well beyond the expiry
date of the product were extracted and subjected to the
optimised HPLC method as described herein. Four main
degradation-product peaks were observed in addition to
those of the cloxacillin and ampicillin at retention times of
1.3, 1.6, 3.2 and 6.6 min.
In an attempt to identify the peaks, ampicillin and cloxacillin were subjected to alkaline hydrolysis in 0.01 tool/1
N a O H at ambient temperature and HPLC performed after
1.5 h. Ampicillin gave rise to three new peaks at retention
times of 1.4, 1.7, and 2.2 rain with decay of the parent peak.
Cloxacillin gave new peaks at 2.1, 3.1, and 5.6 min again
with decay of the parent peak. The peaks in the degraded
product at 1.3, and 1.6 rain appear to correlate with the two
early peaks in the ampicillin sample subjected to alkaline
hydrolysis. It is probable on chemical grounds that the later
peak (which forms first) is due to the penicilloic acid of
ampicillin and that at 1 . 3 - 1 . 4 min is the penicillenic acid
of ampicillin or the penilloic acid of ampicillin formed by
decarboxylation of the penicilloic acid of ampicillin. The
peak at 3.2 rain from the degraded product seems to correlate with the 3.1 rain peak observed in the alkaline hydrolysis of cloxacillin and is probably the penicilloic acid of
cloxacillin. The peak at 6.6 rain had no parallel in the
alkaline hydrolysis products.

Conclusion
The HPLC method described herein is accurate, precise and
importantly, stability indicating and can be readily applied
to the deterioration of cloxacillin and ampicillin contained
in products such as the veterinary intramammary product
described herein.

Acknowledgements. The encouragement and support of Norbrook

Laboratories is acknowledged.

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56
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