@ Springer-Verlag1991
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
54
facility for detection of relevant degradation products is now
reported.
Experimental
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
% 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
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
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.
References
1.
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26.