Liquid chromatographic determination of ampicillin in bovine and dog plasma by using a tandem solid-phase extraction method

Abstract

The determination of ampicillin in plasma and serum by reversed-phase high-performance liquid chromatography with UV detection suffers from poor selectivity and sensitivity. Currently, the most common approach to overcoming these problems consists of improving the compound's detectability via pre-or postcolumn derivatization. In the method that we describe, however, enhanced selectivity is afforded by sample purification by a tandem solid-phase extraction method (ion-exchange and reversed-phase). This approach permits detection at wavelengths of as low as 210 nm, which results in enhanced sensitivity (detection limit, 0.01 ,ug/ml). A second factor that affects selectivity is the addition to the chromatographic eluent of a crown ether to optimize the separation between ampicillin and polar endogenous plasma constituents. This combination of improved sample pretreatment and a more selective chromatographic system in conjunction with internal standardization forms the basis of a new assay for the quantitation of ampicillin in plasma. The overall recovery of ampicillin was 76.4% + 4.9% (n = 24), and the within-run and between-run coefficients of variation ranged from 1.6 to 7.2%. The method was applied to pharmacokinetic studies in cows and dogs after intramuscular or oral administration of the drug.The determination of ampicillin and other penicillins in plasma by high-performance liquid chromatography (HPLC) has traditionally struggled with problems of selectivity and sensitivity, mainly as a result of poor detectability (12). The molar absorption coefficient of ampicillin at its 'max (e = 3,036 M-1 cm-' at 257 cm) (7) is too low to afford sufficient sensitivity. Pre-and postcolumn reactions have been reported to convert the compound either to a derivative that displays more useful light-absorbing properties (nmax, 300 to 330 nm) (2, 3,12,17) or to a fluorescent breakdown product (10, 13). Alternatively, UV detection is carried out at 220 to 230 nm, which is more sensitive but less selective than detection at 257 nm (1,4,6,8,16). However, the loss of selectivity by these methods is not compensated for by a superior sample pretreatment. The latter merely consists of ultrafiltration (6) or a simple deproteinization of plasma without further cleanup of the extract (1,4,8,16). By the latter methods, sensitivity is further restricted by the dilution of the sample. Neither double-phase nor solid-phase methods have been reported for the extraction and concentration of ampicillin from plasma.The method described in this report approaches the basic problems of sensitivity and selectivity in ampicillin quantitation assays from the standpoint of sample pretreatment rather than detection. To this end, a tandem solid-phase extraction procedure was developed. The procedure not only ensures sufficient sample purification to permit sensitive detection at wavelengths of as low as 210 nm but also permits a twofold concentration of the sample. This new approach forms the basis of a reversed-phase HPLC method * Corresponding author.

Liquid chromatographic determination of amoxicillin concentrations in bovine plasma by using a tandem solid-phase extraction method

“…The choice of cefadroxil, the cephalosporin equivalent of amoxicillin, as an internal standard has the same rationale as the use of cephalexin in our ampicillin assay (11). Both compounds behave in a similar way in the assay, as evidenced by their comparable recoveries (Table 3) and the equal slopes of the standard curves obtained by analyzing spiked samples and those obtained by direct injection (Table 2).…”

“…In less selective assays that use UV detection, this quantity is usually limited to 2.5 to 20 ,ul and the detection limits are on the order of 0.5 ,ug/ml (3,4,7,13,14). The limit of detectability for the method described here was about 0.1 jig/ml, which is a factor of 10 higher than the corresponding value for our ampicillin assay (11). The difference was mainly due to the presence of the small endogenous plasma peak that coeluted with amoxicillin.…”

“…A total of 20 samples, including 5 standards and 1 control sample, could be analyzed in a normal working day. Because of the complexity of the sample pretreatment, the present method has the same limitations as our corresponding ampicillin assay (11) in terms of sample throughput. Advantages of the method include its minimal technical requirements (no need for chemical or electrochemical derivatization or column switching) and HPLC column lifetime.…”

“…In our previous ampicillin assay (11), enhanced selectivity and sensitivity were not derived from improved detection but from the use of a tandem solid-phase purification and concentration of the sample. The performance of a single solid-phase extraction method is apparently insufficient to separate amoxicillin efficiently from interfering plasma constituents (6,7).…”

“…A sensitive ampicillin assay with detection in the low UV region (210 nm) requires special approaches for sample pretreatment and chromatography, to eliminate the interference from polar plasma constituents (11). Amoxicillin exhibits a maximum atA230, but it is considerably more polar than ampicillin and, consequently, is more susceptible to such interferences.…”