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.
We report a means of determining amoxicillin in bovine plasma by liquid chromatography with UW detection at 235 nm. Purification and concentration of extracts were accomplished by a tandem solid-phase extraction procedure with two reversed-phase columns. Separation of amoxicillin from interferences was improved by the incorporation of a crown ether in the solvent systems used both for the solid-phase extraction and the final high-pressure liquid chromatography. Cefadroxil was added as an internal standard. The average recovery of amoxicillin from plasma (n = 23) was 78.2 + 3.0%, and the within-run and between-run coefficients of variation ranged from 1.8 to 7.0%. The detection limit was estimated at 0.1 ,ug/ml. This method was used to determine amoxicillin in bovine plasma after intramuscular administration of the drug.The high-pressure liquid chromatographic (HPLC) determination of amoxicillin in plasma represents a greater analytical challenge than does HPLC determination of ampicillin. 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. To improve the selectivity of amoxicillin assays, the compound can be chemically (1, 2, 5, 9) or electrochemically (8) converted to derivatives that absorb UV light at higher wavelengths (1, 5) or that fluoresce (2, 8, 9).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). The principle of two consecutive solid-phase extractions was maintained in the amoxicillin assay reported here. However, the original approach devised for ampicillin did not prove to be transferable to amoxicillin. The modified method uses two different reversed-phase extractions rather than one ion-exchange and one reversed-phase step. A key factor for obtaining the required degree of chromatographic selectivity both on the extraction columns and in the final HPLC separation is the incorporation of a crown ether in the respective solvent systems.MATERIALS AND METHODS Chemicals and reagents. Amoxicillin trihydrate and cefadroxil (for structural formulas, see Fig. 1) were purchased from Sigma (St. Louis, Mo.). HPLC-grade acetonitrile was from ROMIL (Loughborough, United Kingdom). Crown ether (18-crown-6) was obtained from Janssen Chimica (Beerse, Belgium). Bond Elut CH (cyclohexyl) and C18 (500 * Corresponding author. mg, 2.8 ml) came from Baker (Phillipsburg, N.J.) or Analytichem (Harbor City, Calif.). All other reagents were analytical grade and were obtained from UCB (Braine-l'Alleud, Bel...
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