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...