High-performance liquid chromatographic assay of ampicillin, amoxicillin and ciclacillin in serum and urine using a pre-column reaction with 1,2,4-triazole and mercury(II) chloride

Abstract

High-performance liquid chromatographic methods have been developed for the determination of ampicillin (ABPC), amoxicillin (AMPC) and ciclacillin (ACPC) in serum and urine. The methods involve acetylation of these aminopenicillins with acetic anhydride in aqueous solutions (pH 9.0) at ambient temperature for 3 min followed by reaction with 2 M 1,2,4-triazole and 10^–3 M mercury(II) chloride in solution (pH 9.0) at 60 °C for 10 min. The resulting products were separated on a C₁₈ column following stabilisation in an eluent containing sodium thiosulphate. They were detected at 328 nm for ABPC and AMPC and 327 nm for ACPC. The methods have been applied to assays of these aminopenicillins in serum and urine. The procedures, which permit the accurate determination of aminopenicillin concentrations in serum down to 0.05 µg ml^–1 for AMPC and 0.1 µg ml^–1 for ABPC and ACPC, are specific to intact penicillins without interference from the corresponding penicilloates. At an aminopenicillin concentration of 1 µg ml^–1 in serum, the within- and between-run precisions were 0.97–3.51% and 2.07–3.55%, respectively.