High-performance liquid chromatographic assay of clavulanate in human plasma and urine by fluorimetric detection

Abstract

A high-performance liquid chromatographic method with fluorimetric detection has been developed for the determination of clavulanate in human urine and plasma. Clavulanate in plasma samples was ultrafiltered using YMT membrane and reacted with benzaldehyde in phosphate buffer solution (pH 3.8) at 100°C for 20 min. Clavulanate in urine samples was filtered with a polyacrylate membrane after ten-fold dilution, and reacted under the same conditions as those for plasma samples. The fluorescent product thus formed from clavulanate was separated from ordinary components of plasma and urine on a reversed-phase C 18 column followed by fluorimetric detection (λ ex = 386 nm, λ em = 460 nm). The within- and between-run precisions were of the order of 4.02% ( n = 10) and 6.23% ( n = 5) for plasma samples at a level of 0.67 μg/ml. The detection limit was as low as 10 ng/ml in plasma samples with a 50-μl injection. Coexisting ticarcillin, amoxicillin or 1-amino-4-hydroxy-butan-2-one (which is a metabolite of clavulanate in rat and dog) did not interfere in the clavulanate assay.