Determination of Tiopronin in Human Plasma by SPE then Reversed-Phase HPLC–UV

Abstract

An accurate, simple and sensitive method based on reversed-phase high-performance liquid chromatography with UV detection has been developed for determination of tiopronin (TP) in human plasma. TP in plasma was reacted with p-bromophenacyl bromide (p-BPB) to give the TP-p-BPB adduct and this derivative was then extracted from the plasma on a silica gel cartridge. Potential interfering compounds were removed by washing with water, and the TP-p-BPB adduct was then eluted with acetonitrile. The organic phase obtained was evaporated to complete dryness under a stream of nitrogen. The residue was dissolved in acetonitrile and this solution was injected on to a reversed-phase ODS HPLC column. The mobile phase was usually the ternary mixture acetonitrile–water–trifluoroacetic acid, 40:59.88:0.12 (v/v). The retention times of TP-p-BPB and the internal standard adduct were 14.4 and 17.9 min, respectively. No interfering peaks were encountered in several blank plasma samples examined. The limit of detection for TP was 12 ng mL⁻¹. Extraction recovery exceeded 70%. The calibration plot for the TP derivative was linear in the range 40−4000 ng mL⁻¹, regression coefficient 0.9989, and the coefficient of the variation of the points of the calibration plot was below 10%. The method was validated appropriately and successfully applied to the determination of TP in human plasma.