Determination of tiopronin in rat plasma by HPLC following fluorescent derivatization

A sensitive,rapid method for determining reduced tiopronin concentration in rat plasma has been developed by using a high-performance liquid chromatography (HPLC) technique in conjunction with the derivatizing agent N-(1-pyrenyl)maleimide (NPM). The analytes were separated on a Kromasil C₁₈ column (250 mm×4.6 mm, 5 μm) using 0.2% glacial acetic acid aqueous solution including 0.015 mol·L^-1 KH₂PO₄ and acetonitrile (56∶44) as a mobile phase at a flow-rate of 0.8 mL·min^-1, and fluorescence detection wavelength were set at λ_ex=340 nm and λ_em=375 nm, the column temperature was 30 ℃. The calibration curve was found to be linear over a range of 0.1-10.0 μg·mL^-1, the limit of quantitation was 0.1 mg·L^-1. The coefficients of the variation for the within-run and between-run precisions ranged from 5.3% to 10.8% and 7.0% to 10.8%, respectively. The percentage of absolute recovery ranged from 73.7% to 79.7%. The method was used to determine the concentration of tiopronin in rat plasma after a single intragastric administration of 25 mg·kg^-1 tiopronin to 6 healthy male Wistar rats. The pharmacokinetic process was fitted to a two-compartment model. The method has been successfully applied to the determination of tiopronin in rat plasma.