Reverse-phase high performance liquid chromatography for the determination of tiopronin in human plasma after derivatization with p-bromophenacyl bromide

摘要

In the study,we developed a simple,rapid and sensitive method for the determination of tiopronin(TP)in human plasma,which was based on derivatization with p-bromophenacyl bromide(p-BPB)followed by liquid-liquid extraction and reverse-phase HPLC-UV detection.For the first time,the p-BPB was introduced into the derivatization of TP.The thiol group of TP was trapped withp-BPB to form a TP-p-BPB adduct,which can be very suitable for UV detection.From acidified plasma samples,the derivatized TP was extracted with 5 mL dichloromethane.Effective chromato-graphic separation was achieved using a C18 column(DIAMONSIL 150mm x 4mm i.d.,5 mu m)based on an acetonitrile-water-trifluoroacetic acid(40:59.88:0.12,v/v/v)elution at a flow-rate of 1 mL/min.The IS and the derivatized TP were detected at 263 nm.No endogenous substances were found to interfere.The limit of quantification for derivatized TP(TP-p-BPB)in plasma was 40 ng/mL.The calibration curve for the derivatized TP showed linearity in the range 0.04-4 mu g/mL with a regression coefficient corresponding to 0.9991 and the coefficient of the variation of the points of the calibration curve being lower than 10%.Extraction recoveries of the derivatized TP in plasma were greater than 72%.The method was suitably validated and successfully applied to determination of TP in human plasma samples.