RESUMEN
Aim To establish a LC-MS/MS method for the determination of pirfenidone(BT)and its major metabolite 5-carboxy-pirfenidone(SBT)in human plasma.Methods Human plasma samples containing BT and SBT,as well as their corresponding deuterium-labeled internal standards pirfenidone-d5(dBT)and 5-carboxy-pirfenidone-d5(dSBT),were precipitated using methanol.Chromatographic separation was carried out on an Agilent ZORBAX SB C18(3.0 mm×100 mm,3.5 μm)column with the mobile phase of water(0.5％formic acid)and acetonitrile(50/50).The detection of analytes was performed on a tandem mass system equipped with an electrospray ionization source in positive ion mode using multiple-reaction monitoring.The MS/MS ion transitions monitored were m/z 185.958→77.1 for BT,m/z 215.944→77.0 for SBT,m/z 190.965→81.1 for dBT and m/z 220.948→99.1 for dSBT.Results There was no remarkable interference in blank solvent,plasma,and there was no mutual interference between analytes or internal standards.The proposed method showed good linearity over the concentration range of 0.020 59～25.14 mg·L-1 for BT and 0.016 73～20.42 mg·L-1 for SBT.The intra-batch and inter-batch precision and accuracy were proved to be acceptable.Human samples kept stable after 4 h at room temperature,the three freeze-thaw cycles and 10,29 and 52 days at-70 ℃,and the processed samples remained stable after 24 h in the autosampler.The average extraction recovery and matrix effect were precise,reproducible and acceptable.Conclusion Our current LC-MS/MS method is proved to be sensitive,accurate and convenient,and could be suitable for the clinical pharmacokinetic studies of BT-related preparations.