LC-MS/MS法测定人血浆中吡非尼酮及其代谢产物的浓度

摘要

目的 建立LC-MS/MS法测定人血浆中吡非尼酮(pirfenidone,BT)及其主要代谢产物5-羧基吡非尼酮(5-carboxy-pirfenidone,SBT)的含量.方法 血浆样品加入氘代同位素内标吡非尼酮-d5(dBT)和5-羧基吡非尼酮-d5(dSBT),经甲醇沉淀蛋白后进样.液相分离采用Agilent ZORBAX SB C18(3.0 mm×100 mm,3.5 μm)色谱柱,流动相为水(含0.5％甲酸)/乙腈(50/50).质谱检测采用ESI离子源,正离子多反应检测模式检测.BT、SBT、dBT和dSBT的检测目标离子对分别为m/z 185.958/77.1、215.944/77.0、190.965/81.1和220.948/99.1.结果 空白溶剂、空白血浆无干扰,分析物及内标间无相互干扰.血浆中BT和SBT分别在0.020 59～25.14 mg·L-1和0.016 73～20.42 mg·L-1范围内线性关系良好.批内、批间精密度和准确度均在可接受范围内.分析物及内标储备液于4 ℃冰箱中放置156 d,血浆样本于室温放置4 h、-70 ℃冰箱中保存并反复冻融3次、-70 ℃冰箱保存10、29、52 d及样品处理后自动进样器(8 ℃)中放置24 h的情况下均稳定.BT和SBT的平均提取回收率和归一化基质因子均在可接受范围内.结论 本研究所建立的测定BT和SBT血浆药物浓度的LC-MS/MS分析方法具有良好的特异性、准确度、精密度、灵敏度、稳定性和耐用性,适用于人血浆中吡非尼酮及其代谢物的浓度测定及其药动学研究.%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.