LC-MS/MS方法同时检测人血浆中异烟肼、乙胺丁醇和吡嗪酰胺的浓度

目的: 建立同时检测人血浆中3种抗结核药物异烟肼、乙胺丁醇和吡嗪酰胺浓度的LC-MS/MS方法,用于肺结核患者及临床试验中三药血药浓度的测定。 方法: 以对乙酰氨基酚为内标,血浆样品经乙腈沉淀蛋白等处理后检测。采用Agilent ZORBAX SB-Aq色谱柱(2.1 mm×100 mm,3.5 μm)为分析柱,ZORBAX SB-Aq 柱(2.1 mm×12.5 mm,5 μm)为保护柱,以乙腈-5 mmol·L^-1 甲酸铵水溶液(含0.1%甲酸)(8∶92,v/v)为流动相,使用电喷雾离子源(ESI),以正离子多反应监测(MRM)方式进行检测,异烟肼m/z 138.2→121.0,乙胺丁醇m/z 205.2→116.1,吡嗪酰胺m/z 124.1→81.1,对乙酰氨基酚m/z 152.0→110.0。分析时间为5 min。 结果: 血浆中内源性物质对测定无干扰,异烟肼线性范围为0.1~6.0 μg·mL^-1,定量下限(LLOQ)为0.1 μg·mL^-1,乙胺丁醇线性范围为0.1~5.0 μg·mL^-1,定量下限(LLOQ)为0.1 μg·mL^-1,吡嗪酰胺线性范围为1.0~50.0 μg·mL^-1,定量下限(LLOQ)为1.0 μg·mL^-1。日内、日间精密度(RSD)均小于10%,准确率为90.4%~108.7%。 结论: 本方法特异性强,灵敏度高,测定结果可靠,适用于临床血浆样品的高通量分析。

Objective: To establish an LC-MS/MS method for the determination of isoniazid,ethambutol,pyrazinamide in human plasma and to assay the biological samples from clinical trials. Methods: 4-acetamidophenol was used as internal standard.The protein of plasma samples were precipitated with acetonitrile.The supernatant was separated on an Agilent ZORBAX SB-Aq column(2.1 mm×100 mm,3.5 μm) guarded by a ZORBAX SB-Aq column(2.1 mm×12.5 mm,5 μm).The mobile phase consisted of acetonitrile-5 mmol·L^-1 ammonium formate,0.1% formic acid solution(8∶92,v/v).Electrospray ionization(ESI) source was applied and operated in the positive multiple reaction monitoring(MRM) mode.The transition of m/z was 138.2→121.0 for isoniazid,205.2→116.1 for ethambutol,124.1→81.1 for pyrazinamide and 152.0→110.0 for 4-acetamidophenol.Each analysis was completed within 5 min. Results: Chromatograms showed no endogenous interfering peaks with blank samples.The linear calibration curve was obtained over the concentration on range of 0.1-6.0 μg·mL^-1 for isoniazid and the limit of quantification was 0.1 μg·mL^-1,0.1-5.0 μg·mL^-1 for ethambutol and the limit of quantification was 0.1 μg·mL^-1,1.0-50.0 μg·mL^-1 for pyrazinamide and the limit of quantification was 1.0 μg·mL^-1.The inter- and intra-day precision(RSD) was less than 10%.The accuracy was 90.4%-108.7%. Conclusion: The method is specific,sensitive and accurat,and proved to be suitable for the determination of isoniazid,ethambutol,pyrazinamide in human plasma from clinical plasma samples.