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

LC-MS/MS Determination of Isoniazid, Ethambutol and Pyrazinamide in Human Plasma

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（2．1mm×100mm，3．5μm）为分析柱，ZORBAX SB-Aq柱（2．1mm×12．5mm，5μm）为保护柱，以乙腈-5mmol·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／z138．2→121．0，乙胺丁醇m／Z205．2→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5min。结果血浆中内源性物质对测定无干扰，异烟肼线性范围为0．1～6．0μg·ML^-1，定量下限（LLOQ）为0．1μg·ML^-1，乙胺丁醇线性范围为0．1～5．0μg·ML^-1，定量下限（LLOQ）为0．1μg·ML^-1，吡嗪酰胺线性范围为1．0～50．0μg·ML^-1，定量下限（LLOQ）为1．0μg·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 1μ m） guarded by a ZORBAX SB-Aq column（2.1 mm× 12.5 mm, 5 μ m）. 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 was138.2 →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 μ g. 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 μ g . ML^-1, 1.0 - 50.0 μg . ML^-1 for pyrazinamide and the limit of quantification was 1.0 μ g. 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 accurate, and proved to be suitable for the determination of isoniazid, ethambutol, pyrazinamide in human plasma in clinical plasma samples.