HPLC-MS/MS方法同时检测人血浆中异烟肼、利福平、乙胺丁醇、吡嗪酰胺和左氧氟沙星的浓度

摘要

目的：建立同时测定人血浆中5种抗结核药物异烟肼、利福平、乙胺丁醇、吡嗪酰胺和左氧氟沙星浓度的HPLC-MS/MS方法，以便用于肺结核患者该5种药物的血药浓度监测。方法：以对乙酰氨基酚为内标，血浆样品经甲醇沉淀蛋白处理后检测。采用Agilent Poroshell 120SB C18色谱柱（4.6 mm×50 mm，2.7μm）为分析柱，ZORBAX SB C18柱（2.1 mm×12.5 mm，5μm）为保护柱，以0.2%甲醇-0.2%醋酸溶液为流动相，梯度洗脱，使用离子喷雾离子化源（ESI），以正离子多反应监测（MRM）方式进行检测，异烟肼m/z 138.0→121.0，利福平m/z 823.2→791.2，乙胺丁醇m/z 205.2→116.1，吡嗪酰胺m/z 124.1→79.0，左氧氟沙星m/z 362.2→261.1，对乙酰氨基酚m/z152.0→110.0。分析时间为12 min。结果：血浆中内源性物质对测定无干扰，异烟肼、利福平、乙胺丁醇、吡嗪酰胺、左氧氟沙星的线性范围分别为0.20~9.81μg·mL-1、0.60~30.12μg·mL-1、0.10~4.89μg·mL-1、1.01~50.60μg·mL-1、0.10~5.00μg·mL-1。批内、批间精密度（RSD）均小于10%，准确度（RE）均小于10%。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 a method for the simultaneous determination of isoniazid, rifampicin, ethambutol, pyrazinamide and levofloxacin in human plasma by HPLC-MS/MS, and provide the convenience about blood drug monitoring of pulmonary tuberculosis patients.Methods:Acetaminophen was used as internal standard. The proteins of plasma samples were precipitated with methanol. The supernatant fluid was separated on the Agilent Poroshell 120SB C18 column (4.6 mm × 50 mm, 2.7μm) and guarded by the ZORBAX SB C18 column (2.1 mm × 12.5 mm, 5 μm). The mobile phase consisted of 0.2% of methanol and 0.2% of acetic acid in gradient elution. Electrospray ionization (ESI) source was applied and operated in the positive multiple reaction monitoring (MRM) mode. The transition ofm/z was 138.0→121.0 for isoniazid, 823.2→791.2 for rifampicin, 205.2→116.1 for ethambutol, 124.1→79.0 for pyrazinamide, 362.2→261.1 for levofloxacin and 152.0→110.0 for acetaminophen. Each analysis was completed within 12 minutes.Results:Chromatograms showed no endogenous interfering peaks with blank samples. The linear ranges were 0.20– 9.81 μg·mL-1 for isoniazid, 0.60– 30.12 μg·mL-1 for rifampicin, 0.10– 4.89 μg·mL-1 for ethambutol, 1.01– 50.60μg·mL-1 for pyrazinamide, 0.10– 5.00 μg·mL-1 for levofloxacin respectively. The intra-day and inter-day precision (RSDs) were less than 10%. The accuracy (RE) was less than 10%.Conclusion:The method is specific, sensitive and accurate, and proved to be suitable for the simultaneous determination of isoniazid, rifampicin, ethambutol, pyrazinamide and levofloxacin in human plasma from clinical plasma samples.