LC-MS/MS同时测定人血浆中6种抗真菌药物浓度

Simultaneous Determination of Concentration of Six Antifungal Drugs in Human Plasma by LC-MS/MS

关键词:  抗真菌药物  治疗药物监测  血浆  液相色谱-串联质谱

Key words:  antifungal drugs  therapeutic drug monitoring  plasma  LC-MS/MS

摘要:

目的 建立LC-MS/MS方法同时测定人血浆中氟康唑、伏立康唑、泊沙康唑、伊曲康唑、羟基伊曲康唑、卡泊芬净6种抗真菌药物的浓度。方法 向50 μL血浆样本中加入含同位素内标的甲醇沉淀蛋白后稀释进样分析。色谱柱为Kinetex C₁₈（3 mm×100 mm，2.6 μm），流动相为0.1%甲酸-水（含5 mmol·L^-1乙酸铵）和0.1%甲酸-甲醇，梯度洗脱，流速为0.6 mL·min^-1，进样量为5 μL，分析时间为5 min。采用电喷雾离子源，正离子多反应监测模式扫描，用于定量分析的离子对分别为m/z 307.0→220.2（氟康唑）、m/z 350.1→224.1（伏立康唑）、m/z 701.4→683.2（泊沙康唑）、m/z 705.2→392.2（伊曲康唑）、m/z 721.5→408.3（羟基伊曲康唑）、m/z 547.4→131.1（卡泊芬净）。结果 6种抗真菌药物在0.1~20 μg·mL^-1内线性良好（r≥0.995 0），准确度为90.35%~114.94%，仪器精密度RSD均<15%（定量下限处<20%）。提取回收率和基质效应分别为71.99%~90.38%和96.78%~134.17%。稳定性符合生物样本分析方法验证要求。应用本方法测定25例侵袭性真菌感染患者的血浆谷浓度，其中伏立康唑谷浓度0.70~12.94μg·mL^-1，氟康唑4.57~12.64 μg·mL^-1，个体间差异较大。结论 本方法操作简单快捷，准确度和灵敏度高，重现性良好，适用于临床治疗药物监测。

Abstract:

OBJECTIVE To establish a LC-MS/MS method for simultaneous determination of the concentration of six antifungal drugs(fluconazole, voriconazole, posaconazole, itraconazole, hydroxy itraconazole, caspofungin) in human plasma. METHODS The plasma samples(50 μL) were precipitated with methanol containing isotope internal standards. The chromatographic separation was performed on a Kinetex C₁₈ column(3 mm×100 mm, 2.6 μm) using a mobile phase of 0.1% formic acid-water(containing 5 mmol·L^-1 ammonium acetate solution) and 0.1% formic acid-methanol, at a flow rate of 0.6 mL·min^-1. The injection volume was 5 μL and the analysis time was 5 min. The detection of the analytes was performed by electrospray ionization in positive mode by multiple reaction monitoring with the transition of m/z 307.0→220.2(fluconazole), m/z 350.1→224.1(voriconazole), m/z 701.4→683.2(posaconazole), m/z 705.2→392.2(itraconazole), m/z 721.5→408.3(hydroxy itraconazole), m/z 547.4→131.1(caspofungin). RESULTS The linear concentration ranges of calibration curves for six antifungal drugs were 0.1-20 μg·mL^-1(r ≥ 0.995 0). The accuracy ranged from 90.35% to 114.94%. The RSD of instruments precision were less than 15%(20% at the lower limit of quantification). The recovery ranged from 71.99% to 90.38% and the matrix effect was between 96.78% and 134.17%. The stability met the acceptance criteria for bioanalytical method validation. The plasma concentrations of antifungal drugs in 25 patients with invasive fungal infection were determined by this method. Tough concentration of voriconazole ranged from 0.70 to 12.94 μg·mL^-1, and fluconazole ranged from 4.57 to 12.64 μg·mL^-1. Significant variance was found among patients. CONCLUSION This method is simple, accurate, sensitive and has a good reproducibility. It is suitable for clinical therapeutic drug monitoring.