LC-MS/MS法测定大鼠血浆中青蒿素与羟氯喹

Development and Validation of A LC-MS/MS Method for Determination of Artemisinin and Hydroxychloroquine Concentration in Rat Plasma

目的建立一种快速可靠地测定大鼠血浆中青蒿素和羟氯喹浓度的LC-MS/MS法。方法青蒿素用液-液萃取法提取,采用Synergi Hydro-RP C_(18)柱(30 mm×2.0 mm,4μm,80)分析;羟氯喹用蛋白沉淀法提取,采用Agilent Zorbax Bonus-RP柱(100 mm×4.6 mm,3.5μm,80)分析;两者均以0.6%甲酸甲醇和5 mmol·L^(-1)醋酸铵水溶液为流动相进行梯度洗脱,流速0.6 m L·min^(-1)。质谱采用电喷雾电离(ESI)源,正离子扫描,多反应监测(MRM)方式进行检测。结果青蒿素在0.4~1000 ng·m L^(-1),羟氯喹在1~1000 ng·m L^(-1)范围内线性关系良好(r>0.995);青蒿素与羟氯喹的批内、批间精密度和准确度均符合生物样品分析的相关要求;两者提取回收率分别为67.2%~77.0%和73.5%~81.3%。结论该方法快速灵敏、准确可靠,可用于大鼠血浆中青蒿素和羟氯喹浓度的测定。

Objective The research is to establish and validate a LC-MS/MS method for determination of artemisinin and hydroxychloroquine concentration in rat plasma.Methods Artemisinin was extracted from rat plasma using liquid-liquid extraction,and the chromatographic separation was performed using a Synergi Hydro-RP C18 column(30 mm×2.0 mm,4 μm,80 A);Hydroxychloroquine was extracted from rat plasma by protein precipitation,and the chromatographic separation was performed using an Agilent Zorbax Bonus-RP column(100 mm×4.6 mm,3.5 μm,80A).A gradient elution mobile phase for both methods consisted 0.6% formic acid methanol and 5 mmol·L^-1 ammonium acetate aqueous solution at a flow rate of 0.6 m L·min^-1.Detection and quantification were performed using mass spectrometry under the multiple reaction monitoring mode with positive electrospray ionization.Results Both artemisinin and hydroxychloroquine in rat plasma showed good linearity within the range of 0.4-1000 ng·mL^-1 and 1-1000 ng·mL^-1(r > 0.995);the intra-day and inter-day precision and accuracy results for the methods were all within the acceptable criterion across all concentrations.The extraction recovery means of artemisinin and hydroxychloroquine were 67.2%-77.0% and 73.5%-81.3%,respectively.Conclusion The established methods are rapid,sensitive,accurate and reliable,which can be applied to determine artemisinin and hydroxychloroquine concentration in rat plasma.