液相色谱-质谱联用法测定人血浆氯吡格雷的浓度及其药动学

摘要

Objective To establish a liquid chromatography-tandem mass spectrometric ( LC-MS/MS ) for determination of clopidogrel in human plasma and stability of clopidogrel under different conditions,which was used subsequently to investigate the pharmacokinetics of clopidogrel in healthy Chinese volunteers. Methods Clopidogrel-d4 hydrogen sulfate was used as an internal standard. Separation was achieved on a WATERS Xterra® RP18 column (4.6 mm× 100 mm,3.5μm) with a mobile phase consisting of acetonitrile-0.1% formic acid (66:34) at a flow rate of 1.0 mL.min-1 within 3.2 min. ESI source was applied and operated in positive ion mode and multiple reaction monitoring (MRM). Plasma samples were pretreated by acetonitrile precipitation. Results A good linearity of clopidogrel was obtained in the concentration range of ( 5-5 000 ) ng.L-1. The lower limit of quantification was 5 ng.L-1. The intra-and inter-run precisions at three quality control levels were within 1.3%–9.9%,the relative deviation of the assay was within -6.2%-14.3%. The blood samples were stable when chilled with crushed ice ( 0℃) and cold water ( 4℃) ,as well as kept at room temperature ( 20℃) for 40 min. The plasma QC samples were stable at room temperature ( 20 ℃) for 4 h,at -70℃ for 38 days and during three freeze-thaw cycles. Hemolysis in blood sample drawn didn’ t affect the plasma concentration. The determination and the result of incurred sample reanalysis met the requirements. Conclusion A specific, rapid, sensitive and stable LC-MS/MS method is developed and validated for determination of clopidogrel in human plasma. The method is proven to be suitable for study of the pharmacokinetics of clopidogrel in healthy Chinese volunteers after a single oral dose of 75 mg clopidogrel hydrogen sulphate tablet.%目的：建立人血浆中氯吡格雷的液相色谱-质谱联用( LC-MS/MS)定量分析方法，并考察不同条件下氯吡格雷生物样品稳定性，应用于健康受试者体内药动学研究。方法以硫酸氢氯吡格雷-d4作为内标，采用 WATERS Xterra® RP18(4.6 mm×100 mm，3.5μm)色谱柱，流动相为乙腈：水相(0.1％甲酸溶液)(66：34)，流速1.0 mL•min－1，每个样本的分析时间为3.2 min。三重四极杆质谱仪进行多反应监测( MRM)模式，电喷雾离子源( ESI)正离子检测。生物样品预处理采用乙腈直接沉淀蛋白。结果线性范围为5~5000 ng•L－1，最低定量限为5 ng•L－1；批内、批间精密度(RSD)均在1.3％~9.9％，相对偏差(RE)在－6.2％~14.3％。临床采集的全血样品于冰浴(0℃)、水浴(4℃)、室温(20℃)放置40 min的情况下均稳定，血浆质控样品室温(20℃)放置4 h，反复冻融(－70℃)3次及冰冻(－70℃)保存38 d的情况下均稳定；采血时溶血不影响测定准确度；真正样品再分析( ISR)实验准确度符合要求。结论所建立的LC-MS/MS定量分析方法特异、快速、灵敏、稳定，可用于考察健康受试者空腹口服硫酸氢氯吡格雷片75 mg后血浆样品中氯吡格雷的药动学研究。