超高效液相色谱-串联质谱法快速测定人血浆中氯吡格雷及其代谢物浓度

Rapid determination of clopidogrel and its metabolites in human plasma by ultra-high performance liquid chromatography-tandem mass spectrometry

目的建立超高效液相色谱-串联质谱法同时测定人血浆中的氯吡格雷（Clo）及其非活性代谢物（CCAM）和活性代谢物（CATM）的浓度。方法5名健康受试者单剂量口服Clo片300mg，分别经时采集肘静脉血样进行药动学分析。血浆样品沉淀蛋白后经WATERS ACQUITY UPLC HSS T3柱（2．1mm×50mm，1．8μm）分离，流动相为水（含0．1％甲酸）-乙腈（含0．1％甲酸），梯度洗脱。采用电喷雾电离源（ESI）正离子模式、多反应监测（MRM），用于定量分析的离子通道分别为m／z 322．1→211．8（Clo），m／z 356．0→154．9（CATM），m／z 308．3→198．0（CCAM）,m／z 253．1→180．0（内标卡马西平）。结果血浆中Clo、CATM和CCAM线性关系良好（r〉0．99），日内、日间精密度（RSD）小于12．6％，准确度（RE）在-3．6％～7．6％之间。药动学结果显示，健康受试者单剂量口服Clo片300mg后，Clo、CATM和CCAM的ρmax分别为（26．57±24．06）、（38．12±22．80）和（8128．00±1624．58）ng·mL^-1，tmax分别为（1．8±0．8）、（2．0±1．0）和（2．0±1．0）h，AUC0-∞分别为（67．74±48．44）、（212．16±122．58）和（46982．31±14496．05）ng·mL^-1·h。结论本方法操作简便、灵敏度高、分析时间短，适用于Clo药动学研究和常规血药浓度监测。

AIM To establish a sensitive and rapid ultra- high performance liquid chromatographic- tandem mass spectrometric （UPLC-MS/MS） method for simultaneous determination of clopidogrel （Clo）, its active thiol metabolite （CATM） and inactive carboxylic acid metabolite （CCAM） in human plasma. METHODS Venous blood samples for pharmacokinetic measurements were taken at different time points from 5 healthy volunteers after receiving 300 mg dose of clopidogrel. After one-step protein precipitation, the analytes were separated on WATERS ACQUITY UPLC HSS T3 （2.1mm×50mm,1.8μm） column, using a gradient elution program with a mobile phase consisting of 0.1% formic acid water and 0.1% formic acid acetonitrile. An AB QTRAP 4500 tandem mass spectrometer equipped with an electrospray ionization source and operated in positive ion mode was used as detector. The mass transition ion-pairs were m/z 322.1→211.8 （Clo）, m/z 356.0→154.9 （CATM）, m/z 308.3→198.0 （CCAM）, ru/z 253.1→180.0 （IS earbamzepine）. RESULTS The linear calibration curves for Clo, CATM and CCAM showed a good linear relationship. Intra-and inter-day relative standard deviation （RSD） for Clo, CATM and CCAM over the entire concentrations across validation runs were all less than 12.6%, and relative error （RE） ranged from -3.6% to 7.6%. The main pharmacokinefic parameters were as follows： ρmax were （26.57 ±24.06）, （38.12 ±22.80） and （8 128.00 ±1 624.58） ng·mL^-1, t were （1.8 ± 0.8） （2.0± 1.0） and （2.0 ± 1.0） h, AUC0-∞ were （67.74 ±48.44） （212.16 ±122.58） and （46 982.31 ±14 496.05） ng·mL^-1·h for Clo, CATM and CCAM, respectively. CONCLUSION The established UPLC-MS/MS method is simple, sensitive and rapid, which is suitable for pharmacokinetie study and monitoring the plasma concentration of Clo.