HPLC-MS/MS法测定人血浆中华法林对映体浓度及其临床应用

目的： 建立HPLC-MS/MS法测定深静脉血栓患者血浆中华法林对映异构体的浓度。方法： 以华法林-d₅为内标，经甲醇沉淀蛋白后，采用CHIRALPAK?AS-3R手性色谱柱（4.6 mm×150 mm，3 μm）分离样品，流动相为甲醇-含0.1%甲酸的醋酸铵水溶液（95：5）。采用质谱检测系统，电喷雾离子源（ESI源），负离子扫描，多反应监测模式（MRM）监测m/z 307.0→160.8（华法林）及m/z 313.0→160.8（华法林-d₅）。采集77名深静脉血栓患者稳态血样后，以HPLC-MS/MS法测定血浆中华法林对映异构体的浓度，并计算S-华法林与R-华法林浓度的比值（C_S/R）。结果： 华法林的2个对映异构体质量浓度在5~1 500 ng·mL^-1内线性关系良好，定量下限为5 ng·mL^-1。R-华法林批内和批间精密度的RSD为5.0%~8.1%和8.3%~13.4%，提取回收率85.9%~97.7%；S-华法林批内和批间精密度的RSD为6.2%~7.0%和6.8%~15.0%，提取回收率82.4%~93.9%。2个异构体均无基质效应影响，不同条件下的稳定性试验均满足要求。77名患者体内R-华法林与S-华法林稳态血浆药物质量浓度分别为（458.52±226.55）ng·mL^-1和（239.63±152.83）ng·mL^-1，其血浆药物浓度比值的平均值约为0.53±0.34。结论： 本方法快速、简便、可靠，适用于深静脉血栓患者体内华法林对映异构体血药浓度的检测，能够指导临床合理用药。

Objective: To establish a simple,rapid and reliable HPLC-MS/MS method for the determination of the concentration of both warfarin enantiomers in plasmas of patients with deep venous thrombosis. Methods: Warfarin-d₅ used as the internal standard,after precipitating by methanol,the seperation of plasma was accomplished by a CHIRALPAK?AS-3R (4.6 mm×150 mm,3 μm) chiral column with a mobile phase comprising of methanol-ammonium acetate sulution containing 0.1% formic acid(95:5)at a flow rate of 0.6 mL·min -1,and then the detection was performed on the MS system. Detection and quantitation was done in multiple reaction mode(MRM) following the transitions m/z 307.0→160.8 and 307.0→160.8 for warfarin enantiomers and the warfarin-d₅ diastereomers,respectively,in the negative ionization mode. After the steady-state blood samples of 77 patients with deep vein thrombosis were collected,the concentration of warfarin enantiomers in plasma was determined by HPLC-MS/MS method,and the concentration ratio of S-warfarin to R-warfarin (C_S/R) was calculated. Results: The linearity range was 5-1 500 ng·mL^-1 and the lower limit of quantification was 5 ng·mL^-1 for each enantiomers. The intra- and interbatch precisions were in the ranges of 4.99%-8.13% and 8.27%-13.41%,and 6.21%-6.99% and 6.82%-14.99% for R-warfarin and S-warfarin,respectively. The mean extraction recovery rate of both analytes ranged from 82.4% to 97.7%,the effect of the biological matrix was negligible. Both enantiomers were found to be stable under different stability conditions. In 77 patients,the steady plasma concentrations of R-warfarin and S-warfarin were (458.52±226.55) ng·mL^-1 and (239.63±152.83) ng·mL^-1,respectively,with an average C_S/R ratio of about 0.53±0.34. Conclusion: The established HPLC-MS/MS method is rapid,simple and reliable,and is suitable for detecting the plasma concentration of warfarin enantiomers in patients with deep vein thrombosis,and can guide the clinical rational use of drugs.

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