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,3μm)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(CS/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。

Determination of warfarin enantiomers in human plasma by HPLC-MS/MS and its clinical application

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-3 R(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 steadystate 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(CS/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 CS/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.