人血浆中华法林对映体HPLC测定方法建立及其对抗凝疗效的影响

目的: 研究血浆中华法林对映体的浓度对华法林抗凝疗效的影响. 方法: 建立血浆中华法林对映体浓度的立体选择性高效液相色谱法,采用手性-AGP色谱柱(ULTRON ES-OVM 5 μm,150 mm×4.6 mm),保护柱(ULTRON ES-OVM.G 10 mm×4.6 mm),以乙腈-20 mmol·L^-1磷酸盐缓冲液(14:86,pH 5)为流动相,流速1.0 mL·min^-1,检测波长308 nm,柱温30 ℃,内标峰面积法定量计算.对146名心脏瓣膜置换术后使用华法林进行抗凝治疗的患者,服药后11~13 h按上述方法进行华法林对映体血浆浓度的检测. 结果: ①对映体标准曲线范围为75~2500 ng·mL^-1,最低检出限为25 ng·mL^-1,批内、批间RSD均在10%以下,回收率>90%,血浆样品稳定性良好.② R(+)-华法林平均血浆浓度为(635.0±380.7)ng·mL^-1, S(-)-华法林平均血浆浓度为(334.8±237.7)ng·mL^-1; S(-)-华法林血浆浓度与国际标准化比值(INR)值呈显著正相关(r=0.252, P<0.05),随着 S(-)-华法林血浆浓度的升高,华法林的抗凝疗效增强, R(+)-华法林血浆浓度与INR值无显著相关性(r=0.104, P>0.05).INR值在安全范围(1.8~3.0)的125例患者中,有92例(73.6%) S(-)-华法林血浆浓度在(334.8±237.7)ng·mL^-1. 结论: 本研究建立的华法林对映体血浆浓度的检测方法灵敏而可靠,可作为华法林抗凝疗效的监测指标,联合INR值有助于提高抗凝监测的准确性.

Objective: To study the influence of concentration of warfarin enantiomers in human plasma on warfarin anticoagulation. Methods: A stereo selective HPLC method was established to determine warfarin enantiomers in human plasma.The HPLC method employed the chiral-AGP column(150 mm×4.6 mm,5 μm)with a mobile phase of acetonitrile-20 mmol·L^-1 phosphate buffer(14:86,pH 5)at a flow rate of 1.0 mL·min^-1.The UV detection wavelength was 308 nm and the column temperature was 30 ℃.The internal standard peak area method for quantitative calculation was used.One hundred and forty-six Chinese patients after heart valve replacement(HVR)receiving a long-term warfarin maintenance therapy participated in the study.Plasma concentration of warfarin enantiomers at post-dosing 11 to 13 hours were measured by means of the chiral HPLC. Results: ①The range of standard curve of each enantiomer was 75-2500 ng·mL^-1,with the lowest limit of detection of 25 ng·mL^-1.RSDs of within or between-assay were less than 10% and the average recovery rate was >90%.Plasma samples has good stability.②The mean concentration of R(+)- and S(-)-warfarin in plasma were(635.0±380.7)ng·mL^-1 and(334.8±237.7)ng·mL^-1,respectively,indicating that a significant positive relationship between the international normalized ratio(INR)and S(-)-warfarin plasma concentration(r=0.252, P <0.05).With the increase of S(-)-warfarin plasma concentration,the anticoagulant effect was enhanced.There was no significant correlation between R(+)-warfarin plasma concentration with INR values(r=0.104, P>0.05).The results showed that the 125 cases had their INR within the safety range(1.8-3.0),of which 92(73.6%)cases had a S(-)-warfarin level at(334.8±237.7)ng·mL^-1. Conclusion: The method is sensitive and reliable.The plasma concentration of warfarin can serve as a sensitive index in monitoring anticoagulant effect,the combination of warfarin plasma concentration with INR would improve the precision of anticoagulant monitoring.