HPLC法测定大鼠血浆华法林及红参对华法林抗凝作用的影响

Determination of Warfarin in Rats by HPLC and the Effect of Red Ginseng on Anticoagulant of Warfarin

目的:建立和优化HPLC法测定大鼠血浆华法林浓度,并观察红参对大鼠体内华法林抗凝作用的影响,探讨两者是否存在药物相互作用,为临床合理用药提供参考。方法:建立HPLC法测定大鼠血浆华法林浓度,以乙腈∶乙酸水(1%)=70∶30为流动相,等度洗脱。流速为1mL/min,柱温25℃,检测波长308nm,进样量为20μL。全自动凝血分析仪检测血浆PT,计算INR,从而观察红参对华法林抗凝作用的影响。结果:华法林血浆浓度在1.007~50.35μg/mL范围内与峰面积呈良好的线性关系。低、中、高3种浓度的华法林血浆样品提取回收率为85.17%~95.29%,方法回收率分别为97.44%~109.3%,日间、日内RSD均小于10%。同服组和预服组较华法林组的PT值和INR值均有不同程度的延长,其中预服组在D7和D10时PT值和INR值升高具有显著性差异,但两组较华法林组的血药浓度均未见统计学差异。结论:HPLC法能准确、精密、快速、稳定地检测华法林的血药浓度,可用于红参与华法林药物相互作用的研究中测定大鼠血浆中华法林浓度。红参可能增强华法林抗凝效果,增加出血风险,推测与其抑制华法林代谢有关。

Objective To establish and optimize the method for the determination of warfarin levels in rat by HPLC, to observe the effects of red ginseng on anticoagulation of warfarin in rats. And analyzed whether there is drug interaction between red and warfarin, and provide reference for clinical rational drug use. Methods The HPLC method for the determination of warfarin levels in the plasma of the rat was established. The mobile phase was isocratically eluted with acetonitrile: 1% acetic acid water(70∶30, V/V). The analysis was operated at a flow-rate of 1 mL/min with detection wavelength of 308 nm. The column temperature was 25 ℃.The injection volume was 10 μL. Plasma prothrombin time(PT) was measured with an automatic coagulation analyzer, and the international normalized ratio(INR) was calculated. The plasma concentration of warfarin in rats was determined by HPLC, to observe the effects of red ginseng on anticoagulation of warfarin in rats. Results The plasma concentration of warfarin in the range of 1.007~50.35 μg/mL had a good linear relationship with peak area. The recovery rate of low, middle and high concentrations of warfarin plasma samples was 85.17% to 95.29%. The method recovery rate was 97.44% to 109.3%. The RSD during the day and days were less than 10%. The PT and INR of red ginseng co-administrated group and red ginseng pre-administrated group were prolonged in different extents compared with warfarin group. The red ginseng pre-administrated group had significant prolongation of PT value and higher INR value at D7 and D10. The plasma concentrations of warfarin in red ginseng co-administrated and pre-administrated group were no significantly higher than those of warfarin group. Conclusion The HPLC method can detect the plasma concentration of warfarin exactly, fast, and stably. And it can be used in the study of the interaction between red ginseng and warfarin. Red ginseng may enhance the anticoagulant effect of warfarin and increase the risk of bleeding. It was speculated to be related to its inhibition of warfarin metabolism.