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目的:建立血浆中白头翁皂苷B4的分析方法,测定白头翁皂苷B4大鼠血浆蛋白结合率。方法:采用96通道高通量平衡透析系统(HTD 96b)进行透析,利用UPLC-MS/MS测定透析内外液中白头翁皂苷B4的浓度,研究白头翁皂苷B4在大鼠血浆中的血浆蛋白结合率,使用Waters XTerra MS C18色谱柱(2.1 mm×50 mm,5μm),流动相0.1%甲酸水溶液-乙腈梯度洗脱,流速0.7 m L·min~(-1),柱温40℃,进样量5μL。结果:白头翁皂苷B4在5~2 000 ng·L-1线性关系良好,其精密度RSD及准确度均<8.0%,重复性RSD<9.0%,稳定性的RSD均<15%;提取回收率和基质效应均在80%~115%。白头翁皂苷B4在低、中、高(6,12,24 mg·L-1)3个质量浓度下大鼠血浆蛋白结合率分别为(95.32±0.37)%,(94.32±0.63)%,(88.64±0.37)%。结论:白头翁皂苷B4与大鼠血浆具有较强的蛋白结合率,且结合率不具有质量浓度依赖性。
Objective: To establish an HPLC method for the determination of Pulsatilla saponin B4 in plasma and determine the plasma protein binding rate of Pulsatilla saponin B4 in rats. Methods: The 96-well high-throughput dialysis system (HTD 96b) was used for dialysis. The concentration of Pulsatilla saponin B4 in the dialysis solution was determined by UPLC-MS / MS. The plasma protein binding rate of Pulsatilla saponin B4 in plasma was investigated. The mobile phase was eluted with a gradient of 0.1% formic acid in acetonitrile with a flow rate of 0.7 m L · min -1 using a Waters XTerra MS C18 column (2.1 mm × 50 mm, 5 μm) at a column temperature of 40 ° C. The injection volume was 5 μL . Results: Pulsatilla saponin B4 had a good linearity of 5-2000 ng · L-1, RSD accuracy <8.0%, RSD <9.0%, RSD of stability <15%. The extraction recovery And matrix effects are 80% to 115%. The plasma protein binding rates of Pulsatilla saponin B4 at the concentrations of 6, 12 and 24 mg · L-1 were (95.32 ± 0.37)%, (94.32 ± 0.63)% and (88.64 ± 0.37)%. Conclusion: Pulsatilla saponin B4 has a strong protein binding rate with rat plasma, and the binding rate does not have mass concentration dependence.