HPLC-MS/MS法同时测定大鼠血浆中3种蟾皮主要活性成分

建立大鼠血浆中3种蟾皮提取物的HPLC-MS/MS测定方法,并用于大鼠体内的药代动力学研究.采用静脉给药和动脉取血的方式,甲醇直接沉淀蛋白的方法快速制备样品；分析柱为Agilent Zorbax Eclipse XDB-C18(150 mm×4.6mm,5 μm),流动相为乙腈/水(体积比65:35),流速为1.0 mL/min;质谱条件为ESI(+)电离方式,扫描方式为定量的多反应监测(MRM).结果表明,3种提取物分析方法的线性范围均为5～50 000 ng/mL(r＞0.999),样品的检出限均为5ng/mL,日内和日间精密度的相对标准偏差(RSD)≤4.02％,方法的回收率均在78％以上；经此方法测得的药代动力学数据表明,3种提取物在大鼠体内有相似的药代动力学行为.本方法样品预处理快速简便,检测专一灵敏,适合于3种蟾皮提取物的药代动力学研究.

Simultaneous Determination of Three Main Active Bufadienolides of Toad Skin in Rat Plasma by HPLC-MS/MS

An HPLC-MS/MS method for determination of constituents from the skin of the toad Bufo bufo gargarians for their pharmacokinetic study in rat plasma was established.Blood samples were prepared by intravenous administration and withdrawn from jugular vein.And a simplified and rapid protein precipitation with methanol was employed for the sample preparation.The separation was on a Agilent-C18(150 mm×4.6 mm,5 μm) analytical column using a mobile phase of acetonitrile/water(65∶35 by vol.)at a flow rate of 1.0 mL/min.Detection was carried out on a triplequadrupole tandem mass spectrometer in negative ion mode using the multiple reaction monitoring(MRM) via an electrospray ionization(ESI) interface.Calibration curve was linear in range from 5 ng/mL to 50 000 ng/mL with a good correlation(r>0.999).The intra and inter-day precision and the accuracy were all acceptable(RSD≤ 4.02%),and the recovery were above 78%.The pharmacokinetic study show that three main active bufadienolides of toad skin have the similar mean plasma concentration-time curves.The HPLC-MS /MS method for the quantitative determination of three main active bufadienolides of toad skin in rat plasma present simple and fast sample process and sensitive,selective detection,therefore it can be used in their pharmacokinetic studies.