液相色谱串联质谱法测定大鼠血浆中知母皂苷A-Ⅲ质量浓度及其药物代谢动力学研究

Determination of Timosaponin A-Ⅲin Rat Plasma by LC-MS/MS and Its Pharmacokinetic Study

目的建立测定大鼠血浆中知母皂苷A-Ⅲ(TA-Ⅲ)的液相色谱串联质谱(LC-MS/MS)法,并考察其药物代谢动力学(PK)特征。方法色谱柱为Agilent XDB-C_(8)柱(150 mm×2.1 mm,5μm),流动相为乙腈-2 mmol/L乙酸铵(55∶45,V/V),流速为250μL/min,柱温为35℃,进样量为5μL;采用电喷雾离子源,以负离子多离子反应监测方式进行检测。将12只大鼠随机分为知母单煎液组和知母-黄柏(1∶1,m/m)合煎液组,各6只,采用建立的LC-MS/MS法测定两组大鼠血浆中TA-Ⅲ的浓度(以人参皂苷Rg_(2)为内标)。结果TA-Ⅲ质量浓度在0.3~3000 ng/mL范围内与TA-Ⅲ和内标峰面积比值线性关系良好,定量下限为0.3 ng/mL,精密度、准确度、提取回收率、基质效应、残留效应、稳定性试验结果均符合方法学要求。与知母单煎液组比较,知母-黄柏(1∶1,m/m)合煎液组大鼠TA-Ⅲ的血药浓度-时间曲线下面积增大,0-∞的平均滞留时间(MRT_(0-∞))和消除半衰期(t_(1/2))均显著延长(P<0.01)。结论该方法操作简便、结果准确可靠,可用于测定大鼠体内TA-Ⅲ的血药浓度。知母-黄柏(1∶1,m/m)合煎液促进了TA-Ⅲ的吸收,可延长其MRT_(0-∞)和t_(1/2)。

Objective To establish a liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the determination of timosaponin A-Ⅲ(TA-Ⅲ)in the rat plasma,and to investigate its pharmacokinetic(PK)characteristics.Methods The chromatographic column was Agilent XDB-C_(8) column(150 mm×2.1 mm,5µm),the mobile phase was acetonitrile-2 mmol/L ammonium acetate(55∶45,V/V),the flow rate was 250µL/min,the column temperature was 35℃,and the injection volume was 5µL.The detection was performed with the electrospray ionization(ESI)and negative ion-multiple reaction monitoring mode.Twelve rats were randomly divided into the Anemarrhenae Rhizoma decoction group and the Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex(1∶1,m/m)decoction group,with six rats in each group.The established LC-MS/MS method was used to determine the drug concentration of TA-Ⅲin the two groups with ginsenoside Rg_(2) as the internal standard.Results The linear range of TA-Ⅲwas 0.3-3000 ng/mL,and the lower limit of quantitation was 0.3 ng/mL.The results of precision,accuracy,extraction recovery,matrix effect,residual effect and stability tests all met the requirements of methodology.Compared with the those in the Anemarrhenae Rhizoma decoction group,the area under the curve of TA-Ⅲin the Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex(1∶1,m/m)decoction group increased,and its mean residence time at 0-∞(MRT_(0-∞))and half life(t_(1/2))significantly lengthened(P<0.01).Conclusion The method is simple,accurate and reliable,which can be used for the determination of the drug concentration of TA-Ⅲin rats.The Anemarrhenae Rhizoma-Phellodendri Chinensis Cortex(1∶1,m/m)decoction can promote the absorption of TA-Ⅲand the lengthening of its MRT_(0-∞)and t_(1/2).