LC-IT-MS/MS法测定大鼠体内商陆皂苷甲浓度及其药动学研究

Determination of EsA in rat plasma by LC-IT-MS/MS method and its application to pharmacokinetic study

目的:建立测定SD大鼠体内商陆皂苷甲(EsA)血药浓度的高效液相-离子阱串联质谱方法;研究EsA在SD大鼠体内的药动学特征。方法:采用Diamonsil C18色谱柱(50 mm×2.1 mm,3μm),流动相为甲醇-水(含0.1%冰醋酸)(70∶30),流速为0.2 mL.min-1,采用ESI源,正离子检测模式,以人参皂苷Rg1为内标,血浆样品经正丁醇液液萃取后进样分析,测定大鼠血浆中EsA浓度,BAPP软件计算主要药动学参数。结果:在选定的色谱条件下,EsA和内标分离良好,没有内源性物质干扰。EsA在5～500 ng.mL-1范围内线性良好(r=0.998 3),最低定量限为5 ng.mL-1,提取回收率大于70%,日内和日间精密度小于10%。SD大鼠灌胃给予EsA 15 mg.kg-1后,其主要药动学参数AUC0～24 h为(1 013.85±82.73)ng.h.mL-1,AUC0-∞为(1 076.31±92.70)ng.h.mL-1,t1/2为(6.16±0.63)h,Cmax为(218.80±38.33)ng.mL-1,Tmax为(0.8±0.1)h。结论:本方法简便快捷、灵敏准确;本研究所获得的EsA在SD大鼠体内的药动学参数为EsA的临床应用提供了依据。

Objective： To develop an LC-IT-MS/MS method for the determination of esculentoside A （EsA） in rat plasma and study the pharmacokinetics of EsA in SD rats. Methods：After liquid-liquid extraction （LLE） with n-buthanol, the analyte EsA and Rgl （internal standard） were separated on a Diamonsil C18 （50 mm× 2. 1 mm, 3μm） column with the mobile phase of methanol-water containing 0.1% acetic acid （70： 30） at a flow rate of 0.2 mL· min^-1.An ion trap mass spectrometer equipped with an electrospray ionization source performed in positive mode was used as the detector. The plasma concentration of EsA was determined by the developed LC-IT- MS/MS method, and its pharmacokinetic parameters were calculated by BAPP software. Results： Under the de- scribed LC-IT-MS/MS conditions, a good separation of EsA and IS was achieved and no significant endogenous interferences were observed. The calibration curves showed a good linearity over the range of 5 -500 ng·mL^-1 with an average correlation coefficient of 0. 998 3. The lower limit of quantitation of this method was 5 ng· mL^- 1. The extraction recovery of EsA from rat plasma was over 70%. Inter- and intra- precisions were all satisfactory as shown by RSDs lower than 10%. The main pharmacokinetic parameters after a single oral administration of 15 mg· kg^-1 of EsA were summarized as follows：AUC0-24h was （1 013.85 ±82.73） ng·h·mL^-1, AUC0-∞ was （1 076.31 ±92.70） ng·h·mL^-1, t1/2was （6.16±0.63） h, C was （218.80＋38.33）ng·h·mL^-1, Tmax was （0.8±0.1） h. Conclusion： The developed method is simple, rapid, sensitive enough and has been successfully applied to the pharmaeokinetic study of EsA in SD rats.