HPLC-MS/MS法同时测定大鼠体内醋酸亮丙瑞林和内源性睾酮的药动学和药效学研究

Simultaneous determination of leuprolide acetate and endogenous testosterone using HPLC-MS/MS in rats: application in pharmacokinetics and pharmacodynamics study

目的:建立高效液相色谱-串联质谱(HPLC-MS/MS)方法同时测定大鼠血浆中的亮丙瑞林和睾酮的浓度,对亮丙瑞林缓释微球在大鼠体内的药动学和药效学进行研究。方法:采用替代分析物法,以睾酮-D3代替睾酮进行测定。大鼠血浆采用96孔板处理,使用AerisTMPEPTIDE XB-C18柱,采用阿拉瑞林和睾酮-13C分别作为亮丙瑞林和睾酮的内标。流动相为0.1%甲酸-甲醇梯度洗脱,质谱系统采用电喷雾离子源(ESI源)、多反应监测模式(MRM)进行正离子扫描,建立了HPLC-MS/MS方法同时测定亮丙瑞林及睾酮的方法。结果:亮丙瑞林和睾酮-D3分别在0.02~30和0.05~30 ng·mL^-1范围内呈现良好的线性关系,亮丙瑞林和睾酮-D3的定量下限(LLOQ)分别为0.02和0.05 ng·mL^-1,精密度分别在2.7%~8.0%和3.9%~10.2%之间,准确度分别在98.2%~106.0%和96.1%~103.3%之间,SD大鼠皮下注射亮丙瑞林(抑那通)1.875 mg·kg^-1后的主要药动学参数分别为Cmax:(31.43±6.76)ng·mL^-1,AUC0-t:(503.81±62.91)ng·h·mL^-1,AUC0-∞:(516.52±63.34)ng·h·mL^-1。睾酮的主要药动学参数为Cmax:(17.28±3.77)ng·mL^-1,AUC0-t:(38.91±5.64)ng·h·mL^-1,AUC0-∞:(120.8±66.26)ng·h·mL^-1。结论:本方法灵敏度高、操作简便,适用于大鼠体内亮丙瑞林和睾酮的同时测定,可为亮丙瑞林微球的制剂工艺开发及临床研究提供支持及指导。

Objective:This study aims to establish a method for simultaneous determination of leuprolideand testosterone by LC-MS/MS and to characterize the pharmacokinetics and pharmacodynamics of slow-releaseform leuprolide acetate biodegradable microspheres in rats.Methods:The method by using testosterone-D3as asurrogate analyte,96-well format plates and liquid chromatography-tandem mass spectrometry was developed usingAerisTMPEPTIDE XB-C18columns with a gradient mobile phase of 0.1%formic acid/methanol.Alarelin was applied as an internal standard (IS) for leuprolide and testosterone-13C3 for testosterone or testosterone-D3.Theeluates introduced into the ESI source which was operated in positive ionization mode.The multiple reactionmonitoring (MRM) transitions were monitored for the analytes and IS.Results:The method was validated in theconcentration range of 0.02~30.0 ng·mL^-1 for leuprolide and 0.05~30.0 ng·mL^-1for testosterone-D3,respectively.The lower limit of quantification was 0.02 ng·mL^-1 for leuprolide and 0.05 ng·mL^-1 for testosterone.The precisionwas within 2.7%~8.0%for leuprolide and 3.9%~10.2%for testosterone.The accuracy was within 98.2%~106.0% for leuprolide and 96.1%~103.3%for testosterone.The main pharmacokinetic parameters aftersubcutaneous injection of ENANTONE (1.875 mg·kg^-1) in SD rats for leuprolide wereCmax:(31.43±6.76) ng·mL^-1,AUC0-t:(503.81±62.91) ng·h·mL^-1,AUC0-∞:(516.52±63.34) ng·h·mL^-1,and the parameters fortestosterone wereCmax:(17.28±3.77) ng·mL^-1,AUC0-t:(38.91±5.64) ng·h·mL^-1,AUC0-∞:(120.8±66.26) ng·h·mL^-1.Conclusion:A rapid,sensitive,and selective LC-MS/MS method was developed for simultaneousdetermination of leuprolide and endogenous testosterone in rat plasma.The analytical methodology will be helpful indeveloping optimal delivery system and clinical research of leuprolide.