LC-MS/MS法测定恒河猴血浆中巴替非班的浓度及药代动力学研究

Determination of batifeiban in macaque plasma by LC-MS/MS and its pharmacokinetic study

目的:建立恒河猴血浆中巴替非班浓度的高效液相-串联质谱(Liquid Chromatography-TandemMass Spectrometry,LC-MS/MS)测定法,并研究其在恒河猴体内的药代动力学。方法:取恒河猴血浆200μL,加入含2μg.mL-1内标依替巴肽的乙腈-甲醇(70∶30)混合溶剂600μL沉淀蛋白,取上清液吹干,残留物加100μL流动相复溶,取上清液进行LC-MS/MS分析。色谱柱为ODS C18柱(150 mm×4.6 mm,5μm),流动相为甲醇-20 mmol.L-1甲酸铵(50∶50),流速为0.4 mL.min-1;质谱采用电喷雾离子化,正离子检测,巴替非班和内标的选择性检测离子分别为m/z 818.3→632.4和m/z 832.0→646.2。结果:巴替非班的线性范围为25～5 000 ng.mL-1,最低定量下限(lower limit of quantitation,LLOQ)为25 ng.mL-1,准确度、精密度及回收率均符合要求。结论:本方法专属性强,检测限低,灵敏度高,线性关系良好,方法简便快捷,适用于巴替非班临床前药代动力学研究。

Objective： To develop a LC-MS/MS method for determination of batifeiban in macaque plasma, and to investigate the pharmacokinetics of batifeiban. Methods： Plasma samples 200μL were treated with 600 μL mixture of acetonitrile and methanol （70： 30, containing 2μg·mL（-1） of eptifibatide）. After centrifugation to precipitate the proteins, the supernatant was evaporated under nitrogen gas flow at 40℃. Residues were dissolved in 100 μL of mobile phase and centrifuged at 12 000 r·min^（-1） for 5 min. Thereafter, 20 μL aliquots were injected into the LC-MS/MS for analysis. Separation was performed on a Diamonsil C18 column （ 150 mm x 4.6 mm, 5μm）. The mobile phase was composed of methanol and 20 mmol·L^（-1） ammonium formate （50： 50）. The selected reaction monitoring （SRM） transitions were performed at m/z 818.3→632.4 for batifiban and m/z 832.0→646.2 for eptifibatide （IS） in the positive ion mode with electrospray ionization （ESI） source. Results： Calibration curve was linear over the concentration range of 25 - 5 000 ng· mL^（-1）. The lower limit of quantification was 25 ng·mL^（-1）. The mean recovery was （65.7±4.9）%. The intra- and inter-day precisions were less than 5.3% and 14.0% , respectively, and the accuracy was within ±7.5%. Batifiban was stable in possible conditions of storing and han- dling. Conclusion： The method has been validated, and can be applied to determine the plasma concentration of batifiban for a pharmacokinetic study.