异丙酚在人肝微粒体中葡萄糖醛酸化代谢的方法学研究

Study of Propofol Glucuronidation Metabolism in Human Liver Microsomes

目的建立UPLC测定异丙酚及其葡萄糖醛酸化代谢产物的方法,并考察异丙酚在体外人肝微粒体中的葡萄糖醛酸化代谢酶促动力参数。方法采用B Eclipse Plus C18色谱柱(1.8μm,2.1×50mm),以乙腈和纯水为流动相进行梯度洗脱;流速为0.35m L·min-1,进样体积15μL,检测波长220 nm;柱温40℃。采用体外微粒体药物代谢酶孵育法考察异丙酚葡萄糖醛酸化代谢酶促动力学参数。结果异丙酚在15~1200μm,异丙酚葡糖糖醛酸化代谢产物在0.15~50μm范围内呈良好的线性关系,精密度与稳定性均良好。异丙酚在人肝微粒体中米氏常数(Km)和最大反应速度(Vm ax)分别为127.30μM、1.39nmol/(min·mg),药物清除率(CLint)10.92μL/(min·mg)。结论本方法简便、快速、准确,可用于考察异丙酚在体外的葡萄糖醛酸化代谢,异丙酚是UGT1A9的特异性底物,可推广用于考察UGT1A9相关的药物间相互作用。

OBJECTIVE To establish a reliable analytical method for the study of propofol glucuronidation metabolism in pooled human liver microsomes by UPLC system and determine the kinetics of the enzymic reaction. METHODS Propofol and propofol glucuronide （PG） were separated by B Eclipse Plus C18 column （2. 1mm × 50mm, 1.8μm） with ingredient elution using a mobile phase consisted of acetonitrile and aqua pura. The detection wavelength was set at 220nm,the flow rate at 0. 35mL · min-1 and the volume of sample was 20μL,the column tem- perature at 40℃. The enzyme activity was performed in pooled human liver microsomes in vitro. RESULTS The calibration curves of propofol and PG were linear over a concentration of 15 - 1200μm and 0. 15 - 50μm （r 〉 0. 99 ）, respectively. The precision and stability of this method can meet the demand for analysis. The kinetic parameters of propofol metabolized to PG were determined by Michaelis-Menten,the values of Km ,Vmax and CLint were 127. 30μM, 1.39nmol/（min · mg） and 10. 92μL/（min · mg） ,respectively. CONCLUSION The method is convenient,sensitive, accurate, and successfully applied for analyzing propofol glucuronidation metabolism in vitro. Propofol was used as the substrates to determined the glucuronidation activities of UGT1A9, this method can be used to provide platform for evaluation of UGT1A9 related dru-drug interactions.