LC-MS/MS研究新型酪氨酸酶抑制剂UP302在大鼠肝微粒体中代谢的酶动力学

 目的 建立并验证测定大鼠肝微粒体孵育液中UP302含量的高效液相色谱-串联质谱法，研究UP302在大鼠肝微粒体中代谢的酶动力学。方法 经大鼠肝微粒体孵育的UP302样品，经色谱柱分离，电喷雾离子化负离子检测，扫描方式为选择反应监测，用于定量分析的离子反应分别为m/z 301.1→135.2（UP302）和m/z 252.9→132.0（内标大豆苷元）。考察不同孵育时间、底物浓度和微粒体浓度对UP302代谢的影响，确定最佳反应条件。结果 标准曲线线性范围为0.1～20 μmol·L-1，线性关系良好，日内日间准确度在86.42%～112.59%，日内日间精密度均小于9.09%，回收率在100.50%～109.91%之间。确定了酶动力学参数，最大反应速度V_max为196.08 μmol·L-1·min-1·g-1 ，米氏常数K_m为14.98 μmol·L-1，内在代谢清除率CL_int为13.09 mL-1·min-1·g-1。结论 该检测方法快速、专属、灵敏度高，可满足酶动力学检测要求。

Study on Metabolism Enzyme Kinetics of a Natural Novel Tyrosinase Inhibitor UP302 in Rat Liver Microsome by LC-MS/MS

OBJECTIVE To establish and validate a LC-MS/MS method for the determination of UP302 in rat liver microsome， and study the enzyme kinetics of UP302 metabolism in rat liver microsome. METHODS The LC-MS/MS method with mass transitions of m/z 301.1→135.2 for UP302 and 252.9→132.0 for internal standard daidzein was employed to determine UP302 incubated in rat liver microsome at negative electrospray ionization mode. Different incubation conditions， such as incubation time， substrate concentration and microsome concentration， were optimized. RESULTS The linear range of the calibration curve was 0.1-20 μmol·L-1 with good linear relationship. The intra- and inter-day accuracy was 86.42%-112.59%， and RSDs were less than 9.09%. The enzyme kinetic parameters were as followsmaximum reaction speed V_max of 196.08 μmol·L-1·min-1·g-1， K_m of 14.98 μmol·L-1， and CL_int of 13.09 mL-1·min-1·g-1. CONCLUSION The established LC-MS/MS method was fast， simple， specific， sensitive and successfully applied to investigate the enzyme kinetics of UP302 metabolism in rat liver microsome.