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目的考察木瓜蛋白酶水解血中安定及其代谢物蛋白结合物的酶解条件,提高安定的提取率。方法采用正交试验确定酶解的最优条件,检材经蛋白酶水解,固相萃取后,应用LC-MS/MS方法进行检测,运用保留时间和MRM(多离子反应监测)方式来对血中安定及其代谢物进行定性定量分析。结果安定、去甲安定、去甲异安定、羟基安定和去甲羟基安定的最佳酶解条件分别为55℃,2.5h,pH7.0,8000U;50℃,1h,pH7.5,8000U;50℃,1h,pH7.5,8000U;50℃,1.5h,pH7.5,8000U;50℃,1.5h,pH7.5,8000U。结论酶水解后的血液中安定及其代谢物检出量明显增加。
Objective To investigate the enzymatic hydrolysis conditions of diazepam and its metabolites conjugate in papain hydrolysates and improve the extraction rate of diazepam. Methods The optimal conditions for enzymolysis were determined by orthogonal test. After the samples were hydrolyzed by protease and detected by LC-MS / MS, retention time and MRM (multi-ion reaction monitoring) Stability and its metabolites qualitative and quantitative analysis. Results The best conditions of diazepam, diazepam, diazepam and norpazepam were 55 ℃, 2.5h, pH7.0, 8000U; 50 ℃, 1h, pH7.5, 8000U; 50 ° C, 1 h, pH 7.5, 8000U; 50 ° C, 1.5 h, pH 7.5, 8000U; 50 ° C, 1.5 h, pH 7.5, 8000U. Conclusions The stability of diazepam and its metabolites detected in enzymatic hydrolysis increased significantly.