血中安定及其代谢物的酶水解研究

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。

Determination of diazepam and its metabolites in blood by enzyme hydrolysis and high performance liquid chromatography mass spectrometry

Objective To investigate the conditions for enzyme-hydrolysis of the protein conjugates of diazepam and its metabolites catalyzed by enzyme in the blood and to improve the rate of extraction. Methods An orthogonal design was used to define the optimal conditions for enzyme hydrolysis in the experiment. The samples were hydrolyzed by papain, extracted by solidphase extraction and detected by LC-MS/MS. Qualitative and quantitative analysis were obtained by retention time （Rt） and Multiple Reaction Monitoring （MRM） of LC-MS/MS. Results The optimal conditions for enzyme-hydrolysis of diazepam, nordazepam, lofendazam, temazepam and oxazepam were 55℃, 2. 5h, pHT. 0,8000U of the enzyme, 50℃, lh, pHT. 5,8000U of the enzyme, 50℃, lh,pH7.5,8000U of the enzyme, 50℃, 1.5h, pH7. 5,8000U of the enzyme, 50℃, 1. 5h, pHT. 5,8000U of the enzyme, respectively. Conclusion The contents of diazepam and its metabolites increased when amount of papain was added into the same blood sample.