摘要
目的 :考察 β 葡萄糖醛酸苷酶水解尿液中地西泮代谢物的结合物的酶解条件。 方法 :实验设计采用正交表L9( 34)。取尿样 2mL ,加内标 ,再加不同量的 β 葡萄糖醛酸苷酶 ,在不同温度、不同酶解时间下水解 ,然后用正己烷 二氯甲烷 ( 5∶3) 5mL提取 ,挥干提取液后用 5 0 μL甲醇溶解 ,10 μL进样 ,采用高效液相色谱法检测。 结果 :对水解产物峰面积与内标物峰面积的比值进行方差分析 ,发现水解尿液中去甲地西泮、替马西泮、奥沙西泮的葡萄糖醛酸苷结合物的最佳酶解条件分别为 5 5℃ ,5h ,5 0 0 0U酶量 ;5 5℃ ,2h ,10 0 0U酶量 ;5 5℃ ,2h ,5 0 0 0U酶量。结论 :水解 2mL尿液中地西泮代谢物的结合物的最佳条件为 5 5℃ ,5h ,5 0 0 0U酶量。
Objective:To study the conditions for enzyme-hydrolysis of the conjugates of diazepam metabolites catalyzed by β-glucuroridase in the unine. Methods:An orthogonal design on the basis of table L 9(34)was used in the experiment. An internal standard and appropriate amount of β-glucuronidase were added to 2 mL of urine sample, which was then incubated at different temperatures and in different time periods for hydrolysis. The products were extracted with 5 mL of n-hexane: dichloromethane(5∶3) and the extract was dried by volatilization. The residue was dissolved with 50 μL of methanol, and 10 μL of which were added onto the HPLC system. Results:It was shown by analyses of variance of the ratios of peak areas of hydrolyzates to those of internal standards that the optimal conditions for enzyme-hydrolysis of urine glucuronide conjugates of nordiazepan, temazepam and oxazepam were 55℃, 5 h, 5 000 units of β-glucuronidase, 55℃, 2 h, 1,000 U of the enzyme, and 55℃, 2 h, 5,000 U of the enzyme, respectively. Conclusion:The optimal condition for enzyme-hydrolysis of the conjugates of diazepam metabolites in 2 mL of urine was shown to be 55℃, 5 h, and 5,000 units of β-glucuronidase.
出处
《医药导报》
CAS
2003年第6期374-377,共4页
Herald of Medicine
关键词
地西泮
代谢物
酶水解
高效液相色谱法
Diazepam
Metabolite
Enzyme-hydrolysis
High performance liquid chromatography
