摘要：The optimum conditions for the enzymatic hydrolysis of α -hydroxytriazolam ( α -OHTRZ)-glucuronide, one of the major metabolites of triazolam in human urine, were determined. β -Glucuronidases from Escherichia coli ( E. coli ), bovine liver, Helix pomatia ( H. pomatia ) and Patella vulgata ( P. vulgata ) were used, and the parameters studied were amounts of enzyme used, temperature and pH range. α -OHTRZ was extracted with hexane/dichloromethane (1 : 1, v/v) and quantified using a high-performance liquid chromatography with a UV detector set at 230 nm. A preliminary study showed that β -glucuronidase from H. pomatia gave a poor recovery compared with the other three enzymes. The optimal conditions (amounts of enzymes, temperature and pH range) for 1 ml of urine were as follows; β -glucuronidase from E. coli (100 U, 37°C, pH 5.5-7.8), bovine liver (100 U, 45°C, pH 5.0-5.5), and P. vulgata (300 U, 60°C, pH 3.8-4.5). Among these enzymes, β -glucuronidase from E. coli was the most effective for the hydrolysis of α -OHTRZ-glucuronide in terms of efficiencies and the wide pH range tolerated. Incubation for 90 min with β -glucuronidase from E. coli was sufficient for hydrolysis of α -OHTRZ-glucuronide at clinical dose. α -OHTRZ-glucuronide in human urine can be hydrolyzed rapidly and effectively using this method.
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