Optimized Conditions for the Enzymatic Hydrolysis of α-Hydroxytriazolam-Glucuronide in Human Urine

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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℃, pH 5.5-7.8), bovine liver (100 U, 45℃, pH 5.0-5.5), and P. vulgata (300 U, 60℃, 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.
2004-06-01