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J.Health Sci., 50(3), 286-289, 2004
Optimized Conditions for the Enzymatic Hydrolysis of
alpha-Hydroxytriazolam-Glucuronide in
Human Urine
Kenji Tsujikawa,* Kenji Kuwayama, Tatsuyuki Kanamori, Yuko Iwata,
Yoshihito Ohmae, Hiroyuki Inoue,
and Tohru Kishi
National Research Institute of Police Science, 6-3-1,
Kashiwanoha, Kashiwa, Chiba 277-0882, Japan
The optimum conditions for the enzymatic hydrolysis of
alpha-hydroxytriazolam (alpha-OHTRZ)-glucuronide, one of the major metabolites of triazolam in
human urine, were determined. beta-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. alpha-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 beta-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; beta-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, beta-glucuronidase from E.
coli was the most effective for the hydrolysis of
alpha-OHTRZ-glucuronide in terms of efficiencies and the wide pH range
tolerated. Incubation for 90 min with beta-glucuronidase
from E. coli was sufficient for hydrolysis of
alpha-OHTRZ-glucuronide at clinical dose.
alpha-OHTRZ-glucuronide in human urine can be hydrolyzed rapidly and effectively
using this method.
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