Comparison of the rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucuronide and temazepam-glucuronide catalyzed by E-coli, beta-D-glucuronidase using the on-line benzodiazepine screening immunoassay on the Roche/Hitachi 917 analyzer

The catalytic rates of hydrolysis of lorazepam-glucuronide, oxazepam-glucur onide, and temazepam-glucuronide when catalyzed by E. Coli. beta -glucuroni dase both in phosphate buffer and buffered drug-free urine were compared as well as the pH dependence of enzyme activity. In 50 mM phosphate buffer lo razepam-glucuronide has the highest turnover rate of 3.7 s(-1) with an asso ciated K-m of about 100 muM, followed by oxazepam-glucuronide, which has a turnover rate of 2.4 s(-1) with an associated K-m of 60 muM. Temazepam-gluc uronide has the lowest rate of 0.94 s(-1) with an associated K-m of 34 muM. In buffered drug-free urine, a similar trend was observed. In addition, an optimal pH for beta -glucuronidase was determined to be between 6 and 7 wh en the enzyme hydrolyzes the benzodiazepine conjugates in buffered drug-fre e urine. Effects of temperature and incubation time were also examined. It can be concluded that the electron donating or withdrawing of the individua l benzodiazepine structure may play an important role in the reactivity of the lorazepam-glucuronide, oxazepam-glucuronide and temazepam-glucuronide c atalyzed by beta -glu-curonidase. This is consistent with other observation s made for monosubstituted phenyl-beta -glucuronides by Wang et al. (1).