Investigation of commercially available recombinant and conventional β-glucuronidases to evaluate the hydrolysis efficiencies against O-glucuronides and N-glucuronides in urinary drug screening.

Purpose: To achieve the rapid analysis of drug metabolites in urine, we examined the differences in the hydrolysis efficiencies against O-glucuronide and N-glucuronide by two commercially available glucuronidases and three commercially available recombinant ones.

Methods: The metabolites analyzed included oxazepam-O-glucuronide, amitriptyline-N-glucuronide, and diphenhydramine-N-glucuronide. Hydrolysis was performed using commercially available five enzymes at two different temperatures, and the reaction progress was monitored for up to 360 min. The amount of hydrolyzed product was quantified using liquid chromatography-tandem mass spectrometry.

Results: Although no enzyme selectivity was observed for the hydrolysis of O-glucuronide, the hydrolysis efficiency against N-glucuronide varied significantly, depending on the enzyme and reaction temperature. Among the enzymes evaluated, IMCSzyme 3S and the enzyme derived from E. coli demonstrated superior hydrolysis of N-glucuronides under optimal conditions. For IMCS RT, good results were also obtained by adding twice the amount of enzyme specified.

Conclusions: Suitable enzymes and hydrolysis conditions were determined for the rapid and systematic screening of drug metabolites in human urine. These findings are expected to streamline the analytical workflow and reduce the need for tedious sample preprocessing.