A novel reductive transformation of oxazepam to nordiazepam observed during enzymatic hydrolysis.

beta-Glucuronidase is an enzyme often employed to de-conjugate beta-glucuronides during urinary drug testing for benzodiazepines. It is commonly accepted that use of beta-glucuronidase is a preferred method of hydrolysis over acid-catalyzed hydrolysis, which is known to induce benzodiazepine degradation and transformation. Literature to date, however, has not reported any cases of benzodiazepine transformation initiated by commercial beta-glucuronidase products. In this study, urine specimens containing either oxazepam or oxazepam glucuronide were incubated with beta-glucuronidase enzymes obtained from Escherichia coli, Helix pomatia, and Patella vulgata under various incubation conditions. After liquid-liquid extraction, the extract was analyzed by both liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry for the presence of benzodiazepines. All three enzyme preparations examined were capable of reducing oxazepam or oxazepam glucuronide into nordiazepam (desmethyldiazepam). Nordiazepam formation was positively correlated with incubation temperature, incubation time, oxazepam concentration, and enzyme concentration. Under all enzymatic hydrolysis conditions investigated, the percentage of nordiazepam formation is < 2.5% relative to the amount of oxazepam present in the system. The findings of this study have both clinical and forensic implications, and it is clear that the detection of nordiazepam in biological samples subjected to testing involving enzyme-catalyzed hydrolysis should be interpreted with care.