Reduction of temazepam to diazepam and lorazepam to delorazepam during enzymatic hydrolysis

Abstract

It has been previously reported that treatment of urinary oxazepam by commercial β-glucuronidase enzyme preparations, from Escherichia coli, Helix pomatia and Patella vulgata, results in production of nordiazepam (desmethyldiazepam) artefact. In this study, we report that this unusual reductive transformation also occurs in other benzodiazepines with a hydroxyl group at the C3 position such as temazepam and lorazepam. As determined by liquid chromatography-mass spectrometry analysis, all three enzyme preparations were found capable of converting urinary temazepam into diazepam following enzymatic incubation and subsequent liquid-liquid extraction procedures. For example, when H. pomatia enzymes were used with incubation conditions of 18 h and 50 °C, the percentage conversion, although small, was significant--approximately 1% (0.59-1.54%) in both patient and spiked blank urines. Similarly, using H. pomatia enzyme under these incubation conditions, a reductive transformation of urinary lorazepam into delorazepam (chlordesmethyldiazepam) occurred. These findings have both clinical and forensic implications. Detection of diazepam or delorazepam in biological samples following enzyme treatment should be interpreted with care.

Optimisation of Benzodiazepine Immunoassay Using <i>β</i>-Glucuronidase Enzymatic Hydrolysis: A Comparison of Five Different <i>β</i>-Glucuronidase Enzymes

“…A study found that enzymatic hydrolysis using β-glucuronidase enzymes (Escherichia coli, Helix pomatia, and Patella vulgate) caused < 2.5% nordiazepam formation that was relative to the amount of oxazepam present in the system [15]. This unusual reductive transformation also occurs in other benzodiazepines with a hydroxyl group at the C3 position and converting temazepam into diazepam and lorazepam into delorazepam by about 1% [16]. These findings are suggesting the detection of nordiazepam, diazepam or delorazepam in biological samples subjected to testing involving enzyme-catalyzed hydrolysis should be interpreted with care.…”

Fast Determination of Benzodiazepines in Human Urine via Liquid-Liquid Extraction with Low Temperature Partitioning and LC-HRMS

“…Acid and enzymatic hydrolyses of the urine specimens are used to convert the analytes to their free forms and to improve detection [21], but these procedures present a number of drawbacks, such as producing artifacts and consuming time [6][7][8][9]. In this work, a hydrolysis reaction was not produced; nevertheless, the proposed method presented characteristics suitable to clinical and forensic applications involving the use of benzodiazepines, where the concentration of the unchanged compound is appropriate.…”

“…Recently, the literature has reported that hydrolysis methods employing β-glucuronidase may produce artifacts [8]. Moreover, these procedures are time consuming, varying from 1 to 18 hours [9].…”