Rapid Enzymatic Hydrolysis Using a Novel Recombinant β-Glucuronidase in Benzodiazepine Urinalysis

Abstract

Only trace amounts of parent benzodiazepines are present in urine following extensive metabolism and conjugation. Thus, hydrolysis of glucuronides is necessary for improved detection. Enzyme hydrolysis is preferred to retain identification specificity, but can be costly and time-consuming. The assessment of a novel recombinant β-glucuronidase for rapid hydrolysis in benzodiazepine urinalysis is presented. Glucuronide controls for oxazepam, lorazepam and temazepam were treated with IMCSzyme™ recombinant β-glucuronidase. Hydrolysis efficiency was assessed at 55°C and at room temperature (RT) using the recommended optimum pH. Hydrolysis efficiency for four other benzodiazepines was evaluated solely with positive patient samples. Maximum hydrolysis of glucuronide controls at 5 min at RT (mean analyte recovery ≥ 94% for oxazepam and lorazepam and ≥ 80% for temazepam) was observed. This was considerably faster than the optimized 30 min incubation time for the abalone β-glucuronidase at 65°C. Mean analyte recovery increased at longer incubation times at 55°C for temazepam only. Total analyte in patient samples compared well to targets from abalone hydrolysis after recombinant β-glucuronidase hydrolysis at RT with no incubation. Some matrix effect, differential reactivity, conjugation variability and transformation impacting total analyte recovery were indicated. The unique potential of the IMCSzyme™ recombinant β-glucuronidase was demonstrated with fast benzodiazepine hydrolysis at RT leading to decreased processing time without the need for heat activation.

Simultaneous quantitative analysis of polyphenolic compounds in human plasma by liquid chromatography tandem mass spectrometry

“…Then, 10 μL of each internal standard solution at 100 μg/mL was added to plasma. A total of 480 μL of β-glucuronidase enzyme, 10% v/v in sodium acetate (140 mM, pH 4.8) was added to plasma, which was vortexed and incubated for 16 h at 37 • C [32]. After incubation, 20 μL methanol was added to the sample, which was then centrifuged (3000× g; 5 min; 4 • C) before being applied to a pre-conditioned Bond Elut C18 500-mg cartridge (Alltech, Associates, Australia) and washed with 2 mL water: methanol: acetic acid solution (90:10:0.1).…”

Opiate & Benzodiazepine Confirmations: To Hydrolyze or Not to Hydrolyze is the Question

“…Results from several studies have concluded that parent drug recoveries for specific benzodiazepine drugs were increased, based on the incorporation of hydrolysis in sample preparation (16)(17)(18)(19)(20)(21)(22)(23). Hydrolysis reactions have been mediated by β-glucuronidase enzymes from abalone entrails, Helix pomatia (type H-3), Helix aspersa, Patella vulgata (type L-II), Escherichia coli, Chlamys opercularis, and bovine β-glucuronidase (16)(17)(18)(19)(20)(21)(22)(23). Some hydrolysis comparison studies have demonstrated that recombinant β-glucuronidase enzyme was more efficient than other enzyme sources by affording a rapid hydrolysis (5 min) at room temperature (16,17).…”

“…Hydrolysis reactions have been mediated by β-glucuronidase enzymes from abalone entrails, Helix pomatia (type H-3), Helix aspersa, Patella vulgata (type L-II), Escherichia coli, Chlamys opercularis, and bovine β-glucuronidase (16)(17)(18)(19)(20)(21)(22)(23). Some hydrolysis comparison studies have demonstrated that recombinant β-glucuronidase enzyme was more efficient than other enzyme sources by affording a rapid hydrolysis (5 min) at room temperature (16,17). The recombinant source had a maximal hydrolysis recovery of >94% for oxazepam and lorazepam and >80% for temazepam (17).…”

“…Some hydrolysis comparison studies have demonstrated that recombinant β-glucuronidase enzyme was more efficient than other enzyme sources by affording a rapid hydrolysis (5 min) at room temperature (16,17). The recombinant source had a maximal hydrolysis recovery of >94% for oxazepam and lorazepam and >80% for temazepam (17). Recombinant β-glucuronidase was also 50% more effective in hydrolyzing a-hydroxyalprazolam glucuronide than abalone β-glucuronidase (17).…”

“…The recombinant source had a maximal hydrolysis recovery of >94% for oxazepam and lorazepam and >80% for temazepam (17). Recombinant β-glucuronidase was also 50% more effective in hydrolyzing a-hydroxyalprazolam glucuronide than abalone β-glucuronidase (17). Other sources of β-glucuronidase enzymes tend to have longer incubation times at increased temperatures for optimal hydrolysis efficiency.…”