title: 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.
authors.affiliation: Ameritox, Ltd., 486 Gallimore Dairy Road, Greensboro 27409, NC, USA ayodele.morris@ameritox.com.
authors.forename: Ayodele A
authors.initials: AA
authors.lastname: Morris
authors.affiliation: Ameritox, Ltd., 486 Gallimore Dairy Road, Greensboro 27409, NC, USA.
authors.forename: Scot A
authors.initials: SA
authors.lastname: Chester
authors.affiliation: Ameritox, Ltd., 486 Gallimore Dairy Road, Greensboro 27409, NC, USA.
authors.forename: Erin C
authors.initials: EC
authors.lastname: Strickland
authors.affiliation: Ameritox, Ltd., 486 Gallimore Dairy Road, Greensboro 27409, NC, USA.
authors.forename: Gregory L
authors.initials: GL
authors.lastname: McIntire
mesh_terms: D001569:Benzodiazepines / Q000652:urine*; D002851:Chromatography, High Pressure Liquid; D005966:Glucuronidase / Q000378:metabolism*; D020719:Glucuronides / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D008140:Lorazepam / Q000378:metabolism; D010076:Oxazepam / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D013693:Temazepam / Q000378:metabolism; D016482:Urinalysis / Q000379:methods*
chemical_list: D020719:Glucuronides; D001569:Benzodiazepines; D010076:Oxazepam; D013693:Temazepam; D005966:Glucuronidase; D008140:Lorazepam