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

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

Ayodele A Morris, Scot A Chester, Erin C Strickland, Gregory L McIntire

J Anal Toxicol. 2014 Oct;38(8):610-4.

PMID: 25217555

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9001450-A	Fast β-Glucuronidase, Recombinant		9001-45-0	Price
IAR424866	β-Glucuronidase from abalone			Price

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