Rapid enzymatic hydrolysis using a novel recombinant ß-glucuronidase in benzodiazepine urinalysis.

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 ß-glucuronidase for rapid hydrolysis in benzodiazepine urinalysis is presented. Glucuronide controls for oxazepam, lorazepam and temazepam were treated with IMCSzyme™ recombinant ß-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 ß-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 ß-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 ß-glucuronidase was demonstrated with fast benzodiazepine hydrolysis at RT leading to decreased processing time without the need for heat activation.

Determination of tapentadol (Nucynta®) and N-desmethyltapentadol in authentic urine specimens by ultra-performance liquid chromatography-tandem mass spectrometry.

Urine specimens from pain management patients dosed with Nucynta (Tapentadol) were confirmed for the presence of tapentadol and N-desmethyltapentadol using ultra-performance liquid chromatography-tandem mass spectrometry to minimize sample preparation and urine volume requirements. The linearity of the method for both tapentadol and N-desmethyltapentadol demonstrated correlation coefficients (R²) above 0.99 and linear ranges from 50 to 500,000 ng/mL for tapentadol and 100 to 500,000 ng/mL for N-desmethyltapentadol. The intraday precision of the assay for both analytes ranged from 2.2 to 6.9% over three concentrations; the interday precision for both analytes ranged from 1.2 to 8.4%. The limits of quantitation were 50 and 100 ng/mL for tapentadol and N-desmethyltapentadol, respectively, and the upper limit of linearity for both analytes was determined to be 500,000 ng/mL. Urine samples were collected within 24 h of dosing with tapentadol and shipped overnight to the laboratory. Samples were hydrolyzed with acid prior to analysis to measure total (unconjugated and conjugated) tapentadol and N-desmethyltapentadol. Further investigation into characterization of metabolites was performed by using a hybrid quadrupole-time-of-flight mass spectrometer in lieu of suitable analytical reference standards. The presence of significant N-desmethyltapentadol glucuronide was demonstrated for the first time.