Validation of benzodiazepine β-glucuronide primary reference materials for hydrolysis and quality assurance controls

Abstract

The major metabolite of hydroxylated benzodiazepines is the β-glucuronide metabolite, thus a hydrolysis step is included in benzodiazepine analyses in urine. We evaluated the new Alltech ( R, S) oxazepam and lorazepam β-glucuronide primary reference materials as hydrolysis controls and for use in proficiency testing. Using HPLC analysis and GC/MS analysis following acid and β-glucuronidase hydrolysis, we found that the lorazepam β-glucuronide material was >95% pure, but the oxazepam β-glucuronide material was only 54.0% pure. Recovery after hydrolysis with β-glucuronidase at 60 °C for two hours and 22 °C for 24 h was compared. There was no difference in the recovery of either drug incubated under both conditions. Using HPLC analysis to separate the R and S isomers of the glucuronides, we found that the S isomer hydrolyzes faster than the R isomer, but at optimal conditions hydrolysis of both isomers is complete. Alltech's glucuronide materials can be valuable as hydrolysis controls in method development and routine benzodiazepine analyses, but each laboratory must validate the purity of the materials and determine the acceptable references ranges for in-house controls made from these materials. The reference materials were also evaluated for possible use as quality control material for urine drug screening. The material had to be added at 130% of the reported cutoff concentration to produce a positive result on the Triage ™ Panel for Drugs of Abuse. For the Triage ™ Plus TCA Panel for Drugs of Abuse lorazepam β-glucuronide was added at 154% of the cutoff concentration, but addition of the oxazepam β-glucuronide at >200% of the reported cutoff concentration did not produce a positive result. Discrepancies may be due to preferential binding of antibodies to the S isomer, because the Alltech glucuronide materials do not contain the isomers in the same ratio as that excreted in human urine.