Bellet N.F., Galloway R.

Boehringer Mannheim Corporation, Research & Development Department, 2400 Bisso Lane, Concord, CA 94524, USA

The addition of ß-glucuronidase directly to the CEDIA DAU Benzodiazepine assay reagents results in a very sensitive homogeneous enzyme immunoassay for the screening for benzodiazepines in urine samples. By adding ß-glucuronidase directly to the R1 reagent the need to individually treat samples with ß-glucuronidase prior to analysis is eliminated. During the first 5 minute incubation the glucuronide conjugated benzodiazepine metabolites are cleaved to yield products that are much more immunoreactive than the conjugated benzodiazepines. Cross-reactivity to oxazepam glucuronide increases from 0.2% to 26%, cross-reactivity to temazepam glucuronide increases from 1.5% to 27%, and cross-reactivity to lorazepam glucuronide increases from 1.1% to 47%. Sensitivity to actual samples containing lorazepam is increased about 20 fold. GC/MS confirmed benzodiazepine positive urine samples containing oxazepam, temazepam, flunitrazepam, or clonazepam are increased 20 % to 1500 % depending on the drug and the degree of conjuation of the parent drug in the sample.
This additional sensitivity to samples containing benzodiazepines requires a reliable and sensitive confirmation method. Two GC/MS procedures are used to obtain more complete coverage of the potential benzodiazepines. For both methods the samples are first treated with ß-glucuronidase (H. Pomatia). The first GC/MS procedure includes derivitization with MtBSTFA (1% tBDMCS) and GC/MS of tert-butyldimethylsilane derivatives. This procedure detects lorazepam, oxazepam, nor-diazepam, temazepam, 2-OH-ethylflurazepam, alpha-OH-triazolam, and alpha-OH-alprazolam. The second GC/MS procedure includes derivatization with BSTFA (1%TMCS) and GC/MS of trimethylsilane derivatives. This procedure detects 7-amino-flunitrazepam, 7-amino-clonazepam, and 7-amino nitrazepam and uses 7-amino-clonazepam-D4 as internal standard for quantitation.
The combination of a highly sensitive immunoassay screening method and a more complete GC/MS confirmation procedure results in a much more sensitive and convenient method for detecting the presence of benzodiazepines in urine.