Enzymatic Hydrolysis Improves the Sensitivity of Emit Screening for Urinary Benzodiazepines

Abstract

Screening for benzodiazepines is routinely performed by immunochemical analysis, such as enzyme immunoassay. However, the wide range of therapeutic and toxic concentrations of the different benzodiazepines and their extensive metabolism and conjugation hamper the design of an immunoassay protocol that can detect all benzodiazepines and their corresponding urinary metabolites. Multiple commercially available immunoassay methods exist, but false-negative (FN) results are still encountered, even with some frequently prescribed benzodiazepines. Several earlier investigations focused on determining how a particular benzodiazepine reacts in the different types of immunoassays, but only a few investigators have tried to improve the detection of some benzodiazepines, either by incorporating an enzymatic hydrolysis step before screening (1)(2) or by adding β-glucuronidase to the immunoassay reagents (3). These studies were performed with supplemented samples or with samples collected after controlled intake of therapeutic doses of one single benzodiazepine by healthy volunteers. We evaluated the relevance of hydrolyzing urine samples before screening with the Emit® d.a.u.TM Benzodiazepine Assay by analyzing a large number (n = 530) of authentic patient urine samples, reflecting actual practice. All urine samples were screened before (Emit) and after (Emit-H) enzymatic hydrolysis and subsequently analyzed by a sensitive gas chromatographic–mass spectrometric (GC-MS) method specifically developed for the analysis of benzodiazepines as a reference method (4)(5). The Emit d.a.u. Benzodiazepine Assay reagent sets were from Syva Co. (Dade Behring) and were used on a Cobas Mira Instrument (Roche) according to the instructions of the manufacturer. The applied hydrolysis, solid-phase extraction, and derivatization procedures were validated for alprazolam, α-hydroxyalprazolam, 4-hydroxyalprazolam, flunitrazepam, 7-aminoflunitrazepam, desmethylflunitrazepam, flurazepam, hydroxyethylflurazepam, N -desalkylflurazepam, ketazolam, lorazepam, lormetazepam, oxazepam, triazolam, and α-hydroxytriazolam. Because oxazepam and nordiazepam …