Automated on-line hydrolysis of benzodiazepines improves sensitivity of urine screening by a homogeneous enzyme immunoassay

Abstract

Benzodiazepines are used therapeutically as antidepressant, anxiolytic, anticonvulsant, hypnotic, muscle relaxant, and preanesthetic agents. Moreover, benzodiazepines are often abused for their euphoric effects. Consequently, benzodiazepines are commonly detected in toxicological analyses. Immunoassays of benzodiazepines are plagued by several factors that make reliable detection difficult. Complicated metabolic pathways, including N-dealkylation, C-hydroxylation, and glucuronidation, effectively reduce the parent compound to more polar, and thus easily excreted, metabolites. Some of these metabolites are pharmacologically active; in some cases, they are present in higher concentrations and have a longer elimination half-life than the parent compound (1)(2). Many, especially the glucuronide conjugates, which appear at high concentrations in the urine, do not readily cross-react with commercial immunoassays. In contrast, other benzodiazepine metabolites demonstrate high cross-reactivity, depending on the immunoassay utilized