Improved cross-reactivity to α OH triazolam in the BMC CEDIA DAU urine benzodiazepine assay

Immunoassays designed to detect use of older benzodiazepines such as oxazepam or diazepam often cannot detect triazolam use because of the low doses of triazolam administered, rapid biotransformation to metabolites with poor cross-reactivites, and the small amount of α OH triazolam glucuronide excreted in the urine. Previous studies have demonstrated that certain immunoassays have high cross-reactivity to at OH triazolam but are unable to detect therapeutic triazolam use in urine. The objectives of this study were to characterize the immunoreactivity toward α OH triazolam in the reformulated cloned enzyme donor immunoassay (CEDIA) drug abuse urine benzodiazepine assay and to measure the immunoreactivity of urine specimens from subjects who were administered single oral doses of triazolam. α OH triazolam standards were prepared in drug-free urine and the new CEDIA assay gave a positive result at concentrations from 100 to 200 ng/ml, which indicates an eight-fold improvement in CEDIA cross-reactivity to α OH triazolam standards in the reformulated CEDIA assay. With a 200 ng/ml cut- off, 4/30 of the urine specimens screened positive for benzodiazepines without enzymatic hydrolysis and 6/30 after enzymatic hydrolysis. When using an in-house 100 ng/ml nitrazepam cut-off calibrator, 10/30 urine specimens were positive in the reformulated CEDIA assay without hydrolysis and 22/30 were positive with enzymatic hydrolysis before screening.