Comparative evaluation of five immunoassays for the analysis of Alprazolam and Triazolam metabolites in urine: Effect of lowering the screening and GC-MS cut-off values

This study included evaluation of five commercially available immunoassays for the detection of alprazolam and triazolam metabolites in urine following single oral doses of these drugs. The products investigated were the EMIT® d.a.u.(TM) assay, EMIT® II assay, Abbott TDx® (FPIA) assay, Bio Site TRIAGE(TM) device, and the Boehringer Mannheim/Microgenics CEDIA® assay for urinary benzodiazepines. Urine specimens were also analyzed quantitatively by gas chromatography-mass spectrometry. Percent cross- reactivity was assessed by analysis of drug free urine containing drug standards at concentrations ranging from 100 to 10,000 ng/mL. The drug standards analyzed were α-OH-alprazolam, α-OH-triazolam, and α-OH- alprazolam glucuronide. The effect of lowering the screening cut-off value to 100 ng/mL, lowering the confirmation cut-off value to 50 and 25 ng/mL and the use of β-glucuronidase hydrolysis prior to analysis was also studied. Lowering the screening cut-off value and using enzymatic hydrolysis prior to screening increased the positive detection rate for benzodiazepines with the EMIT d.a.u. assay and fluorescence polarization immunoassay (FPIA). The TRIAGE device gave the lowest percent cross-reactivity in the analysis of the drug standards and gave negative results in all urine specimens analyzed following ingestion of alprazolam and triazolam.