A study was conducted to compare the performance of the OnLine and OnTrak immunoassays for benzodiazepines with gas chromatographic-mass spectrometric (GC-MS) analysis in detecting flunitrazepam (FNP) and its metabolites in human urine. Urine was collected over a 72-h period from six individuals (four male and two female) who had taken a single oral dose of either 1 or 4 mg of FNP. The OnTrak assay was run at a 100-ng/mL cutoff of nordiazepam (NDP), and the OnLine assay was run with a standard curve from zero to 200 ng/mL of NDP with and without β-glucuronidase treatment. Each sample was analyzed by GC-MS using FNP, 7-amino-FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP as standards with β-glucuronidase treatment. The specimens from the 1-mg dose did not yield a positive result by immunoassay over the 72-h collection period. Specimens from the 4-mg dose did yield positive results in both immunoassays. The time of the first positive result ranged from 4 to 12 h, and the time to the last positive result ranged from 18 to 60 h. Treatment of the samples with β-glucuronidase increased the OnLine values between 20 and 60%, but it did not appreciably increase the detection time. GC-MS analysis showed no detectable levels of FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP. However, all samples collected past time zero showed detectable levels of 7-amino-FNP (> 2 ng/mL) with peak concentrations at 12-36 h. The peak levels of 7-amino-FNP by GC-MS paralleled the peak levels of the immunoassay response. The amount of 7-amino-FNP metabolite quantitated by GC-MS, however, accounted for only 15-20% of the total immunoassay crossreactive FNP metabolite
