The objective of this study was to develop an immunoassay which would be capable of detecting flunitrazepam and/or cross-reacting metabolites in urine and comparing the results with those obtained by gas chromatoghraphy /mass spectroscopy (GC/MS).

Doses of RohypnolTM varying between 0.5 mg and 4 mg were given to volunteers and urine collected for up to two weeks post ingestion. These samples were analysed by an ELISA which was developed using an antibody raised to flunitrazepam and a drug-enzyme conjugate prepared by attaching 7-ammoflunitrazepam to horseradish peroxidase. Significant levels of flunitrazepam and/or cross-reacting metabolites were detected in urine for up to one week after ingestion. The immunoassay is selective with only diazepam cross-reacting at a level of 1000 ug/L.