A sensitive immunoassay for flunitrazepam and metabolites.

The objective of this study was to develop an immunoassay that would be capable of detecting flunitrazepam and/or cross-reacting metabolites in urine and comparing the results with those obtained by gas chromatography-mass spectrometry. Doses of Rohypnol varying between 0.5 and 4 mg were given to volunteers, and urine was collected for up to two weeks postingestion. These samples were analyzed by an ELISA that was developed using an antibody raised to flunitrazepam and a drug-enzyme conjugate prepared by attaching 7-aminoflunitrazepam 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 microg/L.

The detection and quantitation of 7-aminoflunitrazepam, the major urinary metabolite of flunitrazepam, by gas chromatography/mass spectrometry.

A sensitive gas chromatography/mass spectrometry method for the detection and quantitation of 7-aminoflunitrazepam, the major urinary metabolite of flunitrazepam, is described. The method is based upon solvent extraction followed by derivatisation with methyl-bis-trifluoroacetamide (MBTFA), to give a trifluoroacetyl derivative. A profile of 7-aminoflunitrazepam urinary concentrations following ingestion of 0.5 to 4 mg oral doses is reported. The method has been found to be reproducible and can be used for the confirmation of flunitrazepam administration.

Simple preparation of the major urinary metabolites of flunitrazepam and nitrazepam.

An alarming increase in the misuse/abuse of nitrobenzodiazepine derivatives, especially flunitrazepam, prompted us to establish reliable analytical protocols for their routine detection. Whilst the parent drugs are readily available from a number of commercial sources, it was found difficult to obtain samples of the corresponding amino metabolites which were required as analytical standards. This lead us to develop the straightforward synthetic protocol described here, to convert the readily available parent drugs, namely flunitrazepam and nitrazepam, to their respective 7-amino derivatives. The method requires minimum laboratory facilities. It involves the reduction of the nitro functionality in the parent drug to an amino group using tin (II) chloride under mild conditions, using ultrasonication at room temperature. The method is simple and should give toxicology laboratories better access to these much needed compounds.

A comparison of five commercial immunoassays for the detection of flunitrazepam and other benzodiazepines in urine.

Five commercially available immunoassay test kits (SYVA EMIT(R) d.a.u(TM), SYVA EMIT(R) II assay, Abbott FPIA, Cozart Auto-Lyte(R) and Roche Abuscreen(R) Online(TM), all used for the benzodiazepine group of drugs) were evaluated for their ability to detect flunitrazepam, its major urinary metabolite, 7-aminoflunitrazepam, and several other benzodiazepines at serial dilutions (final concentration 25-1000 ng/ml) in drug-free urine and in urines following oral administration of flunitrazepam (1-3 mg). For comparison, gas chromatography/mass spectrometry was used to measure urinary levels of 7-aminoflunitrazepam. Levels of drug detected in the study were compared with the cross-reactivities presented by the manufacturers for each individual kit. One to three mg doses of flunitrazepam were taken by volunteers and levels excreted in urine analysed over several hours. A positive response was obtained in several samples from volunteers who had taken 2 mg or 3 mg doses, but not a 1 mg dose. Thirty-five clinical samples from the individuals suspected of benzodiazepine abuse were also examined. The results were not consistent among the kits evaluated.We conclude that the test kits evaluated in this study do not detect flunitrazepam reliably, due primarily to their poor sensitivities.