Performance characteristics of 7-aminoflunitrazepam specific enzyme-linked immunosorbent assays

With 7-aminoflunitrazepam (7-amino-FM2)-specific ELISAs now readily available from several commercial sources (e.g., Cozart Bioscience, Immunalysis, this study was conducted to evaluate the performance characteristics of these products when applied to the two-step testing protocol as commonly practiced in today's workplace drug-testing programs. Cross-reacting characteristics of these two assays toward a list of 25 benzodiazepines were evaluated. These assays were then applied to the analysis of urine specimens collected from patients treated with flunitrazepam (FM2) and/or other benzodiazepines. Resulting data were evaluated against gas chromatography-mass spectrometry (GC-MS) test data to ascertain corresponding cutoffs suitable for the two-step immunoassay/GC-MS testing strategy. Both Cozart and Immunalysis ELISAs are highly specific to 7-amino-FM2, with the latter reagent generating slightly higher responses. Diazepam and FM2 (parent compound) are the only compounds with significant cross-reacting characteristics. With the ELISA reagents' optimal dynamic ranges set between 0 and 25 ng/mL, urine specimens should be diluted by a factor of 5 prior to ELISA testing. If 30 ng/mL 7-amino-FM2 is adapted as the GC-MS cutoff, the corresponding ELISA cutoffs range is approximately 100-200 (or 20-40 when diluted by a factor of 5) ng/mL. Reagent lot and specimen characteristics (with or without the presence of cross-reacting compounds) affect the correlation of data derived from ELISA and GC-MS tests.