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.

Improved screen and confirmation test of 7-aminoflunitrazepam in urine specimens for monitoring flunitrazepam (Rohypnol) exposure.

Confirmed and alleged misuses of flunitrazepam (FM2, Rohypnol) have brought about serious interest in the development of an analytical methodology that can be effectively used for preliminary screen and confirmatory test of FM2 (or its metabolites) in urine specimens under high-volume settings. Reported methods do not serve this need well for the following reasons: (1) common benzodiazepine (BZ) immunoassays (IAs) have broad cross-reactivities toward widely prescribed BZs (and their metabolites) and are therefore likely to generate an unacceptable number of false positives and (2) because FM2 is typically used at low doses (1-4 mg), IAs with low cross-reactivities toward FM2 (and its metabolites) are likely to generate false-negative results. In this current study, a familiar and effective two-step IA/gas chromatography-mass spectrometry (GC-MS) approach is successfully developed and applied to clinical specimens. Cross-reacting characteristics of the following BZ IAs toward various BZs (and their metabolites) are evaluated focusing on their effectiveness in serving as the preliminary test reagent in a two-step testing protocol: TDx, Beckman, CEDIA, Roche Cobas Integra, Emit II Plus, and Cozart ELISA. Although other IAs show broad cross-reactivities toward various BZs and their metabolites, diazepam is the only non-FM2 derived compound that exhibits noticeable cross-reactivity toward Cozart ELISA reagent. Cross-reactivity data and data derived from studies conducted on a limited number of clinical specimens demonstrate that, when used to monitor FM2 exposure in a large population group (including those exposed to other BZs), Cozart ELISA has the potential of being as effective as (or better than) those currently used in various workplace drug-testing programs for monitoring respectively targeted drugs. Data derived from this study further suggest that 50 ng/mL apparent 7-aminoflunitrazepam (Cozart ELISA) and 30 ng/mL free 7-aminoflunitrazepam (GC-MS) are potentially effective preliminary test and confirmation test cut-offs. To maximize efficiency, it is further suggested that urine specimens are first diluted by a factor of 5 for the preliminary test in which a 10-ng/mL 7-aminoflunitrazepam standard is used as the assay's cut-off standard.