title: Performance characteristics of 7-aminoflunitrazepam specific enzyme-linked immunosorbent assays.
abstract: 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.
authors.affiliation: Institute of Forensic Medicine, Ministry of Justice, Taipei, Taiwan.
authors.forename: Dong-Liang
authors.initials: DL
authors.lastname: Lin
authors.forename: Rea-Ming
authors.initials: RM
authors.lastname: Yin
authors.forename: Cheng-Hsing
authors.initials: CH
authors.lastname: Chen
authors.forename: Ya-Lei
authors.initials: YL
authors.lastname: Chen
authors.forename: Ray H
authors.initials: RH
authors.lastname: Liu
mesh_terms: D014151:Anti-Anxiety Agents / Q000652:urine*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D005445:Flunitrazepam / Q000031:analogs & derivatives* / Q000652:urine; D005554:Forensic Medicine; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015813:Substance Abuse Detection / Q000379:methods*; D017132:Workplace
chemical_list: D014151:Anti-Anxiety Agents; D005445:Flunitrazepam; C036343:7-aminoflunitrazepam