Improved Screen and Confirmation Test of 7-Aminoflunitrazepam in Urine Specimens for Monitoring Flunitrazepam (Rohypnol) Exposure

Abstract

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.

Electroanalytical Sensing of Flunitrazepam Based on Screen Printed Graphene Electrodes

“…Flunitrazepam can be analysed via traditional analytical methodologies such as Immunoassay, Liquid Chromatography-Mass Spectroscopy (LC-MS) [6][7][8][9][10] or Gas Chromatography-Mass Spectroscopy (GC-MS) [11][12][13] which have successfully been applied for their quantification in biological matrixes. Of note is the analysis of oral fluid (saliva) by Wang et al [12] who found that typical benzodiazepine immunoassays have a broad cross reactivity towards widely prescribed benzodiazepines and found that due to the low dosage of the drug these systems lead to an unacceptable number of false negatives. More recently, Moore et al [7] described a methodology where benzodiazepines in oral fluid samples were analyzed using the Quantisal collection device and quantified via Solid Phase Extraction and LC-MS.…”

An Overview on Total Analytical Methods for the Detection of 1,4-Benzodiazepines

“…Cozart ELISA was proved to be more effective than Beckman and CEDIA reagents in an improved screen and confirmation test of urine samples for preliminary detection of flunitrazepam exposure. It is because Beckman is least sensitive to detect the presence of 7-aminoflunitrazepam, and CEDIA is though sensitive most its cross-reacting characteristics toward other benzodiazepines substantially reduces its effectiveness for monitoring flunitrazepam [116]. Utilizing a bioconjugate of gold nanoparticles and enzyme-labeled antibody as a signal probe increases the sensitivity of a traditional ELISA for 7-ANZP by nearly 20 times [117].…”

“…The four immunoassay urine screening kits; Roche Benzodiazepine PlusKIMS assay, Microgenics CEDIA Benzodiazepine assay, Microgenics CEDIA high sensitivity assay with β-glucuronidase, and Microgenics DRI reagent ready Benzodiazepine assay, were evaluated to investigate their performance in forensic urine analysis [122][123][124]. In all cases confirmatory tests have been done either by HPLC-MS [108,120,122,123] or GC-MS method [10,112,[114][115][116].…”