Cross-reactivities and structure-reactivity relationships of six benzodiazepines to EMIT(®) immunoassay

Benzodiazepines are among the most frequently prescribed drugs due to their sedative, hypnotic, anxiolytic, muscle relaxant and antiepileptic properties. Considering the high consumption of benzodiazepines worldwide, there is increased potential for addiction and abuse in cases of crime, driving under the influence of drugs, suicide and drug-facilitated sexual assault (DFSA). For these reasons, this class of drugs and their metabolites are frequently present in both clinical and forensic cases. In a forensic toxicology laboratory, typical screening analysis for benzodiazepine involves various immunoassay screening methods. The present study investigates the cross-reactivity profiles of six benzodiazepines not included in the manufacturer's instructions (3-hydroxy-flunitrazepam, 7-amino-nitrazepam, brotizolam, delorazepam, pinazepam, α-hydroxy-midazolam) to EMIT(®) II Plus Benzodiazepine Assay. Pinazepam, delorazepam and brotizolam are the most reactive molecules, while the other ones present a very low cross-reactivity. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm the concentrations of the spiked urines for immunoassay test and to make a comparison between the quantitative results of the different methods. Structure-reactivity relationships to EMIT(®) II Plus Benzodiazepine Assay were also evaluated. This paper draws attention to the problem of careless use of immunoassay tests for forensic purposes as they may provide false positive and/or negative results.