Evaluation of enzyme‐linked immunosorbent assay screening kits for the detection of nitazene analogs

Nitazene analogs are a highly potent class of novel psychoactive substances (NPS) that were first identified in forensic casework in the United States in 2019. While enzyme-linked immunosorbent assay (ELISA) remains a prevalent screening tool in forensic toxicology laboratories, no nitazene-specific ELISA kits are commercially available, supporting the use of high-resolution mass spectrometry (HRMS) methods as a more adaptable alternative for screening. However, even with the growth in popularity of HRMS techniques, it is important to understand the cross-reactivity of novel substances, such as nitazenes, with routinely used ELISA kits. Cross-reactivity is particularly important for forensic toxicology casework since it can impact the reliability of screening results, potentially leading to non-detection of novel substances or false-positive identifications in the presence of non-target analytes. This study tested the cross-reactivity of seven nitazene analogs (4'-OH nitazene, 5-methyl etodesnitazene, isotonitazene, metodesnitazene, N-piperidinyl etonitazene, N-pyrrolidino etonitazene, and protonitazene) in whole blood using 13 commercial ELISA kits from three manufacturers. Various drug/class kits were selected based on reported or potential co-positivity with nitazenes (opiates, opioids, fentanyl) or by structural similarities (LSD, zolpidem). Across tested concentrations for the seven selected analytes, none of the tested kits produced a signal sufficient for a positive result, confirming that their presence at these levels does not compromise the screening specificity of the target analytes. However, these findings highlight the need for laboratories to adopt mass spectral-based screening methods like HRMS or advocate for the development of nitazene-specific ELISA kits for effective nitazene analog screening.