A Forward-Thinking Approach to Addressing the New Synthetic Opioid 2-Benzylbenzimidazole Nitazene Analogues by Liquid Chromatography Tandem Quadrupole Mass Spectrometry (LC-QQQ-MS).

Novel psychoactive substances (NPS) continue to represent a threat to public health and safety. The number of new drugs in the latest emergent synthetic opioid class - the 2-benzylbenzimidazole analogues - also called the nitazenes - have begun to dominate the current novel synthetic opioid (NSO) subclass of NPS. We describe a liquid chromatography tandem quadrupole mass spectrometry (LC-QQQ-MS) method for the quantification of nine analogues and/or metabolites of drugs in this series: isotonitazene, metonitazene, protonitazene, etonitazene, clonitazene, flunitazene, N-desethyl isotonitazene, 5-amino isotonitazene, and 4'-hydroxy nitazene in human whole blood, urine, and tissue. Samples were prepared for analysis using a basic liquid-liquid extraction. Chromatographic separation was achieved using a C-18 analytical column. Multiple reaction monitoring (MRM) mode was used for detection. The calibration range for the analytes was 0.5-50 ng/mL (except for 5-amino isotonitazene which was 1.0-50 ng/mL). The limit of detection was 0.1 ng/mL, and the limit of quantitation was 0.5 ng/mL. The method had no carryover or interferences. Ionization enhancement was observed but this did not affect quantitation. All analytes passed the method validation assessment. Authentic human samples suspected of containing NSO's were obtained from medical examiner and coroner offices, as well as partnering forensic toxicology laboratories. Isotonitazene was confirmed in 92 blood samples, and its metabolites were confirmed across various matrices. Metonitazene (n=35), flunitazene (n=5), protonitazene (n=3), etodesnitazene (n = 2), and butonitazene (n=1) were also detected in these cases. These newly emerging 2-benzylbenzimidazole analogues were commonly found in combination with NPS benzodiazepines and opioids (e.g., flualprazolam, fentanyl). Nitazene analogues are potent esoteric drugs that may not be identified during routine toxicological screening and specialized assays based on sensitive instrumentation are needed to accurately characterize these novel synthetic opioids.