A Forward-Thinking Approach to Addressing the New Synthetic Opioid 2-Benzylbenzimidazole Nitazene Analogs 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 analogs—also called the nitazenes—has begun to dominate the current new synthetic opioid (NSO) subclass of NPS. We describe a liquid chromatography–tandem quadrupole mass spectrometry method for the quantification of nine analogs and/or metabolites of drugs in this series: isotonitazene, metonitazene, protonitazene, etonitazene, clonitazene, flunitazene, N-desethyl isotonitazene, 5-amino isotonitazene and 4ʹ-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 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 did not affect quantitation. All analytes passed the method validation assessment. Authentic human samples suspected of containing NSOs were obtained from a 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 cases. These newly emerging 2-benzylbenzimidazole analogs were commonly found in combination with NPS benzodiazepines and opioids (e.g., flualprazolam, fentanyl). Nitazene analogs 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 NSOs.