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

Abstract

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.

First identification, chemical analysis and pharmacological characterization of N-piperidinyl etonitazene (etonitazepipne), a recent addition to the 2-benzylbenzimidazole opioid subclass

“…Quantitative analysis of N-piperidinyl etonitazene in authentic biological samples via LC-HRMS revealed concentrations of 1.21 ng/mL in serum and 0.51 ng/mL in urine. These concentrations are comparable with reported nitazene analogue concentrations from forensic casework samples in the sub and low ng/mL range (Walton et al 2021) (Vandeputte & Krotulski et al, in press). The presence of low ng/mL Npiperidinyl etonitazene concentrations in biological samples highlights the need for highly sensitive analytical methods for drug detection and quantification.…”

“…A total of 73 different NSOs has been formally notified to the EU Early Warning System (EWS) on new psychoactive substances (NPS) between 2009 and 2021, with more than half being reported in the last 4 years (EMCDDA 2021). While newly emerging opioids present with progressively diverse chemical structures (UNODC 2020), opioids with a 2-benzylbenzimidazole core have recently become increasingly prevalent (Ujváry et al 2021;Vandeputte et al 2021b;Walton et al 2021).…”

Identification of etazene (etodesnitazene) metabolites in human urine by LC‐HRMS

“…Reduction of the nitro group on the benzimidazole moiety has also been described for these three NSOs; however, this is not possible for etazene as it lacks a nitro group on its benzimidazole moiety. Moreover because of this, 4 0 -hydroxy nitazene, the considered universal metabolite of the nitazene analogs 20 is not applicable as a biomarker for etazene exposure. To the best of the authors' knowledge, this is the first time nitazene glucuronide conjugates have been detected.…”

Interpol Review of Drug Analysis 2019-2022

“…2021 identification and full chemical characterization of “etonitazepyne” or “N-pyrrolidino etonitazene” (2-(4-ethoxybenzyl)-5-nitro-1-(2-(pyrrolidin-1-yl)ethyl)-1H-benzo[d]imidazole), a potent NPS opioid of the 5-nitrobenzimidazole class by GC-MS, HRAM LC-MS/MS, H-1 NMR, and FTIR [ 707 ]; review [ 708 ]; ten nitazenes and four metabolites were synthesized, analytically characterized via HPLC-DAD and LC-QTOF-MS [ 709 ]; 2022 in-depth chemical analysis of } Etonitazepipne (N-Piperidinyl etonitazene) in powder via different techniques (LC-HRMS, GC-MS, UHPLC-DAD, FT-IR and pharmacological characterization [ 710 ]; LC-MS/MS method for the quantification of analogues and/or metabolites of drugs in the nitazene series (isotonitazene, metonitazene, protonitazene, etonitazene, clonitazene, flunitazene, N-desethyl isotonitazene, and 5-amino isotonitazene) [ 711 ].…”