High-throughput quantification of emerging ''nitazene'' benzimidazole opioid analogs by microextraction and UHPLC-MS/MS.

Abstract

Benzimidazole opioids, often referred to as nitazenes, represent a subgroup of new psychoactive substances with a recent increase in fatal overdoses in the US and Europe. With a variety of analogs emerging on the illicit drug market, forensic labs are challenged to identify these potent drugs. We here present a simple quantitative approach for the determination of nine nitazene analogs, namely, clonitazene, etodesnitazene, etonitazene, etonitazepyne, flunitazene, isotonitazene, metodesnitazene, metonitazene, and protonitazene in whole blood using liquid-phase microextraction and electromembrane extraction in a 96-well format and liquid chromatography tandem mass spectrometry. Green and efficient sample preparation was accomplished by liquid-phase microextraction in a 96-well format and resulted in high extraction yields for all analytes (> 81%). Here, blood diluted with buffer (1:1, %v) was extracted from a donor compartment across a thin organic liquid membrane and into an aqueous acceptor solution. The acceptor solution was collected and directly injected into the analysis platform. Chromatographic separation was accomplished with a biphenyl column, allowing for baseline separation of the structural isomers isotonitazene and protonitazene before detection by multiple reaction monitoring. The validation was performed according to Scientific Working Group of Forensic Toxicology guidelines. The calibration range was from 0.5 to 50 nM (except for protonitazene and clonitazene from 0.1 nM) with good linearity and limits of detection down to 0.01 nM. An AGREEprep assessment was performed to evaluate sample preparation greenness, with a final score of 0.71. Nitazenes represent a current threat to public health, and analytical methods that cover a wide range of these analogs are limited. The here described method may assist in the detection of nitazenes in whole blood and prevent these substances from being missed in post-mortem investigations.