The UHPLC-MS/MS method for the determination of 26 synthetic benzimidazole opioids (nitazene analogs) with isomers separation.

The global opioid crisis is a public health emergency characterized by the widespread misuse of opioid drugs, leading to high rates of addiction, overdose, and death. Initially driven by the over-prescription of opioid painkillers, it has evolved into a worldwide epidemic exacerbated by the rise of potent synthetic opioids. Benzimidazole opioids ('nitazenes') are one group within this class of compounds that have garnered increasing attention in recent years due to their addictive potential and growing presence in illicit drug markets. Because of their high potency, low concentrations in biological samples are expected, implying the need for very sensitive and selective methods. In the presented paper, an UHPLC-QqQ-MS/MS method was developed for the determination of 26 nitazenes in biological samples (including metabolites), with simultaneous separation of structural isomers. Biological samples were prepared using liquid-liquid extraction (LLE) at pH 9, and quantification was performed using MRM mode. Metonitazene-d was used as the internal standard. Precision and accuracy did not exceed 14.9 % and ± 14.1 %, respectively. Recovery and matrix effect values were 80.6-120.4 % and ± 20.4 %. The method achieved a remarkably low limit of quantification (LOQ), with values of 50 pg/mL for flunitazene and 10 pg/mL for all other compounds. Furthermore, MS/MS (QqQ) spectra were collected for all compounds, enabling the evaluation of fragmentation pathways and characteristic ions for different structural analogs. The knowledge regarding the fragmentation mechanisms of these compounds, along with the ESI-MS spectra provided in this paper, can impact the future identification of newly emerging nitazene analogs.