UHPLC-MS/MS-based analysis of 17 nitazenes in human hair for practical forensic casework with simultaneous separation of 6 groups of isomers.

Nitazenes, a new class of potent synthetic opioids characterized by a 2-benzyl-benzimidazole structural core, have emerged on the illicit drug market following fentanyl. Compared to traditional opioids, nitazenes pose heightened risks of respiratory depression and central nervous system suppression. Due to the ease of modification of its parent nucleus, abusers and dealers can rapidly synthesize new structural analogs that fall outside the drug control catalog. Consequently, multiple isomers of nitazenes have emerged, causing significant interference in forensic practices. As an increasing number of poisoning and fatality cases were associated with nitazenes, there is an urgent need for developing an analytical method that can effectively address the challenges currently encountered in their identification in forensic practices. In this study, we established and validated a simple and high-efficiency ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for detecting 17 nitazenes in human hair. All analytes were separated in 6.5 min on a biphenyl column with simultaneous separation of six groups of isomers. Method validation demonstrated linear calibration over the range of 5-1000 pg/mg (r > 0.995), with limits of detection (LODs) ranging from 1 to 15 pg/mg and limits of quantification (LOQs) ranging from 5 to 20 pg/mg. The method also exhibited precision (<15 %) and accuracy ( ± 15 %), along with excellent recovery, acceptable matrix effects, and good stability at 4 ℃ for at least 72 h. When applied to authentic cases, this method successfully identified two positive hair samples and N,N-dimethylamino etonitazene was detected at concentrations of 1528.5 pg/mg and 463.9 pg/mg respectively. This method provides technical support for the rapid and accurate detection of nitazenes, better meeting the needs of anti-drug operations and forensic practice.