Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS²) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS². Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.

过去十年中，药品市场上出现了大量新型精神活性物质（NPS），对公共健康和社会造成了严重威胁。尽管它们含量丰富且具有潜在毒性，但有关其代谢的信息却很少，而这对于临床和法医学目的来说是至关重要的信息。NPS代谢可以使用体外模型进行研究，例如肝微粒体、细胞质、肝细胞等。使用体外模型形成的NPS代谢物的初步结构阐明通常使用液相色谱结合高分辨率串联质谱进行。 LC-HRMS ² ) 采用碰撞诱导解离 (CID) 作为裂解方法。然而，CID 产生的热激发离子可能不足以明确鉴定代谢物或其完整表征。电子激活解离 (EAD) 是一种相对较新的裂解方法，可用于裂解单电荷离子，可提供补充的结构信息，可用于进一步提高代谢物鉴定的可信度。本研究的目的是比较 CID 和 EAD 作为表征和鉴定合成卡西酮位置异构体及其代谢物的裂解方法。使用 LC-HRMS 通过 CID 和 EAD 方法研究了 2-甲基乙卡西酮 (2-MEC)、3-甲基乙卡西酮 (3-MEC) 和 4-甲基乙卡西酮 (4-MEC) 的体外代谢²。初步鉴定出 4、7 和 6 种代谢物分别参与 2-MEC、3-MEC 和 4-MEC 的代谢。在此，首次报道了3-MEC和2-MEC的代谢。与 CID 相比，EAD 产物离子质谱显示出不同的碎片模式，其中在 EAD 中观察到独特且丰富的产物离子，但在 CID 中未观察到。更重要的是，某些 EAD 专有产物离子在某些代谢物的结构阐明中发挥着重要作用。这些结果凸显了 EAD 碎片在代谢物鉴定工作流程中可以发挥的重要作用，与 CID 相比，通过提供额外的碎片数据，从而增强药物代谢物结构阐明的信心。