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Nitazenes, a class of new psychoactive substances, have emerged as a significant public health and safety concern due to their widespread abuse. While various detection methods, particularly mass spectrometry, have been developed for these substances, there is limited information regarding their fragmentation pathways and isomeric identification. This knowledge is crucial for drug analysis and forensic toxicology. Among the mass spectrometry techniques, collision-induced dissociation (CID) is commonly used for analyzing the fragmentation of analytes; however, its fragmentation pattern may not be sufficient for complete characterization or differentiation of isomers. Electron-activated dissociation (EAD), a fragmentation technique, provides complementary data to CID by generating distinct fragment ions that aid in the identification and characterization of small molecules. This study aims to characterize and identify 17 kinds of nitazenes using CID and EAD in combination with liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS). The chromatographic and mass spectrometric behaviors of these compounds were examined, and the fragments were analyzed, with a particular focus on the differences between isomers under CID and EAD modes. Notably, EAD generated more detailed fragmentation profiles than CID, revealing unique fragmentation pathways and characteristic fragment ions. In addition, the doubly charged ions of nitazenes were identified in the EAD spectra. Based on the CID and EAD fragmentation pathways of nitazenes, a novel substance was identified in a seized sample. These findings underscore the value of CID and EAD in enhancing forensic toxicology workflows by providing complementary fragmentation data that improve the identification and characterization of novel and unknown compounds.

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