Structural Characterization of Nitazene Analogs Using Electrospray Ionization–Tandem Mass Spectrometry (ESI–MS/MS)

Nitazene analogs are potent novel synthetic opioids (NSOs) that are becoming increasingly common and pose a threat to the public because of their fentanyl-like effects. Although 12 nitazene analogs are currently classified as Schedule I under the U.S. Controlled Substances Act, novel analogs continue to emerge, making their identification in forensic laboratories exceedingly difficult. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) is commonly utilized in toxicology laboratories and is becoming more common for seized drug analysis, particularly for compounds less suited for gas chromatography-electron ionization-mass spectrometry (GC-EI-MS). This study provides a comprehensive structural characterization of 38 representative nitazene analogs using LC-ESI-MS/MS instrumentation, including the proposed fragmentation mechanisms that lead to the formation of diagnostic product ions, enabling analog differentiation. General fragmentation pathways and mechanisms are proposed for all nitazene analogs, including inductive cleavages and molecular rearrangements. Overall, the most common product ions for nitazene analogs are derived from the substitutions to the amine or benzyl moieties, such as m/z 100, m/z 72, m/z 44, and m/z 107. However, the presence of different substitutions shifts the observed product ions. For example, recently occurring piperidine or pyrrolidine rings produce diagnostic product ions at m/z 112 and m/z 98, respectively. Therefore, modification to the core nitazene structure produces different diagnostic product ions, which can be used to identify existing and novel analogs. This study provides a comprehensive assessment of the fragmentation behavior of nitazene analogs under ESI-MS/MS conditions, which provides the basis for identifying new structural modifications in novel nitazene analogs.