Endogenous Fluorescence Turn-On of Nitro-Containing Nitazenes via In Situ Reduction for Rapid Detection

Nitazenes, a rapidly expanding class of highly potent synthetic opioids, have raised global concerns due to their high abuse potential and detrimental effects on both health and social stability. The fluorescence detection of nitazenes remains challenging, primarily due to the limited specificity of exogenous probe-based methods, and very few fluorescence-based assays have been reported for these compounds to date. Herein, we report an endogenous fluorescence turn-on strategy for the rapid detection of nitro-containing nitazenes via precise modulation of their intrinsic fluorescence through in situ chemical reduction. Using Na2S2O4 as the reducing agent, the nitro group is converted to an electron-donating amino group, which restores the conjugation-driven emission and triggers a remarkable fluorescence turn-on response. The proposed mechanism was supported by fluorescence lifetime analysis, liquid chromatography-high-resolution mass spectrometry/tandem mass spectrometry (LC-HRMS/MS), and time-dependent density functional theory (TD-DFT) calculations. The method successfully detected four representative nitazenes (etonitazene, metonitazene, protonitazene, and isotonitazene) with a detection limit as low as 12.57 nM, achieving analytical performance comparable to that of HPLC. Furthermore, the practical applicability was validated in complex biological matrices, including 50% saliva and 50% urine. This endogenous fluorescence activation strategy offers distinct advantages of simplicity, rapidity, and probe-free reaction-triggered signal generation, which would open new possibilities for the direct fluorescence detection of emerging psychoactive substances featuring reducible nitroaromatic groups, thereby holding great promise for on-site forensic analysis and public security screening.