Stable Isotopic Labeling and Automated Reactivity-Directed Approach Accelerating Discovery of Toxic Disinfection...

Reactivity-directed analysis facilitates prioritization of toxic disinfection byproducts (DBPs), yet its data processing remains laborious and inefficient. Herein, we developed an automated approach integrating stable isotopic labeling with reactivity-directed principles. Normal and deuterated glutathione (GSH) probes were used to generate paired isotopic signatures enabling precise localization of reactive DBPs. Customized bioinformatics tools were employed to efficiently extract paired GSH-DBP adducts. The workflow allowed rapid automated processing of large data sets within minutes while maintaining high coverage and accuracy. When applied to chlorinated and chloraminated water samples, 255 adducts were screened from >50 000 raw features, leading to annotation of 202 DBPs, including 193 newly reported. GSH reaction mechanism analysis attributed 65 chlorinated and 132 unsaturated DBPs to nucleophilic substitution and addition, respectively. Notably, 5 sulfur-containing DBPs enabled thiol oxidation, indicating a new GSH reaction pathway. Among annotated DBPs, chlorination favored unsaturated carbonyls, while chloramination generated more nitrile/nitro group DBPs. Toxicity predictions verified that annotated DBPs exhibited toxicities comparable to or greater than those of regulated DBPs. Prevalent toxic DBPs exhibited a high molecular weight or structural characters, such as benzene rings, carbonyls, and Cl/N functionalization. This approach significantly accelerated toxic DBP discovery, and the findings provided new insights into toxicity drivers in disinfected waters.