Fragmentation pattern-based nontargeted screening strategy uncovered novel halogenated nucleotides in drinking water

Halogenated nucleotides have been reported as emerging disinfection byproducts in drinking water and the identification of halogenated nucleotides is a challenging but worthwhile endeavor due to their high risk, diverse chemical structures and scarcity of chemical standards. To improve the recognition efficiency of halogenated nucleotides in complex environmental matrices with multiple halogen ions, a fragmentation pattern-based nontargeted screening strategy was developed, which combines halogen isotope features and characteristic product ions to identify halogenated nucleotides within extensive datasets from high-resolution mass spectrometry. The data were preliminary screened based on the halogen isotope features of chemicals with varying halogen atom counts, followed by further identification using shared fragments with m/z values of 78.96, 96.97 and 211.00 Da. We tentatively identified 72 halogenated nucleotides, including 9 brominated, iodinated and mixed halogen-substituted nucleotides that were annotated and reported for the first time. The effectiveness of the nontargeted identification strategy was demonstrated through manual verification of parent and product ions. Additionally, by comparing the Gibbs free energies of reactions (ΔG) of forming chlorinated, brominated and iodinated nucleotides, chlorinated nucleotides were revealed to exhibit the greatest formation potential, followed by brominated and iodinated nucleotides. 8-Chloroadenosine 5'-monophosphate was selected for the cytotoxicity test to evaluate its potential biological hazards and proven to manifest relatively high cytotoxicity. This work provides a new fragmentation pattern-based screening strategy for the effective identification of unknown halogenated nucleotides. This method is anticipated to demonstrate broad applications in the nontargeted identification of halogenated nucleotides in diverse environmental samples.