Are We Characterizing the True Peaks of Disinfection Byproducts? Establishment of a Rapid and Accurate 14/15 N-HaloFormula Methodology for FT-ICR-MS

Ultrahigh-resolution mass spectrometry offers exceptional resolving power, yet formula assignment, particularly for nitrogenous disinfection byproducts (N-DBPs), remains challenged by the limited isotopic signature of ¹⁵N and elemental ambiguities. We developed HaloFormula, a software for high-confidence formula assignment of dissolved organic matter (DOM) and DBPs. The workflow integrates candidate selection from a newly constructed formula database, dynamic in situ generation of an isotope matrix database, and formula assignment based on spectral similarity indices, achieving high computational efficiency and assignment accuracy. By incorporating a dual-isotope labeling strategy using equimolar ¹⁴N/¹⁵N-labeled monochloramine, HaloFormula enhances assignment accuracy and enables differentiation of nitrogen origins. The method was validated across diverse FT-ICR-MS data sets, including DOM and model compounds treated by chlor(am)ination and UV-based processes. HaloFormula assigned 1,875 formulas for a Suwannee River Fulvic Acid data set, of which 937 exhibited ¹³C isotopes. For halogenated compounds, over 900 Cl-containing formulas were assigned, with more than 1,400 isotopic peaks exhibiting accurate isotopic distributions. Up to 17 isotopologues were detected for individual N-DBPs due to enhanced ¹⁵N detection from the dual-isotope labeling strategy, enabling unambiguous formula assignment and nitrogen origin tracking. This novel methodology is readily adaptable for elucidating nitrogen sources in other systems and extendable to broader elemental analyses.