Characteristic Fragment-Based Nontarget Identification of Thiol-Reactive Compounds in Disinfected Wastewater Using High-Resolution Mass Spectrometry: Focus on α,β-Unsaturated Carbonyls

Thiol-reactive compounds, byproducts of wastewater chlorination, are under scrutiny for their high potential toxicity in water reuse applications. To date, a limited number of compounds have been identified in disinfected wastewater. In this study, we proposed a framework for comprehensively screening and identifying thiol-reactive compounds using a thiol probe combined with a nontarget high-resolution mass spectrometry (HRMS) analysis workflow, in which we monitored a fragment ion (C₅H₈NO₃S^-). To specifically target α,β-unsaturated carbonyls, this approach was developed and validated for 15 model α,β-unsaturated carbonyl compounds. Then, the technique was applied to analyze chlorinated municipal wastewater effluent, and 58 tentative thiol-reactive compounds were detected. We predicted the formulas of the 30 highest abundance α,β-unsaturated carbonyls. Among these, we used commercially available standards to validate our approach and quantify seven α,β-unsaturated carbonyls, namely, acrylic acid, 2,6-di<i>tert</i>-butyl-1,4-benzoquinone, <i>N</i>-acetyl-<i>p</i>-benzoquinone imine, <i>trans</i>-2-hexenal, 3-methyl-2-cyclopenten-1-one, dibutyl maleate and parthenolide, with concentrations ranging from <0.017-4.28 μg/L. These seven α,β-unsaturated carbonyls were identified as contributors to the toxicity of chlorinated wastewater, making up 4% of its overall cytotoxicity. This study demonstrated the characteristic fragment-based nontarget analysis as an effective approach to screen and identify thiol-reactive α,β-unsaturated carbonyls in treated wastewater.