Effluent organic matter (EfOM), a major ozone consumer during wastewater ozonation, is a complex mixture of natural and anthropogenic organic molecules. Ozonation of EfOM adds to molecular complexity by introducing polar and potentially mobile ozonation byproducts (OBPs). Currently, nontargeted direct infusion (DI) ultrahigh resolution mass spectrometry (e.g. FT-ICR-MS) is used to study OBPs but requires sample extraction, limiting the accessible polarity range of OBPs. To better understand the impact of ozonation on EfOM and the formation of polar OBPs, nonextracted effluent was analyzed by direct injection onto a reversed-phase liquid chromatography system (RP-LC) online hyphenated with an FT-ICR-MS. Over four times more OBPs were detected in nonextracted EfOM compared to effluent extracted with solid phase extraction and measured with DI-FT-ICR-MS (13817 vs 3075). Over 1500 highly oxygenated OBPs were detected exclusively in early eluting fractions of nonextracted EfOM, indicating polar OBPs. Oxygenation of these newly discovered OBPs is higher than previously found, with an average molecular DBE-O value of −3.3 and O/C ratio of 0.84 in the earliest eluting OBP fractions. These polar OBPs are consistently lost during extraction but may play an important role in understanding the environmental impact of ozonated EfOM. Moreover, 316 molecular formulas classified as nonreactive to ozone in DI-FT-ICR-MS can be identified with LC-FT-ICR-MS as isomers with varying degrees of reactivity, providing for the first time experimental evidence of differential reactivity of complex organic matter isomers with ozone.

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<p>Ultrahigh-resolution mass spectrometry like FT-ICR MS has greatly expanded our view of the complexity and reactivity of complex dissolved organic matter (DOM) in the environment. However, both the structural characterization and (semi)-quantification DOM are methodologically not fully resolved, despite recent advances with coupling liquid chromatography (LC) to FT-ICR-MS that allows isomeric separation. Likewise, matrix effects and the existence of multiple isomers with largely varying – but unknown – ionization efficiencies that are simultaneously ionized and detected in direct infusion (DI)-FT-ICR MS prevents (semi)-quantification of DOM compounds and has limited the comparability of samples. Finally, common normalization strategies applied for non-targeted DOM analysis with DI cannot be easily transferred to LC-type DOM data. Here we present a new method that combines a post-column infusion of internal standard (PCI-IS) with LC-FT-ICR MS in order to reduce and compensate matrix effects as well as to provide a robust and reliable way to normalize MS peak intensities and compare samples measured with LC-FT-ICR MS. To this end, DOM samples were analyzed and the peak intensity data normalized by the internal standard and other frequently used normalization methods (e.g. sum of intensity, base peak), and absolute intensity indicating that PCI-IS normalization provides superior precision and accuracy. The potential of this method to provide semi-quantitative information on polarity fractions of DOM is assessed by testing the precision, accuracy, and linearity of PCI-IS normalization of model compounds spiked into DOM samples. Our results indicate that the use of LC separation reduced matrix effects (as compared to DI) and, in combination with the internal standard, improved to potential to obtain reliable peak intensity information.</p>