Intermediate products during natural organic matter biogeochemical processing, a case study inducing photo transformations followed by liquid chromatography-ultrahigh resolution mass spectrometry

Not only the quality but also the fate of natural organic matter (NOM) is of broad environmental interest. NOM reactivity is insufficiently understood due to a lack of experiments with high temporal resolution, particularly with respect to polarity of NOM molecules. By coupling ultrahigh performance liquid chromatography (UHPLC) with ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), insights into the polarity (hydrophilic versus hydrophobic) of NOM compounds can be obtained.

Previous studies addressing NOM reactivity often focused on the fate of molecules – suggesting whether a molecule was degraded, a new molecule was produced, or if the molecule remain unchanged i.e., was resistant to the process of interest. By high time resolution, the reaction time course of molecules can be tracked. Of particular interest is, whether intermediate products can be observed, meaning their abundance initially increases and then decreases during an experiment. In this study, we irradiated a sample from a wastewater treatment plant with light, capturing data with a temporal resolution of 8 time points.

Full profile UHPLC-FT-ICR-MS chromatograms were segmented into 13 one-minute wide segments. Molecular formulas were assigned up to masses of 1000 Da by allowing at most two nitrogen atoms and one sulfur as non-oxygen hetero atoms.

The main findings indicate that the average (from 13 segments) number of intermediate products (800) was in the same order of magnitude as degraded components (1000). The average number of resistant molecules was lower (250), while the number of products was one order of magnitude smaller (65) compared to the intermediate products. More polar CHNO products and intermediate products were detected compared to CHO, which exhibited a higher number of products, degraded MFs and intermediate products with decreasing polarity. The polarity resolution, as evidenced by chromatographic retention, revealed that the same MF can have different reactivity.

From approximately 10,000 assigned MFs more than 2,000 were identified both as degraded and intermediate product, depending on their polarity. About 100 MFs were classified as both products and intermediate products. Notably, at least three reactivity types – product, intermediate product and degraded – could be assigned to about 90 MFs.

In conclusion, the investigation of DOM reactivity requires a combination of high mass resolution (FT-ICR-MS), chromatographic / polarity resolution and high temporal or spatial resolution within the experiment time setup.