Enhancing Molecular Characterization of Dissolved Organic Matter by Integrative Direct Infusion and Liquid Chromatography Nontargeted Workflows

Dissolved organic
matter (DOM) in aquatic systems is a highly heterogeneous
mixture of water-soluble organic compounds, acting as a major carbon
reservoir driving biogeochemical cycles. Understanding DOM molecular
composition is thus of vital interest for the health assessment of
aquatic ecosystems, yet its characterization poses challenges due
to its complex and dynamic chemical profile. Here, we performed a
comprehensive chemical analysis of DOM from highly urbanized river
and seawater sources and compared it to drinking water. Extensive
analyses by nontargeted direct infusion (DI) and liquid chromatography
(LC) high-resolution mass spectrometry (HRMS) through Orbitrap were
integrated with novel computational workflows to allow molecular-
and structural-level characterization of DOM. Across all water samples,
over 7000 molecular formulas were calculated using both methods (∼4200
in DI and ∼3600 in LC). While the DI approach was limited to
molecular formula calculation, the downstream data processing of MS2
spectral information combining library matching and in silico predictions
enabled a comprehensive structural-level characterization of 16% of
the molecular space detected by LC-HRMS across all water samples.
Both analytical methods proved complementary, covering a broad chemical
space that includes more highly polar compounds with DI and more less
polar ones with LC. The innovative integration of diverse analytical
techniques and computational workflow introduces a robust and largely
available framework in the field, providing a widely applicable approach
that significantly contributes to understanding the complex molecular
composition of DOM.