Introduction

Structural characterization of dissolved organic matter (DOM) is essential for understanding its role in the global carbon cycle. 1 Recent studies have revealed distinct multidimensional features in DOM from diverse aquatic environments, along with consistent structural patterns across samples. Recently, we demonstrated that using tandem FT-ICR MS approaches integrating precursor and fragment ion analysis enables structural comparisons of DOM molecular networks. 2 We extend these methods to investigate fulvic acids, focusing on their structural organization, molecular connectivity, and fragmentation behavior across various fulvic acid reference materials.

Methods

DOM fulvic acid (FA) standards were used to generate ultrahigh-resolution mass spectrometry data using a 21T ESI-FT-ICR MS (National High Magnetic Field Laboratory, Tallahassee, FL). Higher energy collision dissociation (HCD) was performed for unsupervised structural classification based on fragmentation pathways.

Results

The assigned formulas are dominated by CHO species (~35%) and CHON (~35%), followed by CHOS (~17%), and CHONS (~11%). The processing of each broadband MS resulted in ~40,000 peaks with signal-to-noise ratios larger than 6σ. The molecular formula annotation resulted in ~13,000 chemical formulas per sample after applying established DOM compositional constraints (C4−50H4−100 N0−3O0−25S0−2, S/N > 6, error < 1ppm, O/C < 1, 0.3 < H/C < 2.5, and double bond equivalent minus oxygen (DBE−O) < 10.). Of these, nearly 9,000 molecular formulas are common to all three fulvic acid samples, highlighting a substantial shared molecular core. Close to ~4,000 formulas are shared exclusively between SRFA2 and SRFA3, reflecting their similar origin and composition. In contrast, PPFA exhibited a lower number of exclusively shared molecular formulas, especially with SRFA2 (~200). Shared molecular formulas predominantly occupy regions associated with lignin-like structures, suggesting conserved structural motifs across different fulvic acid reference materials.

Conclusions

These findings reveal a compositionally coherent molecular landscape across the fulvic acid samples. Together, these results underscore both the structural importance of dissolved organic matter and the source-dependent variability that differentiates fulvic acid reference materials.