Characterization of the chemical composition of soil humic acids using Fourier transform ion cyclotron resonance mass spectrometry

The composition of humic acids (HAs) with varying degrees of humification isolated from 10 common Japanese soils was characterized using negative ion mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at 12 T. In particular, attention was paid to polynuclear aromatic components, which are more associated with the refractory nature of HAs and their resistance to biodegradation in soil than single C ring aromatic entities, such as lignin-like components, and aliphatic functionalities. Thousands of peaks were observed in the m/z range of 200–700, and molecular formulas were assigned to 817–2457 peaks in each sample. The molecular formulas having H/C and O/C ratios similar to those of lipid, protein, and other aliphatics with low double bond equivalents (DBE) of 0–7 were generally observed across the m/z range of 200–700. Although there were a number of molecular formulas having H/C and O/C values similar to those of lignin across the wide m/z range in the HAs with a low degree of humification, most lignin-like molecular formulas in the larger m/z range (450–650) or irrespective of m/z were lacking in the HAs with middle and high degrees of humification, respectively. These observations suggest a longer residence time for lignin monomers/dimers (and their derivatives; m/z 200–400) than larger lignin oligomers (m/z 450–650) in HA structural domains. The number of molecular formulas having H/C and O/C values similar to condensed aromatics increased with increasing degree of humification. The m/z and DBE values of condensed aromatic-like molecular formulas in the HAs with a lower degree of humification were <500 and 10–25, respectively, whilst the ranges expanded to 600 and 30–33, respectively, in the highly-humified black HAs. Kendrick mass defect analysis using a carboxyl group as the characteristic functional group found that 31, 73, and 39 molecular formulas had chain-type, net-type, and biphenyl-type condensed aromatic acids, respectively, as possible structures. Summed peak magnitudes of the condensed aromatic-like molecular formulas, in particular those with higher DBE values (>17) expected to have at least 5 rings, increased with increasing degree of humification and aromatic C content (evaluated by solid-state 13C nuclear magnetic resonance spectroscopy), suggesting their contribution to the intrinsic recalcitrance of HAs in soil.