Chromatography Coupled to High-Resolution Mass Spectrometry Analysis Reveals Flaws in van Krevelen Compound Class...

High-resolution mass spectrometry (HRMS) is a powerful tool for determining molecular formulas in dissolved organic matter (DOM). In the biogeochemical field, it is common to assign DOM molecular formulas to prospective chemical classes based on elemental ratios alone, with these classes based on known biomolecules or empirical classifications (e.g., carboxyl-rich alicyclic molecules). However, accurate identification of molecular origin remains challenging due to DOM's extreme isomeric diversity and the inability of HRMS to provide information about chemical connectivity, calling into question the accuracy of assignment or quantification. To address the lack of chemical connectivity gained from HRMS alone, we used liquid chromatography-HRMS to determine whether retention behavior is consistent with biochemical class assignment. Problematically, all examined classes displayed very broad retention profiles, indicating diversity of chemical functionality within their defined elemental ratios. Furthermore, retention behavior matched poorly with known biochemical class hydrophobicity data, particularly for "carbohydrates" and "peptides". Additionally, differences in retention profiles of various compound classes and molecular formulas between terrestrial and marine samples show that molecular formulas are constituted by different groups of isomers between environments. We recommend an overhaul of standard practice in the use of HRMS data for biogeochemical investigation of DOM, relying less on assignment to prospective classes and more on chemical understanding, which can be developed through the implementation of chromatography and tandem MS methods using modern equipment and workflows.