Improvement of Electrospray Ionization Response Linearity and Quantification in Dissolved Organic Matter Using Synthetic Deuterated Internal Standards

Aquatic dissolved organic matter (DOM) is an ultracomplex mixture of compounds covering a wide range of masses and with an unknown extent of isomeric complexity, making its structural elucidation and quantification highly challenging. Electrospray ionization high-resolution mass spectrometry (ESI-HRMS) has advanced DOM analysis, but accurate concentration determination remains limited by the lack of a response factor correction. Here, we address this limitation by introducing novel deuterated compounds as internal standards that mimic DOM structures. Using a d5-labeled compound free of isobaric interferences in DOM, we assessed ionization suppression in various aquatic sample extracts and improved concentration-based linearity in a coastal DOM reference material. Our results show that deuterated carboxylic acid-rich standards enable "pseudoquantification" by correcting for ionization suppression and instrument drift. Applying this approach, we estimate that DOM consists of 20-30% acids in river, coastal, and deep-ocean reference samples using an Orbitrap system. The same samples were estimated to contain 11-24% acids using 15T FT-ICR-MS, highlighting platform differences. Additionally, we establish a ∼1 ng L-1 feature detection limit for DOM compounds in seawater via a standard LC-MS gradient method. These findings demonstrate that deuterated standards provide a simple, practical way to improve DOM pseudoquantification, enhancing our understanding of its chemical composition in environmental studies.