Quantification of dissolved carboxylate- and phosphate-containing marine metabolites via aniline derivatization with liquid chromatography-tandem mass spectrometry

Metabolites dissolved in seawater are important analytical targets that act as substrates for microbial growth, mediate microbial interactions, and connect microbes to global elemental cycles. However, measuring dissolved metabolites is challenging due to their low concentrations (pM – nM) within a complex and high salt matrix (mM). Dissolved metabolites are often extracted from seawater prior to analysis, but standard extraction methods poorly capture polar metabolites, which have a higher affinity for seawater than typical extraction resins. To address these challenges, we optimized a method for derivatizing carboxylate- and phosphate-containing metabolites using aniline and 1-ethyl-2-(3-dimethylaminopropyl)carbodiimide (EDC), followed by solid-phase extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. Aniline derivatization reduced sample volume requirements by almost three orders of magnitude, improved extraction efficiency and chromatographic separation, and enhanced quantification with analyte-paired internal standards created using isotopically labeled aniline. With this method, we measured a suite of 47 dissolved metabolites with picomolar to nanomolar limits of detection, 19 of which cannot be measured in seawater by pre-existing extraction or derivatization methods. Where comparisons are possible, aniline derivatization yields comparable results to previously measured values. Finally, we tested this method by quantifying dissolved metabolites in two cultures of marine phytoplankton and in field samples from coastal and open ocean sites, demonstrating new possibilities for characterizing the role of marine microbes and their metabolites in global carbon cycling.