Quantification of dissolved metabolites in environmental samples through cation‐exchange solid‐phase extraction paired with liquid chromatography–mass spectrometry

Abstract

Small, biologically produced, organic molecules called metabolites play key roles in microbial systems where they directly mediate exchanges of nutrients, energy, and information. However, the study of dissolved polar metabolites in seawater and other environmental matrices has been hampered by analytical challenges including high inorganic ion concentrations, low analyte concentrations, and high chemical diversity. Here we show that a cation‐exchange solid‐phase extraction (CX‐SPE) sample preparation approach separates positively charged and zwitterionic metabolites from seawater and freshwater samples, allowing their analysis by liquid chromatography–mass spectrometry. We successfully extracted 69 known compounds from an in‐house compound collection and evaluated the performance of the method by establishing extraction efficiencies (EEs) and limits of detection (pM to low nM range) for these compounds. CX‐SPE extracted a range of compounds including amino acids and compatible solutes, resulted in very low matrix effects, and performed robustly across large variations in salinity and dissolved organic matter concentration. We compared CX‐SPE to an established SPE procedure (PPL‐SPE) and demonstrate that these two methods extract fundamentally different fractions of the dissolved metabolite pool with CX‐SPE extracting compounds that are on average smaller and more polar. We use CX‐SPE to analyze four environmental samples from distinct aquatic biomes, producing some of the first CX‐SPE dissolved metabolomes. Quantified compounds ranged in concentration from 0.0093 to 49 nM and were composed primarily of amino acids (0.15–16 nM) and compatible solutes such as trimethylamine N‐oxide (0.89–49 nM) and glycine betaine (2.8–5.2 nM).

Seasonal measurements of the nitrogenous osmolyte glycine betaine in marine temperate coastal waters

“…Targeted analyses for osmolytes reveal higher concentrations in ice influenced regions of Antarctic waters (Dall'Osto et al 2017 and higher concentrations in coastal regions compared to the open ocean (Airs and Archer 2010;Keller et al 2004;Beale and Airs 2016). Recently, Sacks et al (2022) developed a method to determine a wide range of metabolites from the dissolved phase, including GBT, in marine and freshwater samples. Nevertheless, this limited knowledge has come from only a handful of studies or data points.…”

Ammonification by kelp associated microbes increases ammonium availability

“…Amino acid concentrations are rarely estimated in coastal areas, and we do not have measurements in the locales where our experiments were performed. Instead, we assumed a concentration of 1 μM amino acids in seawater, based on previous ocean studies [29][30][31]. Our enrichment achieved 525,430‰ 15 N, compared to the natural amount of 15 N that we are assuming is present in seawater with a concentration of 1 μM amino acids.…”

Benzoyl Chloride Derivatization Advances the Quantification of Dissolved Polar Metabolites on Coral Reefs

“…To overcome some of these limitations, alternative sample preparation approaches have been developed, allowing for specific chemical species to be retained, concentrated, and analyzed. Specifically, alternative SPE resins (i.e., cation exchange) and chemical derivatization − have recently been shown to improve the analysis of otherwise elusive polar compounds. While the development of these methodologies has been described, their widespread application to various marine ecosystems remains limited.…”