Evaluation of extraction solvents for untargeted metabolomics in characterizing Antarctic cryoconite hole dissolved...

Cryoconite holes, a dynamic habitat in Antarctica and other glaciers, are glacial biological hotspots with high biogeochemical turnover rates, significantly influencing carbon and nitrogen cycling within climate-sensitive glacial ecosystems. They are found on glaciers worldwide, highlighting their global importance, and are likely to become more widespread as glaciers continue to retreat. Despite their global occurrence and sensitivity to climate warming, untargeted metabolomic studies of their dissolved organic matter (DOM) remain scarce, largely due to very low biomass, harsh sample matrices, and the absence of optimized extraction methods.

We evaluated combinations of water, methanol (MeOH), and acetonitrile (ACN) to optimize the extraction of chemically diverse polar and non-polar metabolites from cryoconite holes. The individual extraction with organic-rich MeOH: Water (70:30 v/v) and in parallel two-solvent extractions with MeOH: Water (70:30 v/v) and ACN: MeOH: Water (40:40:20 v/v) yielded the greatest number and diversity of metabolites. Combining RP with HILIC chromatography provided the highest number of unique metabolites. This dual-LC and ionization-polarity combination increased the detection of metabolic features by 46.9% and 25.5% in single- and two-solvent combinations, respectively, compared to RP alone.

This study delivers the first comprehensive untargeted metabolomics workflow specifically optimized for the uniquely extreme environment of glacial cryoconite holes. By capturing both extracellular DOM and intracellular metabolites from cold-adapted microbial communities, the method reveals the complex metabolic landscape of these challenging cold-ecosystem niches. By characterizing these metabolic signatures, this research provides a foundation for future studies into metabolic interactions across polar and glacial environments.