Abstract
Sample preparation for metabolomics does not follow a standardized procedure, often involving various sampling and storage methods, with solvent storage, such as in ethanol, sometimes used when immediate freezing is not possible or feasible. While convenient, solvent storage raises concerns about passive metabolite extraction from tissues-a potentially valuable but overlooked process for isolating biomolecules. Thus, while chemical storage conditions are suboptimal for metabolomics analysis, there exists untapped potential for passive extraction of valuable metabolites. This study investigated the potential for metabolite diffusion into ethanol during storage of Thyone aurea, a little-known sea cucumber species endemic to the Western Coastlines surrounding Southern Africa. Samples from Saldanha Bay were collected during summer and winter and, due to the time lapse between collection and analysis, were stored in 100% ethanol. Untargeted analysis on the crude ethanolic storage extract using 1H-NMR metabolomics and full-scan UPLC-QTOF-MS revealed significant metabolite diffusion, with compounds linked to energy metabolism, immune response, osmoregulation, and tissue integrity. Seasonal variation showed higher compound abundance in summer. These results underscore the potential impact of solvent storage during metabolomics studies and highlight the need to assess both storage solvents and tissues in such analyses. In addition, the presence of unidentifiable compounds suggests storage ethanolic extracts from T. aurea as a promising source of novel metabolites, warranting further research to explore its biochemical and therapeutic potential.