Impact of preparation methods and storage conditions for optimization of the fecal metabolome storage stability

Untargeted fecal metabolomics has gained a higher scientific interest in the past decade, due to the increased importance of the gut microbiome metabolism. It is highly sensitive to preanalytical variations. However, the impact of sample collection, storage, and preparation on the metabolome composition remains insufficiently studied. In this study, we have systematically evaluated the effects of two sample preparation protocols: 5% DMSO/water solvation, followed by solvent substitution to methanol, referred to as the double-liquid extraction (DLE) protocol in this study, and methanol homogenization with FastPrep lysing matrices. Additionally, we examined long-term storage at -80 °C in a freezer, including fresh and freeze-dried samples, with and without methanol, at various stages of sample preparation. Global feature coverage and sensitivity analyses revealed that efficient mechanical homogenization is critical for maximizing metabolite recovery, particularly for intracellular and microbially derived compounds (enterolactone, allolithocholic acid). However, complete depletion of water and extraction in pure methanol resulted in a reduced feature coverage and lower signal intensities, particularly for polar metabolites such as proline and tyrosine. In the case of a freeze-drying step, reintroduction of water during sample preparation substantially improved extraction efficiency, underscoring the importance of maintaining controlled hydration of the fecal matrix to improve the solubility of the metabolite mixture.