Optimization of fecal sample homogenization for untargeted metabolomics

Fecal samples are highly complex and heterogeneous, containing materials at various stages of digestion. The heterogeneity and complexity of feces make stool metabolomics inherently challenging. The level of homogenization influences the outcome of the study, affecting the metabolite profiles and reproducibility; however, there is no consensus on how fecal samples should be prepared to overcome the topographical discrepancy and obtain data representative of the stool as a whole.Various combinations of homogenization conditions were compared to investigate the effects of bead size, addition of solvents and the differences between wet-frozen and lyophilized feces.The homogenization parameters were systematically altered to evaluate the solvent usage, bead size, and whether lyophilization is required in homogenization. The metabolic coverage and reproducibility were compared among the different conditions.The current work revealed that a combination of mechanical and chemical lysis obtained by bead-beating with a mixture of big and small sizes of beads in an organic solvent is an effective way to homogenize fecal samples with adequate reproducibility and metabolic coverage. Lyophilization is required when bead-beating is not available.A comprehensive and systematical evaluation of various fecal matter homogenization conditions provides a profound understanding for the effects of different homogenization methods. Our findings would be beneficial to assist with standardization of fecal sample homogenization protocol.