BackgroundMetabolomics analysis of human stool samples is of great interest for a broad range of applications in biomedical research including early detection of colorectal neoplasms. However, due to the complexity of metabolites there is no consensus on how to process samples for stool metabolomics measurements to obtain a broad coverage of hydrophilic and hydrophobic substances.MethodsWe used frozen stool samples (50 mg) from healthy study participants. Stool samples were processed after thawing using eight different processing protocols and different solvents (solvents such as phosphate-buffered saline, isopropanol, methanol, ethanol, acetonitrile and solvent mixtures with or without following evaporation and concentration steps). Metabolites were measured afterwards using the MxP Quant 500 kit (Biocrates). The best performing protocol was subsequently applied to compare stool samples of participants with different dietary habits.ResultsIn this study, we were able to determine up to 340 metabolites of various chemical classes extracted from stool samples of healthy study participants with eight different protocols. Polar metabolites such as amino acids could be measured with each method while other metabolite classes, particular lipid species (better with isopropanol and ethanol or methanol following a drying step), are more dependent on the solvent or combination of solvents used. Only a small number of triglycerides or acylcarnitines were detected in human faeces. Extraction efficiency was higher for protocols using isopropanol (131 metabolites>limit of detection (LOD)) or those using ethanol or methanol and methyl tert-butyl ether (MTBE) including an evaporation and concentration step (303 and 342 metabolites>LOD, respectively) than for other protocols. We detected significant faecal metabolite differences between vegetarians, semivegetarians and non-vegetarians.ConclusionFor the evaluation of metabolites in faecal samples, we found protocols using solvents like isopropanol and those using ethanol or methanol, and MTBE including an evaporation and concentration step to be superior regarding the number of detected metabolites of different chemical classes over others tested in this study.