Gut microbiota metabolism made simple: the SIMPLIFY extraction protocol - Poster Metabolomics 2024 (Osaka, Japan)

SIMPLIFY is a single extraction protocol compatible with several LC-qToF-MS methods for comprehensively analysing gut microbiota metabolites. As a proof-of-concept, we applied this protocol to analyse faecal reference material and faecal inoculum obtained from in vitro colonic fermentation of vegetables and dietary fibres, these later serving as proxies for analysing food and food ingredients, respectively. The faecal inoculum was obtained from in vitro colonic fermentation from different donors at distinct time points. The proposed extraction protocol, which consisted of a simple monophasic solvent system composed of methanol, isopropanol, and methyl tert-butyl ether was further utilized for non-targeted analysis of both endogenous and exogenous metabolites including primary and secondary bile acids, amino acids, terpenoids and polyphenols. The solvent system also provides a good recovery for lipids compared to a standard lipidomics extraction protocol. Furthermore, we demonstrated that the solvent system is suitable for targeted analysis of short-chain fatty acids, TCA cycle-related metabolites, and tryptophan metabolites upon derivatization. Using SIMPLIFY, we significantly reduced the sample amount needed for extraction, minimized overall analysis time and cost, and demonstrated its suitability for easy automation, thus representing a versatile and efficient approach for comprehensive analysis of gut microbiota metabolism.