Untargeted and Semi-Targeted Metabolomics Approach for Profiling Small Intestinal and Fecal Metabolome Using High-Resolution Mass Spectrometry

The gut microbiome is a complex ecosystem varying along different gut sections, consisting of metabolites from food, host, and microbes. Microbially derived metabolites like bile acids and short chain fatty acids interact with host physiology. Current studies often use fecal samples, which don′t fully represent the upper gut due to stratification. To sample the proximal gut microbiome, endoscopic methods or new non invasive devices are used. We developed an approach combining untargeted and semi−targeted metabolomics using a QExactive Plus Orbitrap mass spectrometer to profile gut metabolites. We initially selected forty nine key metabolites based on specific criteria, validated them through repeatability tests, and created a compound database with TraceFinder software. Our workflow enables molecule annotation in untargeted studies while validating thirty seven metabolites in semi−targeted analyses. This method, applied to clinical trial samples, shows promise in discovering new gut metabolites.