A Dual UHPLC-HRMS-Based Fecal Metabolomics and Lipidomics Analysis and Automated Data Processing Pipeline for Comprehensive Gut Phenotyping

In recent years, feces has surfaced as the matrix of choice for investigating the gut microbiome-health axis because of its non-invasive sampling and the unique reflection it offers of an individual’s lifestyle. In cohort studies where the number of samples required is large, but availability is scarce, a clear need exists for high-throughput analyses. Such analyses should combine a wide physicochemical range of molecules with a minimal amount of sample and resources, and downstream data processing workflows that are as automated and time efficient as possible. We present a dual fecal extraction and UHPLC-HR-Q-Orbitrap-MS-based workflow that enables widely targeted and untargeted metabolome and lipidome analy-sis. A total of 836 in-house standards were analyzed, of which 360 metabolites and 132 lipids were consequently detected in feces. Their targeted profiling was validated successfully with respect to repeatability (78% CV<20%), reproducibility (82% CV<20%) and linearity (81% R2>0.9), while also enabling holistic untargeted fingerprinting (15 319 features, CV<30%). To automate targeted processing, we optimized an R-based targeted peak extraction (TaPEx) algorithm relying on a database comprising retention time and mass-to-charge ratio (360 metabolites and 132 lipids), with batch-specific quality control curation. The latter was benchmarked towards vendor-specific targeted and untargeted software and our IPO/XCMS-based untargeted pipeline in Lifelines Deep cohort samples (n = 97). TaPEx clearly outperformed the untargeted approaches (81.3 vs. 56.7-66.0% compounds detected). Finally, our novel dual fecal metabolomics-lipidomics-TaPEx method was successful-ly applied to Flemish Gut Flora Project cohort (n = 292) samples, leading to a sample-to-result time reduction of 60%.