A Dual UHPLC-HRMS-Based Fecal Metabolomics and Lipidomics Analysis and Automated Data Processing Pipeline for Comprehen-sive 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 is large, but sample availability scarce, a clear need exists for high-throughput analyses. Such analyses should com-bine 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 analysis. 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%.