Dual UHPLC-HRMS Metabolomics and Lipidomics and Automated Data Processing Workflow for Comprehensive High-Throughput Gut Phenotyping

In recent years, feces has surfaced as the matrix ofchoice forinvestigating the gut microbiome-health axis because of its non-invasivesampling and the unique reflection it offers of an individual'slifestyle. In cohort studies where the number of samples requiredis large, but availability is scarce, a clear need exists for high-throughputanalyses. Such analyses should combine a wide physicochemical rangeof molecules with a minimal amount of sample and resources and downstreamdata processing workflows that are as automated and time efficientas possible. We present a dual fecal extraction and ultra high performanceliquid chromatography-high resolution-quadrupole-orbitrap-mass spectrometry(UHPLC-HR-Q-Orbitrap-MS)-basedworkflow that enables widely targeted and untargeted metabolome andlipidome analysis. A total of 836 in-house standards were analyzed,of which 360 metabolites and 132 lipids were consequently detectedin feces. Their targeted profiling was validated successfully withrespect to repeatability (78% CV < 20%), reproducibility (82% CV< 20%), and linearity (81% R (2) >0.9),while also enabling holistic untargeted fingerprinting (15,319 features,CV < 30%). To automate targeted processing, we optimized an R-basedtargeted peak extraction (TaPEx) algorithm relying on a database comprisingretention time and mass-to-charge ratio (360 metabolites and 132 lipids),with batch-specific quality control curation. The latter was benchmarkedtoward vendor-specific targeted and untargeted software and our isotopologueparameter optimization/XCMS-based untargeted pipeline in LifeLinesDeep cohort samples (n = 97). TaPEx clearly outperformedthe untargeted approaches (81.3 vs 56.7-66.0% compounds detected).Finally, our novel dual fecal metabolomics-lipidomics-TaPExmethod was successfully applied to Flemish Gut Flora Project cohort(n = 292) samples, leading to a sample-to-resulttime reduction of 60%.