Untargeted Fecal Metabolomic Analysis

Untargeted fecal metabolomic analysis was performed by Metabolon, Inc. (Durham, NC) as previously described^{63 (link)–65 (link)}. Metabolites were extracted from lyophilized and homogenized samples using methanol extraction. Extracts were analyzed by an ACQUITY ultra-performance liquid chromatographer (Waters, Milford, CA) and a ThermoFisher Scientific Q-Exactive high resolution mass spectrometer interfaced with a heated electrospray ionization (HESI-II) source and Orbitrap mass analyzer (ThermoFisher Scientific, Waltham, Massachusetts) operated at 35,000 mass resolution. The scan range covered 70–1000 m/z. Metabolite identification was performed by automated comparison of the ion features in the experimental samples to a reference library. Chemical standard entries included retention time, molecular weight (m/z), preferred adducts, and in-source fragments as well as associated MS spectra, and were curated by visual inspection for quality control using software developed at Metabolon. Peaks were quantified using the area-under-the-curve. Compounds were corrected for inter-day variation by registering the medians to equal 1.00 and normalizing each data point proportionally. Missing values were imputed with the observed minimum for each compound.