Mass spectrometry (MS)-based metabolomics analyses for fecal samples v1

This protocol describes the mass spectrometry–based metabolomics workflow used in the study “Metabolic reprogramming of the infant gut by bifidobacteria-based probiotics drives exclusion of antibiotic-resistant pathobionts” by Bargheet et al. (2026). Fecal metabolomic profiling was performed using complementary targeted and untargeted LC–MS/MS approaches. Samples collected in OMNImet•GUT tubes were processed by centrifugation and spin filtration, and metabolite signals were normalized to estimated dry weight. Targeted quantification of short-chain fatty acids and organic acids was performed after derivatization with 3-nitrophenylhydrazine using UPLC coupled to a triple quadrupole mass spectrometerin multiple reaction monitoring mode. Quantification was based on isotopically labeled internal standards and external calibration curves. Untargeted metabolomic profiling was performed using hydrophilic interaction chromatography coupled to high-resolution Orbitrap mass spectrometry with data-dependent acquisition. Quality control procedures included pooled QC samples, internal standards, blanks, and drift monitoring throughout acquisition. Raw data were processed using MZmine for feature detection and alignment, followed by molecular annotation with SIRIUS and molecular networking in GNPS. Statistical analyses and normalization were performed in MetaboAnalyst. Together, this workflow enabled quantitative assessment of targeted microbial metabolites and high-resolution profiling of global metabolomic changes in fecal samples.