Untargeted LC–MS/MS-Based Metabolomics Across Clinical Stages of Fecal Microbiota Transplantation

Abstract

Background: Fecal microbiota transplantation (FMT) has emerged as a therapeutic strategy to restore gut microbial balance in gastrointestinal disorders. While alterations in gut microbiota and metabolites are known to influence disease progression and treatment outcomes, longitudinal metabolomics data following FMT remain limited.

Methods: Untargeted metabolomics was performed using a Thermo Scientific Q Exactive LC–MS/MS system. Fecal samples from three time points—healthy donor, pre-FMT, and post-FMT—were collected and analyzed. Fecal samples (0.1 g wet; 20–40 mg freeze-dried) were processed using an ACN/MeOH (8:2, v/v) extraction at −20 °C, followed by concentration (≤30 °C) and reconstitution for LC–MS/MS analysis in positive and negative ionization modes. Heptadecanoic acid served as the internal standard.

Results: Distinct metabolomic profiles were observed across clinical stages. Multivariate analysis revealed clear separation between pre- and post-FMT samples. Differential metabolite analysis indicated changes in multiple pathways, particularly amino acid and lipid metabolism, suggesting treatment-related metabolic shifts alongside disease-associated alterations.

Conclusion: This study demonstrates the feasibility of longitudinal metabolomics profiling in FMT and reveals systemic metabolic changes across treatment stages. These findings provide mechanistic insights into host–microbiome–metabolite interactions and highlight the potential of metabolomics for identifying biomarkers of therapeutic response in FMT.