Development of a multi-targeted metabolomics platform for semi-quantification of faecal metabolites: a...

Objectives: Targeted metabolomic analysis of faecal samples has been limited by narrow chemical coverage. Here, we established a multiplexed, triple quadrupole mass spectrometry (TQMS)-based targeted metabolomics workflow. This workflow allows accurate detection and semi-quantification of diverse faecal metabolites and provides a methodological platform for studying host-microbiome metabolic interactions. Methods: Faecal metabolomes from germ-free (GF) mice, ex-germ-free (Ex-GF) mice, and human participants were analysed using TQMS-based targeted metabolomics. The analysis comprised multiple methods targeting amino acids and their derivatives, carbohydrates, short-chain fatty acids, bile acids, lipid mediators, and phospholipids. Results: In total, 607 low-molecular-weight metabolites in 44 chemical categories were detected and semi-quantified. Faecal metabolomes of GF and Ex-GF mice were analysed, uncovering 341 intestinal microbiome-dependent metabolites. A proof-of-concept analysis using faecal samples from five patients with colorectal cancer demonstrated the successful application of this platform to human clinical material, highlighting its strong potential for future disease-oriented metabolomic investigations. Conclusion: We developed a multi-targeted faecal metabolomics platform that substantially expands the chemical space accessible to targeted analysis. This workflow provides a methodological foundation for future large-scale and translational studies.