Development of a multi-targeted metabolomics platform for semi-quantification of faecal metabolites: a proof-of-concept analysis in human faeces

  • Microbiome Res Rep. 2026 Mar 26;5(1):7. doi: 10.20517/mrr.2025.85.
Kayo Ikuta  1 ,  Akihiro Kunisawa  2  1 ,  Itaru Dekio  1 ,  Arisa Ito  3 ,  Qiuyi Wang  2  3 ,  Kazuhiro Kawamura  2  3  4 ,  Masaki Yamada  2 ,  Sumi Nakamura  5  6 ,  Yoshihiro Hayakawa  2 ,  Takuma Higurashi  7 ,  Junko Iida  2  3 ,  Eiichiro Fukusaki  3  4 ,  Toru Suzuki  8  9 ,  Mitsuharu Matsumoto  5  3
Affiliations
  • 1. These authors contributed equally to this work.
  • 2. Shimadzu Corporation, Kyoto 604-8511, Japan.
  • 3. The University of Osaka and Shimadzu Analytical Innovation Research Laboratories, The University of Osaka, Osaka 565-0871, Japan.
  • 4. Department of Biotechnology, Graduate School of Engineering, The University of Osaka, Osaka 565-0871, Japan.
  • 5. Dairy Science and Technology Institute, Kyodo Milk Industry Co. Ltd, Tokyo 190-0182, Japan.
  • 6. BC LAB, Osaka 541-0047, Japan.
  • 7. Department of Gastroenterology and Hepatology, Yokohama City University School of Medicine, Yokohama 236-0004, Japan.
  • 8. Leicester NIHR Biomedical Research Centre and BHF Centre of Research Excellence, University of Leicester, Leicester LE1 7RH, UK.
  • 9. The Institute of Medical Science, The University of Tokyo, Tokyo 108-0071, Japan.
PMID: 42016721 DOI: 10.20517/mrr.2025.85
Abstract

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.

Keywords
Intestinal microbiome; faeces; germ-free mice; metabolome; triple quadrupole mass spectrometry.
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