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

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 / metabolome / triple quadrupole mass spectrometry / germ-free mice

References

[1] Ghosh TS,Shanahan F,O'Toole PW. The gut microbiome as a modulator of healthy ageing Nat Rev Gastroenterol Hepatol. 2022 19 565 84 PMC9035980

[2] Karu N,Deng L,Slae M.et al. A review on human fecal metabolomics: Methods, applications and the human fecal metabolome database Anal Chim Acta. 2018 1030 1 24

[3] Chen MX,Wang SY,Kuo CH,Tsai IL. Metabolome analysis for investigating host-gut microbiota interactions J Formos Med Assoc. 2019 118 Suppl 1 S10 22

[4] Pope R,Visconti A,Zhang X.et al. Faecal metabolites as a readout of habitual diet capture dietary interactions with the gut microbiome Nat Commun. 2025 16 10051 PMC12678775

[5] Franzosa EA,Sirota-Madi A,Avila-Pacheco J.et al. Gut microbiome structure and metabolic activity in inflammatory bowel disease Nat Microbiol. 2019 4 293 305 PMC6342642

[6] Vich Vila A,Hu S,Andreu-Sánchez S.et al. Faecal metabolome and its determinants in inflammatory bowel disease Gut. 2023 72 1472 85 PMC10359577

[7] Vich Vila A,Zhang J,Liu M,Faber KN,Weersma RK. Untargeted faecal metabolomics for the discovery of biomarkers and treatment targets for inflammatory bowel diseases Gut. 2024 73 1909 20 PMC11503092

[8] Yasuda S,Okahashi N,Tsugawa H.et al. Elucidation of Gut Microbiota-Associated Lipids Using LC-MS/MS and 16S rRNA Sequence Analyses iScience. 2020 23 101841 PMC7721639

[9] Wei R,Li G,Seymour AB. High-throughput and multiplexed LC/MS/MRM method for targeted metabolomics Anal Chem. 2010 82 5527 33

[10] Lu W,Bennett BD,Rabinowitz JD. Analytical strategies for LC-MS-based targeted metabolomics J Chromatogr B Analyt Technol Biomed Life Sci. 2008 871 236 42 PMC2607197

[11] Sarmad S,Viant MR,Dunn WB.et al. ; UK Consortium on Metabolic Phenotyping (MAP/UK). A proposed framework to evaluate the quality and reliability of targeted metabolomics assays from the UK Consortium on Metabolic Phenotyping (MAP/UK) Nat Protoc. 2023 18 1017 27

[12] Ribbenstedt A,Ziarrusta H,Benskin JP. Development, characterization and comparisons of targeted and non-targeted metabolomics methods PLoS ONE. 2018 13 e0207082 PMC6237353

[13] Costea PI,Zeller G,Sunagawa S.et al. Towards standards for human fecal sample processing in metagenomic studies Nat Biotechnol. 2017 35 1069 76

[14] Mori H,Maruyama T,Yano M,Yamada T,Kurokawa K. VITCOMIC2: visualization tool for the phylogenetic composition of microbial communities based on 16S rRNA gene amplicons and metagenomic shotgun sequencing BMC Syst Biol. 2018 12 Suppl 30 PMC5861490

[15] Wahlström A,Sayin SI,Marschall HU,Bäckhed F. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism Cell Metab. 2016 24 41 50

[16] Collins SL,Stine JG,Bisanz JE,Okafor CD,Patterson AD. Bile acids and the gut microbiota: metabolic interactions and impacts on disease Nat Rev Microbiol. 2023 21 236 47 PMC12536349

[17] Pang Z,Chong J,Zhou G.et al. MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights Nucleic Acids Res. 2021 49 W388 96 PMC8265181

[18] Wakita Y,Shimomura Y,Kitada Y,Yamamoto H,Ohashi Y,Matsumoto M. Taxonomic classification for microbiome analysis, which correlates well with the metabolite milieu of the gut BMC Microbiol. 2018 18 188 PMC6240276

[19] Schwaiger-Haber M,Stancliffe E,Arends V,Thyagarajan B,Sindelar M,Patti GJ. A workflow to perform targeted metabolomics at the untargeted scale on a triple quadrupole mass spectrometer ACS Meas Sci Au. 2021 1 35 45 PMC8377714

[20] Zheng X,Qiu Y,Zhong W.et al. A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids Metabolomics. 2013 9 818 27 PMC3756605

[21] Liao Z,Zhang S,Liu W.et al. LC-MS-based metabolomics analysis of Berberine treatment in ulcerative colitis rats J Chromatogr B Analyt Technol Biomed Life Sci. 2019 1133 121848

[22] Nagatomo R,Okada Y,Ichimura M,Tsuneyama K,Inoue K. Application of 2-picolylamine derivatized ultra-high performance liquid chromatography tandem mass spectrometry for the determination of short-chain fatty acids in feces samples Anal Sci. 2018 34 1031 6

[23] Li H,Liu Y,Zhang X.et al. Medium-chain fatty acids decrease serum cholesterol via reduction of intestinal bile acid reabsorption in C57BL/6J mice Nutr Metab (Lond). 2018 15 37 PMC5987598

[24] Matsumoto M,Kunisawa A,Hattori T.et al. Free D-amino acids produced by commensal bacteria in the colonic lumen Sci Rep. 2018 8 17915 PMC6297366

[25] Song Y,Song Q,Li J.et al. An integrated platform for directly widely-targeted quantitative analysis of feces part II: an application for steroids, eicosanoids, and porphyrins profiling J Chromatogr A. 2016 1460 74 83

[26] Matsumoto M,Kibe R,Ooga T.et al. Impact of intestinal microbiota on intestinal luminal metabolome Sci Rep. 2012 2 233 PMC3380406

[27] Ling CW,Miao Z,Xiao ML.et al. The association of gut microbiota with osteoporosis is mediated by amino acid metabolism: multiomics in a large cohort J Clin Endocrinol Metab. 2021 106 e3852 64

[28] Mishima E,Fukuda S,Mukawa C.et al. Evaluation of the impact of gut microbiota on uremic solute accumulation by a CE-TOFMS-based metabolomics approach Kidney Int. 2017 92 634 45

[29] Deda O,Virgiliou C,Orfanidis A,Gika HG. Study of fecal and urinary metabolite perturbations induced by chronic ethanol treatment in mice by UHPLC-MS/MS targeted profiling Metabolites. 2019 9 232 PMC6836053

[30] Trošt K,Ahonen L,Suvitaival T.et al. Describing the fecal metabolome in cryogenically collected samples from healthy participants Sci Rep. 2020 10 885 PMC6972823

[31] Louis P,Flint HJ. Formation of propionate and butyrate by the human colonic microbiota Environ Microbiol. 2017 19 29 41

[32] Nagatake T,Kunisawa J. Emerging roles of metabolites of ω3 and ω6 essential fatty acids in the control of intestinal inflammation Int Immunol. 2019 31 569 77 PMC6736389

[33] Hudson JA,Morvan B,Joblin KN. Hydration of linoleic acid by bacteria isolated from ruminants FEMS Microbiol Lett. 1998 169 277 82

[34] Kishino S,Takeuchi M,Park SB.et al. Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition Proc Natl Acad Sci U S A. 2013 110 17808 13 PMC3816446

[35] Miyamoto J,Mizukure T,Park SB.et al. A gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, ameliorates intestinal epithelial barrier impairment partially via GPR40-MEK-ERK pathway J Biol Chem. 2015 290 2902 18 4317025

[36] Simopoulos AP. An Increase in the Omega-6/Omega-3 fatty acid ratio increases the risk for obesity Nutrients. 2016 8 128 PMC4808858

[37] Shinohara M,Serhan CN. Novel endogenous proresolving molecules: essential fatty acid-derived and gaseous mediators in the resolution of inflammation J Atheroscler Thromb. 2016 23 655 64 PMC7399282

[38] Ungaro F,Rubbino F,Danese S,D'Alessio S. Actors and Factors in the Resolution of Intestinal Inflammation: Lipid Mediators As a New Approach to Therapy in Inflammatory Bowel Diseases Front Immunol. 2017 8 1331 PMC5660440

[39] Serhan CN,Chiang N,Dalli J. The resolution code of acute inflammation: Novel pro-resolving lipid mediators in resolution Semin Immunol. 2015 27 200 15 PMC4515371

[40] Jaén RI,Sánchez-García S,Fernández-Velasco M,Boscá L,Prieto P. Resolution-based therapies: the potential of lipoxins to treat human diseases Front Immunol. 2021 12 658840 PMC8102821

[41] Zhao Z,Ning J,Bao XQ.et al. Fecal microbiota transplantation protects rotenone-induced Parkinson's disease mice via suppressing inflammation mediated by the lipopolysaccharide-TLR4 signaling pathway through the microbiota-gut-brain axis Microbiome. 2021 9 226 PMC8597301

[42] Zhang X,Coker OO,Chu ES.et al. Dietary cholesterol drives fatty liver-associated liver cancer by modulating gut microbiota and metabolites Gut. 2021 70 761 74 PMC7948195

[43] Sato Y,Atarashi K,Plichta DR.et al. Novel bile acid biosynthetic pathways are enriched in the microbiome of centenarians Nature. 2021 599 458 64