Oral Presentation Royal Australian Chemical Institute National Congress 2026

Development of a novel analytical method for faecal untargeted metabolomics using gas chromatography-mass spectrometry for simultaneous analysis of both volatile and non-volatile compounds in human faeces. (138150)

Angelina Nikitkina 1 , Hayley Abbiss 1 , Stacey Reinke 1 , Claus Christophersen 1 , David Broadhurst 1
  1. Edith Cowan University, Perth, PERTH, Australia

At present, metabolomic analysis of faeces is gaining significant interest, as the composition of the faecal metabolome reflects gut microbiome state, while also linking human health and disease with symbiotic microorganisms inhabiting the gastrointestinal tract [1-4]. A typical sample processing methodology for faecal metabolomics involves extraction of molecules of interest using different compositions of polar or non-polar solvents followed by an evaporation step and reconstitution in trimethylsilyl derivatising agent [5-9]. Trimethylsilylation is an effective derivatisation method which is extensively applied in gas-chromatography-based metabolomics to reduce polarity of compounds and increase volatility, enabling their elution without thermal degradation and enhancing ionization efficiency [5, 9-11]. However, the evaporation step inevitably leads to the loss of volatile metabolites [5, 12], which possess significant biological relevance for investigating host–microbiome interactions [13-15]. The objective of this research was to develop a novel, broad spectrum, optimised, and reproducible analytical methodology for GC-MS enabling analysis of both volatile and non-volatile compounds from human faeces. A set of 35 reference standards representing fatty acid metabolism, amino acid metabolism, carbohydrate metabolism, indole metabolism, lipid oxidation metabolism and volatile sulphur compounds spiked into a synthetic faecal sample was used to optimise and evaluate the analytical procedure. Acetonitrile was used to precipitate proteins, extract volatile compounds from faeces and as a derivatisation medium. Different salting-out strategies were tested to achieve efficient water removal for successful trimethylsilyl derivatisation, thereby eliminating the need for an evaporation step. Finally, the developed method will be applied to human faecal samples and benchmarked against a conventional sample preparation protocol for trimethylsilylation of faecal metabolites to assess coverage and reproducibility, and these results will be presented.  

 

References

 

  1. Puig-Castellví, F., et al., Advances in the integration of metabolomics and metagenomics for human gut microbiome and their clinical applications. TrAC Trends in Analytical Chemistry, 2023. 167.
  2. Conley, T.E., et al., Microbiome-driven IBS metabotypes influence response to the low FODMAP diet: insights from the faecal volatome. eBioMedicine, 2024. 107.
  3. Karu, N., et al., A review on human fecal metabolomics: Methods, applications and the human fecal metabolome database. Anal Chim Acta, 2018. 1030: p. 1-24.
  4. Sun, B., et al., Integrative plasma and fecal metabolomics identify functional metabolites in adenoma-colorectal cancer progression and as early diagnostic biomarkers. Cancer Cell, 2024. 42(8).
  5. Mojsak, P., et al., The role of gut microbiota (GM) and GM-related metabolites in diabetes and obesity. A review of analytical methods used to measure GM-related metabolites in fecal samples with a focus on metabolites' derivatization step. Journal of pharmaceutical and biomedical analysis, 2020. 191.
  6. Jain, A., et al., An untargeted fecal and urine metabolomics analysis of the interplay between the gut microbiome, diet and human metabolism in Indian and Chinese adults. Scientific Reports 2019 9:1, 2019-06-24. 9(1).
  7. Yin, S., et al., Optimization of GC/TOF MS analysis conditions for assessing host-gut microbiota metabolic interactions: Chinese rhubarb alters fecal aromatic amino acids and phenol metabolism. Analytica Chimica Acta, 2017/12/01. 995.
  8. Trošt, K., et al., Describing the fecal metabolome in cryogenically collected samples from healthy participants. Scientific Reports, 2020 Jan 21. 10(1).
  9. Gao, X., E. Pujos-Guillot, and J.-L. Sébédio, Development of a Quantitative Metabolomic Approach to Study Clinical Human Fecal Water Metabolome Based on Trimethylsilylation Derivatization and GC/MS Analysis. Analytical Chemistry, July 14, 2010. 82(15).
  10. Deda, O., et al., Sample preparation optimization in fecal metabolic profiling. Journal of Chromatography B, 2017/03/15. 1047.
  11. López-Bascón, M.A., et al., Comprehensive analysis of pig feces metabolome by chromatographic techniques coupled to mass spectrometry in high resolution mode: Influence of sample preparation on the identification coverage. Talanta, 2019/07/01. 199.
  12. Zhang, S., H. Wang, and M.-J. Zhu, A sensitive GC/MS detection method for analyzing microbial metabolites short chain fatty acids in fecal and serum samples. Talanta, 2019/05/01. 196.
  13. Dalis, C., et al., Volatile Organic Compound Assessment as a Screening Tool for Early Detection of Gastrointestinal Diseases. 2023. 11(7).
  14. Zhang, V.R.-Y., et al., Volatile organic compounds as potential biomarkers of irritable bowel syndrome: A systematic review. Neurogastroenterology & Motility, 2023. 35(7).
  15. Bond, A., et al., Volatile organic compounds emitted from faeces as a biomarker for colorectal cancer. Alimentary Pharmacology & Therapeutics, 2019. 49(8).