Simultaneous Analysis of Hydrophilic Metabolites in Feces Using a Single Quadrupole Mass Spectrometer

Significance of the Topic

Analysis of hydrophilic metabolites in feces provides critical insights into host–microbiome interactions, disease mechanisms and nutritional effects. Mapping a broad range of polar compounds can reveal biomarkers for intestinal health, drug response and functional food efficacy.

Objectives and Overview of the Study

This study aimed to establish a simple, high-throughput method for simultaneous profiling of hydrophilic metabolites in fecal samples using a single quadrupole LC-MS system. By leveraging a combined extraction and multivariate data package, the work seeks to enable comprehensive metabolomic analysis with minimal instrumentation complexity.

Methodology

Sample Collection and Preparation:

• Feces from five Japanese macaques collected at three distinct field locations, immediately frozen at –80 °C.
• 100 mg of sample extracted with 700 µL phosphate-buffered saline, vortexed, cooled, centrifuged and ultrafiltered (MWCO 3 kDa).
• Filtrate diluted tenfold in water containing 10 µM MES internal standard.

Data Analysis:

• Peak area ratios calculated against the internal standard.
• Principal component analysis performed using the Multi-omics Analysis Package to identify characteristic metabolites by sampling site.

Used Instrumentation

• Nexera XR HPLC system with Shim-pack GIST PFPP column (2.1 × 150 mm, 3 µm).
• LCMS-2050 single quadrupole mass spectrometer equipped with a DUIS dual ESI/APCI ion source (mass range m/z 2–2000).
• HPLC gradient: 0.1% formic acid in water/acetonitrile, 0.25 mL/min (ramped to 0.5 mL/min).
• MS: SIM mode with 143 events in positive and negative ionization; nebulizing gas 2.5 L/min; desolvation 500 °C; DL 250 °C.

Results and Discussion

The method targeted 143 polar metabolites and detected 66 compounds across amino acids, organic acids, nucleosides and nucleotides. Principal component analysis revealed clustering by collection site:

• Location A (S1): elevated 4-aminobutyric acid, carnitine and choline.
• Location B (S2, S3): enrichment of purine derivatives (xanthine, hypoxanthine).
• Location C (S4, S5): higher levels of organic acids (citric and caffeic acids).

These variations likely reflect dietary or environmental differences influencing microbial metabolism.

Benefits and Practical Applications

The single quadrupole LC-MS approach offers:

• Cost-effective operation and simplified maintenance compared to triple quadrupole platforms.
• Rapid, broad-spectrum metabolite coverage suited for routine laboratory use.
• Integrated software tools for streamlined multivariate analysis and biomarker discovery.

Future Trends and Potential Applications

Expanding single quadrupole metabolomics could drive advances in microbiome research, nutritional science and clinical diagnostics. Integration with other omics layers, automation of sample preparation and miniaturized platforms may further increase throughput and applicability in field and point-of-care settings.

Conclusion

This work demonstrates that a single quadrupole LC-MS system, combined with straightforward sample preparation and multivariate data analysis, can effectively profile a wide range of hydrophilic fecal metabolites. The approach balances performance with accessibility, supporting broader adoption in microbiota and metabolomics studies.

Reference

• S. Tsuchida, T. Hattori, A. Sawada, K. Ogata, J. Watanabe, K. Ushida: Fecal metabolite analysis of Japanese macaques in Yakushima by LC-MS/MS and LC-QTOF-MS, J Vet Med Sci. 83(6):1012–1015 (2021).