Analysis of Short-Chain Fatty Acids and Organic Acids in Feces Using a Single Quadrupole Mass Spectrometer

Importance of the Topic

The analysis of short-chain fatty acids (SCFAs) and organic acids in fecal samples provides critical insights into gut microbial metabolism and its influence on host health. SCFAs such as acetate, propionate and butyrate play key roles in energy homeostasis, immune regulation and gut barrier function. Sensitive and straightforward analytical approaches are essential for advancing research in gut microbiota, disease diagnostics and dietary interventions.

Objectives and Study Overview

This study demonstrates a single-quadrupole liquid chromatography–mass spectrometry (LC-MS) method for simultaneous quantification of six SCFAs and sixteen central organic acids in monkey feces. By applying derivatization and a compact LCMS-2050 system, the authors aim to improve detection sensitivity and reproducibility versus conventional LC alone. Fecal samples S1–S5 were collected from three geographic locations and analyzed to explore sample variability and multivariate patterns.

Methodology

Sample Collection and Storage

• Feces from five monkeys at locations A, B and C were frozen onsite and stored at –80 °C.
• Collection intervals ranged from immediate freezing to within one day.

Sample Pretreatment

• 100 mg feces was extracted with phosphate-buffered saline and methanol, mixed, cooled and centrifuged.
• Supernatant underwent ultrafiltration (3 kDa cutoff) to remove macromolecules.
• Derivatization employed 3-nitrophenylhydrazine, pyridine and EDC, with 2-ethylbutyric acid as an internal standard, incubated 30 min.

Used Instrumentation

• Liquid Chromatograph: Shimadzu Nexera XR series with Mastro 2 C18 column (2.0 mm × 150 mm, 3 µm).
• Mass Spectrometer: Shimadzu LCMS-2050 single-quadrupole system equipped with DUIS™ dual ion source (ESI/APCI), mass range m/z 2–2000, SIM mode (23 events).
• Mobile Phase: 0.1% formic acid in water (A) and acetonitrile (B), gradient elution at 0.35 mL/min, injection volume 3 µL.
• Data Analysis: Shimadzu Multi-omics Analysis Package for multivariate statistics.

Key Results and Discussion

A total of 21 SCFAs and organic acids were detected with %RSD values below 5% for most analytes, confirming high reproducibility. Comparison to LC-only analysis showed enhanced sensitivity and detection of isomeric pairs (isobutyric/butyric, isovaleric/valeric acids). Principal component analysis separated sample S1 from others by elevated levels of pyruvate, lactate and α-ketoglutarate and reduced acetate, propionate and butyrate. Samples S2 and S3 grouped closely, reflecting similar diets. Samples S4 and S5 exhibited distinct profiles along PC2, with S5 enriched in citric and isocitric acids. Hierarchical clustering supported these findings, indicating that dietary variation and microbial composition drive metabolite differences.

Benefits and Practical Applications

• High sensitivity for low-abundance SCFAs and organic acids improves detection limits compared to LC methods.
• Capability to resolve structural isomers enhances metabolite profiling accuracy.
• Compact single-quadrupole LC-MS system is cost-effective and user-friendly for laboratories without advanced MS expertise.
• Derivatization strategy increases retention on reversed-phase columns and boosts MS response.

Future Trends and Potential Applications

Integration of this approach with broader multi-omics workflows can deepen understanding of host-microbiome interactions. The method may be adapted to human clinical samples, dietary intervention studies and environmental microbiology. Automation of sample preparation and high-throughput screening platforms will accelerate biomarker discovery. Advances in data analytics and machine learning could refine multivariate interpretations and predictive modeling of gut health.

Conclusion

This work establishes a robust, reproducible and sensitive single-quadrupole LC-MS platform for comprehensive analysis of SCFAs and organic acids in feces. Its simplicity, affordability and performance make it suitable for diverse research and quality-control laboratories. Widespread adoption of this method is expected to advance gut microbiota studies and facilitate new discoveries in microbial metabolite profiling.

Reference

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